leupeptins has been researched along with leupeptin* in 893 studies
8 review(s) available for leupeptins and leupeptin
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Natural products from thioester reductase containing biosynthetic pathways.
Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products. Topics: Alkaloids; Azabicyclo Compounds; Benzodiazepinones; Biological Products; Biosynthetic Pathways; Cyclization; Depsipeptides; Dipeptides; Indoles; Lactams; Leupeptins; Lysine; Multigene Family; Peptide Synthases; Polyketide Synthases; Protein Domains | 2018 |
Peroxisome degradation in mammals.
This review summarizes the historical aspects of the study of peroxisome degradation in mammalian cells. Peroxisomes have diverse metabolic roles in response to environmental changes and are degraded in a preferential manner, by comparison with cytosolic proteins. This review introduces three hypotheses on the degradation mechanisms: (a) the action of the peroxisome-specific Lon protease; (b) the membrane disruption effect of 15-lipoxygenase; and (c) autophagy that sequesters and degrades the organelles by lysosomal enzymes. Among these hypotheses, autophagy is now recognized as the most important mechanism for excess peroxisome degradation. One of the most striking characteristics of peroxisomes is that they are markedly proliferated in the liver by the administration of hypolipidemic drugs and industrial plasticizers. The effects of these substances were fully reversed after withdrawal of the substances, and most of the excess peroxisomes were selectively degraded and recovered to a normal number and size. Autophagic degradation of peroxisomes has been examined using this characteristic phenomenon. Excessive peroxisome degradation that occurs after cessation of hypolipidemic drugs has been extensively investigated biochemically and morphologically. The evidence shows that the degradation of excess peroxisomes and peroxisomal enzymes is inhibited by 3-methyladenine (3-MA), a specific inhibitor of autophagy. Furthermore, in liver-specific autophagy-deficient mice, rapid removal of peroxisomes was exclusively impaired, and degradation of peroxisomal enzymes was not detected. Thus, the significant contribution of autophagic machinery to peroxisomal degradation in mammals was confirmed. However, the important question of the mechanism for the selective recognition of peroxisomes by autophagosomes remains to be fully elucidated. Topics: Animals; Arachidonate 15-Lipoxygenase; Autophagy; Cells, Cultured; Half-Life; Humans; Hypolipidemic Agents; Leupeptins; Mammals; Peroxisomes; Protease La; Ubiquitination | 2011 |
Calpain inhibitors as therapeutic agents in nerve and muscle degeneration.
It seems plausible to hypothesize that in all forms of neurodegeneration or other forms of tissue degeneration, a common pathway exists that, when deciphered, could lead to our understanding of a variety of diseases that result in tissue necrosis, as well as offer potential for therapeutic intervention. In recent years progress toward elucidating this common pathway has been accelerated through the studies of a number of laboratories, including our own, on the role of the protease calpain in this process. Thus, in a variety of disorders, such as stroke, spinal cord injury, traumatic nerve injury, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, muscular dystrophy, cataract formation, unregulated calpain proteolysis, initiated via dysregulation of calcium ion homeostasis, participates in the pathogenesis and is a potentially unifying mechanistic event. In order to demonstrate the feasibility of the approach we have taken in using the calpain inhibitor leupeptin as a therapeutic agent, I will describe two areas of research in which we have been engaged over the past 20 years. One is our long-standing interest in muscular dystrophy. The other is of more recent vintage, and involves the use of calpain inhibitors to protect sensory hair cells and spiral ganglion neurons from damage associated with acoustic trauma, this latter in collaboration with Dr. R. Salvi at SUNY-Buffalo and Dr. A. Shulman at SUNY-Downstate. Topics: Animals; Glycoproteins; Hair Cells, Auditory; Haplorhini; Hearing Loss, Noise-Induced; Leupeptins; Mice; Mice, Inbred C57BL; Muscular Dystrophy, Animal; Nerve Degeneration; Spiral Ganglion | 1999 |
Transport and expression of HLA class-II glycoproteins.
Topics: B-Lymphocytes; Cell Line; Endocytosis; Endopeptidases; Gene Expression Regulation; Genes, MHC Class II; HLA-D Antigens; Humans; Leupeptins; Membrane Glycoproteins; Protein Processing, Post-Translational | 1990 |
Quantitative aspects of cellular autophagocytosis. Morphometric and cell fractionation studies.
The mode of action of agents and treatments known to induce the appearance of autophagic vacuoles (AV) were studied in mouse liver, pancreatic and seminal vesicle cells by morphometric evaluation of the time-dependent changes in the volume fraction of AVs following suppression of sequestration by cycloheximide. Rapid decrease of the AV compartment was observed in cells of animals pretreated with agents including Triton X-100, pilocarpine, leupeptin and estron acetate. The half-life of AVs, estimated from the decay, range between 5.3 to 8.7 min, which does not deviates from normal steady state values. In contrast, no regression or very slow decay was seen in cells of animals pretreated with methylamine, chloroquine and vinblastine (VBL). We think that the main mechanisms responsible for the enlargement of the AV compartment in these experiments are: stimulation of the sequestration under the effect of agents which maintain the half-life of AVs within the physiologic range of 5-10 min and accumulation of AVs due to their prolonged lifetime under the effect of acidotropic agents and VBL. However, our data suggest than in addition to the accumulatory effect on AVs, stimulation of sequestration may also play a role in the enlargement of the AV compartment after treatment with VBL. While it was not possible to isolate a relatively pure fraction of early AVs from the liver, a light and a dense AV fraction was successfully purified in our laboratory from the pancreas treated with either VBL or neutral red. Preliminary enzymological and morphological evidence is presented that the light AVs are of autophagosomal, whereas the dense ones are of autolysosomal nature. Topics: Animals; Autophagy; Cell Compartmentation; Cell Fractionation; Chloroquine; Leupeptins; Liver; Lysosomes; Male; Methylamines; Pancreas; Phagocytosis; Rats; Seminal Vesicles; Vacuoles; Vinblastine | 1989 |
"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.
The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for i Topics: Animals; Breast; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Drug Resistance; Endometrium; Endopeptidases; Female; Humans; Kidney; Leukemia; Leupeptins; Liver; Lysine; Macromolecular Substances; Male; Molybdenum; Osmolar Concentration; Protein Conformation; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone; Substrate Specificity; Ultracentrifugation; Uterine Neoplasms | 1986 |
Calcium-dependent proteolytic stimulation of adenylate cyclase in platelets from spontaneously hypertensive rats.
Abnormalities of platelet aggregation and cyclic nucleotide metabolism are present in hypertension. We observed a greater increase in the level of cyclic adenosine monophosphate (AMP) after prostaglandin E1 (PGE1) stimulation and a lack of decrease of this cyclic nucleotide by epinephrine in platelets from spontaneously hypertensive rats (SHR) as compared to normotensive rats. The difference in cyclic AMP production between SHR and control rats in response to PGE1 is dependent upon platelet exposure to calcium. Since calcium and cyclic AMP are closely related and are both abnormally regulated in hypertension, we have studied the effect of calcium on adenylate cyclase activity. We show here that two forms of endogenous calcium-dependent proteases (membrane-bound and soluble) stimulate the basal activity and the hormonal responsiveness of adenylate cyclase. The sensitivity of calcium-dependent proteolytic control of adenylate cyclase to very-low concentrations of calcium indicates that the regulation may be physiologically important. Furthermore, calcium exerts a greater influence on platelet adenylate cyclase from SHR than on that from normotensive rats. The adenylate cyclase defect seems to be located in the membrane fraction and may, therefore, result from an increase in the activity of the membrane-bound calcium-protease or may be intrinsic to adenylate cyclase itself. The exact site that is sensitive to proteolysis remains to be established. Topics: Adenylyl Cyclases; Alprostadil; Animals; Blood Platelets; Calcium; Calmodulin; Cyclic AMP; Enzyme Activation; Epinephrine; Humans; Hypertension; Leupeptins; Peptide Hydrolases; Platelet Aggregation; Prostaglandins E; Rats; Rats, Inbred Strains; Renin | 1984 |
Are proteinase inhibitors potentially useful in tumor therapy?
Within the last 15 years a vast literature has arisen, which associates increased levels of proteinase activity with most in vitro transformed malignant cells and many tumor cells in vivo. As a consequence, proteinase inhibitors have been widely proposed as potential candidates for therapeutic use. The present review shows that in some studies proteinase inhibitors produced significant anti-tumor effects, while in most other studies only limited effects or no effects were observed. In some instances, opposite, or tumor enhancing, effects by proteinase inhibitors were observed. The reasons for the lack of a clear-cut success of proteinase inhibitors in tumor therapy may be: (1) proteinases may not be crucially involved in tumor growth and spread; (2) proteinases which may be crucially involved have not yet been identified; (3) lack of potent inhibitors with appropriate specificity, or use of inappropriate inhibitors or regimens. Topics: Animals; Antipain; Aprotinin; Blood Coagulation; Cathepsin B; Cathepsin D; Cathepsins; Endopeptidases; Humans; Leupeptins; Microbial Collagenase; Neoplasms; Pancreatic Elastase; Plasminogen Activators; Protease Inhibitors | 1982 |
885 other study(ies) available for leupeptins and leupeptin
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Mechanism of Microbial Metabolite Leupeptin in the Treatment of COVID-19 by Traditional Chinese Medicine Herbs.
Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (M Topics: Animals; Chlorocebus aethiops; COVID-19 Drug Treatment; Ecosystem; Humans; Leupeptins; Medicine, Chinese Traditional; SARS-CoV-2; Vero Cells | 2021 |
Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.
Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP. Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Autophagy; Cell Death; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Hepatocytes; JNK Mitogen-Activated Protein Kinases; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mitochondria; Proteins | 2021 |
Strong Binding of Leupeptin with TMPRSS2 Protease May Be an Alternative to Camostat and Nafamostat for SARS-CoV-2 Repurposed Drug: Evaluation from Molecular Docking and Molecular Dynamics Simulations.
The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial. Topics: Antiviral Agents; Benzamidines; COVID-19; COVID-19 Drug Treatment; Drug Repositioning; Esters; Guanidines; Humans; Leupeptins; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; SARS-CoV-2; Serine Endopeptidases | 2021 |
Thrombin alters the synthesis and processing of CYR61/CCN1 in human corneal stromal fibroblasts and myofibroblasts through multiple distinct mechanisms.
Previous research in our laboratory indicated that prothrombin and other coagulation enzymes required to activate prothrombin to thrombin are synthesized by the cornea and that apoptotic human corneal stromal cells can provide a surface for prothrombin activation through the intrinsic and extrinsic coagulation pathways. The purpose of the work reported here is to study the role of thrombin activity in the regulation of matricellular protein Cyr61 (CCN1) produced by wounded phenotype human corneal stromal fibroblasts and myofibroblasts.. Stromal cells from human donor corneas were converted to defined wounded phenotype fibroblasts and myofibroblasts with fetal bovine serum, followed by basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGFβ-1), respectively, and stimulated with varying concentrations (0-10.0 units (U)/ml) of thrombin from 1-7 h. Cyr61 transcript levels were determined using reverse transcriptase-PCR (RT-PCR) and quantitative PCR (qPCR) while protein forms were analyzed using western blot data. Protease activities were characterized via protease class-specific inhibitors and western blot analysis. Thrombin activity was quantified using the fluorogenic peptide Phe-Pro-Arg-AFC. Protease-activated receptor (PAR) agonist peptides-1 and -4 were used to determine whether cells increased Cyr61 through PAR signaling pathways. The PAR-1 antagonist SCH 79797 was used to block the thrombin cleavage of the receptor. PCR data were analyzed using MxPro software and western blot data were analyzed using Image Lab™ and Image J software. Student. In cultured human corneal stromal fibroblasts and myofibroblasts, thrombin regulates Cyr61 through two mechanisms: 1) thrombin increases the Cyr61 expression at the message and protein levels, and 2) thrombin increases the activation of a leupeptin-sensitive protease that stimulates the cleavage of Cyr61 into N- and C-terminal domain populations in or near the thrombospondin type-1 domain. Generation of Cyr61 peptides during corneal injury stimulation may reveal additional functions of the protein, which modulate corneal wound healing activities or decrease activities of the full-length Cyr61 form. Topics: Alternative Splicing; Cell Differentiation; Corneal Stroma; Culture Media, Conditioned; Cysteine-Rich Protein 61; Fibroblast Growth Factor 2; Fibroblasts; Hirudins; Humans; Leupeptins; Myofibroblasts; Primary Cell Culture; Proteolysis; Pyrroles; Quinazolines; Receptors, Proteinase-Activated; RNA, Messenger; Signal Transduction; Stromal Cells; Thrombin; Transforming Growth Factor beta1 | 2020 |
In vitro effect of a novel protease inhibitor cocktail on Toxoplasma gondii tachyzoites.
Toxoplasmosis is a zoonotic disease and a global food and water-borne infection. The disease is caused by the parasite Toxoplasma gondii, which is a highly successful and remarkable pathogen because of its ability to infect almost any nucleated cell in warm-blooded animals. The present study was done to demonstrate the effect of protease inhibitors cocktail (PIC), which inhibit both cysteine and serine proteases, on in vitro cultured T. gondii tachyzoites on HepG2 cell line. This was achieved by assessing its effect on the invasion of the host cells and the intracellular development of T.gondii tachyzoites through measuring their number and viability after their incubation with PIC. Based on the results of the study, it was evident that the inhibitory action of the PIC was effective when applied to tachyzoites before their cultivation on HepG2 cells. Pre-treatment of T.gondii tachyzoites with PIC resulted in failure of the invasion of most of the tachyzoites and decreased the intracellular multiplication and viability of the tachyzoites that succeeded in the initial invasion process. Ultrastructural studies showed morphological alteration in tachyzoites and disruption in their organelles. This effect was irreversible till the complete lysis of cell monolayer in cultures. It can be concluded that PIC, at in vitro levels, could prevent invasion and intracellular multiplication of Toxoplasma tachyzoites. In addition, it is cost effective compared to individual protease inhibitors. It also had the benefit of combined therapy as it lowered the concentration of each protease inhibitor used in the cocktail. Other in vivo experiments are required to validate the cocktail efficacy against toxoplasmosis. Further studies may be needed to establish the exact mechanism by which the PIC exerts its effect on Toxoplasma tachyzoites behavior and its secretory pathway. Topics: Analysis of Variance; Animals; Aprotinin; Culture Media, Serum-Free; Cysteine Proteinase Inhibitors; Drug Combinations; Hep G2 Cells; Humans; Leucine; Leupeptins; Mice; Microscopy, Electron, Transmission; Organelles; Pilot Projects; Protease Inhibitors; Serine Proteinase Inhibitors; Statistics, Nonparametric; Sulfones; Toxoplasma | 2020 |
Topics: Aprotinin; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Leupeptins; Protein Folding; Protein Processing, Post-Translational; Protein Stability; Proteolysis; RNA Interference; RNA, Small Interfering; S-Nitrosoglutathione; S-Nitrosothiols; Ubiquitin-Protein Ligases; Ubiquitination | 2019 |
Effects of Five Substances with Different Modes of Action on Cathepsin H, C and L Activities in Zebrafish Embryos.
Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene-baseline toxicity; rotenone-an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)-an inhibitor of ATP synthesis; and tebuconazole-a sterol biosynthesis inhibitor) on Topics: Animals; Cathepsin C; Cathepsin H; Cathepsin L; Cysteine Proteinase Inhibitors; Embryo, Nonmammalian; Leupeptins; Zebrafish | 2019 |
Autophagosome immunoisolation from GFP-LC3B mouse tissue.
We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4-5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The post-nuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis. Topics: Animals; Autophagosomes; Biomarkers; Green Fluorescent Proteins; Leupeptins; Mice, Transgenic; Microtubule-Associated Proteins; Organ Specificity | 2019 |
Measurement of Bulk Autophagy by a Cargo Sequestration Assay.
The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.Bulk autophagy can be measured directly by a cargo sequestration assay, using a cargo marker representative of total cytoplasm. The assay described here measures the sequestration and accumulation of the ubiquitous cytosolic protein lactate dehydrogenase (LDH) in the sedimentable autophagic vacuoles of cells incubated with an inhibitor of intravacuolar degradation such as bafilomycin or leupeptin. Electrodisruption of the plasma membrane followed by centrifugal sedimentation of the "cell corpses" (which retain their organelles in an intact state, bound to the cytoskeleton) is a convenient, efficient, and reproducible way to separate the small fraction of sequestered, sedimentable LDH from the major pool of cytosolic LDH. Topics: Animals; Autophagosomes; Autophagy; Cell Culture Techniques; Cytosol; Electroporation; Enzyme Assays; Humans; L-Lactate Dehydrogenase; Leupeptins; Lysosomes; Macrolides | 2019 |
Measuring Nonselective and Selective Autophagy in the Liver.
Administration of leupeptin, a specific inhibitor of lysosomal cysteine proteinases, to starved rats or mice inhibits autolysosomal protein degradation and results in accumulation of autolysosomes in their livers. Immunoblotting of liver homogenates to examine autophagic flux in vivo reveals elevated levels of the selective autophagy substrate p62 and the autophagosomal membrane protein LC3-II in the livers of leupeptin-treated animals. Percoll density gradient centrifugation can be used to isolate autolysosomes from the livers of untreated and leupeptin-treated animals. Moreover, autolysosomes can be examined for the presence of sequestered cytoplasmic proteins as well as degradation intermediates. Topics: Animals; Autophagosomes; Autophagy; Centrifugation, Density Gradient; Cysteine Proteases; Cysteine Proteinase Inhibitors; Leupeptins; Liver; Lysosomes; Male; Mice; Mice, Inbred C57BL; Models, Animal; Rats; Rats, Wistar | 2019 |
Analysis of Chaperone-Mediated Autophagy.
Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. These proteins are identified by a chaperone that targets them to lysosomes. There, they are translocated into the organelle lumen through a lysosomal membrane receptor/translocation complex. CMA plays an important role in maintaining cellular proteostasis by eliminating damaged and altered proteins. CMA also participates in the control of the cellular energetic balance through recycling of amino acids resulting from lysosomal proteolysis of the substrate proteins. Lastly, due to the intrinsic protein selectivity of CMA, this type of autophagy exerts regulatory functions by mediating timely degradation of key cellular proteins that participate in processes such as lipid and glucose metabolism, cell cycle, DNA repair, and cellular reprogramming, among others. Dysfunctional CMA occurs with age and has now been described in a growing list of human pathologies such as metabolic disorders, neurodegeneration, cancer, immunodeficiency, and diabetes. In this chapter, we describe current methodologies to quantitatively analyze CMA activity in different experimental models. Topics: Ammonium Chloride; Animals; Autophagy; Biological Assay; Female; Leupeptins; Liver; Lysosomes; Male; Mice; Mice, Inbred C57BL; Molecular Chaperones; Proteolysis; Rats; Rats, Wistar | 2019 |
Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems - Diffusion through an inhibitor barrier.
A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer. Topics: Antipain; Diffusion; Dose-Response Relationship, Drug; Gelatin; Kinetics; Leupeptins; Models, Biological; Oligopeptides; Particle Size; Peptide Hydrolases; Serine Proteinase Inhibitors; Spectrophotometry; Structure-Activity Relationship; Surface Properties | 2018 |
Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin.
Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca Topics: Allosteric Regulation; Allosteric Site; Calcium; Cations, Divalent; Crystallography, X-Ray; Kallikreins; Kinetics; Leupeptins; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Structure, Tertiary; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Zinc | 2018 |
Controlled cortical impact-induced neurodegeneration decreases after administration of the novel calpain-inhibitor Gabadur.
One aspect of secondary injury in traumatic brain injury is the marked increase in intracellular calcium and resultant over-activation of the calcium-dependent neutral cysteine protease calpain. Gabadur is a novel protease inhibitor with calpain-inhibition properties formulated from the classic protease inhibitor leupeptin linked to a pregabalin carrier. This construction allows the entire compound to cross the blood-brain barrier after peripheral administration to better target the site of injury. In this study, a single intraperitoneal dose of Gabadur was administered immediately following controlled cortical impact injury in rats. Neocortical slices were examined at 48 h post-injury via Fluoro-Jade B staining, revealing an improvement in cortical neurodegeneration in Gabadur treated rats. Levels of detrimental active calpain-2 measured via western blot were also decreased in rats receiving Gabadur. This data supports the benefit of targeted protease inhibition in the treatment of traumatic brain injury. Topics: Animals; Brain; Brain Injuries, Traumatic; Calpain; Disease Models, Animal; Glycoproteins; Leupeptins; Molecular Structure; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Pregabalin; Rats, Sprague-Dawley | 2018 |
Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide.
Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca Topics: Calcium; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; DNA Transposable Elements; Isoenzymes; Leucine; Leupeptins; Muscle Proteins; Protein Conformation; Proteolysis; Recombinant Proteins | 2018 |
Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.
Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity. Topics: Animals; Antipain; Ascomycota; Carboxypeptidases; Chiroptera; Leupeptins; Mycoses; Syndrome | 2018 |
Gelatin Zymography Using Leupeptin for the Detection of Various Cathepsin L Forms.
Zymography is a highly sensitive method to assess the activities as well as molecular weights of enzymes in crude biological fluids and tissue extracts. Cathepsin L is a lysosomal cysteine proteinase that is optimally active at slightly acidic pH and is highly unstable in alkaline solutions such as electrode buffer (pH 8.3). Large amounts of cathepsin L are secreted by various cancer cells, where it promotes invasion and metastasis. Leupeptin is a tight-binding inhibitor of cysteine proteinases, and its complex with cathepsin L is stable in alkaline solutions. Moreover, leupeptin can be easily removed from the complex because it is a reversibly binding inhibitor. In addition, leupeptin is too small to influence the electrode migration distance of the complex with cathepsin L on a sodium dodecyl sulfate-polyacrylamide gel. Here, a novel gelatin zymography technique that employs leupeptin to detect pro-, intermediate, and mature cathepsin L forms on the basis of their gelatinolytic activities is described. Further, the differences in the glycosylation, phosphorylation, and processing statuses of lysosomal and secreted cathepsin L forms isolated from cultured HT 1080 cells are demonstrated using this method. Topics: Cathepsin L; Cells, Cultured; Enzyme Assays; Gelatin; Humans; Leupeptins; Lysosomes; Phosphorylation | 2017 |
Pioglitazone Enhances Cytosolic Lipolysis, β-oxidation and Autophagy to Ameliorate Hepatic Steatosis.
Non-alcoholic fatty liver disease closely contributes to the development of obesity and insulin resistance. Even though pioglitazone has been reported to effectively lessen hepatic steatosis in human studies, its molecular mechanism remains unclear. This study is designed to investigate the regulation of cytosolic lipolysis, β-oxidation and autophagy by pioglitazone in a mice model of high fat diet (HFD) and cell model incubated with palmitic acid. Our results revealed hepatic steatosis was apparently induced by HFD and it was significantly reversed by pioglitazone. The serum insulin and hepatic triglyceride content was significantly decreased by co-administered pioglitazone with HFD. Hepatic expression of cytosolic-lipolysis related proteins (ATGL, HSL), β-oxidation (CPT-1A) and autophagy-related proteins (ATG7, LC3, LAL) was significantly enhanced by pioglitazone. Knockdown PPARα/PPARγ in AML12 cells significantly and proportionally reduced the expressions of ATGL, CPT-1A and LC3II, which was induced by pioglitazone. Furthermore, facilitation of the autophagic flux by pioglitazone was obviously blocked by lysosomal inhibitor, leupeptin, to demonstrate accumulation of the LC3II and intracellular lipid in AML12 cells. Our results demonstrated that pioglitazone attenuating the hepatic steatosis may be mediated by enhancing cytosolic lipolysis, β-oxidation and autophagy in a PPARα and PPARγ dependent manner. Topics: Animals; Autophagy; Cell Line; Diet, High-Fat; Disease Models, Animal; Humans; Insulin; Leupeptins; Lipolysis; Male; Mice; Non-alcoholic Fatty Liver Disease; Oxidation-Reduction; Palmitic Acid; Pioglitazone; PPAR alpha; PPAR gamma; Triglycerides | 2017 |
Global ischemia induces lysosomal-mediated degradation of mTOR and activation of autophagy in hippocampal neurons destined to die.
The mammalian target of rapamycin (mTOR) is a key regulator of cell growth, autophagy, translation, and survival. Dysregulation of mTOR signaling is associated with cancer, diabetes, and autism. However, a role for mTOR signaling in neuronal death is not well delineated. Here we show that global ischemia triggers a transient increase in mTOR phosphorylation at S2448, whereas decreasing p-mTOR and functional activity in selectively vulnerable hippocampal CA1 neurons. The decrease in mTOR coincides with an increase in biochemical markers of autophagy, pS317-ULK-1, pS14-Beclin-1, and LC3-II, a decrease in the cargo adaptor p62, and an increase in autophagic flux, a functional readout of autophagy. This is significant in that autophagy, a catabolic process downstream of mTORC1, promotes the formation of autophagosomes that capture and target cytoplasmic components to lysosomes. Inhibitors of the lysosomal (but not proteasomal) pathway rescued the ischemia-induced decrease in mTOR, consistent with degradation of mTOR via the autophagy/lysosomal pathway. Administration of the mTORC1 inhibitor rapamycin or acute knockdown of mTOR promotes autophagy and attenuates ischemia-induced neuronal death, indicating an inverse causal relation between mTOR, autophagy, and neuronal death. Our findings identify a novel and previously unappreciated mechanism by which mTOR self-regulates its own levels in hippocampal neurons in a clinically relevant model of ischemic stroke. Topics: Acetylcysteine; Adenine; AMP-Activated Protein Kinases; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Beclin-1; Cells, Cultured; Hippocampus; Ischemia; Leupeptins; Lysosomes; Male; Microtubule-Associated Proteins; Neurons; Phosphorylation; Rats; RNA Interference; Sirolimus; TOR Serine-Threonine Kinases | 2017 |
Flumazenil decreases surface expression of α4β2δ GABAA receptors by increasing the rate of receptor internalization.
Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM-100 μM, IC50=∼1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM)+THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil. Topics: Botulinum Toxins, Type A; Cell Membrane; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Dynamins; Flumazenil; GABA Agonists; gamma-Aminobutyric Acid; HEK293 Cells; Humans; Hydrazones; Immunohistochemistry; Isoxazoles; Leupeptins; Membrane Potentials; Patch-Clamp Techniques; Receptors, GABA-A; Recombinant Proteins; Transfection | 2016 |
The fine tuning of metabolism, autophagy and differentiation during in vitro myogenesis.
Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation. Topics: Ammonium Chloride; Animals; Autophagy; Beclin-1; Cell Differentiation; Cells, Cultured; Leupeptins; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Microscopy, Confocal; Microtubule-Associated Proteins; Mitochondria; Multiprotein Complexes; Muscle Fibers, Skeletal; Myoblasts; RNA Interference; RNA, Small Interfering; Satellite Cells, Skeletal Muscle; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53 | 2016 |
Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal "metastability" in primary hepatocytes determining in vitro cell survival.
Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds. Topics: Adenine; Aldehydes; Animals; Apoptosis; Autophagy; Culture Media, Conditioned; Endocytosis; Epidermal Growth Factor; Female; Ferritins; Glutathione; Glutathione Disulfide; Hepatocytes; Intracellular Membranes; Iron Chelating Agents; Leupeptins; Liver; Lysosomes; Molecular Imaging; Permeability; Primary Cell Culture; Protease Inhibitors; Protein Aggregates; Rats; Rats, Inbred F344 | 2015 |
Autophagic flux determination in vivo and ex vivo.
Autophagy is a highly dynamic process that mediates the degradation of cellular constituents inside lysosomes. It is characterized by the formation of autophagosomes, double membrane organelles that engulf cytosolic components and organelles and degrade their contents upon fusion with lysosomes. Upregulation of autophagy in response to specific stimuli can be determined by evaluating autophagic flux. This is achieved by comparing the number of autophagosomes in the absence and presence of lysosomal inhibitors. While the determination of autophagic flux in isolated cells is well-documented, few studies have described its determination in tissues or in vivo. Here, we describe the evaluation of autophagic flux both in vivo and ex vivo in several tissues, after treatment with lysosomal inhibitors and exposure to classical autophagy-inducing stimuli. This method uses LC3 lipidation, as determined by Western blot, fluorescence microscopy and flow cytometry. Our findings demonstrate that autophagic flux can be evaluated in vivo and ex vivo in several tissues. Topics: Animals; Autophagy; Cerebellum; Leupeptins; Liver; Lysosomes; Mice; Microscopy, Fluorescence; Retina | 2015 |
Comparative response of rat and rabbit conceptuses in vitro to inhibitors of histiotrophic nutrition.
Histiotrophic nutrition via the visceral yolk sac is an essential nutritional pathway of the rodent conceptus, and inhibition of this pathway may cause growth retardation, malformations, and death in rodent embryos. Morphologic differences among species during early development indicate that the visceral yolk sac histiotrophic nutrition pathway may be of lesser importance in nonrodent species, including humans. Here, comparative studies were conducted with inhibitors of different steps in the visceral yolk sac histiotrophic nutrition pathway to determine whether the rabbit is similarly responsive to the rat. Early somite stage New Zealand White rabbit and Crl:CD(SD) rat conceptuses (gestation day 9, rabbits; gestation day 10, rats) were exposed for 48 hr to three different histiotrophic nutrition pathway inhibitors using whole embryo culture techniques, after which they were evaluated for growth and malformations. Cubilin antibody, an inhibitor of endocytosis, reduced growth and development and increased malformations in both rat and rabbit embryos, although the rabbit appeared more sensitive. Leupeptin, a lysosomal cysteine protease inhibitor, also impaired growth and development and increased malformations in rat embryos, while in the rabbit it induced malformations and a slight decrease in morphology score but had no effect upon growth. Trypan blue, an inhibitor of endocytosis and endosome maturation, affected all measures in both species to a similar degree at the highest concentration (2500 μg/ml), but rat embryos responded to a greater extent at lower concentrations. Although the specific adverse outcomes appear to be different, these results demonstrate that rabbits, like rats, are sensitive to inhibitors of the histiotrophic nutrition pathway. Topics: Animal Nutritional Physiological Phenomena; Animals; Antibodies; Embryo, Mammalian; Embryonic Development; Female; Fetus; Leupeptins; Rabbits; Rats, Sprague-Dawley; Receptors, Cell Surface; Staining and Labeling; Trypan Blue | 2015 |
Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.
Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known. Topics: Benzamidines; Benzylamines; Binding, Competitive; Fluorine; Kinetics; Leupeptins; Ligands; Magnetic Resonance Spectroscopy; Monte Carlo Method; Trypsin | 2015 |
A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance.
Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC. Topics: Amino Acid Sequence; Animals; Biological Transport; Cell Membrane; Computer Simulation; Cysteine Proteinase Inhibitors; Drug Resistance; Gene Expression Regulation; Genome, Protozoan; Leupeptins; Models, Biological; Molecular Sequence Data; Mutation; Plasmodium falciparum; Protein Structure, Tertiary; Protozoan Proteins | 2015 |
Amino acid starvation induced by protease inhibition produces differential alterations in redox status and the thiol proteome in organogenesis-stage rat embryos and visceral yolk sacs.
The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26-h period (GD10-GD11) and 50 μM over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual tissues following leupeptin treatment. Analysis of the thiol proteome showed few alterations to specific pathways mapped to the Kyoto Encyclopedia of Genes and Genomes Pathway database, but did reveal significant increases in concentrations of proteins associated with glycolysis/gluconeogenesis in the VYS and decreased concentrations proteins associated with ribosome biogenesis and function in the EMB. A subset of proteins elevated by >2-23-fold in the VYS were identified as serum (blood) proteins and r Topics: Amino Acids; Animals; Culture Media; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Glutathione; Leupeptins; Lysosomes; Organogenesis; Oxidation-Reduction; Protease Inhibitors; Proteolysis; Proteome; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sulfhydryl Compounds; Yolk Sac | 2015 |
Measurement of allergen-specific secretory IgA in stool of neonates, infants and toddlers by protection against degradation of immunoglobulins and allergens.
Measurement of secretory immunoglobulin A (SIgA) level is important to monitor various disease conditions. However SIgA in gut mucosa is degraded by pancreatic proteases and proteolytic enzymes from enteric microbiota. Currently, there is no reliable quantitation method that measures allergen-specific SIgA levels in stool of neonates, infants and toddlers.. Allergen-specific SIgA levels in stool of 10 healthy neonates, infants and toddlers aged 0 to 36 months were measured by our new allergen microarray with densely carboxylated arms on a glass slide chip.. Protease activities in stool of 3-day-old neonates were low and no degradation of SIgA, IgA and allergens was detected. However, immunofluorescence signals of SIgA, IgA and allergen on the chip were markedly reduced by stool extracts obtained from infants and toddlers aged more than one month in dose- and time-dependent manners. Such reduction was almost completely inhibited by serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and partly by leupeptin, but not by a variety of other protease inhibitors tested.. Allergen-specific SIgA levels in stool of neonates, infants and toddlers under 36 months of age could be analyzed using protease inhibitors, including PMSF and leupeptin. Topics: Allergens; Child, Preschool; Feces; Humans; Immunoglobulin A, Secretory; Infant; Infant, Newborn; Leupeptins; Phenylmethylsulfonyl Fluoride; Protein Array Analysis; Serine Proteinase Inhibitors | 2015 |
Catalytic autoantibodies against myelin basic protein (MBP) isolated from serum of autistic children impair in vitro models of synaptic plasticity in rat hippocampus.
Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD. Topics: Adolescent; Animals; Autistic Disorder; Brain; Child; Child, Preschool; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Hippocampus; Humans; Immunoglobulin A; In Vitro Techniques; Leupeptins; Long-Term Potentiation; Male; Myelin Basic Protein; Patch-Clamp Techniques; Proteolysis; Rats; Rats, Sprague-Dawley; Synaptic Transmission | 2015 |
Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.
The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear.. This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats.. A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats.. Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 μg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05).. The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption. Topics: Adhesins, Bacterial; Alveolar Bone Loss; Animals; Bacteroidaceae Infections; Canavalia; Canavanine; Chlorhexidine; Chromatography, High Pressure Liquid; Cystatins; Cysteine Endopeptidases; Disease Progression; Epithelial Cells; Gingipain Cysteine Endopeptidases; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; KB Cells; Leupeptins; Male; Microbial Sensitivity Tests; Mouth Mucosa; Phytotherapy; Plant Extracts; Porphyromonas gingivalis; Rats; Rats, Wistar; Specific Pathogen-Free Organisms | 2014 |
Inhibition of calpains fails to improve regeneration through a peripheral nerve conduit.
Intramuscular injection of the calpain inhibitor leupeptin promotes peripheral nerve regeneration in primates (Badalamente et al., 1989 [13]), and direct positive effects of leupeptin on axon outgrowth were observed in vitro (Hausott et al., 2012 [12]). In this study, we applied leupeptin (2mg/ml) directly to collagen-filled nerve conduits in the rat sciatic nerve transection model. Analysis of myelinated axons and retrogradely labeled motoneurons as well as functional 'CatWalk' video analysis did not reveal significant differences between vehicle controls and leupeptin treated animals. Therefore, leupeptin does not improve nerve regeneration via protease inhibition in regrowing axons or in surrounding Schwann cells following a single application to a peripheral nerve conduit suggesting indirect effects on motor endplate integrity if applied systemically. Topics: Action Potentials; Animals; Calpain; Cysteine Proteinase Inhibitors; Leupeptins; Male; Muscle, Skeletal; Nerve Regeneration; Neural Conduction; Rats, Sprague-Dawley; Sciatic Nerve | 2014 |
Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.
Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit β2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions. Topics: Animals; Histones; Hydrolysis; Kinetics; Leupeptins; Molecular Weight; Peptides; Proteasome Endopeptidase Complex; Proteolysis; Rabbits; Trypsin | 2014 |
Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons.
Recent studies have demonstrated that human tau can be secreted by neurons and non-neuronal cells, an event linked to the propagation of tau pathology in the brain. In the present study, we confirmed that under physiological conditions, one tau-positive band was detected in the culture medium with an anti-tau antibody recognizing total tau and the Tau-1 antibody directed against unphosphorylated tau. We then examined whether tau secretion was modified upon insults. Tau secretion was increased by starvation [Earle's Balanced Salt Solution (EBSS)], inhibition of lysosomal function (leupeptin) and when both of these conditions were superimposed, this combined treatment having the most important effects on tau secretion. Interestingly, the pattern of tau secretion was distinct from that of control neurons when neurons were treated either with EBSS alone or EBSS + leupeptin. In these conditions, three tau-positive bands were detected in the culture medium. Two of these three bands were immunoreactive to Tau-1 antibody revealing that at least two tau species were released upon these treatments. Collectively, our results indicate that insults such as nutrient deprivation and lysosomal dysfunction observed in neurodegenerative diseases could result in an increase of tau secretion and propagation of tau pathology in the brain. Topics: Animals; Cells, Cultured; Cerebral Cortex; Culture Media; Leupeptins; Lysosomes; Mice, Inbred C57BL; Neurons; Phosphorylation; Primary Cell Culture; Protein Processing, Post-Translational; tau Proteins | 2014 |
Nostosins, Trypsin Inhibitors Isolated from the Terrestrial Cyanobacterium Nostoc sp. Strain FSN.
Two new trypsin inhibitors, nostosin A (1) and B (2), were isolated from a hydrophilic extract of Nostoc sp. strain FSN, which was collected from a paddy field in the Golestan Province of Iran. Nostosins A (1) and B (2) are composed of three subunits, 2-hydroxy-4-(4-hydroxyphenyl)butanoic acid (Hhpba), L-Ile, and L-argininal (1) or argininol (2). Nostosins A (1) and B (2) exhibited IC50 values of 0.35 and 55 μM against porcine trypsin, respectively, suggesting that the argininal aldehyde group plays a crucial role in the efficient inhibition of trypsin. Molecular docking of nostosin A (1) (449 Da), leupeptin (426 Da, IC50 0.5 μM), and spumigin E (610 Da, IC50 < 0.1 μM) with trypsin suggested prominent binding similarity between nostosin A (1) and leupeptin but only partial binding similarity with spumigin E. The number of hydrogen bonds between ligands and trypsin increased according to the length and size of the ligand molecule, and the docking affinity values followed the measured IC50 values. Nostosin A (1) is the first highly potent three-subunit trypsin inhibitor with potency comparable to the known commercial trypsin inhibitor leupeptin. These findings expand the known diversity of short-chain linear peptide protease inhibitors produced by cyanobacteria. Topics: Inhibitory Concentration 50; Iran; Leupeptins; Molecular Structure; Nostoc; Oligopeptides; Trypsin Inhibitors | 2014 |
Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis.
Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases. Topics: Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Caspases; Cell Line, Tumor; Chelating Agents; Chromatin; Copper Sulfate; Dizocilpine Maleate; Gene Expression Regulation; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Leupeptins; Mice; Neoplasm Proteins; Neurons; Nifedipine; Oxidation-Reduction; Oxidative Stress; Protease Inhibitors; Protein Kinase Inhibitors; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Teratocarcinoma | 2014 |
Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition.
Topics: Biological Transport, Active; Cell Line, Tumor; Chloroquine; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Neurons; Vitamin B 12 | 2014 |
Profiling drug-induced cell death pathways in the zebrafish lateral line.
Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while D-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades. Topics: Animals; Antioxidants; Benzamidines; Calpain; Caspase Inhibitors; Cell Death; Cells, Cultured; Cisplatin; Cross-Linking Reagents; Gentamicins; Guanidines; Hair Cells, Auditory, Inner; Lateral Line System; Leupeptins; Methionine; Neomycin; Oligopeptides; Protease Inhibitors; Protein Synthesis Inhibitors; Reactive Oxygen Species; Zebrafish | 2013 |
Inhibition of proteolysis in histiotrophic nutrition pathways alters DNA methylation and one-carbon metabolism in the organogenesis-stage rat conceptus.
Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM), is biosynthesized from methionine and adenosine triphosphate by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 μM leupeptin in whole embryo culture for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. After 6 h on GD11, the 100-μM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels and inhibited Mat2a specific activity, all of which produced a significant 52% reduction of SAM in the VYS. The 50- and 100-μM leupeptin treatments significantly decreased global methylation levels by 6%-9% in EMB and by 11%-15% in VYS following both 6- and 26-h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation. Topics: Animals; Base Sequence; Carbon; DNA Methylation; Embryo Culture Techniques; Epigenesis, Genetic; Female; Leupeptins; Methionine Adenosyltransferase; Micronutrients; Molecular Sequence Data; Nutritional Physiological Phenomena; Organogenesis; Promoter Regions, Genetic; Proteolysis; Rats; Rats, Sprague-Dawley; S-Adenosylmethionine | 2013 |
Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.
Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein. Topics: Androgens; cdc25 Phosphatases; Cell Cycle Proteins; Cell Line, Tumor; Dihydrotestosterone; Humans; Leucine; Leupeptins; Lysosomes; Male; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Androgen; Transcriptome; Up-Regulation | 2013 |
LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.
To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease.. Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.. The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites.. LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis. Topics: Adhesins, Bacterial; Adult; Aged; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cathelicidins; Cysteine Endopeptidases; Cysteine Proteases; Cysteine Proteinase Inhibitors; Dental Plaque; Enzyme-Linked Immunosorbent Assay; Gingipain Cysteine Endopeptidases; Gingival Crevicular Fluid; Humans; Leupeptins; Middle Aged; Peptide Fragments; Periodontitis; Periodontium; Porphyromonas gingivalis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tosyllysine Chloromethyl Ketone | 2013 |
Sorting of the FGF receptor 1 in a human glioma cell line.
Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor growth in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, 15 % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts. Topics: Caveolins; Cell Line, Tumor; Cell Shape; Clathrin; Endocytosis; Endosomes; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Glioma; Humans; Leupeptins; Ligands; Luminescent Proteins; Lysosomal Membrane Proteins; Lysosomes; Phosphorylation; Protein Transport; Receptor, Fibroblast Growth Factor, Type 1; Time Factors; Transfection | 2013 |
Dephosphorylation reduces passage of ovalbumin antigen through intestinal epithelial Caco-2 cell monolayers.
Allergenic potential of food proteins is associated with stability to gastric and pancreatic digestive enzymes. However, much attention has not been focused on intracellular digestion of protein antigens during the passage through intestinal epithelia. We report here the degradation and survival of a bis-phosphorylated protein, ovalbumin (OVA), in the course of passage through Caco-2 cell monolayers cultured on porous membrane. SDS-PAGE in combination with phosphoprotein staining showed that OVA, which had passed through the cell layers, was almost intact in its polypeptide chain but partly dephosphorylated. By contrast, quantitative analysis using ELISA indicated that complete dephosphorylation in advance by an alkaline phosphatase markedly reduced the OVA passage. The reduced passage was restored in the presence of cathepsin inhibitors, leupeptin and pepstatin-A. Moreover, the complete dephosphorylation increased susceptibility of OVA to in vitro digestion with cathepsin B, which cleaved near an OVA phosphorylation site, Ser345. The susceptibility of OVA to lysosomal proteases may affect its passage through the intestinal epithelia, leading to determination of allergic sensitization and elicitation in egg allergy. Topics: Alkaline Phosphatase; Animals; Antigens; Caco-2 Cells; Cathepsins; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Humans; Intestinal Mucosa; Leupeptins; Mice; Mice, Inbred Strains; Ovalbumin; Pepstatins; Phosphorylation; Protease Inhibitors | 2013 |
Porphyromonas gingivalis promotes monocyte migration by activating MMP-9.
The migration of monocytes into the local environment is crucial for their maturation into macrophages or osteoclasts in the pathogenesis of periodontal disease. The objective of this study was to investigate the role and mechanisms mediated by Porphyromonas gingivalis in promoting the migration of monocytes by regulating MMP-9 and TIMP-1 expression.. Human THP1 monocytes were treated with culture supernatant derived from P. gingivalis (ATCC 33277) for 24 h. Zymography, western blot analysis and quantitative PCRs were performed to analyse protein and mRNA levels of MMP-9. Protein and mRNA levels of TIMP-1 from monocytes treated with or without P. gingivalis were determined as well. Transwell migration assay was carried out to analyse the effect of P. gingivalis on the migration of human peripheral blood CD14-positive monocytes. An MMP inhibitor (GM6001) and a proteinase inhibitor (leupeptin) were used to determine the role of MMP-9 in P. gingivalis supernatant- and lipopolysaccharide-induced monocyte migration.. In zymography and western blot, an 82 kDa band of active MMP-9 emerged in P. gingivalis-treated monocyte culture media in a dose-dependent manner, in addition to the MMP-9 proenzyme (92 kDa) band expressed in control cell culture media. P. gingivalis supernatant increased both the protein and the mRNA levels of MMP-9 and TIMP-1. P. gingivalis supernatant, but not its lipopolysaccharide, increased the migratory ability of CD14-positive monocytes. The increased migratory ability of P. gingivalis-treated monocytes was partly inhibited by leupeptin (200 μg/mL) and completely antagonized by the MMP inhibitor GM6001 (100 nm). Lipopolysaccharide of P. gingivalis increased protein and mRNA levels of MMP-9 in monocytes, but had no effect on the migratory ability or MMP-9 activation.. P. gingivalis supernatant increased the migratory ability of monocytes, in part, by increasing activation and expression of MMP-9. Topics: Cathepsins; Cell Culture Techniques; Cell Line; Cell Movement; Chemotaxis, Leukocyte; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Precursors; Humans; Leupeptins; Lipopolysaccharide Receptors; Lipopolysaccharides; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Monocytes; Porphyromonas gingivalis; Protease Inhibitors; Tissue Inhibitor of Metalloproteinase-1 | 2012 |
Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle.
The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat. Topics: Animals; Caspase Inhibitors; Chickens; Desmin; Egtazic Acid; Glycoproteins; Leupeptins; Meat; Muscle Proteins; Myofibrils; Oligopeptides; Pectoralis Muscles; Postmortem Changes; Proteolysis; Troponin T | 2012 |
Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in adult sensory neurons by increased recycling.
Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1-4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro. Topics: Animals; Axons; Cell Membrane; Cell Movement; Endocytosis; Fibroblast Growth Factor 2; Ganglia, Spinal; Leupeptins; Lysosomes; Macrolides; Monensin; PC12 Cells; Protein Transport; Rats; Receptor, Fibroblast Growth Factor, Type 1; Sensory Receptor Cells; Signal Transduction | 2012 |
Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans.
Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria.. Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay.. Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans.. The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs. Topics: Aggregatibacter actinomycetemcomitans; Anti-Bacterial Agents; Bacterial Adhesion; Cathepsins; Chromatography, Affinity; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Epithelial Cells; Fusobacterium nucleatum; Humans; Immunoglobulin A, Secretory; Kallikreins; Lactoferrin; Leupeptins; Microscopy, Confocal; Mouth Mucosa; Porphyromonas gingivalis; Saliva; Salivary Cystatins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors | 2012 |
Activity-dependent cleavage of the K-Cl cotransporter KCC2 mediated by calcium-activated protease calpain.
The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling. Topics: Action Potentials; Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Ionophores; Calpain; Cycloheximide; Cysteine Proteinase Inhibitors; Dipeptides; Dizocilpine Maleate; Dose-Response Relationship, Drug; Emetine; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; In Vitro Techniques; Ionomycin; K Cl- Cotransporters; Leupeptins; Magnesium; Male; Membrane Potentials; N-Methylaspartate; Patch-Clamp Techniques; Protein Synthesis Inhibitors; Pyramidal Cells; Rats; Rats, Wistar; Statistics, Nonparametric; Symporters; Valine | 2012 |
Altered T-cell responses by the periodontal pathogen Porphyromonas gingivalis.
Several studies support an association between the chronic inflammatory diseases periodontitis and atherosclerosis with a crucial role for the periodontal pathogen Porphyromonas gingivalis. However, the interplay between this pathogen and the adaptive immune system, including T-cells, is sparsely investigated. Here we used Jurkat T-cells to determine the effects of P. gingivalis on T-cell-mediated adaptive immune responses. We show that viable P. gingivalis targets IL-2 expression at the protein level. Initial cellular events, including ROS production and [Ca(2+)](i), were elevated in response to P. gingivalis, but AP-1 and NF-κB activity dropped below basal levels and T-cells were unable to sustain stable IL-2 accumulation. IL-2 was partially restored by Leupeptin, but not by Cathepsin B Inhibitor, indicating an involvement of Rgp proteinases in the suppression of IL-2 accumulation. This was further confirmed by purified Rgp that caused a dose-dependent decrease in IL-2 levels. These results provide new insights of how this periodontal pathogen evades the host adaptive immune system by inhibiting IL-2 accumulation and thus attenuating T-cell proliferation and cellular communication. Topics: Adhesins, Bacterial; Calcium; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression; Gingipain Cysteine Endopeptidases; Host-Pathogen Interactions; Humans; Interleukin-2; Jurkat Cells; Leupeptins; Luciferases; Microscopy, Confocal; NF-kappa B; Porphyromonas gingivalis; Proteolysis; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Transcription Factor AP-1 | 2012 |
The balance between receptor recycling and trafficking toward lysosomes determines synaptic strength during long-term depression.
The strength of excitatory synaptic transmission depends partly on the number of AMPA receptors (AMPARs) at the postsynaptic surface and, thus, can be modulated by membrane trafficking events. These processes are critical for some forms of synaptic plasticity, such as long-term potentiation and long-term depression (LTD). In the case of LTD, AMPARs are internalized and dephosphorylated in response to NMDA receptor activation. However, the fate of the internalized receptors upon LTD induction and its relevance for synaptic function is still a matter of debate. Here we examined the functional contribution of receptor recycling versus degradation for LTD in rat hippocampal slices, and their correlation with receptor dephosphorylation. We observed that GluA1 undergoes sequential dephosphorylation and degradation in lysosomes after LTD induction. However, this degradation does not have functional consequences for the regulation of synaptic strength, and therefore, for the expression of LTD. In contrast, the partition of internalized AMPARs between Rab7-dependent trafficking (toward lysosomes) or Rab11-dependent endosomes (recycling back toward synapses) is the key factor determining the extent of synaptic depression upon LTD induction. This sorting decision is related to the phosphorylation status of GluA1 Ser845, the dephosphorylated receptors being those preferentially targeted for lysosomal degradation. Altogether, these new data contribute to clarify the fate of AMPARs during LTD and emphasize the importance of membrane sorting decisions to determine the outcome of synaptic plasticity. Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Arabidopsis Proteins; Biophysics; Carrier Proteins; Cysteine Proteinase Inhibitors; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Green Fluorescent Proteins; GTP-Binding Proteins; Hippocampus; Leupeptins; Long-Term Potentiation; Long-Term Synaptic Depression; Lysosomes; Male; N-Methylaspartate; Neurons; Organ Culture Techniques; Protein Transport; Rats; Rats, Wistar; Receptors, AMPA; Statistics, Nonparametric; Synapses; Transfection | 2012 |
Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition.
Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an α and γ subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating α1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment. Topics: Animals; Benzodiazepines; Binding Sites; Cell Membrane; Endocytosis; Inhibitory Postsynaptic Potentials; Leupeptins; Neural Inhibition; Neurons; Protein Subunits; Protein Transport; Rats; Receptors, GABA-A; Synapses | 2012 |
Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability.
The ubiquitin-proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signaling, protein transport, DNA repair and stress responses. Hampered proteasome activity leads to the accumulation of polyubiquitylated proteins, endoplastic reticulum (ER) stress and even cell death. The ability of chemical proteasome inhibitors (PIs) to induce apoptosis is utilized in cancer therapy. During PI treatment, misfolded proteins accrue to cytoplasmic aggresomes. The formation of aggresome-like structures in the nucleus has remained obscure. We identify here a nucleolus-associated RNA-protein aggregate (NoA) formed by the inhibition of proteasome activity in mammalian cells. The aggregate forms within the nucleolus and is dependent on nucleolar integrity, yet is a separate structure, lacking nucleolar marker proteins, ribosomal RNA (rRNA) and rRNA synthesis activity. The NoAs contain polyadenylated RNA, conjugated ubiquitin and numerous nucleoplasmic proteasome target proteins. Several of these are key factors in oncogenesis, including transcription factors p53 and retinoblastoma protein (Rb), several cell cycle-regulating cyclins and cyclin-dependent kinases (CDKs), and stress response kinases ataxia-telangiectasia mutated (ATM) and Chk1. The aggregate formation depends on ubiquitin availability, as shown by modulating the levels of ubiquitin and deubiquitinases. Furthermore, inhibition of chromosome region maintenance 1 protein homolog (CRM1) export pathway aggravates the formation of NoAs. Taken together, we identify here a novel nuclear stress body, which forms upon proteasome inactivity within the nucleolus and is detectable in mammalian cell lines and in human tissue. These findings show that the nucleolus controls protein and RNA surveillance and export by the ubiquitin pathway in a previously unidentified manner, and provide mechanistic insight into the cellular effects of PIs. Topics: Acetylcysteine; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line; Cell Nucleolus; Checkpoint Kinase 1; Cyclin-Dependent Kinases; Cyclins; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Humans; Leucine; Leupeptins; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Messenger; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin | 2011 |
A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).
A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM. Topics: Animals; Cathepsin B; Cercaria; Cysteine Proteases; Cysteine Proteinase Inhibitors; Dithioerythritol; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Ethylmaleimide; Histocytochemistry; Hydrogen-Ion Concentration; Hydroxymercuribenzoates; Leupeptins; Trematoda | 2011 |
Induction of IL-13 production and upregulated expression of protease activated receptor-1 by RANTES in a mast cell line.
RANTES is a potent chemoattractant for various important inflammatory cells such as eosinophils, memory T cells and mast cells. It has been long recognized as a crucial player in the pathogenesis of allergy. However, little is known of its effects on cytokine secretion and protease activated receptor (PAR) expression in mast cells. In the present study, we examined potential influence of RANTES on IL-13 and IL-12 release from P815 cells and PAR expression on P815 cells by using flow cytometry analysis, quantitative real-time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that RANTES induced up to 2.2-fold increase in IL-13, but not IL-12 release from P815 cells. Blocking antibodies against RANTES and CCR5 diminished RANTES induced IL-13 release. Furthermore, RANTES upregulated expression of PAR-1, PAR-2 and PAR-3 mRNAs, but enhanced only PAR-1 protein expression. At 1 ng/ml, RANTES can abolish tryptase induced IL-13 release, but enhance trypsin, tryptase and thrombin induced PAR-1, -2 and -4 expression. LY204002 abolished RANTES induced IL-13 release, indicating an Akt cell signaling pathway may be involved in the event. In conclusion, RANTES can stimulate IL-13 release from mast cells through a CCR5 and Akt cell signaling pathway dependent mechanism. It can also enhance trypsin, tryptase and thrombin-induced expression of PARs in mast cells. RANTES may contribute to modulation of IL-13 production and PAR expression in mast cells, through which participates in the mast cell related inflammation. Topics: Animals; Cell Line; Chemokine CCL5; Flow Cytometry; Humans; Interleukin-12; Interleukin-13; Leupeptins; Mast Cells; Mice; Oligopeptides; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thrombin; Trypsin; Tryptases; Up-Regulation | 2011 |
Gilenya (FTY720) inhibits acid sphingomyelinase by a mechanism similar to tricyclic antidepressants.
The immunomodulator drug Gilenya (FTY720), marketed as the first oral sphingosine-1-phosphate receptor (S1P-R) modulator for treatment of Multiple Sclerosis (MS) also inhibits lysosomal acid sphingomyelinase (ASMase). Treatment of cultured cells for 24 h with FTY720 (up to 10 μM) inhibited ASMase by >80% and this could be reversed by pre-treatment with the cathepsin protease inhibitor leupeptin (5 μM). In contrast, neutral sphingomyelinase activity was unaffected and sphingosine-1-phosphate treatment had no effect on ASMase. RT-PCR revealed no inhibition of ASMase mRNA and there was no direct (in vitro) inhibition of ASMase by either FTY720 or FTY720-phosphate. This suggests that its mechanism of inhibition is similar to that of tricyclic anti-depressants such as desipramine, which are also amphiphilic cationic drugs. Both Desipramine and FTY720 treatment reduced ASMase without significant inhibition of other lysosomal hydrolases but most hydrolases showed increased secretion (up to a 50% increase) providing more evidence of lysosomal disruption by these drugs. Topics: Animals; Antidepressive Agents, Tricyclic; Cathepsins; Cell Line, Tumor; Cells, Cultured; Cysteine Proteinase Inhibitors; Desipramine; Enzyme Inhibitors; Fibroblasts; Fingolimod Hydrochloride; Humans; Hydrolases; Immunosuppressive Agents; Leupeptins; Mice; Neurons; Propylene Glycols; Sphingomyelin Phosphodiesterase; Sphingosine | 2011 |
Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss.
The therapeutic efficiency of cochlear infusion of two anti-apoptotic substances: a potent calpain inhibitor, leupeptin and a caspase inhibitor, z-VAD-FMK was evaluated in guinea pigs after a gunshot noise-induced trauma (170 dB SPL). A preliminary study showed that hair cell apoptosis appeared within 7 days of the noise trauma. For each animal, one of the cochleae was perfused directly starting 1 h after the trauma with leupeptin or z-VAD-FMK for 7 days via a mini-osmotic pump whereas the other cochlea was untreated. ABR threshold shifts were measured over a 14-day recovery period. The functional hearing study was supplemented by histological analysis. Two days after the trauma significant differences were observed between threshold shifts in the z-VAD-FMK-treated and the non-treated ears. Cochleograms showed that hair cell losses were significantly lower in z-VAD-FMK-treated ears. Regarding the leupeptin treatment, no significant difference between treated and non-treated ears was observed. This work indicates that early direct infusion of z-VAD-FMK into the cochlea accelerates hearing recovery and reduces hair cell loss after gunshot noise-induced trauma. These results suggest that the gunshot noise-induced trauma may involve the caspase pathway rather than the calpain pathway in the apoptotic process. Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cysteine Proteinase Inhibitors; Firearms; Guinea Pigs; Hair Cells, Auditory; Hearing Loss, Noise-Induced; Infusions, Parenteral; Leupeptins; Time Factors | 2011 |
Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion.
Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. Topics: Cell Fusion; Cell Line; Furin; Gene Expression Regulation, Viral; Humans; Leupeptins; Mutation; Peptide Hydrolases; Severe acute respiratory syndrome-related coronavirus; Trypsin; Virus Internalization | 2011 |
Dendroaspis natriuretic peptide and the designer natriuretic peptide, CD-NP, are resistant to proteolytic inactivation.
Designer natriuretic peptides (NPs) represent an active area of drug development. In canine and human studies, the designer natriuretic peptide CD-NP demonstrated more desirable therapeutic potential than recombinant B-type NP (BNP), which is known as nesiritide and is approved for treatment of acute decompensated heart failure. However, why CD-NP is more effective than BNP is not known. We previously reported that CD-NP is a poorer activator of human guanylyl cyclase-A (GC-A) and a better activator of human guanylyl cyclase-B than BNP. Here, guanylyl cyclase bioassays were used to compare the susceptibility of CD-NP verses ANP, BNP, CNP and DNP to inactivation by human kidney membranes. The half time (t(1/2)) for CD-NP inactivation was increased by factors of 13, 3 and 4 compared to ANP, BNP and CNP, respectively, when measured in the same assay. Surprisingly, DNP failed to undergo complete inactivation and was the most degradation resistant of the peptides tested. The neutral endopeptidase (NEP) inhibitor, phosphoramidon, blocked inactivation of CNP and CD-NP, but not BNP or DNP. In contrast, the general serine and cysteine protease inhibitor, leupeptin, completely blocked the degradation of BNP and CD-NP, but did not block CNP inactivation unless phosphoramidon was included in the assay. Thus, NPs with shorter carboxyl tails (ANP and CNP) are degraded by phosphoramidon-sensitive proteases and NPs with extended carboxyl tails (BNP, DNP and CD-NP) are resistant to NEP degradation and degraded by leupeptin-sensitive proteases. We conclude that DNP and CD-NP are highly resistant to proteolysis and that proteolytic resistance contributes to the beneficial cardiovascular properties of CD-NP. We suggest that this property may be exploited to increase the half-life of NP-based drugs. Topics: Atrial Natriuretic Factor; Cells, Cultured; Cysteine Proteinase Inhibitors; Elapid Venoms; Glycopeptides; HEK293 Cells; Humans; Hydrolysis; Intercellular Signaling Peptides and Proteins; Kidney; Leupeptins; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Neprilysin; Peptides; Receptors, Atrial Natriuretic Factor; Serine Proteinase Inhibitors | 2011 |
Characterization of macroautophagic flux in vivo using a leupeptin-based assay.
Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice. Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Dose-Response Relationship, Drug; Heterozygote; Homeostasis; Lactosylceramides; Leupeptins; Liver; Lysosomes; Male; Mice; Mice, Inbred C57BL; Spleen; Time Factors; Tissue Distribution | 2011 |
Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain.
To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease. Topics: Amino Acid Sequence; Ammonia; Animals; Autophagy; Betaine-Homocysteine S-Methyltransferase; Cell Survival; Cysteine Endopeptidases; Hepatocytes; Humans; L-Lactate Dehydrogenase; Leupeptins; Lysosomes; Male; Mass Spectrometry; Models, Biological; Molecular Sequence Data; Peptide Fragments; Proteolysis; Rats; Rats, Wistar; Sequence Analysis, Protein; Time Factors | 2011 |
Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells.
Development of malaria parasites within vertebrate erythrocytes requires nutrient uptake at the host cell membrane. The plasmodial surface anion channel (PSAC) mediates this transport and is an antimalarial target, but its molecular basis is unknown. We report a parasite gene family responsible for PSAC activity. We used high-throughput screening for nutrient uptake inhibitors to identify a compound highly specific for channels from the Dd2 line of the human pathogen P. falciparum. Inheritance of this compound's affinity in a Dd2 × HB3 genetic cross maps to a single parasite locus on chromosome 3. DNA transfection and in vitro selections indicate that PSAC-inhibitor interactions are encoded by two clag3 genes previously assumed to function in cytoadherence. These genes are conserved in plasmodia, exhibit expression switching, and encode an integral protein on the host membrane, as predicted by functional studies. This protein increases host cell permeability to diverse solutes. Topics: Amino Acid Sequence; Crosses, Genetic; Erythrocytes; High-Throughput Screening Assays; Humans; Ion Channels; Leupeptins; Molecular Sequence Data; Mutation; Permeability; Plasmodium falciparum; Protozoan Proteins; Sequence Alignment | 2011 |
Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction.
The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR-PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles. Topics: Endoplasmic Reticulum; Endoplasmic Reticulum, Smooth; Endosomal Sorting Complexes Required for Transport; Endosomes; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Leupeptins; Lysosomes; Microscopy, Electron; Multivesicular Bodies; Mutation; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; RNA, Small Interfering; Transfection | 2010 |
Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste.
Endogenous proteases, among them cysteine-type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko.. The gel strength of kamaboko obtained by both one-step (50 degrees C, 2 h) and two-step (50 degrees C, 2 h + 80 degrees C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E-64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E-64 and leupeptin. The gel strength of two-step heated kamaboko was increased from 12 to 110 and 130 g cm(-2) by E-64 and leupeptin respectively at a concentration of 0.2 g kg(-1) surimi.. Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi. Topics: Animals; Cathepsin L; Cysteine Proteases; Electrophoresis, Polyacrylamide Gel; Fish Products; Fish Proteins; Gels; Hot Temperature; Hydrolysis; Leucine; Leupeptins; Perciformes; Protease Inhibitors | 2010 |
Lysosomal proteolysis is the primary degradation pathway for cytosolic ferritin and cytosolic ferritin degradation is necessary for iron exit.
Cytosolic ferritins sequester and store iron, consequently protecting cells against iron-mediated free radical damage. However, the mechanisms of iron exit from the ferritin cage and reutilization are largely unknown. In a previous study, we found that mitochondrial ferritin (MtFt) expression led to a decrease in cytosolic ferritin. Here we showed that treatment with inhibitors of lysosomal proteases largely blocked cytosolic ferritin loss in both MtFt-expressing and wild-type cells. Moreover, cytosolic ferritin in cells treated with inhibitors of lysosomal proteases was found to store more iron than did cytosolic ferritins in untreated cells. The prevention of cytosolic ferritin degradation in MtFt-expressing cells significantly blocked iron mobilization from the protein cage induced by MtFt expression. These studies also showed that blockage of cytosolic ferritin loss by leupeptin resulted in decreased cytosolic ferritin synthesis and prolonged cytosolic ferritin stability, potentially resulting in diminished iron availability. Lastly, we found that proteasomes were responsible for cytosolic ferritin degradation in cells pretreated with ferric ammonium citrate. Thus, the current studies suggest that cytosolic ferritin degradation precedes the release of iron in MtFt-expressing cells; that MtFt-induced cytosolic ferritin decrease is partially preventable by lysosomal protease inhibitors; and that both lysosomal and proteasomal pathways may be involved in cytosolic ferritin degradation. Topics: Cell Line; Cytosol; Ferric Compounds; Ferritins; Humans; Iron; Leupeptins; Lysosomes; Mitochondria; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Quaternary Ammonium Compounds | 2010 |
Human B-type natriuretic peptide is not degraded by meprin A.
B-type natriuretic peptide (BNP) combats cardiac stress by reducing blood pressure and ventricular fibrosis. Human BNP is inactivated by unknown cell surface proteases. N-terminal cleavage of mouse BNP by the renal protease meprin A was reported to increase inactivating degradation by a second protease named neprilysin. Since the sequence surrounding the meprin A cleavage site in BNP differs between species, we tested whether meprin A degrades human BNP. Using a recently developed proteolytic bioassay, the ability of various protease inhibitors to block the inactivation of BNP was measured. In rat kidney membranes, inhibitors of meprin A or neprilysin partially or completely blocked inactivation of rat BNP(1-32) when added individually or in combination, respectively. In contrast, neither inhibitor alone or in combination prevented the inactivation of human BNP(1-32) by human kidney membranes. Leupeptin, a serine protease inhibitor, totally blocked inactivation of human BNP by human membranes, substantially blocked the inactivation of rat BNP(1-32) by human membranes, but had no effect on the inactivation of rat BNP(1-32) by rat kidney membranes. Purified neprilysin reduced the bioactivity of rat BNP(1-32) and human BNP. Digestion with both meprin and neprilysis caused the greatest reduction in rat BNP(1-32) but had no effect on the bioactivity of human BNP(1-32). We conclude that meprin A does not degrade BNP in humans and should not be considered a pharmacologic target of the natriuretic peptide system. Topics: Animals; Blood Pressure; Endopeptidases; Humans; Hydrolysis; Kidney; Leupeptins; Metalloendopeptidases; Natriuretic Peptide, Brain; Neprilysin; Peptide Hydrolases; Protease Inhibitors; Rats; Serine Proteases | 2010 |
Collagenolytic activity in sonic extracts of Tannerella forsythia.
The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract.. The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography.. The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced.. The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9. Topics: Bacteroides; Calcium Chloride; Cathepsins; Cell Line, Tumor; Chelating Agents; Collagen Type I; Cysteine; Cysteine Proteinase Inhibitors; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Ethylmaleimide; Gelatinases; Humans; Iodoacetamide; Iodoacetic Acid; Leucine; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 2010 |
Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.
The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species. Topics: Animals; Cysteine Proteinase Inhibitors; Glutathione; Glutathione Disulfide; Heart Arrest, Induced; Leupeptins; Male; Microscopy, Electron, Transmission; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Rats; Rats, Wistar; Time Factors | 2010 |
Leupeptin-based inhibitors do not improve the mdx phenotype.
Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways. Topics: Animals; Biomarkers; Calcium-Binding Proteins; Calpain; Creatine Kinase; Cysteine Proteinase Inhibitors; Diaphragm; Disease Models, Animal; Dose-Response Relationship, Drug; Genotype; Leupeptins; Mice; Mice, Inbred mdx; Muscle Contraction; Muscle Strength; Muscular Dystrophy, Duchenne; Necrosis; Phenotype; Proteasome Endopeptidase Complex; Time Factors | 2010 |
Autolytic activity of human calpain 7 is enhanced by ESCRT-III-related protein IST1 through MIT-MIM interaction.
Calpain 7, a mammalian ortholog of yeast Cpl1/Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESCRT-III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mmN-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7(C290S)-Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system. Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Biocatalysis; Blotting, Western; Calpain; Catalytic Domain; Cysteine Proteinase Inhibitors; Endosomal Sorting Complexes Required for Transport; Ethylmaleimide; Glutathione Transferase; Green Fluorescent Proteins; HEK293 Cells; Humans; Hydrolysis; Immunoprecipitation; Leucine; Leupeptins; Mutation; Oncogene Proteins; Protein Binding; Recombinant Fusion Proteins; Transfection | 2010 |
The 2,2',4,4',5,5'-hexachlorobiphenyl-enhanced degradation of connexin 43 involves both proteasomal and lysosomal activities.
One of the toxic effects of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) is the acute inhibition of gap junctional intercellular communication (GJIC), an event possibly associated with tumor promotion. The model NDL-PCB-2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-induces a sustained GJIC inhibition in rat liver epithelial WB-F344 cells. As this effect might be related to deregulation of connexin 43 (Cx43) synthesis, trafficking, or degradation, we investigated the impact of PCB 153 on these events. Although PCB 153 had no effect on Cx43 mRNA levels, it induced a gradual loss of Cx43 protein and significantly decreased the amount of gap junction plaques in plasma membrane. PCB 153 contributed to extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent accumulation of hyperphosphorylated Cx43-P3 form, thus indicating that ERK1/2 activation by PCB 153 might contribute to its effects on Cx43 internalization or degradation. Inhibition of either proteasomes or lysosomes with their specific inhibitors largely restored total Cx43 protein levels, thus suggesting that both proteasomes and lysosomes may participate in the PCB 153-enhanced Cx43 internalization and degradation. However, neither the proteasomal nor the lysosomal inhibitors restored normal GJIC or number/size of gap junction plaques. Finally, PCB 153 also interfered with restoration of gap junction plaques following the inhibition of Cx43 transport to plasma membrane. Taken together, multiple modes of action seem to contribute to downregulation of Cx43 in PCB 153-treated rat liver epithelial cells. The enhanced degradation of Cx43, together with persistent inhibition of GJIC, might contribute to tumor-promoting effects of NDL-PCBs. Topics: Analysis of Variance; Animals; Cell Communication; Cell Line; Cell Membrane; Connexin 43; Extracellular Signal-Regulated MAP Kinases; Gap Junctions; Leupeptins; Liver; Lysosomes; Metabolic Networks and Pathways; Polychlorinated Biphenyls; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats | 2009 |
Calpain-1 is required for hydrogen peroxide-induced myotube atrophy.
Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress. Topics: Animals; Calpain; Caspase 3; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Hydrogen Peroxide; Leupeptins; Mice; Muscle Proteins; Muscular Atrophy; Myoblasts, Skeletal; Oxidative Stress; RNA Interference; Sarcomeres; Superoxide Dismutase; Time Factors; Transfection | 2009 |
Structures of falcipain-2 and falcipain-3 bound to small molecule inhibitors: implications for substrate specificity.
Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 A crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 A crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at "gatekeeper" positions, may explain the observed kinetic differences between these two closely related enzymes. Topics: Animals; Catalytic Domain; Crystallization; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Kinetics; Leupeptins; Models, Molecular; Plasmodium falciparum; Substrate Specificity | 2009 |
G.B. Quan et al., Inhibition of high glucose-induced erythrocyte phosphatidylserine exposure by leupeptin and disaccharides, Cryobiology 56 (1) (2008) 53-61.
Topics: Cell Membrane Permeability; Disaccharides; Erythrocytes; Glucose; Humans; Leupeptins; Lipid Peroxidation; Malondialdehyde; Phosphatidylserines; Thiobarbiturates; Thiobarbituric Acid Reactive Substances | 2009 |
Role of oxidative stress in ischemia-reperfusion-induced alterations in myofibrillar ATPase activities and gene expression in the heart.
Ischemia-reperfusion (IR) in the heart has been shown to produce myofibrillar remodeling and depress Ca2+ sensitivity of myofilaments; however, the mechanisms for these alterations are not clearly understood. In view of the role of oxidative stress in cardiac dysfunction due to IR, isolated rat hearts were subjected to global ischemia for 30 min followed by a 30-minute period of reperfusion. IR was found to induce cardiac dysfunction, as reflected by depressed LVDP, +dP/dt, and -dP/dt, and elevated LVEDP, and to reduce myofibrillar Ca2+-stimulated ATPase activity. These changes were simulated by perfusing the hearts with a mixture of xanthine plus xanthine oxidase, which is known to generate oxyradicals. The alterations in cardiac function and myofibrillar Ca2+-stimulated ATPase in IR hearts were attenuated by pretreatment with antioxidants (superoxide dismutase plus catalase, and N-acetylcysteine) and leupeptin, an inhibitor of Ca2+-dependent protease. The levels of mRNA for myosin heavy chain isoforms (alpha-MHC and beta-MHC) and myosin light chain (MLC1) were depressed in IR hearts. These changes in gene expression due to IR were prevented upon perfusing the hearts with superoxide plus catalase, with N-acetylcysteine, or with leupeptin. The results suggest that oxidative stress due to IR injury and associated proteolysis play an important role in inducing changes in myofibrillar Ca2+-stimulated ATPase activity and gene expression in the heart. Topics: Acetylcysteine; Animals; Antioxidants; Calcium-Transporting ATPases; Cardiac Myosins; Catalase; Enzyme Inhibitors; Gene Expression Regulation; In Vitro Techniques; Leupeptins; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Myofibrils; Myosin Heavy Chains; Myosin Light Chains; Oxidative Stress; Perfusion; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Ventricular Dysfunction, Left; Ventricular Pressure; Xanthine; Xanthine Oxidase | 2009 |
The comparison of parathyroid hormone degradation effect by various protease inhibitors in blood specimen.
The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples.. Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured.. PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4 degrees C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 micromol/L leupeptin, 10 micromol/L E-64, and 10 micromol/L EDTA, respectively, in the samples stored at room temperature.. The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail. Topics: Aprotinin; Blood Specimen Collection; Edetic Acid; Female; Humans; Leucine; Leupeptins; Male; Parathyroid Hormone; Protease Inhibitors; Time Factors | 2009 |
2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits beta-catenin-dependent transcription in liver epithelial cells.
The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion. Topics: Adherens Junctions; Animals; Axin Protein; beta Catenin; Blotting, Western; Cadherins; Cell Line; Cysteine Proteinase Inhibitors; Epithelial Cells; gamma Catenin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Leupeptins; Liver; Lysosomes; Phosphorylation; Polychlorinated Biphenyls; Rats; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells; Transcription, Genetic | 2009 |
HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients.
Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients. Topics: Adolescent; Adult; Antibodies, Catalytic; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; HIV Infections; HIV Integrase; Humans; Hydrolysis; Leupeptins; Male; Pepstatins; Protease Inhibitors; Sulfones; Young Adult | 2009 |
Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.
The strength of synaptic inhibition depends partly on the number of GABA(A) receptors (GABA(A)Rs) found at synaptic sites. The trafficking of GABA(A)Rs within the endocytic pathway is a key determinant of surface GABA(A)R number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABA(A)Rs. We demonstrate that the targeting of synaptic GABA(A)Rs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the gamma2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the gamma2 subunit retards the lysosomal targeting of GABA(A)Rs and is sufficient to block the loss of synaptic GABA(A)Rs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions. Topics: Animals; Cerebral Cortex; Leupeptins; Lysosomes; Microscopy, Confocal; Neurons; Protein Subunits; Rats; Receptors, GABA-A; Synapses; Ubiquitin | 2009 |
CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization.
CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy. Topics: Autophagy; bcl-X Protein; Caspase 8; Caspases; CD11b Antigen; Cell Survival; Chemokine CCL2; Cytokines; Humans; Interleukin-6; Leucine; Leukocytes, Mononuclear; Leupeptins; Lipopolysaccharide Receptors; Macrophages; Monocytes; Proto-Oncogene Proteins c-bcl-2 | 2009 |
Neuronal apoptosis and autophagy cross talk in aging PS/APP mice, a model of Alzheimer's disease.
Mechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved alpha-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration. Topics: Alzheimer Disease; Animals; Apoptosis; Autophagy; Blotting, Western; Brain; Caspase 3; Cysteine Proteinase Inhibitors; Disease Models, Animal; Enzyme Activation; Female; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intraventricular; Leupeptins; Male; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Neurons; Receptor Cross-Talk | 2008 |
Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes.
Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis. Topics: Adenine; Adenosine Triphosphate; Animals; Autophagy; Cell Shape; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Glucose; Leupeptins; Male; Microtubule-Associated Proteins; Myocytes, Cardiac; Necrosis; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Sirolimus; Time Factors; Vacuoles | 2008 |
The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.
Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation. Topics: Adhesins, Bacterial; Apyrase; Aspirin; Blood Platelets; Calcium Signaling; Cyclooxygenase Inhibitors; Cysteine Endopeptidases; Epinephrine; Gingipain Cysteine Endopeptidases; Humans; In Vitro Techniques; Leupeptins; Nephelometry and Turbidimetry; Oligopeptides; Peptide Fragments; Platelet Aggregation; Platelet Aggregation Inhibitors; Porphyromonas gingivalis; Protease Inhibitors; Pyrroles; Quinazolines; Receptors, Proteinase-Activated; Virulence; Yohimbine | 2008 |
Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment.
We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment. Topics: Animals; Blotting, Western; Culture Media; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Hemiptera; Hydrogen-Ion Concentration; Leucine; Leupeptins; Microscopy, Fluorescence; Protozoan Proteins; Reducing Agents; Salivary Proteins and Peptides; Solanum lycopersicum; Trypanosomatina | 2008 |
Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABA(B) receptors in cortical neurons.
Endocytosis is considered as an important mechanism for regulating cell surface numbers and thereby signaling strength of G protein-coupled receptors. Currently, little is known about the endocytotic pathways of GABA(B) receptors in neurons. Here we report that GABA(B) receptors are constitutively internalized presumably via clathrin-dependent endocytosis in cultured cortical neurons. Colocalization of GABA(B) receptors with endosomal marker proteins indicated sorting of GABA(B) receptors from early endosomes to recycling endosomes and to lysosomes. Cell surface biotinylation experiments revealed fast constitutive recycling of GABA(B) receptors as the predominant pathway that was accelerated by the GABA(B) receptor agonist baclofen. Finally, degradation of GABA(B) receptors in lysosomes was demonstrated by their intracellular accumulation upon inhibition of lysosomal proteases and by blocking recycling which resulted in the redirection of receptors to lysosomes for degradation. These data imply rapid constitutive - agonist-accelerated - recycling of GABA(B) receptors presumably via clathrin-coated pits and their final targeting to lysosomes for degradation. Topics: Animals; Baclofen; Biomarkers; Cell Line; Cells, Cultured; Cerebral Cortex; Clathrin; Cysteine Proteinase Inhibitors; Endocytosis; Endosomes; Female; GABA Agonists; GABA-B Receptor Agonists; Humans; Ionophores; Leupeptins; Lysosomes; Monensin; Neurons; Pregnancy; Rats; Rats, Wistar; Receptors, GABA-B | 2008 |
Inhibition of high glucose-induced erythrocyte phosphatidylserine exposure by leupeptin and disaccharides.
High glucose can lead to serious phosphatidylserine exposure of erythrocytes which may influence the protective effect of glucose on lyophilization of erythrocytes. In this study, caspase activation has not occurred during phosphatidylserine exposure of erythrocytes. However, leupeptin can efficiently inhibit phosphatidylserine exposure of erythrocytes induced by high glucose. With increase of the leupeptin concentrations, the percentages of cells with exposed phosphatidylserine were decreased steadily. In addition, trehalose and sucrose can significantly inhibit phosphatidylserine exposure and cell shrinkage of erythrocytes induced by high glucose through increasing tolerance to osmotic shock. When the disaccharide concentrations were more than 100 mM, the percentages of cells with exposed phosphatidylserine were similar to those of control cells. Moreover, addition of disaccharides in the glucose buffer can result in high osmotic pressure which may facilitate uptake of glucose and disaccharides into erythrocytes and higher cellular glucose and disaccharide concentrations can provide more protection for lyophilized erythrocytes. Although disaccharides can increase the osmotolerance and decrease the phosphatidylserine exposure of erythrocytes exposed to high glucose, whether disaccharides can prevent phosphatidylserine exposure of lyophilized erythrocytes still needs further researches. Topics: Caspase 3; Caspase 8; Cell Size; Erythrocytes; Freeze Drying; Glucose; Hemolysis; Humans; Leupeptins; Lipid Peroxidation; Phosphatidylserines; Sucrose; Trehalose | 2008 |
Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density.
The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH(4)Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation. Topics: Ammonium Chloride; Animals; Cells, Cultured; Chloroquine; Fluorescent Antibody Technique; Humans; Intracellular Fluid; Leupeptins; Lysosomes; Mice; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Time Factors | 2008 |
Calpain inhibition protects spinal motoneurons from the excitotoxic effects of AMPA in vivo.
Microdialysis perfusion of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in rat lumbar spinal cord produces severe motoneuron damage and consequently hindlimb paralysis. Here we studied the time course of the AMPA-induced neurodegenerative changes and motor alterations, and the protective effect of leupeptin, an inhibitor of calpain, a Ca(2+)-activated protease. Paralysis occurs at 4-6 h after AMPA perfusion, but cresyl violet staining showed that motoneuron damage starts at about 3 h and progresses until reaching 50% neuronal loss at 6 h and 90% loss at 12 h. In contrast, choline acetyltransferase (ChAT) immunohistochemistry revealed that the enzyme is already decreased at 30 min after AMPA perfusion and practically disappears at 3 h. Microdialysis coperfusion of leupeptin with AMPA prevented the motor alterations and paralysis and remarkably reduced both the decrement in ChAT immunoreactivity and the loss of motoneurons. We conclude that an increased Ca(2+) influx through Ca(2+)-permeable AMPA receptors activates calpain, and as a consequence ChAT content decreases earlier than other Ca(2+)-dependent processes, including the proteolytic activity of calpain, cause the death of motoneurons. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amyotrophic Lateral Sclerosis; Animals; Calpain; Cysteine Proteinase Inhibitors; Excitatory Amino Acid Agonists; Leupeptins; Male; Motor Neurons; Rats; Rats, Wistar; Rotarod Performance Test; Spinal Cord | 2008 |
Target peptide sequence within infectious human immunodeficiency virus type 1 does not ensure envelope-specific T-helper cell reactivation: influences of cysteine protease and gamma interferon-induced thiol reductase activities.
Recent clinical trials have shown that the presence of a robust human immunodeficiency virus type 1 (HIV-1)-specific T-cell response may not be sufficient to prevent or control HIV-1 infection. Studies of antigen processing in the context of infectious HIV-1 are therefore warranted. Envelope-specific, major histocompatibility complex class II-restricted murine T-cell hybridomas were tested for responsiveness to splenic antigen-presenting cells exposed to HIV-1-infected GHOST cells. Interleukin-2 assays showed that the presence of a peptide within HIV-1 did not ensure the reactivation of peptide-specific T cells. Further experiments defined the impact of gamma interferon-induced thiol reductase and cysteine proteases on the processing of HIV-1 peptides. The results highlight potential influences of peptide context on T-cell reactivation by HIV-1 and encourage the continued study of antigen processing as support for improved vaccine design. Topics: Animals; Antigen Presentation; Cysteine Endopeptidases; env Gene Products, Human Immunodeficiency Virus; Erythrocyte Membrane; Histocompatibility Antigens Class II; HIV Infections; HIV-1; Humans; Hybridomas; Interferon-gamma; Interleukin-2; Leupeptins; Lymphocyte Activation; Mice; Oxidoreductases; Oxidoreductases Acting on Sulfur Group Donors; T-Lymphocytes, Helper-Inducer | 2008 |
Endosomal proteases influence the repertoire of MAGE-A3 epitopes recognized in vivo by CD4+ T cells.
Little is known about the repertoire of MAGE-A3 CD4(+) T-cell epitopes recognized in vivo by neoplastic patients and how antigen processing influences epitope formation. Here, we first show that MAGE-A3-specific CD4(+) T cells are present in the blood of advanced melanoma patients. MAGE-A3(111-125), MAGE-A3(191-205), and MAGE-A3(281-300) were recognized by 7, 6, and 5 of the 11 patients tested, respectively. MAGE-A3(146-160) and MAGE-A3(171-185) were also recognized in two and one cases, whereas no recognition of MAGE-A3(161-175) and MAGE-A3(243-258) was observed. Cytokines produced were mainly interleukin 5 and/or granulocyte macrophage colony-stimulating factor, suggesting impairment of productive polarized Th1 responses. Secondly, proteases inhibitors were used to modulate in vitro the recognition by CD4(+) T-cells clones of dendritic cells loaded with MAGE-A3-expressing cell lysates. We found that formation of MAGE-A3(111-125) depended on both leupeptin-sensitive and pepstatin-sensitive proteases. In contrast, we found that MAGE-A3(161-175), which was never recognized ex vivo, was formed by leupeptin but destroyed by pepstatin-sensitive proteases. Collectively, our results show that (a) anti-MAGE-A3 CD4(+) T-cell immunity develops in vivo in neoplastic patients and is focused toward immunodominant epitopes, (b) the response in advanced disease is skewed toward a Th2 type, and (c) endosomal/lysosomal proteases in dendritic cells influence the repertoire of the epitopes recognized. Topics: Antigen Presentation; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; Dendritic Cells; Endosomes; Epitopes; Humans; Immunophenotyping; Leukocytes, Mononuclear; Leupeptins; Models, Biological; Neoplasm Proteins; Pepstatins; Peptide Hydrolases; Peptides; Th2 Cells | 2008 |
Tryptase activates TGFbeta in human airway smooth muscle cells via direct proteolysis.
Transforming growth factor beta (TGFbeta) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFbeta bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFbeta from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFbeta activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFbeta activation without affecting levels of total TGFbeta. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH(2). Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFbeta via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma. Topics: Benzamidines; Bronchi; Guanidines; Humans; Leupeptins; Myocytes, Smooth Muscle; Oligopeptides; Receptor, PAR-2; Transforming Growth Factor beta; Trypsin Inhibitors; Tryptases | 2008 |
Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons.
Basic fibroblast growth factor (FGF-2) is up-regulated in response to a nerve lesion and promotes axonal regeneration by activation of the tyrosine kinase receptor fibroblast growth factor receptor 1 (FGFR1). To determine the effects of elevated FGFR1 levels on neurite outgrowth, overexpression was combined with lysosomal inhibition of receptor degradation. In pheochromocytoma (PC12) cells, FGFR1 overexpression resulted in flattened morphology, increased neurite outgrowth and activation of extracellular signal-regulated kinase (ERK) and AKT. Degradation of FGFR1 was inhibited by the lysosomal inhibitor leupeptin and by the proteasomal inhibitor lactacystin. In rat primary adult neurons, FGFR1 overexpression enhanced FGF-2-induced axon growth which was further increased by co-treatment with leupeptin. Lysosomal inhibition of receptor degradation concomitant with ligand stimulation of neurons overexpressing FGFR1 provides new insight in tyrosine kinase receptor-mediated promotion of axon regeneration and demonstrates that adult sensory neurons express sub-optimal levels of tyrosine kinase receptors for neurotrophic factors. Topics: Acetylcysteine; Animals; Blotting, Western; Cysteine Proteinase Inhibitors; Fibroblast Growth Factors; Ganglia, Spinal; Green Fluorescent Proteins; Leupeptins; Ligands; Lysosomes; Neurites; PC12 Cells; Pheochromocytoma; Rats; Receptor, Fibroblast Growth Factor, Type 1; Reverse Transcriptase Polymerase Chain Reaction; Sensory Receptor Cells; Signal Transduction | 2008 |
TrkA receptor endolysosomal degradation is both ubiquitin and proteasome dependent.
Gaps in our knowledge exist regarding the degradation of the tropomyosin-regulated kinase A (TrkA) receptor after addition of neurotrophin, nerve growth factor (NGF). TrkA is rapidly and transiently ubiquitinated upon addition of NGF. Here, we demonstrate that the polyubiquitin tag plays a definitive role in receptor sorting. Treatment of PC12 cells with lactacystin prevented NGF-dependent deubiquitination and degradation of TrkA. However, treatment with methylamine, bafilomycin or leupeptin, did not prevent NGF-dependent deubiquitination but blocked the degradation of TrkA. Employing co-immunoprecipitation, biochemical fractionation and confocal microscopy, the kinetics of receptor trafficking post-internalization was observed to occur as a sequel from endosome/multivesicular body, proteasomes, culminating with degradation in the lysosomes. The trafficking of the polyubiquitin-deficient TrkA receptor mutant K485R was impaired and likewise failed to degrade revealing that the receptor escapes degradation. The interaction of TrkA with proteasomes was confirmed by purification and co-immunoprecipitation. We provide evidence that proteasomal deubiquitinating enzymes trim K63-ubiquitin chains from the TrkA receptor prior to its delivery to lysosomes for degradation. Taken together, our results reveal the existence of a novel proteasome-dependent step in receptor degradation. Topics: Animals; Endosomes; Humans; Kinetics; Leupeptins; Lysosomes; Macrolides; Methylamines; Mutation; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Receptor, trkA; Subcellular Fractions; Ubiquitin | 2008 |
Changes in the plasmodial surface anion channel reduce leupeptin uptake and can confer drug resistance in Plasmodium falciparum-infected erythrocytes.
Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants. Topics: Animals; Antimalarials; Biological Transport, Active; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Drug Resistance; Erythrocytes; Genes, Protozoan; Humans; In Vitro Techniques; Ion Channels; Leupeptins; Malaria, Falciparum; Plasmodium falciparum; Protozoan Proteins | 2008 |
Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.
The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain. Topics: Animals; Calcium; Calpain; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Cytoskeleton; Electric Stimulation; Extracellular Space; Immunohistochemistry; In Vitro Techniques; Leupeptins; Male; Mice; Mice, Inbred BALB C; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Signal Transduction | 2008 |
Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogen-induced cholestasis.
Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17alpha-ethinylestradiol administration (5 mg x kg body wt(-1).day(-1) for 5 days) to rats, the bile flow was reduced by 58% (P < 0.05). By subcellular fractionation and immunoblotting analysis, we found that 34 kDa AQP8 was significantly decreased by approximately 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, 17alpha-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 +/- 1 vs. 57 +/- 2 microm/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogen-induced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes. Topics: Animals; Aquaporins; Cell Membrane; Cell Membrane Permeability; Cholestasis; Cysteine Proteinase Inhibitors; Down-Regulation; Estradiol; Estrogens; Ethinyl Estradiol; Gene Expression; Hepatocytes; Leupeptins; Liver; Lysosomes; Male; Rats; Rats, Wistar; Water | 2007 |
Synchronization of cell death in a dinoflagellate population is mediated by an excreted thiol protease.
Regulated programmed cell death (PCD) processes have been documented in several phytoplankton species and are hypothesized to play a role in population dynamics. However, the mechanisms leading to the coordinated collapse of phytoplankton blooms are poorly understood. We showed that the collapse of the annual bloom of Peridinium gatunense, an abundant dinoflagellate in Lake Kinneret, Israel, is initiated by CO2 limitation followed by oxidative stress that triggers a PCD-like cascade. We provide evidences that a protease excreted by senescing P. gatunense cells sensitizes younger cells to oxidative stress and may consequently trigger synchronized cell death of the population. Ageing of the P. gatunense cultures was characterized by a remarkable rise in DNA fragmentation and enhanced sensitivity to H2O2. Exposure of logarithmic phase (young) cultures to conditioning media from stationary phase (old) cells sensitized them to H2O2 and led to premature massive cell death. We detected the induction of specific extracellular protease activity, leupeptin-sensitive, in ageing cultures and in lake waters during the succession of the P. gatunense bloom. Partial purification of the conditioned media revealed that this protease activity is responsible for the higher susceptibility of young cells to oxidative stress. Inhibition of the protease activity lowered the sensitivity to oxidative stress, whereas application of papain to logarithmic phase P. gatunense cultures mimicked the effect of the spent media and enhanced cell death. We propose a novel mechanistic framework by which a population of unicellular phytoplankton orchestrates a coordinated response to stress, thereby determine the fate of its individuals. Topics: Age Factors; Animals; Apoptosis; Biological Evolution; Biomass; Carbon Dioxide; Cysteine Endopeptidases; Dinoflagellida; DNA Fragmentation; Hydrogen Peroxide; Leupeptins; Oxidative Stress; Protozoan Proteins; Signal Transduction | 2007 |
Isolation and characterization of a novel acid proteinase, tropiase, from Candida tropicalis IFO 0589.
A novel acid proteinase (Tropiase) was isolated from Candida tropicalis IFO 0589 by DE52-cellulose, and DEAE-Cosmogel column chromatographies. The purified tropiase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme preparation had a molecular weight of 23,900, isoelectric point of pH 5.1, optimum pH range of 7 to 9 and possessed 208 amino acid residues. The enzyme hydrolyzed casein, fibrinogen, keratin and collagen. The purified tropiase demonstrated hemorrhagic and capillary permeability-increasing activities. Inhibition of tropiase occurred with leupeptin and N-bromosuccinimide, however, no inhibition was observed with alpha(2)-macroglobulin, soybean trypsin inhibitor, benzamidine-HCl or diisopropyl fluorophosphate. Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Bromosuccinimide; Candida tropicalis; Caseins; Chromatography; Electrophoresis; Fibrinogen; Guinea Pigs; Hydrolysis; Isoelectric Point; Leupeptins; Molecular Weight | 2007 |
Peptide degradation is a critical determinant for cell-penetrating peptide uptake.
Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated. Topics: Androstadienes; Animals; Carrier Proteins; Cell Membrane; Cell-Penetrating Peptides; Chloroquine; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Cytochalasin B; Deoxyglucose; Endocytosis; Leupeptins; Membrane Glycoproteins; Models, Biological; Nocodazole; Oligopeptides; Peptide Fragments; Peptides; Perforin; Phenanthrolines; Phenylmethylsulfonyl Fluoride; Pore Forming Cytotoxic Proteins; Protease Inhibitors; Protein Transport; Sodium Azide; Wortmannin | 2007 |
Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations.
Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome. Topics: Acetylcysteine; Adrenoleukodystrophy; Animals; ATP Binding Cassette Transporter, Subfamily D, Member 1; ATP-Binding Cassette Transporters; CHO Cells; Cricetinae; Cricetulus; Fibroblasts; Humans; Leupeptins; Mutation, Missense; Proteasome Inhibitors; Subcellular Fractions | 2007 |
Babesia bovis: effects of cysteine protease inhibitors on in vitro growth.
In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite's invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite's replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite. Topics: Animals; Babesia bovis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Erythrocytes; Leucine; Leupeptins | 2007 |
Endothelial gap junctions are down-regulated by arsenic trioxide.
We investigated the effect of As(2)O(3), an anti-cancer drug, on endothelial gap junctions. Human aortic endothelial cells (HAEC) were treated with As(2)O(3) at 1, 10, 100, and 1000 ng/ml and the cells were examined to evaluate the expression of connexin43 (Cx43) and to assess gap-junction communication. Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) were measured to assess for endothelial dysfunction. Male Sprague-Dawley rats were given intravenous As(2)O(3) (200 mug/kg/day) or saline for 4 weeks, after which aortic endothelial gap junctions, eNOS, and circulating NO levels were evaluated. We found that HAEC Cx43 transcripts and gap junctions were reduced and gap-junction communication was attenuated by As(2)O(3). This decrease of Cx43 gap junctions was prevented by the addition of protease inhibitors. At a dose of 100 ng/ml of As(2)O(3), eNOS was reduced at 48 h, but NO was markedly reduced by 1 h. In animals treated with As(2)O(3), endothelial gap junctions comprising Cx37, Cx40, or Cx43 were all reduced in amount, while eNOS and circulating NO levels remained unchanged. In both in vitro and in vivo rat experiments, endothelial gap junctions were consistently reduced by As(2)O(3), unlike the response of eNOS and NO, which were decreased in cells but not in the rat aortic endothelium. The reduction in Cx43 involved both down-regulation at the transcriptional level and increased degradation. These findings indicate that gap-junction communication in the vascular endothelium is inhibited by treatment with As(2)O(3). Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blotting, Western; Cell Line; Connexin 43; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Gap Junctions; Humans; Immunohistochemistry; Leupeptins; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Oxides; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; von Willebrand Factor | 2007 |
Anthrax protective antigen cleavage and clearance from the blood of mice and rats.
Bacillus anthracis protective antigen (PA) is an 83-kDa (PA83) protein that is cleaved to the 63-kDa protein (PA63) as an essential step in binding and internalizing lethal factor (LF). To assess in vivo receptor saturating PA concentrations, we injected mice with PA variants and measured the PA remaining in the blood at various times using PA83- and PA63-specific enzyme-linked immunosorbent assays. We found that both wild-type PA (WT-PA) and a receptor-binding-defective mutant (Ub-PA) were cleaved to PA63 independent of their ability to bind cells. This suggested a PA-acting protease activity in the blood. The protease cleaved PA at the furin cleavage sequence because furin site-modified PA mutants were not cleaved. Cleavage measured in vitro was leupeptin sensitive and dependent on calcium. Cell surface cleavage was important for toxin clearance, however, as Ub-PA and uncleavable PA mutants were cleared at slower rates than WT-PA. The cell binding-independent cleavage of PA was also verified by using Ub-PA (which is still cleaved) to rescue mice from toxin challenge by competitively binding circulating LF. This mutant was able to rescue mice even when given 12 h before toxin challenge. Its therapeutic ability was comparable to that of dominant-negative PA, which binds cells but does not allow LF translocation, and to the protection afforded through receptor clearance by WT-PA and uncleavable receptor binding-competent mutants. The PA cleavage and clearance observed in mice did not appear to have a role in the differential mouse susceptibility as it occurred similarly in lethal toxin (LT)-resistant DBA/2J and LT-sensitive BALB/cJ mice. Interestingly, PA63 was not found in LT-resistant or -sensitive rats and PA83 clearance was slower in rats than in mice. Finally, to determine the minimum amount of PA required in circulation for LT toxicity in mice, we administered time-separated injections of PA and LF and showed that lethality of LF for mice after PA was no longer measurable in circulation, suggesting active PA sequestration at tissue surfaces. Topics: Animals; Anthrax; Antidotes; Antigens, Bacterial; Bacterial Toxins; Blood; Calcium; Coenzymes; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Leupeptins; Metabolic Clearance Rate; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Mutation; Peptide Hydrolases; Protein Binding; Rats; Rats, Inbred F344; Survival Analysis | 2007 |
Cloning, site-directed mutagenesis and expression of cathepsin L-like cysteine protease from Uronema marinum (Ciliophora: Scuticociliatida).
A cysteine protease gene (ScCtL) homologous to the cathepsin L genes was isolated from a cDNA library of the scuticociliate parasite (Uronema marinum). To express the ScCtL recombinant protein in heterologous system, 17 codons were redesigned to conform to the standard eukaryotic genetic code using PCR-based site-directed mutagenesis. The synthetic U. marinum procathepsin L (proScCtL) was expressed at high levels in E. coli BL21 (DE3) with pGEX-4T-1 vector, and successfully refolded and purified into a functional and enzymatically active form. The optimal pH for protease activity was found to be 4.5. Like any typical cysteine protease, the enzyme was inhibited by E-64 and leupeptin. A dot-blot immunoassay was conducted in an attempt to determine the reaction abilities and sensitivity of the anti-proScCtL polyclonal antibody to the cytosol and to the membrane fraction from the scuticociliate. Our results suggest that the biochemical characteristics of the recombinant ciliate proScCtL protein are similar to that of the cathepsin L-like cysteine protease, and that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form. Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA, Protozoan; Enzyme Stability; Escherichia coli; Gene Expression; Gene Library; Hydrogen-Ion Concentration; Leucine; Leupeptins; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligohymenophorea; Protein Folding; Protozoan Proteins; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid | 2007 |
Structural basis of the zinc inhibition of human tissue kallikrein 5.
Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 A resolution were obtained with and without Zn2+. While the overall structure and the specificity of S1 pocket for basic side-chains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn2+. Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn2+. Instead, Zn2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metal-binding rat trypsin, rotate out of its triad position to provide the third co-ordination site of the bound Zn2+, rendering Zn2+-bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn2+. The His96-99-57 triad is thus suggested to be responsible for the Zn2+-mediated inhibition of hK5 catalysis. Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Humans; Kallikreins; Kinetics; Leupeptins; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Substrate Specificity; Zinc | 2007 |
A novel 51-kDa fragment of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase formed in the stroma of chloroplasts in dark-induced senescing wheat leaves.
The degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was studied. A novel 51-kDa fragment was detected in leaf crude extracts and in chloroplast lysates from leaves with dark-induced senescence. Further studies showed that the 51-kDa fragment was found in the reaction solution with stroma fraction but not in that with the chloroplast membrane fraction and in the chloroplast lysates from mature wheat leaves. The reaction of producing the 51-kDa fragment was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1,10-phenanthroline and EDTA. The N-terminal sequence analysis indicated that the LSU was cleaved at the peptide bond between Lys-14 and Ala-15. In addition, a 50-kDa fragment of LSU formed obviously at pH 6.0-6.5 was detected in the crude extracts of leaves with dark-induced senescence but was not found in lysates of chloroplasts. The degradation was prevented by AEBSF, leupeptin and transepoxysuccinyl-l-leucylamido (4-guanidino) butane (E-64). The results obtained in this study imply that the appearance of the 51-kDa fragment could be because of the involvement of a new senescence-associated protease that is located in the stroma of chloroplasts in senescing wheat leaves. Topics: Chloroplasts; Darkness; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Leupeptins; Pepstatins; Phenanthrolines; Plant Leaves; Protein Subunits; Protons; Ribulose-Bisphosphate Carboxylase; Sulfones; Temperature; Triticum | 2007 |
Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway.
Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [3H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca2+ mobilization in HBF. The HAT-induced increase in Ca2+ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts. Topics: Aged; Bronchi; Butadienes; Calcium; Cell Proliferation; Chromatography, Gel; DNA; Fibroblasts; Humans; Leupeptins; Lung; MAP Kinase Signaling System; Middle Aged; Nitriles; Receptor, PAR-2; Serine Endopeptidases; Sputum; Tryptases | 2006 |
Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.
In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector. Topics: Animals; Antibodies, Protozoan; Antipain; Cell Division; Cells, Cultured; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Detergents; Flow Cytometry; Heteroptera; Immunohistochemistry; Iodoacetamide; Leucine; Leupeptins; Membrane Proteins; Microscopy, Electron; Octoxynol; Plant Proteins; Polyethylene Glycols; Protozoan Proteins; Salivary Glands; Trypanosomatina | 2006 |
Potent pruritogenic action of tryptase mediated by PAR-2 receptor and its involvement in anti-pruritic effect of nafamostat mesilate in mice.
The pruritogenic potency of tryptase and its involvement in anti-pruritic effect of intravenous nafamostat mesilate (NFM) were studied in mice. An intradermal injection of tryptase (0.05-1 ng/site) elicited scratching in ICR mice, while chymase was without effects at doses of 0.05-50 ng/site. The dose-response curve of tryptase action was bell-shaped and the effect peaked at 0.1 ng/site (approximately 0.7 fmol/site). NFM (10 mg/kg) inhibited scratching induced by tryptase but not by histamine and serotonin. NFM (1-10 mg/kg) produced the dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 microg/site). The inhibition by NFM (10 mg/kg) was abolished in mast cell-deficient (WBB6F1 W/W(V)) mice, but not in wild-type (WBB6F1 +/+) mice. NFM (10 mg/kg) suppressed tryptase activity in the mouse skin. Proteinase-activated receptor-2 (PAR-2) neutralizing antibody (0.1 and 1 microg/site) and the PAR-2 antagonist FSLLRY (10 and 100 microg/site) inhibited scratching induced by tryptase (0.1 ng/site) and compound 48/80 (10 microg/site). These results suggest that mast cell tryptase elicits itch through PAR-2 receptor and that NFM inhibits itch-associated responses mainly through the inhibition of mast cell tryptase. Topics: Animals; Antibodies; Antipruritics; Benzamidines; Dose-Response Relationship, Drug; Extravasation of Diagnostic and Therapeutic Materials; Guanidines; Histamine; Histamine H1 Antagonists; Injections, Intradermal; Leupeptins; Male; Mast Cells; Mice; Mice, Inbred ICR; Mice, Mutant Strains; Oligopeptides; p-Methoxy-N-methylphenethylamine; Pruritus; Receptor, PAR-2; Serine Endopeptidases; Serotonin; Skin; Terfenadine; Time Factors; Tryptases | 2006 |
X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.
Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed. Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Humans; Isoenzymes; Leupeptins; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Quaternary; Recombinant Proteins; Sequence Alignment; Serine Endopeptidases; Tryptases | 2006 |
Mast cell beta-tryptase selectively cleaves eotaxin and RANTES and abrogates their eosinophil chemotactic activities.
Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by beta-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of beta-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified beta-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human beta-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. beta-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with beta-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of beta-tryptase on RANTES. These findings suggest that mast cell beta-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles. Topics: Asthma; Cells, Cultured; Chemokine CCL11; Chemokine CCL5; Chemokines, CC; Chemotaxis; Eosinophils; Gene Expression Regulation; Humans; Leupeptins; Mast Cells; Recombinant Proteins; RNA, Messenger; Serine Endopeptidases; Tryptases | 2006 |
Release of iron from ferritin requires lysosomal activity.
How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. In these studies, we investigated whether this occurs in other cell types and whether lysosomes are required. Release of ferritin-Fe was induced with desferoxamine (DFO) in (59)Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. The half-lives for ferritin-(59)Fe and protein were parallel (23, 16, and 11 h for the hepatic, Caco2, and K562 cells, respectively). Co-treatment with 180 microM Fe, leupeptin, chymostatin, or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except for a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis. Topics: Animals; Caco-2 Cells; Chloroquine; Deferoxamine; Enterocytes; Erythroid Cells; Ferritins; Hepatocytes; Humans; Immunoelectrophoresis; Iron; K562 Cells; Leupeptins; Lysosomes; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Rats | 2006 |
Remodeling of connexin 43 in the diabetic rat heart.
In the Streptozotocin-induced diabetic rat heart, a decrease in the conductivity and suppression of electrical cell-to-cell coupling were observed. To clarify this mechanism, the present study was performed to investigate alterations of the gap junction connexin 43 (Cx43) using immunoblotting, immunohistochemistry, electron-microscopic analyses. An enhanced activation of PKCepsilon, an augmentation of PKCepsilon-mediated phosphorylation of Cx43, a decrease in the total amount of Cx43, a reduction in the area of immunoreactive particles for Cx43 at the intercalated disk, distribution of Cx43 to cell periphery or cytoplasm and the internalization approximately annular profiles of the gap junction were all characteristically recognized in the diabetic heart. Such abnormalities in the expression of Cx43 were alleviated by treatment with either lysosomal (NH(4)Cl, Leupeptin) or proteasomal inhibitor (ALLN). These results suggest that the PKCepsilon-mediated hyperphosphorylation of Cx43 makes Cx43 vulnerable to proteolytic degradation and that a decrease in the conductivity in the diabetic heart is also caused by a decrease in the number of gap junction channels due to an acceleration of the proteolytic degradation of Cx43. The remodeling of Cx43 induced by the activation of PKCepsilon may therefore contribute to the formation of the arrhythmogenic substrate in the diabetic heart. The cardioprotective effect of the remodeling of Cx43 by PKCepsilon is discussed. Topics: Animals; Cell Communication; Connexin 43; Diabetes Mellitus, Experimental; Electrophysiology; Gap Junctions; Heart; Immunoblotting; Immunohistochemistry; Leupeptins; Male; Microscopy, Electron; Myocardium; Phosphorylation; Protein Isoforms; Protein Kinase C; Rats; Rats, Wistar; Streptozocin | 2006 |
Leupeptin, a calpain inhibitor, protects inner ear hair cells from aminoglycoside ototoxicity.
Inner ear hair cells play a major role in the auditory pathway that converts sound stimulation into electrical signals, and then into a neural code. However this function is often lost by aminoglycoside ototoxicity. The injury of inner ear hair cells from aminoglycoside treatment is considered apoptosis, and caspase is an important participant in the apoptosis pathway in many organs. It has been reported that calpain, a calcium-dependent protease, is essential for mediation and promotion of cell death. The purpose of the present study was to investigate effects of caspase and calpain inhibitors on the inner ear hair cells after aminoglycoside treatment, and to explore the cell death pathway. Cochlea explant cultures were prepared from mice of postnatal 6 days, cultured with neomycin and/or protease inhibitors, and then stained with phalloidin-fluorescein isothiocyanate (phalloidin-FITC), which was used as a marker to identify surviving hair cells. We demonstrated that neomycin (0.1-1 mM) reduced the number of outer hair cells in a dose-dependent manner. Furthermore, we showed that leupeptin, a calpain inhibitor, significantly protects against the neomycin-induced loss of outer hair cells, whereas a caspase inhibitor was effective only against a lower concentration of neomycin (0.2 mM). Using the TdT-mediated dUTP-biotin nick and labeling method, we also found that a calpain inhibitor, but not a caspase inhibitor, prevents apoptotic DNA fragmentation after treatment with 1 mM neomycin. These results suggest that calpain, rather than caspase, may be responsible for apoptosis induced by aminoglycoside. Thus, leupeptin may prevent hearing loss from aminoglycoside ototoxity. Topics: Animals; Animals, Newborn; Anti-Bacterial Agents; Apoptosis; Biomarkers; Calpain; Cell Survival; Cochlea; Dose-Response Relationship, Drug; Drug Antagonism; Hair Cells, Auditory, Inner; Leupeptins; Mice; Mice, Inbred C57BL; Neomycin; Organ Culture Techniques; Phalloidine; Protein Synthesis Inhibitors | 2006 |
Calpain inhibitors delay injury-induced apoptosis in adult mouse spinal cord motor neurons.
Here, we investigated the effect of calpain inhibitors on apoptosis in organotypic adult spinal cord slices from mice. An increase in calpain I immunoreactivity was found in the nuclei of motor neurons from slices cultured for 30 min. After 4 h, the immunopositive motor neurons exhibited apoptotic changes including nuclear and chromatin condensation. Eight hours after excision, most motor neurons showed nuclear apoptotic features. Two calpain inhibitors, leupeptin and calpain inhibitor XI, inhibited apoptosis in the motor neurons while the caspase inhibitor Z-VAD.fmk had no effect. Leupeptin, but not calpain inhibitor XI and Z-VAD.fmk, also inhibited nucleosomal DNA fragmentation. These results suggest the involvement of calpain I in the induction of apoptosis in motor neurons of adult spinal cord and that apoptosis can be triggered independent of caspase activation. Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Calpain; Caspases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Glycoproteins; Immunohistochemistry; Leupeptins; Mice; Motor Neurons; Nerve Degeneration; Organ Culture Techniques; Spinal Cord; Spinal Cord Injuries; Time Factors | 2006 |
Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4.
Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1(deltaLLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2. Topics: Adenosine Triphosphatases; Animals; ATPases Associated with Diverse Cellular Activities; Carrier Proteins; Cells, Cultured; Chlorocebus aethiops; Cholesterol; COS Cells; Endosomal Sorting Complexes Required for Transport; Endosomes; Fibroblasts; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Glycoproteins; Mutant Proteins; Niemann-Pick C1 Protein; Protein Binding; Protein Processing, Post-Translational; Repressor Proteins; Skin; Ubiquitin; Vesicular Transport Proteins | 2006 |
Heterologous expression, purification, refolding, and structural-functional characterization of EP-B2, a self-activating barley cysteine endoprotease.
We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, alpha2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 A resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue. Topics: Amino Acid Sequence; Crystallography, X-Ray; Cysteine Endopeptidases; Enzyme Activation; Escherichia coli; Gene Expression; Glutens; Hordeum; Hydrogen-Ion Concentration; Kinetics; Leupeptins; Molecular Sequence Data; Pepsin A; Protein Folding; Protein Precursors; Protein Structure, Quaternary; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Trypsin | 2006 |
Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase.
A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH. Topics: Amino Acid Sequence; Animals; Cathepsin L; Cathepsins; Chromatography, Gel; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; L-Lactate Dehydrogenase; Leucine; Leupeptins; Lysosomes; Male; Molecular Sequence Data; Rats; Rats, Wistar; Sequence Alignment | 2006 |
Perturbed interactions of mutant proteolipid protein/DM20 with cholesterol and lipid rafts in oligodendroglia: implications for dysmyelination in spastic paraplegia.
Missense mutations in the human PLP1 gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus-Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLP(A242V) and DM20(A242V) (jimpy-msd in mice), associated with severe PMD-like phenotype in vivo, were not only retained in the ER but also interfered with oligodendroglial process formation. In contrast, glial cells expressing SPG-2-associated PLP(I186T) or DM20(I186T) (rumpshaker in mice) developed processes, and mutant PLP/DM20 reached a late endosomal/lysosomal compartment. Unexpectedly, PLP/DM20 with either substitution exhibited impaired cholesterol binding, and the association with lipid raft microdomains was strongly reduced. Turnover analysis demonstrated that mutant PLP was rapidly degraded in oligodendroglial cells, with half-lives for PLP > PLP(I186T) > PLP(A242V). Protein degradation was specifically sensitive to proteasome inhibition, although PLP/DM20(I186T) degradation was also affected by inhibition of lysosomal enzymes. We conclude that, in addition to ER retention and unfolded protein response (UPR) induction, impaired cholesterol binding and lipid raft association are characteristic cellular defects of PLP1-missense mutations. Mutant protein is rapidly cleared and does not accumulate in oligodendroglial cells. Whereas UPR-induced cell death governs the PMD phenotype of the msd mutation, we propose that impaired cholesterol and lipid raft interaction of the rsh protein may contribute to the dysmyelination observed in SPG-2. Topics: Animals; Blotting, Western; Cells, Cultured; Cholesterol; Cricetinae; Cricetulus; Cysteine Proteinase Inhibitors; Gene Expression; Immunohistochemistry; Immunoprecipitation; Leupeptins; Membrane Microdomains; Mice; Mice, Neurologic Mutants; Mutant Proteins; Myelin Proteolipid Protein; Nerve Tissue Proteins; Oligodendroglia; Protein Transport; Subcellular Fractions; Time Factors; Transfection | 2006 |
Plasminogen/plasmin regulates c-fos and egr-1 expression via the MEK/ERK pathway.
In this study, we showed that plasminogen (Plg) and plasmin (Pla) bind to lysine-binding sites on cell surface and trigger a signaling pathway that activates the mitogen-activated protein kinase (MAPK) MEK and ERK1/2, which in turn leads to the expression of the primary response genes c-fos and early growth response gene egr-1. Our data show that the Plg/Pla-stimulated steady-state mRNA levels of both genes reached a maximum by 30 min and then returned to basal levels by 1h. The gene induction was sensitive to both pharmacological and genetic inhibition of MEK. Leupeptin, a serine protease inhibitor, suppressed Pla but not Plg-induced c-fos and egr-1 expression, emphasizing the role played by the serine protease activity associated with Pla. Pre-incubation with cholera toxin completely blocked the Plg/Pla-induced gene expression, suggesting that another signaling pathway, which recruits G protein-coupled receptors, may also be involved. Furthermore, Plg/Pla also stimulated AP-1 and EGR-1 DNA-binding activities, which were abrogated by pharmacological inhibition of MEK. Altogether, these results suggest that Plg/Pla stimulates c-fos and egr-1 expression via activation of the MEK/ERK pathway. Topics: Animals; Binding Sites; Blotting, Northern; Blotting, Western; Cell Line; Cholera Toxin; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; Early Growth Response Protein 1; Enzyme Activation; Fibrinolysin; Flavonoids; Genes, Dominant; Humans; Immediate-Early Proteins; Leupeptins; Lysine; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Plasminogen; Protein Binding; Proto-Oncogene Proteins c-fos; RNA; RNA, Messenger; Serine Endopeptidases; Signal Transduction; Time Factors; Transcription Factors | 2005 |
The interaction of human tryptase-beta with small molecule inhibitors provides new insights into the unusual functional instability and quaternary structure of the protease.
Human tryptase-beta (HTbeta) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTbeta rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTbeta as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTbeta (si-HTbeta) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTbeta rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTbeta (4-5 microM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTbeta concentration (0.1 microM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTbeta is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers. Topics: Aprotinin; Binding, Competitive; Chromatography, Gel; Enzyme Reactivators; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Hydrolysis; Leupeptins; Protein Structure, Quaternary; Recombinant Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Sulfones; Tryptases | 2005 |
Schistosoma mansoni: functional proteasomes are required for development in the vertebrate host.
Proteasomes are multi-subunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Here, we report for the first time biochemical evidence for the existence of a ubiquitin-proteasome proteolytic pathway in this parasite. Proteasomes from both cercariae and adult worms exhibited a high preference for hydrolysis of the substrate Suc-LLVY-AMC, although in the cercariae extract the rate of hydrolysis was 50% lower when compared to adult worms extracts. The same difference in proteasome activities was observed when endogenous proteins were broken down in the presence of ATP and ubiquitin. Additionally, accumulation of high molecular weight conjugates was observed when cercariae were pre-incubated with proteasome inhibitors. Finally, we present evidence that during experimental schistosomiasis, proteasome inhibitors were able to reduce the number of lung stage schistosomula, reduce the worm burden and consequently decrease the egg output in infected mice. Topics: Adenosine Triphosphate; Animals; Biomphalaria; Coumarins; Host-Parasite Interactions; Hydrolysis; Leupeptins; Lung; Mice; Mice, Inbred BALB C; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Schistosoma mansoni; Schistosomiasis mansoni; Ubiquitin | 2005 |
Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway.
Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels. Topics: Acetylcysteine; Alkaloids; Blotting, Western; Cell Line; Cell Membrane; Cysteine Proteinase Inhibitors; Cytosol; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Enzyme Inhibitors; Glycosylation; Green Fluorescent Proteins; Humans; Immunoprecipitation; Leupeptins; Long QT Syndrome; Mutation; Potassium Channels; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Ribonucleases; Subcellular Fractions; Time Factors; Transfection; Ubiquitin | 2005 |
Induction of cell death in rat small intestine by ischemia reperfusion: differential roles of Fas/Fas ligand and Bcl-2/Bax systems depending upon cell types.
Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0-24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage. Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Caspases; Cytochromes c; Fas Ligand Protein; Immunohistochemistry; In Situ Nick-End Labeling; Intestine, Small; Leupeptins; Membrane Glycoproteins; Microscopy, Electron; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury | 2005 |
Alpha-synuclein-positive structures induced in leupeptin-infused rats.
Abnormal accumulation of alpha-synuclein is regarded as a key pathological step in a wide range of neurodegenerative processes, not only in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) but also in multiple-system atrophy (MSA). Nevertheless, the mechanism of alpha-synuclein accumulation remains unclear. Leupeptin, a protease inhibitor, has been known to cause various neuropathological changes in vivo resembling those of aging or neurodegenerative processes in the human brain, including the accumulation of neuronal processes and neuronal cytoskeletal abnormalities leading to neurofibrillary tangle (NFT)-like formations. In the present study, we administered leupeptin into the rat ventricle and found that alpha-synuclein-positive structures appeared widely in the neuronal tissue, mainly in neuronal processes of the fimbria and alveus. Immunoelectron microscopic study revealed that alpha-synuclein immunoreactivity was located in the swollen axons of the fimbria and alveus, especially in the dilated presynaptic terminals. In addition colocalization of alpha-synuclein with ubiquitin was rarely observed in confocal laser-scan image. This is the first report of experimentally induced in vivo accumulation of alpha-synuclein in non-transgenic rodent brain injected with a well-characterized protease inhibitor by an infusion pump. The present finding suggests that the local accumulation of alpha-synuclein might be induced by the impaired metabolism of alpha-synuclein, which are likely related to lysosomal or ubiquitin-independent proteasomal systems. Topics: alpha-Synuclein; Animals; Cysteine Proteinase Inhibitors; Fornix, Brain; Hippocampus; Infusion Pumps, Implantable; Injections, Intraventricular; Leupeptins; Nerve Tissue Proteins; Neurons; Rats; Rats, Wistar; Secretory Vesicles; Synucleins | 2005 |
Testing the "garbage" accumulation theory of ageing: mitotic activity protects cells from death induced by inhibition of autophagy.
Imperfect autophagic degradation of oxidatively damaged macromolecules and organelles (so-called biological "garbage") is considered an important contributor to ageing and consequent death of postmitotic (non-dividing) cells, such as neurons and cardiac myocytes. In contrast, proliferating cells apparently escape senescence by a continuous dilution and repair of damaged structures during division. Postmitotic ageing can be mimicked and studied in cultures of potentially dividing cells if their mitotic activity is inhibited. To test the "garbage accumulation" theory of ageing, we compared survival of density-dependent growth-arrested (confluent) and proliferating human fibroblasts and astrocytes following inhibition of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent fibroblast cultures to 3MA for two weeks resulted in a significantly increased proportion of dying cells compared to both untreated confluent cultures and dividing cells with 3MA-inhibited autophagy. Similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or leupeptin-exposed cultures, dying cells were overloaded with undegraded autofluorescent material. The results support a key role of biological lysosomal "garbage" accumulation in the triggering of ageing and death of postmitotic cells, as well as the anti-ageing role of cell division. Topics: Adenine; Aging; Astrocytes; Autophagy; Cell Line; Cell Survival; Cellular Senescence; Cysteine Proteinase Inhibitors; Fibroblasts; Humans; Leupeptins; Mitosis; Phosphoinositide-3 Kinase Inhibitors | 2005 |
Transient expression of polymeric immunoglobulin receptor in human adenocarcinoma cell line HT-29.
Human polymeric immunoglobulin receptor (pIgR) protein was expressed in the adeno-carcinoma cell line HT-29 using a recombinant vaccinia virus transfection method. The pIgR protein was detected as 110- and 120-kDa bands by immunoprecipitation after metabolic labeling. PIgR was released as a free secretory component into the culture supernatant and was detected as a 110-kDa band. PIgR cleavage was investigated by adding the proteinase inhibitor leupeptin or protein kinase C activator PMA. Consistent with previous observations in the Madin Darby canine kidney cell system, cleavage of pIgR was inhibited by leupeptin and enhanced by PMA stimulation, thus indicating that it is regulated by common mechanisms. This experimental system should be very useful for pIgR investigation. Topics: Culture Media; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; HT29 Cells; Humans; Immunoprecipitation; Leupeptins; Protein Kinase C; Protein Transport; Receptors, Polymeric Immunoglobulin; Secretory Component; Tetradecanoylphorbol Acetate; Transfection; Vaccinia virus | 2005 |
B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways.
Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes. Topics: Animals; Antigens, CD; Centrifugation, Density Gradient; CHO Cells; Cricetinae; Detergents; Factor VIII; Furin; Glycosylation; Immunoblotting; Immunoprecipitation; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Microscopy, Confocal; Microscopy, Fluorescence; Neuraminidase; Octoxynol; Peptide Fragments; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Sucrose; Transfection; Tunicamycin | 2005 |
Leupeptin-sensitive proteases involved in cell survival after X-ray irradiation in human RSa cells.
Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing. Topics: Apoptosis; Blotting, Western; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Radiation; Drug Resistance, Neoplasm; Fibrinolysis; Flow Cytometry; G2 Phase; Humans; Leupeptins; Peptide Hydrolases; Radiation Tolerance; Tumor Cells, Cultured; X-Rays | 2005 |
Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development.
Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei. Topics: Analysis of Variance; Animals; Gametogenesis; Leupeptins; Mice; Mice, Inbred BALB C; Phenanthrolines; Phenylmethylsulfonyl Fluoride; Plasmodium berghei; Protease Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Zygote | 2005 |
Enzymatic measurement of tryptase-like protease release from isolated perfused guinea pig heart during ischemia-reperfusion.
To elucidate the details of tryptase release from the heart during ischemia-reperfusion (I/R), we attempted the enzymatic measurement of tryptase release from the isolated guinea pig heart perfused by the Langendorff mode I/R model. Tryptase-like activity in the effluent was monitored by the hydrolysis of L-Pyr-Gly-Arg-MCA. Tryptase-like protease and histamine were rapidly released from heart during ischemia within 10 min. After reperfusion, tryptase-like protease levels decreased, achieving stabilization. The tryptase-like protease activity in the effluent was inactivated by serine protease inhibitors. The pattern of inhibition was similar to those of guinea pig and human lung tryptase. In conclusion, tryptase was released into the coronary effluent during ischemia, but not during reperfusion in guinea pig heart. Topics: Animals; Creatine Kinase; Cysteine Proteinase Inhibitors; Female; Guinea Pigs; Histamine Release; In Vitro Techniques; Leupeptins; Myocardial Reperfusion Injury; Myocardium; Serine Endopeptidases; Tryptases | 2005 |
Tryptase increases proliferative activity of human conjunctival fibroblasts through protease-activated receptor-2.
Tryptase that is released by mast cell degranulation has recently been thought to play a key role in wound healing in allergic bronchitis. Conjunctival fibroblasts secrete mediators and extracellular matrices that could exacerbate inflammation and papillary formation in allergic conjunctivitis. This study was conducted to investigate the effect of tryptase on the proliferation of conjunctival fibroblasts and studied whether this effect was mediated by protease-activated receptor (PAR)-2.. Conjunctival fibroblasts were cultured with or without tryptase (0.1 ng/mL to 1.0 microg/mL), and the proliferation rate was assessed after 48 hours. The effects of tryptase inhibitors (leupeptin, benzamidine) and a PAR-2 agonist (SLIGKV) were examined. The existence of PAR-2 mRNA and protein in conjunctival fibroblasts was examined by RT-PCR and Western blot analysis, respectively. The existence of PAR-2 in cultured conjunctival fibroblasts and conjunctival papillae from patients with vernal keratoconjunctivitis, as well as conjunctival tissue from normal subjects was examined by immunohistochemistry.. Conjunctival fibroblast proliferation was upregulated by tryptase in a dose-dependent manner (P < 0.001). Leupeptin and benzamidine inhibited tryptase-induced fibroblast proliferation (P < 0.05), and SLIGKV mimicked tryptase's effect. PAR-2 mRNA and protein were detected in cultured conjunctival fibroblasts using RT-PCR and Western blot analysis. PAR-2 immunoreactivity in both the cultured conjunctival fibroblasts and in stromal cells in excised conjunctival tissues was observed.. Tryptase increased conjunctival fibroblast proliferation and this response appeared to be mediated by PAR-2. Mast cells are the most likely source of tryptase in the conjunctiva and may play an important role in chronic exacerbations with conjunctival papillary formation in allergic conjunctivitis. Topics: Benzamidines; Blotting, Western; Cell Proliferation; Cells, Cultured; Conjunctiva; Dose-Response Relationship, Drug; Fibroblasts; Humans; Immunoenzyme Techniques; Keratoconjunctivitis; Leupeptins; Mast Cells; Oligopeptides; Receptor, PAR-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases; Serine Proteinase Inhibitors; Tryptases; Up-Regulation | 2005 |
High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection.
The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay. Topics: Cathepsin B; Chromatography, Liquid; Dipeptides; Drug Stability; Hot Temperature; Leucine; Leupeptins; Online Systems; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tea | 2005 |
Human airway trypsin-like protease increases mucin gene expression in airway epithelial cells.
Human airway trypsin-like protease (HAT) is a serine protease found in sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor-2 (PAR-2). Results from this study show that HAT treatment also enhances mucus production by the airway epithelial cell line NCI-H292 in vitro. Histologic examination showed that HAT enhances mucous glycoconjugate synthesis, whereas the PAR-2 agonist peptide (PAR-2 AP) has no such effect. HAT, but not PAR-2 AP, enhances MUC2 and MUC5AC gene expression 23-fold and 32-fold, respectively. The proteolytic activity of HAT is required to enhance MUC5AC gene expression; the addition of the inhibitors of trypsin-like protease activity of HAT, aprotinin and leupeptin, abolishes its enhancing effect. AG1478, anti-epidermal growth factor receptor (anti-EGFR)-neutralizing antibody, and anti-amphiregulin (AR)-neutralizing antibody all inhibited the stimulatory effect of HAT. Furthermore, HAT increases AR gene expression and subsequent AR protein release, whereas PAR-2 AP shows no such effects. These results indicate that HAT enhances mucin gene expression through an AR-EGFR pathway, and PAR-2 is not sufficient for or does not directly cause HAT-induced mucin gene expression. Thus, HAT might be a possible therapeutic target to prevent excessive mucus production in patients with chronic airway diseases. Topics: Amphiregulin; Aprotinin; Dose-Response Relationship, Drug; EGF Family of Proteins; Epithelial Cells; ErbB Receptors; Gene Expression Regulation; Glycoconjugates; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Leupeptins; Mucin 5AC; Mucin-2; Mucins; Protease Inhibitors; Quinazolines; Receptor, PAR-2; Respiratory Mucosa; Respiratory System; Serine Endopeptidases; Signal Transduction; Time Factors; Tumor Cells, Cultured; Tyrphostins | 2004 |
Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2.
The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition. Topics: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Cloning, Molecular; Cysteine Endopeptidases; Disulfides; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Immunoblotting; Kinetics; Leupeptins; Molecular Sequence Data; Plasmodium; Plasmodium falciparum; Protein Folding; Protein Structure, Tertiary; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Homology, Amino Acid; Tyrosine | 2004 |
Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis.
The association of periodontitis with atherosclerosis has been suggested from epidemiological studies. Recently, we have reported that macrophages stimulated by Porphyromonas gingivalis formed foam cells in the presence of low-density lipoproteins (LDL). In this study, we examined the direct interactions between LDL and P. gingivalis.. We investigated the aggregation of LDL with P. gingivalis and its outer membrane vesicles (OMVs), degradation of the apo B-100 protein of LDL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, as well as the effects of protease inhibitors or activators on the mobility of LDL by agarose gel shift assays. The binding of P. gingivalis or its OMVs with LDL was demonstrated by western blot analysis. We also examined whether or not the aggregated LDL induced foam cell formation from murine macrophages.. LDL was aggregated in a dose-dependent manner with P. gingivalis and its OMVs. Moreover, degradation of the apo B-100 protein of LDL was directly demonstrated in the presence of P. gingivalis or its OMVs. Furthermore, the gel shift assays indicated that the mobility of LDL was increased in the presence of P. gingivalis. This alteration was attenuated in the presence of the protease inhibitors TLCK and leupeptin and increased in the presence of reducing agents. Moreover, LDL was bound to specific proteins of P. gingivalis suggesting that these proteins may also play a role in aggregation. Finally, the aggregated LDL induced murine macrophages to form foam cells.. These results suggest that P. gingivalis may stimulate foam cell formation, in part, by aggregating LDL by proteolysis of apo B-100. Topics: Adhesins, Bacterial; Animals; Apolipoprotein B-100; Apolipoproteins B; Arteriosclerosis; Cell Line; Cell Membrane; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Foam Cells; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Leupeptins; Lipoproteins, LDL; Macrophages; Mice; Periodontitis; Porphyromonas gingivalis; Protein Synthesis Inhibitors; Tosyllysine Chloromethyl Ketone; Vacuoles | 2004 |
Protease inhibitor leupeptin attenuates myocardial stunning in rat heart.
In order to test the hypothesis that cardiac protein degradation contributes to the pathogenesis of myocardial stunning, the effect of protease inhibitor leupeptin on the postischemic hemodynamics and metabolic functioning was measured in isolated rat hearts.. Isolated rat hearts were perfused in Langendorff mode in the presence or absence of leupeptin (10 Kg/ml for 10 min.), and then subjected to 20 min. of normothermic ischemia and 30 min. of reperfusion. Aortic pressure, cardiac output, coronary flow (CF), global oxygen consumption (MVO2), carbon dioxide and [H+] release, and [Ca2+] uptake were investigated.. Hearts pretreated with leupeptin exhibited better postischemic return of systolic, diastolic and developed aortic pressure and faster return of CF to preischemic values during reperfusion. MVO2 and CO2 release were lower in this group in the 10th and 15th min. of reperfusion and [Ca2+] uptake higher in the 5th and 15th min. of reperfusion. Leupeptin protects the heart from myocardial stunning, which is consistent with the idea that proteases contribute to the pathogenesis of this phenomenon. Topics: Animals; Blood Pressure; Calcium; Carbon Dioxide; Cardiac Output; Coronary Circulation; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; In Vitro Techniques; Leupeptins; Male; Myocardial Contraction; Myocardial Stunning; Oxygen Consumption; Perfusion; Rats; Rats, Wistar | 2004 |
Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization.
Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration. Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Fusion; Cell Line; Cell Movement; Clone Cells; Cysteine Proteinase Inhibitors; Cytoskeleton; Dipeptides; Dose-Response Relationship, Drug; Glucosidases; Intracellular Signaling Peptides and Proteins; Leupeptins; Membrane Proteins; Mice; Muscle Fibers, Skeletal; Myoblasts; Myristoylated Alanine-Rich C Kinase Substrate; Oligopeptides; Phosphoproteins; Stress Fibers | 2004 |
The effect of class-specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma.
B-type natriuretic peptide (BNP) is a cardiac hormone that regulates hemodynamic equilibrium. In the circulation, its activity is controlled by proteolytic factors. Accurate measurement of BNP in a patient's plasma may be affected by degradation due to proteolysis.. We report on the identification and performance of classes of protease inhibitors that stabilize BNP in plasma.. Using the Bayer ADVIA Centaur BNP assay, we measured the effect of arginine, serine and/or specific kallikrein protease inhibitors (PIs) on exogenous spiked or endogenous BNP in patient plasma.. Compared to controls without inhibitor, all PIs were capable, to varying degrees, of retarding the rate of proteolytic degradation. The kallikrein-specific inhibitor, D-Phe-Phe-Arg-chloromethylketone (PPACK II) was most effective as a single constituent and was able to eliminate BNP degradation in patient samples for up to 6-10 days when stored at 2-8 degrees C.. The stability of BNP was markedly increased in the presence of kallikrein-specific PPACK II and a broad spectrum of serine PIs. Use of these compounds offers a simple method of extending sample handling and storage of plasma samples containing BNP. Topics: Amino Acid Sequence; Antipain; Epitopes; Humans; Kallikreins; Leupeptins; Molecular Sequence Data; Natriuretic Peptide, Brain; Protease Inhibitors; Serine Proteinase Inhibitors; Substrate Specificity | 2004 |
A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.
The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146, 1933-1940] were grown on horse blood/agar for 14 days and examined for the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis. Topics: Adhesins, Bacterial; Agar; Animals; Cysteine Endopeptidases; Gingipain Cysteine Endopeptidases; Hemagglutinins; Horses; Leupeptins; Oxyhemoglobins; Pigments, Biological; Porphyromonas gingivalis; Protoporphyrins; Spectrophotometry, Ultraviolet; Spectroscopy, Mossbauer; Spectrum Analysis, Raman; Time Factors | 2004 |
Partial and delayed release of troponin-I compared with the release of lactate dehydrogenase from necrotic cardiomyocytes.
Although the troponins are the serum proteins most frequently used nowadays to diagnose myocardial infarction, controversy continues about whether troponins are released later from infarcted myocardium than the cytoplasmic enzymes used previously, like lactate dehydrogenase (LDH). The present study compared the release kinetics of troponin-I (TnI) and LDH from necrotic cardiomyocytes in vitro. Cardiomyocytes prepared from neonatal rat ventricles were grown for 3 days. A total of 126 cultures were subjected to metabolic inhibition to induce cell necrosis. At various time intervals cells and media were collected for quantitative analysis of LDH activity and TnI concentration. Mean (+/-SD) LDH activity and TnI content of nine cultures at time t=0 were 2.07+/-0.30 U and 1.52+/-0.30 micro g per culture, respectively. Release of LDH from necrotic cardiomyocytes preceded release of TnI by about 60 min. The quantity of LDH released from the cultures after 210 min was 83.2+/-10.0%, whereas that of TnI released after 210 min was always less (33.8+/-22.2%). Cytochemical assessment of necrotic cardiomyocytes showed TnI-positive cells that were poor in LDH. The delay of TnI release relative to LDH release may be explained by slow dissociation of TnI molecules from myofilaments and/or formation of TnI degradation products that are undetected by the currently used ELISA assay. Topics: Animals; Antimetabolites; Calpain; Cysteine Proteinase Inhibitors; Deoxyglucose; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; In Vitro Techniques; L-Lactate Dehydrogenase; Leupeptins; Male; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Sodium Cyanide; Troponin I | 2004 |
Structural basis of the unusual stability and substrate specificity of ervatamin C, a plant cysteine protease from Ervatamia coronaria.
Ervatamin C is an unusually stable cysteine protease from the medicinal plant Ervatamia coronaria belonging to the papain family. Though it cleaves denatured natural proteins with high specific activity, its activity toward some small synthetic substrates is found to be insignificant. The three-dimensional structure and amino acid sequence of the protein have been determined from X-ray diffraction data at 1.9 A (R = 17.7% and R(free) = 19.0%). The overall structure of ervatamin C is similar to those of other homologous cysteine proteases of the family, folding into two distinct left and right domains separated by an active site cleft. However, substitution of a few amino acid residues, which are conserved in the other members of the family, has been observed in both the domains and also at the region of the interdomain cleft. Consequently, the number of intra- and interdomain hydrogen-bonding interactions is enhanced in the structure of ervatamin C. Moreover, a unique disulfide bond has been identified in the right domain of the structure, in addition to the three conserved disulfide bridges present in the papain family. All these factors contribute to an increase in the stability of ervatamin C. In this enzyme, the nature of the S2 subsite, which is the primary determinant of specificity of these proteases, is similar to that of papain, but at the S3 subsite, Ala67 replaces an aromatic residue, and has the effect of eliminating sufficient hydrophobic interactions required for S3-P3 stabilization. This provides the possible explanation for the lower activity of ervatamin C toward the small substrate/inhibitor. This substitution, however, does not affect the binding of denatured natural protein substrates to the enzyme significantly, as there exist a number of additional interactions at the enzyme-substrate interface outside the active site cleft. Topics: Amino Acid Sequence; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Stability; Evolution, Molecular; Leupeptins; Molecular Sequence Data; Papain; Plant Proteins; Protein Folding; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Tabernaemontana | 2004 |
Inhibition of calpains, by treatment with leupeptin, improves motoneuron survival and muscle function in models of motoneuron degeneration.
The effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Exposure of primary rat motoneurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) is an established in vitro model of excitotoxic motoneuron death. Here we show that leupeptin treatment improved motoneuron survival following exposure to AMPA (50 microM). Application of leupeptin (100 microM) to AMPA treated cultures rescued many motoneurons so that 74% (+/-3.4 S.E.M., n=5) survived compared with only 49% (+/-2.4 S.E.M., n=5) in untreated cultures. The effect of treatment with leupeptin on motoneuron survival and muscle function was also examined in vivo. In 3 day-old rats, the sciatic nerve was crushed and at the time of injury, a silicon implant containing leupeptin was inserted onto the lumbar spinal cord. The effect on long-term motoneuron survival and muscle function was assessed 12 weeks after injury. The results showed that there was long-term improvement in motoneuron survival in the leupeptin treated group. Thus, in untreated animals 12 weeks after nerve crush only 30% (+/-2.8. S.E.M., n=3) of sciatic motoneurons survived compared with 43% (+/-1.5 S.E.M., n=3) in the leupeptin-treated group. This improvement in motoneuron survival was reflected in a significant improvement in muscle function in the leupeptin-treated group. For example in the soleus muscle of treated rats 20.8 (+/-1.40 S.E.M., n=5) motor units survived compared with only 14.6 (+/-1.21 S.E.M., n=5) in untreated animals. Thus, treatment with leupeptin, a calpain inhibitor, rescues motoneurons from cell death and improves muscle function following nerve injury. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Calpain; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; Excitatory Amino Acid Agents; Female; Immunohistochemistry; Isometric Contraction; Leupeptins; Male; Microtubule-Associated Proteins; Motor Neuron Disease; Motor Neurons; Muscle Fatigue; Muscle Fibers, Skeletal; Muscle, Skeletal; Myosins; Nerve Crush; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Spinal Cord; Staining and Labeling; Time Factors | 2004 |
Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles.
Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles. In this study, we investigated the capacity of P. gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells. SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors. Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P. gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface. However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA. Treatment of macrophage-like cells with various concentrations of P. gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS. This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment. Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P. gingivalis vesicles. A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation. This study provides evidence that P. gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor. Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P. gingivalis, and other periodontopathogens, to evade the host immune system mechanisms. Topics: Adhesins, Bacterial; Blotting, Western; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Escherichia coli; Gingipain Cysteine Endopeptidases; Gingival Crevicular Fluid; Hemagglutinins; Humans; Leupeptins; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophages; Periodontitis; Porphyromonas gingivalis; Tumor Necrosis Factor-alpha; Virulence Factors | 2004 |
[Effects of protease inhibitors on intestinal absorption of insulin].
To study the effects of protease inhibitors on the large and small intestinal absorption of insulin in rats and to explore the mechanism of various protease inhibitors in different intestinal regions.. The intestinal absorption of insulin was evaluated by its hypoglycemic effect and serum insulin level using an in situ loop method with the washing treatment.. Administration of insulin alone did not decrease the glucose level at either intestinal region with or without the washing treatment. With the unwashing treatment, there were no hypoglycemic effects in small intestinal loop when coadministration of insulin with protease inhibitors. With the washing treatment, the biological effects of insulin were amplified a little in small intestinal loop; obvious hypoglycemic effects were found in large intestinal loop with or without the washing treatment. The effectiveness of protease inhibitors was susceptible to their categories, concentrations and activities of proteolytic enzymes in different regions. The efficacy order of various protease inhibitors for enhancing hypoglycemic response of insulin was: leupeptin > sodium glycocholate > bacitracin > bestatin > cystatin; the percutaneous enhancement effects were observed in the presence of either sodium glycocholate or bacitracin.. Coadministration of protease inhibitors could increase the insulin efficacy more effectively in the large intestine than in the small intestine. Topics: Animals; Bacitracin; Biological Availability; Blood Glucose; Colon; Glycocholic Acid; Insulin; Intestinal Absorption; Intestine, Small; Leupeptins; Male; Protease Inhibitors; Random Allocation; Rats; Rats, Wistar | 2004 |
Calpain inhibitors improve myocardial dysfunction and inflammation induced by endotoxin in rats.
Excessive activation of calpains has been implicated in the pathophysiology of inflammation, trauma, and ischemia reperfusion injury. Here, we investigated the effects of calpain inhibition on myocardial dysfunction and inflammation induced by endotoxin in rats. Rats were treated i.v. with endotoxin (10 mg/kg) or endotoxin plus calpain inhibitors and were then prepared after 4 h for myocardial contractility assessment, detection of endothelium leukocyte interactions, and plasma TNF-alpha, nitrite/nitrate, and endocan levels. Compared with vehicle-treated rats, hearts from endotoxin-treated rats had reduced systolic performance that was partially prevented by calpain inhibitors, i.e., acetyl-leucyl-leucyl-arginal (leupeptin), carbobenzoxy-valyl-phenylalanial (calpain inhibitor III), and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN). Leupeptin and calpain inhibitor III reduced plasma TNF-alpha levels in endotoxin-treated rats. ALLN reduced plasma TNF-alpha and nitrite/nitrate levels in endotoxin-treated rats. Endotoxin treatment increased mesenteric venule leukocyte rolling (10 +/- 3 leukocytes/min vs. 44 +/- 10 leukocytes/min; P < 0.01) and adhesion (2 +/- 2 leukocytes/min vs. 15 +/- 3 leukocytes/min; P < 0.01), which was reduced by calpain inhibitors. Attenuation of leukocyte endothelium interactions observed in calpain inhibitor-treated rats with sepsis was associated with increases in plasma anti-adhesion molecule endocan. In conclusion, calpain inhibitors improved endotoxin-induced cardiac dysfunction, which may be attributed to the modulation of endothelium leukocyte interactions in the inflamed vasculature. Topics: Animals; Cardiomyopathies; Endotoxins; Glycoproteins; Heart; In Vitro Techniques; Inflammation; Injections, Intravenous; Leukocyte Rolling; Leupeptins; Male; Myocardial Contraction; Myocardium; Nitrates; Nitrites; Rats; Rats, Sprague-Dawley; Shock, Septic; Tumor Necrosis Factor-alpha | 2004 |
The sarcoplasmic reticulum proteins are targets for calpain action in the ischemic-reperfused heart.
Ca(2+) overload and free-radical injury are two mutually non-exclusive phenomena suggested to cause myocardial ischemia-reperfusion (IR)-induced contractile dysfunction; however, the mechanisms underlying their effects are not clear. One possible mechanism is the proteolytic modification of proteins by Ca(2+)-dependent proteases, such as calpains, which are activated during Ca(2+) overload that occurs in IR. The sarcoplasmic reticulum (SR) plays a central role in mediating cardiac contractility and therefore any impairment in SR function will induce cardiac contractile dysfunction. We therefore investigated the possibility whether SR proteins were the target for calpain action in IR. Langendorff-perfused rat hearts were subjected to IR in the presence and absence of leupeptin, a calpain inhibitor and the effects of calpain inhibition was examined on cardiac performance, SR function, and its regulation by protein phosphorylation as well as expression of SR Ca(2+)-cycling and -regulatory proteins. Our results show a depression in cardiac contractile function and activation of calpain during IR. Treatment with leupeptin recovered cardiac contractile function and attenuated calpain activity in IR hearts. The cardioprotection observed upon leupeptin treatment was associated with improved SR function and regulation. The recovery in SR function and regulation was consistent with prevention of IR-induced decrease in the expression of key SR Ca(2+)-handling and -regulatory proteins. Our results suggest that a downregulation of SR proteins by calpain may be a mechanism by which Ca(2+) overload causes cardiac contractile dysfunction during IR. Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Calsequestrin; Cyclic AMP-Dependent Protein Kinases; Cytosol; Down-Regulation; Leupeptins; Male; Myocardial Contraction; Myocardium; Perfusion; Phosphorylation; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Time Factors | 2004 |
Multiple cleavage sites for polymeric immunoglobulin receptor.
Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist. Topics: Animals; Cells, Cultured; Cricetinae; Endoplasmic Reticulum; Golgi Apparatus; Humans; Leupeptins; Mutagenesis, Site-Directed; Receptors, Polymeric Immunoglobulin; Secretory Component; Structure-Activity Relationship; Transfection; Vaccinia virus | 2004 |
Myosin Va is proteolysed in rat cerebellar granule neurons after excitotoxic injury.
Cerebellar granule neurons when exposed to glutamate die through an excitotoxic mechanism induced by overactivation of glutamate receptors. This kind of cell death is mediated by an overload of intracellular calcium involving calpain activation, a Ca2+ -dependent intracellular cysteine protease, among other intracellular responses. On the other hand, class V myosins are proteins that move cargo along actin filaments and one of its members, myosin Va, is involved in vesicles transport. Here we studied the effect of excitotoxicity on myosin Va in cultured cerebellar granule neurons. Western blot analysis of control cultures shows a band corresponding to myosin Va as well as an 80 kDa band corresponding to its proteolytic product by calpain. When cells are exposed to glutamate (500 microM), kainate (100 microM) or NMDA (150 microM) during 3-24 h, the proteolytic processing of myosin Va is markedly increased. This proteolysis is inhibited by leupeptin (100 microM) and calpain inhibitor I (50 microM). These inhibitors also significantly improve the morphological appearance of the neurons possibly through the preservation of the cytoskeleton integrity. Our results suggest that myosin Va is a target for calpain I during an excitotoxic injury and could lead to a new area of research to address the participation of molecular motors in neurotoxicity. Topics: Analysis of Variance; Animals; Animals, Newborn; Blotting, Western; Cell Death; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Drug Interactions; Excitatory Amino Acids; Glycoproteins; Leupeptins; Myosin Heavy Chains; Myosin Type V; Neurons; Rats; Tetrazolium Salts; Thiazoles; Time Factors | 2004 |
The function of a trypsin-like enzyme in the saliva of Euplectrus separatae larvae.
Larvae of the gregarious ectoparasitoid, Euplectrus separatae, a species that parasitizes Pseudaletia separata, migrate from the dorsal to the ventral side of the host larva for pupation 7 days after parasitization. The parasitized host larvae die after the migration. The body mass of the parasitoid larvae increases while that of the host larva drastically decreases. Most of the tissue in the dead host larvae completely collapses. In this study, we examined the cause of host death and how the tissues collapse. Artificial removal of all parasitoid larvae before their migration on day 7 rescued the host larvae, but removal after parasitoid migration did not rescue the hosts. Tissues of the dead host larvae were completely liquefied. Injection of saliva from day 7 parasitoid larvae into host larvae killed the host larvae. High activity of a trypsin-like enzyme was detected in the saliva of day 7 parasitoids. Though phospholipase B and hyaluronidase were also detected in the saliva, commercial phospholipase B and hyaluronidase did not kill the hosts, whereas an injection of commercial trypsin was lethal. The trypsin-injected hosts showed the same tissue collapse as noted in parasitized and saliva-injected hosts. Leupeptin, a trypsin inhibitor, reduced mortality when injected into day 7 hosts (parasitoids were removal following migration). These observations suggest that the day 7 parasitoid larvae release saliva containing a trypsin-like enzyme to digest the host tissues following migration. Topics: Animals; Dose-Response Relationship, Drug; Endopeptidase Clp; Host-Parasite Interactions; Hyaluronoglucosaminidase; Larva; Leupeptins; Matrix Metalloproteinases; Moths; Phospholipases; Saliva; Spectrometry, Fluorescence; Trypsin; Trypsin Inhibitors; Wasps | 2004 |
Substrate-dependent modulation of the mechanism of factor XIa inhibition.
Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site. Topics: Aprotinin; Binding Sites; Enzyme-Linked Immunosorbent Assay; Factor IX; Factor XIa; Leupeptins; Models, Biological; Protease Inhibitors | 2004 |
Regulation of muscle Cav1.1 channels by long-term depolarization involves proteolysis of the alpha1s subunit.
The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the alpha1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 microM) and leupeptin (50 microM). In contrast, long-term depolarization had no effect on beta1, the auxiliary subunit of alpha1s. The levels of mRNAs coding the alpha1s and the beta1 subunits were measured by RNase protection assays. Neither the content of the alpha1s nor the beta1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the alpha1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease. Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Leupeptins; Membrane Potentials; Muscle, Skeletal; Patch-Clamp Techniques; Protease Inhibitors; Protein Subunits; Ranidae; RNA, Messenger | 2004 |
Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site.
The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family. Topics: Amino Acid Sequence; Animals; Calpain; Catalytic Domain; Cathepsin K; Cathepsins; Crystallization; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Glycoproteins; Leucine; Leupeptins; Molecular Sequence Data; Papain; Protein Structure, Tertiary; Rats | 2004 |
Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.
In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks. Topics: Animals; Antipain; Chromogenic Compounds; Escherichia coli; Fluorescent Dyes; Hydrogen-Ion Concentration; Ixodidae; Leucine; Leupeptins; Pepstatins; Phenylmethylsulfonyl Fluoride; Recombinant Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Substrate Specificity; Temperature | 2004 |
[Effect of tryptase inhibitors on histamine release from human colon mast cells].
To investigate the effect of tryptase inhibitors (TPIs) on histamine release from human colon mast cells.. Human mast cells were prepared by digestion of colon tissue and with collagenase and hyaluronidase, cultured with four kinds of TPIs, leupeptin, protamine, TLCK, and lactoferrin for 15 min at 37 degrees Celsius respectively. A glass fibre-based fluorometry assay was used to detect histamine in mast cell suspension.. 200 mmol/L leupeptin and 100 mmol/L protamine were able to stimulate histamine release from colon mast cells, while TLCK and lactoferrin did not. All TPIs inhibited anti-IgE-induced histamine release in a concentration dependent manner, and the inhibitory rates were 48.7%, 36.7%, 40.2% and 34.1%, respectively. However preincubation of TPIs with mast cells for 20 min at 37 degrees Celsius before adding anti-IgE had little effect on anti-IgE induced histamine release. All TPIs were able to inhibit calcium ionophore (CI)-induced histamine release, and the maximum inhibition rate was between 25%-32%. Inhibition on histamine release by leupeptin and TLCK obviously enhanced when colon mast cells were preincubated with them for 20 min before adding CI. However, under the same condition, protamine failed to inhibit histamine release.. We prove for the first time that TPIs inhibit anti-IgE-and CI-induced histamine release from human colon mast cells, suggesting that it is possible to treat inflammatory bowel disease or other mast cell-related diseases by using TPIs. Topics: Cells, Cultured; Colon; Histamine Release; Humans; Lactoferrin; Leupeptins; Mast Cells; Protamines; Protease Inhibitors; Serine Endopeptidases; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tryptases | 2004 |
Transferrin-oligomers as potential carriers in anticancer drug delivery.
To investigate if the cross-linking of transferrin receptor (TfR) induced by Tf-oligomers alters the endocytosis of receptor-ligand complexes in cultured tumor cells and hence increases intracellular drug release.. An average of 3.5 Tf molecules per aggregate were cross-linked either by using homobifunctional linker (1, 11-bis-maleimidotetraethyleneglycol) [Tf(3.5-BM(PEO)4)] or heterobifunction linker [succinimidyl 4-(-p-maleimidophenyl)-butyrate] (Tf(3.5-SMPB)). Cell surface binding and competition experiments with 125I-Tf for TfR binding were studied to demonstrate that Tf-oligomers maintain specificity of the TfR-binding. To determine the degradation of Tf-oligomers in TfR-mediated endocytosis, cultured tumor cells were pulsed for 15 min with 125I-Tf-oligomers and chased for 2 h at 37 degrees C in the presence of excess unlabeled Tf. The chase medium was subjected to TCA precipitation to separate the intact and degraded Tf. To investigate if the alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form, methotrexate (MTX) was conjugated to Tf-oligomer (Agg-Tf-MTX) and its antiproliferative activity was compared with monomeric-Tf-MTX (Mono-Tf-MTX) in human colon carcinoma (Caco-2) cells, human breast adenocarcinoma (MCF-7) cells, wild-type Chinese hamster ovary (CHO) cells, and MTX-resistant CHO (CHO-MTX-RII) cells.. TfR-mediated degradation of Tf-oligomers was higher than that of monomeric Tf in both Caco-2 and MCF-7 cells. The IC50 of Agg-Tf-MTX was lower than that of Mono-Tf-MTX in both tumor cell lines. The IC50 of MTX and Mono-Tf-MTX in CHO-MTX-RII cells was higher than that in wild-type CHO cells, whereas the Agg-Tf-MTX was almost identical in both the resistant and wild-type cells.. Cross-linking of TfR induced by oligomeric Tf binding alters the intracellular trafficking of Tf-TfR complexes, redirects them out of the recycling pathway, and targets them to intracellular degradation in cultured tumor cells. The alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form. Consequently, Agg-Tf-MTX is more effective than Mono-Tf-MTX as a TfR-mediated antiproliferative agent in tumor cells, as well as in MTX-resistant transport deficient cells. Therefore, Tf-oligomers are potentially effective TfR-targeting carriers for intracellular delivery of anticancer drugs. Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Drug Carriers; Drug Delivery Systems; Endocytosis; Humans; Indicators and Reagents; Leupeptins; Methotrexate; Polymers; Receptors, Cell Surface; Receptors, Transferrin; Transferrin | 2004 |
A cysteine-activated protease isolated from Todarodes pacificus squid degrades collagen below its denaturation temperature.
Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25 degrees C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen alpha chain. The purified enzymes degraded squid collagen at 25 degrees C, which is 2.5 degrees lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20 degrees C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2-3 degrees C below the denaturation temperature. Topics: Amino Acid Sequence; Animals; Cattle; Collagen; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Decapodiformes; Hydrogen-Ion Concentration; Leucine; Leupeptins; Molecular Sequence Data; Molecular Weight; Muscles; Octopodiformes; Protein Denaturation; Snails; Species Specificity; Temperature | 2004 |
Impact of cysteine proteinase inhibition in midgut fluid and oral secretion on fecundity and pollen consumption of western corn rootworm (Diabrotica virgifera virgifera).
Cysteine proteinases predominate in the midgut fluid (MF) and oral secretion (OS) of adult western corn rootworm (WCR) based on their mild acidic pH optima (pH 6.0), enhanced activities after treatment with thiol reducing agents, and inhibition by selective cysteine proteinase inhibitors (PIs). Four cysteine PIs including E-64, calpeptin, calpain inhibitor II, and leupeptin (also a serine PI) strongly inhibited azocaseinolytic activity in a dose-dependent manner in both the MF and OS. The most significant effect on adult female WCR of cysteine PI consumption with corn pollen was the reduction in fecundity, but female survival was not apparently affected. Mean fresh weights for all PI-fed females were also lower than control groups. All PI-fed groups [E-64, calpain inhibitor I (Cal I) and leupeptin] had a significantly lower daily egg production than respective corn pollen-fed controls. E-64 was more potent than leupeptin and Cal I on inhibiting fecundity, which correlates with their relative anti-proteinase potency in vitro. E-64, Cal I, and leupeptin at 1.5-2 nmol/beetle/day reduced fecundity down to 25-45% of control values. Reduced egg production by PI-fed beetles results from a combination of the direct inhibition of protein digestion and a post-ingestive negative feedback mechanism, which reduces food intake. The supplement of ten essential amino acids into the E-64-treated pollen enhanced up to 3.7-fold the number of eggs laid compared to the E-64-fed group without these amino acids, suggesting that egg production is dependent on the supply of essential amino acids from corn pollen proteolysis. Topics: Animals; Coleoptera; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Digestive System; Eating; Female; Fertility; Leucine; Leupeptins; Male; Oligopeptides; Pollen; Zea mays | 2003 |
Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187.
The involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment. Topics: Animals; Calcimycin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Ionophores; Leucine; Leupeptins; Paragonimus; Parasitology; Reproduction; Serine Proteinase Inhibitors; Sodium Cholate | 2003 |
Expression, purification, and inhibition of in vitro proteolysis of human AMPD2 (isoform L) recombinant enzymes.
AMP deaminase (AMPD) is a multigene family in higher eukaryotes whose three members encode tetrameric isoforms that catalyze the deamination of AMP to IMP. AMPD polypeptides share conserved C-terminal catalytic domains of approximately 550 amino acids, whereas divergent N-terminal domains of approximately 200-330 amino acids may confer isoform-specific properties to each enzyme. However, AMPD polypeptides are subject to limited N-terminal proteolysis during purification and subsequent storage at 4 degrees C. This presents a technical challenge to studies aimed at determining the structural and functional significance of these divergent sequences. This study describes the recombinant overexpression of three naturally occurring human AMPD2 proteins, 1A/2, 1B/2, and 1B/3, that differ by N-terminal extensions of 47-128 amino acids, resulting from the use of multiple promoters and alternative splicing events. A survey of protease inhibitors reveals that E-64 and leupeptin are able to maintain the subunit structure of each AMPD2 protein when they are included in extraction and storage buffers. Gel filtration chromatography of these three purified AMPD2 enzymes comprised of intact subunits reveals that each migrates faster than expected, resulting in observed molecular masses significantly greater than those predicted for native tetrameric structures. However, chemical crosslinking analysis indicates four subunits per AMPD2 molecule, confirming that these enzymes have a native tetrameric structure. These combined results suggest that AMPD2 N-terminal extensions may exist as extended structures in solution. Topics: AMP Deaminase; Baculoviridae; Blotting, Western; Chromatography, Gel; Cross-Linking Reagents; Dimerization; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; Humans; Leupeptins; Peptides; Plasmids; Precipitin Tests; Protein Isoforms; Protein Structure, Tertiary; Recombinant Fusion Proteins; Recombinant Proteins; Temperature; Transfection | 2003 |
The CD8+ cell noncytotoxic anti-HIV response can be blocked by protease inhibitors.
CD8+ cells from healthy HIV-infected individuals can suppress HIV replication in infected CD4(+) cells without killing the cells. This CD8+ cell noncytotoxic antiviral response (CNAR), observed by coculture of CD8+ cells with infected CD4+ cells, is associated with secretion of a CD8+ cell antiviral factor (CAF). In attempts to identify CAF, we discovered that certain protease inhibitors, particularly leupeptin, can block, by up to 95%, the anti-HIV activity in CD8+ cell culture fluids as well as inhibit CNAR. The effect is dose-dependent and is observed in up to 70% of the CAF and CNAR assays by using fluids and cells from several different subjects. Pretreatment of CD8+ cells with leupeptin reduces CNAR, further supporting an inhibitory effect on a CD8+ cell product. This inhibitory activity of protease inhibitors does not affect cell growth, expression of activation antigens, or viability of either CD8+ cells or the infected CD4+ cells. The results suggest that a part of the CD8+ cell noncytotoxic response involves the activity of a protease or a protein that interacts with protease inhibitors. Proteolysis of a CD8+ cell product(s) may be involved. This observation offers a promising approach for identifying the mechanism of CNARCAF activity. Topics: Antiviral Agents; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; HIV; HIV Infections; Humans; In Vitro Techniques; Leupeptins; Protease Inhibitors; Virus Replication | 2003 |
Neuroprotection of vestibular sensory cells from gentamicin ototoxicity obtained using nitric oxide synthase inhibitors, reactive oxygen species scavengers, brain-derived neurotrophic factors and calpain inhibitors.
In order to devise a new treatment for inner ear disorders, the efficacy of a nitric oxide synthase inhibitor (L-N(G)-nitroarginine methylester [L-NAME]), a radical scavenger (D-methionine), a neurotrophin (brain-derived neurotrophic factor [BDNF]) and a calpain inhibitor (leupeptin) for protection from hair cell damage was investigated.. The effects of these drugs on gentamicin-induced production of nitric oxide (NO) and reactive oxygen species (ROS) were studied by means of the fluorescence indicators 4,5-diaminofluorescein diacetate and dihydrotetramethylrosamine. The effect on gentamicin-induced vestibular hair cell damage was examined by using an in vitro LIVE/DEAD system.. L-NAME inhibited the production of NO, D-methionine and BDNF restricted the production of ROS and leupeptin inhibited neither NO nor ROS. All the drugs used limited the vestibular hair cell damage caused by gentamicin. The combinations L-NAME + BDNF, L-NAME + leupeptin and D-methionine + BDNF had a significantly stronger preventive effect on hair cell damage.. It is suggested that combined treatment with a radical inhibitor and either a neurotrophin or calpain inhibitor may help to treat inner ear disorders more effectively. Topics: Animals; Brain-Derived Neurotrophic Factor; Calpain; Cell Survival; Free Radical Scavengers; Gentamicins; Guinea Pigs; Hair Cells, Vestibular; In Vitro Techniques; Leupeptins; Methionine; Microscopy, Fluorescence; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Reactive Oxygen Species | 2003 |
Calpain-mediated AQP2 proteolysis in inner medullary collecting duct.
Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes. Topics: Animals; Aquaporin 2; Aquaporin 6; Aquaporins; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cysteine Proteinase Inhibitors; Dextrans; Dihydrotachysterol; Dose-Response Relationship, Drug; Down-Regulation; Endosomes; Flow Cytometry; Immunoblotting; Immunohistochemistry; Kidney Tubules, Collecting; Leupeptins; Male; Protein Binding; Rats; Rats, Sprague-Dawley; Trypsin | 2003 |
Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions.
Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins. Topics: Autophagy; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Fibroblasts; Humans; Hydrolysis; Kinetics; Lactones; Leupeptins; Lysosomes; Multienzyme Complexes; Proteasome Endopeptidase Complex; Proteins; Scintillation Counting | 2003 |
A nonintrinsic regional basis for increased infrarenal aortic MMP-9 expression and activity.
This investigation was undertaken to determine whether intrinsic or regional factors at different anatomic sites of the aorta affect expression and activity of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).. Aortas from Sprague-Dawley rats (n = 22) were divided into arch, descending thoracic, and infrarenal abdominal segments. Specimens were stimulated with interleukin-1beta (IL-1beta) (2 ng/mL) for 72 hours. In separate experiments, syngeneic aortic segments were transplanted from the thoracic or abdominal aortas of donor rats into the infrarenal aortic position of recipient rats (n = 12 each). At 4 weeks, aortas from rats who had received transplants were harvested, sectioned into arch, thoracic, and transplanted thoracic or transplanted abdominal segments, and stimulated with IL-1beta. Reverse transcriptase polymerase chain reaction, zymography, and reverse zymography were performed to assess MMP-9, MMP-2, and TIMP-1 in all aortic segments. Differences were assessed with analysis of variance (ANOVA) and post-hoc Tukey test.. In control rats, abdominal segments had significantly higher MMP-9 expression compared with arch and thoracic segments (P <.002). Total MMP-9 activity was also higher in abdominal segments (P <.02). In rats who received transplants, transplanted thoracic (P <.004) and transplanted abdominal (P <.05) segments demonstrated upregulation of MMP-9 expression, compared with control arch and thoracic segments. Zymography documented increased total MMP-9 activity in transplanted thoracic (P <.03) and transplanted abdominal (P <.04) segments versus arch and thoracic segments. No significant difference in MMP-9 expression was found between control abdominal, transplanted thoracic, or transplanted abdominal segments. No significant differences in MMP-2 or TIMP-1 expression or activity were demonstrated in either control or transplanted segments.. These data demonstrate that variations in aortic MMP-9 expression and activity result from regional factors affecting the aorta rather than intrinsic aortic wall differences. Increases in abdominal aortic MMP-9 may contribute to the predilection for aneurysm to develop in the infrarenal aorta. Topics: Actins; Animals; Aorta, Abdominal; Aorta, Thoracic; Aortic Aneurysm, Abdominal; Blood Pressure; Chelating Agents; Disease Models, Animal; Edetic Acid; Gelatin; Gelatinases; Heart Rate; Leupeptins; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Models, Cardiovascular; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1 | 2003 |
Endostatin induces autophagic cell death in EAhy926 human endothelial cells.
Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and suppresses neovascularization and tumor growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Electron microscopic analysis revealed that endostatin induced formation of numerous autophagic vacuoles in endothelial in 6 to 24 h after treatment. Moreover, there was only a 2- to 3-fold increase in intracellular reactive oxygen species after endostatin treatment. Endostatin-induced cell death was not prevented by antioxidants (vitamin C, vitamin E, or propyl gallate) or caspase inhibitors, suggesting that the increase of oxidative stress or the activation of caspases may not be the crucial factors in the anti-angiogenic mechanism of endostatin. However, the cytotoxicity of endostatin was significantly reduced by 3-methyladenine (a specific inhibitor of autophagy) and serine and cysteine lysosomal protease inhibitors (leupeptin and aprotinin). Taken together, these results suggest that in human endothelial cells: (1) endostatin predominantly causes autophagic, rather than apoptotic, cell death, (2) endostatin-induced autophagic cell death occurs in the absence of caspase activation and through an oxidative-independent pathway, and (3) endostatin-induced "autophagic cell death" or "type 2 physiological cell death" is regulated by serine and cysteine lysosomal proteases. Topics: Acridine Orange; Adenine; Animals; Antineoplastic Agents; Apoptosis; Aprotinin; Blotting, Western; Caspases; Cell Death; Cells, Cultured; CHO Cells; Cloning, Molecular; Cricetinae; Cysteine; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Endostatins; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Flow Cytometry; Glutathione Transferase; Humans; Immunohistochemistry; Leupeptins; Lysosomes; Microscopy, Electron; Microscopy, Phase-Contrast; Oxidative Stress; Reactive Oxygen Species; Recombinant Proteins; Serine; Time Factors | 2003 |
Intracellular expression of CD69 in endometrial and peripheral T cells represents a useful marker in women with recurrent miscarriage: modulation after allogeneic leukocyte immunotherapy.
To characterize in fertile women and women with recurrent spontaneous abortions (RSA) the expression and functional status of T cells expressing the CD69 molecule.. We analyzed by flow cytometry in peripheral blood and endometrium from fertile and RSA women, the surface and cytoplasmic expression of CD69 on gated T cells. In addition, we investigated by three-color flow cytometry the expression of cytokines, and subsets of memory T cells.. In T cells, CD69 was restricted to the intracellular compartment with a higher frequency in RSA than in fertile women (68.2 +/- 12% versus 23.7 +/- 22%, P < 0.001, and 20 +/- 9.5% versus 2.1 +/- 3.8%, P < 0.005, in endometrium and peripheral blood, respectively). In contrast, the number of interferon-gamma+ (IFN-gamma+) secreting cells was higher (16 +/- 5% versus 6 +/- 1%) in fertile women. All 11 RSA women alloimmunized with parental leukocytes reached values of CD3 +/- CD69+ cells similar to those observed in fertile women.. CD69 might represent a useful marker in the diagnosis and the follow up of RSA patients. Topics: Abortion, Habitual; Adult; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Biomarkers; CD3 Complex; Cysteine Proteinase Inhibitors; Cytokines; Endometrium; Female; Flow Cytometry; Follow-Up Studies; Humans; Immunophenotyping; Immunotherapy; Lectins, C-Type; Leupeptins; Lymphocyte Activation; Lymphocyte Transfusion; Male; Pregnancy; T-Lymphocyte Subsets; T-Lymphocytes; Transplantation, Homologous | 2003 |
Calpain and caspase inhibitors protect vestibular sensory cells from gentamicin ototoxicity.
Apoptosis may play an important role in the mechanism of aminoglycoside ototoxicity. Caspases and calpains are regarded to be important factors in the regulation of cell death in the inner ear. We hypothesized that caspase or calpain inhibitors would protect hair cells from aminoglycoside ototoxicity.. In order to test this hypothesis we carried out a pilot study to determine if gentamicin (GM) would induce caspase and calpain immunolabeling in guinea pig hair cells Having confirmed this we carried out the main experiment using guinea pig vestibular organ culture to determine if caspase and calpain would protect the hair cells from GM ototoxicity.. Immunoreactivity for caspase-3 and m-calpain was detected in the vestibular sensory cells and ganglia after GM treatment. Both caspase and calpain inhibitors protected hair cells against gentamicin ototoxicity.. It is suggested that inhibition of apoptosis is essential in order to block aminoglycoside ototoxicity. Topics: Animals; Anti-Bacterial Agents; Apoptosis; Calpain; Caspase Inhibitors; Cathepsins; Cysteine Proteinase Inhibitors; Gentamicins; Guinea Pigs; Hair Cells, Vestibular; Leupeptins; Oligopeptides; Organ Culture Techniques; Pilot Projects | 2003 |
Differential DNA synthesis in response to activation of protease-activated receptors on cultured guinea-pig tracheal smooth muscle cells.
Both thrombin and tryptase have been shown to induce smooth muscle cell proliferation in vitro. We have used cultured primary guinea-pig tracheal smooth muscle in order to define pharmacologically the receptors involved in this effect. Tryptase, a protease-activated receptor (PAR)-2 agonist, induced DNA synthesis up to the second passage of the cells, thereafter the response waned. In contrast, thrombin, a PAR-1 agonist, and the PAR-1 activating peptide (SFLLRN) induced DNA synthesis starting from the third passage only. Thrombin and tryptase responses were dose-dependently inhibited by leupeptin. The selective PAR-2 activating peptide (SLIGRL-NH(2)) was unable to induce DNA synthesis in cells from passages 1 to 6. In agreement with the functional data, mRNA expression for PAR-1 was increased in cells in later passages. In contradiction with the functional data, however, equal mRNA expression for PAR-2 was found in all passages. These results suggest that thrombin induces guinea-pig tracheal smooth muscle DNA synthesis through activation of PAR-1. However, the differential effect of tryptase and SLIGRL-NH(2) suggests that tryptase might exert some of its effect via a non-PAR-2 receptor. Topics: Actins; Animals; Cell Division; Cells, Cultured; DNA; Guinea Pigs; Humans; Leupeptins; Muscle, Smooth; Peptides; Protease Inhibitors; Receptor, PAR-1; Receptor, PAR-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases; Thrombin; Thymidine; Trachea; Tryptases | 2003 |
Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media.
To investigate how bone cells respond to mechanical stimuli, we subjected osteoblastic cells to fluid flow. We and others already reported that in a culture system of osteoblast-like cells, ERK1/2, Shc, and other proteins were tyrosine-phosphorylated by medium flow and the early response gene, egr-1 or c-fos mRNA, increased. These are the same as events found after stimulation by various growth factors. Moreover, because there were also reports suggesting that growth factor signaling is involved in the responses to mechanical stimuli, we examined the change in epidermal growth factor (EGF) receptor in the cells exposed to medium flow. The results demonstrated that EGF receptor protein increased after exposure to medium flow. This increase did not occur without serum in media, and the addition of EGF restored it. Furthermore, leupeptin blocked this increase. These results suggest that degradation of EGF-occupied EGF receptor by leupeptin-sensitive protease(s) in endosomes decreased with exposure to medium flow. This was presumed to participate, at least in part, in signaling of fluid flow. Topics: Bone and Bones; Bone Development; Culture Media, Conditioned; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Leupeptins; Osteoblasts; Peptide Hydrolases; Physical Stimulation; Protease Inhibitors; Protein Transport; Signal Transduction; Stress, Mechanical; Tumor Cells, Cultured; Up-Regulation | 2003 |
The calpain-calpastatin system and the calcium paradox in the isolated perfused pigeon heart.
To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca(2+) requirements for half and maximum activities are 220 microM and 2 mM, respectively. In the absence of Ca(2+) the protease is strongly activated by Mn(2+) or Sr(2+). In the presence of Ca(2+), Mn(2+) and Sr(2+) exhibit a synergistic effect; Mg(2+) and Ba(2+) have no effect, whereas Co(2+), Ni(2+) and Cd(2+) completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca(2+) depletion, during Ca(2+) repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 microM Mn(2+) or Ba(2+), in the Ca(2+)-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca(2+) depletion, produces two main peaks of ñ150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2(nd) min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart. Topics: Animals; Antipain; Barium; Calcium; Calcium-Binding Proteins; Calpain; Cobalt; Columbidae; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethylmaleimide; Heart; In Vitro Techniques; Kinetics; Leucine; Leupeptins; Magnesium; Manganese; Molecular Weight; Myocardium; Nickel; Phenylmethylsulfonyl Fluoride; Strontium; Time Factors | 2003 |
Characterization of a new p94-like calpain form in human lymphocytes.
Human circulating PBMC (peripheral blood mononuclear cells) contain three calpain isoforms distinguishable on the basis of their chromatographic properties. Two of these proteases belong to the ubiquitous calpain subfamily, corresponding to the classical mu- and m-calpain forms. The third, which shows peculiar activating and regulatory properties, is an alternatively spliced calpain 3 (p94) form. This new calpain differs from calpain 3 in that it has lost IS1 insertion and exon 15, a lysine-rich sequence regarded as a nuclear translocation signal. PBMC p94-calpain undergoes activation and inactivation without the accumulation of a low-Ca2+-requiring form that is typical of the classical activation processes of mu- and m-calpain. Furthermore, it differs from the ubiquitous forms in that it displays a lower sensitivity to calpastatin. On the basis of these selective properties, it can be postulated that PBMC p94-calpain can be activated in response to specific stimuli that are not effective on the other calpain isoenzymes. The enzyme is preferentially expressed in B- and T-lymphocytes, whereas it is poorly expressed in natural killer cells and almost undetectable in polymorphonuclear cells. This distribution might reflect the specific function of this protease, which is preferentially present in cells devoted to the production of the humoral, rather than to the cellular, immune response. Topics: Calcium; Calpain; Catalysis; Cloning, Molecular; Cysteine Proteinase Inhibitors; DNA, Complementary; Enzyme Activation; Erythrocytes; Humans; Immunoblotting; Isoenzymes; Leupeptins; Lymphocytes; Neutrophils; Sequence Analysis, DNA | 2003 |
Calpain-2 as a target for limiting prostate cancer invasion.
Mortality and morbidity of prostate cancer result from extracapsular invasion and metastasis. This tumor progression depends on active cell motility. Previous studies have shown that calpain-regulated rear detachment enabling forward locomotion is required for cell migration initiated by growth factor and adhesion receptors. Therefore, we asked whether calpain would be a target for limiting tumor progression, using as our model the PA DU-145 human prostate carcinoma cell line and a highly invasive subline, wild-type DU-145, derived from it. In vitro, the calpain-specific inhibitor CI-I (ALLN) and the preferential-but-less-specific inhibitor leupeptin decreased transmigration of both cell lines across a Matrigel barrier. These calpain inhibitors limited epidermal growth factor-induced motility but did not alter the growth rate of the tumor cells, as expected. Antisense down-regulation of the growth factor-activated calpain-2 (m-calpain) isoform also reduced transmigration and cell motility. These in vitro findings were then buttressed by in vivo studies, in which i.p. DU-145 tumor xenografts were treated with leupeptin. Tumor invasion into the diaphragm was reduced by leupeptin treatment for both the PA and wild-type DU-145 cells (from 1.7 to 0.78 for the parental line and 2.3 to 1.2 for the invasive derivative, respectively). Tumor cells of both types engineered to express calpain-2 antisense constructs also demonstrated a similar 50% reduced invasiveness in vivo. Finally, we found by gene expression survey of 53 human prostate tumors and 23 normal prostates that calpain was not up-regulated in relationship to invasiveness or metastatic activity, consistent with expectation from the biological role of this effector. Taken together, these results strongly suggest that epigenetic activation of calpain plays an important role in the invasion of human prostate cancer and that it can be targeted to reduce tumor progression. Topics: Animals; Calpain; Cell Movement; Cysteine Proteinase Inhibitors; Down-Regulation; ErbB Receptors; Glycoproteins; Humans; Leupeptins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Oligonucleotides, Antisense; Prostatic Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays | 2003 |
Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors.
To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similar experimental systems.. Human lung tryptase and human skin chymase were purified by a similar procedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacryl gel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined by enzyme assays.. The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native protease inhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain, benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsin inhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymatic activity of chymase. Native protease inhibitors alpha 1-antitrypsin and secretory leukocyte protease inhibitor (SLPI) inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymatic activity. All the 3 inhibitors had weak inhibitory actions on tryptase.. The protease inhibitors tested had relatively good selectivity to either tryptase or chymase. Topics: alpha 1-Antitrypsin; Chymases; Humans; Leupeptins; Lung; Mast Cells; Protease Inhibitors; Serine Endopeptidases; Skin; Tryptases | 2003 |
Calpain and caspase-3 inhibitors reduce infarct size and post-ischemic apoptosis in rat heart without modifying contractile recovery.
Overactivation of proteases play a key role in the development of ischemia reperfusion (IR) myocardial injury. Calpains are calcium-dependent cysteine proteases and have been implicated in post-ischemic cell death. Moreover, activation of caspases, another family of proteases, represents an important step in the apoptotic process. We investigated the effect of leupeptin and calpain inhibitor-1 (CAI-1), two calpain inhibitors and of a caspase-3 inhibitor, Ac-DEVD-CHO, on functional recovery, myocardial infarct size and apoptosis in isolated rat hearts (Langendorff technique) subjected to 30 min of global ischemia and 120 min of reperfusion. Each inhibitor was added to the perfusion medium 10 min before ischemia and during the first 30 min of reperfusion. IR was associated with mechanical dysfunction and myocardial infarction. Apoptosis induced by this sequence was demonstrated by DNA ladder and TUNEL staining. Whereas leupeptin, CAI-1 or Ac-DEVD-CHO did not modify post-ischemic function, they significantly reduced infarct size and cardiomyocyte positive TUNEL staining. Our findings suggest that calpain and caspase-3 inhibitors may protect heart from the development of cell death induced by IR; this effect could be due, at least in part, to the reduction of apoptosis. However, in our experimental conditions, these inhibitors did not afford improvement of post-ischemic myocardial function. Topics: Animals; Apoptosis; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Glycoproteins; Heart; Ischemia; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Muscle Contraction; Myocardium; Rats; Reperfusion | 2003 |
Sustained-release delivery of leupeptin in the chinchilla: hearing results.
Transtympanic medical therapy is becoming an increasingly popular modality for the treatment of "inner-ear disorders." While investigators continue to examine the best dosing paradigms for gentamicin in the treatment of Ménière's disease and for steroids in the treatment of hearing loss, they have also begun to focus on the use of other agents. In particular, transtympanic therapy has been advocated as a plausible route for the treatment of tinnitus. Transtympanic therapy for tinnitus is not new, and a number of groups have reported success in the past. Despite this success, a number of laboratories have been focusing on newer agents that might yield higher success rates in the treatment of tinnitus and other inner-ear disorders. Many of these agents could have systemic side effects when delivered in high enough doses; therefore, they are ideal candidates for transtympanic administration. The goal of this study is to begin to define the effects of one of these agents--leupeptin, a calpain antagonist--on the normal inner ear of an animal model. In this investigation, we demonstrate the effects of sustained-release delivery of leupeptin (2.5 micrograms/ml) on the hearing of chinchillas. The medicine produced no hearing loss at the early time points but did produce some hearing loss at later time points. We discuss these results and begin to outline the next steps in the investigation of this agent. Topics: Animals; Auditory Threshold; Calpain; Chinchilla; Cysteine Proteinase Inhibitors; Delayed-Action Preparations; Dose-Response Relationship, Drug; Ear, Inner; Evoked Potentials, Auditory, Brain Stem; Hearing Loss; Leupeptins; Models, Animal; Perilymph; Time Factors | 2003 |
Matrix metalloprotease 2-mediated activation of Ca(2+)-ATPase by superoxide radical (O2*-) in plasma membrane of bovine pulmonary vascular smooth muscle.
The role of the matrix metalloprotease-2 (MMP-2) in regulating Ca(2+)-ATPase activity in bovine pulmonary artery smooth muscle plasma membranes during treatment with the O2*- generating system, hypoxanthine (HPX) plus xanthine oxidase (XO) has been studied. The smooth muscle membranes possess matrix metalloprotease (MMP) activity in gelatin zymogram, having an apparent molecular mass of 72 kDa; the activity is inhibited by the tissue inhibitor of metalloprotease-2 (TIMP-2). Since both protease and MMP-2 have same molecular mass and are inhibited by TIMP-2, it may, therefore, be suggested that the protease is the MMP-2. Treatment of the smooth muscle membrane suspension with the O2*- generating system stimulates MMP-2 activity, as evidenced by an apparent increase in the intensity of the protease activity. O2*- also enhances [14C]-gelatin degradation and Ca(2+)-ATPase activity. The increase in MMP activity, assessed by [14C]-gelatin degradation and Ca(2+)-ATPase activity are inhibited upon pretreatment with superoxide dismutase (SOD). The O2*- triggered MMP and Ca(2+)-ATPase activities in the membrane are found to be inhibited by TIMP-2. The stimulation of the MMP and Ca(2+)-ATPase activities remain unaffected by the inhibitors of serine, thiol and cysteine groups of proteases such as phenylmethylsulfonylfluoride (PMSF), Bowman Birk inhibitor (BBI), chymostatin, N-ethylmaleimide, leupeptin, antipain and pepstatin. Adding pure bovine MMP-2 to the smooth muscle membrane suspension causes an increase in Ca(2+)-ATPase activity, but the pretreatment with TIMP-2 inhibits the increase in the enzyme activity. Topics: Animals; Antipain; Calcium-Transporting ATPases; Cattle; Cell Membrane; Ethylmaleimide; Free Radicals; Hypoxanthine; Leupeptins; Lung; Matrix Metalloproteinase 2; Muscle, Smooth; Muscle, Smooth, Vascular; Oligopeptides; Oxygen; Pepstatins; Phenylmethylsulfonyl Fluoride; Superoxides; Tissue Inhibitor of Metalloproteinase-2; Trypsin Inhibitor, Bowman-Birk Soybean | 2002 |
Natural killer-like cells in the sheep: functional characterization and regulation by pregnancy-associated proteins.
Natural killer (NK) cells represent an important component of the innate immune system. In ruminants there are few reports regarding presence or characterization of NK cells. Although absence of expression of major histocompatibility complex proteins on ovine trophoblast makes it potentially a target for NK cells, little is known about regulation of NK cells by products of pregnancy in sheep. Objectives of the present study were to determine whether cells with characteristics of NK cells exist in preparations of ovine peripheral blood lymphocytes (PBL) and endometrial epithelial cells (EEC) and to determine regulation of such cells by two pregnancy-associated molecules with immunoregulatory properties (ovine uterine serpin [OvUS] and interferon-tau [IFN-tau]). Ovine PBL and EEC lysed a putative NK target cell, the BHV-1 infected D17 cell, and lysis by both types of cells was neutralized by antibody against a molecule called function-associated molecule (FAM) expressed on NK cells of several species. Moreover, inhibitors that interfere with perforin-mediated lysis blocked NK-like activity of PBL. The NK-like lytic activity of PBL and EEC was inhibited by OvUS, whereas ovine and bovine IFN-tau significantly enhanced NK-like activity of PBL. In conclusion, NK-like activity present in preparations of ovine PBL and EEC is mediated by FAM(+) cells, is dependent on processes that involve perforin processing, and is regulated by OvUS and IFN-tau. Inhibition of NK-like activity of PBL and EEC by OvUS is consistent with a role for OvUS in protecting the conceptus from maternal cytotoxic lymphocytes. Stimulation of lysis by IFN-tau implies the existence of other inhibitory mechanisms during early pregnancy to prevent NK cell-mediated destruction of the conceptus. Topics: Animals; Antibodies, Monoclonal; Dogs; Endometrium; Epithelial Cells; Female; Humans; Interferon Type I; Killer Cells, Natural; Leupeptins; Lymphocytes; Membrane Glycoproteins; Membrane Proteins; Pepstatins; Perforin; Pore Forming Cytotoxic Proteins; Pregnancy; Pregnancy Proteins; Protease Inhibitors; Serpins; Sheep; Tumor Cells, Cultured | 2002 |
Involvement of proteases in the action of IFN-gamma on WISH cells.
This study investigates the possible involvement of serine proteases in interferon-gamma (IFN-gamma) activity on WISH cells. It was observed that inhibition of (3)H-thymidine incorporation induced by IFN-gamma was abrogated by the serine protease inhibitors Nalpha-tosyl-L-lysyl-chloromethane and soybean trypsin inhibitor, both of which act mainly on trypsin. Phenylmethyl sulfonyl fluoride also had a partial inhibitory effect. Other protease inhibitors specific to the cysteine, the aspartic, and the metalloprotease families were not effective. Kinetic analysis revealed that a trypsin-like protease is involved in IFN-gamma activity for up to 7 h. Trypsin-like activity induced by IFN-gamma was detected in the particulate fraction but not in the cytosolic fraction, whereas chymotrypsin activity was not enhanced in either the cytosolic or particulate fractions under similar conditions. Following separation on a gelatin substrate gel, two trypsin-like protease activities located in the particulate fraction were found to increase in response to IFN-gamma treatment. Hence, it seems that a specific membrane-associated trypsin-like protease activity induced by IFN-gamma may play a role in the action of the cytokine on thymidine incorporation in WISH cells. Topics: Amnion; Cell Division; Cell Line; Cell Membrane; DNA Replication; Humans; Interferon-gamma; Leupeptins; Lysine; Pepstatins; Phenylalanine; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Endopeptidases; Serine Proteinase Inhibitors; Subcellular Fractions; Thiorphan; Tosyllysine Chloromethyl Ketone; Trypsin Inhibitor, Bowman-Birk Soybean | 2002 |
Attenuation of the virulence of Porphyromonas gingivalis by using a specific synthetic Kgp protease inhibitor.
The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens. Topics: Adhesins, Bacterial; Animals; Bacteroidaceae Infections; Cysteine Endopeptidases; Disease Models, Animal; Gingipain Cysteine Endopeptidases; Hemagglutinins; Hemolysis; Humans; Leupeptins; Mice; Mice, Inbred BALB C; Mutation; Pigments, Biological; Porphyromonas gingivalis; Protease Inhibitors; Virulence | 2002 |
In vitro antioxidant properties of calpain inhibitors: leupeptin and calpain inhibitor-1.
The aim of this study was 1/ to assess the efficacy of calpain inhibitors: leupeptin and calpain inhibitor-1, to inhibit calpain activity in vitro, 2/ to measure the scavenging abilities of these compounds against free radicals. The efficacy of calpain inhibitors to block calpain activity was tested with azocasein as a substrate for calpain. Leupeptin and calpain inhibitor-1 inhibited calpain activity in the same range of concentrations, the IC50 being 0.14 microM and 0.09 microM, respectively. We measured the antioxidant properties of leupeptin and calpain inhibitor-1 using the allophycocyans assay after identification offree radical species produced by the complex H2O2 + Cu(++). Electron paramagnetic resonance (EPR) spin-trapping studies performed by using DMPO showed that a quartet signal (hyperfine couplings aN = aH = 14.9 G) arisen from DMPO-OH was formed. We found a correlation between leupeptin concentration and oxygen radical absorbance capacity (ORAC) values (r2 = 0.975) indicating an in vivo antioxidant capacity. In contrast, calpain inhibitor-1 showed no protection. In conclusion, our findings indicate that leupeptin and calpain inhibitor-1 are equipotent inhibitors on calpain activity but exhibit diffrent antioxidant efficacy. Topics: Antioxidants; Calpain; Electron Spin Resonance Spectroscopy; Glycoproteins; Kinetics; Leupeptins; Phycocyanin | 2002 |
Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.
We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. Topics: Benzomorphans; Cell Line; Cell Membrane; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Ligands; Protein Binding; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu | 2002 |
Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles.
The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro. A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 x 10(4) cells be stirred with 1.35 x 10(8) platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R. Topics: Adhesins, Bacterial; Antithrombin III; Arginine; Blood Platelets; Cell Membrane; Chromatography; Cysteine; Cysteine Endopeptidases; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Leupeptins; Platelet Aggregation; Porphyromonas gingivalis | 2002 |
Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity.
Calpains, a family of calcium-activated proteases that breakdown proteins, kinases, phosphatases and transcription factors, can promote cell death. Since leupeptin, a calpain inhibitor, protected against hair cell loss from acoustic overstimulation, we hypothesized that it might protect cochlear and vestibular hair cells against gentamicin (GM) ototoxicity. To test this hypothesis, mouse organotypic cultures from the cochlea, maculae of the utricle and the crista of the semicircular canal (P1-P3) were treated with different doses of GM (0.1-3 mM) alone or in the presence of leupeptin (0.1-3 mM). The percentage of outer hair cells (OHCs) and inner hair cells (IHCs) decreased with increasing doses of GM between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced GM-induced damage to IHCs and OHCs; this protective effect was dose-dependent. GM also significantly reduced hair cell density in the crista and utricle in a dose-dependent manner between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced hair cell loss in the crista and utricle for GM concentrations between 0.1 and 3 mM. These results suggest that one of the early steps in GM ototoxicity may involve calcium-activated proteases that lead to the demise of cochlear and vestibular hair cells. Topics: Animals; Anti-Bacterial Agents; Calpain; Cochlea; Dose-Response Relationship, Drug; Gentamicins; Hair Cells, Auditory; Hair Cells, Auditory, Inner; Hair Cells, Auditory, Outer; Leupeptins; Mice; Microscopy, Electron; Organ Culture Techniques; Saccule and Utricle; Semicircular Canals; Vestibule, Labyrinth | 2002 |
Opening of ligand-gated cation channel families by calpain inhibitors.
The class of Ca2+-permeable cation channels is composed of large families with six transmembrane segments including transient receptor potential, vanilloid receptor (VR), polycystin, epithelial calcium channels and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins made us believe that the blockade of calpain opens the silent channels. Using 1 microM of blockers in whole cellular patch pipette fill we measured currents of Chinese hamster ovary cells transfected by VR-like 1 and 2, polycystin-2, or a MLS-like new member (MLS3S). Significant conductance of every clone with a characteristic rectification by blockers was demonstrated. The permeability of Ca2+ to them is similar to that reported. Western blot suggested that blockers did not affect the assembly of the protein but enabled its cleavage. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function. Topics: Amino Acid Sequence; Animals; Calcium; Calcium Channels; Calcium-Binding Proteins; Calpain; Cell Membrane Permeability; CHO Cells; Cloning, Molecular; Cricetinae; Cysteine Proteinase Inhibitors; Electrophysiology; Ion Channel Gating; Leupeptins; Ligands; Membrane Proteins; Molecular Sequence Data; Patch-Clamp Techniques; Receptors, Drug; Transfection; TRPP Cation Channels | 2002 |
Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization, degradation, and mRNA destabilization.
Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA. Topics: Adrenergic beta-1 Receptor Antagonists; Adrenergic beta-2 Receptor Antagonists; Adrenergic beta-Agonists; Amino Acid Sequence; Animals; Blotting, Western; Cell Line; Cricetinae; Cyclic AMP; Down-Regulation; Humans; Isoproterenol; Leupeptins; Molecular Sequence Data; Pepstatins; Protease Inhibitors; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; RNA Stability; RNA, Messenger; Thionucleotides | 2002 |
Stage-specific antimalarial activity of cysteine protease inhibitors.
Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis. Topics: Animals; Antimalarials; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Gene Expression Regulation, Developmental; Hemoglobins; Hydrolysis; Leucine; Leupeptins; Plasmodium falciparum; Time Factors | 2002 |
Immune-complex assays for tyrosine protein kinases.
Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune-complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the Basic Protocol of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS-PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high-throughput assay that evaluates only exogenous substrate phosphorylation. Topics: Adenosine Triphosphate; Animals; Aprotinin; Autoradiography; Caseins; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Humans; Immunoprecipitation; Leupeptins; Peptides; Phenylmethylsulfonyl Fluoride; Phosphopyruvate Hydratase; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rabbits; Signal Transduction; Sodium Fluoride; Structure-Activity Relationship; Time Factors; Vanadates | 2001 |
Role of calcium-dependent protease(s) in globulization of isolated rat lens cortical fiber cells.
To investigate the role of calcium-activated proteases in calcium-dependent disintegrative globulization of isolated rat lens cortex fiber cells.. Rat lens fiber cells were isolated and plated on coverslips at the bottom of a temperature-controlled chamber. The fiber cells were incubated with 10 microM protease substrate, (t-butoxycarbonyl-leu-met-7-amino-4-chloromethylcoumarin:BOC-Leu-M et- CMAC) and the proteolytic activity in the fiber cells was determined by observing the increase in fluorescence, using an excitation wavelength of 360 nm, and measuring emission at 410 nm. Free intracellular calcium was measured using the cell-permeable calcium indicator Fluo-3-AM, and the globulization time (T(g)) was determined using image analysis.. T:(g) of fiber cells superfused with Ringer's solution containing 2 x 10(-)(3) M, 10(-)(6) M, and 10(-)(8) M [Ca(2+)](o) were: 24.7 +/- 1.3, 53.0 +/- 2.8, and more than 120 minutes, respectively. A significant increase in T:(g) ( approximately 95 minutes) was observed when the fibers were preincubated with acetoxymethyl ester of 1,2-bis (2-amino-phenoxy) ethane N:, N:, N:, N:-tetra-acetic acid (BAPTA-AM) to buffer changes in [Ca(2+)](i), or the protease substrate to competitively inhibit degradation of cellular proteins. In the presence of Ringer's solution containing 2 x 10(-)(3) M [Ca(2+)](o) and 0.5 mM of the cysteine protease inhibitor, leupeptin, T:(g) increased to 100 minutes, without affecting [Ca(2+)](i). The proteolytic activity of fiber cells in Ringer's solution containing 10(-)(6) M and 2 x 10(-)(3) M [Ca(2+)](o) increased by approximately 7- and 12-fold, respectively, compared with sucrose-EDTA solution or Ringer's solution containing 10(-)(8) M [Ca(2+)](o). This increase in proteolytic activity was inhibited by leupeptin.. Elevation of calcium in the medium results in a proportionate increase in [Ca(2+)](i) and the proteolytic activity in isolated lens fiber cells. The increase in the proteolytic activity is accompanied by an increase in the rate of globulization of the fiber cells. Inhibition of the proteolytic activity by leupeptin increases T:(g) without affecting the gain in [Ca(2+)](i). These results suggest that globulization of isolated fiber cells in physiological salt solutions is mediated by Ca(2+)-activated protease(s). Topics: Aniline Compounds; Animals; Calcium; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Fluorescent Dyes; Lens Cortex, Crystalline; Leupeptins; Organelles; Rats; Rats, Sprague-Dawley; Time Factors; Xanthenes | 2001 |
Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+).
Previously we showed that the redox active Cu(2+) was much more effective than Cd(2+) at inducing reactive oxygen species ("ROS") formation in hepatocytes and furthermore "ROS" scavengers prevented Cu(2+)-induced hepatocyte cytotoxicity (Pourahmad and O'Brien, 2000). In the following it is shown that hepatocyte cytotoxicity induced by Cu(2+), but not Cd(2+), was preceded by lysosomal membrane damage as demonstrated by acridine orange release. Cytotoxicity, "ROS" formation, and lipid peroxidation were also readily prevented by methylamine or chloroquine (lysosomotropic agents) or 3-methyladenine (an inhibitor of autophagy). Hepatocyte lysosomal proteolysis was also activated by Cu(2+), but not Cd(2+), as tyrosine was released from the hepatocytes and was prevented by leupeptin and pepstatin (lysosomal protease inhibitors). Cu(2+)-induced cytotoxicity was also prevented by leupeptin and pepstatin. A marked increase in Cu(2+)-induced hepatocyte toxicity also occurred if the lysosomal toxins gentamicin or aurothioglucose were added at the same time as the Cu(2+). Furthermore, destabilizing lysosomal membranes beforehand by preincubating the hepatocytes with gentamicin or aurothioglucose prevented Cu(2+)-induced hepatocyte cytotoxicity. It is proposed that Cu(2+)-induced cytotoxicity involves lysosomal damage that causes the release of cytotoxic digestive enzymes as a result of lysosomal membrane damage by "ROS" generated by lysosomal Cu(2+) redox cycling. Topics: Acridine Orange; Adenine; Animals; Aurothioglucose; Cadmium; Cell Death; Chloroquine; Copper; Endopeptidases; Enzyme Activation; Gentamicins; Leupeptins; Lipid Peroxidation; Liver; Lysosomes; Male; Methylamines; Monensin; Oxidation-Reduction; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species | 2001 |
Mechanisms mediating Porphyromonas gingivalis gingipain RgpA-induced oral mucosa inflammation in vivo.
Suffusion of gingipain RgpA (GRgpA) elicited a significant concentration-dependent increase in the clearance of macromolecules from in situ hamster cheek pouch which was attenuated by NPC 17647, a selective bradykinin B(2) receptor antagonist. Leupeptin and a mixture of proteinase inhibitors also attenuated GRgpA-induced responses. These data indicate that GRgpA elicits plasma exudation from in situ oral mucosa in a catalytic site-dependent fashion by elaborating bradykinin. Topics: Adhesins, Bacterial; Animals; Bradykinin; Capillary Permeability; Catalytic Domain; Cricetinae; Cysteine Endopeptidases; Gingipain Cysteine Endopeptidases; Hemagglutinins; Indomethacin; Leupeptins; Male; Mesocricetus; Mouth Mucosa; Periodontitis; Receptor, Bradykinin B2; Receptors, Bradykinin | 2001 |
Leupeptin-induced appearance of partial fragment of betaine homocysteine methyltransferase during autophagic maturation in rat hepatocytes.
A cytosolic enzyme, betaine homocysteine methyltransferase (BHMT), and its partial fragments were discovered as autolysosomal membrane proteins from rat liver in the presence of leupeptin [Ueno et al. (1999) J. Biol. Chem. 274, 15222-15229]. The present study was undertaken to further characterize the transport and processing of BHMT from cytosol to autolysosome and to test if the fragment can be used as an in vitro probe for the maturation step of macroautophagy. Upon subcellular fractionation, BHMT (p44) was found in all fractions, while its 32-kDa fragment (p32) was found only in the mitochondrial-lysosomal (ML) fraction. Incubation of isolated hepatocytes with leupeptin induced time-dependent accumulation of p32 in the ML fraction from 30 to 90 min after the start of incubation. However, chloroquine completely inhibited the appearance of p32, indicating that the processing from p44 to p32 is lysosomal. Incubation with Bafilomycin A(1), a vacuolar H(+)-ATPase inhibitor, together with leupeptin, led to linear accumulation of p44, but not of p32. The p44 accumulation rate was calculated to be 4.9%/h, which was comparable to autophagic sequestration rate. The distribution of p44 within the ML fraction turned out to be dual, i.e., the membrane-surface attached and luminal/sedimentable forms. Amino acids and 3-methyladenine, both of which specifically suppress macroautophagy, inhibited the accumulation of p32 as well as of p44. Finally, energy-dependent appearance of p32 was demonstrated during incubation of postnucler supernatant fractions, making it possible to establish an in vitro assay system. All the results strongly support the idea that BHMT is taken up and degraded to p32 through the macroautophagic pathway, and that p32 could be a novel probe for the maturation of macroautophagy. Topics: Animals; Autophagy; Betaine-Homocysteine S-Methyltransferase; Chloroquine; Cytosol; Hepatocytes; In Vitro Techniques; Leupeptins; Lysosomes; Male; Methyltransferases; Molecular Probes; Peptide Fragments; Proton-Translocating ATPases; Rats; Rats, Wistar; Vacuolar Proton-Translocating ATPases | 2001 |
Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.
One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity. Topics: Adhesins, Bacterial; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Gingipain Cysteine Endopeptidases; Hemagglutinins; Histatins; Leupeptins; Matrix Metalloproteinase Inhibitors; Periodontal Diseases; Protease Inhibitors; Salivary Proteins and Peptides | 2001 |
Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.
Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme. Topics: Amino Acid Sequence; Binding, Competitive; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Histatins; Humans; Kinetics; Leupeptins; Molecular Sequence Data; Salivary Glands; Salivary Proteins and Peptides | 2001 |
Suppression of gingival inflammation induced by Porphyromonas gingivalis in rats by leupeptin.
In this study, we developed a procedure to produce gingivitis in rats by inoculation of Porphyromonas gingivalis and studied the contribution of the bacterial cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp), to the pathology in the gingiva. To adhere the bacterium to periodontal tissues, a cotton thread was inserted between the first and second molar of right maxillary sites of rats. Rats in group A were administered with vehicle alone after bacterial (strain W83) inoculation. In group B, the bacteria were inoculated in combination with leupeptin, a potent inhibitor of Rgp and Kgp, and then leupeptin alone was administered the week after. Rats in group C were administered leupeptin for 6 weeks after bacteria inoculation. All left maxillary gingiva in three groups showed no inflammatory changes. Right maxillary gingiva of group A showed most of the clinical landmarks of gingivitis. Leupeptin exhibited only a little inhibitory effect on this gingivitis in group B, whereas it had a strong inhibitory effect on the inflammation in group C. These results suggest that P. gingivalis-induced gingivitis is attributable to Rgp and Kgp and that leupeptin is more effective in the late phase than the early stage of gingivitis. Topics: Adhesins, Bacterial; Animals; Bacteroidaceae Infections; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Disease Progression; Gingipain Cysteine Endopeptidases; Gingivitis; Hemagglutinins; Leupeptins; Male; Maxilla; Porphyromonas gingivalis; Rats; Rats, Wistar; Virulence | 2001 |
Association between bcl-x(L) and 5-lipoxygenase activating protein (FLAP) levels in IL-3-dependent FL5.12 cells.
The expression of 5-lipoxygenase activating protein (FLAP) in murine hematopoietic FL5.12 cells that are transfected to overexpress bcl-x(L) is less than in control cells. In addition, the withdrawal of IL-3 from the bcl-x(L) overexpressing cells, but not control cells, leads to the rapid loss of FLAP even though these cells, in contrast to control cells, do not undergo apoptosis (Datta et al., J. Biol. Chem. 273, 28163-28169 [1998]). The mechanism(s) underlying these observations is not known. Basal FLAP mRNA levels were actually 2.8-fold higher in bcl-x(L) than control cells indicating that this difference does not have a transcription basis. In addition, an examination of FLAP mRNA levels in response to withdrawal of IL-3 revealed a 2-3-fold increase after 4 and 8 h relative to time-matched samples in both control and bcl-x(L) overexpressing cells. This further indicates that the decrease in FLAP levels in bcl-x(L) overexpressing cells is not related to transcription and suggests an attempt at compensation perhaps in response to increased FLAP degradation/turnover. A proteolytic mechanism was explored by examining the effect of the general caspase inhibitor Boc-D-FMK, and the non-caspase protease inhibitors phenylmethylsulfonyl fluoride (PMSF), pepstatin and leupeptin, on the loss of FLAP in bcl-x(L) overexpressing cells subsequent to IL-3 withdrawal. All inhibitors provided some protection from the loss of FLAP, with PMSF being the most effective, actually increasing FLAP levels above those seen in untreated cells. Given the absence of apoptosis in bcl-x(L) cells, it appears that protease activation is an effect that can accompany a variety of cellular perturbations. The functional consequences of a loss of FLAP in growth-factor deprived cells overexpressing bcl-x(L) is not known. However, these data continue to suggest some link between bcl-x(L) and FLAP. Topics: 5-Lipoxygenase-Activating Proteins; bcl-X Protein; Blotting, Northern; Blotting, Western; Carrier Proteins; Caspase Inhibitors; Cells, Cultured; Cloning, Molecular; Enzyme Inhibitors; Hematopoietic Stem Cells; Interleukin-3; Leupeptins; Membrane Proteins; Pepstatins; Phenylmethylsulfonyl Fluoride; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Transfection | 2001 |
Possible involvement of EBV-mediated alpha-fodrin cleavage for organ-specific autoantigen in Sjogren's syndrome.
A cleavage product of alpha-fodrin may be an important organ-specific autoantigen in the pathogenesis of Sjogren's syndrome (SS), but the mechanisms of alpha-fodrin cleavage remain unclear. Since EBV has been implicated in the pathogenesis of SS, we determined whether EBV activation could induce the SS-specific 120-kDa autoantigen alpha-fodrin. ZEBRA mRNA expression, a marker for activation of the lytic cycle of EBV, was found in the salivary gland tissues from SS patients, but not in those from control individuals. ZEBRA-expressing lymphoid cells were also found in the SS glands in double-stained immunohistochemistry. Furthermore, a significant link between production of Abs against 120-kDa alpha-fodrin and reactivated EBV Ag was found in sera from patients with SS, but not in those from control individuals. EBV-activated lymphoid cells showed specific alpha-fodrin cleavage to the expected 120-kDa fragments in vitro. Pretreatment with caspase inhibitors inhibited cleavage of alpha-fodrin. Thus, an increase in apoptotic protease activities induced by EBV reactivation may be involved in the progression of alpha-fodrin proteolysis in the development of SS. Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aprotinin; Autoantigens; Carrier Proteins; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Herpesvirus 4, Human; Humans; Hydrolysis; Leucine; Leupeptins; Microfilament Proteins; Molecular Weight; Organ Specificity; Pepstatins; Sjogren's Syndrome; Trans-Activators; Tumor Cells, Cultured; Viral Proteins; Virus Activation | 2001 |
Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells.
The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein. Topics: Animals; Baculoviridae; Cell Line; Cysteine Proteinase Inhibitors; Genetic Vectors; Insecta; Leucine; Leupeptins; Recombinant Proteins; Transfection | 2001 |
Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus.
Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25 degrees C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment. Topics: Amino Acid Sequence; Animals; Antipain; Arginine; Benzoylarginine Nitroanilide; Enzyme Stability; Fishes; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Leupeptins; Molecular Sequence Data; Molecular Weight; Trypsin; Trypsin Inhibitors; Viscera | 2001 |
Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system.
The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes.. (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles. Topics: Ammonium Chloride; Animals; Cathepsin B; Cell Line; Cysteine Proteinase Inhibitors; Endocytosis; Endosomes; Epididymis; Ferritins; Fibroblasts; Glycoproteins; Humans; Immunohistochemistry; Intracellular Membranes; Leupeptins; Lysosomes; Male; Mice; Mice, Transgenic; Sandhoff Disease; Saposins; Tay-Sachs Disease; Testis | 2001 |
NO donors inhibit Leishmania infantum cysteine proteinase activity.
Nitric oxide (NO) releasing drugs (e.g., glyceryl trinitrate) were successfully used in the treatment of cutaneous leishmaniasis in man. In the present study, the effect of NO donors on the catalytic activity of the cysteine proteinase from promastigotes of Leishmania infantum, an agent of Old World visceral and cutaneous leishmaniases, is reported. In particular, one equivalent of NO, released by the NO donors S-nitrosoglutathione, glyceryl trinitrate, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, 3-morpholinosydnonimine, S-nitrosoacetylpenicillamine and sodium nitroprusside, inhibited one equivalent of the parasite cysteine proteinase. As expected, NO-deprived compounds did not affect the catalytic activity of the parasite cysteine proteinase. Furthermore, the absorption spectrum of the (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide-treated inactive L. infantum enzyme displayed a maximum in the 330-350 nm wavelength range. The reducing agents dithiothreitol and L-ascorbic acid completely prevented parasite cysteine proteinase inhibition by NO, fully restored the catalytic activity, and reversed the NO-induced absorption spectrum of the inactive enzyme. Moreover, S-nitrosoacetylpenicillamine displayed a leishmanicidal effect, inhibiting the cysteine proteinase activity in vivo. As expected, the NO-deprived compound N-acetylpenicillamine did not affect significantly the parasite viability and the enzyme activity in vivo. These data suggest that the L. infantum cysteine proteinase undergoes NO-mediated S-nitrosylation, thereby representing a possible mechanism of antiparasitic host defence. Topics: Animals; Ascorbic Acid; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dithiothreitol; Glutathione; Kinetics; Leishmania infantum; Leupeptins; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Nitroglycerin; Nitroprusside; Nitroso Compounds; Penicillamine; Protozoan Proteins; S-Nitrosoglutathione | 2001 |
Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor beta chain to late endocytic compartments.
Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor beta chain (IL2Rbeta) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rbeta was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rbeta, with little effect on its internalization. In addition, we show that a fraction of IL2Rbeta can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rbeta targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rbeta were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rbeta toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing. Topics: Acetylcysteine; Antimalarials; Cell Line; Chloroquine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endocytosis; Humans; Immunoblotting; Leupeptins; Microscopy, Fluorescence; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Sorting Signals; Protein Subunits; Receptors, Interleukin-2; Recombinant Fusion Proteins; Transfection; Ubiquitins | 2001 |
Heat-induced proteasomic degradation of HSF1 in serum-starved human fibroblasts aging in vitro.
The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation. Topics: Cells, Cultured; Cellular Senescence; Culture Media, Serum-Free; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Female; Fibroblasts; Heat Shock Transcription Factors; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Binding; Transcription Factors | 2001 |
Studies on the aminopeptidase activities of Porphyromonas gingivalis.
Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV). Topics: Adhesins, Bacterial; Aminopeptidases; Anti-Bacterial Agents; Carbon Radioisotopes; Cathepsins; Chromogenic Compounds; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptidyl Peptidase 4; Gingipain Cysteine Endopeptidases; Guanidines; Hemagglutinins; Humans; Hydroxamic Acids; Leucine; Leupeptins; Mutation; Oligopeptides; Peptides; Porphyromonas gingivalis; Protease Inhibitors; Radiopharmaceuticals | 2001 |
Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.
Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT. Topics: Acetylcysteine; Adenine; Alcohol Dehydrogenase; Animals; Calpain; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dihydrotestosterone; Electrophoresis, Polyacrylamide Gel; Hepatocytes; Leupeptins; Liver; Lysosomes; Male; Multienzyme Complexes; Precipitin Tests; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Ubiquitins | 2001 |
Electrostatic recognition between enzyme and inhibitor: interaction between papain and leupeptin.
Electrostatic forces are involved in a wide variety of molecular interactions that are of biological interest, including, among others, DNA-Protein interactions, protein folding, and the interactions between enzymes and their substrates and inhibitors. In this work, the interaction between papain and an inhibitor, leupeptin, is analyzed from the point of view of their electrostatic interaction. The computations enable one to suggest that negatively charged amino acids located in the region of the active site are responsible for creating an environment that enables efficient binding of the inhibitor. This binding occurs despite the fact that the net global charge of both molecules is positive; an explanation for this apparent contradiction is proposed. Topics: Binding Sites; Computer Simulation; Cysteine Proteinase Inhibitors; Leupeptins; Macromolecular Substances; Models, Chemical; Models, Molecular; Papain; Protein Binding; Static Electricity | 2001 |
Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : understanding the basis of differential inhibition and the role of water.
This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK) and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI) of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition.. The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition.. The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn.H2O.Sn) of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design. Topics: Amino Acid Chloromethyl Ketones; Binding Sites; Chymopapain; Computer Simulation; Cysteine Endopeptidases; Leupeptins; Macromolecular Substances; Plant Proteins; Plants; Protease Inhibitors; Protein Binding; Water | 2001 |
Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.
Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia. Topics: Adhesins, Bacterial; alpha-Amylases; Arginine; Bacterial Adhesion; Bacterial Proteins; Cysteine Endopeptidases; Endopeptidase K; Fimbriae Proteins; Gingipain Cysteine Endopeptidases; Hemagglutinins; Hot Temperature; Humans; Lectins; Leupeptins; Lysine; Mutation; Plant Proteins; Porphyromonas gingivalis; Prevotella intermedia; Tosyllysine Chloromethyl Ketone; Trypsin Inhibitors | 2001 |
Human muscle protein degradation in vitro by eosinophil cationic protein (ECP).
To clarify the role of tissue eosinophils in and around inflammatory foci, we purified eosinophil cationic protein (ECP) and examined its effect on muscle protein degradation in vitro. Eosinophil cationic protein was purified from the buffy coat of blood from healthy volunteers. Myofibrillar, soluble sarcoplasmic, and membrane-associated cytoskeletal proteins were fractionated from latissimus dorsi muscle obtained by orthopedic procedures done on a patient with no neurologic abnormalities. After incubation of these fractions with purified ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed. Eosinophil cationic protein degraded the myofibrillar proteins, especially the myosin heavy chain (MHC) and alpha-actinin. It also degraded membrane-associated cytoskeletal proteins dystrophin and spectrin, whereas soluble sarcoplasmic proteins did not undergo proteolysis. Quantitative analysis of the MHC degradation showed that the ECP reaction was dose-dependent and that the optimal pH was 7.0. Protein degradation was not inhibited by heparin or the protease inhibitors leupeptin, E-64, and pepstatin A. Our results suggest that ECP functions in the degradation of myofibrillar and membrane-associated cytoskeletal proteins, indicating that tissue eosinophils have a specific role in muscle fiber degradation in some myopathies associated with numerous tissue eosinophils, such as eosinophilic myositis, eosinophilic myalgia syndrome, and eosinophilic endocardial disease. Topics: Actinin; Blood Proteins; Cysteine Proteinase Inhibitors; Cytoskeleton; Dose-Response Relationship, Drug; Dystrophin; Eosinophil Granule Proteins; Eosinophils; Fibrinolytic Agents; Heparin; Humans; In Vitro Techniques; Inflammation Mediators; Leupeptins; Muscle Proteins; Myofibrils; Myosin Heavy Chains; Myositis; Pepstatins; Protease Inhibitors; Ribonucleases; Sarcoplasmic Reticulum; Spectrin; Zinc | 2001 |
Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.
Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention. Topics: Animals; Apoptosis; Calcium; Caspase 3; Caspases; Cysteine Endopeptidases; Death Domain Receptor Signaling Adaptor Proteins; DNA-Binding Proteins; Erythrocytes; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Macrophage Activation; Mice; Mitochondria; Models, Biological; Oligopeptides | 2001 |
Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.
Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate. Topics: Animals; Anti-Bacterial Agents; Biological Transport; Chromatography, Gel; Enzyme Inhibitors; Heparan Sulfate Proteoglycans; Hepatocytes; Leupeptins; Lysosomes; Macrolides; Male; Rats; Subcellular Fractions; Sulfur Radioisotopes; Temperature; Tritium; Vinblastine | 2001 |
Characterization of an important enzymatic component in collagenase that is essential for the effective digestion of the human and porcine pancreas.
Recent clinical results from Edmonton have demonstrated the feasibility of achieving normoglycemia in type I diabetic patients by islet transplantation. One of the key issues in obtaining this success was transplanting sufficient numbers of islets by sequential transplants. Although the development of semipurified endotoxin-free Clostridium histolyticum-derived collagenase (Liberase) has improved islet yields from the human pancreas, batch-to-batch variation and loss of activity with time still hampers progress in obtaining consistent islet preparations. In order to define key components of crude collagenase, a panel of monoclonal antibodies (McAbs) was raised against crude collagenase. Monoclonal antibodies were generated by fusions between splenocytes of BALB/c mice immunized with Boheringer P collagenase and the myeloma cell line NS-0. These monoclonal antibodies were used as probes to study molecular differences between effective and ineffective collagenase batches using Western blotting. Two monoclonal antibodies (LDS71 and LDS81) were raised and characterized as recognizing separate epitopes on a 125-kDa component. Western blotting indicated that the 125-kDa band was rapidly broken down by storage or by dialysis in the presence of dithiothreitol. However, this breakdown could be prevented by the addition of leupeptin (a protease inhibitor) to the dialysis buffer. On testing fractions at 5-min intervals from the "Ricordi" digestion circuit during porcine and human pancreas digestion, the 125-kDa component was rapidly broken down in relatively ineffective collagenase batches but in effective batches was present throughout the digestion process. The correlation between the presence of the 125-kDa band and effectiveness of pancreas digestion suggests that this may be a key component in the formulation of C. histolyticum collagenase. Topics: Animals; Antibodies, Monoclonal; Cell Separation; Clostridium; Cysteine Proteinase Inhibitors; Enzyme Stability; Epitopes; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Leupeptins; Mice; Mice, Inbred BALB C; Microbial Collagenase; Molecular Weight; Pancreas; Swine | 2001 |
Delay of muscle degeneration and necrosis in mdx mice by calpain inhibition.
Inhibition of muscle degeneration by the tripeptide calpain inhibitor, leupeptin, was tested in vivo in a dystrophin-deficient mdx murine model. In a short-term control study, intramuscular administration of leupeptin for 30 days inhibited muscle degeneration as assessed by histologic analysis. Calpain inhibition could be correlated with retention of myofiber size and our results suggest that this may be a promising treatment modality in human Duchenne muscular dystrophy. Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Histocytochemistry; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Microscopy, Electron; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Necrosis | 2000 |
Proteasome involvement and accumulation of ubiquitinated proteins in cerebellar granule neurons undergoing apoptosis.
We investigated the potential role of the ubiquitin proteolytic system in the death of cerebellar granule neurons induced by reduction of extracellular potassium. Inhibitors of proteasomal function block apoptosis if administered at onset of this process, but they do not exert such effect when added 2-3 hr later. The same inhibitors also prevent caspase-3 activity and calpain-caspase-3-mediated processing of tau protein, suggesting that proteasomes are involved upstream of the caspase activation. Although the proteasomes seem to play an early primary role in programmed cell death, we found that with progression of apoptosis, during the execution phase, a perturbation in normal ubiquitin-proteasome function occurs, and high levels of ubiquitinated proteins accumulate in the cytoplasm of dying cells. Such accumulation correlates with a progressive decline of proteasome chymotrypsin and trypsin-like activities and, to a lower extent, of postacidic-like activity. Both intracytoplasmic accumulation of ubiquitinated proteins and decline of proteasome function are reversed by the pan-caspase inhibitor Z-VAD-fmk. The decline in proteasome function is accompanied by, and likely attributable to, a marked and progressive decline of deubiquitinating activities. The finding that the proteasomes are early involved in apoptosis and that ubiquitinated proteins accumulate during this process prospect granule neurons as a model system aimed at correlating these events with neurodegenerative diseases. Topics: Acetylcysteine; Animals; Apoptosis; Cell Division; Cell Survival; Cells, Cultured; Cerebellum; Culture Media, Serum-Free; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Leucine; Leupeptins; Multienzyme Complexes; Neurons; Oligopeptides; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rats; Rats, Wistar; Ubiquitins | 2000 |
Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II.
Newly synthesized major histocompatibility complex class II molecules (MHC-II) are transported to MHC-II-containing endosomal and lysosomal compartments (MIICs) for the degradation of associated invariant chain and peptide loading. Subsequently MHC-II is transported to the plasma membrane, in part through direct fusion of MIICs with the plasma membrane. In search of potential alternative pathway(s) we studied the 3-dimensional structure of MIICs and the subcellular distribution of MHC-II by immuno electronmicroscopy on whole-mount preparations and cryosections of Mel JuSo cells. Intracellular MHC-II and invariant chain mainly localized to lamp-1 positive compartments suggesting that the majority of MHC-II exits the endocytic tract at lysosomes. Clathrin-coated lattices and buds were found to be associated with these organelles, but MHC-II was not found to be enriched in the clathrin-coated domains. Moreover, leupeptin, a drug that interferes with Ii-processing and delays delivery of newly synthesized MHC-II to the plasma membrane, was not found to decrease the relative amount of MHC-II in clathrin-coated areas. Together these data indicate clathrin-mediated exit site(s) from lysosomes but suggest that they do not selectively recruit mature MHC-II, consistent with the notion that transport to the plasma membrane occurs independently of the cytoplasmic domains of the MHC-II (&agr;) and (beta) chains. Topics: Antigens, Differentiation, B-Lymphocyte; Biological Transport, Active; Cell Compartmentation; Cell Line; Clathrin; Coated Pits, Cell-Membrane; Histocompatibility Antigens Class II; Humans; Leupeptins; Lysosomes; Microscopy, Immunoelectron | 2000 |
Intracellular protein degradation and autophagy in isolated pancreatic acini of the rat.
Simultaneous investigation of protein degradation and autophagy of isolated exocrine pancreatic cells is carried out here for the first time in a systematic way by a complex biochemical, morphological and morphometrical approach. Protein degradation proceeds with a decreasing rate of 4-1.5 per cent per h over a 4-h period indicating a comparatively low degradation capacity. Cells in freshly isolated acini do not contain autophagic vacuoles but the latter appear within an hour in vitro and their quantity remains close to a steady state during the subsequent 3 h. Both traditional inhibitors of the autophagic-lysosomal pathway, e.g. vinblastine, leupeptin, and lysosomotropic amines together with the recently introduced 3-methyladenine, inhibit degradation to a similar maximal extent, offering the possibility of the estimation of the ratio of lysosomal/non-lysosomal degradation. In pancreatic acinar cells autophagic sequestration is unaffected and protein degradation is inhibited inside secondary lysosomes by leupeptin and lysosomotropic amines, while 3-methyladenine prevents the formation of autophagosomes. Vinblastine seems to act by inhibiting the fusion of autophagosomes with lysosomes and there is no evidence for the stimulation of autophagic sequestration by vinblastine in the present system. The effect of inhibitors of protein breakdown on protein synthesis is variable and does not correlate with their influence on degradation. Amino acids strongly stimulate protein synthesis, but in contrast to what is found in liver cells, they do not seem to affect protein degradation or autophagy significantly, thus indicating major regulatory differences of these processes between pancreatic acinar cells and hepatocytes. Topics: Adenine; Amines; Amino Acids; Animals; Autophagy; Cells, Cultured; Enzyme Inhibitors; Kinetics; Leupeptins; Lysosomes; Male; Microscopy, Electron; Pancreas; Protein Biosynthesis; Proteins; Rats; Rats, Long-Evans; Vinblastine | 2000 |
Identification of soluble forms of lectin-like oxidized LDL receptor-1.
Lectin-like oxidized LDL receptor-1 (LOX-1) is a type II membrane protein belonging to the C-type lectin family molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. In this study, we show that soluble forms of LOX-1 are present in conditioned media of cultured bovine aortic endothelial cells (BAECs) and CHO-K1 cells stably transfected with LOX-1 cDNA. Immunoblot analysis of conditioned media from TNF-alpha-activated BAECs and CHO-K1 cells stably expressing LOX-1 revealed that soluble LOX-1 has an approximate molecular mass of 35 kDa. In TNF-alpha-activated BAECs, cell-surface expression of LOX-1 precedes soluble LOX-1 production. Cell-surface biotinylation followed by immunoprecipitation and immunoblotting showed that soluble LOX-1 in cell-conditioned media is derived from LOX-1 expressed on the cell surface. Production of soluble LOX-1 was inhibited by PMSF, suggesting that PMSF-sensitive proteases may be involved in this process. Purification of soluble LOX-1 by high-performance liquid chromatography and N-terminal amino acid sequencing of soluble LOX-1 identified the 2 cleavage sites between Arg(86)-Ser(87) and Lys(89)-Ser(90), which were located in the membrane proximal extracellular domain of LOX-1. The data demonstrate that cell-surface LOX-1 can be cleaved at 2 different sites and transformed into soluble forms. Further studies may explore therapeutic and diagnostic applications of soluble LOX-1 in atherosclerotic diseases. Topics: Amino Acid Sequence; Animals; Aorta; Aprotinin; Arteriosclerosis; Biotinylation; Cattle; Cell Membrane; CHO Cells; Cricetinae; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Endopeptidases; Endothelium, Vascular; Glycoproteins; Lectins; Leupeptins; Lipoproteins, LDL; Membrane Proteins; Molecular Sequence Data; Pepstatins; Protease Inhibitors; Protein Structure, Tertiary; Receptors, LDL; Receptors, Oxidized LDL; Serine Proteinase Inhibitors; Solubility; Tosyl Compounds; Tosyllysine Chloromethyl Ketone; Tumor Necrosis Factor-alpha | 2000 |
Accumulation and degradation in the endoplasmic reticulum of a truncated ER-60 devoid of C-terminal amino acid residues.
The accumulation and degradation in the endoplasmic reticulum (ER) of a truncated ER-60 protease, from which the C-terminal 89 amino acid residues have been deleted (K 417 ochre), was examined. K 417 ochre overexpressed in COS-1 cells is not secreted into the medium, but accumulates as insoluble aggregates in non-ionic detergent without degradation in unusual clump membrane structures. K 417 ochre, stably expressed, forms soluble aggregates in non-ionic detergent and is distributed in the reticular structures of ER. Under these conditions, K 417 ochre is not secreted into the medium but is degraded with a half-life time of more than 8 h. Since K 417 ochre/C all S, in which all the Cys residues of K 417 ochre are replaced by Ser, also forms aggregates, an inter-disulfide bond appears unnecessary for aggregation. In both types of aggregates, Ig heavy chain binding protein, calnexin, glucose regulated protein 94, calreticulin, ERp72, and protein disulfide isomerase are scarcely found. Since degradation of the stably expressed K 417 ochre was not inhibited by lactacystin, leupeptin, NH(4)Cl, or cytocharasin B, but was inhibited by N-acetyl-leucyl-leucyl-norleucinal, the self-aggregated abnormal protein in the lumen of ER is assumed to be degraded by an unknown protease system other than proteasome, lysosome or autophagy. Topics: Acetylcysteine; Animals; Calcium-Binding Proteins; Calnexin; Calreticulin; Carrier Proteins; Cell Membrane; COS Cells; Culture Media; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochalasin B; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Half-Life; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Leupeptins; Membrane Proteins; Molecular Chaperones; Protein Disulfide-Isomerases; Recombinant Proteins; Ribonucleoproteins | 2000 |
Suppression of endothelin-converting enzyme-1 during buccal mucosal ulcer healing: effect of chronic alcohol ingestion.
Among the factors affecting the efficiency of soft oral tissue healing is endothelin-1 (ET-1), a potent vasoactive peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1). We investigated the expression of ECE-1 during buccal mucosal ulcer healing in rats maintained for 5 weeks on alcohol containing or control diet. The mucosal activity of ECE-1, characterized by sensitivity to phosphoramidon, was associated with microsomal fraction and showed an elevated (3.1-fold) level in the alcohol diet group. Moreover, the ulcer onset in the alcohol group was reflected in a 39% greater expression of ECE-1 activity, and was accompanied by a 1.4-fold greater increase in TNF-alpha and a 2.5-fold greater enhancement in epithelial cell apoptosis. While in both groups the ulcer healing was associated with a decrease in buccal mucosal expression of ECE-1, as well as a decline in TNF-alpha and apoptosis, the changes were significantly slower in the alcohol diet group and manifested by a 40% delay in healing. Thus, chronic alcohol ingestion leads to up-regulation of ECE-1 expression, induction of TNF-alpha, and triggering apoptotic events that delay the mucosal repair. Topics: Animals; Apoptosis; Aspartic Acid Endopeptidases; Edetic Acid; Endothelin-Converting Enzymes; Epithelial Cells; Ethanol; Leupeptins; Metalloendopeptidases; Mouth Mucosa; Oral Ulcer; Pepstatins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Time Factors; Tosyl Compounds; Tumor Necrosis Factor-alpha; Up-Regulation; Wound Healing | 2000 |
Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation.
Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes. Topics: Acetylcysteine; Animals; Antigen Presentation; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Cell Line, Transformed; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epitopes, T-Lymphocyte; Hepatitis B e Antigens; Histocompatibility Antigens Class I; HIV Envelope Protein gp160; HIV-1; Humans; Hydrolysis; Leupeptins; Metalloendopeptidases; Mice; Mice, Inbred BALB C; Multienzyme Complexes; Pepstatins; Peptide Fragments; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocytes, Cytotoxic | 2000 |
Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes.
The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes. Topics: Adult; Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Calpain; Captopril; Cathepsins; Chymotrypsin; Cysteine Proteinase Inhibitors; Humans; Leucine; Leupeptins; Lisinopril; Lymphocyte Activation; Male; Oligopeptides; Peptidyl-Dipeptidase A; Protease Inhibitors; T-Lymphocytes | 2000 |
Suppression of autophagy by ethionine administration in male rat liver in vivo.
The administration of ethionine to male rats decreased the liver adenosine triphosphate (ATP) levels to about 20% of that in control rats and prevented leupeptin-induced intralysosomal accumulation of cytosolic proteins, including lactate dehydrogenase sequestered into lysosomes by autophagy, and leupeptin-induced increase of lysosomal density. These actions of ethionine were reversed by the subsequent administration of adenine plus methionine. All these findings show that the administration of ethionine to male rats suppresses the autophagic sequestration of intracellular proteins into lysosomes, probably due to ethionine-induced ATP depletion. Topics: Adenine; Adenosine Triphosphate; Animals; Antimetabolites; Centrifugation, Density Gradient; Cysteine Proteinase Inhibitors; Cytosol; Ethionine; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Male; Phagosomes; Rats; Rats, Wistar | 2000 |
Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality.
Several small-scale Chinese hamster ovary (CHO) suspension cultures were grown in perfusion mode using a new acoustic filtration system. The separation performance was evaluated at different cell concentrations and perfusion rates for two different CHO cell lines. It was found that the separation performance depends inversely on the cell concentration and perfusion rate. High media flow rates as well as high cell concentrations resulted in a significant drop in the separation performance, which limited the maximal cell concentration achievable. However, packed cell volumes of 10% to 16% (corresponding to 3 to 6. 10(7) cells/mL) could be reached and were maintained without additional bleeding after shifting the temperature to 33 degrees C. Perfusion, up to 50 days, did not harm the cells and did not result in a loss of performance of the acoustic filter as often seen with other perfusion systems. Volumetric productivities in perfusion mode were 2- to 12-fold higher for two cell lines producing two different glycoproteins when compared to fed-batch or batch processes using the same cell lines. Product concentrations were in the range of 20% to 80% of batch or fed-batch culture, respectively. In addition, using the protease-sensitive product rhesus thrombopoietin, we could show that cultivation in perfusion mode drastically reduced proteolysis when compared to a batch culture without addition of protease inhibitors such as leupeptin. Topics: Acoustics; Animals; Blotting, Western; Cell Culture Techniques; Cell Survival; CHO Cells; Cricetinae; Filtration; Leupeptins; Macaca mulatta; Perfusion; Tissue Plasminogen Activator | 2000 |
The mitogenic activity of fibroblast growth factor-1 correlates with its internalization and limited proteolytic processing.
The fibroblast growth factor-1 (FGF-1) mitogenic signal transduction pathway is not well characterized, and evidence indicates that FGF-1 binding to and activation of cell-surface receptors is not solely sufficient for a full mitogenic response. Although initiation of the phosphorylation signaling cascades are likely important in FGF-1-induced mitogenic signaling, there appear to be additional signaling requirements. In this study, we demonstrate that FGF-1 internalization and subsequent processing correlates with the mitogenic potential of the growth factor on NIH 3T3 cells. Using site-directed mutants of FGF-1 and inhibitors of the endocytic and degradative pathways, we provide evidence for growth factor internalization and exposure to an acidic environment as necessary components of FGF-1-induced mitogenesis. In addition, a protease-sensitive event(s) appears critical for a complete mitogenic response to FGF-1, whereas, this protease sensitivity was not detected under the same conditions for serum-stimulated mitogenesis. Therefore, proteolytic modification of internalized FGF-1 may result in the activation of additional, intracellular signaling events. Topics: 3T3 Cells; Animals; Antiviral Agents; Cell Nucleus; Chlorpromazine; Cysteine Proteinase Inhibitors; Cytoplasm; Cytosol; Dopamine Antagonists; Endocytosis; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Filipin; Humans; Leupeptins; Ligands; Mice; Mitogens; Signal Transduction | 2000 |
Involvement of cysteine proteases in bFGF-induced angiogenesis in guinea pig and rat cornea.
Overexpression and activation of matrix metalloprotease (MMP) have been implicated in angiogenesis. However, the involvement of cysteine proteases, such as calpains (EC 34.22.17), is obscure. Thus, the purpose of this experiment was to study the involvement of cysteine proteases in angiogenesis induced by basic fibroblast growth factor (bFGF) in guinea pig and rat corneas using cysteine protease inhibitors. Sustained-release polymers containing bFGF were implanted into guinea pig and rat corneas to induce angiogenesis. For treatment of corneal angiogenesis, polymers containing cysteine protease inhibitors, leupeptin or SJA6017, were also implanted into corneas. Using the slit lamp, the corneas were observed for nine days after polymer implantation. Soluble proteins and albumin levels were used as markers of corneal injury by angiogenesis. bFGF induced angiogenesis in guinea pig and rat corneas. In guinea pig cornea, wet weight, the amount of soluble protein and albumin was highest at four days after bFGF-containing pellet implantation. In rat cornea, the amount of soluble protein and albumin was highest at six days, and wet weight increased within four days. One hundred nmole of leupeptin showed a tendency to reduce bFGF-induced angiogenesis in guinea pig cornea, and 10 nmole of SJA6017 was effective in reducing bFGF-induced angiogenesis in rat cornea, although SJA6017 showed a stronger effect than leupeptin. Ten nmole of SJA6017 significantly reduced the number of new blood vessels. These data suggested involvement of cysteine proteases in angiogenesis in guinea pig and rat cornea. Topics: Albumins; Animals; Cornea; Corneal Neovascularization; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Delayed-Action Preparations; Dipeptides; Eye Proteins; Fibroblast Growth Factor 2; Guinea Pigs; Leupeptins; Male; Organ Size; Rats | 2000 |
Fibroblast growth factors are translocated to the nucleus of human endothelial cells in a microtubule- and lysosome-independent pathway.
Exogenous acidic and basic fibroblast growth factors undergo rapid nuclear translocation in human umbilical vein endothelial cells. When nuclear translocation reaches saturation, more than 70% of the internalized growth factors are in the nuclear fraction. Lysosomal inhibitors, such as leupeptin and chloroquine, and microtubule inhibitors including colchicine and 2-methoxyl-beta-estradiol neither increase nor decrease nuclear translocation. The results suggest that nuclear translocation of fibroblast growth factors does not require cytosolic accumulation or lysosomal processing and that the transportation of exogenous growth factors across the cytoplasm is independent of microtubules. Topics: Biological Transport, Active; Cell Nucleus; Cells, Cultured; Chloroquine; Colchicine; Cytosol; Endothelium, Vascular; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Humans; Kinetics; Leupeptins; Lysosomes; Microtubules; Subcellular Fractions | 2000 |
Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.
Atrial natriuretic peptide (ANP) is a cardiac hormone essential for the regulation of blood pressure. In cardiac myocytes, ANP is synthesized as a precursor, pro-ANP, that is converted to biologically active ANP by an unknown membrane-associated protease. Recently, we cloned a transmembrane serine protease, corin, that is highly expressed in the heart. In this study, we examine effects of corin on pro-ANP processing. Our results show that recombinant human corin converts pro-ANP to ANP and that the cleavage in pro-ANP by corin is highly sequence specific. Our findings suggest that corin is the long-sought pro-ANP-converting enzyme and that the corin-mediated pro-ANP activation may play a role in regulating blood pressure. Topics: Animals; Aprotinin; Atrial Natriuretic Factor; Benzamidines; Catalysis; Cell Line; COS Cells; Cricetinae; Gene Expression; Humans; Leupeptins; Membrane Proteins; Protein Precursors; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Structure-Activity Relationship; Trypsin Inhibitors | 2000 |
Increased activity of calcium leak channels caused by proteolysis near sarcolemmal ruptures.
Dystrophin, a 427 kD membrane-associated structural protein in muscle cells, is thought to confer strength to the myofiber sarcolemma and protect the membrane from rupture during the stresses of contraction. Dystrophin is absent in muscle cells from Duchenne muscular dystrophy (DMD) patients and mdx mice, a DMD model. Dystrophic muscle membranes undergo more frequent transient, nonlethal tears than normal cell membranes, especially during exercise. In addition, the mean open probability of a background ("leak") calcium channel is higher in dystrophic muscle cells, which leads to higher intracellular free calcium levels. Because elevated calcium levels may contribute to the eventual necrosis of muscle cells in DMD, we examined the possibility that the history of sarcolemmal rupture at a specific location on the membrane affects the open probability of nearby calcium leak channels. Membrane ruptures left by the excision of cell-attached patch-clamp electrodes were used to mimic natural tears. Patches made within 5 microns of excision sites contained channels with a fourfold greater mean open probability than channels in patches 50 microm away from ruptures. The increased leak channel activity near ruptures was seen continuously through the duration of the recordings and was not seen if the rupture was made in the presence of the protease inhibitor leupeptin. Calcium background channels proteolytically activated near ruptures, perhaps in a calcium-dependent manner, may thus be the lasting consequence of the weaker dystrophic sarcolemma, leading to chronically raised intracellular free calcium, increased calcium-dependent proteolysis and, eventually, necrosis. Topics: Animals; Calcium Channels; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Leupeptins; Mice; Muscle, Skeletal; Sarcolemma | 2000 |
Subtilisin increases macromolecular efflux from the oral mucosa.
The purpose of this study was to determine whether subtilisin, a potent serine proteinase derived from Bacillus species contaminating smokeless tobacco, increases macromolecular efflux from the oral mucosa and, if so, whether local elaboration of bradykinin mediates this response. Using intravital microscopy, I found that suffusion of subtilisin elicits significant, concentration-dependent leaky site formation and an increase in the clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the in situ hamster cheek pouch (P<0.05). Heat-inactivated subtilisin had no significant effects on macromolecular efflux. Subtilisin-induced responses were significantly attenuated by Hoe 140 and NPC 17647, two structurally distinct selective bradykinin B(2) receptor antagonists, but not by des-Arg(9)-[Leu(8)]bradykinin, a selective bradykinin B(1) receptor antagonist, or CP-96,345, a selective neurokinin-1 receptor antagonist. Aprotinin, but not leupeptin, significantly attenuated subtilisin-induced increase in macromolecular efflux. Indomethacin had no significant effects on subtilisin-induced responses. Collectively, these data indicate that subtilisin increases the macromolecular efflux from the in situ hamster cheek pouch in a catalytic-site-dependent fashion through local elaboration of bradykinin. This response does not involve the stimulation of local afferent nerves or the production of prostaglandins. Topics: Animals; Aprotinin; Biphenyl Compounds; Bradykinin; Bradykinin Receptor Antagonists; Cricetinae; Cyclooxygenase Inhibitors; Cysteine Proteinase Inhibitors; Indomethacin; Leupeptins; Macromolecular Substances; Male; Mesocricetus; Mouth Mucosa; Neurokinin-1 Receptor Antagonists; Serine Proteinase Inhibitors; Subtilisin | 2000 |
[Demonstration of secondary free radicals and the role of calpain in functional changes associated with the myocardial ischemia-reperfusion sequence].
The aim of this study was to investigate the role of secondary free radicals and calpain, a calcium-activated cysteine protease, in the development of reperfusion injury in the heart. The time course of radical generation was assessed directly by Electron Paramagnetic Resonance (EPR) and spin trapping with N-ter butyl-alpha-phenylnitrone (PBN), in isolated perfused rat heart subjected to 30 minutes of global ischemia and 30 minutes of reperfusion. The effect of leupeptin, a calpain inhibitor, was assessed on postischemic dysfunction. The antioxidant properties of leupeptin were also investigated by using allophycocyanin, a fluorescent protein sensitive to oxidative stress generated by the H2O2 + Cu++ system. Moreover, we measured the capacities of leupeptin to scavenge hydroxyl (.OH) and superoxide (O2-.) radicals using EPR technique. Our results show that myocardial reperfusion is associated with an increase of alkyl, alkoxyl free radicals release; the administration of catalase 5.10(5) UI/L significantly reduces this release, but didn't improve the postischemic contractile function of the heart. In our study leupeptin 50 microM possess, in vitro, antioxidant properties and scavenging abilities against .OH and O2-., in return leupeptin does not influence the cardiac functions during reperfusion period. In conclusion, our results confirm that myocardial reperfusion induces an important production of secondary free radicals associated with contractile dysfunction. The role of calpain in myocardial ischemia-reperfusion injury remains to be clarified 1) by assessing the activities of calpain and calpastain, its main endogenous inhibitor, during these periods, 2) by measuring the ability of leupeptin in inhibiting the calpain dependent proteolysis. Topics: Animals; Antioxidants; Calcium-Binding Proteins; Calpain; Catalase; Cathepsins; Cyclic N-Oxides; Cysteine Proteinase Inhibitors; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Leupeptins; Magnetic Resonance Spectroscopy; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Nitrogen Oxides; Oxidative Stress; Phycocyanin; Rats; Rats, Wistar; Spin Labels; Superoxides; Time Factors | 2000 |
Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang.
Bacillus sp. strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques. The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF, but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN', but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. Topics: Bacillus; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Fermentation; Fibrinolysis; Food Analysis; Korea; Leupeptins; Molecular Weight; Subtilisins | 2000 |
Changes in the Ca2+-activation characteristics of demembranated rat single muscle fibres after prolonged incubation at room temperature in the presence and absence of DTT and protease inhibitors.
Prolonged incubation (24 h) of chemically skinned rat muscle preparations in rigor solutions at room temperature and in the absence of reducing agents and protease inhibitors modified the Ca2+-activation characteristics of the contractile machinery. In the absence of reducing agents and protease inhibitors, the contraction threshold for incubated fibres was shifted to lower Ca2+ concentrations and the steepness of the steady-state force-pCa (-log10)[Ca2+]) curve was decreased compared to that of control muscle fibres. Mean myosin ATPase activity under these conditions was significantly lowered by a factor of 2.7. Fibres incubated in the presence of 10 mM dithiothreitol (DTT) and protease inhibitors (100 microM pepstatin A/200 microM leupeptin) produced a maximum Ca2+-activated force per cross-sectional area that compared favourably with that of freshly dissected muscle fibres and there were no changes in the other contractile activation characteristics. Intermediate responses were obtained when fibres were incubated in the presence of either DTT or protease inhibitors. MgATPase activities of incubated preparations increased significantly following the addition of protease inhibitors and/or DTT to the incubation medium. Taken together, these results suggest that in the presence of DTT and protease inhibitors, most contractile properties are maintained at levels seen in fresh mechanically skinned fibres. The extended viability of this preparation and its closely related properties with fresh muscle fibres make it a useful model for experiments requiring longer term incubations with biological agents. Topics: Adenosine Triphosphatases; Animals; Calcium; Dithiothreitol; Histological Techniques; Leupeptins; Male; Muscle Contraction; Muscle Fibers, Skeletal; Myofibrils; Osmolar Concentration; Pepstatins; Protease Inhibitors; Rats; Rats, Long-Evans; Temperature; Time Factors | 2000 |
Il-1-related cytokine responses of nonimmune skin cells subjected to CEES exposure with and without potential vesicant antagonists.
Sulfur mustard provokes an acute inflammatory response in skin. To determine if keratinocytes regulate this response and whether three potential vesicant antagonists can counteract adverse changes, specimens of EpiDerm (MatTek Corp., Ashland, MA), a human skin model of differentiating keratinocytes, were exposed 2 h to humidified air with or without 2-chloroethyl ethyl sulfide (CEES, 1.72-1.73 mg/L/min) with or without 10 mM niacinamide, a poly (ADP-ribose) polymerase (PARP) inhibitor, 25 microM CGS9343B (calmodulin antagonist), or 8.4 mM leupeptin (cysteine protease inhibitor). After a 22-h incubation, levels of interleukin-1 alpha (IL-1alpha), its receptor antagonist (IL-1Ra), soluble type II receptor (sIL-1RII) and prostaglandin-E(2) (PGE(2)) were determined. Methylthiazole tetrazolium (MTT) viability tests and histological observations were also conducted. PGE(2) levels were abundant but unaffected by CEES regardless of antagonist presence. Total amounts (media plus lysate) of IL-1alpha, IL-1Ra, and sIL-1RII were reduced with CEES irrespective of antagonist. CEES promoted the release of IL-1Ra. Exposure of EpiDerm to CEES in the presence of the vesicant antagonists did not improve viability or counteract histological damage. We conclude CEES depresses total IL-1alpha and related cytokines, does not affect PGE(2) release, and adverse changes associated with CEES-exposed EpiDerm are not ameliorated by these particular antagonists. Dramatically increased (5- to 10-fold) release of IL-1Ra may provide a useful marker for cytotoxicity. The high level of IL-1Ra and increased release with injury suggest a primary function in down-regulating IL-1 inflammatory responses in skin. Topics: Benzimidazoles; Biomarkers; Calmodulin; Cells, Cultured; Cysteine Proteinase Inhibitors; Dinoprostone; Drug Synergism; Enzyme Inhibitors; Humans; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Irritants; Keratinocytes; Leupeptins; Mustard Gas; Niacinamide; Poly(ADP-ribose) Polymerase Inhibitors; Receptors, Interleukin-1; Receptors, Interleukin-1 Type II; Sialoglycoproteins | 2000 |
A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.
Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. Topics: Animals; Cells, Cultured; Cerebellum; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; Fluorometry; Glutamic Acid; Leupeptins; Neurons; Protease Inhibitors; Pterins; Rats; Rats, Wistar; Temperature; Xanthine Oxidase; Xanthopterin | 2000 |
Inhibition of apoptosis in cultured porcine granulosa cells by inhibitors of caspase and serine protease activity.
Protease inhibitors were used to test the hypothesis that caspases and other proteases were active during apoptosis in cultured porcine granulosa cells. Cells isolated from 3 to 6 mm follicles were cultured for 24 h in Dulbecco's modified Eagles medium: Hams F12 (1:11 containing 1% fetal bovine serum. Final inhibitor concentrations, added in 10 microL of dimethylsulfoxide, were 0, 1, 5, 25 and 125 microM. Cells with compromised plasma membrane integrity, identified by uptake ethidium homodimer, increased during culture in the absence of inhibitors from 37% to 43%. Apoptotic (A0) cells, identified by DNA fluorescence flow cytometry, increased (P < 0.05) from 1.7% to 29%. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) at 125 microM was lethal increasing (P < 0.05) cells with compromised membranes to 92%. In response to TPCK, A0 cells decreased from 55% to 1.2%; progesterone and estradiol production were decreased by 94% and 98%, respectively. The general caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoro methylketone, decreased (P < 0.05) A0 cells linearly from 33% to 3 % between 0 and 125 microM without significant effect on steroidogenesis or on the percentage of cells with compromised plasma membranes. Other inhibitors only had a marginal effect on apoptosis; concentrations of > or = 1 microM decreased (P < 0.05) A0 cells from 29% to 18% to 21% and had no significant effect on membrane integrity or steroid production. We conclude that caspases are associated with apoptosis in cultured porcine granulosa cells. Death induced by TPCK was through a non-apoptotic mechanism. Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane; Cysteine Proteinase Inhibitors; DNA Fragmentation; Electrophoresis, Agar Gel; Estradiol; Female; Flow Cytometry; Granulosa Cells; Leucine; Leupeptins; Microscopy, Fluorescence; Phenylmethylsulfonyl Fluoride; Progesterone; Radioimmunoassay; Regression Analysis; Serine Proteinase Inhibitors; Swine; Tosylphenylalanyl Chloromethyl Ketone | 2000 |
MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M.
We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4+ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulumlocalized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4+ T cells that would not otherwise be activated. Topics: Acetylcysteine; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; Antimalarials; Brefeldin A; CD4-Positive T-Lymphocytes; Chloroquine; Coculture Techniques; Cysteine Proteinase Inhibitors; Cytosol; Dose-Response Relationship, Drug; Endocytosis; Endoplasmic Reticulum; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Genes, MHC Class II; Humans; Hybridomas; Leupeptins; Major Histocompatibility Complex; Mice; Muramidase; Plasmids; Primaquine; Protein Synthesis Inhibitors; Protein Transport; Ribonucleases; Transfection; Tumor Cells, Cultured | 2000 |
Exposure to lysosomotropic amines and protease inhibitors retard corneal endothelial cell migration along the natural basement membrane during wound repair.
Regulation of cell migration along the natural basement membrane during wound repair in the organ culture corneal endothelium was investigated using various lysosomotropic amines and protease inhibitors. Following a circular transcorneal freeze injury, cells within the area die and expose the underlying basement membrane (Descemet's membrane). During normal wound repair, cells traverse this expanse and repopulate the region by approximately 48 h postinjury. During this time, acid phosphatase histochemistry revealed distinct alterations in the lysosomal population of cells that were adjacent to, and migrated into, the wound region. To explore whether relationships may exist between changes in the lysosome population and cell migration, injured endothelia were organ cultured in the presence of either methylamine or chloroquine, two lysosomotropic amines. Methylamine significantly retarded cell translocation (85%) into the injury zone when compared to nontreated controls. In comparison, chloroquine was less effective in restricting injury-induced cell migration and propylamine, also a lysosomotropic amine, had no influence on the repair process. In addition, two serine/thio protease inhibitors, leupeptin and antipain, were both able to impede cell translocation during wound repair by 85 and 52%, respectively, whereas soybean trypsin inhibitor, a serine protease inhibitor, exhibited no inhibitory effect on the repair process. Similarly, incubating injured tissues in either 1,10-phenanthroline or phosphoramidon, both metalloproteinase inhibitors, did not prevent endothelial cell movement nor wound repair. Results indicate that corneal endothelial cell migration along the natural basement membrane is dependent on protease function. Although the precise nature of the proteases involved has yet to be ascertained, results indicate that lysosomal enzymes may have a distinct role in corneal endothelial cell movement along the natural basement membrane during wound repair. Topics: Amines; Animals; Antipain; Basement Membrane; Cell Movement; Chloroquine; Culture Techniques; Descemet Membrane; Endopeptidases; Endothelium, Corneal; Freezing; Leupeptins; Lysosomes; Metalloendopeptidases; Methylamines; Propylamines; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Trypsin Inhibitor, Kunitz Soybean; Wound Healing | 1999 |
Reversible accumulation of lipofuscin-like inclusions in the retinal pigment epithelium.
The amounts of autofluorescent lysosomal storage bodies, known as lipofuscin, increase during senescence in the retinal pigment epithelia (RPEs) of mammalian eyes. This increase in lipofuscin content may result from a failure of the RPE to dispose of any lipofuscin constituents once they have formed. Alternatively, the RPE may eliminate lipofuscin but at a rate insufficient to prevent its accumulation. Experiments were conducted to distinguish between these two possibilities.. Albino rats were given intravitreal injections of the protease inhibitor leupeptin, which induces a rapid accumulation of lipofuscin-like inclusions in the RPE. The amount of these inclusions in the RPE was monitored as a function of time after the leupeptin treatment with quantitative ultrastructural analysis. In addition, the intensity of lipofuscin-specific fluorescence in the RPE was monitored over the same time period with the use of quantitative microfluorometry. These parameters were also followed in untreated control eyes of age-matched animals.. A single leupeptin injection resulted in a rapid massive accumulation of electron-dense inclusion bodies in the RPE. These inclusions appeared to be derived primarily from phagocytosed photoreceptor outer segments. Accompanying the accumulation of these inclusions was a significant increase in lipofuscin-specific fluorescence in the RPE. Over a 12-week period after the leupeptin treatment, the amounts of inclusion material and the fluorescence intensities returned to normal levels.. These findings suggest that the age-related increase in RPE lipofuscin content results from an imbalance in the rates of lipofuscin formation and disposal rather than from a complete absence of a disposal mechanism. The results imply that turnover of lipofuscin constituents may be rapid relative to the animals' life span. Thus, it may be possible to slow or reverse the age-related increase in RPE lipofuscin content by either inhibiting the processes involved in lipofuscin formation or enhancing the disposal processes. Topics: Aging; Animals; Eye Proteins; Inclusion Bodies; Injections; Leupeptins; Lipofuscin; Male; Microscopy, Fluorescence; Pigment Epithelium of Eye; Rats; Rats, Inbred F344; Vitreous Body | 1999 |
Tyrosine kinases play a permissive role in glucose-induced insulin secretion from adult rat islets.
The role(s) played by protein tyrosine kinases (PTKs) in the regulation of insulin secretion from pancreatic beta cells is not clear. We have examined the effects of glucose, the major physiological insulin secretagogue, on the tyrosine phosphorylation state of islet proteins, and assessed beta cell insulin secretory responses in the presence of PTK inhibitors. Under basal conditions islets contained many proteins phosphorylated on tyrosine residues, and glucose (20 mM; 5-15 min) was without demonstrable effect on the pattern of tyrosine phosphorylation, in either the absence or presence of the protein tyrosine phosphatase (PTP) inhibitor, sodium pervanadate (PV). PV alone (100 microM) increased tyrosine phosphorylation of several islet proteins. The PTK inhibitors genistein (GS) and tyrphostin A47 (TA47) inhibited islet tyrosine kinase activities and glucose-, 4alpha ketoisocaproic acid (KIC)- and sulphonylurea-stimulated insulin release, without affecting glucose metabolism. GS and TA47 also inhibited protein serine/threonine kinase activities to a limited extent, but had no effect on Ca2+, cyclic AMP- or phorbol myristate acetate (PMA)-induced insulin secretion from electrically permeabilised islets. These results suggest that PTK inhibitors exert their inhibitory effects on insulin secretion proximal to Ca2+ entry and it is proposed that they act at the site of the voltage-dependent Ca2+ channel which regulates Ca2+ influx into beta cells following nutrient- and sulphonylurea-induced depolarisation. Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Genistein; Glucose; Insulin; Insulin Secretion; Ion Transport; Islets of Langerhans; Isoflavones; Leupeptins; Phenylmethylsulfonyl Fluoride; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tyrphostins; Vanadates | 1999 |
A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation.
Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation. Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antigen Presentation; beta 2-Microglobulin; Down-Regulation; Endocytosis; Enzyme Inhibitors; Gene Expression Regulation, Viral; Genes, Viral; Histocompatibility Antigens Class I; Immunohistochemistry; Leupeptins; Lysosomes; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Muromegalovirus; Transfection; Viral Proteins | 1999 |
Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization.
Egg activation in cross-fertilization between Xenopus eggs and Cynops sperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified from Cynops sperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation in Xenopus eggs. We inseminated unfertilized Xenopus eggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained from Xenopus sperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease in Xenopus sperm is involved in fertilization, most likely by participating in egg activation. Topics: Animals; Aprotinin; Calcium; Cathepsin B; Cell Division; Coumarins; Electrophysiology; Endopeptidases; Fertilization; Kinetics; Leupeptins; Male; Oligopeptides; Oocytes; Protease Inhibitors; Salamandridae; Spermatozoa; Substrate Specificity; Xenopus | 1999 |
Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.
We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization. Topics: Amino Acid Chloromethyl Ketones; Antithrombins; Arachidonic Acid; Blood Platelets; Calcium; Enzyme Activation; Hirudins; Humans; Ionomycin; Ionophores; Leukocyte Elastase; Leupeptins; Peptide Fragments; Phospholipases A; Phospholipases A2; Platelet Glycoprotein GPIb-IX Complex; Receptor, PAR-1; Receptors, Thrombin; Thrombin; Trypsin | 1999 |
Leupeptin protects sensory hair cells from acoustic trauma.
Calpains, a family of calcium activated proteases, promote the breakdown of cellular proteins, kinases, phosphatases and transcription factors. Calpain inhibitors attenuate some neurodegenerative processes in certain cell types. Here we show that leupeptin, a potent calpain inhibitor, protects the sensory hair cells in the inner ear from acoustic overstimulation (48 h, 100 or 105 dB SPL, octave band noise at 4 kHz). Acoustic overstimulation caused a significant increase in calpain immunolabeling in the sensory epithelium suggesting a possible role in noise-induced cochlear degeneration. Infusion of leupeptin into the inner ear significantly reduced the amount of sensory cell loss from acoustic overstimulation. However, leupeptin did not protect against hair cell loss from the ototoxic drug, carboplatin. Topics: Animals; Antineoplastic Agents; Calpain; Carboplatin; Chinchilla; Enzyme Inhibitors; Hair Cells, Auditory; Hearing Disorders; Hearing Loss, Noise-Induced; Immunohistochemistry; Leupeptins; Organ of Corti | 1999 |
Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum.
Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX. Topics: Amiloride; Animals; Antimalarials; Bicarbonates; Biological Transport; Chloroquine; Erythrocyte Membrane; Erythrocytes; Hemin; Hemoglobins; Humans; Hydrogen-Ion Concentration; Kinetics; Leupeptins; Plasmodium falciparum; Sodium-Hydrogen Exchangers; Verapamil | 1999 |
Metabolism and degradation products of recombinant human insulin-like growth factor-I in lysosomes of rat kidney.
1. The degradation of recombinant human insulin-like growth factor-I (rhIGF-I) by purified lysosomes of rat kidney was examined in vitro. The peptide structures of the 13 degradation products were deduced from the sequence analysis and the molecular mass. Rat kidney lysosomal cathepsins efficiently cleave rhIGF-I to two chain peptides, like insulin. The cleavages mainly occur at the C-peptide/A-chain junction, D-peptide/A-chain junction and B21-22 or B22-23. 2. The effect of inhibitors on the lysosomal degradation of rhIGF-I was examined semiquantitatively by the rate of formation of the degradation products. The degradation of rhIGF-I was almost completely inhibited by the lysosomal cysteine protease inhibitors, leupeptin and leucine chloromethyl ketone, and a serine protease inhibitor, phenylmethylsulphonyl fluoride. On the other hand, the degradation was enhanced by the addition of a reducing agent, glutathione. Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Glutathione; Humans; Insulin; Insulin-Like Growth Factor I; Kidney; Leupeptins; Lysosomes; Male; Microscopy, Electron; Molecular Sequence Data; Peptide Fragments; Proinsulin; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sequence Analysis | 1999 |
Novel gelatinolytic activities in rat organs.
Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs. Topics: Animals; Cattle; Enzyme Inhibitors; Gelatin; Leupeptins; Male; Metalloendopeptidases; Phenanthrolines; Phenylmercuric Acetate; Rats; Rats, Wistar; Serine Endopeptidases | 1999 |
Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient. Model system for revealing the intracellular transport of normal and mutated lysosomal enzymes.
Fibroblastic cell lines derived from a galactosialidosis patient, stably expressing the chimaeric green fluorescent protein variant (EGFP) gene fused to the wild-type and mutant human lysosomal protective protein/cathepsin A (PPCA) cDNA, were first established as a model system for revealing the sorting and processing of lysosomal enzymes and for investigating the molecular bases of their deficiencies. In the cell line expressing the wild-type PPCA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to the C-terminus of the 54 kDa PPCA precursor) was produced, then processed into the mature 32/20 kDa two-chain form free of the EGFP domain. The intracellular cathepsin A, alpha-N-acetylneuraminidase and beta-galactosidase activities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and strong fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was distributed throughout the EGFP-PPwild cell line. Bafilomycin A1, a potent inhibitor of vacuolar ATPase and intracellular acidification, induced the distribution of Golgi-like perinuclear fluorescence throughout the living and fixed cells, in which only the 81 kDa product was detected. After removal of the agent, time-dependent transport of the chimaeric protein from the Golgi apparatus to the prelysosomal structure in living cells was monitored with a confocal laser scanning microscope system. Leupeptin caused the distribution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latter agent also dose-dependently induced an increase in the intracellular amount of the 81 kDa product containing the EGFP domain and inhibited the restoration of cathepsin A activity in the EGFP-PPwild cells after the removal of bafilomycin A1. In parallel, both the mature two-chain form and PPCA function disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteolytic processing of the PPCA precursor/zymogen, quenching of the fluorescence, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A1184-->G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patie Topics: Amino Acid Sequence; Anti-Bacterial Agents; Base Sequence; Biological Transport; Carboxypeptidases; Cathepsin A; Cell Line; DNA, Complementary; Fibroblasts; Green Fluorescent Proteins; Humans; Leupeptins; Luminescent Proteins; Lysosomal Storage Diseases; Lysosomes; Macrolides; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Mutation; Protein Processing, Post-Translational; Recombinant Fusion Proteins | 1999 |
Modulation of major histocompatibility complex protein expression by human gamma interferon mediated by cysteine proteinase-adhesin polyproteins of Porphyromonas gingivalis.
Cysteine proteinases have been emphasized in the virulence of Porphyromonas gingivalis in chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-gamma). Treatment with the cysteine proteinases resulted in a rapid shift in the apparent molecular size of IFN-gamma from 17 to 15 kDa, as shown by Western blot analysis, a response which also occurred in the presence of serum. Further, glycosylated natural IFN-gamma from human leukocytes and unglycosylated recombinant IFN-gamma from Escherichia coli were both digested by the cysteine proteinases. Immunoblot analysis indicated that cleavage within the carboxyl terminus of recombinant IFN-gamma correlated with the loss of induction of MHC class II expression as monitored by analytical flow cytometry. No hydrolysis of MHC class II molecules or human IFN-gamma receptor by these proteinases was detected by Western blot analysis. These findings suggest that P. gingivalis cysteine proteinases may alter the cytokine network at the point of infection through the cleavage of IFN-gamma. Degradation of IFN-gamma could have important consequences for the recruitment and activation of leukocytes and therefore may contribute significantly to the destruction of the periodontal attachment. Topics: Adhesins, Bacterial; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endothelium, Vascular; Epitopes, B-Lymphocyte; Gingipain Cysteine Endopeptidases; Hemagglutinins; HLA-DR Antigens; Humans; Interferon-gamma; Leupeptins; Porphyromonas gingivalis; Proteins; Recombinant Proteins | 1999 |
MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.
Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated. Topics: Animals; Antigen Presentation; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; beta-N-Acetylhexosaminidases; Binding Sites; Catalysis; Cell Compartmentation; Endocytosis; Histocompatibility Antigens Class II; Horseradish Peroxidase; Immune Sera; Intracellular Fluid; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Membrane Glycoproteins; Mice; Microscopy, Immunoelectron; Peptides; Pinocytosis; Receptors, Antigen, B-Cell; Receptors, Antigen, T-Cell; Receptors, Transferrin; Subcellular Fractions; Tumor Cells, Cultured | 1999 |
Involvement of interleukin-1beta in the mechanism of human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120-induced apoptosis in the neocortex of rat.
The effect of subchronic intracerebroventricular injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to seven consecutive days) on interleukin-1beta expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (300 ng, given intracerebroventricularly for up to seven days) -treated animals (n=6), interleukin-1beta immunoreactivity increased in the brain cortex and hippocampus of rats (n=6) receiving a single injection of the viral protein 24 h before analysis with more substantial increases being observed in these regions of the brain (n=6) after seven days treatment. Double-labelling immunofluorescence experiments support a neuronal and, possibly, a microglial cell origin for gp120-enhanced interleukin-1beta expression. Transmission electron microscopy analysis of brain tissue sections revealed that combination treatments (given intracerebroventricularly daily for seven days) with gp120 (100 ng) and interleukin-1 receptor antagonist (80 ng) or with the interleukin converting enzyme inhibitor II (100 pmol), but not with leupeptin (100 pmol), prevented apoptotic death of rat (n=6/group) brain cortical cells typically elicited by the viral protein. These data demonstrate that gp120 enhances interleukin-1beta expression in the brain and this may be involved in the mechanism underlying apoptosis induced by gp120 in the brain cortex of rat. Further support to this hypothesis comes from the evidence that intracerebroventricular injection of murine recombinant interleukin-1beta (200 U, given daily for seven consecutive days) produces DNA fragmentation in the brain cortex of rat (n=6). Interestingly, the latter treatment enhanced nerve growth factor level in the hippocampus but not in the cerebral cortex and this coincides with a similar effect recently reported in identical brain areas of rats treated likewise with gp120. In conclusion, the present data demonstrate that treatment with gp120 enhances interleukin-1beta expression and this participates in the mechanism of apoptotic cell death in the brain cortex of rat. By contrast, in the hippocampus, gp120-enhanced interleukin-1beta expression elevates nerve growth factor that may prevent or delay apoptosis in this plastic region of the rat brain. Topics: Animals; Apoptosis; Body Temperature; Cattle; Citrulline; Gene Expression Regulation; HIV Envelope Protein gp120; Humans; In Situ Nick-End Labeling; Injections, Intraventricular; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Kinetics; Leupeptins; Male; Microglia; Neocortex; Rats; Rats, Wistar; Recombinant Proteins; Serum Albumin, Bovine; Sialoglycoproteins; Time Factors | 1999 |
Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.
Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place. Topics: Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Cell Line; Endosomes; Histocompatibility Antigens Class II; Horseradish Peroxidase; Humans; Leupeptins; Lysosomes; Microscopy, Fluorescence; Peroxidases; Precipitin Tests; Transferrin | 1999 |
Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.
Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines. Topics: Animals; Cell Line; Cell Membrane Permeability; Chlorides; Cysteine Proteinase Inhibitors; DNA-Directed RNA Polymerases; HeLa Cells; Humans; Intracellular Membranes; Leucine; Leupeptins; Mice; Murine hepatitis virus; Rabbits; RNA, Viral; Staining and Labeling; Time Factors; Viral Proteins; Zinc Compounds | 1999 |
The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein.
Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism. Topics: Anemia, Megaloblastic; Animals; Antibodies; Apolipoprotein A-I; Case-Control Studies; Chloroquine; Chromatography, Affinity; Dog Diseases; Dogs; Endocytosis; Epithelial Cells; Female; Humans; Iodine Radioisotopes; Kidney; Leupeptins; Lipoproteins, HDL; Malabsorption Syndromes; Male; Rats; Rats, Wistar; Receptors, Cell Surface; Reference Values; Syndrome; Vitamin B 12 Deficiency; Yolk Sac | 1999 |
Lipofuscin turnover.
Topics: Animals; Cysteine Proteinase Inhibitors; Inclusion Bodies; Injections; Leupeptins; Lipofuscin; Pigment Epithelium of Eye; Rats | 1999 |
Enzymatic modulation of cell volume in C6 glioma cells.
We monitored the volume of C6 glioma cells in suspension using a Coulter Counter and exposed the cells to micromolar or nanomolar levels of collagenase or clostripain. In 13 experiments, type IV collagenase (310 units ml-1; approximately 3 microM L-1) decreased the volume by 8-12%, 8 min after addition. In 13 of 21 experiments, the volume decrease was followed by a volume regulatory increase (VRI) back to control levels in the continued presence of collagenase. The shrinkage evoked by type IV collagenase was eliminated by heat-inactivation of the enzyme preparation. A highly purified collagenase (type VII) at the same concentration evoked a relatively minor decrease in volume. A well-known contaminating protease present in type IV collagenase, clostripain, which specifically cleaves arginyl peptide bonds, evoked a 7 +/- 2% shrinkage (100 nM L-1, 7 experiments). Clostripain did not evoke a volume regulatory increase. The initial velocity of shrinkage evoked by clostripain (0.0012 pL min-1, 0.0034 pL min-1, 0.0132 pL min-1; 1 pL = 10(-12) liters) scaled with its concentration (1 nM L-1, 10 nM L-1, 100 nM L-1). The effect of clostripain was inhibited by heat-inactivation of the enzyme. Leupeptin, an inhibitor of clostripain, prevented the decrease in volume evoked by clostripain. The activity of stretch-activated ion channels was unaffected by type IV collagenase. Barium, cesium, amiloride, DIDS, or bumetanide failed to block the shrinkage evoked by type IV collagenase. These results demonstrate that clostripain, present in crude collagenase enzyme preparations, causes the shrinkage, and that C6 glioma cells can undergo a volume regulatory increase at virtually constant osmotic pressure. In addition, cleavage of a cell surface moiety, which contains arginine, and possibly proline, causes shrinkage. This moiety may be part of the extracellular or intracellular matrix providing mechanical support to the cells. VRI reflect actions of another substance in the type IV crude collagenase preparations, on a receptor independent of the arg-pro moiety. The enzymatic modulation of glioma cell volume by these two receptors may reflect a new mechanism by which such cells, and possibly other glia, regulate their contact area and interactions with other cells in the central nervous system. Topics: Brain Neoplasms; Cell Size; Collagenases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Glioma; Humans; Ion Channels; Leupeptins; Matrix Metalloproteinase 9; Mechanoreceptors; Peptide Hydrolases; Tumor Cells, Cultured | 1999 |
Differential presentation of glutamic acid decarboxylase 65 (GAD65) T cell epitopes among HLA-DRB1*0401-positive individuals.
Glutamic acid decarboxylase 65 (GAD65) is one of the major autoantigens in type 1 diabetes. We investigated whether there is variation in the processing of GAD65 epitopes between individuals with similar HLA backgrounds and whether the processing characteristics of certain immunogenic epitopes are different in distinct APC subpopulations. Using DR401-restricted T cell hybridomas specific for two immunogenic GAD65 epitopes (115-127 and 274-286), we demonstrate an epitope-specific presentation pattern in human B-lymphoblastoid cell lines (B-LCL). When pulsed with the GAD protein, some DRB1*0401-positive B-LCL, which presented GAD65 274-286 epitope efficiently, were unable to present the GAD65 115-127 epitope. However, all B-LCL presented synthetic peptides corresponding to either GAD epitope. In addition, when pulsed with human serum albumin, all cell lines gave equal stimulation of a DR4-restricted human serum albumin-specific T hybridoma. GAD65-transfected cell lines displayed the same presentation phenotype, showing that lack of the presentation of the 115-127 epitope was not due to inefficient uptake of the protein. Blood mononuclear adherent cells, B cells, or dendritic cells derived from the same individual displayed the same presentation pattern as observed in B cell lines, suggesting that the defect most likely is genetically determined. Therefore, individual differences in Ag processing may result in the presentation of distinct set of peptides derived from an autoantigen such as GAD65. This may be an important mechanism for the deviation of the immune response either into a regulatory pathway or into an inflammatory autoimmune reactivity. Topics: Antigen Presentation; Antigen-Presenting Cells; B-Lymphocytes; Cell Line, Transformed; Cell Lineage; Epitopes, T-Lymphocyte; Glutamate Decarboxylase; HLA-DR Antigens; HLA-DRB1 Chains; Humans; Leupeptins; Lymphocyte Activation; Pepstatins; Protease Inhibitors; Transfection | 1999 |
Ceramide-induced apoptosis occurs independently of caspases and is decreased by leupeptin.
Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; fas Receptor; Female; Humans; Leupeptins; Necrosis; Sphingosine; Tumor Cells, Cultured; U937 Cells | 1999 |
Stabilizing neuromuscular contacts reduces motoneuron death caused by paralysis of muscles in neonatal rats.
Transient paralysis of the soleus muscle in neonatal rats leads to permanent muscle weakness, loss of muscle fibres and motoneuron death. Application of leupeptin, an inhibitor of a calcium-activated neutral protease, to the neuromuscular junction is known to enhance the maintenance of neuromuscular contacts during development and axonal sprouting. Here, we show that treatment of soleus muscles with leupeptin as they recover from a period of paralysis rescues motoneurons that would otherwise die. The number of motoneurons to the soleus muscle was established by retrograde labelling with horseradish peroxidase eight to 10 weeks after recovery from paralysis. There were only 38.4 (+/-2.8 S.E.M., n=5) motoneurons innervating the soleus muscle that had been paralysed with alpha-bungarotoxin, compared to 58.2 (+/-3.1 S.E.M., n=5) to the control untreated soleus. Thus, the number of motoneurons to the soleus muscle on the alpha-bungarotoxin-treated side was 66.9% (+/-6.2% S.E.M., n=5) of the control side. In those animals where paralysis of the soleus muscle was followed three days later by treatment with leupeptin, the number of labelled motoneurons on the treated side of the spinal cord was 61.5 (+/-4.6 S.E.M., n=4) and that on the contralateral untreated control side was 59 (+/-3.8 S.E.M., n=4). This improvement in motoneuron survival in the leupeptin-treated animals is also confirmed by counts of the number of motor units in the soleus muscle obtained by recording muscle tension. In animals that had their soleus muscles paralysed at birth, only 21 (+/-0.7 S.E.M., n=5) motor units were present, compared to 30 motor units in control muscles. When the paralysed soleus muscle was subsequently treated with leupeptin, the number of remaining motor units in the muscle was 29.8 (+/- 1.0 S.E.M., n=5). In addition, the force output of the soleus muscles that had undergone a period of neonatal paralysis was calculated for both the NaCl- and leupeptin-treated animals. The results showed that paralysis at birth results in a reduction in weight and force output of the soleus muscle, which is not improved following treatment with leupeptin. This study shows that application of leupeptin to the soleus muscle after alpha-bungarotoxin-induced paralysis rescues motoneurons to the soleus that would otherwise die. This effect is most likely due to stabilization of their neuromuscularjunctions. Topics: Animals; Animals, Newborn; Bungarotoxins; Calpain; Cell Count; Cell Survival; Cholinergic Antagonists; Isometric Contraction; Leupeptins; Motor Neurons; Muscle, Skeletal; Neuromuscular Junction; Neurotoxins; Paralysis; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic | 1999 |
Is lipofuscin eliminated from cells?
Topics: Fibroblasts; Heart; Humans; Inclusion Bodies; Leupeptins; Lipofuscin; Myocardium; Pigment Epithelium of Eye | 1999 |
A role for the proteasome in the light response of the timeless clock protein.
The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop in Drosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells. Topics: Acetylcysteine; Animals; Biological Clocks; Cells, Cultured; Circadian Rhythm; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Darkness; Drosophila; Drosophila Proteins; Feedback; Insect Proteins; Leucine; Leupeptins; Light; Multienzyme Complexes; Neurons; Phosphorylation; Phosphotyrosine; Protease Inhibitors; Proteasome Endopeptidase Complex; Ubiquitins | 1999 |
Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller).
Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OC-I delta 86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OC-I delta 86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs. The synthetic cysteine proteinase inhibitors E-64 and leupeptin also significantly reduced slug weight gain (by at least 40%) and digestive gland cysteine proteinase activity when administered in an artificial diet, indicating that their antimetabolic effects are due to direct inhibition of gut proteolytic activity. These results suggest that transgenic crop plants expressing phytocystatins could be used to suppress the growth rates of slug populations in the field. Topics: Animals; Arabidopsis; Cystatins; Cysteine Proteinase Inhibitors; Leucine; Leupeptins; Mollusca; Plant Leaves; Plants, Genetically Modified; Recombinant Proteins | 1999 |
Effect of the selective and non-selective cysteine protease inhibitors on the intracellular processing of interleukin 6 by HEPG2 cells.
The effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxy-succinyl-L-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH2F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of 125I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of 125I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 microM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles. Topics: Cathepsin B; Cathepsins; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Dipeptides; Humans; Interleukin-6; Intracellular Fluid; Iodine Radioisotopes; Isotope Labeling; Ketones; Leucine; Leupeptins; Recombinant Proteins; Time Factors; Tumor Cells, Cultured | 1999 |
Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes.
The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome. Topics: Adrenocorticotropic Hormone; Agouti Signaling Protein; alpha-MSH; Animals; Binding, Competitive; Endocytosis; Intercellular Signaling Peptides and Proteins; Iodine Radioisotopes; Leupeptins; Lymphocytes; Lysosomes; Male; Monensin; Protein Binding; Proteins; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin; Receptors, Melanocortin; Spleen; Temperature | 1999 |
Arginine vasopressin transport and metabolism in the pigmented rabbit conjunctiva.
The purpose of this study was to evaluate the transepithelial transport and metabolism of arginine vasopressin (AVP) in the pigmented rabbit conjunctiva, both in the absence and presence of protease inhibitors. The apparent permeability coefficient, P(app), for 3H-AVP was determined in the modified Ussing chamber, and AVP metabolites were monitored by reversed phase HPLC using a C18 column. At 50 nM donor 3H-AVP, the P(app) in the mucosal-to-serosal (ms) direction was about five times higher than that in the opposite direction. Excess (0.1 mM) AVP decreased the P(app) for labelled AVP in the mucosal-to-serosal (ms) direction by about 50%. However, intact AVP transport showed neither concentration nor direction dependence. HPLC analysis revealed two subspecies of 3H-AVP in the receiver fluid and virtually no degradation products in the donor fluid following 3 h flux experiments. 3H-AVP transported in the ms direction underwent extensive hydrolysis (73%), which was decreased by 33% with mucosal application of 2 mM camostat mesylate (an aminopeptidase inhibitor) or by 27% with 0.5 mM leupeptin (a serine protease inhibitor). By contrast, 3H-AVP transported in the serosal-to-mucosal (sm) direction resulted in only 37% hydrolysis, and mucosal application of either inhibitor did not significantly affect the P(app) for intact AVP. These data suggest that intact AVP transport in the conjunctiva may be mediated mostly by passive diffusion and enzymatic degradation of AVP may be mediated by proteolytic enzymes present on the mucosal side of the conjunctiva. Topics: Animals; Arginine Vasopressin; Biological Transport; Chromatography, High Pressure Liquid; Conjunctiva; Esters; Gabexate; Guanidines; In Vitro Techniques; Leupeptins; Male; Protease Inhibitors; Rabbits; Tritium | 1998 |
Smokeless tobacco-exposed oral keratinocytes increase macromolecular efflux from the in situ oral mucosa.
The purpose of this study was to determine whether supernatants of cultured human oral keratinocytes (HOK) exposed to an aqueous extract of smokeless tobacco (STE) increase macromolecular efflux from the oral mucosa in vivo and, if so, whether bradykinin mediates in part this response. Subconfluent monolayers of HOK were incubated with STE or media, and supernatants were collected 24, 48, and 72 h thereafter. Using intravital microscopy, we found that suffusion of supernatants of STE- but not media-exposed HOK elicited significant concentration- and time-dependent increases in efflux of fluorescein isothiocyanate-labeled dextran (mol mass 70 kDa) from the in situ hamster cheek pouch (P < 0.05). These effects were significantly attenuated by HOE-140 and NPC-17647 but not by des-Arg9, [Leu8]-bradykinin. Proteolytic activity was increased in supernatants of STE- but not media-exposed HOK. However, a mixture of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid had no significant effects on HOK supernatant-induced responses. Collectively, these data suggest that oral keratinocytes modulate smokeless tobacco-induced increase in macromolecular efflux from the in situ oral mucosa in part by elaborating proteases that may account for local bradykinin production. Topics: Animals; Bradykinin; Bradykinin Receptor Antagonists; Cells, Cultured; Cheek; Cricetinae; Dextrans; Fluorescein-5-isothiocyanate; Humans; Keratinocytes; Kinetics; Leupeptins; Male; Mesocricetus; Microcirculation; Mouth Mucosa; Plants, Toxic; Tobacco, Smokeless | 1998 |
Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter.
We have studied the involvement of proteolytic pathways in the regulation of the Na/Pi cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, L-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (i) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/Pi cotransport and (ii) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/Pi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the Na/Pi cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes. Topics: Acetylcysteine; Animals; Carrier Proteins; Cell Compartmentation; Cells, Cultured; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Kidney; Leupeptins; Lysosomes; Microscopy, Confocal; Multienzyme Complexes; Opossums; Parathyroid Hormone; Phosphates; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Sodium-Phosphate Cotransporter Proteins; Sodium-Phosphate Cotransporter Proteins, Type II; Symporters | 1998 |
Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway.
Presentation of the Mtv-1 superantigen (vSag1) to specific Vbeta-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II alphabeta dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells. Topics: Animals; Antigen Presentation; Antigens, Viral; B-Lymphocytes; Biological Transport; Blotting, Western; Cell Membrane; Centrifugation, Density Gradient; Exocytosis; Hexosaminidases; Histocompatibility Antigens Class II; Leupeptins; Lysosomes; Mice; Sodium Dodecyl Sulfate; Superantigens; Tumor Cells, Cultured | 1998 |
Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin.
Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatus SMF13 by ammonium sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 degrees C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation. Topics: Amino Acid Sequence; Chromatography; Edetic Acid; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Hydrolysis; Leucine; Leupeptins; Metalloendopeptidases; Microscopy, Electron, Scanning; Molecular Weight; Peptide Fragments; Phenanthrolines; Streptomyces; Substrate Specificity | 1998 |
Carboxy-terminal truncation of long-tailed amyloid beta-peptide is inhibited by serine protease inhibitor and peptide aldehyde.
The 42/43-residue amyloid beta-peptide (Abeta) is widely believed to play a major role in Alzheimer's disease. The present study shows that the rat brain contains a carboxypeptidase that efficiently deletes three amino acids from Abeta1-43. The carboxypeptidase activity in the brain was completely inhibited by 1 mM phenylmethylsulfonyl fluoride, suggesting the protease is a serine carboxypeptidase. The carboxy-terminal truncation of Abeta1-43 was moderately inhibited by carbobenzoxy-Leu-leucinal, carbobenzoxy-Leu-Leu-leucinal, and carbobenzoxy-Leu-Leu-norvalinal, and weakly by antipain. The present data suggest that the serine carboxypeptidase contributes to the generation of short-tailed Abeta peptides and is important in the intracellular clearance of Abeta1-42/43 in brains. Topics: Amino Acids; Amyloid beta-Peptides; Animals; Antipain; Brain; Carboxypeptidases; Chloromercuribenzoates; Edetic Acid; Leupeptins; p-Chloromercuribenzoic Acid; Pepstatins; Peptide Fragments; Phenylmethylsulfonyl Fluoride; Rats; Serine Proteinase Inhibitors | 1998 |
On the degradability and exocytosis of ceroid/lipofuscin in cultured rat cardiac myocytes.
The accumulation of lipofuscin (LF)--a polymeric, electron-dense, autofluorescent substance--within postmitotic cells is a characteristic manifestation of aging. It is generally believed that LF is undegradable and formed due to peroxidative alterations of various macromolecules under intralysosomal autophagic degradation. We report here that a short-term exposure of cultured neonatal rat cardiac myocytes to the thiol protease-inhibitor leupeptin, causes an accumulation of numerous electron-dense autophagic lysosomes within the cells. Although very similar to LF by ultrastructure, these inclusions do not display LF-specific, yellow-orange autofluorescence when excited with blue light. Moreover, they rapidly disappear from the cells upon re-establishment of normal culture conditions. In contrast, prolonged leupeptin treatment results in an accumulation of dense lysosomes that also show LF-typical autofluorescence. This autofluorescent material remains in the cells after the end of leupeptin action. The results suggest that: (i) a certain amount of time is needed for autophagocytosed material to become peroxidized, autofluorescent and undegradable, i.e. to acquire properties typical of LF; (ii) protease-inhibition by itself does not lead to LF-formation but rather allows the prolonged time needed for oxidative modification of autophagocytosed material; (iii) mature LF is probably not subjected to either degradation or exocytosis. Topics: Animals; Cells, Cultured; Ceroid; Exocytosis; Leupeptins; Lipofuscin; Myocardium; Rats; Rats, Sprague-Dawley; Time Factors | 1998 |
The unusual active site of Gal6/bleomycin hydrolase can act as a carboxypeptidase, aminopeptidase, and peptide ligase.
The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin. Topics: Amino Acid Sequence; Aminopeptidases; Binding Sites; Carboxypeptidases; Cloning, Molecular; Crystallography, X-Ray; Cysteine Endopeptidases; DNA Primers; Escherichia coli; Kinetics; Leupeptins; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Synthases; Point Mutation; Protein Conformation; Recombinant Proteins; Sequence Deletion | 1998 |
Deterioration of connectin/titin and nebulin filaments by an excess of protease inhibitors.
We studied the effect of protease inhibitors at a high concentration on connectin and nebulin filaments in myofibrils. Calpastatin domain I at 0.1 mM bound to connectin and nebulin filaments, and deteriorated their physico-chemical properties; the calcium-binding ability of connectin and nebulin filaments was suppressed, the susceptibility of both filaments to trypsin was markedly decreased, and the resting tension of mechanically skinned fibers was increased by 2.5 times that of the control at a sarcomere length of 3.6 microns. This indicates that the connectin filaments were made more rigid. The same phenomenon was observed from the treatment of skinned fibers with 1 mM leupeptin whose resting tension was increased to 2 times the control value. Microscopically, both protease inhibitors induced dense aggregation and disappearance of the regular striation of myofibrils due to their non-specific binding to many myofibrillar proteins. The use of excess calpastatin domain I and leupeptin should therefore be avoided in physiological and biochemical studies on connectin and nebulin filaments, as well as on myofibrils. Topics: Animals; Calcium-Binding Proteins; Connectin; Cysteine Proteinase Inhibitors; Leupeptins; Muscle Proteins; Myofibrils; Protein Kinases; Rabbits | 1998 |
Purification and characterisation of a tripeptidyl aminopeptidase I from rat spleen.
A tripeptidyl aminopeptidase I with an M(r) of 47,000 Da has been purified from rat spleen. The N-terminal sequence of the enzyme and internal sequences did not resemble that of any known protein. The enzyme cleaves tripeptides from synthetic substrates provided that the N-terminus is unsubstituted and the amino acid in the P1 position is not charged. The enzyme also cleaves small peptides (angiotensin II and glucagon) releasing tripeptides but does not appear to demonstrate any preference for amino acids on either side of the cleavage site. The enzyme had maximum activity at pH 4 but was unstable above pH 7. Rat spleen tripeptidyl peptidase I was not inhibited by classical inhibitors of serine, cysteine, aspartate or metalloproteinases. The peptidase was potently inhibited by a series of substrate-based tripeptidyl chloromethyl ketones (Ki's of 10(-6)-10(-8) M). Inhibition was rapid and reversible. This mode of inhibition is different to the interaction between chloromethyl ketones and cysteine or serine peptidases. These tripeptidyl chloromethyl ketones were also inhibitors of bone resorption using an in vitro assay suggesting that a tripeptidyl peptidase is involved in the degradation of bone matrix proteins. Topics: Aminopeptidases; Animals; Bone Resorption; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Isoflurophate; Leupeptins; Protease Inhibitors; Rats; Serine Proteases; Spleen; Tripeptidyl-Peptidase 1 | 1998 |
Submandibular salivary proteases: lack of a role in anti-HIV activity.
Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity. Topics: Anti-HIV Agents; Aprotinin; Benzamidines; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Edetic Acid; Egtazic Acid; Endopeptidases; Ethylmaleimide; HIV-1; Humans; Isoflurophate; Leupeptins; Metalloendopeptidases; Norleucine; Parotid Gland; Pepstatins; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Saliva; Serine Proteinase Inhibitors; Submandibular Gland; Trypsin Inhibitor, Bowman-Birk Soybean | 1998 |
Cholesterol sulfate inhibits proteases that are involved in desquamation of stratum corneum.
We previously reported that desmosomes play a key role in the adhesion of corneocytes, and their digestion by two types of serine proteases leads to desquamation. Patients with recessive X-linked ichthyosis show hyperkeratosis attributable to desmosomes, associated with an increased content of cholesterol sulfate (CS) and an increased thickness of stratum corneum. In this study, therefore, we examined the possibility that CS provokes the abnormal desquamation, acting as a protease inhibitor. Scaling was induced on mice after topical application of chymostatin and leupeptin. Visible scale was also observed on mice after topical application of CS. We found that the stratum corneum thickness of CS-treated mice was increased in comparison with that of vehicle-treated mice. The thickness of the epidermis and the labeling index with proliferating cell nuclear antigen from CS-treated mice was almost the same as that from vehicle-treated mice. Moreover, in the stratum corneum of CS-treated mice, the content of desmosomes was higher than that in vehicle-treated mice. CS also inhibited the protease-induced cell dissociation of human stratum corneum sheets. In vitro, CS competitively inhibited both types of serine protease: the Ki for trypsin was 5.5 x 10(-6) M and that for chymotrypsin was 2.1 x 10(-6) M. These results indicate that CS retards desquamation by acting as a protease inhibitor. Thus, accumulation of stratum corneum in recessive X-linked ichthyosis may be a result of the inhibition by excessive CS of proteases involved in the dissolution of desmosomes, required for desquamation of the stratum corneum. Topics: Animals; Cholesterol Esters; Epidermis; Humans; Ichthyosis, X-Linked; Leupeptins; Male; Mice; Mice, Hairless; Oligopeptides; Serine Proteinase Inhibitors | 1998 |
Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes.
Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes. Topics: Ammonia; Animals; Asparagine; Endocytosis; Endosomes; Gold; Leupeptins; Liver; Male; Microscopy, Electron; Phagosomes; Propylamines; Rats; Rats, Wistar | 1998 |
Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4(+) T cells.
The induction of an efficient CD4(+) T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4(+) T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10(4) transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation. Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Division; Cell Line, Transformed; Chloramphenicol O-Acetyltransferase; COS Cells; Genes, Viral; Herpesvirus 4, Human; Histocompatibility Antigens Class II; Leupeptins; Viral Core Proteins; Viral Structural Proteins | 1998 |
Endocytosed ricin and asialoorosomucoid follow different intracellular pathways in hepatocytes.
Earlier studies have suggested that fluid phase endocytosis in rat hepatocytes takes place via a clathrin-independent mechanism [1,2]. This observation suggests that a relatively large amount of plasma membrane outside coated pits may be involved in hepatic endocytosis. Ricin, which binds to galactose residues on glycoproteins and glycolipids, has, in this report, been used as a general marker for the plasma membrane of hepatocytes. The endocytosis of ricin was compared with that of asialoorosomucoid (AOM) which is taken up exclusively via clathrin-coated pits. Hypertonic medium has been shown to inhibit uptake via coated pits more effectively than clathrin-independent uptake [3-5]. It was found, in this study, that the addition of 100 mM sucrose to the incubation medium inhibited the uptake of 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) more extensively than that of 125I-tyramine-cellobiose-ricin (125I-TC-ricin), compatible with the notion that the two probes are internalised via different mechanisms. Subcellular fractionation experiments indicated that 125I-TC-ricin entered a denser endocytic organelle than that receiving 125I-TC-AOM. To determine whether the separation of the two probes was due to a different transport kinetics (i.e. that 125I-TC-ricin is transported more rapidly to a later, denser compartment than 125I-TC-AOM) the cells were incubated at 18 degreesC to allow a slower internalisation/transport of the labelled probes. The results obtained showed, again, that the early endosomes containing 125I-TC-ricin were significantly denser than those containing 125I-TC-AOM. We also employed the horseradish peroxidase (HRP)-diaminobenzidine (DAB) density shift technique of Courtoy et al. [6] to determine whether 125I-TC-ricin and 125I-TC-AOM were in separate endosomes early after their uptake. The results showed that early endosomes containing 125I-TC-AOM were density shifted whereas those containing 125I-TC-ricin were unaffected by the density shift procedure. The use of probes labelled with 125I-TC allowed us to identify compartments involved in the degradation of 125I-TC-AOM and 125I-TC-ricin, by measuring acid soluble radioactivities in the gradient fractions. It was found that 125I-TC-ricin was degraded mainly in endosomes, whereas 125I-TC-AOM, as expected, was degraded mainly in lysosomes. Topics: Animals; Asialoglycoproteins; Centrifugation, Density Gradient; Culture Media; Endocytosis; Iodine Radioisotopes; Leupeptins; Liver; Male; Orosomucoid; Osmolar Concentration; Rats; Rats, Wistar; Ricin; Subcellular Fractions; Vinblastine | 1998 |
Vasopressin-induced disruption of actin cytoskeletal organization and canalicular function in isolated rat hepatocyte couplets: possible involvement of protein kinase C.
The effect of vasopressin (VP) on canalicular function and hepatocellular morphology, with particular regard to actin cytoskeletal organization and the concomitant plasma membrane bleb formation, was studied in isolated rat hepatocyte couplets. VP induced the concentration-dependent formation of multiple plasma membrane blebs as well as simultaneous impairment in both canalicular vacuolar accumulation (cVA) and retention (cVR) of the fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), which evaluate couplet secretory function and tight-junction integrity, respectively. These effects were mimicked by the protein kinase C (PKC) activator, phorbol dibutyrate (PDB), but not by the protein kinase A (PKA) activator, dibutyryl-cAMP. VP-induced bleb formation and canalicular dysfunction were fully prevented by the protein kinase inhibitor, H-7, but not by the PKA inhibitor, KT5720, further suggesting a specific role of PKC. VP-induced alterations were also prevented by pretreatment with the Ca2+-buffering agent, BAPTA/AM, but not with the calmodulin-dependent protein kinase II antagonist, calmidazolium. Neither the Ca2+-activated neutral protease inhibitor, leupeptin, nor the antioxidants, alpha-tocopherol or deferoxamine, were able to prevent either VP-induced plasma membrane blebbing or canalicular dysfunction. The Ca2+-ionophore, A23187, mimicked the VP-induced alterations, but its harmful effects were completely prevented by H-7. Bleb formation induced by VP and PDB was accompanied by an extensive redistribution of filamentous actin from the pericanalicular area to the cell body, and this effect was fully prevented by H-7. These results suggest that VP-induced canalicular and cytoskeletal dysfunction is mediated by PKC and that classical (Ca2+-dependent) PKC appear to be involved because intracellular Ca2+ is required for VP to induce its harmful effects. Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Antioxidants; Bile Canaliculi; Bucladesine; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cells, Cultured; Chelating Agents; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Imidazoles; Indoles; Leupeptins; Liver; Male; Microscopy, Electron, Scanning; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrroles; Rats; Rats, Wistar; Tight Junctions; Vacuoles; Vasopressins | 1998 |
Protease inhibitor coinfusion with amyloid beta-protein results in enhanced deposition and toxicity in rat brain.
Amyloid beta-protein, Abeta, is normally produced in brain and is cleared by unknown mechanisms. In Alzheimer's disease (AD), Abeta accumulates in plaque-like deposits and is implicated genetically in neurodegeneration. Here we investigate mechanisms for Abeta degradation and Abeta toxicity in vivo, focusing on the effects of Abeta40, which is the peptide that accumulates in apolipoprotein E4-associated AD. Chronic intraventricular infusion of Abeta40 into rat brain resulted in limited deposition and toxicity. Coinfusion of Abeta40 with the cysteine protease inhibitor leupeptin resulted in increased extracellular and intracellular Abeta immunoreactivity. Analysis of gliosis and TUNEL in neuron layers of the frontal and entorhinal cortex suggested that leupeptin exacerbated Abeta40 toxicity. This was supported further by the neuronal staining of cathepsin B in endosomes or lysosomes, colocalizing with intracellular Abeta immunoreactivity in pyknotic cells. Leupeptin plus Abeta40 caused limited but significant neuronal phospho-tau immunostaining in the entorhinal cortex. Intriguingly, Abeta40 plus leupeptin induced intracellular accumulation of the more toxic Abeta, Abeta42, in a small group of septal neurons. Leupeptin infusion previously has been reported to interfere with lysosomal proteolysis and to result in the accumulation of lipofuscin, dystrophic neurites, tau- and ubiquitin-positive inclusions, and structures resembling paired helical filaments. Coinfusion of Abeta40 with the serine protease inhibitor aprotinin also increased diffuse extracellular deposition but reduced astrocytosis and TUNEL and was not associated with intracellular Abeta staining. Collectively, these data suggest that an age or Alzheimer's-related defect in lysosomal/endosomal function could promote Abeta deposition and DNA fragmentation in neurons and glia similar to that found in Alzheimer's disease. Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Antibodies, Monoclonal; Aprotinin; Cathepsins; Female; Frontal Lobe; Glial Fibrillary Acidic Protein; Gliosis; In Situ Nick-End Labeling; Leupeptins; Lysosomes; Microscopy, Confocal; Neuroglia; Neurons; Neurotoxins; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Serine Proteinase Inhibitors; Thalamus | 1998 |
Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II.
A decrease of proximal tubular reabsorption of phosphate (Pi), which can be provoked by parathyroid hormone (PTH) or by a high Pi-diet, has been shown to correlate with a decrease of the number of type II Na/Pi-cotransporters residing in the brush border membrane. While both PTH and a high Pi-diet lead to an internalization of type II cotransporters, the further cellular routing of internalized cotransporters has not been established unequivocally.. To prevent lysosomal degradation, rats were treated with leupeptin prior to the injection of PTH or feeding acutely with a high Pi-diet. Kidney cortex were recovered and used for immunohistochemistry. In parallel, brush border membranes and lysosomes were isolated and analyzed by Western blotting.. Under both conditions (PTH and high Pi-diet), a strong overlap of internalized type II cotransporters with the late endosomes/lysosomes was observed by immunohistochemistry. In agreement, the content of type II Na/Pi-cotransporters was increased in lysosomes isolated from the corresponding tissues.. These results suggest that in proximal tubular cells type II Na/Pi-cotransporters internalized due to the action of PTH and acute high Pi-diet are routed to the lysosomes, and likely do not enter a recycling compartment. Topics: Animals; Carrier Proteins; Diet; Immunohistochemistry; Kidney Tubules, Proximal; Leupeptins; Lysosomes; Microvilli; Parathyroid Hormone; Phosphates; Rats; Sodium; Sodium-Phosphate Cotransporter Proteins; Sodium-Phosphate Cotransporter Proteins, Type II; Symporters | 1998 |
Differential toxicity of protease inhibitors in cultures of cerebellar granule neurons.
Involvement of proteases has been postulated in several neurodegenerative processes. Accordingly, protease inhibition has been proposed as a potential therapeutic tool to limit damage in some neuropathological states. The timed turn-over of proteins is, however, an essential biochemical process and its prolonged block may be dangerous to the cell. We report here data on toxicity consequent to 24-h exposure of cerebellar granule neurons in culture to inhibitors of different classes of proteases. Inhibition of calpains (calcium-activated cysteine proteases) resulted in dose-dependent neuronal death which largely occurred through apoptotic process. Leupeptin, an inhibitor acting on a broad spectrum of cellular serine proteases, was less toxic but resulted in definite morphological alteration of the cells. On the contrary, inhibitors of caspases, proteases belonging to the ICE (interleukin 1-beta converting enzyme) family, did not apparently damage granule neurons upon exposure for 24 h to high concentrations (up to 200 microM) of two inhibitors specific for ICE (Ac-YAVD-CHO) and CPP-32 (Ac-DEVD-CHO), respectively. These results suggest that inhibition of proteases that are activated by stressful stimuli but are not essential for the normal functioning of healthy cells, as it is likely the case for caspases, may not be harmful to neurons. Instead, the potential risks and side effects of prolonged inhibition of proteases such as calpains, that regulate the disposal and the turn-over of key cellular proteins, should be carefully tested in the assessment of possible neuroprotective roles. Topics: Animals; Apoptosis; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Dipeptides; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Leupeptins; Neurons; Oligopeptides; Protease Inhibitors; Rats; Rats, Wistar | 1998 |
Dexamethasone stimulates proteasome- and calcium-dependent proteolysis in cultured L6 myotubes.
The effect of dexamethasone on protein degradation and the involvement of different proteolytic pathways were examined in cultured L6 myotubes. Treatment of the cells with dexamethasone resulted in an approximately 20% increase in protein degradation at a hormone concentration of 10(-7) to 10(-6) M. By using various proteolytic blockers, evidence was found that the dexamethasone-induced increase in protein breakdown mainly reflected energy-proteasome-dependent proteolysis and to a lesser extent calcium-dependent protein breakdown. In contrast, the hormone treatment did not increase lysosomal proteolysis. mRNA levels for cathepsin B, ubiquitin, and the proteasome subunit C3 were increased by dexamethasone. The results suggest that glucocorticoids stimulate calcium and energy-proteasome-dependent muscle proteolysis and that changes in mRNA levels for proteolytic enzymes do not necessarily reflect the involvement of different proteolytic pathways. Topics: Adenosine Triphosphate; Animals; Calcium; Calpain; Cathepsin B; Cathepsin D; Cells, Cultured; Chloroquine; Cysteine Endopeptidases; Deoxyglucose; Dexamethasone; Leucine; Leupeptins; Methylamines; Mifepristone; Multienzyme Complexes; Muscle, Skeletal; Proteasome Endopeptidase Complex; Proteins; Rats; Tyrosine; Ubiquitins | 1998 |
Effect of chloroquine and leupeptin on intracellular accumulation of amyloid-beta (A beta) 1-42 peptide in a murine N9 microglial cell line.
Murine N9 microglia accumulated A beta from media containing 0.67 microM A beta within 6 h. In N9 and in primary rat microglia, chloroquine, which disrupts lysosomal pH, increased A beta-induced accumulation of A beta, particularly A beta1-42. Leupeptin similarly enhanced A beta accumulation. The scavenger receptor antagonist fucoidan did not affect acute chloroquine-dependent A beta1-42 accumulation, demonstrating uptake of non-aggregated A beta. After prolonged incubations, chloroquine enhanced A beta multimer (8-12 kDa) accumulation, an effect inhibited by fucoidan. Disruptions of the lysosomal system enhance A beta and its multimer formation. Despite negligible effects of fucoidan on initial A beta uptake, chronic exposure inhibits multimer accumulation, demonstrating a role for scavenger receptor in multimer accumulation. Topics: Amyloid beta-Peptides; Animals; Biological Transport; Cell Line; Cells, Cultured; Chloroquine; Kinetics; Leupeptins; Membrane Proteins; Mice; Microglia; Peptide Fragments; Polysaccharides; Rats; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class B | 1998 |
Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex.
The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex. Topics: Biological Transport; Brefeldin A; Cell Compartmentation; Cell Line; Cell Membrane; Endoplasmic Reticulum; Golgi Apparatus; Leupeptins; Lysosomes; Platelet Glycoprotein GPIb-IX Complex; Protein Processing, Post-Translational; Recombinant Proteins; Transfection | 1998 |
[Cell to cell transfer of dense bodies induced by intracisternal infusion of leupeptin in rat cerebellum--quantitative and ultrastructural studies].
Previous studies have demonstrated that rats treated with a proteinase inhibitor, leupeptin (Leu), show an accumulation of lipofuscin (Lf)-like dense bodies in the neurons. The present study examines the quantitative and ultrastructural changes in the Lf-like dense bodies of Purkinje (P) cells and Bergmann (B) glia over 20 days following a 4-day regimen of Leu administration by intracisternal infusion. Toluidine blue staining revealed considerable Lf-like granules in both types of cells 1 day after Leu infusion. However, these granules had almost disappeared by 20 days after infusion. Quantitative analysis revealed a decrease in the amount of accumulated Lf-like granules in P cells whereas the granules in B glia increased 10 days after Leu infusion. Electron microscopic examination revealed the presence of Lf-like dense bodies on the plasma membranes of both the P cells and B glia. The present results suggest that Leu-induced dense bodies in P cells are transferred to B glia, indicating a possible route for the removal of Lf. Topics: Animals; Cerebellum; Cisterna Magna; Histocytochemistry; Leupeptins; Lipofuscin; Male; Neuroglia; Protease Inhibitors; Purkinje Cells; Rats; Rats, Sprague-Dawley | 1998 |
Degradation of serum albumin by rat liver and kidney lysosomes.
Lysosomes were isolated from the livers and from the kidneys of rats treated or not treated with the cysteine proteinase inhibitor leupeptin, and the levels of the intralysosomal serum albumin of the leupeptin-treated rats were compared with those of the saline-treated control rats. Leupeptin caused an intralysosomal accumulation of albumin in vivo because of its potent inhibition of lysosomal protein degradation. In fact, the lysosomes isolated from the livers and kidneys of leupeptin-treated rats almost completely lost their ability to degrade rat albumin in vitro. These findings show that the lysosomes are subcellular sites of the degradation of unlabeled serum albumin in these tissues. They also suggest that cysteine proteinases sensitive to leupeptin are involved in the lysosomal degradation of albumin. Albumin was degraded by total lysosomal enzymes in vitro. It was also degraded by the lysosomal extract being devoid of cathepsins H and J, prepared from rat kidney. The degradation of albumin by total lysosomal enzymes in vitro was greatly suppressed by a cysteine proteinase inhibitor, cystatin alpha, with no inhibition of cathepsins B and L. It was slightly suppressed by N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prol ine (CA-074), a selective inhibitor of cathepsin B, and by pepstatin, an inhibitor of cathepsin D, whereas it was markedly suppressed by a combination of cystatin alpha and either CA-074 or pepstatin. These and associated findings show that cystatin alpha-sensitive cysteine proteinase(s), which is distinct from cathepsins B, H, L, and J, and cathepsins B and D are involved in the lysosomal degradation of albumin. Topics: Animals; Cathepsins; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Electrophoresis, Polyacrylamide Gel; Kidney; Leupeptins; Liver; Lysosomes; Male; Pepstatins; Rats; Rats, Wistar; Serum Albumin | 1998 |
Cysteine proteases and cysteine protease inhibitors in non-small cell lung cancer.
In this study we investigated the levels of two lysosomal cysteine protease proteins cathepsin B (CB) and cathepsin L (CL) and the levels of three cysteine protease inhibitor proteins stefin A (SFA), stefin B (SFB) and cystatin C (CNC) in squamous-cell lung carcinoma (SQCLC) and matched lung parenchyma specimens and examined the inhibition of CB and cathepsin C (CC) activities by endogenous inhibitors in extracts from SQCLC, lung adenocarcinoma (LAC) and lung parenchyma specimens. We found that Stage I SQCLCs contained significantly increased levels of CB protein, CB activity and SFA protein as compared to matched lungs. Neither the levels of CL protein nor the levels of SFB protein nor the levels of CNC protein in Stage I SQCLCs and the lungs were significantly different, but the levels of CB and CL proteins as well as the levels of SFA and SFB proteins showed significant positive correlation in SQCLCs. In SQCLCs as well as in the lungs the level of SFB protein was significantly higher than the level of SFA protein or the level of CNC protein. In the lungs the levels of SFA protein and CNC protein revealed a weak negative correlation trend. In extracts from SQCLCs the level of SFA protein showed a weak negative correlation with the residual CB activity (i.e. the activity remaining after extract preincubation) whereas in extracts from the lungs the level of CNC protein displayed a weak negative correlation trend with the residual CB activity and with the residual CC activity. We observed that SQCLCs and LACs contained not only a significantly increased activity of CB but also a significantly higher inhibitory potential against the activity of endogenous CB as compared to matched lungs. Leupeptin, a small inhibitor of CB, was capable to protect CB in lung carcinoma and lung parenchyma extracts from preincubation-induced inhibition, revealing an active-site directed and competitive nature of CB inhibition by endogenous cystatins. Ultrafiltration passaged protein preparations of nominal Mr < or = 30,000 obtained from extracts of SQCLCs inhibited significantly higher quantities of activity of purified bovine spleen CC than did such protein preparations from matched lungs. Reaction courses of purified bovine spleen CC that had been preincubated with such protein preparations resembled those of endogenous CC from SQCLC and lung extracts showing a slow steady-state approach. These observations and the relaxation kinetics of CC from SQCLC and lung extracts sugges Topics: Adenocarcinoma; Adult; Aged; Animals; Carcinoma, Squamous Cell; Cathepsin B; Cathepsin C; Cathepsin L; Cathepsins; Cattle; Cystatin A; Cystatin B; Cystatin C; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Humans; Kinetics; Leupeptins; Lung; Lung Neoplasms; Lysosomes; Male; Middle Aged; Sensitivity and Specificity | 1998 |
Tissue plasminogen activator mediates reverse occlusion plasticity in visual cortex.
Preventing visual input to one eye (monocular deprivation) in early postnatal development reduces cortical responses to stimulation of the deprived eye, with a significant loss of thalamocortical connections. These effects are reversible by opening the deprived eye and closing the previously open eye (reverse occlusion). We show that intracortical blockade of tissue plasminogen activator or plasmin selectively prevents recovery of cortical function and thalamic neuron size during reverse occlusion, without affecting the monocular deprivation response. Therefore, a proteolytic cascade consisting of plasmin generated by tissue plasminogen activator may selectively mediate reverse-occlusion-induced cortical plasticity, perhaps via structural remodeling of axons. Topics: Animals; Animals, Newborn; Cats; Cell Size; Fibrinolysin; Geniculate Bodies; Leupeptins; Neuronal Plasticity; Plasminogen Activators; Sensory Deprivation; Tissue Plasminogen Activator; Vision, Monocular; Visual Cortex | 1998 |
Persyn, a member of the synuclein family, influences neurofilament network integrity.
Topics: Calcium; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Endopeptidases; gamma-Synuclein; Leupeptins; Neoplasm Proteins; Nerve Tissue Proteins; Neurofilament Proteins; Neurons, Afferent | 1998 |
Expression of cathepsin B-like enzyme activity in cell lysate from cultured human normal keratinocytes.
In the present study, we have found that the cell lysate from cultured human normal keratinocytes from foreskin (HFKs) hydrolyzed alpha-N-benzoyl-DL-arginine beta-naphthylamide (BANA), and the BANA hydrolysis occurred most under conditions of 37 degrees C and pH 6.0. This activity was strongly inhibited by leupeptin, which is an inhibitor to cathepsin B. These results suggested that the cell lysate from cultured HFKs contained cathepsin B-like enzyme activity. This is the first report to demonstrate that cathepsin B-like enzyme activity was expressed in the cell lysate from human normal keratinocytes. Topics: Benzoylarginine-2-Naphthylamide; Cathepsin B; Cells, Cultured; Child; Cysteine Proteinase Inhibitors; Humans; Hydrolysis; Keratinocytes; Leupeptins; Male | 1998 |
Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells.
Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[alpha 1-2]Man[alpha 1-2]Man[alpha 1-3][Man alpha 1-6]Man[beta 1-4] GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 microM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes. Topics: Anti-Bacterial Agents; Bacterial Proteins; Biological Transport; Carbohydrate Sequence; Carcinoma, Hepatocellular; Cell Fractionation; Cell Membrane Permeability; Cytosol; Enzyme Inhibitors; Golgi Apparatus; Humans; Leupeptins; Liver Neoplasms; Lysosomes; Macrolides; Mannans; Mannose; Molecular Sequence Data; Oligosaccharides; Proton-Translocating ATPases; Streptolysins; Swainsonine; Tumor Cells, Cultured | 1997 |
A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins.
Two populations of rat liver lysosomes can be distinguished on the basis of their density. A major difference between these populations is that one contains the heat shock cognate protein of 73 kDa (hsc73) within the lysosomal lumen. The lysosomal fraction containing hsc73 exhibits much higher efficiencies in the in vitro uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, two well established substrates of the selective lysosomal pathway of intracellular protein degradation. Preloading of the lysosomal population that is devoid of lumenal hsc73 with hsc73 isolated from cytosol activated the selective transport of substrate proteins into these lysosomes. Furthermore, treatment of animals with leupeptin, an inhibitor of lysosomal cathepsins, or 88 h of starvation also increased the amount of hsc73 within their lysosomal lumen, and these in vivo treatments also activated the selective transport of substrate proteins in vitro. Thus, the hsc73 located within lysosomes appears to be required for efficient uptake of cytosolic proteins by these organelles. The difference in hsc73 content between the lysosomal populations appears to be due to differences in their ability to take up hsc73 combined with differences in the intralysosomal degradation rates of hsc73. The increased stability of hsc73 in one population of lysosomes is primarily a consequence of this lysosomal population's more acidic pH. Topics: Adenosine Triphosphate; Animals; Cell Compartmentation; Cytosol; Glyceraldehyde-3-Phosphate Dehydrogenases; Heat-Shock Proteins; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Leupeptins; Liver; Lysosomes; Male; Microscopy, Electron; Rats; Rats, Wistar; Ribonuclease, Pancreatic | 1997 |
Production of an extracellular trypsin-like protease by the fungal plant pathogen Verticillium dahliae.
The plant pathogenic fungus Verticillium dahliae produced extracellular alkaline protease activity when grown in liquid medium supplemented with a protein source. A serine protease was purified 80-fold in a single step, using cation-exchange chromatography, from the filtrate of cultures grown with skim milk as a protein source. N-terminal amino acid sequence analysis of the 30-kDa protein (VDP30) that copurified with the serine protease activity suggested that VDP30 is a trypsin-like protein. The purified enzyme hydrolyzed the synthetic substrate N alpha-benzoyl-DL-arginine p-nitroanilide hydrochloride (BAPNA), and the activity on BAPNA was inhibited by leupeptin, further verifying the trypsin-like nature of the enzyme. Topics: Amino Acid Sequence; Animals; Benzoylarginine Nitroanilide; Biomass; Chromatography, Ion Exchange; Culture Media; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Leupeptins; Milk; Mitosporic Fungi; Molecular Sequence Data; Sequence Homology, Amino Acid; Serine Endopeptidases; Serine Proteinase Inhibitors; Time Factors | 1997 |
Ii chain controls the transport of major histocompatibility complex class II molecules to and from lysosomes.
Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting alpha beta dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A(b) alpha beta dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A(b) class II molecules in lysosomes. In leupeptin, I-A(b) dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A(b) dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages. Topics: Animals; Antigens, Surface; Biological Transport; Cell Compartmentation; Cell Membrane; Dimerization; Electrophoresis; Histocompatibility Antigens Class II; Leupeptins; Lymphoma; Lysosomes; Mice; Microscopy, Immunoelectron; Protein Structure, Tertiary; Tumor Cells, Cultured | 1997 |
Memory disturbance and hippocampal degeneration induced by continuous intraventricular infusion of a protease inhibitor, leupeptin.
Effects of a protease inhibitor, leupeptin, on the memory function and the morphological changes in the hippocampus were examined in rats. The leupeptin was infused by an implanted-osmotic minipump into the lateral ventricle of the rats for 14 days. The acquisition and the maintenance of memory were evaluated by a step-down passive avoidance task. The control rats, infused with an artificial cerebral spinal fluid, showed good retention for the passive avoidance training for 21 days after training. The leupeptin-treated rats showed good retention for 7 days following training; however, pronounced impaired retention was observed on day 10 and thereafter. These rats were accompanied by a degeneration of the dentate gyrus in the histological examinations on Days 14 and 21. The granule cells in the dentate gyrus of the hippocampus appeared much more eosinophilic pyknotic. Numerous eosinophilic spherical structures of the cell processes were seen in the neuropil beneath the granule cell layer. Electron microscopic examination disclosed a marked accumulation of lipofuscin-like granules in the perikaryon of the cells and in the dendrites and the axons. These findings suggest that the memory impairment is closely related to the degeneration of the dentate gyrus in the hippocampus in the leupeptin-treated rats. Topics: Animals; Avoidance Learning; Axons; Cerebral Ventricles; Cytoplasmic Granules; Dendrites; Dentate Gyrus; Hippocampus; Infusions, Parenteral; Leupeptins; Lipofuscin; Memory; Memory Disorders; Motor Activity; Nerve Degeneration; Neurons; Rats; Rats, Wistar | 1997 |
Selective increased presentation of type II collagen by leupeptin.
Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII. Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Brefeldin A; Collagen; Cyclopentanes; Dimerization; Epitopes; Histocompatibility Antigens Class II; Immunophenotyping; Leupeptins; Lymphoma, B-Cell; Mice; Mice, Inbred DBA; Protease Inhibitors; Protein Synthesis Inhibitors; Sodium Dodecyl Sulfate; Tumor Cells, Cultured | 1997 |
Thrombin receptor-activating peptide releases arachidonic acid from human platelets: a comparison with thrombin and trypsin.
The serine proteases thrombin and trypsin are both powerful platelet agonists that act by cleaving the terminal portion of the thrombin receptor and allowing the new C-terminal to auto-stimulate the receptor. Synthetic peptides, termed thrombin receptor-activating peptides (TRAPs), have been shown to mimic many of the effects of thrombin. Here we have compared the effects of inhibitors on platelet aggregation and [14C]-arachidonic acid release in response to thrombin, trypsin and TRAP. Pretreatment of human platelets with BW755C (80 microM), which inhibits both cyclooxygenase and lipoxygenase, blocked trypsin (15-20 nM)- or TRAP (4-6 microM)-induced aggregation, but not thrombin (0.06-0.1 U/ml)-induced aggregation. The protease inhibitor leupeptin (10 micrograms/ml) abolished trypsin-induced aggregation and returned [14C]-arachidonic acid release from [14C]-arachidonic acid-prelabeled platelets to control levels. In contrast, leupeptin did not affect either aggregation or [14C]-arachidonic acid release in platelets stimulated by TRAP. Thrombin-induced aggregation and [14C]-arachidonic acid release were only partially inhibited by leupeptin. These data are consistent with the activation of platelets by both trypsin and TRAP occurring via the proteolytic receptor, whereas thrombin-induced platelet activation appears to occur by a dual mechanism of action. One component of thrombin-induced platelet activation is by a proteolytic action on the moderate-affinity receptor. This effect is sensitive to inhibition by leupeptin and is mimicked by trypsin and TRAP. The other component of thrombin is nonproteolytic and may occur by an action at a high-affinity receptor such as glycoprotein lb. Topics: Arachidonic Acid; Blood Platelets; Humans; Leupeptins; Peptide Fragments; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin; Trypsin | 1997 |
Differences between fluid-phase endocytosis (pinocytosis) and receptor-mediated endocytosis in isolated rat hepatocytes.
To characterize possible differences between the fluid-phase endocytosis (pinocytosis) of bovine serum albumin and the receptor-mediated endocytosis of asialo-orosomucoid (AOM) in isolated rat hepatocytes, both probes were conjugated to radioiodinated tyramine-cellobiose, [125I]TC. The use of these conjugates made it possible to measure the uptake and intracellular distribution of the intact proteins as well as of their acid-soluble, membrane-impermeant degradation products. [125I]TC-albumin was taken up at a very low rate (0.5%/h) compared to [125I]TC-AOM (45%/h), suggesting that neither membrane adsorption nor membrane permeation compromised its suitability as a fluid-phase marker. Sucrose gradient analysis indicated that both probes sequentially entered light endosomes (1.11 g/ml), dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml), but [125I]TC-albumin traversed the endocytic compartments more rapidly than [125I]TC-AOM, and was partially degraded intralysosomally already after 15 min. The microtubule inhibitor, vinblastine, had a stronger inhibitory effect on the uptake and degradation of [125I]TC-AOM (80% and 95%, respectively) than on the uptake and degradation of [125I]TC-albumin (50% and 70%, respectively). In the presence of vinblastine, [125I]TC-AOM was retained both in light and dense endosomes, whereas [125I]TC-albumin was retained in dense endosomes only, suggesting that the early steps of fluid-phase endocytosis were less critically dependent on microtubular function than the early steps of receptor-mediated endocytosis. A perturbant of vacuolar pH, propylamine, inhibited the degradation of both probes strongly (75-100%), as would be expected from its lysosomotropic effect. Propylamine also inhibited endocytic uptake, with a stronger effect on [125I]TC-AOM uptake (95% inhibition) than on [125I]TC-albumin uptake (60% inhibition), probably reflecting a reduction in endosomal acidity, reduced receptor-ligand dissociation and diminished recycling of free asialoglycoprotein receptors to the cell surface in addition to a general trapping of membrane in swollen vacuoles. A protein phosphatase inhibitor, okadaic acid, strongly (80-100%) inhibited the uptake and degradation of both [125I]TC-albumin and [125I]TC-AOM. An inhibitor of lysosomal proteinases, leupeptin, strongly suppressed the degradation of both probes and moderately reduced the uptake of [125I]TC-AOM, whereas the uptake of [125I]TC-albumin was unaffected. In contrast, an inhibitor of Topics: Animals; Asialoglycoprotein Receptor; Asialoglycoproteins; Autophagy; Cell Separation; Cellobiose; Endocytosis; Iodine Radioisotopes; Leupeptins; Liver; Male; Okadaic Acid; Orosomucoid; Pinocytosis; Propylamines; Rats; Rats, Wistar; Receptors, Cell Surface; Serum Albumin, Bovine; Subcellular Fractions; Tyramine; Vinblastine | 1997 |
Differentiation-induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT-29 cell line.
Enterocyte-like differentiated HT-29 colon carcinoma cells were shown to contain far higher intracellular levels of activity of lysosomal cathepsins B, D, and L than their undifferentiated counterparts. In the latter, inhibition of lysosomal functions by leupeptin or ammonium chloride led to a marked increase in the cell-associated activity of the three cathepsins. High levels of pro-cathepsins B, D, and L were found in the culture media of both HT-29 cell populations. Ammonium chloride and chloroquine, which are known to impair the mannose-6-phosphate-dependent trafficking of lysosomal-targeted proteins, did not increase the secretion of the three cathepsins in either undifferentiated or differentiated cultures of HT-29 cells. Analyses by cell fractionation revealed heterogeneities with regard to the density and the content of lysosomal cathepsins between the two cell populations. Leupeptin induced the accumulation of mature lysosomal cathepsins B and L in light density organelles in undifferentiated HT-29 cells. Altogether, these data demonstrate that (1) the expression and subcellular distribution of cathepsins B, D, and L in HT-29 cells are influenced by their state of enterocytic differentiation, (2) the segregation of lysosomal cathepsins is largely inefficient in this tumor cell line and does not increase upon differentiation, and (3) the mannose-6-phosphate-receptor-dependent pathway plays a minor role in the sorting of the three cathepsins, both in undifferentiated and enterocytic-differentiated HT-29 cells. Topics: Ammonium Chloride; Cathepsin B; Cathepsin D; Cathepsin L; Cathepsins; Cell Differentiation; Colon; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endopeptidases; HT29 Cells; Humans; Leupeptins; Lysosomes; Mannosephosphates | 1997 |
Thrombin regulates nerve growth factor secretion from vascular, but not bladder smooth muscle cells.
The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet. We have been examining the cellular processes of NGF delivery and secretion in smooth muscle. NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media. Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied. The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs. The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required. The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C. These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle. Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult. Topics: Animals; Aorta; Aprotinin; Cells, Cultured; Hirudins; Leupeptins; Muscle, Smooth; Muscle, Smooth, Vascular; Nerve Growth Factors; Protease Inhibitors; Protein Kinase C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar; Thrombin; Trypsin; Urinary Bladder | 1997 |
Intestinal epithelial cells use two distinct pathways for HLA class II antigen processing.
Intestinal epithelial cells express a low level of HLA class II molecules constitutively, with elevated levels seen in the setting of mucosal inflammation including inflammatory bowel disease. The ability of intestinal epithelial cells to act as antigen presenting cells for alphabeta CD4(+) T lymphocytes was examined through a molecular analysis of the HLA class II antigen processing pathway. We have shown that intestinal epithelial cells contain abundant constitutive levels of the cathepsin proteases proven to function in HLA class II mediated antigen presentation. Activation of these cells by gamma-IFN induced the expression of invariant chain and HLA-DM alphabeta, thus facilitating the formation of compact, SDS-stable HLA- DR alphabeta heterodimers. Using HLA-DR-restricted T cells and retroviral mediated gene transfer of HLA-DR alleles into the intestinal epithelial cell lines HT-29 and T84, we demonstrated efficient antigen processing and presentation to CD4(+) T lymphocytes in the presence of the proinflammatory cytokine gamma-IFN. The class II processing pathway and presentation in the presence of gamma-IFN was indistinguishable from that observed with a conventional antigen presenting cell. Antigen processing also occurred in intestinal epithelial cells in the absence of gamma-IFN, and in contrast to that seen after stimulation with gamma-IFN, required high concentration of antigen and was not inhibited by the protease inhibitor leupeptin. These data suggest the use of two distinct pathways of HLA class II antigen processing in enterocytes with differential immunomodulatory properties in the presence or absence of mucosal inflammation. Topics: Cathepsin B; Cathepsin H; Cathepsin L; Cathepsins; CD4-Positive T-Lymphocytes; Colonic Neoplasms; Cysteine Endopeptidases; Dimerization; DNA Primers; Endopeptidases; Histocompatibility Antigens Class II; HLA-D Antigens; HLA-DQ Antigens; HLA-DR Antigens; Humans; Interferon-gamma; Intestinal Mucosa; Leupeptins; Polymerase Chain Reaction; Protease Inhibitors; Recombinant Proteins; Transfection; Tumor Cells, Cultured | 1997 |
Utilization of intracellular ferritin iron for hemoglobin synthesis in developing human erythroid precursors.
Ferritin (Ft) plays an important role in cellular iron metabolism. It can store substantial amounts of iron in a nontoxic soluble form. However, its ability to donate iron for cellular needs, in particular for hemoglobin (Hb) synthesis in human erythroid cells, is still controversial. We studied the role of intracellular Ft-iron in Hb synthesis and the involvement of lysosomal proteolysis in iron release from Ft. Ft-iron release and its subsequent incorporation into heme was investigated in normal human erythroid precursors developing in culture. Dual staining flow cytometry with antibody (Ab)-specific for Ft and for Hb showed a decrease in cellular Ft content in erythroid cells during their maturation. Cellular Ft-iron participation in heme synthesis was studied by labeling cells with 59Fe. Cells were incubated with 59Fe-labeled human diferric transferrin (Tf), then chased, and intracellular radioiron distribution between Ft and Hb was determined on subsequent days by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or Ft immunoprecipitation and heme extraction. On day 6, most of the 59Fe accumulated in Ft. Thereafter, a progressive decrease of radioiron in Ft and a corresponding increase of the label in Hb was observed. Inhibition of heme synthesis with succinylacetone caused radioiron to remain in Ft and prevented its redistribution. Addition of unlabeled diferric Tf to the culture medium did not prevent radioiron from appearing in Hb. Chloroquine repression of lysosomal function prevented radio-iron redistribution between Ft and Hb. Inhibition of proteolysis by chymostatin and/or leupeptin led to Ft-protein accumulation in the cells and also prevented radioiron transfer from Ft to Hb. The results of the present study suggest that intracellular Ft donates iron for heme synthesis and that proteolytic Ft degradation in a lysosomal-like compartment is necessary for iron release and its transfer to heme. Topics: Autoradiography; Cells, Cultured; Chloroquine; Enzyme-Linked Immunosorbent Assay; Erythroblasts; Erythroid Precursor Cells; Ferritins; Flow Cytometry; Heme; Hemoglobins; Humans; Iron; Iron Radioisotopes; Kinetics; Leupeptins; Oligopeptides; Protease Inhibitors | 1997 |
Serine protease inhibitors block neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin.
To address the role of a plausible protease cascade in daunorubicin-triggered apoptosis, we evaluated the effect of cell-permeant protease inhibitors on its signal transduction pathway. Treatment of U937 and HL-60 cells with 0.5-1 microM of the chemotherapeutic drug daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. DNA fragmentation and the classical morphological features of apoptosis were observed within 4-6 h. Pretreatment of cells with the serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (20 microM) or dichloroisocoumarin (20 microM) for 30 min inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. Other cell-permeant protease inhibitors such as pepstatin, leupeptin, and antipain had no such effect. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. Daunorubicin-induced NF-kappaB activation was inhibited by dichloroisocoumarin but not by N-tosyl-L-phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibitors of serine proteases can act upstream of ceramide in drug-triggered apoptosis and that neutral sphingomyelinase activation is either directly or indirectly serine protease dependent. Topics: Antibiotics, Antineoplastic; Antipain; Apoptosis; Ceramides; Coumarins; Daunorubicin; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; HL-60 Cells; Humans; Hydrolysis; Isocoumarins; Leupeptins; NF-kappa B; Pepstatins; Protease Inhibitors; Serine Proteinase Inhibitors; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingomyelins; Tosylphenylalanyl Chloromethyl Ketone; Tumor Cells, Cultured | 1997 |
Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.
Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides. Topics: Amino Acid Sequence; Animals; Antigens, Differentiation, B-Lymphocyte; Cathepsin D; Cathepsins; Cricetinae; Cysteine Proteinase Inhibitors; Histocompatibility Antigens Class II; Leupeptins; Mice; Molecular Sequence Data; Polymorphism, Genetic; Rabbits | 1997 |
Interaction of combinations of drugs, chemosensitizers, and peptides with the P-glycoprotein multidrug transporter.
P-Glycoprotein functions as an ATP-driven efflux pump for hydrophobic natural products and peptides, and gives rise to resistance to multiple chemotherapeutic drugs. The inhibition of colchicine transport via P-glycoprotein by various compounds was determined in a plasma membrane vesicle model system. A chemotherapeutic drug (vinblastine) and several chemosensitizers (verapamil, reserpine, cyclosporin A) and hydrophobic peptides (N-acetyl-leucyl-leucyl-methioninal, leupeptin, pepstatin A, valinomycin) were examined, both as individual species and as combinations of compounds. The median effect analysis was used to determine the concentration of each combination required to produce a median effect, Dm, as well as the sigmoidicity of the concentration-effect plot, m. The combination of cyclosporin A and verapamil was the only one established to be mutually nonexclusive, whereas several mutually exclusive pairs of compounds were identified. The combination index, CI, was calculated for several combinations of drugs, chemosensitizers, and peptides, and used to ascertain whether effects were synergistic, antagonistic, or additive. Some combinations (vinblastine/verapamil; verapamil/valinomycin) showed antagonism over the entire concentration range. Other combinations (valinomycin/N-acetyl-leucyl-leucyl-methioninal; cyclosporin A/verapamil) displayed both synergism and antagonism over different regions of the CI plot. Many combinations of compounds displayed additive interactions over most of the CI plot. The median effect analysis may be helpful in identifying potentially useful additive or synergistic combinations of compounds for reversal of Pgp-mediated drug resistance. Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line; Cell Membrane; Colchicine; Cyclosporine; Drug Interactions; Drug Resistance, Multiple; Leupeptins; Oligopeptides; Peptides; Reserpine; Valinomycin; Vinblastine | 1997 |
MHC class II-associated invariant chain-induced enlarged endosomal structures: a morphological study.
The major histocompatibility complex class II-associated invariant chain is believed to direct newly synthesized class II to endocytic compartments. Invariant chain synthesized at high levels in transiently transfected cells induces formation of large vesicular structures. We have examined the effect of stable expression of invariant chain in human fibroblasts by light and electron microscopy. Invariant chain expression dramatically modified endocytic compartments and induced the formation of greatly enlarged structures. These modifications were not lethal. Ultrastructurally, at least three morphologically distinct enlarged compartments could be discerned in the cells. These three compartments may represent early and late endosomes and lysosomes. Internalization of anti-invariant chain antibodies shows that invariant chain may reach the large endosomes via rapid internalization from the plasma membrane. Internalized protein remained in the enlarged vesicles for 4-6 h, indicating an invariant chain-induced delay in the pathway to lysosomes. Although the large invariant chain-induced vesicles have not yet been seen in professional antigen-presenting cells, the invariant chain-induced effects may play a role in regulating the endocytic pathway, creating a special environment for MHC class II to bind antigen. Topics: Adenine; Antigens, CD; Brefeldin A; Cell Line; Chloroquine; Cyclopentanes; Endocytosis; Endosomes; Fibroblasts; HLA-DR alpha-Chains; HLA-DR Antigens; Humans; Immunohistochemistry; Leupeptins; Lysosomal Membrane Proteins; Lysosomes; Macromolecular Substances; Membrane Glycoproteins; Receptor, IGF Type 2; Recombinant Proteins; Transfection | 1997 |
Efficient photoaffinity labeling of the rat V1a vasopressin receptor using a linear azidopeptidic antagonist.
We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I )-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 +/- 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 +/- 35 pM) and in COS-7 cells (Kd = 320 +/- 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site. Topics: Affinity Labels; Animals; Antidiuretic Hormone Receptor Antagonists; Azides; Cell Membrane; COS Cells; Electrophoresis, Polyacrylamide Gel; Female; Hydrolysis; Leupeptins; Liver; Magnetic Resonance Spectroscopy; Male; Oligopeptides; Photochemistry; Rats; Rats, Wistar; Receptors, Vasopressin; Recombinant Proteins; Spectrophotometry, Ultraviolet; Spodoptera; Zinc | 1997 |
Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.
Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluorescence and RT-PCR. Trypsin, SLIGRL-NH2 (corresponding to the PAR-2 tethered ligand), mast cell tryptase, and a filtrate of degranulated mast cells stimulated a prompt increase in [Ca2+]i in myocytes. The response to tryptase and the mast cell filtrate was inhibited by the tryptase inhibitor BABIM, and abolished by desensitization of PAR-2 with trypsin. PAR-2 activation inhibited the amplitude of rhythmic contractions of strips of rat colon. This response was unaffected by indomethacin, l-NG-nitroarginine methyl ester, a bradykinin B2 receptor antagonist and tetrodotoxin. Thus, PAR-2 is highly expressed by colonic myocytes where it may be cleaved and activated by mast cell tryptase. This may contribute to motility disturbances of the colon during conditions associated with mast cell degranulation. Topics: Adrenergic beta-Antagonists; Animals; Benzimidazoles; Bradykinin; Calcium; Cells, Cultured; Chymases; Colon; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Fluorescent Antibody Technique, Indirect; Gastrointestinal Motility; In Vitro Techniques; Indomethacin; Inflammation Mediators; Leupeptins; Mast Cells; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitroprusside; Rats; Receptor, PAR-2; Receptors, Cell Surface; Serine Endopeptidases; Substance P; Tetrodotoxin; Time Factors; Trypsin; Tryptases | 1997 |
Myoblast fusion requires fibronectin degradation by exteriorized m-calpain.
We recently reported that when myoblasts fuse, m-calpain could be exteriorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particularly associated to extracellular matrix components. Knowing that the cell surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the phenomenon of fusion via fibronectin cleavage or degradation. Using different digestion experiments, we demonstrated that soluble purified fibronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fibronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a defined medium supplemented by exogenous factors capable of stimulating or inhibiting m-calpain activity. The effects of such factors on rat myoblast fusion and concomitantly on the targeted glycoprotein were analyzed and quantified. When m-calpain activity and the phenomenon of fusion were reduced (defined medium without insulin), the amount of the 220-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calpain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by approximately 67 and approximately 71%, respectively. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and more particularly associated to the extracellular matrix when muscle cells were initially treated by anti-m-calpain IgG. Taken together, these results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or degradation. Topics: Animals; Calpain; Cell Fusion; Cells, Cultured; Cysteine Proteinase Inhibitors; Fibronectins; Leupeptins; Muscle, Skeletal; Peptide Fragments; Protease Inhibitors; Rabbits; Rats; Rats, Wistar | 1997 |
Calcium homeostasis of isolated single cortical fibers of rat lens.
To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+]i during the process of disintegrative globulization.. Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+]i of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+]i in fiber cells exposed to Ringer's solution was measured, and the effects on the increase of [Ca2+]i of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated.. In Hepes sucrose solution (containing approximately 1.5 microM Ca2+), the isolated fiber cells maintained stable values of [Ca2+]i at 99.6+/-10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+]i at a rate of 0.12 min(-1). This increase in [Ca2+]i was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+]i, monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+]i in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+]i in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6+/-0.7 microM (n = 23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+]i in fibers was much slower in the presence of 50 microM verapamil (0.047 min[-1]), in Na+-free (0.086 min[-1]) and in K+-free (0.062 min[-1]) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min[-1]). After 30 minutes, the [Ca2+]i of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7+/-29 nM; n = 7) or Zn2+ (402.6+/-77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995+/-461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+]i. The [Ca2+]i of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+]i in fibers from the outer cortex was approximately three times higher than [Ca2+]i in those from the inner cortex.. Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+]i of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+]i. Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+]i but delay globulization by inhibiting calcium-mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes. Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Calcium Channel Blockers; Fluorescent Dyes; Homeostasis; Imidazoles; Lens Cortex, Crystalline; Leupeptins; Nickel; Organic Chemicals; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence; Verapamil; Xanthenes; Zinc | 1997 |
Fluorometric analysis of endocytosis and lysosomal proteolysis in the rat visceral yolk sac during whole embryo culture.
Using spectrofluorimetry and fluorescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) during whole embryo culture. Rat conceptuses exposed continuously to FITC-albumin had linear increases of both acid-soluble and acid-insoluble FITC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumulated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of fluorescence. During a chase period following a pulse exposure to FITC albumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of proteinase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluorescence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluorescence microscopy of yolk sacs exposed to FITC-albumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and decreased substantially during a chase period. In conceptuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC-fluorescence whose clearance in pulse-chase experiments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125l-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of the effects of embryotoxicants on yolk sac function during whole embryo culture. Topics: Animals; Cell Culture Techniques; Embryo, Mammalian; Endocytosis; Endopeptidases; Enzyme Inhibitors; Female; Fluorescein-5-isothiocyanate; Leupeptins; Lysosomes; Microscopy, Fluorescence; Pregnancy; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Spectrometry, Fluorescence; Yolk Sac | 1997 |
The effect of tacrine and leupeptin on the secretion of the beta-amyloid precursor protein in HeLa cells.
The senile plaque in Alzheimer's disease (AD) consists mainly of the amyloid beta-peptide (A beta) derived from a larger beta-amyloid precursor protein (betaAPP). The majority of betaAPP is processed by either a secretory or lysosomal/endosomal pathway. Soluble derivatives of betaAPP (sAPP) and A beta generated by the proteolytic processing of full-length betaAPP are normally secreted into the conditioned medium of cultured cells. Tacrine, a centrally active potent cholinesterase inhibitor that has been shown to improve cognitive functions in some patients with AD, inhibits the secretion of sAPP. Here we have investigated whether leupeptin, a lysosomal protease inhibitor, could influence this effect of tacrine. We analyzed levels of betaAPP derivatives in cultured HeLa cells by immunoblotting cell lysates and conditioned media using the monoclonal antibody 22C11. Levels of sAPP normally present in conditioned media were severely reduced by treating cells with tacrine. The treatment of cells with tacrine resulted in a small decrease in the intracellular levels of betaAPP. The effect of treating the cells with tacrine did not depend upon the growing state of the cells as a similar effect was observed when the drug was added either during initial plating of the cells or after the attachment of the cells. The effect of tacrine was not affected by preincubating the cells with low serum in the culture medium. The treatment of cells with tacrine plus leupeptin reduced the secretion of sAPP in the medium to the same degree as did the treatment with tacrine alone, suggesting that the tacrine-mediated inhibition of sAPP release may not involve leupeptin-sensitive proteolytic pathways. The results suggest that the inhibitory effect of tacrine on sAPP secretion is not due to the proteolytic cleavage of the holoprotein in the medium. Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Carbachol; Cholinesterase Inhibitors; Culture Media, Conditioned; HeLa Cells; Humans; Immunoblotting; Leupeptins; Parasympathomimetics; Protease Inhibitors; Tacrine | 1997 |
Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C.
1. Hippocampal neurones respond to acute oxygen deprivation (hypoxia) with an inhibition of whole-cell Na+ current (INa), although the mechanism of the inhibition is unknown. Kinases can modulate INa and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia-induced inhibition of INa. 2. Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. INa was recorded at baseline, during exposure to kinase activators (with and without kinase inhibitors), and during acute hypoxia (with and without kinase inhibitors). 3. Hypoxia (3 min) reduced INa to 38.1 +/- 4.5% of initial values, and shifted steady-state inactivation in the negative direction. Hypoxia produced no effect on activation or fast inactivation. 4. Protein kinase A (PKA) activation with 2.5 mM adenosine 3',5'-cyclic adenosine monophosphate, N6,O2-dibutyryl, sodium salt (db-cAMP) resulted in reduction of INa to 62.8 +/- 5.5% without an effect on activation or steady-state inactivation. INa was also reduced by activation of protein kinase C (PKC) with 5 nM phorbol 12-myristate 13-acetate (PMA; to 40.0 +/- 3.7%) or 50 microM 1-oleoyl-2-acetyl-sn-glycerol (OAG; to 46.1 +/- 2.8%). In addition, steady-state inactivation was shifted in the negative direction by PKC activation. Neither the activation curve nor the kinetics of fast inactivation was altered by PKC activation. 5. The response to PKA activation was blocked by the PKA inhibitor N-[2-p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulphonamide (H-89; 30 microM) and by 30 microM of PKA inhibitory peptide PKA5-24 (PKAi). PKC activation was blocked by the kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7; 100 microM), by the PKC inhibitor calphostin C (10 microM) and by 20 microM of the inhibitory peptide PKC19-31 (PKCi). 6. The hypoxia-induced inhibition of INa and shift in steady-state inactivation were greatly attenuated with H-7, calphostin C, or PKCi, but not with H-89 or PKAi. 7. We conclude that hypoxia activates PKC in rat CA1 neurones, and that PKC activation leads to the hypoxia-induced inhibition of INa. Topics: Animals; Cell Hypoxia; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hippocampus; Isoquinolines; Leupeptins; Naphthalenes; Nerve Tissue Proteins; Neurons; Oxygen; Patch-Clamp Techniques; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channels; Sulfonamides | 1997 |
Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases.
The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease. Topics: alpha-Macroglobulins; Animals; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Leupeptins; Protease Inhibitors; Time Factors; Tosyl Compounds; Trypanosoma cruzi | 1997 |
Calcium mediated proteolysis enhances calcium release in skinned L6 myotubes.
The mechanism for calcium (Ca2+) release in heart and skeletal muscle during excitation-contraction coupling is currently unknown. A widely held hypothesis is that a small amount of Ca2+ enters the cell and elicits a larger intracellular release of Ca2+ from the sarcoplasmic reticulum (SR), termed "Ca2+-induced Ca2+-release" (CICR). In addition to its role in excitation-contraction coupling, Ca2+ is also known to activate the cysteine protease calpain, which has been recently found to specifically cleave the ryanodine receptor in vitro. The authors investigated the question of whether Ca2+ sensitive protease activation could account for an apparent CICR. The authors first reproduced the phenomenon of CICR using detergent treated L6 myotubes ("skinned cells"). Leupeptin, a cysteine protease inhibitor, reduced the initial velocity and extent of Ca2+ release from the SR; a similar result was obtained when skinned cells were treated with iodoacetate, a sulfhydryl alkylating agent. Dithiothreitol enhanced both the rate and extent of Ca2+ release. Caffeine-induced Ca2+-release was unaffected by the thiol protease inhibitors or activators. This suggests that a cysteine protease may be responsible, in part, for CICR in vitro. The authors also found that vesicles exposed to Ca2+ to induce CICR were unable to fully reaccumulate Ca2+ a second time. Yet, when caffeine released comparable amounts of Ca2+, the initial Ca2+ level was fully restored. Similarly, leupeptin protected the vesicles from the reaccumulation deficit induced by Ca2+. The authors' findings suggest that proteolysis activated by a Ca2+-sensitive protease may account for the direct in vitro demonstration of CICR; such an effect may more likely reflect a role in apoptosis than excitation-contraction coupling. Topics: Animals; Caffeine; Calcium; Cell Line; Clone Cells; Dithiothreitol; Endopeptidases; Kinetics; Leupeptins; Muscle Contraction; Muscle, Skeletal; Protease Inhibitors; Rats; Sarcoplasmic Reticulum; Signal Transduction | 1997 |
[Modulation of T-cell-mediated immune responses by trichosanthin via antigen processing and presentation].
It has been shown in our previous study that the lymphoproliferation to allo- or soluble antigens could be inhibited when T cells were cocultured with antigen presenting cells (APCs) pulsed with Trichosanthin (Tk), a plant protein purified from a Chinese medicinal herb Trichosanthes kirilowii Maximovich. In this paper, data are presented dealing with the mechanism by which the Tk functioned as a down modulator. APCs of human peripheral blood were first treated with one of inhibitors of antigen processing (chloroquine, leupeptin or cycloheximide), followed by pulsed with Tk, and then added into a T cell culture with stimulators PMA and A 23187. The suppression of lymphoproliferation was observed to be obviously diminished or totally disappeared. In contrast, when CyA was used to replace the Tk for pulse-treatment of APCs that had received the same pretreatment with the inhibitors, no significant change was detected for the suppression, suggesting that the Tk might use a different pathway to induce hyporeactivity and that the pathway was concerned in APC's function of antigen presentation. This view obtained support from our immuno-histochemical examination of the Tk-pulsed APCs. By preparing colloidal gold-labelled Tk (Tk-G particle), we were able to show that the Tk-G, when incubated with APCs and T cells at 37 degrees C for two hours and washed, was visualized under the electronic microscope to be bound to APCs', instead of T cells', membrane surface and internalized cells' into endosomes. And then the Tk-G particles were further identified within lysosomes. In this way, the Tk molecules were subjected to be processed as an external antigen and might be presented to T cells to activate certain T subsets that in turn mediated the immunosuppression. This course was capable of, as expected, being interrupted by antigen processing inhibitor, especially by chloroquine. Topics: Antigen Presentation; Antigen-Presenting Cells; Cell Division; Chloroquine; Humans; Leupeptins; T-Lymphocytes; Trichosanthin | 1997 |
The mutation Gly142-->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes.
While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study are involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142e hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL. Topics: Animals; Base Sequence; Cell Compartmentation; Cells, Cultured; Chlorocebus aethiops; DNA Primers; Fluorescent Antibody Technique, Indirect; Heparin; Humans; Immunohistochemistry; Leupeptins; Lipoprotein Lipase; Lysosomes; Microscopy, Fluorescence; Molecular Sequence Data; Mutagenesis, Site-Directed; Protease Inhibitors; Structure-Activity Relationship; Transfection | 1996 |
Intracellular generation of amyloid beta-protein from amyloid beta-protein precursor fragment by direct cleavage with beta- and gamma-secretase.
Two amyloid beta protein precursor (beta APP) fragments involving Met and 103 amino acids of C-terminus of beta APP (delta NOR-beta) and its KM-NL substitution (delta NL-beta) were expressed in COS-7 cells to clarify the proteolytic cleavages to generate amyloid beta protein (A beta). The 4.5-kD protein, A beta with additional N-terminal amino acids, and 4-kD A beta were directly produced and released from 12.5-kD expression proteins without any production of 11.4-kD C-terminal fragment starting at N-terminus of A beta and 3-kD "p3" A beta derivative. Intracellular 4-kD A beta was also detected. The substitution of KM-NL of beta APP found in Swedish familial Alzheimer's disease (AD) promoted the production of intracellular A beta and its release with no increase in level of 11.4-kD C-terminal fragment. These results suggested the presence of a distinct pathway in which A beta is directly cleaved at both N- and C-termini from beta APP fragment intracellularly to release A beta. Since KM-NL substitution enhanced intracellular A beta generation, this pathway may be associated with amyloidogenesis in AD. Topics: Alzheimer Disease; Ammonium Chloride; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Base Sequence; Cell Line; Chlorocebus aethiops; DNA Primers; Endopeptidases; Humans; Kinetics; Leupeptins; Methylamines; Molecular Sequence Data; Peptide Fragments; Polymerase Chain Reaction; Protease Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Transfection | 1996 |
Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases.
Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase. Topics: Amino Acid Sequence; Animals; Aprotinin; Base Sequence; Benzamidines; Cattle; DNA; Electrochemistry; Enzyme Activation; Enzyme Precursors; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Leupeptins; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides; Rats; Serine Endopeptidases; Serine Proteinase Inhibitors; Substrate Specificity; Thermodynamics; Trypsin; Trypsin Inhibitors | 1996 |
Early proteolytic cleavage with loss of a C-terminal fragment underlies altered processing of the beta-galactosidase precursor in galactosialidosis.
Processing of human beta-galactosidase (beta-GAL) was studied in permanently transfected Chinese hamster ovary (CHO) cells and compared with that in normal cells and in cells from subjects with GM1-gangliosidosis, galactosialidosis and I-cell disease. Biosynthesis of beta-GAL in CHO cells results in the synthesis of an 88 kDa glycosylated and phosphorylated monomer precursor which is enzymically active and is secreted into the medium. Post-translational processing begins at the C-terminal end of the protein and gives rise to structurally related 67 and 64 kDa mature forms. These are subsequently degraded to give several inactive products of which a 50 kDa and a 18 kDa species are prominent. In normal fibroblasts only the 84 kDa precursor is readily detected inside cells, while the 88 kDa precursor is the only form secreted from cells in the presence of ammonium chloride. Processing of the precursor in normal fibroblasts results in the appearance of both the 67 and 64 kDa mature forms, which are also degraded to give 50 and 18 kDa products, as in transfected CHO cells. As affected controls, GM1-gangliosidosis cells showed a general loss of all forms of the enzyme, while in I-cell fibroblasts only the 84 kDa precursor and an 18 kDa degradation form were prominent. In galactosialidosis fibroblasts, taken from two different subjects, processing of beta-GAL was characterized by the respective appearance of intermediate 80 and 72 kDa enzymically inactive polypeptides, at levels lower than the normal amounts of the 67 and 64 kDa mature forms and higher than the normal amounts of degradation products, one of which is of 45 kDa and arises by endoproteolytic cleavage of the 80 kDa polypeptide. Incubation for up to 72 h in medium containing leupeptin, a potent inhibitor of thiol-dependent proteases, resulted in a significantly increased level of beta-GAL activity to near normal levels in fibroblasts from one galactosialidosis subject. Concordant with this, the abundance of the 84 kDa precursor was increased and the levels of the 80 kDa, 45 kDa and 18 kDa digestion products were diminished. However, in fibroblasts from the second galactosialidosis subject, the amount of the abnormal 72 kDa polypeptide was not influenced by leupeptin treatment. Leupeptin treatment did not increase enzymic activity levels in normal, GM1-gangliosidosis or I-cell disease fibroblasts, despite the fact that the production of the 50 kDa and 18 kDa degradation products was blocked in the pre Topics: Animals; beta-Galactosidase; Cells, Cultured; CHO Cells; Cricetinae; Endopeptidases; Enzyme Precursors; Fibroblasts; Gangliosidosis, GM1; Humans; Leupeptins; Lysosomal Storage Diseases; Molecular Weight; Protein Processing, Post-Translational | 1996 |
Generation of VV-hemorphin-7 from globin by peritoneal macrophages.
Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D. Analysis of resulting peptides, by reversed-phase high-performance liquid chromatography (RP-HPLC), showed the release of a bioactive peptide, VV-hemorphin-7. When a carboxyl proteinase inhibitor such as pepstatin A was added, no hemorphin was generated. Our results clearly demonstrated that VV-hemorphin-7 generation was principally due to cathepsin D. This study allowed us to hypothesize a possible pathway for in vivo hemorphins appearance from globin catabolism by macrophages. Topics: Amino Acid Sequence; Amino Acids; Animals; Cathepsin D; Cattle; Cells, Cultured; Female; Globins; Hemoglobins; Hydrolysis; Leupeptins; Lysosomes; Macrophages, Peritoneal; Mice; Molecular Sequence Data; Pepstatins; Peptide Fragments; Protease Inhibitors; Specific Pathogen-Free Organisms | 1996 |
Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes.
Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion. Topics: Animals; Apolipoprotein B-100; Apolipoproteins B; Aprotinin; Cells, Cultured; Chlorides; Endoplasmic Reticulum, Rough; Endoplasmic Reticulum, Smooth; Glycoproteins; Golgi Apparatus; Homeostasis; Kinetics; Leupeptins; Liver; Methionine; Oleic Acid; Oleic Acids; Phenanthrolines; Protease Inhibitors; Rabbits; Sulfur Radioisotopes; Zinc Compounds | 1996 |
Secretion of human intestinal angiotensin-converting enzyme and its association with the differentiation state of intestinal cells.
Human angiotensin I-converting enzyme (ACE) exists in intestinal epithelial cells as a membrane-bound (ACEm) and secretory glycoprotein (ACEsec). The electrophoretic mobilities of ACEsec and ACEm on SDS/polyacrylamide gels are similar; the N-deglycosylated ACEsec and ACEm, in contrast, display slight differences in their apparent molecular masses, indicating that the carbohydrate contents of ACEsec and ACEm are different. Moreover, ACEsec is solely N-glycosylated whereas ACEm is N- and O-glycosylated, assessed by lectin binding studies. Spontaneous release of ACEsec is achieved by incubation of brush border membranes at 37 degrees C. Aprotinin, leupeptin and EDTA partly inhibit the generation of ACEsec, strongly suggesting that ACEsec is generated from ACEm by proteolytic cleavage. The expression levels of ACEsec in the intestine may be associated with the differentiation state of mucosal cells. Thus ACEsec is more abundant than ACEm in immature non-epithelial crypt cells of patients with coeliac disease. Well-differentiated epithelial cells, by contrast, contain predominantly ACEm. The variations in the proportions of cleaved ACEsec in differentiated and non-differentiated cells may be due to varying levels of the cleaving protease. Alternatively, because epithelial cell differentiation is accompanied by alterations in the levels of oligosaccharyltransferases, the results suggest a critical role for the glycosylation pattern of ACEm in its susceptibility to the putative cleaving protease. Topics: Aprotinin; Biopsy; Cell Differentiation; Humans; Intestinal Mucosa; Intestine, Small; Isoenzymes; Lectins; Leupeptins; Microvilli; Peptidyl-Dipeptidase A; Protease Inhibitors | 1996 |
Leupeptin causes an accumulation of lipofuscin-like substances and other signs of aging in kidneys of young rats: further evidence for the protease inhibitor model of aging.
The "protease inhibitor model of aging" has been proposed on the basis of observations that young rat brains, livers, and retinas exposed to a protease inhibitor, leupeptin, accumulate lipofuscin-like substances (LLS) that are similar to age pigment, and also display a variety of other manifestations of aging. In order to validate this hypothesis in more general terms, the present study reports attempts to induce age-like changes in kidney cells of young rats. Male F-344 rats (4-5 weeks of age) were continuously infused intraperitoneally (i.p.) with various doses of leupeptin (1-50 mg/100 g/day) for 2 weeks. Control animals received saline solution and normal aged rats (27-30 months-old) received no treatment. Animals were sacrificed and subjected to histological examination. In kidneys of leupeptin-, but not saline-treated rats, generally round-shaped PAS-positive particles were clearly observed, which were predominantly distributed in proximal convoluted tubules, and which resembled particles in normal aged kidneys. With increasing drug doses, particles tended to become bigger and more numerous. The dominant accumulation of LLS in cells of the proximal convoluted tubules had a fine morphologic configuration that resembled age pigments in old rats. Also, there was a concomitant thickening of the basement membrane that was present in leupeptin-treated and aged kidneys, but not in controls. The results, therefore, support the protease inhibitor model of aging and provide an experimental tool for probing the cellular mechanisms of aging. Topics: Aging; Animals; Kidney; Leupeptins; Lipofuscin; Male; Microscopy, Electron; Models, Biological; Protease Inhibitors; Rats; Rats, Inbred F344 | 1996 |
Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system.
The anticryptosporidial potential of the protease inhibitors alpha-1-antitrypsin (AAT), antipain, aprotinin, leupeptin, methoxysuccinyl-ala-ala-pro-valine chloromethylketone (MAAPVCK), soybean trypsin inhibitor (SBTI), and phenylmethylsulfonyl fluoride (PMSF) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system. Protease inhibitor concentrations of 5, 10, 50, 100, and 500 micrograms/ ml (PMSF at 1, 2, and 3 mM) in RPMI medium were mixed with Cryptosporidium parvum oocysts and used to inoculate BFTE cell monolayers. At 24 hr postinoculation (candlejar/37 C), cells were rinsed with RPMI medium, fixed in methanol, and stained with Giemsa. Parasites were enumerated in cell monolayers by brightfield microscopy. The mean number of parasites counted in each protease inhibitor treatment group was expressed as a percentage of the mean number of parasites counted in an infection control group. Leupeptin and SBTI reduced parasite numbers to 40-50% of the control mean at 500 micrograms/ml: AAT, antipain, and aprotinin reduced parasite numbers to 10-15% at the same concentration. PMSF reduced parasite numbers to 40% of the control mean at 3 mM. MAAPVCK did not significantly inhibit cryptosporidial infection. These findings suggest that a protease component of C. parvum may be essential for host cell infection. Topics: alpha 1-Antitrypsin; Amino Acid Chloromethyl Ketones; Animals; Antipain; Aprotinin; Cattle; Cells, Cultured; Cryptosporidium parvum; Epithelial Cells; Epithelium; Fallopian Tubes; Female; Leupeptins; Phenylmethylsulfonyl Fluoride; Serine Proteinase Inhibitors; Trypsin Inhibitors | 1996 |
Proteinases in renal cell death.
The role of proteinases in renal proximal tubule (RPT) cellular death was examined using specific inhibitors of proteinases. Rabbit RPT suspensions were incubated with antimycin A for 1 h or tetrafluoroethyl-L-cysteine (TFEC) for 4 h in the absence or presence of the specific cysteine proteinase inhibitor L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64), the serine proteinase inhibitors N-p-tosyl-L-lysine chloromethyl ketone (TLCK) or 3,4-dichloroisocoumarin (DCS), the serine and cysteine proteinase inhibitors leupeptin or antipain, or the aspartic proteinase inhibitor pepstatin. E-64 and pepstatin decreased lactate dehydrogenase (LDH) release, a marker of cell death, from RPT exposed either to antimycin A or TFEC. TLCK, DCS, leupeptin, or antipain did not decrease antimycin A- or TFEC-induced cell death. Bromohydroquinone- or t-butylhydroperoxide-induced cell death was not decreased by any of the proteinase inhibitors. Loss of lysosomal membrane potential, indicated by neutral red release, occurred prior to the onset of antimycin A-induced cell death. Extensive inhibition of lysosomal cathepsins B and L by E-64 was correlated with cytoprotection. However, E-64 was only protective after some cell death had occurred. These results suggest that lysosomal cysteine and aspartic proteinases, but not serine proteinases, play a role in RPT cell death induced by antimycin A or TFEC. The observation that E-64 was only protective after some cell death had occurred suggests that lysosomal cathepsins are released from dying cells and subsequently attack the remaining viable cells. Topics: Animals; Anti-Bacterial Agents; Antimycin A; Antipain; Carboxypeptidases; Cathepsin A; Cathepsin B; Cell Death; Coumarins; Cysteine; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Endopeptidases; Hydrocarbons, Fluorinated; Hydroquinones; Isocoumarins; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Leucine; Leupeptins; Lysosomes; Membrane Potentials; Pepstatins; Peroxides; Rabbits; Reactive Oxygen Species; Serine Proteinase Inhibitors; tert-Butylhydroperoxide; Tosyllysine Chloromethyl Ketone | 1996 |
Uncleaved env gp160 of human immunodeficiency virus type 1 is degraded within the Golgi apparatus but not lysosomes in COS-1 cells.
The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS-1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half-life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (< 0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS-1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus. Topics: Animals; Anti-Bacterial Agents; Cell Line; Chlorocebus aethiops; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Gene Products, env; Genes, env; Golgi Apparatus; HIV Envelope Protein gp160; HIV-1; Humans; Kinetics; Leupeptins; Lysosomes; Macrolides; Protein Precursors; Recombinant Proteins; Time Factors; Transfection | 1996 |
Selective uptake and degradation of c-Fos and v-Fos by rat liver lysosomes.
The transcription factor c-Fos is a short-lived protein and calpains and ubiquitin-dependent systems have been proposed to be involved in its degradation. In this report, we consider a lysosomal degradation pathway for c-Fos. Using a cell-free assay, we have found that freshly isolated lysosomes can take up and degrade c-Fos with high efficiency. v-Fos, the oncogenic counterpart of c-Fos, can also be taken up by lysosomes, yet the amount of incorporated protein is much lower. c-Fos uptake is independent of its phosphorylation state but it appears to be regulated by dimerization with differentially phosphorylated forms of c-Jun, while v-Fos escapes this regulation. Moreover, we show that c-Fos is immunologically detected in lysosomes isolated from the liver of rats treated with the protease inhibitor leupeptin. Altogether, these results suggest that lysosomes can also participate in the selective degradation of c-Fos in rat liver. Topics: Animals; Cell Line; Cell-Free System; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Humans; Kinetics; Leupeptins; Liver; Lysosomes; Oncogene Proteins v-fos; Phosphorylation; Protease Inhibitors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Recombinant Proteins; Transfection; Trypsin | 1996 |
Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.
A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx. Topics: Amphotericin B; Animals; Brain; Cells, Cultured; Endocytosis; Iodine Radioisotopes; Leupeptins; Neurons; Rats; Rats, Wistar; Scorpion Venoms; Scorpions; Sodium Channels; Time; Veratridine | 1996 |
tBHP induced in vitro swelling of rat liver mitochondria.
Tert-butyl hydroperoxide induced swelling of freshly isolated rat liver mitochondria was inhibited by butylated hydroxytoluene, butylated hydroxyanisole and alpha-tocopherol by acting at the initial phase. EDTA was more effective than EGTA in reducing the initial swelling and so were desferal and bipyrridyl. Spermine, an allosteric activator of calcium uptake, enhanced swelling whereas lanthanum and ruthenium red, the Ca2+ uniport blockers, reduced it. Inhibition of phospholipase A2 by dibucaine and Ca2+ activated proteases by antipain and leupeptin also reduced t-BHP induced swelling. The data indicate that peroxidative mitochondrial swelling involves an iron mediated initial rapid phase and a subsequent calcium dependent propagation phase. Topics: Animals; Antipain; Deferoxamine; Dibucaine; Edetic Acid; Egtazic Acid; Kinetics; Leupeptins; Male; Mitochondria, Liver; Mitochondrial Swelling; Peroxides; Phospholipases A; Phospholipases A2; Protease Inhibitors; Rats; Rats, Wistar; Reactive Oxygen Species; Spermine; tert-Butylhydroperoxide | 1996 |
Trypsin-like protease of Streptomyces exfoliatus SMF13, a potential agent in mycelial differentiation.
Streptomyces exfoliatus SMF13 sequentially produced leupeptin, leupeptin-inactivating enzyme (LIE) and trypsin-like protease (TLP). TLP was produced upon exhaustion of glucose. Autolysis of mycelium was accompanied by an increase in TLP activity. However, in three bld mutants isolated from S. exfoliatus SMF13 after UV-mutagenesis, mycelium autolysis did not occur, and neither LIE nor TLP was produced, although leupeptin was produced. Production of both LIE and TLP was restored in a spontaneous Spo+ revertant of a bld mutant. In contrast, two whi mutants sequentially produced leupeptin, LIE and TLP. The molecular mass of TLP produced during morphological differentiation was estimated to be 31.8 kDa by SDS-PAGE. The N-terminal amino acid sequence was RVGGTxAAQGNFPFQQxLSM. TLP was competitively inhibited by leupeptin; the inhibition constant was 0.015 microM. TLP effectively hydrolysed the mycelial protein extract of S. exfoliatus SMF13, but the hydrolytic activity was inhibited by leupeptin. It was concluded that morphological differentiation and production of TLP are coordinately regulated, that TLP may function as an enzyme in the metabolism of mycelial proteins, and that the hydrolytic activity of TLP is regulated by autogenous leupeptin in S. exfoliatus SMF13. Topics: Amino Acid Sequence; Endopeptidases; Glucose; Kinetics; Leupeptins; Microscopy, Electron, Scanning; Molecular Sequence Data; Mutation; Oligopeptides; Protease Inhibitors; Sequence Homology, Amino Acid; Streptomyces; Substrate Specificity; Trypsin | 1996 |
Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis.
The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface. Topics: Animals; Antipain; Aprotinin; Cell Line; Cell Membrane; Cell Polarity; Cytoplasmic Granules; Dogs; Endopeptidases; Glycosylphosphatidylinositols; GPI-Linked Proteins; Kidney; Leupeptins; Membrane Glycoproteins; Molecular Weight; Pancreas; Peptide Hydrolases; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Type C Phospholipases | 1996 |
Inhibitors of Ca(2+)-dependent endopeptidases modulate morphine-induced effects in rats.
The effects of inhibitors of Ca(2+)-dependent endopeptidases (antipain and leupeptin) on morphine analgesia, reinforcing properties of morphine and on the development of opiate physical dependence were studied. Male Wistar rats were used. The analgesic action of morphine in the tail-immersion test was increased significantly by combined injection of morphine with antipain or leupeptin. Antipain or leupeptin alone had no analgesic action. The combination of morphine with antipain or leupeptin led to the reduction of morphine-induced place preference and the development of physical dependence. A single injection of antipain diminished the opiate-withdrawal signs in morphine-dependent rats. These results suggest a possible inhibitory effect of antipain or leupeptin on the Ca(2+)-dependent endopeptidases of neurons that mediate analgesia, reinforcing properties of morphine, development of opiate dependence and withdrawal. Topics: Animals; Antipain; Drug Synergism; Leupeptins; Male; Morphine; Morphine Dependence; Pain Measurement; Pain Threshold; Protease Inhibitors; Rats; Rats, Wistar; Substance Withdrawal Syndrome | 1996 |
Induction of apoptosis in primary culture of rat hepatocytes by protease inhibitors.
There is growing evidence that suggests the involvement of intracellular proteases in the process of apoptosis or programmed cell death. In this study, we have demonstrated that leupeptin, a cysteine protease inhibitor, can significantly increase the incidence of both apoptotic nuclear morphology change and internucleosomal DNA fragmentation in primary cultured hepatocytes in the absence of known apoptotic stimuli for hepatocytes. On the other hand, aspartic and serine protease inhibitors showed little or no effects on the apoptotic changes. In addition, we found that the apoptotic changes could be induced by chloroquine, an inhibitor of lysosomal proteolysis, but could not be induced by calpain inhibitors. These data suggest that inhibition of lysosomal cysteine proteases may induce apoptosis in primary cultured hepatocytes. Topics: Animals; Apoptosis; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cathepsins; Cell Nucleus; Cells, Cultured; Chloroquine; Cysteine Endopeptidases; Electrophoresis, Agar Gel; Female; Glycoproteins; Leupeptins; Liver; Lysosomes; Microscopy, Fluorescence; Protease Inhibitors; Rats; Rats, Wistar; Transforming Growth Factor beta | 1996 |
ATP is required in platelet serotonin exocytosis for protein phosphorylation and priming of secretory vesicles docked on the plasma membrane.
Calcium-evoked secretion generally requires the presence of millimolar concentrations of Mg-ATP. We investigated the role of Mg-ATP in the secretion of serotonin from electropermeabilized bovine platelets. The secretion of serotonin was lost within 5 minutes when the Mg-ATP concentration was diluted to less than 0.1 mM, but was maintained when ATP-gamma S (adenosine 5'-O-3-thiotriphosphate) was used instead of ATP. Okadaic acid, a potent inhibitor of protein phosphatase, could also maintain the exocytotic activity even when ATP was diluted. Decrease in the secretory activity was paralleled by a decrease in phosphorylation level of four proteins after dilution of ATP, but the activity was maintained when the thiophosphorylation level of these proteins was maintained. Two of these proteins were digested by a protease, calpain, which has been shown to lead to a loss in the exocytotic activity. Electron microscopic studies showed that calcium did not induce the formation of distinct bridge-like structures between the granule membrane and the plasma membrane in Mg-ATP-diluted cells, previously shown as the structure transiently formed prior to fusion of the two membranes. Anchorage of the secretory dense granules to the plasma membrane and the presence of the amorphous structures between the granules and the plasma membrane were unchanged by dilution of ATP. These results indicate that ATP is not required for the anchorage itself, but is required to prime anchored granules for calcium-triggered secretion. Maintenance of the phosphorylated state of proteins by ATP enables the calcium trigger to form the bridge-like structures preceding membrane fusion events. Topics: Adenosine Triphosphate; Animals; Blood Platelets; Calcium; Calpain; Cattle; Cell Membrane; Culture Media; Cytoplasmic Granules; Exocytosis; Leupeptins; Magnesium; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Proteins; Serotonin | 1996 |
Different effects of modulation of mosquito (Diptera:Culicidae) trypsin activity on the infectivity of two human malaria (Hemosporidia:Plasmodidae) parasites.
Trypsin production in the malaria vector Anopheles tessellatus Theobald peaks between 12 and 21 h after a blood meal. The presence of leupeptin or soybean trypsin inhibitor in a blood meal delayed the onset of maximal trypsin activity. Trypsin inhibitors in an infective blood meal increased the infectivity of Plasmodium vivax Grassi and decreased infectivity of P. falciparum Welch to An tessellatus. The opposite effects of trypsin inhibitors on infectivity of the 2 malaria parasites were attributed to differences in the biology of the parasites within the midgut of the vector, particularly the time of ookinete formation and the requirement for activation of a chitinase. Topics: Animals; Anopheles; Chymotrypsin; Humans; Leupeptins; Plasmodium falciparum; Plasmodium vivax; Rabbits; Trypsin; Trypsin Inhibitors | 1996 |
Two crystal structures of the leupeptin-trypsin complex.
Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57. Topics: Binding Sites; Crystallography, X-Ray; Leupeptins; Oxygen; Protein Conformation; Trypsin | 1996 |
Phorbol ester, but not endotoxin, desensitizes mannan-induced glycogenolysis in the perfused rat liver.
Mannan, a ligand for the mannose/N-acetylglucosamine (GlcNAc) receptor, induces suppression of oxygen consumption and increases glucose production in the perfused rat liver, and repeated infusion of mannan causes desensitization of the responses. In this study, we examined whether activation of Kupffer cells by endotoxin and phorbol ester alters the glycogenolytic responses to mannan. Infusion of lipopolysaccharide (LPS, 10 micrograms/ ml) in the perfusate failed to inhibit the responses to mannan. Intravenous administration of LPS (1 mg/kg) 6 and 24 h before perfusion did not desensitize the responses to mannan, suggesting that the responses through mannose/GlcNAc receptors in the liver are retained even after activation of Kupffer cells by LPS. In contrast, prior infusion of phorbol 12-myristate 13-acetate (PMA, 100 nM) in vitro abolished the glycogenolytic responses to subsequently infused mannan, but not that to norepinephrine (100 nM), while prior infusions of 4-alpha-phorbol 12,13-didecanoate (100 nM), A23187 (50 nM), or forskolin (1 microM) had no effect on the mannan-induced responses. H-7, an inhibitor of protein kinase C, reduced the glycogenolytic responses to mannan, while it failed to restore the desensitization. These results suggest that protein kinase C may be involved in the process of glycogenolysis by mannan, but is unlikely to be involved in the homologous desensitization of the responses. Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Aprotinin; Calcimycin; Colforsin; Enzyme Inhibitors; Escherichia coli; Leupeptins; Lipopolysaccharides; Liver; Liver Glycogen; Male; Mannans; Norepinephrine; Perfusion; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate | 1996 |
Absence of lysosomal cleavage in the cytotoxicity mechanism of an immunoconjugate composed of anti-alpha-fetoprotein monoclonal antibody and vindesine analog.
The effect of lysosomal enzyme inhibition on the cytotoxic activity of an immunoconjugate composed of anti-alpha-fetoprotein monoclonal antibody and vindesine analog (VDS) was studied in vitro using human tumor clonogenic assay (HTCA). Addition of the lysosome enzyme inhibitors, leupeptin and ammonium chloride, to the HTCA system had little influence on the cytotoxicity of this immunoconjugate. In separate experiments, no released VDS was detected by HPLC after incubation with the supernatant of rat liver homogenate without inhibitor. These results show that the immunoconjugate may bypass the lysosomal process and exert its activity as an intact or similar form. Topics: alpha-Fetoproteins; Ammonium Chloride; Animals; Antibodies, Monoclonal; Antineoplastic Agents, Phytogenic; Cell Survival; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Endocytosis; Humans; Immunotoxins; Leupeptins; Liver; Lysosomes; Male; Rats; Rats, Wistar; Tumor Cells, Cultured; Vindesine | 1996 |
The prosequence of procaricain forms an alpha-helical domain that prevents access to the substrate-binding cleft.
Cysteine proteases are involved in a variety of cellular processes including cartilage degradation in arthritis, the progression of Alzheimer's disease and cancer invasion: these enzymes are therefore of immense biological importance. Caricain is the most basic of the cysteine proteases found in the latex of Carica papaya. It is a member of the papain superfamily and is homologous to other plant and animal cysteine proteases. Caricain is naturally expressed as an inactive zymogen called procaricain. The inactive form of the protease contains an inhibitory proregion which consists of an additional 106 N-terminal amino acids; the proregion is removed upon activation.. The crystal structure of procaricain has been refined to 3.2 A resolution; the final model consists of three non-crystallographically related molecules. The proregion of caricain forms a separate globular domain which binds to the C-terminal domain of mature caricain. The proregion also contains an extended polypeptide chain which runs through the substrate-binding cleft, in the opposite direction to that of the substrate, and connects to the N terminus of the mature region. The mature region does not undergo any conformational change on activation.. We conclude that the rate-limiting step in the in vitro activation of procaricain is the dissociation of the prodomain, which is then followed by proteolytic cleavage of the extended polypeptide chain of the proregion. The prodomain provides a stable scaffold which may facilitate the folding of the C-terminal lobe of procaricain. Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Cathepsin B; Computer Simulation; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Precursors; Fruit; Hydrogen Bonding; Leucine; Leupeptins; Models, Molecular; Molecular Sequence Data; Mutation; Oligopeptides; Papain; Plant Proteins; Protein Processing, Post-Translational; Protein Structure, Secondary; Sequence Homology, Amino Acid | 1996 |
HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes.
HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions. Topics: Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Cell Compartmentation; Histocompatibility Antigens Class II; HLA-D Antigens; HLA-DR Antigens; Humans; Leupeptins; Macromolecular Substances; Peptide Fragments; Peptides; Precipitin Tests; Protein Binding | 1996 |
Degradation of lactoferrin by periodontitis-associated bacteria.
The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process. Topics: Aggregatibacter actinomycetemcomitans; Bacteria; Bacteroides; Campylobacter; Capnocytophaga; Enzyme Inhibitors; Fusobacterium nucleatum; Humans; Iodoacetamide; Lactoferrin; Leupeptins; Peptostreptococcus; Periodontitis; Phenylmethylsulfonyl Fluoride; Porphyromonas gingivalis; Prevotella intermedia; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 1996 |
Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport.
P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport. Topics: Affinity Labels; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Biological Transport; CHO Cells; Colchicine; Cricetinae; Dihydropyridines; Drug Resistance, Multiple; Kinetics; Leupeptins; Oligopeptides; Substrate Specificity; Vinblastine | 1996 |
Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood.
Enzyme inhibitors have been compared with conventional anticoagulants for the flow cytometric analysis of live leucocytes in whole blood. At 18 to 20 degrees C in vitro, PPACK (60 microM), hirudin (10 units ml-1), leupeptin (20 microg ml-1) and aprotinin (50 microg ml-1) inhibited blood clotting for 4 h or more, whereas PMSF (8 mg ml-1) or AEBSF (5 mg ml-1) inhibited clotting for only 20 or 40 min, respectively. When labelled with CD11b antibodies and analysed immediately ex vivo at 4 degrees C, the percentages of lymphocytes, monocytes, and polymorphs which stained positively and their mean fluorescence intensities were similar, irrespective into which anticoagulant blood was collected. Less than 1.5-fold increases in expression occurred on monocytes and polymorphs when blood anticoagulated with enzyme inhibitors or conventional anticoagulants was kept at 4 degrees C, or when blood anticoagulated with citrate, heparin, or hirudin was kept at 18 to 20 degrees C for 1 h before labelling and analysis, whereas approximately 2-fold increases in expression occurred in blood kept with K3EDTA, leupeptin, or aprotinin and more than 3-fold increases in blood kept with AEBSF or PPACK at 18 to 20 degrees C for 1 h. Further studies showed that leupeptin could be used effectively as the anticoagulant when investigating functional responses of live leucocytes in whole blood samples by flow cytometry and for the isolation of leucocytes with minimal modulation of adhesion molecules. Topics: Adult; Amino Acid Chloromethyl Ketones; Anticoagulants; Aprotinin; Blood; Blood Cell Count; Citrates; Flow Cytometry; Hirudins; Humans; L-Selectin; Leukocytes; Leukocytes, Mononuclear; Leupeptins; Macrophage-1 Antigen; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phenylmethylsulfonyl Fluoride; Sulfones | 1996 |
Cathepsin B in the growth of colorectal cancer: suppressive effect of leupeptin on the growth of DMH-induced rat colon neoplasm.
Cathepsin B, a thiol protease, has been reported to be involved in cancer progression and metastasis. The suppressive effects of two kinds of protease inhibitors, leupeptin and dietary camostate (FOY-305), on tumorigenesis and progression in 1, 2-dimethylhydrazine (DMH)-induced rat colon neoplasm were examined in relation to tissue cathepsin B activity. Male Donryu rats were treated with leupeptin or FOY-305 during or after the administration of DMH. There were no significant differences in average tumor numbers among all DMH-treated groups. However, the percentage of small tumors was significantly higher in the group in which leupeptin was supplied during DMH administration. This trend was not recognized in the FOY-305-treated groups. The ratio of cathepsin B activity in the tumors to that in the tumor-bearing tissue (T/Tb) was significantly increased with increasing tumor size (P = 0.009). The cathepsin B activity levels in the tumor-bearing mucosa in the groups which received leupeptin or FOY-305 following DMH treatment were both significantly lower than that in the group which received neither protease inhibitor (P = 0.046 and P = 0.0067, respectively). The results obtained indicate that leupeptin may have suppressed tumor growth by lowering the tissue cathepsin B activity. Topics: 1,2-Dimethylhydrazine; Animals; Carcinogens; Cathepsin B; Colonic Neoplasms; Dimethylhydrazines; Esters; Gabexate; Guanidines; Leupeptins; Male; Protease Inhibitors; Rats | 1996 |
Distinct antigen MHC class II complexes generated by separate processing pathways.
The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (Ak). These complexes were also detected on the surface of antigen-presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS-stable, long-lived Ak complexes containing *HEL peptides and, unexpectedly, full-length *HEL. Both SDS-stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA-DM or proteases. SDS-stable *HEL-Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS-unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full-length protein as a product of antigen processing. Topics: Cell Line; Chloroquine; Histocompatibility Antigens Class II; Kinetics; Leupeptins; Protein Binding; Protein Folding; Protein Processing, Post-Translational; Sodium Dodecyl Sulfate | 1996 |
Granulocytic protein p25 is a DNA-binding subunit of protein M(r) = 50,000: subcellular localization, cell and species specificity.
We have previously reported the presence and isolation of the novel protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined. Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigen-Antibody Reactions; Chromobox Protein Homolog 5; DNA-Binding Proteins; Granulocytes; Humans; In Vitro Techniques; Leupeptins; Molecular Sequence Data; Pepstatins; Protease Inhibitors; Reference Values; Species Specificity; Subcellular Fractions | 1996 |
Evidence for the presence of an inactive precursor of human hepatocyte growth factor in plasma and sera of patients with liver diseases.
Human hepatocyte growth factor (hHGF), which is now known to be the same protein as the scatter factor and the tumor cytotoxic factor, is a heterodimeric protein with one heavy chain and one light chain linked together by a disulfide bond, and is thought to be involved in liver regeneration. Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we found that a significant amount of single chain precursor of hHGF (pro-hHGF) was present in plasma of patients with fulminant hepatic failure (FHF) and that normal human serum contained a protease or proteases that convert pro-HGF to a heterodimeric (mature) form of hHGF. We also showed that the processing protease activity for hHGF was suppressed by such serine protease inhibitors as leupeptin, antipain, and aprotinin, and that sera of patients with liver diseases such as fulminant hepatic failure, acute hepatitis, chronic hepatitis, and cirrhosis contained not only pro-hHGF but also the protease. This is the first report showing the presence of pro-hHGF in human blood, and our observations suggest that hHGF is synthesized and secreted from the hHGF-producing cells as an inactive pro-hHGF after hepatic injuries, and the pro-hHGF is then converted to an active heterodimeric form of hHGF in the blood. It is also suggested that plasma of patients with liver diseases contains an active protease or proteases that convert pro-hHGF to a mature form of hHGF. Topics: Animals; Antipain; Aprotinin; Blotting, Western; CHO Cells; Cricetinae; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Hepatocyte Growth Factor; Humans; Leupeptins; Liver Failure, Acute; Macromolecular Substances; Protease Inhibitors; Protein Precursors; Recombinant Proteins; Transfection | 1995 |
Degradation of cystathionine gamma-lyase in rat liver lysosomes: effect of leupeptin treatment.
Highly purified lysosomes from the livers of normal and leupeptin-treated rats were subjected to immunoblot analysis, using antibodies against rat liver cystathionine gamma-lyase. Normal lysosomes showed small amount of single band at about 40 kDa, whereas the leupeptin-treated lysosomes showed large amounts of a major band at about 40 kDa and a minor band at about 35 kDa. These observations suggest that cystathionine gamma-lyase is sequestered into lysosomes at the original subunit molecular weight and is degraded in the lysosome by way of the sequential formation of an intermediate with distinct molecular weight. Topics: Animals; Cystathionine gamma-Lyase; Immunoblotting; Leupeptins; Liver; Lysosomes; Molecular Weight; Rats; Reference Values | 1995 |
Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions.
The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred. In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagosomes) and suggesting that early AVs (autophagosomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-n Topics: Animals; Antigens, CD; Asparagine; Autophagy; Biomarkers; Carbonic Anhydrases; Cattle; Endocytosis; Gold Colloid; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 1; Lysosomes; Male; Membrane Glycoproteins; Microtubules; Protease Inhibitors; Rats; Rats, Wistar; Serum Albumin, Bovine; Superoxide Dismutase; Vacuoles; Vinblastine | 1995 |
How MHC class II molecules reach the endocytic pathway.
We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)-ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide-loaded, SDS-stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B-treated cells, a slow increase (> 20-fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface-disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor-treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans-Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway. Topics: Anti-Bacterial Agents; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Biological Transport; Cell Compartmentation; Cell Membrane; Cells, Cultured; Cold Temperature; Endocytosis; Endopeptidases; Epitopes; Fluorescent Antibody Technique; Histocompatibility Antigens Class II; Hot Temperature; Humans; Leupeptins; Macrolides; Microscopy, Confocal; Microscopy, Immunoelectron; Models, Immunological; Neuraminidase; Sodium Dodecyl Sulfate | 1995 |
Unique specificity of in vitro inhibition of mosquito midgut trypsin-like activity correlates with in vivo inhibition of malaria parasite infectivity.
Synchrony in the egress of Plasmodium ookinetes from the food bolus and enzymatic digestion of the blood meal in the mosquito midgut suggests that digestive enzymes play a role in the successful transmission of malaria parasites. Previously, we found that parasite-produced chitinase is essential for parasite transmission and can be activated by mosquito midgut protease. To determine the suitability of developing a transmission-blocking vaccine directed against mosquito trypsin-like enzyme(s), Aedes aegypti midgut trypsin-like proteases were characterized biochemically and compared to a mammalian trypsin. Mosquito trypsin is more sensitive to inhibition by aprotinin and less sensitive to egg white trypsin inhibitor than is bovine pancreatic trypsin. Soybean trypsin inhibitor and leupeptin inhibit both enzymes to similar extent. Membrane-feeding assays with aprotinin, leupeptin, and egg white trypsin inhibitor revealed a correlation between in vitro inhibition of mosquito trypsin-like activity and transmission-blocking activity. The results suggest a role for mosquito midgut trypsin(s) in malaria parasite development and indicate that the protease(s) is a potential target for blocking malaria transmission. Topics: Aedes; Animals; Aprotinin; Chickens; Female; Insect Vectors; Leupeptins; Plasmodium gallinaceum; Trypsin; Trypsin Inhibitors | 1995 |
Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)-phthalate treatment. III. Fusion of early autophagosomes with lysosomal compartments.
Fusion of early autophagosomes containing peroxisomes with endosomal and lysosomal structures in rat liver cells was investigated. Male Wistar rats were administered di-(2-ethylhexyl)phthalate (DEHP) for 14 days to induce proliferation of peroxisomes, and then the animals were injected intravenously with horseradish peroxidase (HRP)-conjugated asialofetuin to label the lysosomal compartment. Either 30 min or 60 min after the injection, the animals were treated with leupeptin for 20, 40 and 60 min, respectively. Most of autophagic vacuoles containing peroxisomes were stained with diaminobenzidine (DAB) endocytosed HRP-asialofetuin 60 min after leupeptin injection, whereas many of them were negative for DAB reaction 20 min after leupeptin injection. Between 20 to 40 min after leupeptin treatment many autophagic vacuoles fused with DAB-positive lysosomal compartments, including late endosomes. Percoll gradient centrifugation showed that particles containing HRP activity migrated from a density of 1.09 g/ml to that of 1.14 g/ml as the time after leupeptin injection passed. Acid phosphatase activity migrated in the same manner. These results clearly show that early autophagosomes obtain the lysosomal proteinases by fusion with lysosomal compartments, including the late endosomes and that peroxisomes trapped in autophagosomes are degraded by these proteinases. Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Autophagy; Cell Compartmentation; Centrifugation, Density Gradient; Diethylhexyl Phthalate; Fetuins; Horseradish Peroxidase; Leupeptins; Lysosomes; Male; Microbodies; Rats; Rats, Wistar | 1995 |
Antigen-presenting cell lines internalize peptide antigens via fluid-phase endocytosis.
In this report, we present experimental evidence that antigen-presenting cell lines take up peptide antigens in a manner consistent with fluid-phase endocytosis. Using the fluid phase endocytic marker inulin and a mathematical model for fluid phase uptake, we have found a basal uptake rate constant of approximately 0.9-2 microns 3/cell minutes in A20, TA3, and J774 cells. An influenza virus peptide, PB2(303-313), the octapeptide, angiotensin II, an ovalbumin peptide, OVA(323-339), and a guinea pig myelin basic protein peptide, MBP(72-86), have uptake rate constants comparable to inulin, i.e., between 1 and 4 microns3/cell minutes in A20 cells. However, another influenza virus peptide, PB2(146-159), has an uptake rate constant approximately sixfold higher than that found for inulin in A20 cells. We have also determined that the peptide antigens we tested are retained in A20 cells similarly to inulin, with half-times calculated to be from 2 to 13 min as compared to 2 min for inulin. Notably, these results were obtained over short incubation times (up to 20 min) and under conditions that restrict peptide proteolysis and also protein synthesis. We conclude from these studies that peptide antigens enter antigen-presenting cells via fluid-phase endocytosis. Topics: Animals; Antigen-Presenting Cells; Antigens; Aprotinin; Cell Line; Chromatography, Gel; Leupeptins; Mice; Peptides; Pinocytosis | 1995 |
Heparin-binding vitronectin up-regulates latent TGF-beta production by bovine aortic endothelial cells.
Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-beta, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-beta. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-beta by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-beta activity observed when vitronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent TGF-beta with vitronectin in a cell-free system does not result in increased TGF-beta activity. Assays of total TGF-beta levels in heat-treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-beta in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with antibodies specific for latent TGF-beta. These data suggest that vitronectin regulates expression and/or secretion of TGF-beta by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by alpha v beta 3 integrins.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Antibodies; Aorta; Aprotinin; Blotting, Western; Cattle; Cell Line; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Endothelium, Vascular; Extracellular Matrix Proteins; Glycoproteins; Heparin; Kinetics; Leupeptins; Membrane Glycoproteins; Thrombospondins; Transforming Growth Factor beta; Vitronectin | 1995 |
A labile hyperphosphorylated c-Fos protein is induced in mouse fibroblast cells treated with a combination of phorbol ester and anti-tumor promoter curcumin.
Curcumin is a potent inhibitor of tumor promotion, and was shown previously to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity. The c-Fos protein is inducible by TPA and thus is associated with c-Jun to result in an increased AP-1 activity in mouse fibroblast cells. We therefore hypothesized that c-Fos may be one of the targets of curcumin action. In the present study, the effects of curcumin on TPA-induced c-fos mRNA and protein levels were determined by RNA hybridization and western blot analysis, respectively. Curcumin decreases the TPA-induced nuclear abundance of c-Fos protein in spite of the slight super-induction of c-fos mRNA. Upon TPA stimulation, the amount of c-Fos in the quiescent cells increases and reaches maximum at 30 min, and then progressively disappears over a period of 60 min. However, the c-Fos protein seems susceptible to rapid degradation by 45 min if NIH 3T3 cells were treated with TPA in the presence of curcumin. The curcumin-induced hyperphosphorylated forms of c-Fos proteins are significantly more unstable; they entirely disappeared within 40 min after incubation at 37 degrees C. These findings prompted us to suggest that the decrease of c-Fos protein could account for the repressed in vitro DNA binding probably by reducing the Jun/Fos complex formation. Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antineoplastic Agents; Base Sequence; Carcinogens; Curcumin; Gene Expression; Genes, fos; Genes, jun; In Vitro Techniques; Leupeptins; Mice; Molecular Sequence Data; Phosphoproteins; Phosphorylation; Protease Inhibitors; Proto-Oncogene Proteins c-fos; RNA, Messenger; Tetradecanoylphorbol Acetate | 1995 |
The intracellular transport of MHC class II molecules in the absence of HLA-DM.
The HLA-DM alpha and HLA-DM beta genes encode a nonpolymorphic, class II-like molecule that functions by an as yet undefined mechanism in the assembly of processed antigen-HLA class II complexes. Mutant cells that fail to express HLA-DM are deficient in Ag processing. We previously isolated a subcellular compartment in mouse B cells in which functional processed Ag-class II complexes are first formed, referred to as the peptide-loading compartment. Here, evidence is provided that HLA-DM resides in a subcellular compartment with the characteristics of a peptide-loading compartment in a human B lymphoblastoid cell line, but is not required for the intracellular transport of HLA-DR3 molecules to a corresponding compartment in HLA-DM-deficient cells. Thus, the primary defect in HLA-DM-deficient cells does not appear to be a failure in the intracellular trafficking of class II molecules. Topics: Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Biological Transport; Cell Line; Histocompatibility Antigens Class II; HLA-D Antigens; HLA-DR3 Antigen; Humans; Leupeptins; Lymphocyte Activation; Protease Inhibitors; Subcellular Fractions | 1995 |
Inhibitory effect of leupeptin on the intracellular maturation of lysosomal cathepsin L in primary cultures of rat hepatocytes.
To investigate the intracellular processing event for lysosomal cathespin L, we examined the effect of leupeptin, a non-covalent cysteine proteinase inhibitor, on the intracellular processing kinetics of cathepsin L as analyzed by pulse-chase experiments in vivo with [35S]methionine in primary cultures of rat hepatocytes. This revealed that cathepsin L was initially synthesized as proenzyme of molecular weight 39 kDa and the proenzyme was subsequently processed to the mature form of the enzyme, 30 and 25 kDa. In the leupeptin-treated cells, the proteolytic conversion of cellular procathepsin L, of molecular weight 39 kDa, to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 8 h chase periods. Furthermore, the subcellular fractionation experiment demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the dense lysosomal fraction after a 2 h chase period. These results suggest that leupeptin treatment caused significant inhibition of the intracellular maturation of cathepsin L. These findings show that cysteine proteinase plays an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo and that this processing event occurs within the lysosomes. Topics: Animals; Cathepsin L; Cathepsins; Cells, Cultured; Cysteine Endopeptidases; Endopeptidases; Kinetics; Leupeptins; Liver; Lysosomes; Male; Molecular Weight; Rats; Rats, Wistar; Serine Proteinase Inhibitors; Subcellular Fractions; Sulfur Radioisotopes | 1995 |
T cell recognition of major histocompatibility complex class II complexes with invariant chain processing intermediates.
Peptides from the lumenal portion of invariant chain (Ii) spanning residues 80-106 (class II-associated Ii peptide [CLIP]) are found in association with several mouse and human major histocompatibility complex (MHC) class II allelic variants in wild-type and presentation-deficient mutant cells. The ready detection of these complexes suggests that such an intermediate is essential to the MHC class II processing pathway. In this study, we demonstrate that T cells recognize CLIP/MHC class II complexes on the surface of normal and mutant cells in a manner indistinguishable from that of nominal antigenic peptides. Surprisingly, T cell hybrids specific for human CLIP bound to murine MHC class II molecule I-Ab and a new monoclonal antibody 30-2 with the same specificity, recognize two independent epitopes expressed on this peptide/class II complex. T cell recognition is dependent on a Gln residue (position 100) in CLIP, whereas the 30-2 antibody recognizes a Lys residue-at position 90. These two residues flank the 91-99 sequence that is conserved among human, mouse, and rat Ii, potentially representing an MHC class II-binding site. Our results suggest that the COOH-terminal portion of CLIP that includes TCR contact residue Gln 100 binds in the groove of I-Ab molecule. Moreover, both T cells and the antibody recognize I-Ab complexed with larger Ii processing intermediates such as the approximately 12-kD small leupeptin-induced protein (SLIP) fragments. Thus, SLIP fragments contain a CLIP region bound to MHC class II molecule in a conformation identical to that of a free CLIP peptide. Finally, our data suggest that SLIP/MHC class II complexes are precursors of CLIP/MHC class II complexes. Topics: Alleles; Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Binding Sites; Epitopes; Glycine; Histocompatibility Antigens Class II; Humans; Hybrid Cells; Leupeptins; Lysine; Macromolecular Substances; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein Conformation; Rats; Receptors, Antigen, T-Cell; Sequence Alignment; Structure-Activity Relationship; T-Lymphocytes; Transfection | 1995 |
Protection by lysosomal hydrolase inhibitors against cytotoxicity of 2-chloroethylethyl sulfide.
A possible participation of lysosomal hydrolases in the cytotoxicity of 2-chloroethylethyl sulfide in spleen lymphocytes was investigated using inhibitors of lysosomal phospholipases and proteases. Pepstatin (6 microM) and leupeptin (60 microM), inhibitors of lysosomal proteases, raised the viability of lymphocytes exposed to 2-chloroethylethyl sulfide from 63 to 87 and 88% of control, respectively. Serine protease inhibitors showed no significant effect on viability. Aminoglycoside inhibitors of lysosomal phospholipases were also found to prevent the decrease in viability of spleen lymphocytes exposed to 2-chloroethylethyl sulfide, and the effectiveness of these aminoglycosides (30 microM) was as follows: gentamicin > kanamycin > streptomycin, with viability increased to 89, 79 and 67%, respectively. In contrast to a co-operative action between leupeptin and gentamicin, the protection by pepstatin was reduced in the presence of gentamicin. Moreover, the order of the aminoglycosides in terms of the extent to which they antagonized the protective action of pepstatin was the same as their order of efficacy in preventing the cytotoxicity of CEES. It is suggested that inhibitors of lysosomal hydrolases reduce the cytotoxicity of 2-chloroethylethyl sulfide, presumably through lysosomal stabilization in spleen lymphocytes. Topics: Alkylation; Animals; Cell Survival; Cells, Cultured; Drug Interactions; Gentamicins; Kanamycin; Leupeptins; Lysosomes; Mice; Mice, Inbred ICR; Mustard Gas; Oligopeptides; Pepstatins; Phospholipases; Protease Inhibitors; Spleen; Streptomycin; Structure-Activity Relationship; T-Lymphocytes | 1995 |
Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells.
Mice deficient for the major lysosomal aspartic proteinase cathepsin D, generated by gene targeting, develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26 +/- 1. An atrophy of the ileal mucosa first observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. Thymus and spleen undergo massive destruction with fulminant loss of T and B cells. Lysosomal bulk proteolysis is maintained. These results suggest, that vital functions of cathepsin D are exerted by limited proteolysis of proteins regulating cell growth and/or tissue homeostasis, while its contribution to bulk proteolysis in lysosomes appears to be non-critical. Topics: Animals; Atrophy; B-Lymphocytes; Cathepsin D; Chimera; Fibroblasts; Gene Targeting; Ileum; Intestinal Mucosa; Leupeptins; Lysosomes; Male; Mice; Mice, Inbred C57BL; Pepstatins; RNA, Messenger; Specific Pathogen-Free Organisms; Spleen; T-Lymphocytes; Thymus Gland | 1995 |
Kinetic study on the production and degradation of leupeptin in Streptomyces exfoliatus SMF13.
Medium composition and cultivation conditions were constructed for the optimum production of leupeptin by Streptomyces exfoliatus SMF13. The production of leupeptin was related to mycelial growth, being optimum in the cultivation with glucose-excess, phosphate-limited, and casamino acids medium. However, leupeptin-inactivating enzyme (LIE) was produced in the cultivation with glucose-limited, phosphate-excess, and Na-caseinate medium where mycelium degradation was accompanied. LIE was one of the most important factors in optimizing the leupeptin productivity. Kinetic parameters calculated from batch and chemostat cultivations revealed that qlpt was closely related to qs and mu, but qLIE was increased after mu declined to near zero, and followed by kd. Optimum production process of leupeptin was determined with phosphate-limited continuous cultivation, which did not permit LIE production. The maximum productivity (0.24 g l-1 h-1) and production yield (1.64 g leupeptin per g glucose) of phosphate-limited chemostat cultivation were 2.4- and 4-times larger than those of batch cultivation, respectively. This is the first cultivation kinetic analysis for leupeptin production and its inactivation by LIE in relation to mycelium differentiation. Topics: Cell Division; Culture Media; Glucose; Kinetics; Leupeptins; Phosphates; Serine Endopeptidases; Streptomyces | 1995 |
Excystment of Paragonimus westermani metacercariae by endogenous cysteine protease.
To infect definitive or paratenic hosts, metacercariae of Paragonimus westermani should excyst in the host intestine. Optimum conditions for the excystment have been known to be pH 8-9 and a temperature of 40 C. Under these conditions, excystment of P. westermani metacercariae was accelerated in the presence of 1 mM dithiothreitol (DTT). The DTT acceleration was antagonized dose-dependently by cysteine protease inhibitors of L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64, 2-20 microM) or leupeptin (0.1-1 mM), suggesting that certain cysteine proteases of the metacercaria are involved in excystment. Protease activities were detected in excretory-secretory products (ESP) of newly excysted metacercariae. Two distinct proteases were purified by DEAE anion-exchange chromatography of the ESP. While a 27-kDa protease exhibited endodipeptidolytic activity at pH 5-8.5 and remained stable at neutral pH for 3 days, the 28-kDa enzyme was stable at pH 5-7.5, with lower activity at pH 8.5. Both proteases hydrolyzed collagen, fibronectin, and myosin within 1 hr at pH 8. These results suggest that cysteine proteases secreted by P. westermani metacercariae modulate excystment. Topics: Animals; Chromatography, Ion Exchange; Collagen; Coumarins; Cysteine Endopeptidases; Dipeptides; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Fibronectins; Hydrogen-Ion Concentration; Iodoacetamide; Leucine; Leupeptins; Myosins; Oligopeptides; Pancreatic Elastase; Paragonimus; Temperature; Trypsin | 1995 |
Cytoskeletal changes in rat cortical neurons induced by long-term intraventricular infusion of leupeptin.
Neurofibrillary tangles (NFTs), which are composed of paired helical filament (PHF)-like filaments, were induced by the long-term intraventricular infusion of leupeptin, a potent protease inhibitor. The fibrils composing the NFTs were 20 nm in maximal width and had periodic constrictions at 40-nm intervals. They were identical to the PHF that had been found in aged rat neurons. Dystrophic axons filled with mainly tubular structures were also abundantly found in the parietal and temporal isocortices, which were not affected in the acute or subacute phases of leupeptin treatment. An immunohistochemical study using antibodies related to the neuronal cytoskeleton showed that neuronal cytoskeletal changes accompanying ubiquitination occurred in dystrophic axons distributed widely in the isocortex as well as the hippocampal formation. The present findings suggest that long-term administration of leupeptin accelerates the neuronal ageing process in rats and causes other neuronal changes: NFT formation, such as seen in the aged brain or in neurodegenerative diseases including Alzheimer's disease, in addition to accumulation of lipofuscin granules and degeneration of neuronal processes. In other words, some disturbance of the balance between proteases and their inhibitors may play an important role in the neuronal ageing process, and some regulatory intervention in the intraneuronal protease activity may provide a new therapeutic strategy for the neurodegenerative diseases. Topics: Alzheimer Disease; Animals; Cerebral Cortex; Disease Models, Animal; Injections, Intraventricular; Leupeptins; Male; Models, Neurological; Nerve Degeneration; Neurofibrillary Tangles; Parietal Lobe; Rats; Rats, Wistar | 1995 |
Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles.
B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers. Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Biological Transport; Endosomes; Histocompatibility Antigens Class II; Leupeptins; Mice; Protein Conformation; Sodium Dodecyl Sulfate | 1995 |
A protease-dependent TCR-induced death pathway in mature lymphocytes.
Several cysteine and serine protease inhibitors previously shown to block TCR-induced death of 2B4 T hybridoma cells were tested for their ability to block various T lymphocyte apoptotic death systems. TCR-induced death of both peripheral CD4+ and CD8+ T cell blasts was inhibited similarly to the hybridoma, but cell death in these cells induced by anti-Fas, gamma-irradiation, etoposide, or extracellular ATP was not blocked. For T cell lines, cell death induced by CTL or by IL-2 withdrawal was also not inhibited. TCR-induced death of immature CD+8+ thymocytes triggered by culture on immobilized anti-CD3 was not blocked by these protease inhibitors, whereas similar death induced in the resting CD4+8- thymocyte subset under these conditions was inhibited similarly to the T cell blasts. TCR-induced proliferation of the latter subset was modest in the absence of exogeneous IL-2, but was enhanced two- to fourfold by the protease inhibitors. These results show that a protease-dependent death pathway can be triggered by the TCR in mature T cells; similar protease-dependent steps are not common to the TCR-triggered activation pathway or other apoptotic death pathways in these cells. Topics: Animals; Antibodies, Monoclonal; Apoptosis; Calpain; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line; Cytotoxicity, Immunologic; Endopeptidases; Leupeptins; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Protease Inhibitors; Receptors, Antigen, T-Cell; T-Lymphocytes; Thymus Gland | 1995 |
Thiol-protease inhibitors selectively inhibit presentation of antigen incorporated through B cell antigen receptors.
B cells efficiently uptake exogenous antigens through cell surface IgM molecules and present them to specific T cells. Although it is implicated that antigens taken up by surface IgM associate with newly synthesized class II molecules, the precise pathway of the presentation is still unclear. Here we report that thiol-protease inhibitors inhibit the presentation of trinitrophenol-conjugated ovalbumin by a leukemic B cell line, A20.HL, which constitutively expresses trinitrophenol-specific surface IgM although they had only a minimum effect on the presentation of unconjugated ovalbumin. In addition, thiol-protease inhibitors slightly reduced the surface expression of class II molecules. Our results suggest that exogenous antigen taken up through surface IgM is presented by a pathway which involves proteolysis by thiol-proteases and distinct from that for the antigens taken up through non-specific endocytosis. Topics: Antigen Presentation; Antigens; Antipain; Cysteine Proteinase Inhibitors; Immunoglobulin M; Leupeptins; Major Histocompatibility Complex; Pinocytosis; Receptors, Antigen, B-Cell; Tumor Cells, Cultured | 1995 |
Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition.
Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. CANP is thought to be activated by the calcium overload that develops during ischemia. We studied the involvement of CANP in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol/L NaCN + 10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion exchange and affinity chromatography. Then the efficacy of the CANP inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity was tested with the use of fluorescently labeled beta-casein as a substrate. The IC50 for the inhibitors was between 2.1 and 56 mumol/L. Uptake of the inhibitors by intact cells was assessed with the use of 99mTc-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic CANP-specific substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin. During metabolic inhibition, intracellular CANP activity was increased compared with control incubation. The time course of CANP activation was compatible with that of the rise in [Ca2+]i, as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibitor and leupeptin inhibited intracellular CANP activity both during metabolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Animals, Newborn; Calcium; Calpain; Cell Death; Cells, Cultured; Chromatography, Affinity; Chromatography, Ion Exchange; Cysteine Proteinase Inhibitors; Enzyme Activation; Leucine; Leupeptins; Myocardium; Protease Inhibitors; Rats; Rats, Wistar | 1995 |
Physiological roles of leupeptin and extracellular proteases in mycelium development of Streptomyces exfoliatus SMF13.
Streptomyces exfoliatus SMF13 produced leupeptin, chymotrypsin-like protease (CTP), metalloprotease, and trypsin-like protease (TLP) extracellularly. The activity of TLP was specifically inhibited by leupeptin. Production of leupeptin was closely associated with growth but leupeptin was inactivated by leupeptin-inactivating protein (LIP) when growth reached the stationary phase in submerged cultures, or when aerial mycelia started to form on surface cultures. Autolysis of mycelia after the stationary phase in submerged cultures was apparently retarded by the addition of leupeptin; on surface cultures, aerial mycelium formation was clearly retarded by the addition of leupeptin. We propose that CTP participates primarily in utilization of a proteinaceous nitrogen source, that TLP functions as an essential enzyme involved in the metabolism of mycelial protein, that leupeptin inhibits the activity of TLP and that LIP inactivates leupeptin. The cascade of regulatory actions of the compounds, which are produced sequentially during mycelium development, may provide selective advantages in adverse culture conditions. Topics: Amino Acid Sequence; Endopeptidases; Extracellular Space; Glucose; Leupeptins; Metalloendopeptidases; Molecular Sequence Data; Oligopeptides; Protease Inhibitors; Serine Endopeptidases; Streptomyces; Substrate Specificity; Trypsin | 1995 |
Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases.
Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA. Topics: ADP Ribose Transferases; Amino Acid Sequence; Animals; Antigens, Bacterial; Bacterial Toxins; Cathepsin B; CHO Cells; Cricetinae; Diphtheria Toxin; Dose-Response Relationship, Drug; Endopeptidases; Exotoxins; Furin; Leupeptins; Molecular Sequence Data; Peptide Fragments; Pseudomonas aeruginosa Exotoxin A; Substrate Specificity; Subtilisins; Virulence Factors | 1995 |
Acquisition of peptides by MHC class II polypeptides in the absence of the invariant chain.
Association of invariant chain with class II molecules has been suggested to inhibit binding of peptides that are available while the class II complex is present in the endoplasmic reticulum (ER) and subsequently transported to endosomes. We tested HLA-DR-transfected rat2 fibroblast cells lacking expression of invariant chain for their ability to form SDS stable class II dimers indicative of peptide binding in the class II cavity. No SDS-resistant class II dimers originating from short pulse-labeled immunoprecipitates can be identified. Prolonged ER retention of DR polypeptides by Brefeldin A treatment does not induce any stable class II dimers. In pulse-chase experiments, heat labile class II dimers are readily detectable after a 60-min chase, increasing in amounts by 4 h of chase. In vitro incubation of rat2DR cell lysate with DR3-binding peptides converts pulse-labeled class II molecules to SDS-resistant dimers. This indicates the ability of ER-resident DR dimers to bind peptides. Inhibition studies were conducted to define the intracellular site where stable class II complexes are formed in rat2DR cells. The lysosomotropic reagent chloroquine abrogates the appearance of SDS-resistant DR complexes in invariant chain-free rat2DR cells, which is consistent with the impact of chloroquine on peptide loading in other APCs. Leupeptin treatment strongly reduces the amount of heat-labile class II molecules but does not impair peptide loading when DR3-specific peptides were added to viable cells or to cell lysates. This result suggests that leupeptin inhibits intracellular degradation of polypeptides and thereby depletes the endocytic pathway of peptides available for class II binding. Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Differentiation, B-Lymphocyte; Brefeldin A; Cell Line; Chloroquine; Cyclopentanes; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Leupeptins; Molecular Sequence Data; Peptides; Precipitin Tests; Protein Binding; Rats; Sodium Dodecyl Sulfate; Transfection; Tumor Cells, Cultured | 1995 |
In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B.
NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation. Topics: Amino Acid Sequence; Cell Line; Cell Nucleus; Cytosol; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Humans; I-kappa B Proteins; Immunoblotting; Kinetics; Leupeptins; Macromolecular Substances; Molecular Sequence Data; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Phosphorylation; Transcription Factor RelA; Tumor Cells, Cultured | 1995 |
Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins.
The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein. Topics: Amino Acids, Branched-Chain; Animals; Caseins; Cations; Endopeptidases; Hot Temperature; Leupeptins; Multienzyme Complexes; Muscle Proteins; Muscles; Myofibrils; Nephropidae; Oligopeptides; Peptide Fragments; Protease Inhibitors; Substrate Specificity; Troponin | 1995 |
Degradation of proteasomes by lysosomes in rat liver.
Proteasomes are high-molecular-mass multisubunit complexes which are believed, either by themselves or as a part of the 26S proteinase complex, to play a central role in extralysosomal pathways of intracellular protein breakdown. We have addressed the degradation of proteasomes in rat liver, investigating the possible role of lysosomes. Affinity-purified antibodies against rat liver proteasomes were used for immunoblot analysis of isolated lysosomes. Although proteasomes are not found in lysosomes from normally fed rats, they were found to accumulate in lysosomes of rats treated with leupeptin (an inhibitor of lysosomal proteases) and could also be detected in lysosomes isolated from livers of starved (24 h) rats. Proteinase-K treatment of these fractions, as well as immunogold procedures, show that a proportion of the proteasomes are inside lysosomes. Comparison of the amount of proteasomes found in lysosomes by immunoblotting with their experimentally determined half life (8.3 days) is consistent with an important role of these organelles in the degradation of rat liver proteasomes. Nevertheless, these data do not exclude the possibility that some nonlysosomal degradation of proteasome components also occurs. Since proteasomes were localized in autophagic vacuoles, it is likely that they are taken up mainly by nonselective autophagy. However, using an in vitro system, it was found that, under conditions of starvation, proteasomes may also be taken up into lysosomes and degraded via the heat-shock cognate protein of 73 kDa (hsc73)-mediated transport. Topics: Animals; Cysteine Endopeptidases; Fasting; Half-Life; In Vitro Techniques; Leupeptins; Liver; Lysosomes; Male; Microscopy, Immunoelectron; Multienzyme Complexes; Proteasome Endopeptidase Complex; Rats; Rats, Wistar | 1995 |
Plasmodium falciparum: effects of proteinase inhibitors on globin hydrolysis by cultured malaria parasites.
The effects of peptide proteinase inhibitors on globin hydrolysis by cultured malaria parasites were studied. All of the four cysteine proteinase inhibitors evaluated blocked globin hydrolysis, as documented by the development of a morphological abnormality in which parasite food vacuoles filled with undegraded globin and by SDS-PAGE showing that the cysteine proteinase inhibitor-treated parasites accumulated large quantities of globin. The aspartic proteinase inhibitor pepstatin did not block globin hydrolysis by cultured parasites. None of seven antimalarial drugs tested elicited the food vacuole abnormality caused by cysteine proteinase inhibitors, indicating that this morphological alteration was not simply a sign of nonspecific parasite toxicity. Our results indicate that a trophozoite cysteine proteinase is required for initial cleavages of globin by intact malaria parasites. Topics: Animals; Antimalarials; Chymotrypsin; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Fluorescent Dyes; Globins; Humans; Hydrolysis; Leucine; Leupeptins; Oligopeptides; Pepstatins; Plasmodium falciparum; Vacuoles | 1995 |
Trichomonas vaginalis: amoeboid and flagellated forms synthesize similar proteinases.
Topics: Animals; Caseins; Culture Media; Cysteine Proteinase Inhibitors; Diazomethane; Endopeptidases; Leucine; Leupeptins; Lipoproteins; Trichomonas vaginalis | 1995 |
Presentation of endogenous viral proteins in association with major histocompatibility complex class II: on the role of intracellular compartmentalization, invariant chain and the TAP transporter system.
Major histocompatibility complex (MHC) class II-associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously synthesized antigens. We have studied the MHC class II-associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis, it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II-associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non-professional APC. We found that class II-associated presentation of endogenously synthesized membrane associated LCMV-GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II-associated presentation of endogenously synthesized cytosolic LCMV-NP was not observed even in Ii-deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV-GP apparently does not require processing in acidic endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading. Topics: Animals; Antigen Presentation; Antigens, Differentiation, B-Lymphocyte; ATP-Binding Cassette Transporters; Cell Compartmentation; Chloroquine; Drug Resistance; Epitopes; Glycoproteins; Histocompatibility Antigens Class II; Hybridomas; Leupeptins; Lymphocyte Activation; Lymphocytic choriomeningitis virus; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nucleoproteins; T-Lymphocytes; Viral Structural Proteins | 1995 |
Neuromuscular recovery after peripheral nerve repair: effects of an orally-administered peptide in a primate model.
Oral delivery of the tripeptide calpain inhibitor, leupeptin, after median nerve transection and epineural nerve repair in primates (Cebus apella) was studied for its potential benefits to neuromuscular recovery. Results of a controlled, dose-response study indicated that leupeptin was absorbed into plasma by the oral route of administration. When plasma leupeptin concentrations were 3 micrograms/ml or greater, morphologic and functional motor recovery were facilitated after nerve repair. Serial testing in hematology, clotting, and serum biochemistry showed that there were no adverse effects, when leupeptin was administered twice daily for 6 months following nerve repair. These data indicate that leupeptin is an effective and safe pharmaceutic adjunct to nerve repair and may have clinical benefits in humans, where the oral route is a much preferred method of delivery. Topics: Administration, Oral; Animals; Biological Availability; Calpain; Cebus; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Leupeptins; Male; Median Nerve; Microsurgery; Motor Neurons; Muscle, Skeletal; Nerve Regeneration; Neural Conduction; Neuromuscular Junction; Protease Inhibitors; Wound Healing | 1995 |
Stabilisation of neuromuscular junctions by leupeptin increases motor unit size in partially denervated rat muscles.
The effect on inhibiting the calcium activated neutral protease (CANP) by leupeptin on force output and motor unit size of the partially denervated rat EDL muscle was studied. Partial denervation was performed under anaesthesia by section of the L4 ventral ramus in 3- and 18-day-old Wistar rats. Two days after the operation a silicon strip containing the inhibitor of CANP leupeptin was implanted alongside the partially denervated EDL. Two to 3 months later the animals were anaesthetized and the EDL muscles on both sides prepared for tension recording. The results from these recordings show dramatic reduction in force output and muscle weight in animals operated at 3 days and this reduction was less pronounced in muscles treated with leupeptin. The mean force output of individual motor units increases in the leupeptin-treated partially denervated muscle compared to the untreated muscle. The increased fatigue resistance typical of muscles partially denervated at 3 days [37] is less pronounced in the treated muscle. In animals operated at 18 days the individual motor units actually increased in size and the leupeptin treatment had no effect on the partially denervated EDL muscles. The difference between the response to leupeptin of the 3 day and 18 day operated animals could be due to the different patterns of innervation of the muscles at the time of the application of the inhibitor of CANP. Topics: Animals; Calpain; Cell Size; Leupeptins; Motor Neurons; Muscle Denervation; Nerve Endings; Neuromuscular Junction; Rats; Rats, Wistar | 1995 |
Vitamin A incorporation into lipofuscin-like inclusions in the retinal pigment epithelium.
Intravitreal injection of the protease inhibitor leupeptin causes a rapid accumulation of lipofuscin-like autofluorescent inclusions in the retinal pigment epithelium (RPE) of the eye. In vitamin A-deprived animals, similar inclusions form in response to leupeptin treatment, but they do not become autofluorescent. Because vitamin A is necessary to the development of fluorescence, it appears likely that retinoids are directly incorporated into the inclusions. Experiments were conducted to determine whether this is the case. Rats were reared on a diet containing retinoic acid as the only retinoid. Retinoic acid cannot be utilized in visual transduction by the retina. When the eyes had been over 90% depleted of visual cycle retinoids, the animals were given a single intramuscular injection of 3H-all-trans retinol. After 7 days, when visual cycle retinoids had returned to an average of almost 70% of normal, the animals were given an intravitreal injection of leupeptin in each eye. At either 1 day or 7 days after the leupeptin treatment, some of the animals were dark-adapted for at least 12 h. The eyes were enucleated and fixed under dim red light. A region of each retina just superior to the optic nerve head was examined with electron microscopic autoradiography. At both one day and 7 days after the leupeptin treatment, the radiolabel in the RPE was primarily associated with the leupeptin induced inclusion bodies. Label was also present in the photoreceptor outer segments. The localization of vitamin A to the leupeptin-induced inclusions in the RPE strongly suggests that vitamin A is covalently bound to outer segment proteins that have been phagocytosed by the RPE but remain undegraded due to protease inhibition. This bound vitamin A is probably responsible for the autofluorescence of the leupeptin-induced inclusions. Vitamin A is not likely to be bound through a Schiff base linkage, since retinal-Schiff base compounds do not exhibit lipofuscin-like fluorescence. Topics: Animals; Inclusion Bodies; Leupeptins; Lipofuscin; Male; Pigment Epithelium of Eye; Protease Inhibitors; Rats; Rats, Inbred F344; Vitamin A | 1995 |
Inhibition of coronavirus MHV-A59 replication by proteinase inhibitors.
Topics: Animals; Cell Line; Giant Cells; Kinetics; Leupeptins; Mice; Murine hepatitis virus; Protease Inhibitors; RNA-Dependent RNA Polymerase; RNA, Viral; Time Factors; Uridine; Viral Plaque Assay; Virus Replication | 1995 |
Proteolytic conversion of single chain precursor macrophage-stimulating protein to a biologically active heterodimer by contact enzymes of the coagulation cascade.
Human serum macrophage stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. It is a member of the family of kringle proteins, which typically exist in extracellular fluid as single chain precursors that are activated by proteolytic cleavage. In this work, we expressed [35S]cysteine-labeled recombinant pro-MSP in MSP cDNA-transfected Chinese hamster ovary cells and studied proteolytic processing of pro-MSP and the requirement of cleavage for biological activity. In media containing heat-inactivated fetal bovine serum, the protein was secreted as single chain pro-MSP, which was cleaved over a period of hours to the mature heterodimer. Cleavage was prevented by serine protease inhibitors such as leupeptin or aprotinin; it did not occur if cells were cultured in serum-free medium. Nanomolar concentrations of coagulation proteases kallikrein, factor XIIa or factor XIa cleaved pro-MSP to MSP within 30 min. Pro-MSP had no biological activity. After cleavage by kallikrein, biological activity was quantitatively comparable to that of natural MSP isolated from human plasma. These results support our hypothesis that MSP circulates as the biologically inactive precursor and can be activated by enzymes of the intrinsic coagulation cascade. Topics: Animals; Aprotinin; Blood Coagulation Factors; Cells, Cultured; Chemotaxis; CHO Cells; Cloning, Molecular; Cricetinae; Cysteine; Enzyme-Linked Immunosorbent Assay; Factor XIa; Factor XIIa; Growth Substances; Hepatocyte Growth Factor; Humans; Kallikreins; Kinetics; Leupeptins; Macromolecular Substances; Macrophages; Mice; Molecular Weight; Protein Precursors; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Recombinant Proteins; Serine Proteinase Inhibitors; Transfection | 1994 |
Aging and proteolysis of oxidized proteins.
The main objective of this study was to investigate the possible cause(s) of the age-related augmentation of oxidatively damaged proteins in animal tissues. The hypothesis that activity of alkaline proteases, involved in the proteolysis of oxidized proteins, declines during aging was tested in the adult male housefly and further explored in the rat. Alkaline protease activity was measured fluorometrically by the release of trichloroacetic acid-soluble fluorescamine-reactive material from X ray-oxidized bovine serum albumin (BSA). Alkaline protease activity in the housefly was linearly related to the number of protein carbonyl groups. Possible involvement of serine or serine and thiol proteases was deduced from a 70% proteolytic inhibition by aprotinin and a 50% inhibition by leupeptin. Protease activity of houseflies for oxidized or native BSA did not alter with age. In contrast, a varied age-related pattern of protease activity was observed in the tissues of the rat. A comparison of 3-, 13-, and 23-month-old Sprague-Dawley rats indicated no age-related decline in alkaline protease activity in the brain, a 50% decline in the liver, and a 20% decline in the heart during 13 to 22 months. Results of this study suggest that in some species or tissues an age-related increase in the oxidized protein content is primarily due to a corresponding increase in the rate of protein oxidation, while in some other tissues a decline in proteolysis may be a contributory factor. Topics: Aging; Animals; Aprotinin; Endopeptidases; Enzyme Stability; Hot Temperature; Houseflies; Hydrogen-Ion Concentration; Leupeptins; Male; Oxidation-Reduction; Protease Inhibitors; Proteins; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine | 1994 |
Identification of targeting proteinase for rat alpha 1-macroglobulin in vivo. Mast-cell tryptase is a major component of the alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes.
The alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes was purified by a series of column chromatographic steps on concanavalin A-Sepharose, Sephacryl S-300, DEAE-cellulose and TSK gel DEAE-5PW columns. The complex contained no detectable alpha 2-macroglobulin. Studies on the substrate specificity indicated that the complex had tryptase-like activities towards various synthetic substrates, but no elastase, chymotrypsin, cathepsin-B and cathepsin-L activities. The proteinase activity was completely inhibited by di-isopropyl fluorophosphate, leupeptin and antipain, indicating that the proteinase bound to alpha 1-macroglobulin is a serine proteinase. Two protein bands (62 and 59 kDa) of the complex were labelled with [3H]diisopropyl fluorophosphate and both bands cross-reacted with anti-(mast-cell tryptase)antibody. These results suggest that mast-cell tryptase is a major targeting proteinase for alpha 1-macroglobulin in vivo. The main alpha-macroglobulin-proteinase complex in the adjuvant-treated rats was also the alpha 1-macroglobulin-tryptase complex, even though the plasma level of alpha 2-macroglobulin was elevated. Topics: alpha-Macroglobulins; Amino Acid Sequence; Animals; Chymases; Endocytosis; Hydrogen-Ion Concentration; Leupeptins; Liver; Lysosomes; Male; Mast Cells; Molecular Sequence Data; Protease Inhibitors; Rats; Rats, Wistar; Serine Endopeptidases; Substrate Specificity; Tryptases | 1994 |
In vitro stability of endogenous parathyroid hormone-related protein in blood and plasma.
We describe a systematic comparison of the effects of anticoagulants, protease inhibitors and conditions of sample handling on the in vitro stability of endogenous parathyroid hormone-related protein (PTHrP) in blood from patients with hypercalcaemia of malignancy (HM). When blood was separated within 15 min of collection, PTHrP1-86 levels measured by two-site immunoradiometric assay in serum and heparinized plasma were significantly lower than in ethylenediaminetetraacetic acid (EDTA) plasma (P < 0.02). PTHrP was unstable in blood kept at 20 degrees C for 4 h and inclusion of protease inhibitors reduced, but failed to abolish, this instability. In blood collected in the presence of EDTA, inclusion of leupeptin either alone or in combination with pepstatin and aprotinin increased the mean half-time of disappearance from 3.9 to 10.1 and 11.2 h, respectively (P < 0.05). In contrast, when blood containing EDTA was separated within 15 min, PTHrP was stable in plasma at 20 degrees C for at least 4 h. As a result of the instability of PTHrP1-86 immunoreactivity in whole blood at ambient temperatures we advise that for our immunoradiometric assay (IRMA) blood collected in EDTA should be separated within 15 min, and the plasma frozen until assay. Topics: Anticoagulants; Aprotinin; Blood Specimen Collection; Edetic Acid; Heparin; Humans; Hypercalcemia; Immunoradiometric Assay; In Vitro Techniques; Infant; Leupeptins; Neoplasms; Parathyroid Hormone-Related Protein; Pepstatins; Peptide Fragments; Peptides; Protease Inhibitors; Temperature | 1994 |
The role of proteases in stratum corneum: involvement in stratum corneum desquamation.
The effects of protease inhibitors on cell dissociation were studied in vitro in order to examine the involvement of proteases in stratum corneum desquamation. Stratum corneum sheet (peeled from human backs after sunburn) was incubated in a detergent mixture containing 8 mM N,N-dimethyldodecylamine oxide, 2 mM sodium lauryl sulphate and 60 micrograms/ml kanamycin with or without protease inhibitors, and the number of released cells was counted after incubation for 48 h. Cell dissociation was inhibited strongly by antipain or aprotinin, but not at all by N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin, N-ethylmaleimide or pepstatin, which suggests that only serine proteases are associated with desquamation. Furthermore, leupeptin and chymostatin each reduced cell dissociation about half as effectively as aprotinin or antipain, while a mixture of leupeptin and chymostatin prevented stratum corneum dissociation as potently as antipain or aprotinin. In addition, the activity of chymotrypsin-like protease in scaly skin was higher than that in normal skin, as we have previously found for trypsin-like protease. These results suggest that both trypsin-like and chymotrypsin-like serine proteases are involved in stratum corneum desquamation. Topics: Antipain; Aprotinin; Detergents; Endopeptidases; Epidermis; Ethylmaleimide; Humans; Leucine; Leupeptins; Oligopeptides; Pepstatins; Protease Inhibitors; Sunburn | 1994 |
Human chorionic gonadotropin beta-subunit nicking enzymes in pregnancy and cancer patient serum.
hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT Topics: Amino Acid Sequence; Blotting, Western; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Chromatography, Gel; Disulfides; Endopeptidases; Female; Humans; Leupeptins; Macromolecular Substances; Male; Metalloendopeptidases; Molecular Sequence Data; Neoplasms; Peptide Fragments; Phenanthrolines; Pregnancy | 1994 |
Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.
Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P. Topics: Amino Acid Sequence; Ammonium Chloride; Animals; Arsenicals; Cell Line, Transformed; Chloroquine; Colchicine; Endocytosis; Epithelium; Hypertonic Solutions; Immune Sera; Immunohistochemistry; Kinetics; Leupeptins; Microscopy, Fluorescence; Molecular Sequence Data; Monensin; Peptides; Rats; Receptors, Neurokinin-1; Recombinant Proteins; Substance P; Time Factors; Transfection | 1994 |
Cytosolic Ca2+ elevation and calpain inhibitors in HgCl2 injury to rat kidney proximal tubule epithelial cells.
This study assessed HgCl2 injury to proximal tubule epithelial cells as it relates to the concentration of ionized cytosolic Ca2+ ([Ca2+]i) elevation and activation of calpains. Experiments in high and low extracellular Ca2+ concentration ([Ca2+]e) were performed using the calpain inhibitors antipain and leupeptin, and also trypsin inhibitor, methylamine, chloroquine, and ryanodine. Cell killing was time/dose dependent and greater with high [Ca2+]e. After 30 min treatment with 25 microM HgCl2, 19% of cells in low [Ca2+]e were dead while 72% died in high [Ca2+]e. Morphologic changes such as cytoplasmic blebbing were also greater in high [Ca2+]e. Antipain and leupeptin diminished toxicity. Leupeptin did not block Ca2+ entry into cells. Results show that HgCl2 toxicity is correlated with increased [Ca2+]i, and that calpains may mediate the resultant pathological changes. Topics: Animals; Antipain; Calcium; Calpain; Cell Survival; Cells, Cultured; Cytoplasm; Cytosol; Kidney Tubules, Proximal; Leupeptins; Male; Mercuric Chloride; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Rats | 1994 |
Calpain-mediated regulation of AMPA receptors in adult rat brain.
Changes in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain structures. Activation of calpain has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. We report here that preincubation of thin (10 microns) frozen rat brain sections with calcium at physiological temperature changes the immunological properties of AMPA receptors, an effect totally blocked by calpain inhibitors. Immunocytochemistry indicates that in situ calpain activation produces a decreased immunoreactivity for GluR1, and to a lesser extent for GluR2/3, in the neuropil throughout the brain and an increased immunoreactivity in cell bodies, particularly in hippocampus. Western blots of calcium-treated sections suggest that the decreased immunoreactivity for GluR subunits is due to partial proteolysis. These results strongly suggest the involvement of calpain in the regulation of glutamatergic synapses. Topics: Animals; Brain; Calcium; Calpain; Glycoproteins; Immunohistochemistry; Leupeptins; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Tissue Distribution | 1994 |
Endogenous, Ca(2+)-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle.
The association of an endogenous, Ca(2+)-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and beta-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 microM and maximal activity at 10 microM. Mg2+ is also an activator of this enzyme (CI50 = 22 microM). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356-1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383-1400. The RyR cleavage by the Ca(2+)-dependent thiol-protease is prevented in the presence of ATP (1-5 mM) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed. Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Calcium; Calcium Channels; Calpain; Cysteine; Dithiothreitol; Iodoacetamide; Leupeptins; Magnesium; Mercaptoethanol; Mercuric Chloride; Molecular Sequence Data; Molecular Weight; Muscle Proteins; Muscle, Skeletal; Rabbits; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sodium Chloride; Substrate Specificity | 1994 |
Rat myoblast fusion requires exteriorized m-calpain activity.
Our previous studies demonstrated that fibronectin could be proteolyzed by m-calpain during muscle cell differentiation. Recent results indicated also that m-calpain could be exteriorized and more particularly associated to extracellular matrix components. To clarify one of the possible physiological functions of this proteinase during myogenesis, we have analyzed the incidence of added purified m-calpain and calpain inhibitors on the fusion kinetics of cultured myoblasts. Our results provided evidence that at low concentration (0.01 microgram/ml), added m-calpain induces precocious fusion and increases myoblast fusion by 78%. At high concentrations (10 micrograms/ml), the viability of the cells was not affected but the myoblasts were unable to fuse. Leupeptin and calpastatin--potent m-calpain inhibitors--added to the culture medium reduced myoblast fusion by 70%. On the other hand, the addition of monospecific m-calpain polyclonal antibodies to the culture medium induced a 76% decrease of myoblast fusion. In order to trap exteriorized m-calpain, myoblasts were incubated for 24 h with m-calpain antibodies. Following this treatment, nonpermeabilized myoblasts exposed to labeled secondary antibodies showed fluorescent spots scattered at the cell surface. These results strongly support that m-calpain which was involved in myoblast fusion was exteriorized and suggest therefore that this enzyme may play an important role extracellularly. Topics: Animals; Biological Assay; Calcium-Binding Proteins; Calpain; Cell Fusion; Cells, Cultured; Culture Media; Immunoglobulin G; Immunohistochemistry; Kinetics; Leupeptins; Muscle Fibers, Skeletal; Rats; Rats, Wistar | 1994 |
Potentially amyloidogenic fragment of 50 kDa and intracellular processing of amyloid precursor protein in cells cultured under leupeptin.
The principal neuropathological feature of Alzheimer's disease is extracellular deposition of approximately 4-kDa proteinous fragment, designated as beta-amyloid peptides (beta/A4 peptides) derived by proteolytic cleavage from amyloid precursor protein (APP), a large cell-surface receptor-like protein. There has been evidence that APP is proteolytically degraded in the secretory and endosomal/lysosomal pathways. The pathway in which APP is cleaved to generate beta/A4 peptides is still not identified. To clarify the intracellular processing of APP into the generation of beta/A4 peptides, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies in the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa containing full-length beta/A4 peptides were identified in the cultured cells. Immunoblot analysis, biochemical study for specific marker enzyme activity of the fractions obtained from subcellular fractionation, sucrose density gradient centrifugation indicated that the 50-kDa APP fragment was produced in the compartment closely related to endosomal/lysosomal system. Our data suggest that the endosomal/lysosomal pathway is involved in the processing and generation of beta/A4 peptides. Topics: Amyloid; Amyloidosis; Blotting, Western; Cell Line; Centrifugation, Density Gradient; Humans; Immunologic Techniques; Intracellular Membranes; Leupeptins; Molecular Weight; Peptide Fragments; Prion Proteins; Prions; Protein Precursors; Subcellular Fractions | 1994 |
Modulation of lipoxidase activity by urokinase-type plasminogen activator.
Malignant cells show increased urokinase (UK) activity and decreased peroxidation of essential fatty acids (EFA). In order to explore this phenomenon the effect of UK on the lipoxidase activity was spectrophotometrically investigated. Decreased lipoxidase activity was obtained with increased UK concentrations (r = -1.000, p < 0.0001). This proteolytic effect of UK on lipoxidase was eliminated with the addition of the UK inhibitor leupeptin. These results suggest that the increase in UK activity in malignant cells may decrease the lipoxidase activity and thus peroxidation of EFA. The effectiveness of a given EFA in killing cancer cells would therefore depend on the modulation of the lipoxidase activity by the UK-type plasminogen activator. Topics: Amino Acid Sequence; Fatty Acids, Essential; Humans; Kidney; Leupeptins; Lipid Peroxidation; Lipoxygenase; Lipoxygenase Inhibitors; Molecular Sequence Data; Neoplasms; Plant Proteins; Urokinase-Type Plasminogen Activator | 1994 |
Neuromuscular contacts of expanded motor units in rat soleus muscles are rescued by leupeptin.
In the soleus muscle of the rat following section of the L5 ventral ramus (partial denervation) the remaining motor axons increase their territory by sprouting. Nerve sprouts are first seen two to three days after the operation, their number peaks at 10-14 days and subsequently remains at this level. The time course of the initial sprouting in partially denervated muscles is not altered by paralysing the muscles with alpha-bungarotoxin, and the initial extent of the sprouting is, if anything, greater in the paralysed muscles. However, unlike in controls, this level of sprouting is not maintained and neuromuscular contacts are lost when muscles recover from the paralysis. The loss of these contacts can be prevented by treatment of these partially denervated paralysed muscles with leupeptin, an inhibitor of calcium-activated neutral protease. Interestingly, more contacts are rescued when leupeptin is applied 10 days after alpha-bungarotoxin treatment, when sprouting has reached high levels, than at three days, when sprouting has just begun. The neuromuscular connections rescued by leupeptin are functional. Maximum tetanic tension produced by untreated soleus muscles two to five months after partial denervation is 66 +/- 9% of contralateral control muscles, but only 39 +/- 8% when the muscles were paralysed with alpha-bungarotoxin for 12-14 days after partial denervation. However, when partially denervated paralysed muscles were treated with leupeptin three and 10 days after alpha-bungarotoxin treatment their tension output is 74 +/- 3% and 81 +/- 8%, respectively. After partial denervation alone, motor units are twice their normal size. Short-term paralysis with alpha-bungarotoxin prevents this increase in motor unit territory. However, the application of leupeptin to the paralysed muscles rescues neuromuscular contacts, allowing motor unit size to remain expanded, at around 2-2.5-fold. Thus, following recovery from temporary paralysis with alpha-bungarotoxin, there is a sudden withdrawal of neuromuscular contacts and these can be rescued by treatment with leupeptin. Topics: Amino Acid Sequence; Animals; Bungarotoxins; Denervation; Leupeptins; Molecular Sequence Data; Motor Neurons; Muscle Contraction; Muscle Fibers, Skeletal; Muscle, Skeletal; Neuromuscular Junction; Rats; Rats, Wistar; Silicone Elastomers | 1994 |
Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.
Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Amino Acid Sequence; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; CD4-Positive T-Lymphocytes; Chloroquine; Histocompatibility Antigens Class II; Humans; Immunoglobulin Light Chains; Immunotoxins; Leupeptins; Lymphoma, B-Cell; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Peptides; Tetanus Toxin | 1994 |
Increased activities of cathepsin B and other lysosomal hydrolases in fibroblasts and bone tissue cultured in the presence of cysteine proteinases inhibitors.
Leupeptin is an established, reversible inhibitor of cathepsin B, a lysosomal cysteine proteinase. Yet, in rat fibroblasts as well as in foetal mouse calvaria, we observed an increase of the activity of cathepsin B in homogenates of cells and tissue harvested after culture in the presence of leupeptin. This effect was also seen for other lysosomal hydrolases, namely sphingomyelinase, N-acetyl-beta-glucosaminidase, arylsulphatase A and phospholipase A1 in fibroblasts, and beta-glucuronidase in mouse calvaria. In calvaria, antipain, another reversible cysteine proteinase inhibitor, caused a similar effect, whereas E-64, an irreversible inhibitor, was consistently inhibitory of the cathepsin B activity; yet it also caused an increase of beta-glucuronidase activity. The effect of leupeptin in fibroblasts was dose and time-dependent, required the continuous presence of the inhibitor, and was not dependent from protein synthesis. Actually, addition of cycloheximide caused a severe loss of activity of cathepsin B and of sphingomyelinase. In the presence of both cycloheximide and leupeptin, however, these two activities were retained to a value corresponding to that found in excess in cells cultivated with leupeptin alone. The data therefore suggests that leupeptin exerts the effects described in this paper by preventing the degradation of cathepsin B, sphingomyelinase and probably several other lysosomal hydrolases by cysteine proteinases. We therefore propose that cysteine proteinases play a key role in the control of the steady-state levels of these enzymes in normal conditions. Topics: Animals; Bone Resorption; Cathepsin B; Cells, Cultured; Culture Techniques; Cycloheximide; Fibroblasts; Hydrolases; Leupeptins; Mice; Parathyroid Hormone; Protein Biosynthesis; Proteins; Rats; Rats, Wistar; Skull; Time Factors | 1994 |
Inhibition of procollagen I degradation by colligin: a collagen-binding serpin.
Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment. Topics: Amino Acid Sequence; Animals; Biological Transport; Brefeldin A; Carrier Proteins; Cell Line; Cyclopentanes; Endoplasmic Reticulum; Fibroblasts; Fibronectins; Glycoproteins; Humans; Hydrogen-Ion Concentration; Leupeptins; Microsomes, Liver; Molecular Sequence Data; Procollagen; Protease Inhibitors; Rats; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 1994 |
Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.
Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer. Topics: Actins; Amino Acid Sequence; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Cell Membrane Permeability; Complement Inactivator Proteins; Digitonin; Humans; Leupeptins; Membrane Glycoproteins; Molecular Sequence Data; Platelet Aggregation; Platelet Membrane Glycoproteins; Protein Binding; Tetraspanin 29 | 1994 |
beta-Amyloid precursor protein fragments and lysosomal dense bodies are found in rat brain neurons after ventricular infusion of leupeptin.
Infusion of the serine and thiol protease inhibitor, leupeptin, is known to cause a reduction of fast axoplasmic transport, and accumulation of lysosomal dense bodies in neuronal perikarya. We have found these dense bodies in hippocampal and cerebellar neurons contain ubiquitin conjugated proteins. We now demonstrate that these accumulated neuronal lysosomes are labeled by antisera to the cytoplasmic, transmembrane and extracellular domains of beta-amyloid precursor protein (APP) and also that lysosomal APP is fragmented. This in vivo model confirms that neurons can process APP via a lysosomal pathway and that neuronal lysosomes in vivo contain both N-terminal and potentially amyloidogenic C-terminal fragments of APP. We also show that increased APP immunoreactivity after leupeptin treatment is seen first in neurons and later in astrocytes. On recovery from infusion, APP N-terminal immunoreactivity diminishes whilst C-terminal reactivity remains in neurons. These findings are consistent with production in whole brain of potentially amyloidogenic fragments of APP within neuronal lysosomes in perikarya and dendrites implying that neurons may play a role in forming the beta-amyloid of plaques. Topics: Amyloid beta-Protein Precursor; Animals; Antibodies, Monoclonal; Axons; Blotting, Western; Brain; Cytoplasmic Granules; Dendrites; Female; Immunohistochemistry; Injections, Intraventricular; Leupeptins; Lysosomes; Neurons; Peptide Fragments; Rats; Rats, Wistar | 1994 |
Anti-ubiquitin immunoreactivity associates with pyramidal cell death induced by intraventricular infusion of leupeptin in rat hippocampus.
Pathological studies on several neurodegenerative diseases including Alzheimer's disease have revealed common deposition of ubiquitin in many inclusion bodies. This implies a possible association of ubiquitin with neurodegeneration. To address this possibility, we examined histochemically the effect of intraventricular infusion of leupeptin, a thiol proteinase inhibitor, which is known to elevate anti-ubiquitin immunoreactivity in rat Purkinje cells. In the leupeptin-infused rat, an intense anti-ubiquitin immunoreactivity in the cytoplasm of neurons occurred not only in cerebellar Purkinje cells but also elsewhere in a wide area of the rat brain. The increase in the immunoreactivity was followed by a gradual depletion of pyramidal neurons in the hippocampal CA1 and CA3 subfields. The immunoreactive neurons disappeared concurrently. The number of anti-ubiquitin immunoreactive neurons was negatively correlated with that of surviving neurons when the duration of leupeptin infusion was varied. These results suggest that increased anti-ubiquitin immunoreactivity associates with neuronal death in leupeptin-treated rat brain. Topics: Animals; Cell Death; Hippocampus; Immunohistochemistry; Injections, Intraventricular; Learning; Leupeptins; Male; Neurons; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Ubiquitins | 1994 |
Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes.
The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF-mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell. Topics: Acids; Ammonium Chloride; Animals; Chloroquine; Dose-Response Relationship, Drug; Endocytosis; Flagella; Humans; Hydrogen-Ion Concentration; Immunohistochemistry; Intracellular Membranes; Leupeptins; Lipoproteins, HDL; Microscopy, Immunoelectron; Models, Biological; Monensin; Organelles; Protein Binding; Trypanosoma brucei brucei | 1994 |
Impact of lipofuscin on the retinal pigment epithelium: electroretinographic evaluation of a protease inhibition model.
With aging, the retinal pigment epithelium (RPE) becomes increasingly congested with residual debris called lipofuscin. Little is known about the impact of lipofuscin on retinal function, and this was addressed in the present study by examining the influence of RPE debris on electroretinographic (ERG) parameters utilizing an experimental model of lipofuscin accumulation. Pigmented rats were injected intravitreally with the protease inhibitor leupeptin, and examined 1 week later by electroretinogram (ERG) recording and light and electron microscopy. Relative to vehicle-injected controls, leupeptin-treated retinas showed abundant accumulation throughout the RPE cytoplasm of inclusions that resembled lipofuscin. RPE cells filled with this debris showed a marked increase in height and a displacement of melanin from their apical border. Morphological changes in the RPE had no influence on retinal function since ERG threshold, a- and b-wave maximum amplitude, latency and implicit time were not significantly different between leupeptin-treated eyes and controls. Furthermore, leupeptin-induced RPE inclusions did not alter either the rate or extent of ERG dark adaptation. These findings suggest that filling of the RPE cytoplasm with residual debris is not in itself likely to be the cause of functional alterations in the aging eye. Topics: Animals; Dark Adaptation; Disease Models, Animal; Electroretinography; Female; Injections; Leupeptins; Lipofuscin; Pigment Epithelium of Eye; Rats; Retina; Vitreous Body | 1994 |
Transcytosis and processing of intrinsic factor-cobalamin in Caco-2 cells.
Intracellular events within enterocytes following receptor-mediated endocytosis of intrinsic factor-cobalamin (IF-Cbl) are poorly understood. We have examined the fate of IF and Cbl in filter-grown Caco-2 cells which express both IF receptors and transcobalamin II and which transcytose Cbl. Uptake of IF-Cbl from the apical surface leads to the intracellular accumulation of Cbl in a process that reaches an equilibrium between internalization and secretion only after a 20-h continuous incubation. Transcytosed Cbl is detectable in the basolateral medium 4 h after the onset of endocytosis. Cbl is released from the basolateral surface with the same kinetics irrespective of from which cell surface endocytosis of IF-Cbl took place. Following uptake, internalized IF is degraded with a half-time of 4 h. Leupeptin causes a partial block in the proteolysis of IF, an intracellular accumulation of Cbl bound to IF, and a decrease in transcytosis of Cbl. Finally, an analysis of intracellular Cbl during transcytosis shows that free Cbl is present within cells during transcytosis. Topics: Biological Transport; Cell Line; Cell Polarity; Cytoplasm; Endocytosis; Humans; Intestinal Mucosa; Intrinsic Factor; Leupeptins; Vitamin B 12 | 1994 |
Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility.
The effects of in situ postrigor injection (24 h postmortem) of exogenous aspartic, serine, and cysteine proteinase effectors into cylindrical beef longissimus samples on tenderness and myofibrillar protein degradation and integrity were studied. Injection of phenylmethanesulphonylfluoride (PMSF) and pepstatin did not influence shear force or protein degradation measured 8 d postmortem, confirming that neither serine nor aspartic proteinases affect tenderization. Injection of leupeptin, an epoxysuccinyl peptide (E-64), or N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I) blocked tenderization completely, as observed by higher (P < .05) shear force values. A causal relationship between increased toughness and prevented action of the cysteine proteinases was suggested by a concomitant reduction of myofibrillar protein degradation, generally reflected in higher (P < .05) remaining troponin-T and titin amounts and lower (P < .05) levels of 30-kDa peptide, as evaluated by semiquantitative SDS-PAGE. Moreover, parallel to these changes, amounts of salt-soluble myofibrillar protein and semiquantitative concentrations of individual salt-soluble proteins (SDS-PAGE) were also reduced (P < .05). Injection of Triton-X-100 and Ca2+ increased (P < .05) tenderness, as well as myofibrillar protein degradation and solubility, and free Ca2+, whereas EDTA induced the opposite results, indicating an important role for calpains in tenderization. Because cathepsin B, D, H, and L inhibitors did not affect texture or proteolysis, our results suggest that calpains are the main proteases involved in beef tenderization. Topics: Animals; Calcimycin; Calcium; Calpain; Cathepsins; Cattle; Cysteine Proteinase Inhibitors; Diazomethane; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glycoproteins; Leucine; Leupeptins; Male; Meat; Muscle Proteins; Muscles; Octoxynol; Pepstatins; Phenylmethylsulfonyl Fluoride; Postmortem Changes; Solubility | 1994 |
Leupeptin inhibits phospholipases D and C activation in rat hepatocytes.
The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10-1500 microM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C, perhaps at the level of a G protein. Topics: Animals; Cell Membrane; Enzyme Activation; Glycerophospholipids; Guanosine 5'-O-(3-Thiotriphosphate); Leupeptins; Liver; Male; Phosphatidic Acids; Phospholipase D; Rats; Rats, Sprague-Dawley; Type C Phospholipases | 1994 |
Inhibition of peptide binding to DR molecules by a leupeptin-induced invariant chain fragment.
Loading of peptides onto DR molecules was studied by characterizing precursors of the mature peptide-DR complexes expressed at the surface of B cells. Since invariant chain (Ii) prevents binding of peptides by DR molecules, it was speculated that analysis of complexes between DR heterodimers and proteolytic fragments of Ii offers the possibility to examine how DR molecules and peptides assemble. Using a procedure combining a two-step affinity chromatography and gel filtration, we isolated from leupeptin-treated B cells complexes between DR molecules and N-terminal Ii fragments previously called "leupeptin-induced polypeptides" (LIP; Blum and Cresswell, 1988, Proc. natn. Acad. Sci. U.S.A. 85, 3975-3979). It was observed that the most prominent LIP fragment has a relative molecular mass (M(r)) of 16 kDa. In addition, we show that this polypeptide species does not bear N-linked glycans, indicating that this fragment does not extend beyond residue 129 of Ii. Similarly to DR alpha beta heterodimers associated with the full length 33 and 35 kDa Ii forms, DR alpha beta heterodimers associated with LIP fragments are unstable in sodium dodecyl sulfate (SDS) at ambient temperature, whereas mature DR alpha beta heterodimers are resistant to dissociation with SDS. These results are indirect evidence that LIP-DR complexes are devoid of bound peptides. This possibility was supported by showing that LIP-DR complexes fail to bind a radioiodinated tetanus toxin peptide (125I-p2), while DR molecules, which are spontaneously released from complexes with LIP fragments, bind the labeled peptide. These results demonstrate that association with LIP fragments is sufficient to prevent binding of peptides by DR molecules. This notion was further documented by showing that binding of 125I-p2 on DR heterodimers is inhibited by preparations of LIP fragment. By contrast, a soluble recombinant fragment corresponding to the extracytoplasmic region of Ii did not block 125I-p2 binding. The results presented in this study indicate that the cytoplasmic and/or transmembrane region of Ii is required to prevent peptide binding by DR molecules, while the extracytoplasmic portion of Ii, though capable of associating with DR molecules, lacks the capacity to block peptide binding. Topics: Amino Acid Sequence; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Cell Line, Transformed; Histocompatibility Antigens Class II; HLA-DR Antigens; Leupeptins; Molecular Sequence Data; Particle Size; Peptide Fragments; Protein Binding | 1994 |
Cleavage of the epidermal growth factor receptor by a membrane-bound leupeptin-sensitive protease active in nonionic detergent lysates of senescent but not young human diploid fibroblasts.
Numerous studies suggest that epidermal growth factor (EGF) signaling is impaired in nonproliferating senescent human diploid fibroblasts downstream of receptor binding. One possible explanation for these results is that senescent cells possess unique enzymatic activities capable of regulating functional levels of the EGF receptor. To test that hypothesis, nonionic detergent lysates of young and senescent cells were compared for proteolytic activity directed towards the EGF receptor, and a protease that cleaves the 170 kDa EGF receptor was identified in lysates from senescent but not young cells. Although studies presented here were carried out with WI-38 cells, our data indicate that other senescent fibroblasts possess a similar activity. The degradation product immunoprecipitated by a monoclonal antibody specific for an EGF receptor exocytosolic epitope had an approximate molecular weight of 100,000. This product was also detected following cell surface labeling with 125I, and by cross-linking 125I-EGF to intact cells with disuccinimidyl suberate. The proteolytic activity in senescent cell lysates was specifically inhibited by leupeptin and did not require divalent cations; it was also inactivated by aprotic solvents such as dimethylsulfoxide (DMSO) or ethylene carbonate. Interestingly, this protease was not active during ligand-induced intracellular processing of the EGF receptor, suggesting that it does not normally function in endocytic or lysosomal compartments. The susceptibility of the protease to inactivation by cell surface trypsinization is consistent with a plasma membrane localization. Since EGF receptor cleavage is not observed unless senescent cells are solubilized with nonionic detergents, it seems likely that the protease is confined to specialized regions of the plasma membrane. Whether or not the EGF receptor is a physiologic target for this protease is unclear. Its expression at the cell surface is nevertheless significant, since it suggests there are mechanisms for regulating membrane-bound proteins, or biologically active peptides in the extracellular space, in senescent cells that are either absent or inactive in young cells. Topics: Cell Line; Cell Membrane; Cellular Senescence; Detergents; Diploidy; Endopeptidases; ErbB Receptors; Fibroblasts; Humans; Intracellular Membranes; Leupeptins; Ligands; Phosphorylation; Protease Inhibitors; Protein Processing, Post-Translational; Solvents; Stimulation, Chemical; Trypsin | 1994 |
Leupeptin and E-64, inhibitors of cysteine proteinases, prevent gentamicin-induced lysosomal phospholipidosis in cultured rat fibroblasts.
Aminoglycoside antibiotics, such as gentamicin, cause an early lysosomal phospholipidosis in the renal cortex, which is considered as a key event in the onset of acute tubular necrosis induced by these drugs. In a model of primary cultures of embryonic rat fibroblasts which develop typical lysosomal phospholipidosis when incubated with gentamicin (decrease of sphingomyelinase activity; increase in total cells lipid phosphorus; appearance of so-called 'myeloid bodies' in lysosomes), we observed a protective effect exerted by inhibitors of cysteine proteinases (leupeptin, E-64) against this alteration on the basis of both biochemical and morphological criteria. Actually leupeptin and E-64 caused a marked stimulation of sphingomyelinase activity both in control and in gentamicin-treated cells, which we suggest to be the cause of protection. Topics: Animals; Cells, Cultured; Cysteine Proteinase Inhibitors; Fibroblasts; Gentamicins; Leucine; Leupeptins; Lysosomes; Phospholipids; Rats | 1994 |
Internalization and intracellular processing of an anti-B-cell lymphoma monoclonal antibody, LL2.
The successful clinical experience with antibody LL2 (an IgG2a, anti-B-cell lymphoma antibody) in radioimmunodetection and radioimmunotherapy suggests that this antibody may have potential as a carrier of cytotoxic agents. The internalization, cellular trafficking, and catabolism of this antibody in target human Burkitt lymphoma cells (Raji) were investigated. Internalization of intact antibody as well as of the F(ab')2 and Fab' fragments was detected by an FITC-labeled anti-mouse second antibody probe, and evaluated by fluorescence microscopy. Internalization of intact IgG (or the fragments) was observed as early as 5 min after incubation at 37 degrees C. Initially, the internalized antibodies were present as micro-particles inside the cell membrane, and were translocated to the lysosomal compartment within 2 hr. The anatomic location of the internalized antibody, before translocation to the lysosomal compartment, was deduced by comparing the fluorescence images obtained with the antibody to those obtained with fluorescent probes with known cellular distribution in a co-internalization study. A Golgi-like compartment was found to be involved in the translocation of the antibody. Cellular catabolism of the bound antibody was studied by using 125I-labeled antibody on the target cells. At 21 h, 40% of the radioactivity was released into the supernatant as degraded fragments. The observation suggested that the antibody was degraded mainly in the lysosomes, since the degradation was significantly inhibited in the presence of lysosomal inhibitors such as ammonium chloride or leupeptin. Subcellular fractionation of Raji cells after the binding of 125I-labeled LL2 indicated that the antibody was translocated to lysosomes as evidenced by SDS-PAGE. The rate of internalization (Ke) of LL2, and the re-expression of the antigen were determined. The rapid internalization of LL2 and the re-expression of the antigen suggest that this antibody may have potential as a therapeutic immunoconjugate, since it could deliver a higher accumulation of cytotoxic agents into lymphoma cells. Topics: 4-Chloro-7-nitrobenzofurazan; Ammonium Chloride; Antibodies, Monoclonal; Burkitt Lymphoma; Ceramides; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Golgi Apparatus; Humans; Immunoglobulin Fab Fragments; Kinetics; Leupeptins; Lymphoma, B-Cell; Lysosomes; Microscopy, Fluorescence; Tumor Cells, Cultured | 1994 |
Pharmacological interference with tissue hypercatabolism in tumour-bearing rats.
Marked loss of body weight and profound waste of both skeletal muscle and white adipose tissue occur in rats into which the ascites hepatoma Yoshida AH-130 has been transplanted, associated with marked perturbations in the hormonal homoeostasis and the presence of circulating tumour necrosis factor and high plasma levels of prostaglandin E2 [Tessitore, Costelli and Baccino (1993) Br. J. Cancer 67, 15-23]. On the basis of previous findings, the present study examined whether the development of cachexia in this model system could be significantly affected by adrenalectomy or by pharmacological treatments that may interfere with proximal or distal mediators of tissue hypercatabolism. In no instance was tumour growth modified. Medroxyprogesterone acetate, an anabolic-hormone-like drug, was completely ineffective. In adrenalectomized animals, although changes such as the elevation of plasma triacylglycerols and corticosterone were corrected, the general course of cachexia was not modified. A partial prevention of muscle waste was observed with acetylsalicylic acid, a non-steroidal anti-inflammatory drug, or with leupeptin, a proteinase inhibitor. Insulin afforded the most significant preservation of muscle protein and adipose-tissue mass, which were maintained close to control values even 10 days after transplantation. The effects of insulin on gastrocnemius muscle and liver protein content were exerted by slowing down protein turnover, mainly enhancing synthesis. Consistently, the total free amino acid concentration in the gastrocnemius of insulin-treated rats 10 days after tumour transplantation was close to that of controls. Although treatment with insulin decreased plasma corticosterone to normal values, it did not modify the circulating level of tumour necrosis factor. On the whole these data show that it seems possible to prevent, at least in part, the tissue waste that characterizes cancer cachexia by purely pharmacological means. Topics: Adrenalectomy; Animals; Aspirin; Blood Glucose; Body Weight; Cachexia; Cathepsin D; Cholesterol; Endopeptidases; Hormones; Insulin; Leupeptins; Male; Medroxyprogesterone Acetate; Muscle Proteins; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Wistar; Triglycerides | 1994 |
Protein degradation in kidney proximal tubule cell monolayers.
Isolated proximal tubule cells have been labelled with L-[4,5-3H]leucine prior to cell division. Histochemical staining demonstrated the purity of the cultures. The bicarbonate ion or a collagen support was required for cell growth. Different culture growth rates were established by varying these parameters. The proximal tubule marker enzyme, gamma-glutamyl transpeptidase, was expressed throughout the culture period (7-10 days) and the cells undergo a glycolytic shift, shown by an increase in the levels of lactate dehydrogenase. The specific activities of these enzymes were related to the growth conditions. Exponential rates of protein degradation were observed. The uptake of labelled exogenous hepatocyte proteins in proximal tubule cell cultures was completely suppressed in the presence of serum (10%, v/v) showing that endocytosis did not contribute to the observed measurements of intracellular protein degradation. The increased growth rates seen in cultures were accompanied by decreased rates of protein degradation. Use of the inhibitors of proteolysis, leupeptin and ammonium chloride, showed that the decrease was at the lysosomal level. The results suggest that targeting of inhibitors of lysosomal proteolysis, via low-molecular-weight proteins, may be useful in stimulating tubular regeneration in kidney disease. Topics: Ammonium Chloride; Animals; Cells, Cultured; Half-Life; Kidney Tubules, Proximal; Leupeptins; Proteins; Rats; Tritium | 1994 |
Separation of lysosomes and autophagosomes by means of glycyl-phenylalanine-naphthylamide, a lysosome-disrupting cathepsin-C substrate.
In density-gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LDH). Lysosomes were identified by marker enzymes such as acid phosphatase, or by degradation products from 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) loaded into the lysosomes by an intravenous injection in vivo 18 h prior to cell isolation. Autophagosomes and lysosomes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated the possibility of using lysosomal enzyme substrates to achieve selective destruction of lysosomes by swelling. Hepatocytes were first incubated for 2 h at 37 degrees C with vinblastine (50 microM) to obtain an accumulation of autophagosomes (to 3-5-times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl-phenylalanine-2-naphthylamide (GPN), a cathepsin-C substrate, and methionine-O-methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sedimentability of acid phosphatase and a nearly complete loss of 125I-TC-AOM sedimentability in substrate-treated preparations from control or vinblastine-treated cells. The lysosomes of cells treated with leupeptin or asparagine were largely resistant to the action of GPN, probably as a result of interference with cathepsin-C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe, as indicated by a reduction in sedimentable LDH, but GPN had no effect on either autophagosomes or mitochondria. The ability of GPN to selectively destroy lysosomes without affecting the autophagosomes of vinblastine-treated cells should make GPN treatment a useful aid in the purification of rat liver autophagosomes. Topics: Animals; Autophagy; Cathepsin C; Cell Fractionation; Centrifugation; Centrifugation, Density Gradient; Dipeptides; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Male; Methionine; Phagosomes; Rats; Rats, Wistar; Vinblastine | 1994 |
Differential phosphorylation in normal and leukemic granulocytes in response to phorbol 12-myristate 13-acetate.
Granulocytes from the peripheral blood of patients with chronic myeloid leukemia (CML) exhibit a number of functional defects. To explore the relationship of these aberrations to signal transduction, granulocytes from normal subjects and CML patients were labelled with 32Pi, stimulated with phorbol myristate acetate (PMA) and the phosphoproteins (Pps) in the unstimulated and stimulated cells analyzed by 2D-SDS-PAGE followed by autoradiography. Results show that there are six distinct reproducibly phosphorylated proteins referred to as Pp1-Pp6 identifiable in the basal patterns of the resting granulocytes. Amongst these, Pp1 and Pp5 are more intensely phosphorylated and Pp3 is very faint or absent in unstimulated CML cells, relative to the normal granulocytes. On stimulation of normal cells with PMA, Pp1, Pp3, Pp4 and Pp6 exhibit distinct patterns of phosphorylation-dephosphorylation. In the CML cells, however, Pp1 and Pp4 are unresponsive to PMA. We conclude that PKC-mediated functions involving Pp1, Pp3 and Pp4 are most probably defective in CML cells. Topics: Autoradiography; Electrophoresis, Gel, Two-Dimensional; Granulocytes; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leupeptins; Neoplasm Proteins; Phosphoproteins; Phosphorylation; Tetradecanoylphorbol Acetate | 1994 |
Mechanism of trimethylamine-induced inhibition of macromolecular synthesis by mouse embryos in culture.
The effects of trimethylamine (TMA) on uptake mechanisms and lysosomal function were studied in mouse embryos, isolated yolk sacs and limb buds. TMA at 0.75 mM did not inhibit uptake of [14C]sucrose by yolk sacs of day 9 embryos or by day 15 isolated yolk sacs but did inhibit uptake of 125I-labelled bovine serum albumin ([125I]BSA) by day 15 isolated yolk sacs. Concentrations of TMA up to 2.5 mM did not inhibit lysosomal degradation of [125I]BSA by isolated yolk sacs, as judged by the release of trichloroacetic acid (TCA)-soluble radioactivity into the culture media. The inhibition of [125I]BSA uptake induced by TMA was reversible on removal of TMA. When day 8 embryos were cultured in serum containing [3H]leucine-labelled proteins, uptake and incorporation of radioactivity in 0.75 mM TMA-treated embryos was 47 and 44%, respectively, of that in untreated controls. TMA at 0.75 mM did not inhibit the uptake and incorporation of free [3H]leucine into embryonic protein nor the amount of free [3H]leucine taken up or incorporated into protein by day 12 isolated limb buds. It is concluded that the reduced macromolecular synthesis in embryos exposed to TMA is due to an inhibition of receptor-mediated uptake of nutrients by the yolk sac. Topics: Animals; Blood; Culture Techniques; Embryo, Mammalian; Extremities; Female; Insulin; Insulin-Like Growth Factor II; Leucine; Leupeptins; Male; Methylamines; Mice; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Sucrose; Vitamin A; Yolk Sac | 1994 |
Purification and characterization of the 1,200-kDa subfragment of connectin filaments produced by 0.1 mM calcium ions.
When myofibrils prepared from chicken leg muscle were treated with a solution containing 0.1 mM CaCl2 and 30 micrograms/ml of leupeptin, alpha-connectin, which exists as a longitudinal thin filament in a sarcomere, was split into beta-connectin and a 1,200-kDa subfragment. The native subfragment was successfully purified without using any denaturant: It was extracted with 1 M KI solution from the Ca-treated myofibrils and purified by TSKgel G6000PW column chromatography. About 10 mg of the subfragment was yielded from 100 g of starting muscle. Using immunofluorescence microscopy and immunoelectron microscopy, we show here that polyclonal antibodies against the 1,200-kDa subfragment bind to the Z-disk and the epitope, which is about 0.34 micron apart from the Z-disk at a sarcomere length of 2.6 microns; the 1,200-kDa subfragment constitutes the proximal region of connectin filaments. Purified alpha-actinin decorated alpha-connectin and the 1,200-kDa subfragment on nitrocellulose blots of myofibrillar proteins separated by SDS-PAGE. Therefore, we conclude that connectin filaments are anchored to the Z-disk by the binding of the 1,200-kDa subfragment to alpha-actinin. Topics: Actinin; Animals; Calcium Chloride; Carrier Proteins; Chickens; Chromatography, Gel; Connectin; Electrophoresis, Polyacrylamide Gel; Leupeptins; Microscopy, Fluorescence; Molecular Weight; Muscle Proteins; Myofibrils; Peptide Fragments; Protein Binding; Protein Kinases | 1994 |
Stimulation of transcytosis of the polymeric immunoglobulin receptor by dimeric IgA.
The polymeric immunoglobulin receptor (pIgR) is transcytosed from the basolateral to the apical surface of polarized epithelial cells. We have previously shown that phosphorylation of Ser-664 in the cytoplasmic domain of the pIgR is a signal for its transcytosis. We now report that binding of a physiological ligand, dimeric IgA, to pIgR stimulates pIgR transcytosis. This stimulation occurs in both the presence or absence of Ser-664 phosphorylation. We have used three methods to measure transcytosis of the pIgR. (i) The pIgR was biosynthetically labeled and its cleavage to secretory component after transcytosis was measured. (ii) The pIgR was labeled with biotin at the basolateral surface. After transcytosis, release of the biotin-labeled secretory component into the apical medium was measured. (iii) Transcytosis of a ligand bound to the pIgR was measured. All three methods indicated that dimeric IgA stimulates transcytosis of the pIgR. Topics: Animals; Biological Transport; Cell Line; Cell Polarity; Dogs; Epithelium; Immunoglobulin A; Immunoglobulin Fab Fragments; Leupeptins; Mutagenesis, Site-Directed; Phosphoserine; Receptors, Immunologic; Secretory Component; Structure-Activity Relationship; Transferrin | 1994 |
Kinetic studies on phagocytosis and lysosomal digestion of rod outer segments by human retinal pigment epithelial cells in vitro.
Using novel methodology, this study describes the kinetics of rod outer segment (ROS) phagocytosis and digestion by human retinal pigment epithelial (RPE) cells in vitro and examines the effect of certain lysosomal enzyme inhibitors on ROS digestion in these cells. Human RPE cells displayed saturation of phagocytosis with respect to both ROS concentration and time. While surface-binding and ingestion phases of ROS phagocytosis saturated after 24-36 h, the rate of ROS digestion reached a maximal level within 24 h. Increasing the concentration of zinc in the culture medium from 1.9 to 100 microM had no significant effect on ROS digestion. The effects of swainsonine (an alpha-mannosidase inhibitor), pepstatin (an aspartic protease inhibitor), and leupeptin (a cysteine protease inhibitor) were also examined. At 6 h, ROS digestion was reduced 27.3 +/- 15.3% by swainsonine, 69.4 +/- 20.9% by pepstatin, and 77.0 +/- 14.4% by leupeptin. The effect of these inhibitors declined with increasing time. This study is the first to demonstrate the functional importance of cysteine and aspartic proteases in the digestion of ROS by RPE cells in vitro. Topics: alpha-Mannosidase; Animals; Aspartic Acid Endopeptidases; Cattle; Cell Membrane; Cells, Cultured; Cysteine Proteinase Inhibitors; Humans; Kinetics; Leupeptins; Lysosomes; Mannosidases; Microscopy, Electron; Microscopy, Electron, Scanning; Pepstatins; Phagocytosis; Pigment Epithelium of Eye; Rod Cell Outer Segment; Swainsonine; Zinc | 1994 |
Effects of lysosomal protease inhibitors on the degradation of acetylated low density lipoprotein in cultured rat peritoneal macrophages.
The effect of protease inhibitors, leupeptin and pepstatin A, on the metabolism of acetylated low density lipoprotein (acetyl-LDL) was investigated in cultured rat peritoneal macrophages and compared with that of chloroquine. While both leupeptin and pepstatin inhibited the proteolytic degradation of 125I-acetyl-LDL, a combination of both showed an additive effect. Similar to chloroquine, both protease inhibitors diminished [3H] oleate incorporation into cellular cholesteryl[3H] oleate and increased cholesterol content of macrophages. These results suggest that both thiol protease and cathepsin D participate in the physiological degradation of apolipoprotein in macrophages. The inhibition of apolipoprotein degradation seemed to have an effect on cholesterol metabolism in macrophages cultured with acetyl-LDL. Topics: Acetylation; Animals; Cells, Cultured; Cholesterol; Humans; Iodine Radioisotopes; Leupeptins; Lipoproteins, LDL; Lysosomes; Macrophages, Peritoneal; Oleic Acid; Pepstatins; Protease Inhibitors; Radioligand Assay; Rats; Rats, Sprague-Dawley; Tritium | 1994 |
Peptidase modulation of vasoactive intestinal peptide pulmonary relaxation in tracheal superfused guinea pig lungs.
The effects of enzyme inhibitors on vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (PaO) and VIP-like immunoreactivity (VIP-LI) recovery were studied in isolated tracheal superfused guinea pig lungs. In the absence of inhibitors, VIP 0.38 (95% CI 0.33-0.54) nmol/kg animal, resulted in a 50% decrease in PaO and 33% of a 1 nmol/kg VIP dose was recovered as intact VIP. In the presence of two combinations of enzyme inhibitors, SCH 32615 (S, 10 microM) and aprotinin (A, 500 tyrpsin inhibitor units [TIU]/kg) or S and soybean trypsin inhibitor (T, 500 TIU/kg), VIP caused a significantly greater decrease in PaO and greater quantities of VIP were recovered from lung effluent (both P < 0.001). The addition of captopril, (3 microM), leupeptin (4 microM), or bestatin (1 microM) failed to further increase pulmonary relaxation or recovery of VIP-LI. When given singly, A, T, and S did not augment the effects or recovery of VIP. The efficacy of S (a specific inhibitor of neutral endopeptidase [NEP]) and A and T (serine protease inhibitors) thus implicated NEP and at least one serine protease as primary modulators of VIP activity in the guinea pig lung. We sought to corroborate this finding by characterizing the predominant amino acid sites at which VIP is hydrolized in the lung. When [mono(125I)iodo-Tyr10]VIP was offered to the lung, in the presence and absence of the active inhibitors, cleavage products consistent with activity by NEP and a tryptic enzyme were recovered. These data demonstrate that NEP and a peptidase with an inhibitor profile and cleavage pattern compatible with a tryptic enzyme inactivate VIP in a physiologically competitive manner. Topics: Animals; Aprotinin; Captopril; Dose-Response Relationship, Drug; Endopeptidases; Guinea Pigs; Leucine; Leupeptins; Lung; Male; Muscle Relaxation; Muscle, Smooth; Perfusion; Protease Inhibitors; Time Factors; Trachea; Trypsin Inhibitors; Vasoactive Intestinal Peptide | 1993 |
Protective effect of the protease inhibitor leupeptin against myocardial stunning.
To elucidate whether activation of intracellular protease causes the contractile dysfunction of post-ischemic reperfused heart (stunned myocardium), the effect of leupeptin, a cysteine-protease inhibitor, was evaluated in isolated guinea pig hearts. Left ventricular (LV) isovolumic pressure was measured in hearts reperfused after global ischemia (15 min, 37 degrees C). Recovery of developed pressure during reperfusion in hearts treated with 50 microM leupeptin was significantly greater than that in untreated hearts [94.3 +/- 3.2% of control, n = 11 (mean +/- SEM] vs. 78.1 +/- 3.1%, n = 14), and was almost identical to that in nonischemic control (93.5 +/- 1.6%, n = 11). Maximal Ca(2+)-activated pressure, the intact-heart correlate of maximal Ca(2+)-activated force, was also evaluated at the end of experiments during tetani elicited by rapid pacing after exposure to ryanodine. Maximal Ca(2+)-activated pressure in hearts treated with leupeptin (168 +/- 4.6 mm Hg) was significantly higher than in untreated stunned hearts (144.5 +/- 5.7 mm Hg), but significantly lower than in nonischemic control (198.4 +/- 5.5 mm Hg). These results indicate that leupeptin has a protective effect against myocardial stunning. In coupling with previous reports of transient increase in intracellular [Ca2+] during ischemia and/or reperfusion, activation of proteases by Ca2+ overload is suggested to play a significant role in myocardial stunning. Topics: Animals; Calpain; Enzyme Activation; Guinea Pigs; Hemodynamics; In Vitro Techniques; Leupeptins; Male; Myocardial Reperfusion Injury; Protease Inhibitors; Reference Values | 1993 |
Processing of endogenously synthesized hen egg-white lysozyme retained in the endoplasmic reticulum or in secretory form gives rise to a similar but not identical set of epitopes recognized by class II-restricted T cells.
To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment. Topics: Amino Acid Sequence; Animals; Cells, Cultured; Chickens; Egg Proteins; Endoplasmic Reticulum; Epitopes; Histocompatibility Antigens Class II; Leupeptins; Mice; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocytes; Transfection | 1993 |
Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with the oolemma: inhibitor studies using the golden hamster.
The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction. PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes. Topics: Acrosin; Acrosome; Animals; Benzamidines; Cell Membrane; Cricetinae; Female; In Vitro Techniques; Leupeptins; Male; Membrane Fusion; Mesocricetus; Microscopy, Electron; Oocytes; Protease Inhibitors; Sperm-Ovum Interactions; Spermatozoa; Trypsin | 1993 |
Hepatectomy impairs hepatic processing of somatostatin-14.
Somatostatin-14 (SS-14) inhibits the hepatotrophic effect of a variety of growth factors in cultured hepatocytes. We hypothesized that hepatic somatostatin processing might be altered during regeneration. Male Sprague-Dawley rats underwent the intraportal injection of radiolabeled SS-14 after sham or 70% hepatectomy. To study the mechanisms of hepatic SS-14 transport in the rat, the lysosomal enzyme inhibitors, chloroquine and leupeptin, and the microtubule inhibitors, vinblastine and colchicine, were administered 1 to 2 hours prior to the intraportal injection of SS-14. Bile was collected, organs were weighed, and radioactivity was quantitated. The analysis of serial timed collections of bile revealed that, for saline, chloroquine, and leupeptin, peak biliary radioactivity appeared at 20 minutes. Pretreatment with vinblastine and colchicine abolished the 20-minute peak of radioactivity. The appearance of biliary and hepatic iodine 125-SS-14 (125I-[tyr11]-SS-14) at various times after 70% hepatectomy showed a significant decrease starting at 2 hours, which persisted for up to 24 hours. In regenerating liver, both vinblastine and chloroquine decreased 125I-[tyr11]-SS-14 in bile and the liver. In summary, after sham or 70% hepatectomy, vinblastine and colchicine inhibit biliary and increase hepatic 125I-[tyr11]-SS-14 accumulation. After 70% hepatectomy was performed, chloroquine also inhibited 125I-[tyr11]-SS-14 accumulation. We concluded that an important mechanism for hepatic regeneration is decreased responsiveness to SS-14, by decreased SS-14 uptake and increased SS-14 degradation. Topics: Animals; Biological Transport; Chloroquine; Colchicine; Hepatectomy; Iodine Radioisotopes; Leupeptins; Liver; Liver Regeneration; Male; Rats; Rats, Sprague-Dawley; Somatostatin; Vinblastine | 1993 |
Regulation of pathways within cultured epithelial cells for the transcytosis of a basal membrane-bound peroxidase-polylysine conjugate.
A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used as a non-specific adsorptive probe to study transcytosis in MDCK strain I and Caco-2 epithelial cells. As we have shown previously, HRP-PLL transcytosis proceeds via an intracellular, non-lysosomal proteolytic compartment in MDCK cells; yet, this compartment is utilized for transcytosis only in the basal-to-apical direction (Taub, M. E. and Shen, W.-C. J. Cell. Physiol., 150, 283-290, 1992). Using size exclusion chromatography, we demonstrate that the PLL moiety of the conjugate is effectively cleaved during transcytosis, thus releasing free HRP from the apical surface of the cells. Pulse-chase studies indicate that approximately 6% of basolateral surface-associated HRP-PLL conjugate in Transwell-grown cell monolayers enters the basal-to-apical transcytotic pathway. Brief (1 hour) treatment with 160 nM phorbol ester (PMA), a protein kinase C stimulator, elicits a 2-fold increase in the rate and amount of HRP-PLL transcytosis following a 1 hour lag time. Treatment with 1.6 micrograms/ml brefeldin A (BFA) inhibits HRP-PLL transcytosis by approximately 30%; additionally, BFA is able to abolish completely the PMA stimulatory effect. Removal of BFA causes a re-establishment of the normal rate of transcytosis within 2 hours, demonstrating the reversibility of BFA inhibition with respect to HRP-PLL transcytosis. Thus, PMA most likely elicits an increase in the amount of basally internalized conjugate delivered to BFA-sensitive transcytotic compartments.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Biological Transport; Brefeldin A; Cell Polarity; Cyclopentanes; Dose-Response Relationship, Drug; Endocytosis; Epithelium; Horseradish Peroxidase; Leupeptins; Models, Biological; Polylysine; Protease Inhibitors; Tetradecanoylphorbol Acetate; Time Factors | 1993 |
pH-dependent binding constants for the inhibition of thrombin by transition state analogs.
Topics: Amino Acid Sequence; Animals; Antithrombins; Arsenicals; Boronic Acids; Carboxylic Acids; Cattle; Dipeptides; Hydrogen-Ion Concentration; Kinetics; Leupeptins; Molecular Sequence Data; Peptides; Piperidines; Thrombin | 1993 |
Degradation of rat C-reactive protein by macrophages.
Rat C-reactive protein (CRP) is a serum glycoprotein belonging to the 'pentraxin' family of proteins. In this study we have shown the specific binding of 125I-CRP to rat peritoneal macrophages at 4 degrees C. This binding was dependent upon incubation time, CRP and cell concentrations, and was not inhibited by either phosphorylcholine or human IgG. At 37 degrees C, the surface-bound 125I-CRP was internalized and degraded. The degradation of 125I-CRP was measured by the formation of 125I-labelled trichloroacetic-acid-soluble CRP peptides by either precipitation assays or by h.p.l.c. of the incubation medium using a gel-filtration column. Since chloroquine and leupeptin inhibited CRP degradation, it was concluded that degradation of CRP occurred in the lysosomal compartment of the macrophage. There was an absolute requirement for the presence of bivalent cations (Ca2+ and Mg2+) in the incubation medium for the binding and degradation of CRP, which could be inhibited by EDTA but not by phosphorylcholine or human IgG. H.p.l.c. analysis of the medium obtained from incubation of macrophages with 125I-CRP revealed the presence of 125I-labelled low-M(r) peptides, the formation of which was dependent upon incubation time. Topics: Animals; Biological Transport; C-Reactive Protein; Cations, Divalent; Cells, Cultured; Chloroquine; Dose-Response Relationship, Drug; Edetic Acid; Immunoglobulin G; Leupeptins; Macrophages, Peritoneal; Peptides; Phosphorylcholine; Rats; Rats, Sprague-Dawley; Time Factors | 1993 |
Formation of autophagosomes during degradation of excess peroxisomes induced by administration of dioctyl phthalate.
The formation of autophagosomes in rat hepatocytes was investigated during degradation of excess peroxisomes. Rat liver peroxisomes were markedly proliferated by administration of dioctyl phthalate (DEHP) for 2 weeks. When the animals were fed on normal diet for a week further, the number and size of the peroxisomes recovered to normal. The recovery process was confirmed by the assay and immunoblot analysis of acyl-CoA oxidase and catalase. During the recovery process, only a few autophagosomes were noted. However, when leupeptin (2 mg/100 g body weight) was injected into these animals, there was a marked accumulation of autophagosomes in the hepatocytes. Using this as an experimental model, the early stage of the autophagosome formation was analyzed by electron microscopy. Twenty minutes after the injection, isolation membranes surrounding the target organelles appeared. They were characterized by double layers with a narrow cisternal space and were sometimes continuous with the rough endoplasmic reticulum. Between the inner membrane of the isolation membranes and the enclosed organelles, electron-dense bridges were noted. Forty minutes after leupeptin injection, the lumen of the isolation membranes were enlarged and the inner membrane attached to the entrapped material. Enzyme cytochemical staining showed that the isolation membranes were negative for acid phosphatase and thiamine pyrophosphatase, but were strongly positive for glucose-6-phosphatase (G6Pase). The enlarged cisternae of the isolation membranes of the early autophagic vacuoles were in part positive for this enzyme, but gradually became negative with time. Similarly, the G6Pase activity was lost when the inner membrane was degraded. The results suggest 1) that the process of degradation of excess peroxisomes is rapid and carried out by the autophagic system in hepatocytes and 2) that the isolation membranes enclosing the target organelles are derived from the endoplasmic reticulum. Topics: Animals; Autophagy; Diethylhexyl Phthalate; Histocytochemistry; Leupeptins; Liver; Male; Microbodies; Microinjections; Rats; Rats, Wistar; Vacuoles | 1993 |
Polypeptide import and degradation by isolated lysosomes.
We reported previously that lysosomes derived from human diploid fibroblasts import and degrade polypeptides and that these processes are stimulated by ATP and by the heat shock cognate protein of 73 kDa (hsc73). We now report several new aspects of this in vitro proteolytic pathway. (a) Among four polypeptides tested, this pathway appears to be selective for those containing KFERQ-like peptide motifs. (b) Substrate proteins specifically bind to a protein-containing site on lysosomal membranes. (c) Lysosomes derived from serum-deprived cells are twice as active as those from serum-supplemented cells. (d) A portion of intracellular hsc73 is associated with lysosomes, and the amount of lysosomal hsc73 increases in response to serum withdrawal. Additional characterization of this proteolytic pathway not reported previously shows that intact lysosomes are required, the import process is saturable with an apparent Km of 5 microM for RNase S-peptide, and reducing agents activate this lysosomal import and degradation pathway. Topics: Amino Acid Sequence; Biological Transport; Cell Line; Heat-Shock Proteins; Humans; In Vitro Techniques; Leupeptins; Lysosomes; Molecular Sequence Data; Peptide Fragments; Peptides; Ribonuclease, Pancreatic; Ribonucleases; Structure-Activity Relationship | 1993 |
Normal cellular processing of the beta-amyloid precursor protein results in the secretion of the amyloid beta peptide and related molecules.
Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta peptide (A beta). This peptide is derived from a large integral membrane protein, the beta-amyloid precursor protein (beta APP), by proteolytic processing. The A beta has previously been found only in the brains of patients with Alzheimer's disease or advanced aging. We describe here the finding that A beta is produced continuously by normal processing in tissue culture cells. A beta and closely related peptides were identified in the media of cells transfected with cDNAs coding for beta APP in a variety of cell lines and primary tissue cultured cells. The identity of these peptides was confirmed by epitope mapping and radiosequencing. Peptides of a molecular weight of approximately 3 and approximately 4 kDa are described. The 4 kDa range contains mostly the A beta and two related peptides starting N-terminal to the beginning of A beta. In the 3 kDa range, the majority of peptides start at the secretase site; in addition, two longer peptides were found starting at amino acid F(4) and E(11) of the A beta sequence. To identify the processing pathways which lead to the secretion of these peptides, we used a variety of drugs known to interfere with certain cell biological pathways. We conclude that lysosomes may not play a predominant role in the formation of 3 and 4 kDa peptides. We show that an acidic environment is necessary to create the N-terminus of the A beta and postulate that alternative secretory cleavage might result in the formation of the N-terminus of A beta and related peptides. This cleavage takes place either in the late Golgi, at the cell-surface or in early endosomes, but not in lysosomes. The N-terminus of most of the 3 kDa peptides is created by secretory cleavage on the cell surface or within late Golgi. Topics: Amino Acid Sequence; Ammonium Chloride; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Brefeldin A; Cell Line; Colchicine; Cyclopentanes; Golgi Apparatus; Humans; Kidney; Leupeptins; Lysosomes; Molecular Sequence Data; Monensin; Nocodazole; Protein Processing, Post-Translational; Transfection | 1993 |
Routes of excretion of neuronal lysosomal dense bodies after ventricular infusion of leupeptin in the rat: a study using ubiquitin and PGP 9.5 immunocytochemistry.
To determine the rate and routes of removal of lysosomal, lipofuscin-like dense bodies from neurons, the protease inhibitor, leupeptin, was infused into the lateral ventricle of rats for up to nine days. After seven days a number of animals were then allowed to recover. The formation and later disappearance of dense bodies was followed by morphology and immunocytochemistry. After 48 h of infusion lysosomal dense bodies in large numbers appeared in cortical, hippocampal and cerebellar neurons, which also showed increased ubiquitin immunoreactivity, as well as in other cell types. By 3-4 days ubiqutin-immunoreactive dense bodies were equally distributed between neurons and astroglia. After seven to nine days of infusion ubiquitin immunoreactive dense bodies filled neuronal perikarya, dendrites and expanded initial segments of many axons and were abundant in glial processes. All dense bodies studied by electron microscopy were ubiquitin immunoreactive. After four days of recovery dense bodies were markedly fewer in neuronal perikarya, and virtually all were now within glial processes. From 7 to 28 days of recovery, when most neurons appeared normal, lipofuscin bodies remained in axon initial segments and in reduced numbers in glial processes, particularly around blood vessels and beneath the pia of hippocampus and of cerebellar cortex. Thus, neurons probably have a steady passage of short lived proteins through the lysosomal excretory pathway. The observed temporal sequence of events on recovery suggests that secondary lysosomes probably pass rapidly from neuronal perikarya and dendrites to astrocytes and thus to the vascular bed or pia-arachnoid. The mechanism of cell-to-cell transfer is not clear from this study. Topics: Animals; Cerebral Ventricles; Female; Immunohistochemistry; Infusions, Parenteral; Leupeptins; Lysosomes; Microscopy, Electron; Neurons; Rats; Rats, Wistar; Thiolester Hydrolases; Ubiquitin Thiolesterase; Ubiquitins | 1993 |
Release of DR molecules from complexes with invariant chain through the formation of a C-terminal 25 kDa invariant chain fragment.
To investigate how class II major histocompatibility complex (MHC) molecules are released from complexes with invariant chain (Ii), we studied a 25 kDa Ii fragment (p25) detected by Western blotting in affinity chromatographed DR preparations. The p25 species corresponds to the non-transmembrane, C-terminal Ii fragment 107-232. It was determined by gel filtration chromatography that the p25 fragment has a relative molecular mass (M(r)) of 46 kDa, indicating that this Ii fragment is present as dimers in B cell lysate. Two independent approaches were followed to demonstrate that generation of the p25 fragment takes place shortly before, or concomitantly to, loading of class II MHC molecules with antigen fragments. First, it was shown that a fraction of the p25 molecules is resistant to endoglycosidase H digestion, indicating that the p25 polypeptide can exit the endoplasmic reticulum (ER) and is transported at least to the cis-Golgi compartment. Second, treatment of class II MHC-positive B cells with leupeptin blocks the formation of p25, further indicating that this Ii fragment is generated in the endosomal compartment. The role of the p25 Ii species in the assembly of complexes between peptides and DR molecules was then investigated. While the p25 fragment was totally unable to prevent binding of a synthetic tetanus toxin peptide to DR molecules, the full-length Ii species (p33/35) effectively inhibited peptide binding, indicating that, by contrast with the p33/35 species, the p25 fragment does not occlude the peptide binding site of DR molecules. We concluded that the p25 fragment, which is produced by proteolytic cleavage at the N-terminal side of Methionine 107, has a decreased affinity for DR molecules as compared with the p33/35 species. Dissociation of the p25 fragment from DR molecules exposes the peptide binding site, which is thus made accessible for antigen fragments. This model of the complexes between DR and antigen fragments proposes that a stretch of Ii prevents peptide binding by occluding the peptide binding site without directly occupying it. Topics: Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Cell Compartmentation; Cell Line, Transformed; Cells, Cultured; Chromobox Protein Homolog 5; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Golgi Apparatus; Herpesvirus 4, Human; Hexosaminidases; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Leupeptins; Lysosomes; Peptide Fragments; Protein Binding; Protein Conformation; Tetanus Toxin | 1993 |
Effect of high hydrostatic pressure on the conversion of alpha-connectin to beta-connectin.
The factors affecting the conversion of alpha-connectin to beta-connectin induced by pressurization of muscle were investigated over a pressure range from 100 to 400 MPa by using SDS-PAGE and immunoblot analysis. When muscles were exposed to high pressures, the conversion of alpha-connectin to beta-connectin was the most pronounced at a pressure of 300 MPa, and the appearance of 1,200-kDa peptide accompanied by conversion of alpha- to beta-connectin was observed. Connectin was relatively resistant to degradation under a pressure of 400 MPa. The degradative products of beta-connectin reactive with mAb 2D4 were not observed. The effect of high pressure on connectin in isolated myofibrils was similar to that on connectin in muscle. Addition of leupeptin and E-64 to the isolated myofibrils resulted in the prevention of the degradation of connectin at each stage of the pressurization. The ability of calcium-activated protease (calpain) to hydrolyze connectin from alpha to beta gradually declined with increasing pressure. The results indicate that calpain is responsible for the pressure-induced conversion of alpha- to beta-connectin. The rate of this conversion is probably regulated by the pressure-dependent structural change of alpha-connectin and inactivation of calpain. Topics: Animals; Antibodies, Monoclonal; Calcium; Calpain; Connectin; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Hydrostatic Pressure; Leucine; Leupeptins; Molecular Weight; Muscle Proteins; Muscles; Protein Kinases; Rabbits | 1993 |
Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M.
Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology. Topics: Amino Acid Sequence; Blood Proteins; Calpain; Down-Regulation; Erythrocyte Membrane; Glycoproteins; Humans; In Vitro Techniques; Leupeptins; Membrane Proteins; Molecular Sequence Data; Phosphorylation; Protease Inhibitors; Protein Kinase C; Tetradecanoylphorbol Acetate | 1993 |
Substratum acidification and proteinase activation by murine B16F10 melanoma cultures.
Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 +/- 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride. EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line. Topics: Animals; Cathepsin B; Cell Line; Endopeptidases; Enzyme Activation; Fluoresceins; Gels; Hydrogen-Ion Concentration; Leupeptins; Mice; Serum Albumin, Bovine; Tumor Cells, Cultured | 1993 |
Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells.
Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Calcium Channels; GTP-Binding Proteins; Guanosine Diphosphate; Hirudins; In Vitro Techniques; Isradipine; Leupeptins; Membrane Potentials; Muscle, Smooth, Vascular; Portal Vein; Rats; Rats, Wistar; Thionucleotides; Thrombin; Vasoconstriction | 1993 |
Calcium-induced degradation of the inositol (1,4,5)-trisphosphate receptor/Ca(2+)-channel.
Ca(2+)-induced degradation of the neuronal inositol (1,4,5)-trisphosphate receptor, a protein which regulates Ca(2+)-release from intracellular stores, has been examined. The IP3-receptor, immunopurified from rat cerebellum, appeared to be an excellent substrate for purified Ca(2+)-activated neutral protease (calpain). Incubation of membranes or immunopurified IP3-receptor with Ca2+ and cerebellar cytosol also resulted in degradation of the receptor. Two main fragments with approximate molecular masses of 130 and 95 kDa were generated, both of which appeared to derive from the carboxyterminal Ca(2+)-channel-containing part of the protein. These data suggest that activation of the IP3-receptor, by causing increases in intracellular [Ca2+], might result in degradation of the N-terminal, IP3-binding part of the receptor. Topics: Animals; Astrocytes; Calcium; Calcium Channels; Calpain; Cell Membrane; Cerebellum; Chromatography, Affinity; Dipeptides; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leupeptins; Male; Molecular Weight; Neurons; Phosphorylation; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Testis; Tumor Cells, Cultured; Vas Deferens | 1993 |
Proteolysis results in altered leak channel kinetics and elevated free calcium in mdx muscle.
Resting free calcium levels ([Ca2+]i) are elevated in Duchenne human myotubes and mdx mouse muscle and myotubes which lack the gene product dystrophin at the sarcolemma. Increased net muscle protein degradation has been directly related to this elevated [Ca2+]i. The [Ca2+]i rise may result from increased calcium influx via leak channels, which have increased opening probabilities (Po) in dystrophic cells. Dystrophin, therefore, might directly regulate leak channel activity. In intact mdx soleus muscles, protein degradation was reduced to normal levels by leupeptin, a thiol protease inhibitor. In muscle homogenates, leupeptin also abolished calcium-induced increases in protein degradation. When mouse myotubes were cultured in the continuous presence of leupeptin (50 microM), the elevation in mdx resting [Ca2+]i was prevented. Leak channel Po increased with age in mdx myotubes, whereas leupeptin-treated mdx leak channel opening probabilities were always lower or equal to the Po for untreated normal myotubes. These results indicate that increased leak channel activity in dystrophic muscle results in elevated [Ca2+]i levels, but also suggest that dystrophin does not directly regulate channel activity. Instead the results suggest that proteolysis may be responsible for the altered gating of calcium leak channels. The resultant increased channel Po in turn elevates [Ca2+]i, which further increases proteolytic activity in a positive feedback loop, leading to the eventual necrosis of the muscle fibers. Topics: Animals; Calcium; Calcium Channels; Calpain; Cells, Cultured; Clenbuterol; Dose-Response Relationship, Drug; Dystrophin; Feedback; Glycoproteins; Kinetics; Leupeptins; Mice; Mice, Mutant Strains; Muscles; Muscular Dystrophy, Animal | 1993 |
Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli.
Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated. The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 +/- 1.69 vs 93.56 +/- 10.40 nmol/mg protein/min) and was dose dependent. Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa. The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells. Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa. However, STa had no direct role on the enzyme activity. Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes. Topics: Alkaloids; Animals; Bacterial Toxins; Calcium; Calmodulin; Cyclic AMP; Cyclic GMP; Diglycerides; Enterotoxins; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Intestinal Mucosa; Jejunum; Leupeptins; Magnesium; Membrane Proteins; Phosphatidylserines; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Staurosporine | 1993 |
A system for characterizing cellular and molecular events in programmed neuronal cell death.
A model system has been established in which PC12 cells are converted to neuronal-like cells that undergo transcription-dependent cell death following removal of NGF. Nineteen sublines of PC12 cells were tested to establish parameters for making cells dependent on NGF for survival. In most sublines, a relatively small percentage of cells become dependent on NGF for survival, and following removal of NGF, most of the cells begin proliferating in serum-containing medium. In several sublines, however, a significant percentage of cells die following removal of NGF. One of these sublines, PC6-3, can be grown under conditions in which 90% of the cells undergo transcription-dependent cell death following removal of NGF in either serum-free or serum-containing medium. Fourteen hours after removing NGF, 50% of the cells are committed to die, while initial morphological signs of cell death as determined by time-lapse videomicroscopy occur 2-6 hr later and include loss of neurites followed by a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death can be blocked by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF activates an endonuclease that causes nucleosomal laddering of the DNA; however, endonuclease activity does not appear to be required for cell death. In agreement with previous studies (Batistatou and Greene, 1991; Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo transcription-independent cell death when shifted into serum-free medium in the absence of growth factors, all cell lines tested except for one die when cultured in RPMI medium lacking growth factors. DNA fragmentation is a prominent feature of transcription-independent cell death, and death can be blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl. The PC12 model system described here should be useful for identifying cell death genes and for characterizing cellular and molecular events in programmed neuronal cell death. Topics: Animals; Apoptosis; Aurintricarboxylic Acid; Bucladesine; Cell Division; Cell Survival; Dactinomycin; DNA, Neoplasm; Epidermal Growth Factor; Fibroblast Growth Factor 2; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Nerve Growth Factors; Neurons; PC12 Cells; Time Factors; Transcription, Genetic; Video Recording | 1993 |
Structural analysis of proteolytic products of MHC class II-invariant chain complexes generated in vivo.
The MHC class II alpha beta heterodimer associates with invariant (I) chain in the endoplasmic reticulum and remains associated until the complex reaches a post-Golgi compartment. During early stages of transport, I chain blocks peptide binding to alpha beta dimers. I chain is proteolytically cleaved in a post-Golgi compartment releasing alpha beta dimers that can bind antigenic peptides and transport them to the cell surface. Human B lymphoblastoid cell lines grown in leupeptin, a sulfhydryl protease inhibitor, accumulate a partial proteolytic product of the I chain called leupeptin-induced protein (LIP). LIP remains associated with alpha beta dimers. We find, using chemical cross-linking, sucrose gradient sedimentation, and size exclusion chromatography, that the alpha beta LIP complex retains the nine-subunit structure described for alpha beta I complexes. Unlike the alpha beta I complex, in certain detergents the alpha beta LIP nonamer is unstable and dissociates into trimers containing one alpha, beta, and LIP molecule. This finding emphasizes the reported stoichiometry of the alpha beta I complex as a nine-subunit structure comprised of three alpha beta I trimers. Also, these data indicate that the region(s) of I chain necessary for retaining the nonameric structure lie within the LIP fragment, but that domains to the C-terminus of the LIP cleavage site act to further stabilize the nine chain structure. In addition, alpha beta I complexes containing forms of human I chain encoding the p35/p43 N-terminal cytoplasmic extension responsible for endoplasmic reticulum retention can transport to post-Golgi proteolytic compartments where LIP is formed. Topics: Antigens, Differentiation, B-Lymphocyte; Biological Transport; Cell Line; Centrifugation, Density Gradient; Cholic Acids; Chromatography, Gel; Histocompatibility Antigens Class II; Humans; Leupeptins; Molecular Weight; Peptide Fragments | 1993 |
X-ray crystallographic structure of a papain-leupeptin complex.
The three-dimensional structure of the papain-leupeptin complex has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S' subsites; (ii) the 'carbonyl' carbon atom of the inhibitor is covalently bound by the Cys-25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the 'carbonyl' oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln-19 and Cys-25. Topics: Amino Acid Sequence; Binding Sites; Crystallography; In Vitro Techniques; Leupeptins; Models, Molecular; Molecular Sequence Data; Papain; Protein Structure, Tertiary; X-Ray Diffraction | 1993 |
Leupeptin-binding site(s) in the mammalian multicatalytic proteinase complex.
The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called 'trypsin-like' activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the 'trypsin-like' activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding. Topics: Amino Acid Sequence; Animals; Antipain; Binding Sites; Cysteine Endopeptidases; Ethylmaleimide; Leupeptins; Liver; Molecular Sequence Data; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats; Trypsin | 1993 |
beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.
We have analyzed the cellular processing pathways which produce the 4-kDa amyloid beta-peptide (A beta) and a 3-kDa derivative (p3) of the beta-amyloid precursor protein (beta APP) found in conditioned media of tissue culture cells and in cerebrospinal fluid. Pulse-chase experiments reveal that both peptides are secreted in parallel with soluble beta APP (APPs); no precursor-product relation between A beta and p3 was found. The protease inhibitor leupeptin did not influence the production of either peptide. In contrast, the weak base ammonium chloride (NH4Cl) showed a dose-dependent inhibition of A beta production with less decrease in p3. A similar effect was observed using the monovalent ionophore monensin. Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmic reticulum or early Golgi are not sufficient for the production of the small peptides. Deletion of the beta APP cytoplasmic domain, which removes a consensus sequence that probably mediates reinternalization, caused an increase in secretion of both APPs and p3 and did not abolish A beta production. These observations suggest that completely mature beta APP within the late Golgi and/or at the cell surface is a prerequisite for A beta production but processing within the lysosome might not be directly required. p3 appears to derive from the 10-kDa C-terminal stub of beta APP following secretion of APPs. Topics: Ammonium Chloride; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Anti-Bacterial Agents; Base Sequence; Brefeldin A; Cell Line; Cell Membrane; Codon; Colchicine; Cyclopentanes; Golgi Apparatus; Humans; Kidney; Kinetics; Leupeptins; Molecular Sequence Data; Molecular Weight; Monensin; Oligodeoxyribonucleotides; Peptide Fragments; Sequence Deletion; Transfection | 1993 |
Effects of brefeldin A on cleavage of invariant chain to p21 and p10 and the appearance of Ii-freed class II MHC dimers.
Intracellular cleavage of class II MHC-associated Ii to p21 and p10 and the appearance of Ii-freed alpha, beta dimers were concurrent events lasting from 1 to 6 hr after synthesis of alpha, beta, Ii trimers, possibly related to charging of foreign peptides to the class II MHC antigen-binding site. Sequential immunoprecipitations of pulse-chase radiolabeled cells were made four times with anti-Ii monoclonal antibody to remove Ii and alpha, beta, Ii trimers and then with anti-class II antibody to detect the time-dependent appearance of Ii-freed alpha, beta dimers. The cleavage of Ii to p21 and p10 was revealed in leupeptin-treated cells. Cell treatment with Brefeldin A (BFA) was associated with a decrease in Ii-freed alpha, beta dimers, with inhibition of leupeptin-revealed cleavage of Ii to p21 and p10, and with persistence of endoglycosidase H susceptibility of Ii and class II alpha, beta chains. We conclude that in untreated cells, cleavage and release of Ii from class II MHC alpha and beta chains occur after those complexes traverse a BFA-sensitive step in the Golgi apparatus. Topics: Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Binding Sites; Brefeldin A; Cell Compartmentation; Cyclopentanes; Golgi Apparatus; Histocompatibility Antigens Class II; Leupeptins; Tumor Cells, Cultured | 1993 |
Successful 7-day perfusion preservation of the canine kidney.
Topics: Adenosine; Allopurinol; Animals; Benzamidines; Creatinine; Dogs; Female; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Leupeptins; Mersalyl; Mitochondria; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Perfusion; Quinacrine; Raffinose; Solutions; Time Factors | 1993 |
Uptake of ferritin by isolated rat hepatocytes. Effect of metabolic inhibitors and iron.
To further characterize the hepatic endocytic pathway of ferritin, the effects of inhibitors of intracellular dissociation of ligands (monensin 15 microM, chloroquine 400 microM), intracellular proteolysis (leupeptin 100 microM) and iron loading on the endocytosis of 125I-rat liver ferritin were studied in isolated rat hepatocytes. Cell associated radioactivity at 37 degrees C was decreased by 27% with chloroquine and 18% with monensin after 4 h. Cell associated radioactivity increased by 38% with leupeptin at 37 degrees C. Acid soluble radioactivity in the extracellular medium was significantly decreased at 4 h in the leupeptin group, which suggests that leupeptin inhibited the lysosomal degradation of the 125I-ferritin resulting in intracellular accumulation of ligand rather than increased uptake of ferritin. Iron loading of cells (5.4-fold increase in intracellular iron) did not significantly alter the binding or accumulation of 125I-ferritin. The characteristics of the endocytic pathway for ferritin are more similar to the asialoglycoprotein receptor than the transferrin receptor, and the hepatic uptake of ferritin is unaffected in this study by increasing the intracellular iron concentration. Topics: Animals; Antimetabolites; Cell Survival; Chloroquine; Endocytosis; Ferritins; Iodine Radioisotopes; Iron; Leupeptins; Liver; Monensin; Rats; Rats, Wistar; Trypan Blue | 1993 |
Invariant chain retains MHC class II molecules in the endocytic pathway.
During exocytosis of MHC class II, the class II alpha beta heterodimer associates with a third polypeptide termed invariant chain (Ii). Class II and Ii are coordinately processed and transported until proteolytic cleavage of Ii in an acidic compartment immediately before class II surface expression. Although the lysosomotropic agent chloroquine (CQ) prevents dissociation of class II/Ii within the cell, the ultimate fate of these complexes has not been determined. We considered two alternative possibilities. If Ii encodes an intracellular retention signal, then persistent association of Ii with class II could lead to intracellular accumulation of class II/Ii complexes. Alternatively, if Ii does not block further transport of class II, then CQ treatment should result in aberrant expression of class II/Ii complexes at the cell surface. Ltk- and EL4 cells transfected with I-A(d) alone or I-A(d) plus Ii were treated with CQ and examined for changes in surface class II and Ii expression. Anti-Ii mAb surface staining did not increase with prolonged CQ treatment, but a dramatic decrease in surface class II staining was observed. This decrease in class II was observed both with genomic Ii and p31 cDNA transfectants and was rapidly reversed upon drug removal. Accumulation of Ii and class II within treated cells was directly observed by intracellular staining. Similar effects on MHC surface expression were observed with the lysosomotropic agents primaquine and NH4Cl and the cysteine protease inhibitor leupeptin. Ii-negative cells treated in parallel displayed no effect of the lysosomotropic agents or leupeptin on class II surface staining. These results indicate that dissociation of Ii from newly synthesized class II is required for transport of the alpha beta dimer to the cell surface, and suggest that Ii serves to retain class II molecules in a post-Golgi endocytic compartment. Topics: Ammonium Chloride; Animals; Antigens, Differentiation, B-Lymphocyte; Cell Line; Chloroquine; Endocytosis; Histocompatibility Antigens Class II; Leupeptins; Lysosomes; Primaquine | 1993 |
Factors affecting secretory protein production in primary cultures of rat hepatocytes.
Previous studies suggest that protein synthesis in the liver may be influenced by alterations in hepatic proteolysis and gluconeogenesis. Since proteolysis and gluconeogenesis are accelerated in acute stress states (especially when associated with nutrient deprivation), these alterations may substantially affect hepatic protein synthesis, the integrity of which is important for host survival. In the present study, we have investigated albumin secretion and glucose production in primary cultures of rat hepatocytes in response to nutrient-limiting conditions, including amino acid depletion, proteolysis inhibition, and augmented gluconeogenesis. In nonlimiting nutrient culture medium containing 10 times the normal plasma amino acid concentrations, hepatocytes produced 8.05 +/- 1.62 micrograms/plate-hr of albumin. Short-term (5 hr) inhibition of cellular protein degradation with the lysosomal protease inhibitor leupeptin did not influence albumin production, but caused a profound reduction (17-41%) when amino acid supply was reduced to the physiologic range (1.5-0.5 times, respectively). This indicates the need for active proteolysis for the maintenance of secretory protein production during nutrient limitation. Similarly, leupeptin inhibited glucose production by 22-30% at physiologic (1.5 times and 0.5 times, respectively) amino acid concentrations. Additionally, hepatocyte glucose production could be augmented 168% by epinephrine (2 microM) in 10 times medium, but this response was markedly depressed by leupeptin. Similar catecholamine-mediated effects, but of a smaller magnitude, were noted at lower medium amino acid concentrations. These findings indicate that hepatocyte albumin and glucose production are associated with the common factor of active cellular proteolysis, probably through the regulation of amino acid supply. However, protein synthesis exhibits a higher priority, since stimulated hepatocyte glucose production did not substantially alter albumin secretion. Topics: Amino Acids; Animals; Cells, Cultured; Culture Media; Dexamethasone; Epinephrine; Glucagon; Insulin; Kinetics; Leupeptins; Liver; Male; Rats; Rats, Sprague-Dawley; Serum Albumin; Urea | 1993 |
Protective effects of calpain inhibitors against neuronal damage caused by cytotoxic hypoxia in vitro and ischemia in vivo.
The calpains are calcium-dependent intracellular proteases that are activated in a number of pathogenic conditions. We tested the capacities of protease inhibitors, calpain inhibitor I and leupeptin, to protect against the neuronal degeneration caused by cytotoxic hypoxia or transient global cerebral ischemia. Primary neuronal cultures were prepared from embryonic chick telencephalon, and cytotoxic hypoxia was induced by adding 1 mM NaCN to the culture medium for 30 min. Global ischemia was induced in rats by clamping both carotid arteries and lowering the arterial blood pressure to 40 mmHg for 10 min. Both calpain inhibitor I and leupeptin protected neurons against ischemic and hypoxic damage. Neuroprotection was indicated by increased cell viability and protein content in the cultures, and fewer damaged neurons in the hippocampal CA1-subfield. Thus, blockade of proteolysis can protect neurons against cytotoxic and ischemic damage. Topics: Animals; Blood Pressure; Calpain; Cells, Cultured; Glycoproteins; Hypoxia, Brain; Ischemic Attack, Transient; Leupeptins; Male; Neurons; Prosencephalon; Rats; Rats, Wistar | 1993 |
Intermediates in degradation of the erythropoietin receptor accumulate and are degraded in lysosomes.
The erythropoietin receptor (EPO-R) is synthesized in transfected Ba/F3 cells as a major 64-kDa endoglycosidase H (Endo H)-sensitive species, with a single N-linked oligosaccharide, and a minor 62-kDa unglycosylated form. Approximately half of the newly made EPO-R is processed to a mature 66-kDa form with a Golgi-processed Endo H-resistant oligosaccharide, of which only a minor fraction is expressed at the cell surface. Both the Endo H-sensitive and the Endo H-resistant forms of the receptor have a half-life of 45-60 min (Yoshimura, A., D'Andrea, A. D., and Lodish, H. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4139-4143). The mature, Endo H-resistant form of the EPO-R appears to be degraded in lysosomes or in other acidic organelles, since receptor degradation is blocked by treatment with NH4Cl, chloroquine, or leupeptin. A fraction of the Endo H-resistant EPO-R molecules is cleaved, generating two fragments of 46 and 39 kDa. The sizes of these fragments and their reactivities with carboxyl-terminal-specific antibodies indicate that the receptor is cleaved at two sites in the exoplasmic domain, 7 kDa apart, and carboxyl-terminal to the N-glycosylation site. Both fragments are membrane anchored and are probably formed in a late or post-Golgi compartment, since their formation is blocked by incubation of cells at 20 degrees C or by incubation with brefeldin A. These membrane-anchored COOH-terminal fragments are probably degraded in lysosomes or in other acidic vesicles as cell fractionation demonstrates that they colocalize with lysosomes, and similar to the intact EPO-R, their degradation is inhibited by NH4Cl. Finally, double labeling immunofluorescence experiments demonstrate that in NH4Cl-treated cells both intact mature EPO-R and the 46- and 39-kDa fragments accumulate in lysosomes and presumably are normally degraded there. The sensitivity of the EPO-R to endoproteolytic cleavages in its exoplasmic domain may relate to its low surface expression and to its extreme metabolic instability. Topics: 3T3 Cells; Ammonium Chloride; Animals; Cells, Cultured; Chloroquine; Cycloheximide; Endoplasmic Reticulum; Golgi Apparatus; Hexosaminidases; Leupeptins; Lysosomes; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mice; Peptide Fragments; Receptors, Erythropoietin | 1993 |
Clonal viability measurements on Plasmodium falciparum to assess in vitro schizonticidal activity of leupeptin, chloroquine, and 5-fluoroorotate.
Until now, the in vitro activity of potential antimalarial agents has been evaluated primarily by monitoring decreases in parasite proliferation. These traditional assays do not distinguish between compounds that arrest proliferation of parasites and compounds that kill them. In this report, a more complex in vitro cytocidal assay for Plasmodium falciparum is described. This assay measures the clonal viability of P. falciparum after the parasites have been treated with an antimalarial agent. The new assay was used to assess cytocidal activities of three antimalarial agents that work through unrelated mechanisms. Leupeptin, a protease inhibitor, arrested the proliferation of W2 clones of P. falciparum at a MIC of 50 microM, but at least 80% of leupeptin-treated cells were viable as judged by the cytocidal assay. On the other hand, chloroquine at 1 microM, its MIC for W2 cells, not only arrested parasite proliferation but also killed more than 99% of the cells. Earlier studies had shown that treatment of P. falciparum with 100 nM 5-fluoroorotate for 48 h was sufficient to inhibit parasite proliferation and parasite thymidylate synthase but not enough to cause significant incorporation of 5-fluoropyrimidines in parasite nucleic acids. By using the new schizonticidal assay, these conditions were found to be necessary and sufficient to kill all parasites in culture. Results of these studies are consistent with the hypothesis that 5-fluoroorotate-based inactivation of P. falciparum thymidylate synthase triggers a lethal mechanism against malarial parasites. Topics: Animals; Antimalarials; Cell Division; Chloroquine; Drug Resistance; Humans; Leupeptins; Orotic Acid; Plasmodium falciparum | 1993 |
Giant sarcoplasmic reticulum vesicles: a study of membrane morphogenesis.
Rabbit sarcoplasmic reticulum vesicles were fused into giant proteoliposomes in a medium of 0.1 M KCl, 10 mM Tris-maleate, pH 7.0, 10 micrograms ml-1 antipain, 10 micrograms ml-1 leupeptin, 25 IU per ml Trasylol, 3 mM NaN3, 3.75% PEG 1500 and 3% DMSO by brief exposure to 37 degrees C, followed by incubation for 4 h at 25 degrees C. Approximately 5-10% of the sarcoplasmic reticulum elements underwent fusion, forming single-walled spherical vesicles of 1-25 microns diameter, in which the polarity of the native membrane was preserved. The Ca(2+)-stimulated ATPase activity remained essentially unchanged after fusion. On exposure to decavanadate in a Ca(2+)-free medium the spherical vesicles assumed a corrugated appearance with the formation of long ridges separated by deep furrows that eventually pinched off longitudinally and separated into numerous long crystalline tubules of uniform (approximately 0.1 microns) diameter. The vanadate-induced transformation of giant vesicles into tubules implies that the geometry of the sarcoplasmic reticulum membrane is determined by the conformation of the Ca(2+)-ATPase. Topics: Animals; Antipain; Aprotinin; Calcium-Transporting ATPases; Crystallization; Egtazic Acid; Lanthanum; Leupeptins; Liposomes; Membrane Fusion; Membrane Proteins; Microscopy, Electron; Morphogenesis; Muscle Proteins; Proteolipids; Rabbits; Sarcoplasmic Reticulum; Temperature; Vanadates | 1992 |
Differences in alpha 2u-globulins increased in male rat kidneys following treatment with several alpha 2u-globulin accumulating agents: cystein protease(s) play(s) an important role in production of kidney-type-alpha 2u-globulin.
Effects of alpha 2u-globulin accumulating agents on alpha 2u-globulins in rat kidneys were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male animals with decalin (150 mg/kg), 2,2,4-trimethylpentane (50 mg/kg), isophorone (150 mg/kg), d-limonene (150 mg/kg) or 1,4-dichlorobenzene (150 mg/kg) by gavage for 14 consecutive days in each case resulted in a marked intensification of a protein band corresponding to kidney-type-alpha 2u-globulin, with a molecular mass calculated to be approximately 16 kDa. However, intraperitoneal treatment with leupeptin and E-64 (two times 0.07 mmol/kg, for each), well known cystein protease inhibitors, while only slightly increasing this kidney-type-alpha 2u-globulin band, caused the intensification of a approximately 19-kDa molecular mass protein band which was revealed to be a native-type-alpha 2u-globulin by SDS-PAGE and immunoblotting. These results indicated that at least two types of alpha 2u-globulin can be increased in male rat kidney by chemical treatment. Moreover, cystein protease(s) appear(s) to play an important role in the degradation of alpha 2u-globulin and particularly in the conversion of native-type-alpha 2u-globulin to kidney-type-alpha 2u-globulin in rat kidneys. Topics: Alpha-Globulins; Animals; Chlorobenzenes; Cyclohexanones; Cyclohexenes; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Kidney; Leucine; Leupeptins; Limonene; Male; Naphthalenes; Octanes; Rats; Rats, Sprague-Dawley; Terpenes | 1992 |
Protease activities in carp retina.
Protease of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae. The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin. Topics: Amino Acid Sequence; Animals; Carps; Coumarins; Endopeptidases; Hydrogen-Ion Concentration; Leucine; Leupeptins; Molecular Sequence Data; Peptides; Phenanthrolines; Protease Inhibitors; Retina; Serine Endopeptidases; Tosyllysine Chloromethyl Ketone | 1992 |
Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.
The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway. Topics: Animals; Chloroquine; Chromatography, Liquid; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Intrinsic Factor; Kidney Cortex; Leupeptins; Male; Microvilli; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Vitamin B 12 | 1992 |
Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour.
Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family. Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Benzoylarginine Nitroanilide; Chromatography, Gel; Electrophoresis; Enzyme Activation; Enzyme Stability; Female; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Leupeptins; Mammary Neoplasms, Experimental; Methylnitrosourea; Molecular Sequence Data; Rats; Rats, Inbred Strains; Sensitivity and Specificity; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Substrate Specificity; Tissue Extracts | 1992 |
Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events.
Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities. Topics: Leupeptins; Models, Biological; Murine hepatitis virus; Peptide Fragments; Precipitin Tests; Protein Biosynthesis; Protein Precursors; Protein Processing, Post-Translational; Reading Frames; Time Factors; Viral Proteins | 1992 |
Metabolism of omega-conotoxin-sensitive voltage-operated calcium channels in human neuroblastoma cells: modulation by cell differentiation and anti-channel antibodies.
The turnover of voltage-operated calcium channels was studied in two different human neuroblastoma cell lines (IMR32 and SH-SY5Y) using omega-conotoxin. The 125I-omega-conotoxin bound to surface channels was internalized and degraded by the cells in a time- and temperature-dependent manner. The radioactive degradation products released in the medium were all trichloroacetic acid soluble and no longer recognized by anti-omega-conotoxin antibodies. Altering the pH of intracellular organelles with chloroquine and inhibiting lysosomal proteases with leupeptin reduced 125I-omega-conotoxin degradation but had no effect on its internalization. Postlabeling measurements showed that the rates of 125I-omega-conotoxin internalization and degradation were equal to the rate of channel removal from the cell surface after protein synthesis inhibition. The rate of removal of omega-conotoxin binding sites was parallel to the rate of loss of functional channels, as measured by means of the fura-2 technique. Drug-induced differentiation of human neuroblastoma cells slowed down channel internalization and degradation rates, leading to the known increased expression of plasma membrane calcium channels in differentiated cells. On the other hand, both human (from Lambert-Eaton myasthenic patients) and murine (from immunized mice) anti-channel antibodies increased the rates of channel internalization and degradation, leading to channel downregulation. The activity of presynaptic calcium channels is already known to be acutely modulated by a number of different agents (e.g., hormones and neurotransmitters); our studies suggest that a different form of channel modulation (changes in the number of channels due to interference with channel turnover) may be active over a longer time scale in neurons.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Antibodies; Calcium Channels; Cell Differentiation; Chloroquine; Electrophysiology; Humans; Leupeptins; Neuroblastoma; omega-Conotoxins; Peptides, Cyclic; Temperature | 1992 |
Role of cathepsin D in antigen presentation of ovalbumin.
Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339. Topics: Animals; Antigen-Presenting Cells; Cathepsin B; Cathepsin D; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Histocompatibility Antigens Class II; In Vitro Techniques; Interleukin-2; Leupeptins; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Ovalbumin; Pepstatins; T-Lymphocytes | 1992 |
Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells.
Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA. Topics: Ammonium Chloride; beta-N-Acetylhexosaminidases; Carcinoma, Hepatocellular; Chloroquine; Endocytosis; Humans; Kinetics; Leupeptins; Liver Neoplasms; Lysosomes; Pepstatins; Plasminogen Activator Inhibitor 1; Primaquine; Recombinant Proteins; Subcellular Fractions; Tissue Plasminogen Activator; Tumor Cells, Cultured | 1992 |
Protease inhibition causes some manifestations of aging and Alzheimer's disease in rodent and primate brain.
Topics: Aging; Alzheimer Disease; Animals; Brain; Dolichols; Endopeptidases; Humans; Immunologic Techniques; Intermediate Filaments; Leucine; Leupeptins; Lipofuscin; Macaca fascicularis; Male; Protease Inhibitors; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Ubiquitins | 1992 |
Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers.
Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element. Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcium; Cell Line; Chloramphenicol O-Acetyltransferase; Drug Interactions; Enzyme Activation; Gene Expression Regulation, Enzymologic; Ionomycin; Leupeptins; Membrane Potentials; PC12 Cells; Plasmids; Protein Kinase C; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Second Messenger Systems; Sphingosine; Transcription, Genetic; Tyrosine 3-Monooxygenase; Veratridine | 1992 |
Different patterns of protein kinase C redistribution mediated by alpha 1-adrenoceptor stimulation and phorbol ester in rat isolated left ventricular papillary muscle.
1. In rat left ventricular papillary muscle, phenylephrine, an alpha 1-adrenoceptor agonist, had a staurosporine-sensitive positive inotropic effect and increased the particulate-associated protein kinase C (PKC) activity without significant changes in total PKC activity or in cytosolic Ca2+/phospholipid-independent kinase (PKI) activity. 2. A PKC stimulant, phorbol 12,13-dibutyrate (PDBu), decreased contractility and slightly increased PKC activity in the particulate fractions, with a marked decrease and increase in total PKC and PKI activities, respectively. 3. The PDBu-induced negative inotropic response was attenuated by two protease inhibitors, leupeptine and a microbial peptide isolated from Aspergillus japonicus (E-64), which are known to inhibit the conversion of particulate-associated PKC to PKI. 4. Such differences in the patterns of PKC redistribution, i.e. marked increases in particulate PKC and cytosolic PKI activities caused by phenylephrine and PDBu, respectively, may account for the opposite inotropic effects of PKC stimulation by an alpha 1-agonist and a phorbol ester. Topics: Adrenergic alpha-Agonists; Animals; Leucine; Leupeptins; Male; Myocardial Contraction; Papillary Muscles; Phenylephrine; Phorbol 12,13-Dibutyrate; Protease Inhibitors; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha | 1992 |
Modified immunoradiometric assay of parathyroid hormone-related protein: clinical application in the differential diagnosis of hypercalcemia.
We have developed a sensitive, specific solid-phase immunoradiometric assay (IRMA) of parathyroid hormone-related protein (PTH-RP) with use of affinity-purified polyclonal immunoglobulins. Antibodies recognizing PTH-RP(37-74) are immobilized to a polystyrene bead to "capture" analytes from the sample; antibodies to epitopes within the 1-36 amino acid region of PTH-RP are labeled with 125I. This IRMA recognizes PTH-RP(1-74) and PTH-RP(1-86) equivalently, but does not detect N-terminal or C-terminal fragments of PTH-RP, intact human parathyrin (PTH), or fragments of PTH. PTH-RP is not stable in plasma at 3-5 degrees C or room temperature, but a mixture of aprotinin (500 kallikrein units/L) and leupeptin (2.5 mg/L) improves PTH-RP stability in blood samples. In plasma collected in the presence of these protease inhibitors from normal volunteers and patients with various disorders of calcium metabolism, PTH-RP concentrations were above normal (greater than 1.5 pmol/L) in 91% (42 of 46) of patients with hypercalcemia associated with nonhematological malignancy. In plasma from patients with other hypercalcemic conditions (e.g., primary hyperparathyroidism, sarcoidosis, and vitamin D excess), PTH-RP was undetectable. Above-normal concentrations of PTH-RP and total calcium decreased to normal in a patient with an ovarian cyst adenocarcinoma after surgical removal of the tumor. We conclude that PTH-RP is related to and probably the causative agent of hypercalcemia in most patients with cancer, and that measurements of PTH-RP are useful in the diagnosis and management of patients with tumor-associated hypercalcemia. Topics: Aprotinin; Diagnosis, Differential; Drug Stability; Humans; Hypercalcemia; Immunoradiometric Assay; Leupeptins; Neoplasms; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins | 1992 |
Cyclic AMP induces transforming growth factor beta 2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line PC-3.
The standard therapy for advanced prostate cancer is androgen ablation. Despite transitory responses, hormonally treated patients ultimately relapse with androgen-independent disease that is resistant to further hormonal manipulation and cytotoxic chemotherapy. To develop an additional approach to the treatment of advanced prostate cancer, we have been studying the signal transductions controlling the growth of human androgen-independent prostate carcinoma cell lines. We report here that elevation of intracellular cAMP markedly inhibits the growth of the hormone-refractory cell line PC-3. To examine the mechanism of cAMP action in PC-3 cells, we tested the effect of the cAMP analog dibutyryl cAMP (Bt2-cAMP) on the regulation of the potent negative growth factor transforming growth factor beta (TGF-beta). Bt2-cAMP selectively induced the secretion of TGF-beta 2 and not TGF-beta 1 by PC-3 cells. This TGF-beta 2 was shown to be bioactive by using the CCL-64 mink lung cell assay. TGF-beta 1 was not activated despite being present at 3-fold higher concentrations than TGF-beta 2. Northern analysis showed that Bt2-cAMP induced an increase in the five characteristic TGF-beta 2 transcripts and had no effect on the level of TGF-beta 1 or TGF-beta 3 transcripts. TGF-beta 2 induction was only weakly enhanced by cycloheximide and was completely inhibited by actinomycin D. These data show that Bt2-cAMP induces the expression of active TGF-beta 2 by PC-3 prostate carcinoma cells, suggesting a new approach to the treatment of prostate cancer and a new molecular mechanism of cAMP action. Topics: 1-Methyl-3-isobutylxanthine; Aprotinin; Blotting, Northern; Bucladesine; Cell Division; Cell Line; Cyclic AMP; Cycloheximide; Dactinomycin; DNA Replication; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leupeptins; Male; Pentoxifylline; Phenylmethylsulfonyl Fluoride; Prostatic Neoplasms; RNA, Neoplasm; Theophylline; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured | 1992 |
Protease inhibitors as a model for NCL disease, with special emphasis on the infantile and adult forms.
An animal model of NCL disease has been developed with the use of protease inhibitors. Young rats received a continuous infusion of various specific protease inhibitors or of physiological saline into the lateral ventricle of the brain using osmotic mini-pumps. Treatment lasted for 2 weeks, at which time animals were sacrificed and the brains were removed and processed for light or electron microscopic analysis. The thiol protease inhibitors leupeptin and E64C, but not saline or the serine protease inhibitor aprotinin, caused a massive accumulation of ceroid-lipofuscin (CL) in brain cells that bore a strong morphological resemblance to the CL in the infantile and adult forms of NCL disease, and bore similarity to the CL of the late infantile and juvenile forms. Leupeptin also caused the death of cerebellar Purkinje cells, as is seen in the infantile and adult forms of NCL. Further evidence is presented in support of the hypothesis (Ivy et al.: Science 226:985-987, 1984) that decreased or defective lysosomal thiol proteases or their substrates may underly the pathogenesis of at least the infantile and adult forms of NCL disease. Administration of protease inhibitors to the brains of young rats provides an important model for studying the cellular mechanisms of ceroid-lipofuscino-genesis. Topics: Animals; Aprotinin; Ceroid; Cysteine Proteinase Inhibitors; Disease Models, Animal; Dogs; Humans; Leupeptins; Lipofuscin; Lysosomes; Neuronal Ceroid-Lipofuscinoses; Purkinje Cells; Rats; Rats, Inbred Strains | 1992 |
Phenotypic variation of surface antigenic determinants in Trichomonas vaginalis detected by monoclonal antibodies.
We produced a large panel of murine monoclonal antibodies against surface determinants of Trichomonas vaginalis using the hybridoma technique. An immunoenzymatic technique (E.L.I.S.A.) was used to screen positive hybrid cells producing specific antibodies against the protozoan surface. Eleven monoclonal antibodies (Mabs) out of seventy-seven positives were further characterized. We tested antibody reactivity in order to investigate the antigenic variance among 13 different strains of Trichomonas vaginalis of different geographic origin. To elucidate the complexity of antigenic expression in Trichomonas vaginalis, further characterization of the antigenic pattern in our 13 clinical isolates was carried out by immunoblotting techniques. We demonstrate that some monoclonal antibodies react with antigens varying in molecular weight in the different strains tested. We also demonstrate the pivotal role of protozoan proteases in antigenic rearrangement. Topics: Animals; Antibodies, Monoclonal; Antibodies, Protozoan; Antigenic Variation; Antigens, Protozoan; Antigens, Surface; Epitopes; Fluorescent Antibody Technique; Hybridomas; Immunoblotting; Leupeptins; Phenotype; Trichomonas vaginalis | 1992 |
Alterations of the levels of glycoproteins Ib-IX and IIb-IIIa in platelets stored at 22 degrees C.
Platelets stored in CLX blood bags, under normal blood banking conditions, were studied for up to 7 days to determine if changes occurred in the levels of membrane glycoproteins (GP) Ib-IX and IIb-IIIa. Radiolabeled monoclonal antibodies (MAB) were used to estimate the number of glycoprotein molecules on the surface membrane of intact platelets. GP IX and GP IIb-IIIa levels remained essentially unaltered during storage. In contrast, the content of GP Ib at day 7 decreased by 45% of the total when fresh. The aggregation response to ristocetin, which requires GP Ib, was also diminished after 7 days. Addition of protease inhibitors, leupeptin and/or aprotinin did not appear to influence the degradation of this glycoprotein. We conclude that storage at 22 degrees C had deleterious effects on the GP Ib content of platelets. Topics: Antibodies, Monoclonal; Aprotinin; Blood Platelets; Blood Preservation; Cold Temperature; Humans; Leupeptins; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins; Ristocetin | 1992 |
Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes.
Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo. Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Biological Transport; Cells, Cultured; Chloroquine; Fetuins; Kinetics; Kupffer Cells; Leupeptins; Liver; Polyethylene Glycols; Rats; Time Factors | 1992 |
Unique expression of von Willebrand factor by type IIA von Willebrand's disease endothelial cells.
Endothelial cells (EC) were cultured from the umbilical cord of a male neonate whose mother was previously diagnosed with type IIA von Willebrand's disease (vWd). The diagnosis of type IIA vWd in the proband was confirmed by low ristocetin activity and the absence of the highest molecular weight (MW) forms of von Willebrand factor (vWf) in his platelet poor plasma. The vWf of EC cultured from the neonate's umbilical cord differed from that of control EC and the cell line EA.hy926 in two respects. Firstly, the full range of molecular weight forms was present in the patient EC lysate and, secondly, vWf:Ag expression was approximately seven-fold greater than that of control cells. Platelet lysates prepared from other affected members of the type IIA vWd family in the presence or absence of proteolytic inhibitors demonstrated a near normal vWf multimeric distribution. Resistance of these high MW forms to heat degradation was conferred by the presence of proteolytic inhibitors. Moreover, the full plasma vWf multimeric distribution could not be restored by the inclusion of EDTA. N-ethylmaleimide and leupeptin in the anticoagulant during the rapid preparation of platelet poor plasma. These findings lend support to the heterogeneous nature of type IIA vWd and has possible implications in the understanding of the intracellular processes involved in the biosynthesis and storage of the vWf macromolecular complex as well as the pathogenesis of type IIA vWd. Topics: Blood Platelets; Blotting, Western; Cells, Cultured; Edetic Acid; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Ethylmaleimide; Female; Humans; Leupeptins; Male; Molecular Weight; Pedigree; von Willebrand Diseases; von Willebrand Factor | 1992 |
Calcium-activated potassium transport and high molecular weight forms of calpromotin.
Investigations of human red blood cells show that a cytoplasmic protein called calpromotin is involved in the regulation of calcium-activated potassium transport. Calpromotin associates with the membrane in the presence of calcium and undergoes a chemical transformation. High performance gel filtration and gel electrophoresis show that the cytoplasmic and membrane-bound calpromotin can exist in both low and high molecular weight forms. The biochemical properties of the high molecular weight membrane-bound calpromotin are not the same as the high molecular weight cytoplasmic calpromotin. The high molecular weight membrane forms of calpromotin are increased by leupeptin and diminished by iodoacetic acid. Therefore, the leupeptin enhancement and iodoacetic inhibition of calcium-activated potassium transport may involve the high molecular weight forms of membrane-bound calpromotin. Topics: Biological Transport; Blood Proteins; Blotting, Western; Calcium; Cells, Cultured; Chromatography, High Pressure Liquid; Cytoplasm; Erythrocyte Membrane; Humans; Iodoacetates; Iodoacetic Acid; Leupeptins; Molecular Weight; Potassium | 1992 |
Recovery after delayed nerve repair: influence of a pharmacologic adjunct in a primate model.
Inhibition of calpains in skeletal muscle by the tripeptide, leupeptin, after median-nerve transection in the mid-forearm and a delayed nerve repair of 3-weeks duration, was studied in a primate (Cebus apella) model. Results indicated that leupeptin facilitates axon regrowth and neuromuscular recovery after delayed nerve repair. Toxicologic testing showed that leupeptin, administered at 18 mg/kg intramuscularly, twice daily for 24 weeks after delayed nerve repair, did not adversely affect hematology, clotting, blood chemistry, or echocardiogram profiles. These data indicate that leupeptin is an effective and safe adjunct to delayed nerve repair. Topics: Animals; Axons; Calpain; Cebus; Leupeptins; Median Nerve; Muscles; Muscular Atrophy; Nerve Regeneration; Neural Conduction; Time Factors | 1992 |
Effect of lysosomotropic agents on the taurocholate-stimulated biliary excretion of horseradish peroxidase.
The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholate-stimulated biliary excretion of horseradish peroxidase (HRP) was studied in bile fistula rats. HRP (0.5 mg/100 g body wt) was injected into the portal vein during taurocholate (0.4 mumol/min/100 g body wt) or saline infusion. HRP appeared in bile showing both an early (approx. 5 min) and a late (approx. 25 min) excretion peak. The late peak, which represented about 95% of the total HRP excreted, is due to transcellular vesicular transport. The early peak is mainly due to paracellular leakage although a rapid vesicular transport also contributes. Taurocholate infusion significantly increased the biliary output of HRP (both peaks) and of the endogenous lysosomal enzyme acid phosphatase. Pretreatment with chloroquine or leupeptin inhibited the taurocholate-stimulated late excretion of HRP into bile, without affecting its early excretion. The lysosomotropic agents did not affect the biliary excretion of bile salts but significantly inhibited the taurocholate-stimulated biliary excretion of acid phosphatase. The results are consistent with a role of lysosomes in the taurocholate-stimulated major transcellular vesicular transport of HRP into bile. Topics: Acid Phosphatase; Animals; Bile Acids and Salts; Biliary Tract; Chloroquine; Horseradish Peroxidase; Leupeptins; Lysosomes; Male; Rats; Rats, Wistar; Taurocholic Acid | 1992 |
Assembly and secretion of fibrinogen. Degradation of individual chains.
Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation. Topics: Ammonium Chloride; Animals; Cell Line; Chickens; Chloroquine; Fibrinogen; Humans; Kinetics; Leupeptins; Liver; Lysosomes; Macromolecular Substances; Methionine; Sulfur Radioisotopes; Transfection; Tumor Cells, Cultured | 1992 |
The fertilization and development of mouse oocytes following cortical granule discharge in the presence of a protease inhibitor.
The effect of leupeptin, a serine protease inhibitor, on the fertilization and development potential of oocytes stimulated to undergo cortical granule exocytosis has been investigated. An in-vitro bioassay system was used in which mouse oocytes were exposed to calcium ionophore, A23187, in the presence and absence of leupeptin, before their fertilization and development to the blastocyst stage was assessed. We have demonstrated that the presence of leupeptin in the incubation medium, at concentrations of 1 micrograms/ml and 10 micrograms/ml during the first 10 min of cortical granule exocytosis, reversed the ionophore-induced decrease in the capacity of oocytes to fertilize and develop to blastocysts. The induction of exocytosis of cortical granules by calcium ionophore was confirmed using fluorescence microscopy. Using this technique, we also confirmed that leupeptin did not inhibit ionophore-induced cortical granule exocytosis, thus supporting the contention that leupeptin acted upon released cortical exudate. It was concluded that leupeptin acted by inhibiting proteases released into the perivitelline space during the early stages of cortical granule exocytosis. Based on these results it was proposed that leupeptin could be used to prevent premature loss of fertility of human oocytes which are inadvertently activated under in-vitro conditions. Topics: Animals; Blastocyst; Calcimycin; Culture Techniques; Embryo, Mammalian; Embryonic and Fetal Development; Exocytosis; Female; Fertilization; Fertilization in Vitro; Fluorescent Dyes; Insemination, Artificial; Leupeptins; Mice; Oocytes; Superovulation | 1992 |
Opsonized zymosan decreases cytoplasmic motility of alveolar macrophages in dogs.
To examine the mechanisms of changes in alveolar macrophage (AM) activities caused by phagocytic stimulus, we studied the effect of opsonized zymosan (OZ) on cytoplasmic motility (CM) of AM from dog lungs in vitro. Four days after the instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the lower lobe bronchus, AM were harvested by broncho-alveolar lavage. AM were adhered to the bottom of plastic vials (10(6) cells of AM per each vial). Remanent field strength (RFS) from the AM containing Fe3O4 particles was measured immediately after magnetization. RFS decreased with time due to particle rotation (relaxation), which is related to cytoplasmic motility of AM. OZ (1-500 micrograms) decreased lambda 0 (the relaxation rate for the first min) in a concentration-dependent fashion. Neither BW755C (10(-5) M), indomethacin (10(-6) M), leupeptin (10(-5) M), bestatin (10(-5) M), nor superoxide dismutase (1000 U/ml) inhibited OZ (500 micrograms)-induced inhibitory effects on lambda 0, suggesting that cyclooxygenase and lipoxygenase products, serine, thiol enzymes, aminopeptidase and superoxide anion wer not responsible for OZ-induced effects. OZ (500 micrograms) significantly increased the intracellular concentration of Ca2+ (P less than 0.01). Likewise, OZ (500 micrograms)-induced effects on lambda 0 of AM were significantly inhibited by replacement of the medium with a Ca2+ free solution (P less than 0.01). These results imply that opsonized zymosan inhibits cytoplasmic motility of AM via external calcium influx. Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Aminopeptidases; Animals; Calcium; Captopril; Cells, Cultured; Cytoplasm; Dogs; Indomethacin; Leucine; Leupeptins; Macrophages; Phagocytosis; Pulmonary Alveoli; Superoxide Dismutase; Zymosan | 1992 |
Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses.
E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo. Topics: Amino Acid Sequence; Animals; Calcimycin; Calpain; Cataract; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Culture Techniques; Cysteine Proteinase Inhibitors; Dipeptides; Drug Interactions; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Lens, Crystalline; Leucine; Leupeptins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley | 1992 |
A 1H-NMR study of the interactions between rat tissue kallikrein and two peptide inhibitors.
The 1H-NMR spectra have been obtained for rat submandibular kallikrein in the absence and presence of inhibitors. Two competitive inhibitors were investigated, the tripeptide leupeptin (a potent inhibitor with Ki 0.5 microM) and a hexapeptide (a much weaker, substrate-analogue inhibitor with Ki 380 microM). Analysis of the NMR spectra showed that binding of leupeptin to kallikrein led to a change in the conformation of the enzyme, whereas binding of the substrate analogue to the enzyme produced no such change and may have resulted in a conformational change of the inhibitor. Topics: Amino Acid Sequence; Animals; Kallikreins; Leupeptins; Magnetic Resonance Spectroscopy; Molecular Conformation; Molecular Sequence Data; Protein Conformation; Rats; Submandibular Gland | 1992 |
Enhanced ex vivo proteolysis of plasma von Willebrand factor in disseminated intravascular coagulation.
Plasma levels of von Willebrand factor (vWf) are frequently elevated in patients with disseminated intravascular coagulation (DIC). To investigate the qualitative abnormalities of vWf and the possibility of its ex vivo modification in DIC, we analysed the multimeric composition of vWf in citrated plasma from 15 patients with DIC in the presence or absence of serine protease inhibitors (aprotinin and soybean trypsin inhibitor) and/or cysteine protease inhibitors (leupeptin, N-ethylmaleimide and EDTA). The proportion of large vWf multimers in plasma prepared in the presence of cysteine protease inhibitors was higher than those without such inhibitors. The addition of serine protease inhibitors during the preparation of plasma had no effect on the relative amounts of large multimers. The relative proportion of large multimers in plasma prepared without inhibitors and the difference between plasmas prepared with and without cysteine protease inhibitors correlated with plasma plasmin-alpha 2-plasmin inhibitor complex values, but not with other plasma or serum markers of DIC (platelet count, fibrinogen, FDP, D-dimer or thrombin-antithrombin III complex). We conclude that ex vivo proteolysis of plasma vWf occurs frequently in patients with DIC and cysteine protease inhibitors can protect this degradation. Topics: alpha-2-Antiplasmin; Antifibrinolytic Agents; Disseminated Intravascular Coagulation; Edetic Acid; Ethylmaleimide; Fibrinolysin; Humans; Leupeptins; Multienzyme Complexes; Protein Conformation; von Willebrand Factor | 1992 |
The effect of protease inhibitors, leupeptin and E64d, on differentiation of C2C12 myoblasts in tissue culture.
Intracellular calcium levels play an important role in myofibril disintegration and regeneration of muscle fibers. Earlier studies have shown that the calcium activated protease, calpain, is involved in the removal of Z-discs from myofibrils of striated muscle and the tripeptide-aldehyde, leupeptin, which is an inhibitor of calpain, inhibits this activity. In the present communication, we demonstrate that leupeptin and another calpain inhibitor, E64d, inhibit the fusion of mouse skeletal muscle C2C12 myoblasts to form multinucleated myotubes in tissue culture. Topics: Animals; Calcium; Calpain; Cell Differentiation; Cell Fusion; Culture Techniques; Leucine; Leupeptins; Mice; Muscles; Protease Inhibitors | 1992 |
A block in degradation of MHC class II-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment.
Invariant chain (Ii) associated with MHC class II molecule is processed proteolytically via several distinct intermediates during its intracellular transport through endosomal compartments. Leupeptin added to the culture medium blocks processing of Ii, prevents its dissociation from the class II molecules and leads to an intracellular accumulation of a 22 kDa intermediate form of Ii. We show here that leupeptin has a very general effect on protein transport in the endocytic pathway. When added to Mel Juso cells leupeptin reduces the transport of endocytosed material from multivesicular body-like, endosome carrier vesicles (ECV) to the prelysosomal compartment (late endosome) and leads to a concomitant increase in the number of ECV. Our results argue that one effect of leupeptin, related to antigen processing and presentation, is to block transport of antigen and/or MHC class II molecules to prelysosomal compartments. Topics: Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Biological Transport; Endocytosis; Histocompatibility Antigens Class II; Humans; Leupeptins; Tumor Cells, Cultured | 1992 |
Proteolytic activation of a single-chain precursor of hepatocyte growth factor by extracellular serine-protease.
Hepatocyte growth factor (HGF) is biosynthesized as a single-chain precursor (pro-HGF) and is proteolytically processed to a two-chain mature form. When MRC-5 fibroblasts were pulse-radiolabeled under serum-free conditions, pro-HGF was the predominant molecular form of HGF in the culture medium. CHO cells transfected with an expression plasmid containing a full-size human HGF cDNA produced pro-HGF when these cells were cultured in serum-free medium. These findings suggest that HGF is secreted as a pro-form, which is then converted to a two-chain form by extracellular protease. Single-chain HGF exhibited mitogenic activity on cultured hepatocytes, with a potency similar to that of mature HGF, but this activity was remarkably inhibited by leupeptin. We postulate that inactive pro-HGF is converted to an active two-chain form by a leupeptin-sensitive serine-protease expressed by hepatocytes. Neither plasminogen activators nor plasmin showed any processing activity of pro-HGF in vitro. Topics: Animals; Autoradiography; Cell Division; Cell Line; CHO Cells; Cricetinae; Cysteine; DNA; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Hepatocyte Growth Factor; Humans; Leupeptins; Liver; Lung; Methionine; Plasmids; Protein Precursors; Protein Processing, Post-Translational; Rats; Recombinant Proteins; Serine Endopeptidases; Sulfur Radioisotopes; Tissue Plasminogen Activator; Transfection; Urokinase-Type Plasminogen Activator | 1992 |
Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.
Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases. Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Chymotrypsin; Cyanogen Bromide; Cysteine; Cysteine Endopeptidases; Erythrocytes; Ethylmaleimide; Humans; Kinetics; Leupeptins; Macromolecular Substances; Molecular Sequence Data; Multienzyme Complexes; Myocardium; Peptide Fragments; Peptide Mapping; Proteasome Endopeptidase Complex; Trypsin | 1992 |
Turnover of peroxisomal vesicles by autophagic proteolysis in cultured fibroblasts from Zellweger patients.
Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis. Topics: Adenine; Autophagy; Cell Line; Fibroblasts; Fluorescent Antibody Technique; Humans; Hydrolases; Leupeptins; Lysosomes; Membrane Proteins; Microbodies; Zellweger Syndrome | 1992 |
Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol.
The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca(2+)-requiring proteinase required 5-10 microM Ca2+ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25-2.0 microM) in the absence or presence of Ca2+ (5.0 microM) added. The effect of regucalcin, however, was the greater in the absence of Ca2+ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca2+ (5.0 microM). In the absence of Ca2+, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 micrograms/ml), and heavy metals (25 microM cadmium or 25 microM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca(2+)-independent neutral cysteinyl-proteinase. Topics: Animals; Cadmium; Calcium; Calcium-Binding Proteins; Carboxylic Ester Hydrolases; Cytosol; Dose-Response Relationship, Drug; Endopeptidases; Intracellular Signaling Peptides and Proteins; Leupeptins; Liver; Male; Rats; Sulfotransferases; Zinc | 1992 |
Serum proteases alter the antigenicity of peptides presented by class I major histocompatibility complex molecules.
Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed. Topics: Animals; Antigen-Presenting Cells; Cells, Cultured; Cyanogen Bromide; Endopeptidases; In Vitro Techniques; Leucine; Leupeptins; Mice; Molecular Weight; Ovalbumin; Peptides | 1992 |
Stimulation-enhanced 3-O-methylglucose efflux from the frog sartorius: kinetics and properties of the system.
The characteristics of the process by which contraction enhances glucose transport in the frog sartorius were studied. Electrical stimulation increased the permeability of muscles to 3-O-methylglucose (3-O-MeGlc), a nonmetabolizable glucose analogue, increasing efflux as well as uptake. Enhanced efflux was due to an increase in Vmax of the efflux process. A lactacidosis had no effect on basal 3-O-MeGlc efflux, and replacement of media Na+ with Li+ did not affect stimulation-induced uptake. Also, basal and stimulated uptake was not affected by 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. Lastly, N-carbobenzoxy-glycyl-L-phenylalaninamide, which inhibits insulin-enhanced, but not basal, glucose uptake in adipocytes, inhibited both basal and stimulated 3-O-MeGlc fluxes in the frog sartorius. From these findings, we conclude: (1) contraction and exercise enhance glucose transport in muscle by increasing the number of transporters in the plasma membrane, or their turnover, by an unknown process; and (2) basal glucose transport of muscle, unlike that of adipocytes, can not be distinguished from stimulated transport on the basis of its insensitivity to N-carbobenzoxyglycyl-L-phenylalaninamide. Topics: 3-O-Methylglucose; Animals; Calcium; Cycloheximide; Dipeptides; Electric Stimulation; Humans; Kinetics; Leupeptins; Male; Methylglucosides; Muscles; Phloretin; Rana pipiens; Tetradecanoylphorbol Acetate | 1992 |
Intraventricular infusion of leupeptin decreases Bmax of the D2 receptor in the striatum of young rats.
Intraventricular infusion of a thiol protease inhibitor, leupeptin, was previously shown to induce several morphological and immunochemical manifestations of normal and pathological aging in rat brain. The present study attempted to elucidate whether this treatment also perturbs another brain function which declines in aging, dopamine D2 receptor binding in striatum. Intraventricular infusion of leupeptin (0.6 mg per day) for two weeks caused a significant (about 20%) reduction in the binding maximum (Bmax) of dopamine D2 receptors (as examined by [3H] spiperone binding) in the striatum of young male Fischer-344 rats in comparison to (saline-infused) control rats. The apparent Kd values did not differ significantly between the control and leupeptin-treated rat groups. The results suggest that decreased protein turnover may be a factor in the decline in Bmax of D2 receptors during aging. Topics: Animals; Corpus Striatum; Injections, Intraventricular; Leupeptins; Male; Protein Binding; Radioligand Assay; Rats; Rats, Inbred F344; Receptors, Dopamine; Receptors, Dopamine D2; Spiperone | 1992 |
Characterization of different proteolytic activities in Trypanosoma brucei brucei.
The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity. Topics: Amino Acid Sequence; Animals; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dithiothreitol; Endopeptidases; Glycoproteins; Hydrogen-Ion Concentration; Kinetics; Leucine; Leupeptins; Molecular Sequence Data; Protease Inhibitors; Trypanosoma brucei brucei | 1992 |
Lymphoproliferative activity of Pseudomonas exotoxin A is dependent on intracellular processing and is associated with the carboxyl-terminal portion.
Pseudomonas aeruginosa exotoxin A (PE) represents a microbial superantigen that requires processing by accessory cells in order to induce the proliferation of V beta 8-bearing murine T lymphocytes. In this study, we have observed that PE requires intracellular processing by a protease in order to induce lymphoproliferation. Pepstatin A, an inhibitor of acid proteases, inhibited PE-induced lymphoproliferation, whereas leupeptin, an inhibitor of serine and thiol proteases, had no effect on PE-induced lymphoproliferation. A number of mutant forms of PE were examined for their ability to induce lymphoproliferation. The mutant form which lacks amino acids 5 to 224 of the receptor-binding domain, PE43, was capable of inducing murine thymocytes to proliferate in the presence of accessory cells. However, neither PEgly276, a mutant toxin which undergoes a different intracellular processing pattern than wild-type PE, nor PE589, a mutant toxin which lacks amino acids 590 to 613 at the carboxyl terminus, was able to induce thymocyte proliferation. In addition, the lymphoproliferation induced by the PE43 mutant form of PE could also be inhibited by pepstatin A. Therefore, our data indicate that intracellular processing by a proteolytic enzyme which is inhibited by pepstatin A is critical for PE-induced lymphoproliferation. Furthermore, the lymphoproliferative activity of PE is associated with the carboxyl-terminal portion of PE. Topics: ADP Ribose Transferases; Animals; Antigen-Presenting Cells; Bacterial Toxins; Cathepsin D; Endopeptidases; Exotoxins; In Vitro Techniques; Leupeptins; Lymphocyte Activation; Mice; Mice, Inbred Strains; Pepstatins; Peptide Mapping; Protein Processing, Post-Translational; Pseudomonas aeruginosa Exotoxin A; T-Lymphocytes; Thymus Gland; Virulence Factors | 1992 |
Aminopeptidase activity of an antitumor antibiotic, C-1027.
An antitumor antibiotic C-1027, a complex protein consisting of an apoprotein and a non-covalently bound chromophore, showed some aminopeptidase activity, 1/15 (on the basis of activity per mg protein) that of porcine kidney enzyme [E.C. 3.4.11.2] by use of L-phenylalanyl 4-methyl-coumaryl-7-amide as the substrate. Neither the apoprotein alone nor the chromophore alone were active. Amastatin and bestatin but not leupeptin inhibited the activity. The enzyme activity of the holo-antibiotic, as opposed to that of the porcine kidney enzyme, was readily lost by UV irradiation, indicating that the intact structure of the chromophore was needed to maintain the native conformation of the holo-antibiotic. The cytotoxicity of the holo-antibiotic, but not that of the chromophore, to Ehrlich carcinoma cells in vitro was reduced to 1/5 by 1 microgram/ml of amastatin which alone had no effect on cell growth. The porcine aminopeptidase was not cytotoxic at all even at higher concentrations (higher enzyme activities/ml). Amastatin possibly occupied the catalytic domain of the holo-antibiotic, interfering with the binding of the holo-antibiotic with some cell-surface protein(s). Amastatin did not inhibit the holo-antibiotic to cleave isolated DNA. Topics: Aminoglycosides; Aminopeptidases; Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoproteins; Carcinoma, Ehrlich Tumor; Cell Survival; DNA, Neoplasm; Enediynes; Leucine; Leupeptins; Microsomes; Oligopeptides; Peptides; Proteins; Substrate Specificity; Swine; Tumor Cells, Cultured; Ultraviolet Rays | 1992 |
Effect of platelet calpain on normal T-lymphocyte CD43: hypothesis of events in the Wiskott-Aldrich syndrome.
The Wiskott-Aldrich syndrome (WAS) is an inherited disease involving defects of platelets (small size, severe thrombocytopenia due to accelerated destruction) and T lymphocytes (progressive immunodeficiency, lymphopenia). The best-characterized molecular defect is the deficiency and, in some cases, abnormal forms of the T-lymphocyte surface mucin molecule CD43; deficiency of the platelet surface mucin GPIb was observed previously in two of four patients. Neither of these defects is primary, since CD43 and GPIb are encoded by autosomal genes and the disease is X-linked. This study uses cellular biological approaches to explore the possibility that destruction of structurally defective WAS platelets, mimicked experimentally by sonication of normal platelets, plays a role by releasing protease and generating other cellular defects. We show that a protease of normal platelets, identified as Ca(2+)-dependent neutral protease (calpain), which is known to cleave platelet GPIb, also specifically cleaves CD43 on the surface of neighboring desialylated T lymphocytes. The identification of the CD43 cleaving protease was based on its requirement for Ca2+ and inhibition by leupeptin, but not by diisopropylfluorophosphate (DFP). The approximate site of CD43 cleavage was identified by the use of a rabbit antibody. Sensitivity of GPIb to calpain is shown to be sialylation-independent and that of CD43 to be sialylation-dependent, and these findings are explained in terms of molecular structures. These and previous findings are incorporated into a putative mechanism, which explains most of the defects in the WAS. The mechanism suggests that the primary defective molecule in the WAS is unlikely to be a surface glycoprotein, but rather a cytoplasmic molecule with a function in cytoskeletal interactions and/or calcium ion regulation and calpain activation. Topics: Antigens, CD; Blood Platelets; Calcium; Calpain; Humans; Immunoblotting; Leukosialin; Leupeptins; Models, Biological; N-Acetylneuraminic Acid; Neuraminidase; Platelet Membrane Glycoproteins; Sialic Acids; Sialoglycoproteins; Substrate Specificity; T-Lymphocytes | 1992 |
Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.
Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors. Topics: Antipain; Freeze Fracturing; Humans; Intercellular Junctions; Leupeptins; Protein Precursors; Protein Processing, Post-Translational; Tumor Cells, Cultured | 1992 |
Regulation of the functional expression of hexose transporter GLUT-1 by glucose in murine fibroblasts: role of lysosomal degradation.
The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [3H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 microM) and ammonium chloride (10 mM) cause accumulation (both approximately 4-fold) of GLUT-1 protein and a small increase (both approximately 25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 microM) causes accumulation (approximately 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (less than or equal to 5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (approximately 4-fold) and GLUT-1 content (approximately 7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: 3T3 Cells; Animals; Biological Transport; Cell Compartmentation; Chloroquine; Glucose; Hexoses; In Vitro Techniques; Leupeptins; Lysosomes; Membrane Proteins; Mice; Monosaccharide Transport Proteins | 1992 |
Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.
Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease. Topics: Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Biotin; Cell Membrane; Endothelium, Vascular; Humans; Leupeptins; Lysosomes; Molecular Weight; Peptide Fragments; Umbilical Veins | 1992 |
Role of hepatic lysosomes in the degradation of metallothionein.
The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L. Topics: Animals; Cathepsins; Electrophoresis, Polyacrylamide Gel; Leucine; Leupeptins; Liver; Lysosomes; Male; Metallothionein; Pepstatins; Rats; Rats, Inbred Strains | 1992 |
Effects of heparin on osteoclast activity.
The effect of heparin on osteoclastic bone resorption was studied in vitro using the disaggregated osteoclast resorption assay. Bone resorption was assessed by counting the resorption lacunae on bone slices by light microscopy. Low concentrations of heparin (5 micrograms/ml) increased bone resorption by isolated chick and rat osteoclasts. Among other glycosaminoglycans tested at 5 micrograms/ml, only dextran sulfate showed a small but significant stimulation of resorption. Chondroitin sulfates A, B, and C were without effect at 25 and 100 micrograms/ml, whereas resorption was increased by 100 micrograms/ml of heparan sulfate. With chick osteoclasts, which could be maintained in serum-free conditions, a stimulatory effect of heparin was found both in the presence of 5% fetal calf serum and in serum-free media containing insulin, transferrin, and selenium. The magnitude of the heparin-induced increase in resorption was similar in the presence or absence of serum. The stimulation of resorption was associated with an increase in the number of osteoclasts on bone slices. Pretreatment of the bone slices with heparin also enhanced resorption. In time course experiments, 5 micrograms/ml of heparin caused a doubling of chick osteoclast activity index (number of resorption pits per number of osteoclasts) at 12 and 24 h. In 24 h cultures, treatment with 10 micrograms/ml of the arginine-rich basic protein, protamine, 1 microgram/ml of the immunosuppressant, cyclosporine A, or 5 micrograms/ml of the cysteine-proteinase inhibitor, leupeptin, negated the heparin effect on bone resorption. Leupeptin also inhibited basal resorption.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Bone Resorption; Cells, Cultured; Chickens; Chondroitin Sulfates; Culture Media; Cyclosporine; Dextran Sulfate; Heparin; Histamine; Leupeptins; Osteoclasts; Phenytoin; Protamines; Rats | 1992 |
Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate.
We have analyzed the metabolic pathway of maturation of APP751 in stably transfected 293 cells, in the presence of either of the cysteine protease inhibitors leupeptin or E-64. Metabolic labeling, followed by immunoprecipitation at various times in the chase with a rabbit polyclonal antibody (anti-BX6) specific to the carboxyl-terminal end of amyloid precursor protein (APP), revealed the accumulation of a novel approximately 22-kDa carboxyl-terminal fragment (22-CTF) in the inhibitor-treated cells. This fragment, which was not detectable in untreated cells, was immunoprecipitated by four separate antibodies to the carboxyl-terminal region of APP as well as by polyclonal and monoclonal antibodies specific to the first 16 amino acids of the beta-peptide domain. Antibodies to the amino-terminal end of APP do not, however, recognize the fragment. Co-treatment of the inhibitor-treated cells with either of the lysosomotropic agents chloroquine or ammonium chloride completely blocked the generation of this fragment but did not significantly affect APP maturation or secretion. All, however, slowed the intracellular turnover of the cell-associated, approximately 9-kDa carboxyl-terminal fragment (c-CTF) produced during constitutive secretion. Densitometric analyses of these results suggest that this non-secretory pathway of APP degradation, mediated by cysteine proteases in an intracellular acidic compartment, accounts for approximately 70% of total APP metabolism and that a key processing intermediate in this pathway is a 22-kDa, beta-peptide-containing APP carboxyl-terminal fragment. It is possible that inefficient degradation of such an intermediate leads to the formation of aggregating beta-peptide. Topics: Amyloid beta-Protein Precursor; Cell Line; Chloroquine; Cysteine Proteinase Inhibitors; Humans; Kinetics; Leucine; Leupeptins; Molecular Weight; Peptide Fragments; Transfection | 1992 |
Use of antibody/peptide constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation.
Human T cells can express MHC-class II products and were shown to be potential antigen-presenting cells. However, they are unable to capture the antigen and only antigens, which bind to T cell membranes such as the gp120 glycoprotein of HIV, are internalized, processed, and presented by T cells. To better understand the role of T cells as antigen-presenting cells, we established a method which overcomes the lack of antigen capture by T cells. Antigen (tetanus toxoid, TT) or an antigenic peptide of TT (residue 830-843, P2) was coupled to antibodies directed to T cell surface molecules such as CD2, CD4, CD8. Antibody/TT and antibody/P2 constructs stimulated P2-specific T cell clones in the absence of accessory cells, if the antibody recognized a T cell surface structure. Compared to the peptide alone, a 100-500 times lower molar concentration of the antibody/peptide construct was required to achieve a similar proliferative response. T cell stimulation via the constructs involved intracellular processing, as nonspecific, glutaraldehyde fixed T cell lines pulsed with the constructs could present the peptide and processing inhibitors like Leupeptin or Chloroquine inhibited the development of a proliferative response to the constructs. Our data underline the ability of T cells to function as antigen-processing and -presenting cells and show that antibody/antigen or antibody/peptide constructs are able to direct a certain antigen or peptide to a T cell. Antibody/peptide constructs may be interesting tools to better understand antigen processing and to study the consequences of antigen presentation by different cells. Topics: Antigen-Antibody Complex; Antigen-Presenting Cells; Antigens, Bacterial; Chloroquine; Clone Cells; Dose-Response Relationship, Immunologic; Humans; In Vitro Techniques; Leupeptins; Lymphocyte Activation; Peptides; T-Lymphocytes; Tetanus Toxoid | 1992 |
The ability and inability of ATP to stop aluminum from reducing the sodium efflux in unpoisoned barnacle muscle fibers.
A study has been made in single barnacle muscle fibers with the object of determining whether ATP is able to protect the resting Na efflux from the effects of injected aluminum (Al) and whether Al is able to reduce or abolish the stimulatory action of ATP on the efflux. The results of the experiments show that neither ATPMg nor ATPNa2 preinjection stops Al from reducing the basal Na efflux in unpoisoned fibers which undergo a large fall (hypersensitive fibers). Preinjection of Al into such fibers reduces or abolishes the stimulatory response of the Na efflux to ATP injection. In less hypersensitive fibers, however, ATPMg is protective. This is also true of ATPNa2 preinjection in both classes of fibers showing stimulation. Injection of a mixture of AlCl3-ATPNa2 into unpoisoned fibers causes less inhibition than AlCl3 injection. The hypothesis that both ATPMg and ATPNa2 are protective is also supported by the results obtained with ouabain-poisoned fibers: (i) Al injection after ATP fails to reverse the stimulatory response to ATP, while ATP injection after Al exerts only a small or no effect. (ii) Mg2+ injection fails to reverse the stimulatory response to Al injection in poisoned fibers. And (iii) Anti-proteolysis agents e.g. leupeptin and pepstatin, upon preinjection, do not alter the kinetic results obtained by injecting Al into unpoisoned and ouabain-poisoned fibers. Topics: Adenosine Triphosphate; Aluminum; Aluminum Chloride; Aluminum Compounds; Animals; Biological Transport; Chlorides; Drug Antagonism; Leupeptins; Magnesium Chloride; Myofibrils; Pepstatins; Sodium; Thoracica | 1992 |
Dysfunction of nonadrenergic noncholinergic inhibitory system after antigen inhalation in actively sensitized cat airways.
We have investigated whether proteases released during antigen inhalation cause dysfunction of the nonadrenergic noncholinergic inhibitory nervous system (NANCIS). Frequency-response (F-R) studies of NANCIS were performed before and after Ascaris antigen (ASC) inhalation using actively sensitized cats. NANC dilatatory effects were obtained by stimulating bilateral cervical vagi under cholinergic and beta-adrenergic blockade and serotonin-induced bronchoconstriction, and assessed by maximal percent relaxation (rmax) and the frequency causing 50% of maximal relaxation (EF50). ASC inhalation caused a transient increase in pulmonary resistance in all animals. One hour after ASC inhalation, pulmonary resistance returned to the baseline value, but ASC inhalation significantly attenuated NANC inhibitory activities: rmax decreased from 82.2 +/- 4.7 (mean +/- SE) to 64.3 +/- 11.2% (p less than 0.05), and the geometric mean of EF50 increased from 1.7 to 4.3 Hz (p less than 0.05). Dilatatory effects of infused VIP, a possible neurotransmitter of NANCIS, was also attenuated after ASC inhalation. Pretreatment with leupeptin (3 mg/kg) abolished ASC-induced impairment of NANC inhibitory activities. By contrast, dilatatory effects of adrenergic nerve stimulation were not affected by ASC inhalation. These results suggest that NANC inhibitory activities can be impaired after ASC inhalation, and that this impairment of NANCIS may be due to effects of proteases released during allergic reaction. Topics: Administration, Inhalation; Airway Resistance; Animals; Antigens; Ascaris; Bronchi; Cats; Electric Stimulation; Immunization; Leupeptins; Nervous System; Neural Inhibition; Respiratory Hypersensitivity; Serine Proteinase Inhibitors; Vasoactive Intestinal Peptide | 1992 |
Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.
The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis. Topics: Ammonium Chloride; Animals; Autoradiography; Biological Transport; Cells, Cultured; Chromatography, Gel; Cobalt Radioisotopes; Electrophoresis, Polyacrylamide Gel; Epithelium; Intrinsic Factor; Iodine Radioisotopes; Kidney; Leupeptins; Molecular Weight; Opossums; Rats; Vitamin B 12 | 1992 |
Processing of the amyloid protein precursor to potentially amyloidogenic derivatives.
The approximately 120-kilodalton amyloid beta protein precursor (beta APP) is processed into a complex set of 8- to 12-kilodalton carboxyl-terminal derivatives that includes potentially amyloidogenic forms with the approximately 4-kilodalton amyloid beta protein (beta AP) at or near their amino terminus. In order to determine if these derivatives are processed in a secretory pathway or by the endosomal-lysosomal system, (i) deletion mutants that produce the normal set of carboxyl-terminal derivatives and shortened secreted derivatives were analyzed and (ii) the effect of inhibitors of endosomal-lysosomal processing was examined. In the secretory pathway, cleavage of the beta APP occurs at a single site within the beta AP to generate one secreted derivative and one nonamyloidogenic carboxyl-terminal fragment, whereas, in the endosomal-lysosomal system, a complex set of carboxyl-terminal derivatives is produced that includes the potentially amyloidogenic forms. Topics: Ammonium Chloride; Amyloid; Amyloid beta-Protein Precursor; Base Sequence; Cell Line; Endopeptidases; Humans; Leupeptins; Lysosomes; Molecular Sequence Data; Mutagenesis; Peptide Fragments; Transfection | 1992 |
The possible involvement of trypsin-like enzymes in germination of spores of Bacillus cereus T and Bacillus subtilis 168.
Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Arginine; Bacillus cereus; Bacillus subtilis; Benzoylarginine Nitroanilide; Chromogenic Compounds; Hot Temperature; Hydrolysis; Leupeptins; Spores, Bacterial; Tosylarginine Methyl Ester; Tosyllysine Chloromethyl Ketone; Trypsin | 1991 |
Morphological and functional changes in the enterocyte induced by fructose.
In the presence of 10-50 mM-fructose, enterocytes of organ-cultured pig intestinal-mucosal explants fail to glycosylate correctly their newly synthesized microvillar enzymes, and instead degrade them [Danielsen (1989) J. Biol. Chem. 264, 13726-13729]. In the present work, this degradation was shown to occur extremely rapidly as the microvillar enzyme aminopeptidase N (EC 3.4.11.2) was hardly detectable after a 10 min pulse with [35S]methionine. The abnormal biosynthesis of membrane glycoproteins affected both the morphology and the function of the Golgi complex as well as the microvillar membrane. Thus the stack of Golgi cisternae was condensed and devoid of dilated rims, and the secretion of a non-glycosylated protein, apolipoprotein A-1, was almost completely blocked in the presence of fructose, showing that transport through the secretory pathway is disturbed even for proteins unaffected by the defective glycosylation. The microvilli of the brush-border membrane were markedly shortened (by about 40%) in the presence of fructose, and incorporation of newly made actin into the microvillar cytoskeleton was similarly decreased. By affecting membrane glycoprotein synthesis, the common dietary sugar fructose thus profoundly perturbs the exocytic membrane traffic in the enterocyte. Topics: Aminopeptidases; Animals; Apolipoprotein A-I; CD13 Antigens; Cell Fractionation; Electrophoresis, Polyacrylamide Gel; Fructose; Glycosylation; Golgi Apparatus; Intestinal Mucosa; Leupeptins; Mannose; Membrane Proteins; Microscopy, Electron; Microvilli; Organ Culture Techniques; Swine | 1991 |
Soluble fibrin preparations inhibit the reaction of plasmin with alpha 2-macroglobulin. Comparison with alpha 2-antiplasmin and leupeptin.
The kinetics of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M) and leupeptin were studied in the presence of fibrin monomer (Fn) and CNBr fragments of fibrinogen (Fg-CNBr). Active plasmin was detected in continuous and discontinuous assays using the chromogenic substrate D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251). The two 'fibrin-like' preparations functioned as hyperbolic mixed-type inhibitors of S-2251 hydrolysis. The dissociation constants (KF) for the binding of plasmin to Fn and Fg-CNBr were 22 nM and 17 nM respectively. Fn and Fg-CNBr inhibited the reaction of plasmin with alpha 2AP: the extent of inhibition depended on the fibrin concentration. In the presence of 800 nM-Fn or 800 nM-Fg-CNBr, the experimental second-order rate constant (K"app.) was decreased from 2.4 x 10(7) M-1.s-1 to 1.2 x 10(6) and 5.3 x 10(5) M-1.s-1 respectively. The effect of Fn and Fg-CNBr on the rate of plasmin inhibition by alpha 2M was even greater. The k"app. value was decreased from 4.0 x 10(5) M-1.s-1 to 8.0 x 10(2) and 1.3 x 10(3) M-1.s-1 in the presence of 800 nM-Fn and -Fg-CNBr respectively. By contrast, the fibrin preparations caused only a small change in the rate of plasmin inhibition by leupeptin. The maximum change in k"app. was 3-fold. All plasmin inhibition curves were linear, suggesting that free and fibrin-bound forms of plasmin remained in equilibrium during the course of reaction with proteinase inhibitors. Fn and Fg-CNBr had no effect on the reaction of miniplasmin with S-2251, alpha 2AP or alpha 2M. When 125I-plasmin was incubated with Fg-CNBr and then allowed to react with a premixed solution of alpha 2AP and alpha 2M, the Fg-CNBr did not significantly change the percentage of plasmin bound to alpha 2AP. These experiments demonstrate that the reaction of plasmin with alpha 2M is inhibited by the non-covalent binding of plasmin to fibrin. We propose that plasmin bound to the surface of a clot is protected from inhibition by alpha 2M as well as by alpha 2AP. Topics: alpha-Macroglobulins; Chromogenic Compounds; Cyanogen Bromide; Fibrin; Fibrin Fibrinogen Degradation Products; Fibrinogen; Fibrinolysin; Humans; Kinetics; Leupeptins; Oligopeptides | 1991 |
Cysteine proteinase inhibitors and bleomycin-sensitive and -resistant cells.
We have isolated a new human head and neck carcinoma cell line (C-10E) that is highly resistant to BLM (40-fold) when compared to the parental (A-253) cell line. Consonant with BLM resistance in the C-10E cell line, we found that this cell line accumulated 2- to 3-fold less BLM A2 than A-253 cells. Kinetic analyses of BLM A2 association revealed a decreased Vmax for C-10E cells with little change in Ka. Furthermore, the BLM-resistant cell line (C-10E) metabolized BLM A2 to a greater extent than its sensitive counterpart (A-253). Thus, compared to A-253 cells, the C-10E cells exhibited both decreased cellular association and increased metabolism of BLM. Synergistic cytotoxicity was seen when BLM was combined with either E-64 or leupeptin, cysteine proteinase inhibitors known to block BLM metabolism in vitro. E-64 inhibited the metabolism of BLM A2 in both C-10E and A-253 cells, and cellular accumulation of radiolabeled BLM A2 was increased by leupeptin or E-64 in only A-253 cells. These results suggest that both inhibition of drug metabolism and increased drug accumulation contribute to this synergism. Topics: Bleomycin; Carcinoma, Squamous Cell; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Drug Resistance; Drug Synergism; Head and Neck Neoplasms; Leucine; Leupeptins; Tumor Cells, Cultured | 1991 |
The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.
A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis. Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Cathepsin B; Cathepsin D; Dipeptides; Fetuins; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Leupeptins; Liver; Lysosomes; Male; Pepstatins; Proteins; Rats; Rats, Inbred Strains; Thiocyanates | 1991 |
Reversal of rapid axonal transport at a lesion: leupeptin inhibits reversed protein transport, but does not inhibit reversed organelle transport.
The hypothesis that the reversal of rapid axonal transport requires a proteolytic conversion of anterograde transport vesicles was examined using sciatic nerve preparations from Xenopus laevis and leupeptin as an inhibitor of proteolysis. The transport of newly synthesized 35S-labeled proteins was studied with a position-sensitive detector of radiation. Organelle transport in isolated myelinated axons was studied by video microscopy. Leupeptin (0.1-0.4 mM) reduced the anterograde-to-retrograde reversal of protein transport adjacent to an axonal lesion. In experiments in which organelle transport was observed close to lesions in axons maintained in a medium compatible with intracellular function, 1.0 mM leupeptin inhibited neither the anterograde-to-retrograde nor the retrograde-to-anterograde reversal of organelle transport. In addition, in experiments in which conditions approximated those used to study protein transport, organelle transport away from the lesion was not inhibited by 1.0 mM leupeptin. A comparison of the morphology of rapidly transported organelles that underwent anterograde transport to the morphology of those that returned from a lesion (with or without the presence of leupeptin) provided no evidence that a morphological conversion was a necessary step in transport reversal. Topics: Animals; Axonal Transport; Axons; Leupeptins; Methionine; Nerve Crush; Nerve Tissue Proteins; Organelles; Reference Values; Sciatic Nerve; Spinal Cord; Sulfur Radioisotopes; Xenopus laevis | 1991 |
Effect of neutral endopeptidase inhibition on substance-P-induced increase in short-circuit current of canine cultured tracheal epithelium.
We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP. Topics: Amiloride; Animals; Anti-Bacterial Agents; Biological Transport, Active; Captopril; Cells, Cultured; Complement C1; Dogs; Dose-Response Relationship, Drug; Electric Conductivity; Female; Furosemide; Glycopeptides; In Vitro Techniques; Ion Channel Gating; Leucine; Leupeptins; Male; Membrane Potentials; Neprilysin; Substance P; Time Factors; Trachea | 1991 |
Acid proteinases of the fore-gut in metamorphosing tadpoles of Rana catesbeiana.
1. Two types of acid proteinases were found in the adult stomach of the bullfrog, Rana catesbeiana. 2. The first type of enzyme appeared in the developing stomach and esophagus and contained more than two kinds of acid proteinases. 3. These enzymes were identified as pepsin-type enzymes. 4. The second type of enzyme existed from the larva to adult stage and was also present in the adult duodenum. 5. This enzyme was different from pepsin and thought to be cathepsin E. Topics: Animals; Chromatography, Agarose; Digestive System; Endopeptidases; Hydrogen-Ion Concentration; Larva; Leupeptins; Molecular Weight; Pepstatins; Rana catesbeiana | 1991 |
Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis.
Sepsis was produced in rats by implanting into their abdominal cavities fecal pellets containing Escherichia coli (10(2) colony-forming units [CFU]) and Bacteroides fragilis (10(4) CFU). Control rats were implanted with sterile pellets. A febrile response and hyperlactacidemia marked the onset of the septic injury. Control and septic rats were killed 24 and 48 hr after implantations, and posterior leg muscles were removed. Muscles were homogenized to prepare soluble fractions containing calcium-independent lysosomal (cathepsins B and L) and calcium-dependent cytosolic (calpain) proteases. Cathepsin and calpain activities were then assayed using standard procedures. There were no alterations in cathepsins B or L activities during sepsis. Calpain activity in septic muscle was significantly higher than that in control muscles. In vitro calpain sensitivity to Ca2+ was also higher in septic muscle than in controls. The cysteine protease inhibitor leupeptin caused a quantitatively greater inhibition of calpain activity in septic than in control muscles. These data indicate that whereas sepsis has no effect on Ca(2+)-insensitive lysosomal proteases, it is associated with an elevation of the Ca(2+)-dependent cytosolic protease activity. Topics: Animals; Bacteremia; Calcium; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Endopeptidases; Escherichia coli Infections; Leupeptins; Male; Muscles; Rats; Rats, Inbred Strains | 1991 |
Catalytic subunits of the multicatalytic proteinase.
Topics: Animals; Binding Sites; Cysteine Endopeptidases; Ethylmaleimide; Leupeptins; Liver; Macromolecular Substances; Multienzyme Complexes; Proteasome Endopeptidase Complex; Rats; Substrate Specificity; Trypsin | 1991 |
Lysosomotropic agents induce morphological and functional changes in human muscle cells in vitro.
We studied the distribution of lysosomes in differentiating human muscle cells in culture treated with propranolol, leupeptin and chloroquine. Chloroquine treated cells showed a significant vacuolization and an increase of the lysosomal apparatus as assessed by cytochemical analysis of the lysosomal enzyme acid phosphatase and by acridine orange staining. At electron microscopic level, an increase of lysosome-like bodies and disorganization of the contractile apparatus were demonstrated in multinucleated myotubes. These alterations observed in cultured muscle cells suggest that lysosomotropic agents may be harmful. Topics: Cells, Cultured; Chloroquine; Histocytochemistry; Humans; Leupeptins; Lysosomes; Microscopy, Electron; Muscles; Propranolol | 1991 |
Inhibitory effects of recombinant human cystatin C on human coronaviruses.
Cystatin C, a potent inhibitor of cysteine proteases such as papain and cathepsin B, was examined for its effect on human coronaviruses OC43 and 229e. Both viruses were greater than 99% inhibited by 0.1 mM inhibitor. Endpoint titrations showed that inhibiting activity paralleled that of leupeptin, a serine and cysteine protease inhibitor, and indicated that 1 to 2 microM inhibitor, slightly above physiologic levels, was effective. Topics: Antiviral Agents; Cathepsin B; Cells, Cultured; Coronaviridae; Cycloheximide; Cystatin C; Cystatins; Escherichia coli; Humans; Leupeptins; Papain; Recombinant Proteins; Virus Replication | 1991 |
Atrial natriuretic factor inhibits ciliary motility in cultured rabbit tracheal epithelium.
To study the effect of atrial natriuretic factor (ANF) on airway ciliary motility, we measured ciliary beat frequency by a photoelectric method in response to ANF in cultured tracheal epithelial cells from rabbits. Addition of ANF but not [Tyr8]ANF-(5-27) decreased ciliary beat frequency in a dose-dependent fashion; the maximal decrease from the baseline value was 24.1 +/- 1.5% (+/- SE, P less than 0.001), and a half-maximal inhibitory concentration (IC50) was 3 x 10(-12) M. Inhibition of neutral endopeptidase activity by phosphoramidon (10(-6) M) or thiorphan (10(-6) M) potentiated the effect of ANF so that the dose-response curve for ANF was shifted to lower concentrations by approximately 0.5 log units (P less than 0.05, in each case). The inhibition of ciliary motility induced by ANF was not affected by the blockade of arachidonic acid metabolism with indomethacin, piroxicam, or nordihydroguaiaretic acid, but it was blocked by methylene blue, a soluble guanylate cyclase inhibitor, and was potentiated by M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor. The intracellular cGMP levels were increased by ANF, an effect that was further potentiated by phosphoramidon or thiorphan. These results suggest that ANF inhibits ciliary motility presumably through a guanylate cyclase-dependent regulatory pathway and that neutral endopeptidase may play a role in modulating the ANF effect on airway mucociliary transport function. Topics: Animals; Atrial Natriuretic Factor; Captopril; Cells, Cultured; Cilia; Enzyme Inhibitors; Epithelium; Glycopeptides; Indomethacin; Kinetics; Leucine; Leupeptins; Male; Masoprocol; Methylene Blue; Movement; Piroxicam; Purinones; Rabbits; Thiorphan; Trachea | 1991 |
Intracellular catabolism of radiolabeled anti-CD3 antibodies by leukemic T cells.
The endocytosis and intracellular metabolism of radiolabeled anti-CD3 MoAb 64.1 by the malignant human T cell line HPB-ALL were studied using biochemical, morphological, electrophoretic, and chromatographic techniques. Biosynthetically labeled [3H]64.1 and externally radioiodinated 125I-64.1 were similarly internalized and degraded by tumor cells, with approximately 70% of the initially bound radioactivity being released to the culture supernatant as trichloroacetic acid-soluble radioactivity in the first 24 hr of culture. Radiolabeled 64.1 was routed from the cell membrane to endosomes where initial proteolysis began and finally to lysosomes where terminal catabolism to single amino acids occurred. SDS-PAGE demonstrated four major intracellular metabolite species (46, 25, 15, and less than 10 kDa). Thin-layer chromatography demonstrated that greater than 95% of the trichloroacetic acid-soluble radioactivity in culture supernatants was 125I-monoiodotyrosine, indicating that proteases, not deiodinases, were of primary importance in catabolism of 125I-64.1. In the presence of inhibitors of lysosomal function (leupeptin, monensin, and ammonium chloride), 125I-64.1 degradation was impeded, causing prolonged retention of radioactivity in the lysosomal compartment of cells. However, although the pace of catabolism was markedly diminished by these agents, no major changes in the sizes of intermediate metabolites generated were observed. Our results suggest that judicious administration of lysosomal inhibitors (e.g. chloroquine, verapamil, monensin) may significantly enhance retention of radioimmunoconjugates by lymphoid malignancies, improving radioimmunoscintigraphic and radioimmunotherapeutic efforts. Topics: Ammonium Chloride; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Autoradiography; CD3 Complex; Endocytosis; Humans; In Vitro Techniques; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Molecular Weight; Monensin; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocytes; Tumor Cells, Cultured | 1991 |
The N-glycan processing in HT-29 cells is a function of their state of enterocytic differentiation. Evidence for an atypical traffic associated with change in polypeptide stability in undifferentiated HT-29 cells.
When the human colon cancer cells HT-29 undergo enterocytic differentiation, they correctly process their N-glycans, whereas their undifferentiated counterpart are unable to process Man9-8-GlcNAc2 species, the natural substrate of alpha-mannosidase I. As this enzyme is fully active in both HT-29 cell populations, we hypothesize that N-glycoproteins are unable to reach the cis Golgi, the site where alpha-mannosidase I has been localized. We have demonstrated this point by using 1-deoxymannojirimycin, leupeptin, and monensin. In the presence of 1-deoxymannojirimycin, a specific inhibitor of alpha-mannosidase I, differentiated HT-29 cells, as expected, accumulate Man9-8-GlcNAc2 species, whereas in undifferentiated HT-29 cells these compounds continue to be rapidly degraded. In contrast, the use of leupeptin, a specific inhibitor of thiol and serine proteases, leads to the accumulation of these oligosaccharides in undifferentiated HT-29 cells. Monensin, a carboxylic ionophore that perturbs distal Golgi functions, is unable to stabilize these compounds. Therefore, we conclude that N-linked glycoproteins in undifferentiated HT-29 cells rapidly egress from the exocytic pathway to a leupeptin-sensitive degradative compartment without entering a monensin-sensitive compartment. These results favor the hypothesis that a direct pathway should exist between the rough endoplasmic reticulum and a leupeptin-sensitive degradative compartment in undifferentiated HT-29 cells. The emergence of this new pathway could explain why protein stability and N-glycan processing may vary as a function of the state of cell differentiation. Topics: 1-Deoxynojirimycin; alpha-Mannosidase; Biological Transport; Cell Differentiation; Colon; Colonic Neoplasms; Glucosamine; Humans; In Vitro Techniques; Leupeptins; Mannosidases; Membrane Glycoproteins; Monensin; Polysaccharides; Protein Processing, Post-Translational; Tumor Cells, Cultured | 1991 |
Inhibition of proteolysis protects hippocampal neurons from ischemia.
Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons. Topics: Animals; Calpain; Cerebral Ventricles; Electrophysiology; Endopeptidases; Evoked Potentials; Gerbillinae; Hippocampus; Hypoxia; Infusions, Parenteral; Ischemic Attack, Transient; Leupeptins; Neurons; Spectrin; Synapses | 1991 |
Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation.
This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity. Topics: Animals; Blotting, Western; Cells, Cultured; Chloroquine; Cycloheximide; Cysteine Endopeptidases; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Insulin; Insulin Antagonists; Leucine; Leupeptins; Mice; Peptide Fragments; Receptor, Insulin | 1991 |
The generation of immunogenic peptides can be selectively increased or decreased by proteolytic enzyme inhibitors.
The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment. Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Hybridomas; In Vitro Techniques; Leupeptins; Mice; Ovalbumin; Peptides; Protease Inhibitors; Spleen; T-Lymphocytes | 1991 |
Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing.
The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perh Topics: Animals; Antibodies; Blister; Edetic Acid; Gene Expression Regulation; Humans; Immunohistochemistry; Leupeptins; Skin; Skin Physiological Phenomena; Swine; Transforming Growth Factor beta; Wound Healing | 1991 |
Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy.
The naturally occurring peptidyl protease inhibitor leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) has been prepared labeled with 13C at the argininal carbonyl. 13C chemical shift data for the trypsin-leupeptin inhibitor complex in the pH range 3.0-7.6 reveal the presence of two pH-dependent covalent complexes, suggestive of two interconverting diastereomers at the new asymmetric tetrahedral center created by covalent addition of Ser195 to either side of the 13C-enriched aldehyde of the inhibitor. At pH 7 two signals are observable at delta 98.8 and delta 97.2 (84:16 ratio), while at pH 3.0 the latter signal predominates. In the selective proton 13C-edited NOE spectrum of the major diastereomer at pH 7.4, a strong NOE is observed between the hemiacetal proton of the inhibitor and the C2 proton of His57 of the enzyme, thus defining the stereochemistry of the high pH complex to the S configuration in which the hemiacetal oxygen resides in the oxyanion hole. pH titration studies further indicate that the 13C chemical shift of the S diastereomer follows a titration curve with a pKa of 4.69, the magnitude of which is consistent with direct titration of the hemiacetal oxygen. Similar pH-dependent chemical shifts were obtained by using CPMAS 13C NMR, providing evidence for the existence of the same diastereomeric equilibrium in the solid state. Topics: Binding Sites; Hydrogen-Ion Concentration; Leupeptins; Macromolecular Substances; Magnetic Resonance Spectroscopy; Serine Proteinase Inhibitors; Solutions; Trypsin | 1991 |
Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors.
Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP. Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blood Proteins; Calcimycin; Calcium; Calpain; Enzyme Activation; Erythrocytes; Female; In Vitro Techniques; Leucine; Leupeptins; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Rabbits | 1991 |
Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (Matrigel).
We have investigated the adhesive properties and invasiveness of cells of the human ovarian carcinoma line, NIH:OVCAR-3, in vitro. OVCAR-3 cells exhibited a similar rate of adhesion to all substrates tested including laminin, fibronectin, and collagens I and IV. The synthetic peptide YIGSR-NH2, which corresponds to an attachment site in laminin, inhibited the adhesion of the cells to laminin, but not to fibronectin. In contrast, a GRGDS-NH2 peptide blocked adhesion to fibronectin but not to laminin. OVCAR-3 cells invaded and formed branched colonies on Matrigel. Colony formation was retarded by both YIGSR-NH2 and GRGDS-NH2 peptides. Serine protease inhibitors and human recombinant TIMP, the tissue inhibitor of metalloproteases, inhibited ovarian tumor cell invasion while a synthetic collagenase IV inhibitor (SC-44463) had no effect. These studies suggest that metalloproteases other than collagenase IV may be important for the invasive activity of ovarian cancer cells. It is possible that synthetic peptides with antiadhesive cellular activity and certain antiproteases could be used to control the progressive colonization and invasion of peritoneal surfaces by malignant ovarian cancer cells. Topics: Amides; Amino Acid Sequence; Aminocaproates; Antineoplastic Agents; Benzamidines; Biocompatible Materials; Cell Adhesion; Cell Line; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Female; Glycoproteins; Humans; In Vitro Techniques; Laminin; Leupeptins; Metalloendopeptidases; Molecular Sequence Data; Neoplasm Invasiveness; Oligopeptides; Ovarian Neoplasms; Pepstatins; Polyamines; Protease Inhibitors; Proteoglycans; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured; Tyrosine | 1991 |
Effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to oxidase in Chinese hamster V79 cells.
The effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to the free radical-producing xanthine oxidase in Chinese hamster V79 cells have been investigated using a newly developed fluorimetric enzyme assay. Hypoxia caused an increase in xanthine oxidase activity from 25% to 80% of the total activity of xanthine oxidase and dehydrogenase. The ratio returned to normal levels within 24 h of aerobic incubation. Hypoxia caused the release of xanthine oxidase in the medium of V79 cells and an increase in total protein concentration in the medium. There was an early change induced in lipid peroxidation markers and this was inhibited by allopurinol. The effects of glucose deprivation and calcium blockers were also investigated. Fura-2 AM was found to interact with V79 cells, making it impossible to determine intracellular calcium levels in V79 cells by this reagent. Topics: Allopurinol; Animals; Calcium; Cell Line; Cell Membrane; Cricetinae; Cricetulus; Glucose; Hypoxia; Leupeptins; Lipid Peroxidation; Verapamil; Xanthine Dehydrogenase; Xanthine Oxidase | 1991 |
Studies on the antimalarial mode of action of quinoline-containing drugs: time-dependence and irreversibility of drug action, and interactions with compounds that alter the function of the parasite's food vacuole.
The quinoline-containing antimalarial drugs chloroquine, quinine and mefloquine exert an irreversible inhibitory effect on erythrocytic stages of Plasmodium falciparum grown in culture. Inhibition is time- and concentration-dependent and the full effect is observed after 2-6 hours of exposure to the drug. Washing of infected cells after drug exposure in the presence of NH4Cl to accelerate drug efflux, intensifies the inhibitory effect of chloroquine, probably due to the pH-dependent release of highly concentrated drug from the acidic food vacuole of the parasite. When both antimalarials and NH4Cl are present in the culture, drug effect is reduced, as expected from the demonstrable alkalinization of the food vacuole and the consequent reduction in drug accumulation. The protease inhibitor leupeptin inhibits digestion of ingested host cell cytosol, and thus inhibits parasite growth, though reversibly so (Rosenthal et al, J. Clin. Invest. 82 1560-1566 (1988)). Thus, although the antimalarials also inhibit the feeding process, this is not the cause of their irreversible action. Leupeptin is found to be antagonistic to antimalarials' action, suggesting that the drugs form complexes with products of host cell digestion that are responsible for irreversible inhibition of parasite growth. Topics: Ammonium Chloride; Animals; Antimalarials; Drug Combinations; Drug Interactions; Hydrogen-Ion Concentration; Leupeptins; Plasmodium falciparum; Quinolines; Time Factors | 1991 |
Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells.
Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain. Topics: Animals; Axons; Blood; Calpain; Endopeptidases; Hirudins; Leupeptins; Neuroblastoma; Osmolar Concentration; Protease Inhibitors; Thrombin; Tumor Cells, Cultured | 1991 |
Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract.
Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors. Topics: Aged; Autoradiography; Calcium; Carcinoma, Squamous Cell; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Humans; Leupeptins; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Proteins; Tissue Extracts | 1991 |
Myofibrillar proteinase, cathepsin B, and protein breakdown rates in skeletal muscle from septic rats.
Muscle catabolism during sepsis is mainly caused by myofibrillar protein breakdown. The mechanism of this metabolic response is not known. We tested the hypothesis that increased protein breakdown in the extensor digitorum longus (EDL) muscle of septic rats is caused by increased activity of the so-called myofibrillar proteinase, which is a nonlysosomal proteolytic enzyme, and cathepsin B, which is a lysosomal proteinase. Sepsis, induced in male Sprague-Dawley rats (50 to 60 g) by cecal ligation and puncture (CLP), resulted in an approximately 50% increase in myofibrillar proteinase activity and an approximately 30% increase in cathepsin B activity. Concomitantly, both total and myofibrillar protein breakdown rates, measured as release of tyrosine and 3-methylhistidine (3-MH), respectively, by incubated EDL muscles, were substantially elevated. Treatment of septic rats with the mast cell degranulating compound 48/80 or the lysosomal protease inhibitor leupeptin significantly reduced myofibrillar proteinase and cathepsin B activities, but did not affect protein breakdown rates. The results suggest that increased protein breakdown in septic skeletal muscle is associated with, but not caused by, myofibrillar proteinase or cathepsin B activity. The data also support the concept of a mast cell origin of the myofibrillar proteinase activity, but do not suggest an obligatory involvement of mast cell proteinase in increased protein degradation during sepsis. Topics: Animals; Cathepsin B; Endopeptidases; Infections; Leupeptins; Male; Muscle Proteins; Muscles; Myofibrils; Rats; Rats, Inbred Strains; Reference Values | 1991 |
Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome).
The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu. Topics: Amino Acid Sequence; Animals; Antipain; Cattle; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Erythrocytes; Humans; Insulin; Kinetics; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Oxidation-Reduction; Pentamidine; Proteasome Endopeptidase Complex | 1991 |
Effects of calcium on protein turnover of incubated muscles from mdx mice.
Mdx mice have a genetic defect similar to that which causes Duchenne muscular dystrophy in humans. The influence of calcium on muscle protein metabolism of mdx and wild type (C57BL/10) mice was examined in vitro. Incubation of mdx muscles in a medium containing calcium at a concentration of 2.0 mM (but not 0.2 mM) resulted in proteolytic rates that were greater than those of C57BL/10 muscles. At 2.0 mM extracellular calcium, mdx muscle proteolysis was attenuated by thiol protease inhibitors but not by the weak base methylamine. Protein synthetic rates were higher in incubated mdx muscles than in incubated C57BL/10 muscles, but no effect of extracellular calcium concentration was observed in either strain. These data suggest that mdx mice have an abnormality of muscle calcium handling, which results in activation of nonlysosomal proteolytic processes but does not exert acute effects on protein synthetic rate. Topics: Animals; Calcium; Cycloheximide; Kinetics; Leupeptins; Methylamines; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscles; Muscular Dystrophy, Animal; Proteins; Tyrosine | 1991 |
Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium falciparum.
17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium falciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC50 in the order of 10(-7) M. Chymostatin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC50 in the order of 10(-5) M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium falciparum. Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Erythrocytes; Humans; Leucine; Leupeptins; Oligopeptides; Pepstatins; Plasmodium falciparum | 1991 |
Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets.
The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms. Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Blood Platelets; Blotting, Western; Calcium; Cell Compartmentation; Enzyme Activation; In Vitro Techniques; Leupeptins; Molecular Sequence Data; Molecular Weight; Peptides; Phosphorylation; Protein Kinase C; Rabbits; Tetradecanoylphorbol Acetate | 1991 |
Leupeptin causes an accumulation of lipofuscin-like substances in liver cells of young rats.
Leupeptin, a thiol protease inhibitor, has previously been shown to cause a dense accumulation of substances resembling age pigment and called ceroid-lipofuscin, in brain cells of young rats. Thus far, however, attempts to produce age pigments in hepatocytes of normal young rats with protease inhibitor(s) have not been successful. The present study provides the first demonstration that leupeptin induces lipofuscin-like substances in normal young rat hepatocytes. Male Fischer-344 rats (age 4-6 weeks) were continuously infused with leupeptin or saline i.p. for 2 weeks by an osmotic minipump (dosage, 1-50 mg/100 g per day). Liver tissues were then examined by light, fluorescence and electron microscopy. Both hepatocytes and non-parenchymal cells of livers treated with leupeptin, but not saline, showed a dense accumulation of pigments which stained deeply with toluidine blue, were PAS-positive and were brightly autofluorescent. After UV excitation the pigments had an emission spectrum with a broad peak at 480-540 nm extending to 650 nm resembling the spectrum of age pigment from livers of normal aged rats. Electron microscopic examination revealed numerous lipofuscin-like deposits with heterogeneous morphology in the cytoplasm of both hepatocytes and non-parenchymal cells; lipid and myelin-like bodies were also present in hepatocytes. The results indicate that the perturbation of proteolytic activity in liver by leupeptin causes an accumulation of substances which by several criteria resemble lipofuscin. These results thus provide further support for the 'Protease Inhibitor Model of Lipofuscin Formation' as well as a potential experimental model for studying hepatocellular aging processes. Topics: Aging; Animals; Ceroid; Histocytochemistry; Leupeptins; Lipofuscin; Liver; Male; Microscopy, Fluorescence; Rats; Rats, Inbred F344 | 1991 |
Two-stage autolysis of the catalytic subunit initiates activation of calpain I.
Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products. Topics: Amino Acid Sequence; Animals; Calcium; Calcium-Binding Proteins; Calpain; Carbohydrate Sequence; Cattle; Enzyme Activation; Erythrocytes; Leupeptins; Molecular Sequence Data | 1991 |
Study of thiol proteases of normal human skin fibroblasts.
The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose. Topics: Caseins; Cathepsin B; Cathepsin H; Cathepsin L; Cathepsins; Cell Fractionation; Cells, Cultured; Concanavalin A; Coumarins; Cysteine Endopeptidases; Dipeptides; Endopeptidases; Fibroblasts; Humans; Hydrogen-Ion Concentration; Hymecromone; Leupeptins; Lysosomes; Microsomes; Skin | 1991 |
The effect of lysosomal inhibitors on protein degradation in cat hepatocyte monolayers.
1. Isolated cat liver cells were established in monolayer culture and intracellular proteins were labelled for 20 hr with L-[4,5-3H]leucine. 2. The known intralysosomal protein degradation inhibitors such as NH4Cl, leupeptin, cycloheximide and insulin were all effective in decreasing the rate of proteolysis in cat hepatocytes. However, the maximum inhibitory effects of these agents were generally lower when compared to the rat. 3. These results indicated a smaller contribution of the lysosomal pathway towards overall protein degradation in cat liver. Topics: Ammonium Chloride; Animals; Cats; Cells, Cultured; Cycloheximide; Insulin; Leucine; Leupeptins; Liver; Lysosomes; Proteins | 1991 |
Deficiency of kallikrein-like enzyme activities in cerebral tissue of patients with Alzheimer's disease.
We examined the changes in the intracerebral activities, at the time of postmortem autopsy, in patients with Alzheimer's disease. When compared with the control group, the activity of kallikrein-like enzyme was significantly decreased, while prolyl endopeptidase activity increased, in the patients group. Aprotinin inhibited 50% of the activity of the former enzyme at 2 x 10(-7) M. Taken together with the results of a multivariate study, the above findings may indicate that intracerebral kallikrein deficiency plays an important role in the pathogenesis of Alzheimer's disease. Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amino Acid Sequence; Aprotinin; Brain; Endopeptidases; Female; Humans; Kallikreins; Leupeptins; Male; Middle Aged; Molecular Sequence Data; Peptide Hydrolases; Prolyl Oligopeptidases; Serine Endopeptidases | 1990 |
Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: evidence for the role of cell death genes.
Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules). Topics: Animals; Cell Survival; Chick Embryo; Chloroquine; Curare; Cycloheximide; Dactinomycin; Ganglia, Spinal; Gene Expression; Genotype; Leupeptins; Microscopy, Electron; Motor Neurons; Protein Biosynthesis; Puromycin; RNA; Spinal Cord | 1990 |
Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin.
By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC. Topics: Animals; Antigen-Presenting Cells; Aspartic Acid Endopeptidases; B-Lymphocytes; Cathepsins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endopeptidases; Epitopes; H-2 Antigens; In Vitro Techniques; Leupeptins; Macrophages; Mice; Ovalbumin; Pepstatins; Peptide Fragments; T-Lymphocytes, Helper-Inducer | 1990 |
Enhanced proliferation of murine T cell lines following interaction of Poly(Glu60, Phe40) (GPhe) and antigen-presenting accessory cells. III. Possible mechanisms responsible for activity of GPhe.
The random copolymers (Glu80, Phe20)n (GPhe20), (Glu60, Phe40)n (GPhe), and (Glu50, Phe50)n (GPhe50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe20, GPhe, and GPhe50 showed "augmenting" activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the "active" determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM] driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) "GPhe-anti-GPhe" complexes interfered with necessary "processing" of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [3H]thymidine incorporation ([3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be "processed" by APCs to exhibit activity. Topics: Animals; Antibodies; Antigen-Presenting Cells; Cell Division; Cell Line; Cell Membrane; Culture Media; Epitopes; Interleukin-1; Leupeptins; Lymphocyte Activation; Mice; Phenylalanine; Polyglutamic Acid; T-Lymphocytes; Time Factors | 1990 |
Effects of inhibitors on proteinase activities in gels following isoelectric focussing.
Homogenates of rat submandibular glands were electrophoresed on isoelectric focussing gels and bands of proteinase activity demonstrated by means of overlay membranes impregnated with fluorogenic oligopeptide substrates. Inhibition characteristics of individual proteinase bands were tested within the gels and after excision and subsequent elution from the gels. Some bands of activity were resistant to inhibition by pre-incubation of the gel with certain inhibitors, but were subsequently found to be inhibited when tested with the same substrate after elution. These inconsistencies were influenced by the use of different substrates. The results indicate that, though some useful screening information may be obtained by applying inhibitors to the gels, this system does not permit a reliable assessment of inhibition characteristics which require subsequent elution and assay. Topics: Amino Acid Sequence; Animals; Aprotinin; Endopeptidases; Isoelectric Focusing; Leupeptins; Molecular Sequence Data; Oligopeptides; Protease Inhibitors; Rats; Submandibular Gland; Trypsin Inhibitors | 1990 |
Natural killer cell activity of rhesus macaques against retrovirus-pulsed CD4+ target cells.
Rhesus peripheral blood mononuclear cells (PBMC) fail to demonstrate natural killer (NK) activity against the human T-cell lines CEM, CEM x 174, or SUP-T1. However, these cell lines could act as NK-sensitive target cells if they were pulsed with heat-inactivated, whole simian immunodeficiency virus (SIV). The ability of these SIV-pulsed T-cell lines to act as NK-sensitive target cells was directly related to the relative density of CD4 on their surface. Target cell generation was inhibited by preincubation of cell lines with CD4 monoclonal antibody (MAb) with specificity for the SIV binding site. In addition, NK activity was seen against target cells that had been prepared with human immunodeficiency virus type 1 (HIV-1) gp120, nonglycosylated gp120, env A of feline leukemia virus (FeLV), and simian type D retrovirus (SRV). Addition of leupeptin to target cells prior to SIV pulsing did not result in a significant decrease in cytotoxic activity, suggesting that processing is not required for the generation of target cells. The cells that mediate NK activity are nonadherent, do not form rosettes with AET-treated sheep red blood cells (SRBC), and are phenotypically CD16+ and CD8+. NK activity of SIV-infected macaques was significantly decreased against both K562 cells and SIV-pulsed target cells as compared with uninfected animals. However, treatment of PBMC with interleukin-2 (IL-2) resulted in a partial restoration of NK activity. Topics: Animals; CD4 Antigens; CD4-Positive T-Lymphocytes; Cell Line; Cytotoxicity, Immunologic; Female; Humans; Interleukin-2; Killer Cells, Natural; Leupeptins; Macaca mulatta; Male; Retroviridae Infections; Simian Immunodeficiency Virus | 1990 |
Calpain inhibitors block long-term potentiation.
Long-term potentiation (LTP) is a form of synaptic plasticity that serves as a model for certain types of learning and memory. The role of the calcium-activated thiol proteases or calpains in the biochemical mechanism of LTP has been explored. We show that the extracellular application of two newly developed, highly potent calpain inhibitors, N-acetyl-Leu-Leu-norleucinal and N-acetyl-Leu-Leu-methioninal, block LTP in both the Schaffer collateral-CA1 synaptic zone of the rat hippocampal slice and in perforant path-stimulated dentate granule cells in the intact hippocampus. The inhibitors do not affect baseline synaptic transmission and block LTP in the slice preparation if applied before but not after tetanic stimulation. The calpain inhibitor leupeptin is less potent than the above peptides but also blocks LTP if applied at a sufficient concentration. Topics: Animals; Calpain; Glycoproteins; Hippocampus; In Vitro Techniques; Kinetics; Leupeptins; Neuronal Plasticity; Rats; Rats, Inbred Strains; Synapses | 1990 |
Prevention of thromboxane B2-induced hepatocyte plasma membrane bleb formation by certain prostaglandins and a proteinase inhibitor.
Isolated hepatocytes incubated in the presence of thromboxane B2 developed many plasma membrane blebs which are a characteristic feature of toxic or ischaemic cell injury. When hepatocytes were incubated in the presence of both thromboxane B2 and the non-lysosomal proteinase inhibitor, leupeptin, were also well protected from the formation of blebs. This implies that thromboxane B2 is able to activate non-lysosomal proteinases which appear to attack certain cytoskeletal proteins. The data presented are consistent with thromboxane B2 acting as an intermediary in a proposed mechanism of cell injury and death in which elevated cytosolic free Ca2+ levels activate phospholipase A2 and the arachidonic acid cascade. Topics: 16,16-Dimethylprostaglandin E2; Animals; Calcimycin; Calcium; Cell Membrane; Epoprostenol; Leupeptins; Liver; Male; Oligopeptides; Prostaglandins E, Synthetic; Rats; Thromboxane B2 | 1990 |
Endocytosis and degradation of ovine prolactin by Nb2 lymphoma cells: characterization and effects of agents known to alter prolactin-induced mitogenesis.
Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear. Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells. Stationary-phase cells were incubated with [125I]oPRL in Fischer's medium containing horse serum. Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity. Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total [125I]oPRL bound by the cells. Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium. Endocytosis of [125I]oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions. Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added [125I]oPRL was acid soluble. Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented [125I]oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation. Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions. Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration. Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h. Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased [125I]oPRL degradation by 25% during a 4-h incubation. This response was abolished when the cells were exposed simultaneously to PMA. These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C. The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells. Since amiloride also produced major changes in oPRL metabolism, pos Topics: Amiloride; Animals; Calcimycin; Cell Division; Cell Membrane; Chloroquine; Endocytosis; Kinetics; Leupeptins; Lymphoma; Methylamines; Mitosis; Monensin; Prolactin; Rats; Receptors, Prolactin; Sheep; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured | 1990 |
Assembly and GPIIIa content of cytoskeletal core in platelets agglutinated with bovine von Willebrand factor.
The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface-labeled aspirin-treated washed platelets. Binding of ligands to GPIIb-IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained. Topics: Actinin; Actins; Adenosine Diphosphate; Adult; Animals; Blood Platelets; Calpain; Cattle; Cytoskeleton; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Leupeptins; Microfilament Proteins; Molecular Weight; Myosins; Platelet Aggregation; Platelet Membrane Glycoproteins; von Willebrand Factor | 1990 |
Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling.
Lipoprotein lipase (LPL) hydrolyzes triglyceride in plasma lipoprotein primarily while bound to vascular endothelial cells. LPL metabolism by cultured endothelial cells was studied. Purified radioiodinated bovine LPL bound to porcine aortic endothelial cells at 4 degrees C with an association constant of 0.18 x 10(7) m-1. Analysis of the time course of LPL dissociation from endothelial cells at 4 degrees C yielded a dissociation rate constant of 3.9 x 10(-6)s-1. After 1 h at 37 degrees C, 28% of the LPL initially bound to the cell surface was no longer releasable by heparin or trypsin treatments, suggesting that LPL was internalized by the cells. Addition of heparin to the medium or pretreatment of the cells with heparinase markedly reduced the amount of LPL internalized, establishing a requirement for cell surface heparan sulfate proteoglycans in the process. When cells containing internalized LPL were incubated at 37 degrees C, a time-dependent increase in the amount of LPL in the medium and a corresponding decrease in LPL associated with the cells was found. This suggested that internalized LPL was released back into the medium. The catalytic activity, molecular size, and heparin-binding characteristics of the released LPL was similar to native LPL. Addition of either heparin, heparinase, or excess unlabeled LPL to prevent the rebinding of released 125I-LPL to the cell surface increased the amount of 125I-LPL present in the medium, suggesting that there is a process of recycling of 125I-LPL bound to the cell surface. Studies examining the effect of pH on dissociation of LPL from its binding site showed less dissociation of cell surface bound LPL at pH 5.5 compared with pH 7.4 and 8.5. These results suggest that even at acidic pH as in endocytotic vesicles, LPL remains bound to proteoglycans and this may facilitate the recycling of internalized LPL molecules. Topics: Animals; Aorta; Biological Transport; Cattle; Cells, Cultured; Chloroquine; Chondroitin Sulfate Proteoglycans; Cytochalasin B; Endothelium, Vascular; Female; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin; Heparin Lyase; Heparitin Sulfate; Hydrogen-Ion Concentration; Kinetics; Leupeptins; Lipoprotein Lipase; Milk; Monensin; Polysaccharide-Lyases; Protein Binding; Proteoglycans; Swine | 1990 |
Stabilization of rat cardiac sacroplasmic reticulum Ca2+ uptake activity and isolation of vesicles with improved calcium uptake activity.
The Ca2+ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca2+ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0 degrees or 37 degrees, but the Ca2+ uptake activity was relatively stable at 25 degrees. The decay of Ca2+ uptake activity at 0 degrees could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca2+ uptake activity of homogenates kept on ice but not of homogenates kept at 37 degrees. We also found that release of the CSR from the cellular debris required homogenization in high KCl. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mumol/min-mg in the absence of CSR calcium channel blockers and 0.67 mumol/min/mg in the presence of 10 microM ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca2(+)-ATPase rates averaged 1.07 mumol/min/mg and 0.88 mumol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a 'crude' preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca2(+)-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function. Topics: Animals; Calcium; Calcium-Transporting ATPases; Centrifugation, Density Gradient; Dithiothreitol; Dogs; Electrophoresis, Polyacrylamide Gel; Kinetics; Leupeptins; Male; Myocardium; Phenylmethylsulfonyl Fluoride; Potassium Chloride; Rats; Rats, Inbred Strains; Sarcoplasmic Reticulum; Sulfites | 1990 |
Calpain inhibitors improve the recovery of synaptic transmission from hypoxia in hippocampal slices.
Two inhibitors of calcium activated proteases (calpains) were tested for their effects on hypoxia-induced synaptic dysfunction in hippocampal slices. Hypoxic episodes lasting for either one or two minutes beyond the point at which action potentials (fiber volleys) disappeared were used. Leupeptin and calpain inhibitor I had no reliable effects on the rate at which synaptic transmission declined during hypoxia or the time required for loss of action potentials, but both drugs did substantially improve the degree of recovery. Moreover, the percentage of slices meeting an arbitrary criterion for viability after hypoxic treatment was greatly increased by the drug treatment. These results point to the conclusion that proteolysis triggered by calcium influx during hypoxia contributes to pathophysiology. Topics: Animals; Calpain; Cell Hypoxia; Glycoproteins; Hippocampus; In Vitro Techniques; Leupeptins; Rats; Rats, Inbred Strains; Synapses; Synaptic Transmission | 1990 |
Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L-132 cells.
We have used a new approach to test the possible participation of lysosomes in the degradation of long-lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L-132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed by ferritin uptake and electron microscopy. Degradation rates of long-lived proteins increased with the uptake of lysosomal proteases. However, the increased protein degradation of chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells 'starved' of serum (step-down conditions). This approach opens a new way of investigating the degradation of intracellular proteins in cultured cells. Topics: Cathepsins; Cells, Cultured; Chloroquine; Drug Carriers; Endocytosis; Ferritins; Humans; L Cells; Leupeptins; Liposomes; Lysosomes; Membrane Proteins; Microinjections; Microscopy, Electron; Peptide Hydrolases; Protein Biosynthesis; Proteins; Time Factors | 1990 |
Regulation of total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscle incubated flaccid or at resting length.
The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle. Topics: Animals; Insulin; Kinetics; Leupeptins; Male; Methylhistidines; Muscle Proteins; Muscles; Myofibrils; Rats; Rats, Inbred Strains; Tyrosine | 1990 |
Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties.
Enzymatic properties of a protease involved in hatching of mouse embryos were examined. A trypsin-like protease, which most efficiently hydrolyzed t-butoxycarbonyl-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide, was demonstrated in culture medium of mouse hatching embryos. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, N alpha-tosyl-L-lysyl-chloromethane, soybean trypsin inhibitor, and Trasylol, but not or weakly inhibited by p-chloromercuribenzoic acid, EDTA, E-64, pepstatin, chymostatin, and bestatin, suggesting a trypsin-like serine proteinase. The protease activity in the medium gradually elevated during the course of hatching, whereas the embryo-associated activity showed no significant change. Furthermore, pyroglutamyl-Leu-argininal, the strongest inhibitor for the enzyme among peptidyl argininals, all of which are potent trypsin inhibitors, showed the strongest inhibition toward hatching. Thus, a trypsin-like protease secreted from hatching embryos into the culture medium may participate in mouse hatching, probably as a hatching enzyme. Topics: Animals; Blastocyst; Coumarins; Culture Media; Embryo, Mammalian; Leupeptins; Metalloendopeptidases; Mice; Mice, Inbred ICR; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Serine Endopeptidases; Substrate Specificity | 1990 |
Cooperation of lysosomes and inner mitochondrial membrane in the degradation of carbamoyl phosphate synthetase and other proteins.
Carbamoyl phosphate synthetase (CPS) from rat liver is proteolitically inactivated at acid pH by broken lysosomes. Inactivation increases when lysosomes are previously incubated with inner mitochondrial membrane, although this mitochondrial fraction does not inactivate CPS 'per se'. The increased degradation is due to membrane factor(s), most probably mitochondrial proteinase(s), solubilized by lysosomal matrix proteinases, after incubation of the inner mitochondrial membrane fraction with broken lysosomes. This (these ) factor(s) degrade(s) CPS and other proteins in the absence of lysosomal proteinases or when these are inhibited by leupeptin, chymostatin and pepstatin. We have also tested the possible regulation of this degradation and found that ATP and, particularly, acetyl glutamate accelerate the degradation of CPS by the factor(s) liberated from the inner mitochondrial membrane. Topics: Adenosine Triphosphate; Animals; Carbamoyl-Phosphate Synthase (Ammonia); Enzyme Activation; Glutamates; Hydrogen-Ion Concentration; Intracellular Membranes; Leupeptins; Lysosomes; Mitochondria, Liver; Oligopeptides; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Rats; Substrate Specificity | 1990 |
Phalloidin-induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation.
Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels. Topics: Acid Phosphatase; Actins; Animals; Cathepsins; Female; Fructose-Bisphosphate Aldolase; Immunoenzyme Techniques; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Microscopy, Electron; Myosins; Oligopeptides; Phalloidine; Pyruvate Kinase; Rats; Rats, Inbred Strains | 1990 |
Another mechanism for the defect in type III collagen accumulation in Ehlers-Danlos syndrome type IV: increased intracellular degradation of the procollagen.
The nature of type III collagen was examined in the skin and cultured skin fibroblasts from a patient with Ehlers-Danlos syndrome type IV. Although the culture medium contained a much lower amount of Type III collagen than the controls, the cells contained an apparently normal amount of Type III collagen. The patient's Type III procollagen showed no abnormalities in apparent molecular weight, the peptide length as examined by cyanogen bromide cleavage, the genomic DNA size including its C- and N-propeptide portion, mRNA size, or thermal stability; but a pulse-chase study revealed prolonged retention of the type III collagen in the cells. Degradation of Type III procollagen was induced by cell extracts but did not occur in the extracellular space and was inhibited in intact cells by the addition of ammonium chloride or leupeptin to the culture medium. Fluorescent staining showed a characteristic granular deposition of Type III procollagen in the peripheral region of the cytoplasm but no granular deposition of Type I procollagen. These results offer new insight into the mechanism of the decreased amount of Type III collagen in the tissue of patients with Ehlers-Danlos syndrome type IV. Topics: Ammonium Chloride; Biological Transport; Blotting, Southern; Cells, Cultured; Collagen; Ehlers-Danlos Syndrome; Extracellular Space; Fluorescent Antibody Technique; Glycosylation; In Vitro Techniques; Leupeptins; Lysosomes; Peptide Fragments; Procollagen; Skin | 1990 |
Phylogeny of immune recognition: antigen processing/presentation in channel catfish immune responses to hemocyanins.
Studies were conducted to address the role(s) of antigen (Ag) processing/presentation in channel catfish immune responses. Vigorous and specific secondary in vitro proliferative and antibody (Ab) responses were obtained to keyhole limpet and Limulus polyphemus hemocyanins with peripheral blood leukocytes (PBL) from catfish previously primed in vivo with Ag. In addition, such antigen-specific in vitro proliferative and Ab responses were efficiently elicited by antigen-pulsed and subsequently paraformaldehyde-fixed autologous PBL used as putative antigen-presenting cells (APC) but not by APC fixed prior to Ag pulsing. Treatment of these putative APC with lysosomotropic agents, protease inhibitors, or the ionophore monensin prior to or during pulsing with Ag significantly inhibited both in vitro responses. Furthermore, the use of radiolabeled protein indicated that both untreated and inhibitor-treated PBL but not erythrocytes take up Ag; however, only untreated PBL were able to degrade Ag. Immune restriction was indicated by the use of allogeneic PBL as APC in that only strong MLRs were generated with no detectable antibodies produced in vitro. Finally, the employment of isolated leukocyte subpopulations demonstrated that both catfish B (sIg+) lymphocytes and monocytes were efficient Ag presentors. Topics: Ammonium Chloride; Animals; Antibody Formation; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Chloroquine; Fixatives; Hemocyanins; Ictaluridae; In Vitro Techniques; Leupeptins; Lymphocyte Activation; Monensin; Monocytes; Phenylmethylsulfonyl Fluoride | 1990 |
Protein breakdown in submandibular glands rendered hypertrophic by amputation of lower incisor teeth in rats.
Protein breakdown in submandibular glands rendered hypertrophic by amputation of the lower incisor teeth in rats was investigated. Reduced protein breakdown was observed in the hypertrophic gland tissues, and was found to be inhibited by 20 mM epsilon-amino-n-caproic acid, an inhibitor of serine protease, and 50 microM leupeptin, an inhibitor of trypsin, plasmin, papain and cathepsin B, but not by 2 mM PMSF (phenylmethylsulfonyl fluoride), an inhibitor of serine protease, 10 microM pepstatin, an inhibitor of cathepsin D and 20 microM antipain, an inhibitor of cathepsin A and B. These results suggest that some serine proteases and leupeptin-sensitive proteases (presumably cathepsin B) participate in protein breakdown in hypertrophic gland tissues, and that hypertrophy of the submandibular glands is closely related to the reduced protein breakdown in these tissues. Topics: Aminocaproic Acid; Animals; Antipain; Hypertrophy; Incisor; Leupeptins; Male; Mandible; Pepstatins; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Rats; Rats, Inbred Strains; Salivary Proteins and Peptides; Submandibular Gland | 1990 |
Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts.
The lysosomal cysteine proteinase cathepsin L is synthesized in cultured mouse NIH 3T3 cells as a 39 kDa precursor and processed intracellularly into active 29 kDa and 20 kDa + 5 kDa lysosomal forms. Addition to culture media of the peptidyl aldehyde leupeptin, a non-covalent inhibitor of cathepsin L, results in the accumulation of the 20 kDa mature form of the enzyme, resulting in increased activity of cathepsin L as measured in an in vitro assay system in the absence of leupeptin. The more potent irreversible cathepsin L inhibitors benzyloxycarbonyl-Phe-Ala-diazomethane and L-transepoxysuccinyl-L-leucylamino-(4-guanidino)butane, when added to living cells at low concentrations, result in accumulation of all partially processed forms of cathepsin L, especially the 29 kDa form, suggesting that cathepsin L is responsible for its own processing. Exogenous procathepsin L introduced into CHO cells by endocytosis via the mannose 6-phosphate receptor is processed in a manner similar to endogenous procathepsin L. We conclude that the major intracellular pathway for processing of procathepsin L, either endogenous or exogenous, probably requires active cathepsin L. Topics: Animals; Cathepsin L; Cathepsins; Cell Line; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Fibroblasts; Kinetics; Leupeptins; Methionine; Mice; Molecular Weight | 1990 |
Factors influencing the binding of calpain I to human erythrocyte inside-out vesicles.
The mechanism for binding of human erythrocyte calpain I to human erythrocyte inside-out vesicles was studied by immunoelectrophoretic blot analysis. Binding of calpain I to inside-out vesicles was observed both in the absence and presence of Ca2+. Moreover, in the absence of Ca2+, acidic proteins like casein, ovalbumin and calpastatin suppressed while basic proteins like arginase and lysozyme did not affect the binding of calpain I to inside-out vesicles. Here, we propose a model for the binding of calpain to the membrane. Topics: Arginase; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Erythrocyte Membrane; Humans; Immunoelectrophoresis; Immunoglobulins; Leupeptins; Models, Biological; Muramidase; Ovalbumin; Protease Inhibitors | 1990 |
Proteolysis is a critical step in the physiological stop pathway: mechanisms involved in the blockade of axonal regeneration by mammalian astrocytes.
Regenerating axons of adult dorsal roots are stopped by reactive astrocytes at the PNS-CNS junction. While it has been suggested that the astrocytes might pose a physical barrier to axonal growth, based on ultrastructural comparisons of physically blocked and axo-glial endings, it was proposed that astrocytes in the root transitional zone block axonal growth by activating the physiological stop pathway within the growing axon tips. Part of the stop pathway involves the proteolytic breakdown and removal of neurofilaments as they enter the axon endings. Another component involves the establishment of anterograde-to-retrograde conversion for the removal of membranous elements from the axonal endings. Both of these components appear to be dependent upon the activation of proteases within the axon tips. Therefore, to further test our hypothesis we infused, by intrathecal catheterization, the region of the dorsal root transitional zone with the protease inhibitor leupeptin at a time when the majority of regenerating axons have terminated in the region. Ultrastructural analyses after leupeptin treatment revealed axo-glial endings distended by accumulations of neurofilaments and organelles, particularly tubulovesicular profiles. These observations further support the idea that astrocytes, like normal target cells, can activate the physiological stop pathway. Topics: Animals; Astrocytes; Enzyme Inhibitors; Injections, Spinal; Leupeptins; Male; Microscopy, Electron; Nerve Endings; Nerve Regeneration; Nerve Tissue Proteins; Oligopeptides; Rats; Rats, Inbred Strains; Spinal Nerve Roots | 1990 |
Uptake and intracellular processing of galactosyl-neoglycoalbumin by the isolated perfused rat liver.
Topics: 1-Naphthylisothiocyanate; Albumins; Animals; Asialoglycoprotein Receptor; Bile; Biological Transport, Active; In Vitro Techniques; Leupeptins; Liver; Perfusion; Rats; Receptors, Immunologic | 1990 |
Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.
Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover. Topics: Animals; Cell Fractionation; Centrifugation, Density Gradient; Cytosol; Electricity; Female; Half-Life; In Vitro Techniques; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Phagocytosis; Rats; Rats, Inbred Strains; Vacuoles | 1990 |
Identification of proteases that process distinct epitopes on the same protein.
Proteolytic degradation of protein antigens is thought to be a major step in the processing of Ag for presentation to T cells, but the range of proteases involved is unknown. Here we used a large panel of protease inhibitors to determine the role of each of the four classes of proteases in antigen processing. Moreover, we asked whether different proteases were necessary for presentation of different known epitopes, defined by three Th cell clones. For all three epitopes of myoglobin, intracellular thiol proteases such as cathepsins B or L were the only proteases necessary. Furthermore, myoglobin pre-digested with cathepsin B could be presented to all three clones without further processing. Thus, a single protease may be both necessary and sufficient for Ag processing to present the majority of epitopes, at least for myoglobin. This finding provides an explanation of earlier data on the fragments produced from processed myoglobin, and so may contribute to a much needed solution to the long standing problem of predicting where a protein will be cleaved during processing. Topics: Animals; Antigen-Presenting Cells; Antigens, Surface; Cathepsin B; Epitopes; Female; Histocompatibility Antigens Class II; Hydrolysis; Leupeptins; Male; Mice; Myoglobin; Peptide Hydrolases; Protease Inhibitors | 1989 |
Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase.
The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase. Topics: Aprotinin; Binding Sites; DNA Mutational Analysis; Genes, Viral; Leupeptins; Papain; Peptide Hydrolases; Plant Viruses; Substrate Specificity; Viral Proteins; Zinc | 1989 |
Purification and characterization of a protease from Xenopus embryos.
A proteolytic enzyme was purified from Xenopus embryos. The purification procedure consisted of fractionation of an extract of embryos with acetone, gel filtration of Sephadex G-75 and chromatography on carboxymethyl-cellulose and hydroxylapatite. The preparation of enzyme appeared to be homogeneous as judged by electrophoresis in polyacrylamide gels. This protease had a molecular mass of 43-44 kDa and was composed of two subunits with molecular masses of 30 kDa and 13 kDa. The optimal pH of the reaction catalysed by the protease was approximately 4.0. This proteolytic activity was inhibited by antipain, leupeptin and iodoacetic acid; it was not affected by phenylmethylsulfonyl fluoride and pepstatin; and it was enhanced by dithiothreitol. In the presence of RNA, the optimal pH was shifted from pH 4.0 to pH 4.5. The protease was activated by addition of total RNA from Xenopus embryos, by poly(rU) or poly(rG). In contrast, after addition of tRNA or poly(rC), no activation of the protease was observed. Topics: Animals; Antipain; Chromatography; Embryo, Nonmammalian; Enzyme Activation; Iodoacetates; Iodoacetic Acid; Leupeptins; Molecular Weight; Peptide Hydrolases; Polyribonucleotides; RNA; Substrate Specificity; Xenopus | 1989 |
Effects of lovastatin and leupeptin on ceroidogenesis of vitamin E-deficient and -supplemented young rats.
Previous studies in young normal rats have shown that intracerebral administration of the proteinase inhibitor, leupeptin, caused a rapid accumulation of lipofuscin-like pigment in lysosomes of brain cells (Ivy et al., 1984a). On the other hand, we have recently found that the administration of lovastatin, an inhibitor of HMG-CoA reductase, reduced the ceroid-like pigment and dolichol contents in the crushed epididymal fat pad of rats (Porta et al., 1988). In order to study now the possible modulating effects of these enzyme inhibitors on ceroidogenesis associated with vitamin E deficiency, two main groups of weanling Wistar female rats were respectively fed ad libitum a vitamin E-deficient basal diet, or the same diet supplemented with 16 mg% of dl-alpha-tocopherol acetate. The vitamin E-deficient and -supplemented rats were further subdivided and received for 8 weeks their diets alone or with 2, 1, or 0.5 g of lovastatin/kg of diet. Other subgroups were treated with constant peritoneal infusion of 0.5 mg/day of leupeptin by means of osmotic minipumps (Alzet 2002) consecutively implanted at days 15, 30, and 45. Lovastatin treatment to vitamin E-deficient rats was associated with dose-dependent toxicity, resulting in 100%, 75%, and 50% mortality at concentrations of 2, 1, and 0.5 g/kg diet, respectively. This mortality was mainly due to extensive hepatic necrosis. Food intake and growth rates were reduced, while the relative weights of liver, kidneys, spleen, heart and brain, as well as the serum levels of GPT and GOT were significantly increased over the values of the untreated vitamin E-deficient control rats. The volumetric densities of ceroid pigment and the dolichol contents in liver and kidneys were not significantly modified. Lovastatin toxicity was partially prevented by vitamin E supplementation. However, in these supplemented rats, lovastatin treatment did not modify the volumetric densities of hepatic and renal ceroid, although the contents of hepatic and renal dolichol were significantly increased. No correlations could be found between levels of hepatic or renal ceroid and total dolichol content in vitamin E-deficient and supplemented rats. Leupeptin treatment to vitamin E-deficient rats only slightly reduced food intake and growth rates, and did not significantly modify the relative organ weights or the serum levels of cholesterol, GOT and GPT. Although in both vitamin E-deficient and -supplemented rats the leupeptin treatment consistently s Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Ceroid; Cholesterol; Dolichols; Eating; Female; Kidney; Leupeptins; Liver; Lovastatin; Organ Size; Rats; Rats, Inbred Strains; Vitamin E; Vitamin E Deficiency | 1989 |
Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine protease inhibition.
The protease inhibitor, leupeptin, has been previously shown to cause an accumulation of lysosomally associated intracytoplasmic dense bodies resembling lipofuscin when administered intraventricularly to the brains of young rats (Ivy et al., Science, 226:985, 1984). These findings support the idea that lipofuscin formation during normal aging involves perturbed proteolytic degradation. In the current study, we delineate more precisely the proteolytic mechanisms in question and examine the effects of protease inhibition on other tissues and species. Four sets of experiments were done. In the first, rats were administered leupeptin (L; an inhibitor of cysteine and some serine proteases), E-64C (a cysteine protease inhibitor), chloroquine (C; a lysosomal enzyme inhibitor), aprotinin (A; a serine protease inhibitor) or physiological saline (S) into the lateral ventricle of the brain at a constant rate for two weeks using an osmotic mini-pump. In the second set, beagle dogs were administered L or S intraventricularly in a similar fashion. In the third set, young rats received intraocular injections of L, E64C, C, A or S from 1 to 9 days at 24 hr intervals. In the fourth set, young rats and mice were administered L, E-64C or S intraperitoneally via an osmotic mini-pump for 2 or more weeks. The tissue of all animals was processed for light and electron microscopy as well as for fluorescence analysis. In rat brain cells (especially hippocampus and cerebellum), substances which by morphological, histochemical and fluorescence emission criteria resemble lipofuscin, accumulated following L, C or E64C, but not A or S treatment. A similar buildup of lipofuscin-like substance occurred in canine brain following L but not S infusion. In retinal pigment epithelial cells, undigested rod outer segment discs accumulated after L, C or E-64C and possibly A, but not after S injections. With increasing survival times, the accumulated substances became morphologically more similar to lipofuscin. Cells of liver (both hepatocytes and non-parenchymal cells) and kidney (mainly renal tubule cells) also accumulated substances resembling lipofuscin in response to L but not S infusion; other internal organs were differentially affected. These results demonstrate 1) that specific inhibition of cysteine proteases is sufficient to cause accumulation of lipofuscin-like substances in brain and retina and 2) that several species and organ systems are similarly affected by protease inhibition. Topics: Aging; Animals; Brain; Cysteine Proteinase Inhibitors; Dogs; Leupeptins; Lipofuscin; Lysosomes; Mice; Mice, Inbred C57BL; Microscopy, Electron; Rats; Rats, Inbred F344; Rats, Inbred Strains; Retina; Viscera | 1989 |
Morphological, physiological and biochemical alterations in livers of rodents induced by protease inhibitors: a comparison with old livers.
A "Protease inhibitor model of aging" has been proposed primarily based on observations on brain tissues exposed to a thiol protease inhibitor, leupeptin (Ivy et al., 1984a). In order to validate this model in terms of a mechanism of cellular aging, as well as of lipofuscin formation in particular, attempts have been made to induce lipofuscin in hepatocytes in young rodent (rat and mouse) livers by continuous i.p. infusion of two different thiol protease inhibitors, leupeptin and E-64C. With doses of leupeptin higher than 1.0 mg/100g/day for 2 wks, a fine granular lipofuscin-like deposition with distinct yellowish-green fluorescence was induced in young rat hepatocytes. The deposition became greater in degree with increasing leupeptin doses. In Kupffer cells and other endothelial cells, fluorescent granules were also induced. In contrast to rat livers, lipofuscin-like pigments induced in hepatocytes in mice were much less, even with a higher dose (20 mg/100 g/day). E-64C also induced the accumulation of lipofuscin-like pigments at a dose of 5 mg/100 g/day, their characteristics being very similar to those induced by leupeptin, but the accumulation being smaller in degree. The fluorescence of leupeptin induced lipopigments was yellowish-green having a peak around 520 nm in emission profile, closely resembling that observed in old rat livers. The hepatobiliary transport functions such as biliary transport maximum (Tm) for sulfobromophthalain and the biliary recovery of iv injected ouabain which are known to decline with age tended to decline in young (6-wk-old) rats administered with leupeptin at a dose of 5 mg/100 g/day for 2 wks. On the other hand, dolichol concentration in leupeptin treated livers was not increased in comparison to control livers, whereas in old rat livers, the dolichol concentration was more than 2 times greater than in young livers. A clear-dose-dependent deposition of ceroid-lipofuscin induced in young rodent livers by protease inhibitors strongly suggests that the "Protease inhibitor model" is generally valid not only for the brain but for other tissues such as the liver, and for two different thiol protease inhibitors. Topics: Aging; Animals; Biological Transport; Dolichols; Leucine; Leupeptins; Liver; Male; Mice; Mice, Inbred C57BL; Ouabain; Protease Inhibitors; Rats; Rats, Inbred F344; Rats, Inbred Strains; Sulfobromophthalein | 1989 |
An epidermal growth factor-ricin A chain (EGF-RTA)-resistant mutant and an epidermal growth factor-Pseudomonas endotoxin (EGF-PE)-resistant mutant have distinct phenotypes.
H2Oe12 is a mutant HeLa cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of epidermal growth factor (EGF) and the toxic A chain of ricin (RTA). ET-28 is a mutant KB cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of EGF and Pseudomonas exotoxin (PE). In this report we describe the presence or absence, in these mutants, of cross-resistance to the two toxic conjugates and the effects of ammonium chloride, leupeptin, and adenovirus cotreatments on toxin efficacies. ET-28 cells, the EGF-PE-resistant cells, are resistant to both EGF-PE and EGF-RTA. In contrast, H2Oe12 cells, the EGF-RTA-resistant cells, are as sensitive to EGF-PE toxicity as are their parent HeLa cells. Ammonium chloride cotreatment substantially reduces the resistance of H2Oe12 cells to EGF-RTA but has little or no effect on the resistance of ET-28 cells to either EGF-RTA or EGF-PE. Leupeptin has no effect on the toxicity of either chimeric conjugate on any of the four cell lines, effect on the toxicity of either chimeric conjugate on any of the four cell lines, despite its demonstrated ability to inhibit cellular degradation of EGF. In contrast, adenovirus cotreatment enhances the toxicity of EGF-RTA and EGF-PE on all cells tested, and completely nullifies the relative resistance of H2Oe12 and ET-28 cells to these toxic conjugates. H2Oe12 and ET-28 cells appear to be altered in distinct, possibly endosomal, functions. Topics: Adenoviridae; ADP Ribose Transferases; Ammonium Chloride; Bacterial Toxins; Drug Resistance; Epidermal Growth Factor; Exotoxins; HeLa Cells; Humans; Immunotoxins; Leupeptins; Mutation; Phenotype; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Ricin; Virulence Factors | 1989 |
Oleate-induced translocation of protein kinase C to hepatic microsomal membranes.
The incubation of rat liver homogenates in the presence of oleate induces the translocation of protein kinase C from the cytosol to the endoplasmic reticulum membranes. The half-maximal effect was obtained at 0.3 mM oleate. The redistribution of this enzyme induced by oleate was also obtained with purified protein kinase C and hepatic microsomal membranes. This effect seems to be mediated by long-chain fatty acids since translocation was not obtained with esterified derivatives. Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Brain; Calcium; Fatty Acids; Intracellular Membranes; Isoenzymes; Isoquinolines; Leupeptins; Microsomes, Liver; Oleic Acid; Oleic Acids; Phorbol 12,13-Dibutyrate; Phosphatidate Phosphatase; Piperazines; Protein Kinase C; Rats; Spermine | 1989 |
Quantitation of platelet fibrinogen and thrombospondin in Glanzmann's thrombasthenia by electroimmunoassay.
Fibrinogen and thrombospondin are major constituents of human platelet alpha-granules and contribute to cell-cell interactions following their release. Glanzmann's thrombasthenia is characterized by the absence of platelet aggregation and reduced levels of GP IIb-IIIa complexes and platelet fibrinogen. The level of thrombospondin is thought to be normal but has not so far been quantified. Using an electroimmunoassay method adapted from Laurell, we have measured fibrinogen and thrombospondin in platelet extracts of four patients with classical Glanzmann's thrombasthenia and two variants with abnormal platelet aggregation associated with subnormal levels of GP IIb-IIIa complexes. Triton X-100 lysates were prepared in the presence of leupeptin or EDTA to avoid endogenous calcium-dependent protease activation during the solubilization procedure. Platelet fibrinogen was not detected in one patient with type I Glanzmann's thrombasthenia; it was reduced to 5-10% of normal values in two other type I patients and to 65% of normal values in one type II patient. It was normal in patient R.P., a variant of Glanzmann's thrombasthenia with 60% of GP IIb-IIIa complexes but decreased in patient A.P. a newly described variant with 35% of GP IIb-IIIa complexes. These findings support a role for GP IIb-IIIa complexes in the packaging of fibrinogen into alpha-granules. Normal or subnormal amounts of thrombospondin were measured in thrombasthenic platelets. Patient A.P., who was investigated on two different occasions, demonstrated variable levels of thrombospondin. This underlines the need for quantifying this protein when evaluating its expression in this disorder. Topics: Blood Platelet Disorders; Blood Platelets; Detergents; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Humans; Immunoblotting; Immunoelectrophoresis; Leupeptins; Membrane Glycoproteins; Octoxynol; Polyethylene Glycols; Thrombasthenia; Thrombospondins | 1989 |
Action of endogenous proteases on the distribution of tyrosinase isozymes in Harding-Passey mouse melanoma.
A high percentage of the total tyrosinase found in Harding-Passey mouse melanoma occurs as a soluble form. This paper shows that melanosomal tyrosinase can be solubilized by several endogenous proteases to yield active tyrosinase. This enzyme, once proteolytically solubilized, can be further degraded, leading to enzyme inactivation. The nature and specificity of the main proteases involved in the solubilization process change depending on the size and necrosis stage of the tumour. Cathepsin B could be the main protease responsible for the solubilization in small tumours (less than 0.5 g). Large tumours are rich in necrotic cells, and cathepsin D and serine-proteases are the main hydrolytic enzymes involved in the proteolytic action on melanosomes. These results support the view that the high activity of tyrosinase found in the soluble fraction of malignant melanoma is mainly an artefact resulting from degradation of melanosomes by a variety of endogenous proteases, rather than the result of the actual occurrence of high levels of an independent cytosolic isozyme. Topics: Animals; Catechol Oxidase; Cathepsin D; Cytosol; Hydrolysis; Isoenzymes; Leupeptins; Melanocytes; Melanoma, Experimental; Mice; Monophenol Monooxygenase; Pepstatins; Peptide Hydrolases; Phenylmethylsulfonyl Fluoride; Solubility | 1989 |
Neuronal plasminogen activators: cell surface binding sites and involvement in neurite outgrowth.
Sympathetic neurons release both urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). A number of inhibitors of serine proteases have been tested to determine their effects on neurite outgrowth from rat sympathetic neurons. Some inhibitors increase neurite outgrowth while others have little or no effect on outgrowth. Inhibition of plasminogen activator (PA) activity but not other serine protease activity correlates with the increase in neurite outgrowth (uPA, r = 0.89; tPA, r = 0.86; plasmin, r = 0.015; thrombin, r = 0.025). Antibodies that inhibit uPA activity increase neurite outgrowth, while antibodies that bind to uPA but do not inhibit activity do not alter outgrowth. Time-lapse videomicroscopy of neurite outgrowth indicates that about 85% of the neurites increase their rate of outgrowth following exposure to inhibitors of PA. Routinely, 1-2 min after exposure of a growth cone to an inhibitor, there is an increase in lamellipodial activity at the leading edge of the growth cone and a decrease in lamellipodial activity on the sides and base of the growth cone. The increase in the rate of outgrowth combined with the decrease in lamellipodial activity on the sides of the growth cones results in neurites being very long and straight in the presence of inhibitors (persistence time P = 3.7 and 15.3 hr for controls and in the presence of inhibitors of PA, respectively). PAs released from sympathetic neurons and PC12 cells interact with 3 different binding sites on the cell surface: (1) an inhibitor of serine proteases (including uPA and tPA) is bound to the surface via a heparinase-sensitive site; (2) a uPA-selective binding site is present in patches on the bottom surface of PC12 cells; and (3) a tPA-selective binding site with high affinity (KD = 23 +/- 10 nM) and high capacity (340,000 +/- 130,000 sites/neuron) for 125I-tPA is homogeneously distributed over the entire surface. Data in the present study are consistent with PA being involved in neurite outgrowth and open the possibility of other PA-dependent functions occurring when tPA and/or uPA interacts with cell surface binding sites. Topics: Amino Acid Chloromethyl Ketones; Animals; Axons; Benzoates; Binding Sites; Cell Membrane; Cell Survival; Dansyl Compounds; Leupeptins; Neurons; Plasminogen Activators; Sympathetic Nervous System; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator | 1989 |
Intracellular degradation of unassembled asialoglycoprotein receptor subunits: a pre-Golgi, nonlysosomal endoproteolytic cleavage.
The human asialoglycoprotein receptor is a heterooligomer of the two homologous subunits H1 and H2. As occurs for other oligomeric receptors, not all of the newly made subunits are assembled in the RER into oligomers and some of each chain is degraded. We studied the degradation of the unassembled H2 subunit in fibroblasts that only express H2 (45,000 mol wt) and degrade all of it. After a 30 min lag, H2 is degraded with a half-life of 30 min. We identified a 35-kD intermediate in H2 degradation; it is the COOH-terminal, exoplasmic domain of H2. After a 90-min chase, all remaining intact H2 and the 35-kD fragment were endoglycosidase H sensitive, suggesting that the cleavage generating the 35-kD intermediate occurs without translocation to the medial Golgi compartment. Treatment of cells with leupeptin, chloroquine, or NH4Cl did not affect H2 degradation. Monensin slowed but did not block degradation. Incubation at 18-20 degrees C slowed the degradation dramatically and caused an increase in intracellular H2, suggesting that a membrane trafficking event occurs before H2 is degraded. Immunofluorescence microscopy of cells with or without an 18 degrees C preincubation showed a colocalization of H2 with the ER and not with the Golgi complex. We conclude that H2 is not degraded in lysosomes and never reaches the medial Golgi compartment in an intact form, but rather degradation is initiated in a pre-Golgi compartment, possibly part of the ER. The 35-kD fragment of H2 may define an initial proteolytic cleavage in the ER. Topics: Acetylglucosaminidase; Animals; Asialoglycoprotein Receptor; Cell Line; Chloroquine; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fluorescent Antibody Technique; Golgi Apparatus; Half-Life; Leupeptins; Lysosomes; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Microscopy, Fluorescence; Molecular Weight; Monensin; Receptors, Immunologic; Temperature | 1989 |
Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes.
This report presents evidence for diversity in membrane binding sites between three forms of ankyrin: brain ankyrin, erythrocyte ankyrin, and a variant of erythrocyte ankyrin (protein 2.2) present in circulating human erythrocytes that is missing a regulatory domain. These ankyrins were compared with respect to binding to kidney microsomes and exhibited the following behavior. 1) Brain and erythrocyte ankyrin each bind to distinct sites. 2) Protein 2.2 is an activated ankyrin that binds to all of the sites accessible to both brain and erythrocyte ankyrin and, in addition, associates with its own specialized sites. 3) The specificity of these membrane sites for various ankyrins is not absolute but reflects 2.5-10-fold differences in relative affinities. Further evidence that binding sites of different ankyrins share some common features is that the cytoplasmic domain of the erythrocyte anion transporter associates with all three ankyrins and displaces binding of the ankyrin variants to kidney membranes. The differences between erythrocyte and brain ankyrins in association with kidney membranes are likely to have physiological relevance to kidney because immunologically related isoforms of ankyrin are expressed in this tissue: erythroid ankyrin which is restricted to the basolateral domains of two cell types and a brain-related ankyrin expressed in all cells and present on apical as well as basolateral membrane surfaces. An unanticipated observation was the discovery of a membrane-associated ankyrin protease in kidney that is specific for erythrocyte ankyrin and may selectively activate the erythroid isoform of ankyrin. The variety of binding sites within this group of ankyrin proteins supports the idea that ankyrins are capable of linking a number of different membrane proteins to the spectrin-actin skeleton. Topics: Animals; Ankyrins; Binding Sites; Blood Proteins; Brain; Cell Compartmentation; Cell Membrane; Erythrocytes; Fluorescent Antibody Technique; In Vitro Techniques; Kidney; Leupeptins; Membrane Proteins; Microsomes; Molecular Weight; Peptide Hydrolases; Protein Binding; Sheep; Spectrin | 1989 |
Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase.
Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed. Topics: Calcium; Calcium-Transporting ATPases; Calmodulin; Calpain; Erythrocyte Membrane; Glycodeoxycholic Acid; Humans; Kinetics; Leupeptins | 1989 |
The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse.
Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity. Topics: Animals; Calpain; Chediak-Higashi Syndrome; Cysteine Proteinase Inhibitors; Cytotoxicity, Immunologic; Disease Models, Animal; Killer Cells, Natural; Killer Factors, Yeast; Leucine; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Protein Biosynthesis; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate | 1989 |
Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain.
5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation. Topics: Adenosine; Adenosine Diphosphate; Affinity Labels; Antipain; Blood Platelets; Calpain; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Humans; Leupeptins; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protease Inhibitors; Thrombin | 1989 |
Degradation of 125I-glucagon, -pancreatic polypeptide and -insulin by acid saline extract of rat submaxillary gland and their protection by proteinase inhibitors.
The acid saline extract (ASE) of rat submaxillary gland exerts a powerful degrading effect on 125I-glucagon. In order to study the degradation of other 125I-peptides by ASE and the effects of their inhibitors, 125I-pancreatic polypeptide (PP) and 125I-insulin were used together with 125I-glucagon. The degradation studies were done by the trichloroacetic acid (TCA) method or gel filtration. Besides 125I-glucagon, 125I-PP was found to be destroyed by ASE in the ordinary immunoassay system using the TCA method, but 125I-insulin was intact in the presence of ASE. Leupeptin, and to a lesser extent p-chloromercuriphenyl-sulfonic acid (PCMS) and N-ethylmaleimide, inhibited the destruction of 125I-glucagon or -PP under the TCA method. PCMS was especially protective at high concentrations, for example 16 mM. These findings were confirmed by gel filtration of the assay mixture. In the presence of leupeptin (0.4 mM) and PCMS (16 mM), no shift in the peak of labelled glucagon or PP occurred. Thus ASE degrades not only 125I-glucagon but -PP, and thiol proteinase inhibitors have a strong inhibitory action on them. Topics: 4-Chloromercuribenzenesulfonate; Animals; Chemical Precipitation; Chromatography, Gel; Ethylmaleimide; Glucagon; Hydrogen-Ion Concentration; Insulin; Iodine Radioisotopes; Leupeptins; Pancreatic Polypeptide; Protease Inhibitors; Rats; Rats, Inbred Strains; Sodium Chloride; Submandibular Gland; Trichloroacetic Acid | 1989 |
Possibility of Bacteroides gingivalis hemagglutinin possessing protease activity revealed by inhibition studies.
Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and anti-pain inhibited the HA activity at nM order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK), p-chloromercuribenzoate (PCMB), and N-ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding. Topics: Bacteroides; Benzoylarginine Nitroanilide; Hemagglutination Inhibition Tests; Hemagglutinins; Leupeptins; Peptide Hydrolases; Protease Inhibitors | 1989 |
Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway.
The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway. (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins. (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2's) except E2(20K) and E2(230K). Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted. Topics: Adenosine Triphosphate; Animals; Cell Differentiation; Lactoglobulins; Leukemia, Erythroblastic, Acute; Leupeptins; Ligases; Lysosomes; Mice; Peptide Hydrolases; Protease Inhibitors; Reticulocytes; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Ubiquitins | 1989 |
A growth hormone-vesicular stomatitis virus G hybrid protein is rapidly degraded in lysosomes following transport to the cell surface.
We have expressed the hybrid protein, GHG3, in baby hamster kidney cells to study protein turnover. GHG3 contains the cytoplasmic and transmembrane domains of vesicular stomatitis virus G protein linked to the C-terminus rat growth hormone. Turnover of GHG3 was prevented by lysosomal inhibitors (leupeptin, chloroquine, primaquine or monensin), while the accumulated GHG3 was localized to intracellular vesicles, results indicating that degradation occurred in lysosomes. The kinetics of degradation at 34 degrees C were determined in pulse-chase studies of metabolically labeled cells. After a lag period of 1 h, degradation was rapid (t1/2 = 1.25 h). The fate of GHG3 during the lag period was determined by immunofluorescence. We detected GHG3 on the cell surface when growth hormone antiserum was added to the growth medium 90 min prior to fixation and staining. No staining was observed if protein synthesis was inhibited with cycloheximide 90 min prior to the addition of growth hormone antiserum, a result indicating that GHG3 was rapidly removed from the cell surface. Unless the cells were pretreated with cycloheximide, antiserum was also detected in intracellular vesicles, which showed that GHG3 was endocytosed. These data indicate that a pool of GHG3 is transported rapidly to the cell surface, endocytosed and with little or no recycling directed to lysosomes for degradation. Topics: Animals; Cells, Cultured; Chloroquine; Cricetinae; Endocytosis; Growth Hormone; Kidney; Leupeptins; Lysosomes; Membrane Proteins; Monensin; Vesicular stomatitis Indiana virus; Viral Proteins | 1989 |
Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated.
The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P]phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatide acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin). Topics: Adenosine Diphosphate; Humans; In Vitro Techniques; Leupeptins; Phosphatidylinositols; Platelet Activating Factor; Platelet Activation; Receptors, Cell Surface; Receptors, Thrombin; Serotonin; Thrombin; Thromboxane A2; Trypsin | 1989 |
Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts.
The effect of various metalloproteinase-inhibiting compounds on collagen phagocytosis by fibroblasts was studied in cultured periosteal tissue. Evidence is presented indicating that neither anti-collagenase nor anti-stromelysin interfere with the uptake of collagen fibrils from the extracellular space and their intracellular digestion. Similar results were obtained with tissue inhibitor of metalloproteinases (TIMP). In the presence of the proteinase inhibitor leupeptin, a compound which strongly inhibits the intracellular degradation of phagocytosed collagen, a time-dependent increase in the amount of internalized collagen was found. This increase proved to be similar in explants treated as well as in those not treated with the metalloproteinase-inhibiting compounds. It is concluded that enzymes, such as collagenase and stromelysin, do not play a crucial role in the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. If these enzymes are involved it must be prior to these events. Based on the morphometric data the intralysosomal degradation time of collagen was calculated to be about 30 minutes. A comparison with findings in the literature on collagen metabolism in the periodontal ligament of the rat molar suggests that all collagen degraded may pass through the phagolysome pathway during physiological turnover and remodelling. Topics: Animals; Bone and Bones; Collagen; Culture Techniques; Cytochalasin B; Enzyme Inhibitors; Fibroblasts; Immune Sera; Immunoglobulin G; Kinetics; Leupeptins; Matrix Metalloproteinase 3; Metalloendopeptidases; Microbial Collagenase; Microscopy, Electron; Phagocytosis; Rabbits; Tissue Inhibitor of Metalloproteinases | 1989 |
Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease.
The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures. Topics: Animals; Blood Proteins; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Chickens; Egtazic Acid; Enzyme Activation; Erythrocyte Membrane; Immunoblotting; Kinetics; Leupeptins; Macromolecular Substances; Membrane Proteins; Mice; Molecular Weight; Rabbits; Vimentin | 1989 |
Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes.
We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules. Topics: Animals; Asialoglycoproteins; Cells, Cultured; Chloroquine; Colchicine; Dose-Response Relationship, Drug; Galactose; Leupeptins; Liver; Male; Monensin; Oligopeptides; Orosomucoid; Rats; Rats, Inbred Strains; Receptors, Cell Surface | 1989 |
Cobalamin release from intrinsic factor and transfer to transcobalamin II within the rat enterocyte.
To ascertain the mechanism of release of cobalamin (Cbl) from intrinsic factor (IF) and subsequent formation of transcobalamin II (TC-II)-Cbl complex, we studied the intracellular distribution of 57Co-labeled Cbl after its uptake in suckling and adult rats. The amount of Cbl bound to IF, to the IF-Cbl receptor via IF, and to TC-II was determined by immunoprecipitation with monospecific antisera raised to these proteins. IF-Cbl receptor activity was found to be very low in suckling rats up to 12 days after birth. Oral administration of leupeptin in amounts known to alter protein turnover had no effect on the release of Cbl from IF nor did it inhibit the formation of the TC-II-Cbl complex in either adult or suckling animals. However, oral administration of chloroquine resulted in a transient increase in the intestinal concentration of Cbl in both adult and suckling rats and in total inhibition of Cbl released from IF in adults rats. Chloroquine prevented completely the transfer of Cbl to TC-II in adult rats and inhibited the transfer by 50% in suckling rats. These data demonstrate that in adult mucosa utilizing receptor-mediated endocytosis, Cbl is transferred from IF to TC-II. This transfer does not require the IF-Cbl receptor, as it occurs in suckling rats. Finally, transfer of Cbl to TC-II is decreased by a drug that alters vesicular pH. Because Cbl can be released at acid pH from IF, it is proposed that release of Cbl from IF and its transfer to TC-II occurs in an acidic vesicle. Topics: Administration, Oral; Animals; Animals, Suckling; Chloroquine; Gastric Mucosa; Immune Sera; Intestinal Mucosa; Intestines; Intrinsic Factor; Leupeptins; Male; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Transcobalamins; Vitamin B 12 | 1989 |
Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.
N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present. Topics: Animals; Cell Division; Cell Line; Chromatography, Ion Exchange; Cricetinae; Cricetulus; Dipeptides; gamma-Glutamylcyclotransferase; In Vitro Techniques; Leupeptins; Methylamines; Oligopeptides; Proteins; Transglutaminases | 1989 |
The protease inhibitor leupeptin interferes with the development of LTP in hippocampal slices.
The effect of leupeptin, an inhibitor of thiol-proteases, was tested on the induction of long-term potentiation (LTP) in field CA1 of hippocampal slices. Two h of drug application did not produce substantial changes while a greater than 3-h application caused a sizeable reduction in the degree of LTP induced. Leupeptin had no obvious effects on the facilitation of postsynaptic responses occurring within or between the short high frequency bursts used to induce LTP, suggesting that the drug acted on cellular chemistries occurring after the initial physiological events that normally trigger LTP. These results are consistent with the hypothesis that a calcium-activated thiol protease (calpain) is involved in the induction of LTP. Topics: Action Potentials; Animals; Hippocampus; In Vitro Techniques; Leupeptins; Male; Neuronal Plasticity; Oligopeptides; Protease Inhibitors; Rats; Rats, Inbred Strains | 1989 |
Secretory activity as a function of the development and maturation of ameloblasts.
The biosynthetic and secretory activity of rat incisor ameloblasts was studied by grain count analysis of radioautographs at various times following a single injection of either 3H-methionine, 3H-leucine, or 3H-glycine. Experiments were also carried out with leupeptin, a thiol and serine proteinase inhibitor which blocks degradation of proteins within lysosomes. The results from this study indicate that the biosynthetic and secretory activities of ameloblasts increase steadily as the cells differentiate (presecretory stage) and start to form the enamel layer (secretory stage). Secretory activity reaches a peak when the ameloblasts form about one-third of the eventual thickness of the enamel, and remains at this high level until shortly before they start to form the outer and final layers of enamel. Secretory activity then drops rapidly as the cells undergo postsecretory transition, and declines slowly thereafter as the shortened ameloblasts modulate continuously along the surface of the maturing enamel. Ameloblasts appear to biosynthesize more proteins than are secreted. The excess proteins are degraded rapidly in lysosomes and the amino acids reutilized for production of new exportable and/or structural proteins. Topics: Ameloblasts; Animals; Cell Differentiation; Dental Enamel; Dental Enamel Proteins; In Vitro Techniques; Leupeptins; Lysosomes; Male; Rats; Rats, Inbred Strains | 1989 |
Structural and functional aspects of HLA class II glycoproteins and the associated invariant chain.
Topics: Cell Line; Glycoproteins; HLA-D Antigens; Humans; Leupeptins; Molecular Structure; Molecular Weight; Monensin; Peptides; Protein Binding; Protein Conformation | 1989 |
The influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells.
Cell response to irradiation depends on many micro-environmental and intracellular factors. It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle. They also could be involved in cell response to irradiation. In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied. Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth. Immediately after irradiation cathepsin B or leupeptin were added to the growth medium. Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction. Mutation frequency was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG). Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals. The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants. If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency. The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases. As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities. Topics: Animals; Aspartic Acid Endopeptidases; Cathepsin B; Cell Line; Cell Survival; Cysteine Endopeptidases; DNA; DNA Repair; Endopeptidases; Leupeptins; Mutation; Oligopeptides; Serine Endopeptidases | 1989 |
Activation of an insulin-stimulated S6 kinase in 3T3 L1 cell-free extracts by proteolysis.
Using chromatography on a Fast S-Sepharose column, the insulin-stimulated S6 kinase can be resolved from other S6 kinases present in 3T3 L1 cell extracts. Only one S6 kinase is greatly activated by insulin (4-5-fold) and phosphorylates S6 with a high stoichiometry (4-5 mol phosphate per mol S6). This insulin-dependent S6 kinase can be activated in cell-free extracts by incubation with high concentrations of Ca2+. This activation is blocked by protease inhibitors such as leupeptin and is mimicked by trypsin. The stimulation does not require the presence of the protein kinase dependent on phospholipids and calcium (PK-C) in the cell extracts. The level of stimulation produced by proteolysis in the cell extracts is comparable to that reached in vivo by incubation with insulin. Topics: Animals; Calcium; Cell-Free System; Chromatography; Enzyme Activation; Insulin; L Cells; Leupeptins; Mice; Peptide Hydrolases; Phosphates; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases; Trypsin | 1989 |
Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodium falciparum.
Among several protease inhibitors tested, only leupeptin was found to modify qualitatively the processing of P126, a major antigen of the parasitophorous vacuole of Plasmodium falciparum, and to inhibit the release of merozoites. Whereas P126 is normally processed upon merozoite release into 2 polypeptides of 50 and 73 kDa which are discharged in the culture medium, leupeptin treatment led to the recovery of a 56 kDa fragment which was recognized by a monoclonal antibody specific for the 50 kDa polypeptide and of a 73 kDa fragment comigrating with the one obtained in normal culture conditions. Mild trypsinization of the 56 kDa polypeptide gave rise to a 50 kDa product the tryptic fragments of which comigrated with those of the 50 kDa antigen obtained from untreated cultures. Topics: Animals; Antigens, Protozoan; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Humans; Hydrolysis; Immunosorbent Techniques; Leupeptins; Oligopeptides; Peptide Fragments; Plasmodium falciparum; Protease Inhibitors; Trypsin | 1989 |
Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines.
1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction. Topics: Acid Phosphatase; Animals; Cathepsin B; Enzyme Induction; Leucine; Leupeptins; Melanoma, Experimental; Mice; Oligopeptides; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Subcellular Fractions; Tumor Cells, Cultured | 1989 |
Effects of inhibitors of membrane signal peptide peptidase on protein translocation into membrane vesicles.
The effect of the removal of signal peptides after cleavage of precursor molecules by the signal peptidase I was examined in an in vitro translocation system with Escherichia coli membrane vesicles. The translocation of periplasmic alkaline phosphatase precursors was significantly inhibited by the protease inhibitors antipain, elastatinal and leupeptin. Antipain and leupeptin enhanced the translocation of precursors of outer membrane protein OmpA, but inhibited the processing. However, antipain did not inhibit the processing of precursors mediated by signal peptidase I in the soluble form. Moreover, the inhibition by antipain was not due to the disruption of membrane integrity, but occurred during the process of protein translocation. Since these small peptide inhibitors are known to inhibit membrane protease IV, a signal peptide peptidase, these results suggest that the hydrolysis of signal peptides is an important step in the recycles of the overall translocation process, and that the prevention of degradation of signal peptides feedback inhibits the preceding steps in the translocation pathway. Topics: Alkaline Phosphatase; Antipain; Bacterial Outer Membrane Proteins; Endopeptidases; Escherichia coli; Leupeptins; Membrane Proteins; Oligopeptides; Protease Inhibitors; Protein Processing, Post-Translational; Protein Sorting Signals; Serine Endopeptidases | 1989 |
Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy.
Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease. Topics: Autoradiography; Cell Fractionation; Cells, Cultured; Cholesterol Esters; Esterification; Fibroblasts; Humans; Leupeptins; Lysosomes; Monensin; Mucolipidoses; Niemann-Pick Diseases | 1989 |
Degeneration of neuronal processes in rats induced by a protease inhibitor, leupeptin.
Severe degeneration of neuronal processes, including axons and dendrites, as well as accumulation of lipofuscin-like dense bodies have been induced in rats by continuous intraventricular administration by infusion of a protase inhibitor, leupeptin. The aggregation of degenerated processes in neuropils mingled with glial cells and their processes resembled the aggregation of degenerated neurites that are important constituents of the senile plaque of Alzheimer's disease. The present findings provide morphological evidence supporting the hypothesis that protease inhibitors participate in the process of senile plaque formation. Topics: Animals; Central Nervous System; Enzyme Inhibitors; Injections, Intraventricular; Leupeptins; Microscopy, Electron; Nerve Degeneration; Neurotoxins; Oligopeptides; Rats; Rats, Inbred Strains | 1989 |
Cytotoxicities of two disulfide-bond-linked conjugates of methotrexate with monoclonal anti-MM46 antibody.
In studies on (antitumor antibody)-drug conjugates as potential antitumor agents, the amide derivatives of methotrexate (MTX) with cysteine and with 2-mercaptoethylamine (cysteamine) (MTX-Cys and MTX-MEA, respectively) were linked via a disulfide bond with a monoclonal antibody (alpha MM46) to a mouse mammary tumor MM46 with attached 3-(2-pyridyldithio) propionyl groups to give conjugates of MTX with alpha MM46 (MTX-Cys-SS-alpha MM46 and MTX-MEA-SS-alpha MM46, respectively). These two conjugates are both linked by a disulfide bond and are very similar in structure, but MTX-MEA-SS-alpha MM46 showed only weak in vitro cytotoxicity against MM46 cells, whereas MTX-Cys-SS-alpha MM46 had strong cytotoxicity. The cytotoxicity of the latter was comparable to that of the conventional direct MTX-alpha MM46 conjugate prepared with an MTX-active ester. However, this conjugate had a greater selectivity than that of the direct conjugate, calculated as the IC50 (concentration of a conjugate by MTX equivalence required for suppression of the number of viable MM46 cells to 50% of that of the untreated control) for the corresponding nonspecific conjugate divided by the IC50 for the alpha MM46 conjugate. The inhibitory activities of MTX-Cys and MTX-MEA on dihydrofolate reductase were similar. The cytotoxicity of MTX-Cys-SS-alpha MM46 was not affected by thiamine pyrophosphate, an inhibitor of the active transport of MTX across the cell membrane, but was decreased significantly by ammonium chloride, a lysosomotropic amine. However, the cytotoxicity was decreased only to a small extent by leupeptin, an inhibitor of lysosomal cysteine proteases cathepsins B, H, and L. These results suggest that the cytotoxicity is mediated by lysosomes, and may involve lysosomal enzymes other than cathepsins B, H, and L. Topics: Animals; Antibodies, Monoclonal; Carcinoma, Ehrlich Tumor; Complement System Proteins; Cytotoxicity, Immunologic; Disulfides; Folic Acid Antagonists; Immunotoxins; Leupeptins; Methotrexate; Mice; Mice, Inbred C3H; Sulfhydryl Compounds; Thiamine Pyrophosphate | 1989 |
Microinjections of leupeptin in the frontal cortex or dorsal hippocampus block spatial learning in the rat.
Adult male Long-Evans hooded rats were given bilateral microinjections of 18 mM leupeptin or saline through cannulae implanted with tips aimed at the frontal cortex or CA1 or CA3 hippocampal cell fields. Five minutes following injections animals were allowed to complete an eight-arm radial maze. The acquisition criterion required that the animal make 7 correct choices of the first 8, and 8 correct choices of the first 10, for five consecutive sessions. Leupeptin slowed acquisition of the eight-arm radial maze task when injected into the CA1 and CA3 hippocampal fields or the frontal cortex, but did not influence spontaneous activity. These results suggest that earlier reports of the amnestic effect of leupeptin when administered into the lateral cerebral ventricle may have been due to effects within the cortex and hippocampus. The present experiment represents the first attempt to identify behaviorally those brain areas in which leupeptin acts to alter learning. Topics: Animals; Arousal; Brain Mapping; Discrimination Learning; Frontal Lobe; Hippocampus; Injections; Leupeptins; Male; Memory; Mental Recall; Motor Activity; Oligopeptides; Orientation; Rats; Rats, Inbred Strains | 1989 |
Epidermal growth factor-induced truncation of the epidermal growth factor receptor.
NIH-3T3 cells expressing the human epidermal growth factor (EGF) receptor were used in experiments to determine the fate of the EGF receptor in cells continuously exposed to EGF. EGF receptor was immunoprecipitated from cells labeled for 12 h with [35S] methionine in the absence or presence of 10 nM EGF. As expected, a single Mr = 170,000 polypeptide representing the mature EGF receptor was immune-precipitated from control cells. Surprisingly, immune precipitates from EGF-treated cells contained a prominent Mr = 125,000 receptor species, in addition to the Mr = 170,000 mature receptor. The Mr = 125,000 species was shown to be derived from the Mr = 170,000 form by pulse-chase experiments, in which the Mr = 170,000 receptor chased into the Mr = 125,000 form when EGF was included during the chase and by partial proteolysis. Both proteins became extensively phosphorylated on tyrosine residues in immune precipitate kinase assays. Treatment of immune precipitates with endoglycosidase F changed the apparent molecular weight of the Mr = 170,000 receptor to Mr = 130,000 and of the Mr = 125,000 form to Mr = 105,000, indicating that the appearance of the Mr = 125,000 protein was probably due to proteolysis. Antibody against the carboxyl terminus of the mature EGF receptor recognized the Mr = 125,000 protein, whereas antibody against the amino terminus did not. Incubation of cells with leupeptin prior to and during EGF addition inhibited processing to the Mr = 125,000 species. Methylamine and low temperature also inhibited the EGF-induced processing to the Mr = 125,000 form. These data suggest a possible role for proteolysis of the EGF receptor in receptor function. Topics: Animals; Cells, Cultured; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Leupeptins; Methylamines; Mice; Mice, Inbred Strains; Molecular Weight; Thermodynamics | 1989 |
Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain.
Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding. Topics: Algorithms; Arginine; Cathepsin B; Chromatography, High Pressure Liquid; Kinetics; Leupeptins; Magnetic Resonance Spectroscopy; Oligopeptides; Papain; Protease Inhibitors | 1989 |
Leupeptin-mediated alteration of renal phagolysosomes: similarity to hyaline droplet nephropathy of male rats exposed to unleaded gasoline.
alpha 2u-Globulin, a protein of hepatic origin found in the urine of male rats, is accumulated in the kidney cortex during exposure to unleaded gasoline and has been implicated in the development of fuel hydrocarbon-induced nephropathy and renal neoplasia. The principal morphological feature of gasoline-induced nephropathy is accumulation of hyaline droplets (enlarged secondary lysosomes or phagolysosomes) in epithelial cells of the proximal convoluted tubule S1 and S2 segments. Inhibition of cathepsin B (a major lysosomal peptidase) by treatment of male rats with leupeptin causes rapid accumulation of phagolysosomes and alpha 2u-globulin in the kidney very similar to gasoline exposure. Further, the renal cortical subcellular distribution of alpha 2u-globulin, determined with an electron microscopic immunochemical method, is almost totally confined to phagolysosomes following administration of either gasoline or leupeptin. These results, taken together, indicate that the mechanism of nephrotoxicity of gasoline involves inhibition of renal phagolysosomal proteolysis. Topics: Alpha-Globulins; Animals; Gasoline; Kidney; Kidney Diseases; Leupeptins; Male; Oligopeptides; Petroleum; Phagosomes; Proteins; Rats; Rats, Inbred F344 | 1988 |
Enhancement of transformed cell growth in agar by serine protease inhibitors.
We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor. Topics: Antigens, Polyomavirus Transforming; Aprotinin; Cell Line; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Leupeptins; Serine Proteinase Inhibitors; Trypsin Inhibitor, Kunitz Soybean | 1988 |
Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes.
Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium. Topics: Adenosine Triphosphate; Animals; Calcium; Cell Membrane; Electric Conductivity; Fluorides; Heart; Heart Ventricles; Ion Channels; Kinetics; Leupeptins; Potassium; Rats | 1988 |
Regulation of Hermissenda K+ channels by cytoplasmic and membrane-associated C-kinase.
Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage-dependent K+ currents (IA and ICa2+-K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low-molecular-weight protein was changed only within the eyes of conditioned Hermissenda. To examine the role of PKC in causing K+ current changes as well as changes of phosphorylation during conditioning (and possibly other physiologic contexts), we studied here the effects of endogenous PKC activation and exogenous PKC injection on phosphorylation and K+ channel function. Several phosphoproteins (20, 25, 56, and 165 kilodaltons) showed differences in phosphorylation in response to PKC activators applied to intact nervous systems or to isolated eyes. Specific differences were observed for membrane and cytosolic fractions in response to both the phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA) or exogenous PKC in the presence of Ca2+ and phosphatidylserine/diacylglycerol. Type B cells pretreated with DPBA responded to PKC injection with a persistent reduction of K+ currents. In the absence of DPBA, PKC injection also caused K+ current reduction only following Ca2+ loading conditions. However, the direct effect of PKC injection in the absence of DPBA was only to increase ICa2+-K+. According to a proposed model, the amplitude of the K+ currents would depend on the steady-state balance of effects mediated by PKC within the cytoplasm and membrane-associated PKC. The model further specifies that the effects on K+ currents of cytoplasmic PKC require an intervening proteolytic step. Such a model predicts that increasing the concentration of cytoplasmic protease, e.g., with trypsin, will increase K+ currents, whereas blocking endogenous protease, e.g., with leupeptin, will decrease K+ currents. These effects should be opposed by preexposure of the cells to DPBA. Furthermore, prior injection of leupeptin should block or reverse the effects of subsequent injection of PKC into the type B cell. All of these predictions were confirmed by results reported here. Taken together, the results of this and previous studies suggest that PKC regulation of membrane excitability critically depends on it Topics: Animals; Calcium; Cell Membrane; Central Nervous System; Cytoplasm; Electric Conductivity; Ion Channels; Leupeptins; Models, Biological; Mollusca; Nerve Tissue Proteins; Phorbol Esters; Phosphorylation; Photoreceptor Cells; Potassium; Protein Kinase C; Trypsin | 1988 |
Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells.
We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis. Topics: alpha-Fetoproteins; beta-Galactosidase; Cell Count; Cells, Cultured; Humans; Hyperlipoproteinemia Type II; Kidney Tubules, Proximal; Leupeptins; Lipoproteins, LDL; Methylation; Receptors, LDL; Time Factors | 1988 |
Individual class II-restricted antigenic determinants of the same protein exhibit distinct kinetics of appearance and persistence on antigen-presenting cells.
The kinetics of presentation of class II-restricted T cell determinants of influenza virus hemagglutinin (HA) was investigated over a 48-h time course after pulsing of A20 B lymphoma APC with non-replicative virus or isolated HA. At intervals after Ag pulse, APC were fixed with paraformaldehyde to arrest Ag processing and to preserve the expression levels of the presented determinants. Expression of T cell sites at each time point was probed by a panel of BALC/c T hybridomas specific for the HA of influenza A/Puerto Rico/8/34 virus, recognizing either site 1 (residues 111 to 119), site 2 (126 to 138), or site 3 (302 to 313). Characteristic patterns of presentation were observed for each site: sites 2 and 3 achieved maximal expression by 8 h post pulse, but declined thereafter, whereas site 1 presentation continued to increase over time. The quantitative expression of each T cell site was affected by the proteolysis inhibitor leupeptin, resulting in partial inhibition of site 1, complete blocking of site 2, but enhancement of site 3. However, the expression kinetics of sites 1 and 3, which could be observed in the presence of the inhibitor, remained qualitatively unchanged. These observations indicate that some T cell determinants (e.g., HA site 1) may exhibit a greater longevity of expression on APC than other antigenic sites of the same protein. Differences in the persistence of surface expression of distinct T cell sites may be a factor in their relative immunodominance. Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Cell Line; Epitopes; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral; Histocompatibility Antigens Class II; Kinetics; Leupeptins; Mice; Mice, Inbred BALB C; T-Lymphocytes; Viral Proteins | 1988 |
[Phorbol myristate acetate promotes entry of calcium in hypophysial cells GH3 B6 via potential dependent calcium channels].
10 GH3/B6 cells were patched-clamped using a pipette containing NMG as internal cation, 2 mM ATP and 100 microM leupeptin. Whole-cell calcium or barium currents were recorded prior and after PMA (10(-8) or 10(-7) M). PMA increased the inward calcium current at potential levels close to threshold in 8 cells; 7 cells only exhibited an increase in transitory calcium current at potential levels close to threshold; in one cell, both transitory and conventional calcium currents were increased. 2 cells did not respond to PMA. Topics: Animals; Barium; Calcium; Cell Line; Electrophysiology; Ion Channels; Leupeptins; Pituitary Gland; Rats; Tetradecanoylphorbol Acetate | 1988 |
Inhibition of proteolysis blocks anterograde-retrograde conversion of axonally transported vesicles.
To test the hypothesis that proteolysis is required for anterograde-retrograde (A-R) conversion of membranous organelles at axon tips, a new experimental paradigm was developed. By cutting the sciatic nerves of rats, a concentrated population of axon tips was produced, and proteases in the axon tips were locally inhibited by immersing the cut end of the nerve into a solution containing protease inhibitors (E-64 or leupeptin). Membranous organelles were pulse-labeled with [3H]leucine at the nerve cell body, and the amount of retrogradely transported radiolabeled vesicles from the axon tips was quantified with a proximal collection ligature. The results show that protease inhibition decreased the amount of radioactivity that was transported retrogradely from the axon tips and correspondingly increased the amount that remained in the tips. Ultrastructural analyses showed that the protease-inhibited axon tips were greatly distended by 40-80 nm membranous tubules. By contrast, the control axon tips had relatively few of these membranous tubules. These results show that protease inhibition at the axon tip blocks the removal of membranous elements from the axon tips by retrograde transport. We propose that proteolysis is an A-R converting mechanism which is critically required at the axon tip for the conversion of 40-80 nm membranous tubules into retrograde organelles. Apparently, the 40-80 nm membranous tubules are normally transient intermediates in the A-R conversion pathway, and they rapidly accumulate in the axon tip if the mechanisms that convert them into retrograde organelles are blocked. These 40-80 nm tubules also accumulate in certain pathologies and in the aging process.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Axonal Transport; Axons; Leucine; Leupeptins; Male; Microscopy, Electron; Oligopeptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Reference Values; Sciatic Nerve | 1988 |
Characterization of proteolytic and collagenolytic enzymes from the larvae of Lucilia cuprina, the sheep blowfly.
Isoelectric focusing was used to characterize proteolytic enzymes in homogenate and excretory-secretory preparations of the larvae of L. cuprina, the sheep blowfly. Zymogram overlays showed that the larvae produce a number of highly active proteases which have a wide range of isoelectric points and molecular weights. The alkaline and neutral pI proteases were inhibited by phenylmethyl-sulfonylfluoride, leupeptin and aprotinin; this indicated the presence of serine in the active site. Pepstatin and the metal chelating agent ethylenediaminetetraacetic acid had no effect on the activity of any of the proteases. Optimal pH for activity of the proteases was between 7 and 8. In addition, the proteases were found to be heat labile. Digestion of collagen fibrils confirmed the existence of collagenolytic activity in the excretory-secretory enzyme preparations. It is suggested that these enzymes may be involved in the nutrition of the larvae and in the pathogenesis of the lesion on the skin. Topics: Animals; Aprotinin; Collagen; Diptera; Gelatin; Hydrogen-Ion Concentration; Isoelectric Point; Larva; Leupeptins; Microbial Collagenase; Molecular Weight; Peptide Hydrolases; Phenylmethylsulfonyl Fluoride; Protease Inhibitors | 1988 |
Aggregation-related association of lipid with the cytoskeleton of rabbit and human platelets prelabeled with [3H]palmitic acid. Similar effects of adenosine diphosphate- and thrombin-induced aggregation.
To investigate the association of lipid with the cytoskeleton of platelets during aggregation, rabbit and human platelets were isolated and labeled with [3H]palmitic acid; lipid extraction showed approximately 80% in phospholipid. Limited aggregation was induced with ADP or thrombin, and the cytoskeleton was isolated after lysis with 1% Triton X-100, 5 mM EGTA. Cytoskeleton from unactivated platelets had approximately 0.03% of the total label in the platelets, but after aggregation with ADP (2 microM) or thrombin (0.1 U/ml) for 20-30 s, 1.5-8% of the label was with the cytoskeleton. Fibrinogen enhanced aggregation and the association of label with the cytoskeleton; incorporation of label increased exponentially as aggregation proceeded, decreased exponentially during deaggregation, and appeared to be related to the number of sites of contact. Inhibitors that increase cyclic AMP inhibited aggregation and cytoskeletal labeling, but aspirin had no effect. Some experiments were done with DNase I and Ca2+ in the Triton X-100 lysis medium to cause actin depolymerization, under conditions in which the Ca2+-dependent protease activity was inhibited. This greatly reduced the association of label with the cytoskeleton at early time points, but when aggregation had proceeded further, a large proportion of the label was not dissociated by this treatment. These findings, electron microscopy, and the enrichment of the cytoskeleton of aggregated platelets with only some of the membrane proteins that were labeled by the 125I-lactoperoxidase method, indicated that with limited aggregation, the 3H-labeled lipid was mainly associated with the cytoskeleton and not with trapped membrane fragments resulting from incomplete lysis. Since the pattern of cytoskeleton labeling ([3H]palmitate) and the selective association of some membrane proteins with the cytoskeleton/lipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents. Topics: Actin Cytoskeleton; Adenosine Diphosphate; Animals; Blood Platelets; Calcium; Cyclic AMP; Cytoskeleton; Deoxyribonuclease I; Fibrinogen; Humans; In Vitro Techniques; Leupeptins; Lipid Metabolism; Microscopy, Electron; Palmitic Acid; Palmitic Acids; Platelet Aggregation; Platelet Membrane Glycoproteins; Rabbits; Thrombin | 1988 |
Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets.
The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein. Topics: Blood Platelets; Cell Membrane; Chymotrypsin; Endopeptidases; Epinephrine; Ethylmaleimide; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; In Vitro Techniques; Leupeptins; Phosphoric Monoester Hydrolases; Stimulation, Chemical; Thrombin; Trypsin | 1988 |
Inhibition of thrombin-induced platelet activation by leupeptin. Implications for the participation of calpain in the initiation of platelet activation.
Inhibitors of calcium-dependent proteases (calpains) such as leupeptin and antipain have been shown to selectively inhibit platelet activation by thrombin. Based upon this observation, it has been proposed that calpains play a role in the initiation of platelet activation. In the present studies, we have examined the effect of leupeptin on the earliest known event in thrombin-induced platelet activation: the interaction between the agonist, its receptors, and the guanine nucleotide-binding proteins which stimulate phospholipase C (Gp) and inhibit adenylyl cyclase (Gi). We found that leupeptin inhibited thrombin's ability to stimulate phosphoinositide hydrolysis, suppress cAMP formation, and dissociate Gp and Gi into subunits. Leupeptin had no effect, however, on the same responses to other agonists or on thrombin binding to platelets. Although these observations might suggest, as others have concluded, that calpain is involved in the initiation of platelet activation by thrombin, we also found that: 1) substituting platelet membranes for intact platelets and decreasing the free Ca2+ concentration below the threshold required for calpain activation did not diminish the effects of leupeptin on phosphoinositide hydrolysis and cAMP formation, 2) washing the platelets after incubation with leupeptin reversed the effects of the inhibitor, 3) permeabilizing the platelets with saponin did not enhance the inhibitory effects of leupeptin, and 4) leupeptin inhibited the proteolysis of fibrinogen and the hydrolysis of S2238 by thrombin. Similar results in these assays were obtained with antipain. Therefore, our observations suggest that the inhibition of platelet activation by leupeptin is due to a direct interaction with thrombin and need not reflect a role for calpain in the initiation of platelet activation. Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Antipain; Calpain; Dose-Response Relationship, Drug; GTP-Binding Proteins; Humans; Leupeptins; Oligopeptides; Phosphatidic Acids; Phosphatidylinositols; Platelet Aggregation; Thrombin; Virulence Factors, Bordetella | 1988 |
Cytocidal mechanism of TNF: effects of lysosomal enzyme and hydroxyl radical inhibitors on cytotoxicity.
The participation of lysosomal enzymes, hydroxyl radicals, and mitochondrial respiration in the cytocidal effect of TNF on tumor cells was investigated. The cytotoxicity of TNF on L-M cells was clearly reduced by lysosomotropic agents, DMSO (hydroxyl radical scavenger), NDGA (lipoxygenase inhibitor), and sodium azide (mitochondrial respiration inhibitor). The results suggest that lysosomal enzyme and hydroxyl radicals play an important triggering role in the destruction of tumor cells by TNF, and that the process of destruction might require ATP. Topics: Ammonium Chloride; Animals; Chloroquine; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Free Radicals; Hydroxides; Hydroxyl Radical; Leupeptins; Masoprocol; Methylamines; Mice; Muramidase; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha | 1988 |
Fluoride-dependent calcium-induced platelet procoagulant activity shows that calpain is involved in increased phospholipid transbilayer movement.
Treatment of platelets with fluoride (10 mM) was found to result in a transient increase in Ca2+-permeability of the platelet plasma membrane. This phenomenon was used to provide supplementary evidence for the suggestions made earlier (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143; Verhallen et al. (1987) Biochim. Biophys. Acta 903, 206), that cytoskeletal disrupture by calpain is involved in the process leading to transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. This was achieved by relating both calpain activity and exposure of phosphatidylserine with platelet procoagulant activity. It was found that only upon addition of extracellular Ca2+ to fluoride-treated platelets, procoagulant activity, expressed as prothrombinase activity, and calpain activity, estimated from protein patterns after gel electrophoresis, were generated. Both Ca2+-inducible prothrombinase activity and calpain activity followed an identical time-course during incubation with fluoride: after a time-lag of about 10 min they sharply increased towards a peak level. Upon further incubation with fluoride, both activities decreased towards a final plateau, still above basal level. The presence of leupeptin during incubation with fluoride was found to inhibit Ca2+-inducible calpain activity and prothrombinase activity in an identical way. Ca2+-inducible exposure of phosphatidylserine, as determined with extracellular phospholipase A2, showed a similar pattern as Ca2+-inducible calpain activity and prothrombinase activity. From the strict parallelism between prothrombinase activity, calpain activity and exposure of phosphatidylserine, it is concluded that calpain plays an important role in the activation-dependent transbilayer movement of phosphatidylserine during expression of platelet procoagulant activity. It is suggested that degradation of the platelet membrane-skeleton by calpain disturbs the structural organization of the lipid bilayer of the platelet plasma membrane leading to enhanced transbilayer movement of phospholipids and appearance of phosphatidylserine at the platelet outer surface. Topics: Blood Platelets; Calcium; Calpain; Fluorides; Humans; In Vitro Techniques; Leupeptins; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Motion; Phosphatidylserines; Thromboplastin | 1988 |
Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells.
A study of insulin-receptor internalization and recycling was undertaken in primary cultures of rat hepatocytes and a human hepatoma cell line (HepG2). Receptors were quantitated by measuring 125I-insulin binding to partially purified soluble receptor preparations from untreated cells (total receptors) and trypsinized cells (intracellular receptors). In resting HepG2 cells, exposure to insulin results in internalization of insulin receptors, the rate and extent of which is dependent on the insulin concentration. However, receptors do not accumulate inside the cell in proportion to the higher rates of internalization at high concentrations of insulin. This lack of accumulation is explained by much higher recycling rates after exposure to high concentrations of insulin. Similar results were noted for primary cultures of rat hepatocytes. These results imply qualitatively different fates for receptors internalized after exposure to different concentrations of insulin. To further investigate the possibility of different pathways for insulin-receptor internalization and processing, cells in low (1 ng/ml) or high (100 ng/ml) concentrations of insulin were exposed to drugs or treatments known to affect receptor metabolism. Hypotonic shock and hypokalemia, which arrest coated-pit formation, blocked internalization of insulin and insulin receptors at low concentrations of insulin but allowed internalization in response to high concentrations of insulin. The lysosomotropic drugs monensin and chloroquine caused intracellular accumulation of insulin and its receptors internalized at low concentrations of insulin but had a relatively smaller effect on receptors internalized at high concentrations of insulin. All internalization is blocked by 2,4-dinitrophenol. We conclude that high doses of insulin lead to insulin-receptor internalization and recycling through a pathway that is functionally distinct from the pathway taken by receptors internalized by low (physiologic) concentrations of insulin. The pharmacologic experiments raise the possibility that the high-dose pathway, unlike the low-dose pathway, may proceed independently of coated pits and endosomal acidification. Topics: 2,4-Dinitrophenol; Animals; Biological Transport; Biotransformation; Carcinoma, Hepatocellular; Cells, Cultured; Chloroquine; Coated Pits, Cell-Membrane; Dinitrophenols; Humans; Insulin; Leupeptins; Liver; Liver Neoplasms; Monensin; Osmolar Concentration; Potassium; Rats; Receptor, Insulin; Tumor Cells, Cultured | 1988 |
Synthesis of a new cell penetrating calpain inhibitor (calpeptin).
N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor. Topics: Animals; Blood Platelets; Calcimycin; Calpain; Cell Membrane Permeability; Dipeptides; Leupeptins; Microfilament Proteins; Papain; Peptides; Swine | 1988 |
Calcium-activated neutral proteases (calpains) are carbohydrate binding proteins.
Calcium-activated neutral proteases (calpain, EC 3.4.22.17) bind to agarose matrices (Bio-Gel A-150m, Sepharose 4B, and Ultrogel AcA 34) with high affinity in the presence of calcium. 6-O-beta-Galactopyranosyl-D-galactose, a disaccharide which closely resembles the repeating unit of the agarose matrices, completely blocks the binding of calpains and can release agarose-bound enzymes in the presence of calcium. At least 1 microM level of free calcium is required for binding. Other calcium binding proteins, including calmodulin, calpastatin, casein, and neurofilament proteins, fail to bind under the same conditions. Both calpain I and calpain II can be readily purified from crude enzyme preparations by agarose chromatography in the presence of calcium and leupeptin. Agarose-bound enzymes are eluted with calcium-free solutions or can be released in the presence of calcium by 1% Triton X-100, but not by 1 M urea or 20% ethylene glycol. Enzymes eluted from agarose are activated, as evidenced by the appearance of faster migrating forms (76 and 78 kDa) of the 80-kDa catalytic subunit of calpain I upon electrophoresis and by the increased sensitivity of calpain II to activation by micromolar levels of calcium. The electrophoretic migration of the 30-kDa regulatory subunit is, however, unaltered in enzyme fractions eluted from an agarose column. When the enzyme subunits are dissociated in 1 M NaSCN, only the 30-kDa subunit binds to the agarose matrix. Furthermore, neither calpain I nor calpain II binds to agarose when their 30-kDa subunit is autocatalyzed to an 18-kDa fragment, indicating that the NH2-terminal of the 30-kDa subunit is important for the binding of calpains to an agarose matrix. Topics: Calcium; Calcium-Binding Proteins; Calpain; Carbohydrate Metabolism; Chromatography; Disaccharides; Enzyme Activation; Ethylene Glycol; Ethylene Glycols; Leupeptins; Octoxynol; Polyethylene Glycols; Sepharose; Thiocyanates; Urea | 1988 |
The effect of microinfusions of drugs into the accessory olfactory bulb on the olfactory block to pregnancy.
Female mice which have mated and are subsequently exposed to the odour (pheromones) of a strange male undergo hormonal changes resulting in a block to their pregnancy. The fact that the stud male's odours can also block pregnancies, that is other than his own, implies the formation of a memory or some form of recognition process by the female for this male's pheromones at the time of mating. The purpose of this study was to evaluate the effect of microinfusions of drugs which interfere with neural transmission, into the accessory olfactory bulbs. This was carried out immediately after mating over a 4-h period during which the "memory" to the stud male's pheromones is formed. Infusions of the alpha-blocker, phentolamine, blocked the formation of the olfactory memory, while the GABA receptor blocker, bicuculline, itself blocked pregnancy, but was without effect on memory formation. Protein synthesis inhibition or calpain inactivation in the accessory bulb was without effect on memory formation at any of the doses used. These studies demonstrate that GABAergic transmitter blockade in the accessory olfactory bulb at the time of mating can prevent subsequent blastocyst implantation some 3 days later, while alpha-noradrenergic blockade can prevent the formation of an olfactory memory to the stud male. Topics: Animals; Anisomycin; Bicuculline; Dose-Response Relationship, Drug; Female; Leupeptins; Male; Mice; Mice, Inbred BALB C; Microinjections; Olfactory Bulb; Phentolamine; Pheromones; Pregnancy; Pregnancy, Animal; Receptors, Adrenergic, alpha | 1988 |
Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. I. Biochemical studies.
Lesions of the rat entorhinal cortex cause extensive synaptic restructuring and perturbation of calcium regulation in the dentate gyrus of hippocampus. Calpain is a calcium-activated protease which has been implicated in degenerative phenomena in muscles and in peripheral nerves. In addition, calpain degrades several major structural neuronal proteins and has been proposed to play a critical role in the morphological changes observed following deafferentation. In this report we present evidence that lesions of the entorhinal cortex produce a marked increase in the breakdown of brain spectrin, a substrate for calpain, in the dentate gyrus. Two lines of evidence indicate that this effect is due to calpain activation: (i) the spectrin breakdown products observed following the lesion are indistinguishable from calpain-generated spectrin fragments in vitro; and (ii) their appearance can be reduced by prior intraventricular in fusion of leupeptin, a calpain inhibitor. Levels of spectrin breakdown products are increased as early as 4 h post-lesion, reach maximal values at 2 days, and remain above normal to some degree for at least 27 days. In addition, a small but significant increase in spectrin proteolysis is also observed in the hippocampus contralateral to the lesioned side in the first week postlesion. At 2 days postlesion the total spectrin immunoreactivity (native polypeptide plus breakdown products) increases by 40%, suggesting that denervation of the dentate gyrus produces not only an increased rate of spectrin degradation but also an increased rate of spectrin synthesis. These results indicate that calpain activation and spectrin degradation are early biochemical events following deafferentation and might well participate in the remodelling of postsynaptic structures. Finally, the magnitude of the observed effects as well as the stable nature of the breakdown products provide a sensitive assay for neuronal pathology. Topics: Animals; Calmodulin; Calpain; Cerebral Cortex; Enzyme Inhibitors; Hippocampus; Injections, Intraventricular; Leupeptins; Male; Molecular Weight; Rats; Rats, Inbred Strains; Spectrin | 1988 |
Lesions of entorhinal cortex produce a calpain-mediated degradation of brain spectrin in dentate gyrus. II. Anatomical studies.
Lesions of the various afferents to the hippocampus have been widely used to investigate the mechanisms underlying growth and degeneration in adult mammalian CNS. It has been proposed that disturbances in intracellular calcium and activation of calcium-dependent proteases represent key steps in producing come of the consequences of the lesions. In this study, we show that lesions of the entorhinal cortex or of the commissural pathway result in profound changes in the distribution of brain spectrin. At 2 days after lesions of the entorhinal cortex, immunoreactivity to spectrin is markedly increased in the outer molecular layer (OML) of the dentate gyrus; conversely at 2 days after commissural lesions, immunoreactivity to the same antigen is increased in the inner molecular layer. The increase in immunoreactivity to spectrin varies with survival time after lesions of the entorhinal cortex. By 24 h post lesion, the increase is homogeneous across the OML, and becomes more intense by 48 h. Between 1 and 3 weeks the increase is much less than at 48 h and is concentrated at the inner border of the OML. Pretreatment of the animals with the calpain inhibitor leupeptin reduces the increase in spectrin immunoreactivity normally seen 48 h after the lesion of the entorhinal cortex. Changes in the pattern of immunoreactivity to GFAP are very different to that seen with spectrin antibodies and are consistent with the known modifications in astrocytes that follow lesions of hippocampal afferents.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Calpain; Cerebral Cortex; Enzyme Inhibitors; Glial Fibrillary Acidic Protein; Hippocampus; Immunohistochemistry; Injections, Intraventricular; Leupeptins; Male; Nerve Degeneration; Rats; Rats, Inbred Strains; Spectrin; Time Factors | 1988 |
Protease inhibitors suppress the in vitro and in vivo replication of rotavirus.
Rotaviruses are major causes of infectious gastroenteritis in humans and other animals. We found that a variety of protease inhibitors suppressed the replication of the SA-11 strain of rotavirus in MA-104 cell cultures. Three of these compounds, leupeptin, pentamidine, and bis (5-amidino-2-benzimidazolyl) methane (BABIM) also restricted the intestinal replication of the murine strain of rotavirus when protease inhibitor and virus were administered simultaneously to suckling mice. Repeated administration of BABIM resulted in significantly reduced levels of intestinal rotaviral antigen even if administration of the compound was begun as late as 48 h after viral inoculation. Additionally, BABIM-treated animals had significantly less intestinal replication of rotavirus than did placebo-treated controls when placed in a heavily rotavirus-contaminated environment. The use of protease inhibitors represents a novel approach to the control of this important gastrointestinal pathogen and is a potential modality for the prevention and treatment of diseases caused by other enteric viruses, for which proteolytic cleavage is necessary for efficient replication. Topics: Animals; Benzimidazoles; Cell Line; Humans; Leupeptins; Mice; Pentamidine; Protease Inhibitors; Rotavirus; Virus Replication | 1988 |
Inhibition of the proteolysis of rat erythrocyte membrane proteins by a synthetic inhibitor of calpain.
A synthetic inhibitor of calpain protects rat erythrocyte membrane-associated cytoskeletal proteins from proteolytic degradation (IC50 = 1 microM) which occurs when the cells are rendered permeable to Ca++. Leupeptin, a naturally occurring inhibitor of the enzyme, does not afford any protection at concentrations up to 100 microM. Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Membrane Permeability; Cytoskeletal Proteins; Dipeptides; Erythrocyte Membrane; Hydrolysis; Leupeptins; Membrane Proteins; Molecular Weight; Peptide Hydrolases; Protease Inhibitors; Rats; Spectrin | 1988 |
Ca2+-mediated catabolism of human erythrocyte band 3 protein.
Catabolism of human erythrocyte membrane band 3 protein in the presence of Ca2+ was studied. An increase in the amount of a 30 kDa amino terminal fragment of band 3 was observed when erythrocyte membranes were incubated for 30 min with 1 mM Ca2+ in the presence of whole erythrosol. Incubation of the membranes with Ca2+ alone did not result in band 3 breakdown. Generation of the 30 kDa fragment from band 3 was related to the action of a leupeptin-sensitive Ca2+-dependent proteinase in the cytosol. This proteinase was also responsible for the increased production of a 52 kDa and a 70 kDa transmembrane carboxyl terminal fragment of band 3. From the size of the generated fragments, it is deduced that in the presence of Ca2+ and Ca2+-dependent proteinase, band 3 protein is cleaved at the cytoplasm/membrane interface and along its cytoplasmic domain. Topics: Anion Exchange Protein 1, Erythrocyte; Calcium; Calpain; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Humans; Leupeptins; Molecular Weight | 1988 |
Regulation of the breakdown rates of biotin-containing proteins in Swiss 3T3-L1 cells.
1. Degradation rate constants for individual biotin-labelled proteins were measured in Swiss 3T3-L1 adipocytes that had been incubated with inhibitors of autophagy or of lysosomal proteolysis. 2. Inhibitory effects produced by 10 mM-3-methyladenine and a combination of 5 mM-NH4Cl and leupeptin (50 micrograms/ml) were approximately equal. The inclusion of NH4Cl did not significantly enhance the responses to 3-methyladenine, suggesting that autophagy was already maximally inhibited. 3. The extent of inhibition by 3-methyladenine or by the NH4Cl/leupeptin mixture was similar for the cytosolic enzyme acetyl-CoA carboxylase and for the three mitochondrial carboxylases. This inhibition averaged 50%. The breakdown rate of a more-stable 38 kDa biotin-containing mitochondrial protein was more responsive to the inhibitory agents. These results are best explained by mitochondrial proteolysis occurring via a combination of the degradation of whole mitochondria within autophagic vacuoles, supplemented by the selective intramitochondrial breakdown of more labile proteins. 4. A number of intermediate products in the degradation of biotin-containing proteins were detected. Differences in the patterns of radioactivity between these peptides after incubation of cells in the presence of inhibitors of the breakdown process provided evidence that some peptides were produced before autophagy, others as a result of intralysosomal inhibition, while at least one was associated with intramitochondrial proteolysis. Topics: Adenine; Biotin; Cell Line; Electrophoresis, Polyacrylamide Gel; Insulin; Leucine; Leupeptins; Proteins | 1988 |
A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.
To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy. Topics: Animals; Cysteine Endopeptidases; Hemoglobins; Leucine; Leupeptins; Malaria; Microscopy, Electron; Molecular Weight; Plasmodium falciparum; Protease Inhibitors | 1988 |
Evidence for the role of proteinases in uremic catabolism.
Enhanced muscle protein breakdown has been demonstrated in acutely uremic rats by numerous authors. In order to investigate the pathogenetic role of skeletal muscle proteinases leupeptin, a low-molecular weight proteinase inhibitor, was administered intraperitoneally to acutely uremic rats. Twenty-four hours after bilateral nephrectomy, leupeptin-treated animals displayed significantly lowered serum urea levels (-32%), as compared to untreated uremic rats. As a sign of muscle protein breakdown, plasma levels of Nt-methylhistidine, an indicator of myofibrillar protein degradation, were also decreased (-35%) in the uremic animals treated with leupeptin as compared to untreated uremic rats. Finally, leupeptin treatment resulted in a significant inhibition of the myofibrillar alkaline proteinase activity, a proteinase which has been related to various catabolic conditions. These findings suggest that the increased muscle protein breakdown in uremia is caused by enhanced activity of muscular proteinases and that anti-proteolytic agents display favourable effects on the enhanced protein degradation observed in acute uremia. Topics: Acute Disease; Animals; Blood Chemical Analysis; Endopeptidases; Hydrolysis; Leupeptins; Male; Methylhistidines; Muscle Proteins; Nephrectomy; Protease Inhibitors; Rats; Rats, Inbred Strains; Uremia | 1988 |
Import of rat liver mitochondrial transhydrogenase.
The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin. Topics: Animals; Biological Transport; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; In Vitro Techniques; Leupeptins; Liver; Mitochondria, Liver; NADH, NADPH Oxidoreductases; NADP Transhydrogenases; Phenanthrolines; Rats; Rhodamines | 1988 |
The presence of a reduced amount of 32-kd "protective" protein is a distinct biochemical finding in late infantile galactosialidosis.
The biochemical defect underlying the late infantile form of galactosialidosis has been investigated in fibroblasts from two patients presenting with this phenotype. Immunoprecipitation experiments demonstrated that a reduced amount of 32-kd "protective" protein and a normal amount of its precursor are present in late infantile galactosialidosis fibroblasts, while neither of the two polypeptides are detectable in early infantile and juvenile/adult fibroblasts. Leupeptin treatment led to a slight increase in the amount of 54-kd and 32-kd polypeptides in both late-infantile galactosialidosis cell lines. Uptake studies in one of the two cell lines confirmed the hypothesis that a block in the maturation of the protective protein is responsible for the late infantile type of galactosialidosis. This mutation seems to be a distinct finding in all patients affected by this form of the disease. Topics: beta-Galactosidase; Carbohydrate Metabolism, Inborn Errors; Cells, Cultured; Fibroblasts; Galactosidases; Humans; Infant; Leupeptins; Molecular Weight; Neuraminidase; Proteins | 1988 |
Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.
Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes. Topics: alpha-Mannosidase; Antipain; beta-Glucosidase; Biological Transport; Cell Compartmentation; Cysteine Proteinase Inhibitors; Dictyostelium; Golgi Apparatus; Hexosaminidases; Leupeptins; Lysosomes; Mannosidases; Molecular Weight; Protein Processing, Post-Translational; Time Factors | 1988 |
Reduction of muscle protein degradation by leupeptin in acutely uremic rats.
Enhanced muscle protein breakdown has been demonstrated in acutely uremic rats by numerous authors. These findings have been used to explain the clinical signs of muscle wasting and enhanced urea-N appearance, frequently observed in patients suffering from uremia. In order to investigate whether inhibition of skeletal muscle proteinases would have a favourable effect on the extent of muscle protein degradation, leupeptin, a low-molecular-weight proteinase inhibitor, was administered intraperitoneally to acutely uremic rats. 24 h after bilateral nephrectomy, leupeptin-treated animals displayed significantly lowered serum urea levels (-32%), and hence decreased urea-N appearances (-39%) as compared to untreated uremic rats. As a sign of muscle protein breakdown, plasma levels of Nt-methylhistidine, an indicator of myofibrillar protein degradation, were also decreased (-35%) in the uremic animals treated with leupeptin as compared to untreated uremic rats. Finally, leupeptin treatment resulted in a significant inhibition of the myofibrillar alkaline proteinase activity, a proteinase which has been related to various catabolic conditions. These findings suggest that the increased muscle protein breakdown in uremia is caused by enhanced activity of muscular proteinases and that antiproteolytic agents display favourable effects on the enhanced protein degradation observed in acute uremia. Topics: Acute Disease; Animals; Leupeptins; Male; Methylhistidines; Muscle Proteins; Muscles; Nephrectomy; Oligopeptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Urea; Uremia | 1988 |
Subcellular distribution of protein kinase C in rat adrenal glomerulosa cells.
With the aid of a synthetic nonapeptide which is a selective substrate for protein kinase C the activity of this enzyme was determined in the crude cytosolic and particulate fractions of rat adrenal glomerulosa cells. When the cells were sonicated in the presence of Ca2+ chelators 65 per cent of their total protein kinase C activity was found in the cytosolic extract. The treatment of cells with angiotensin II under conditions where the maximal stimulation of inositol-lipid hydrolysis was observed did not cause a statistically significant change in the apparent subcellular distribution of protein kinase C. However, when the cytosolic extract was prepared in the presence of Ca2+ the protein kinase C activity was recovered nearly exclusively from the particulate fraction. Topics: Angiotensin II; Animals; Calcium; Cytosol; Leupeptins; Phenylmethylsulfonyl Fluoride; Phospholipids; Protein Kinase C; Rats; Rats, Inbred Strains; Subcellular Fractions; Tetradecanoylphorbol Acetate; Zona Glomerulosa | 1988 |
Distribution of protein disulfide isomerase in rat hepatocytes.
We investigated quantitatively the distribution of protein disulfide isomerase (PDI) in rat hepatocytes by immunocytochemistry using a post-embedding protein A-gold technique. In hepatocytes, gold particles were mainly localized in the intracisternal space of the rough and smooth endoplasmic reticulum (ER) and nuclear envelopes. Autolysosomes engulfing ER were occasionally densely labeled, especially in rat hepatocytes previously treated with leupeptin in vivo, suggesting that the autophagosome-autolysosome system may be an important route for degradation of PDI. A few gold particles were also found on the plasma membranes. Localization of gold particles on the other subcellular organelles, such as Golgi apparatus, peroxisomes, and nuclear matrix, was sparse and at the control level. The predominant localization of PDI on the intracisternal surface of the ER and nuclear envelope supports a potential role of PDI in the formation of disulfide bonds of nascent polypeptides, thus accelerating formation of the higher-order structure of secretory and membrane proteins and rendering the translocation process irreversible. Topics: Animals; Cell Membrane; Endoplasmic Reticulum; Gold; Immunoblotting; Immunohistochemistry; Isomerases; Leupeptins; Liver; Lysosomes; Male; Microscopy, Electron; Microsomes, Liver; Nuclear Envelope; Protein Disulfide-Isomerases; Rats; Rats, Inbred Strains; Staphylococcal Protein A | 1988 |
Ferritin iron kinetics and protein turnover in K562 cells.
The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes. Topics: Animals; Cell Line; Deferoxamine; Electrophoresis, Polyacrylamide Gel; Ferritins; Iron; Kinetics; Leupeptins; Pentetic Acid; Proteins | 1988 |
Degradation of aspartate aminotransferase in rat liver lysosomes.
Highly purified lysosomes from the normal and leupeptin-treated rat livers were subjected to immunoblot analysis using antibodies against cytosolic and mitochondrial isozymes of aspartate aminotransferase (cAspAT and mAspAT). In the case of cAspAT (subunit M.W. = 46K), the leupeptin-treated lysosomes showed a major band of 46K and a minor band of 36K while normal lysosomes showed a major band of 36K and a minor band of 41K. In the case of mAspAT (subunit M.W. = 44K), the leupeptin-treated lysosomes showed a 44K band and the normal lysosomes showed a 40K band. These observations suggest that both cAspAT and mAspAT are sequestered into lysosomes with the original subunit molecular weights and are degraded in the lysosomes by way of sequential formation of relatively stable intermediates with distinct molecular weights. Topics: Animals; Aspartate Aminotransferases; Blotting, Western; Isoenzymes; Leupeptins; Liver; Lysosomes; Molecular Weight; Rats | 1988 |
Influence of leupeptin on protein and amino-acid metabolism in septic rats.
We investigated the influence of leupeptin (LP) intraperitoneal injection (40 mumol/2 days) on protein and amino-acid metabolism of septic rats (cecal ligation). All septic rats lost weight (-17 +/- 4 g), which was not prevented by LP administration (-24 +/- 1.8 g, n.s.). LP injection evoked weight loss even in normal rats (p less than 0.05 vs controls). Weight loss was accompanied by enhanced urinary nitrogen losses in all three groups. LP reduced food intake for 47% in control rats. Cecal ligation, and also the administration of LP, led to alterations of amino-acid metabolism. The most important changes were found in muscle free amino-acid concentrations with highly decreased levels of free glutamine. A glutamine deficiency is known to be related to a decreased rate of protein synthesis. The proteolytic rate in incubated soleus muscle was increased for 11.5% and even higher in LP-treated septic rats (+22%). It is concluded that the administration of LP cannot reverse protein catabolism in sepsis--possibly because LP does not influence those enzymes or proteases involved in tissue loss, or LP is inactivated by enzymes in rat tissues. Topics: Amino Acids; Animals; Bacterial Infections; Body Weight; Eating; Leupeptins; Liver; Muscles; Oligopeptides; Proteins; Rats; Rats, Inbred Strains; Tyrosine | 1988 |
Increased mortality in septic rats after leupeptin application.
Topics: Animals; Bacterial Infections; Body Weight; Creatinine; Leupeptins; Liver; Male; Muscles; Oligopeptides; Rats; Rats, Inbred Strains; Tyrosine | 1988 |
Inhibition of EGF processing in responsive and nonresponsive human fibroblasts.
We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies (Schaudies et al., 1985) have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells. Incubations in leupeptin, proteolysis of EGF beyond the des-(48-53)-EGF was blocked; however, no large-molecular-weight species were released from the cells under these conditions. Topics: Animals; Cells, Cultured; Chloroquine; Epidermal Growth Factor; Humans; Iodine Radioisotopes; Kinetics; Leupeptins; Lung; Male; Mice; Protein Processing, Post-Translational; Skin | 1988 |
Regulation of the production of a prolactin-like protein (MRP/PLF) in 3T3 cells and in the mouse placenta.
Mitogen-regulated protein (MRP), a heterogeneously glycosylated mouse protein of Mr 34,000, is in the same protein family as prolactin, growth hormone, and placental lactogen. We show here that the level of translatable MRP mRNA is increased in response to fibroblast growth factor. Also, the amount of MRP secreted by 3T3 cells is modulated by the rate of degradation of newly synthesized MRP in the lysosomes. This is indicated by several results. First, agents that inhibit protein degradation by lysosomal proteases selectively increased by 2- to 6-fold the incorporation of [35S]methionine into MRP. These agents are ammonium chloride, the carboxylic ionophores, monensin and nigericin, and two thiol protease inhibitors, leupeptin and antipain. MRP that has already been secreted is not degraded by 3T3 cells. We examined the developmental appearance of MRP using immunofluorescence microscopy and found MRP localized in the mouse placenta between days 9 and 13 of development. The amount of MRP in the placenta drops suddenly after day 13. Whereas the appearance of MRP in the placenta follows the reported appearance of its mRNA, MRP disappears from the placenta more rapidly than its mRNA. On the basis of the results of our studies with cells in culture we propose that the production of MRP in the placenta is regulated similarly to prolactin. Thus we propose that the initial increase in MRP production in the placenta is due to pretranslational regulation by growth factors, and the later rapid decline is due to posttranslational regulation through degradation in the lysosomes. Topics: Ammonium Chloride; Animals; Antipain; Cell Line; Epidermal Growth Factor; Fibroblast Growth Factors; Glycoproteins; Glycosylation; Intercellular Signaling Peptides and Proteins; Kinetics; Leupeptins; Mice; Monensin; Placenta; Prolactin; RNA, Messenger | 1988 |
Selective inhibition of antigen presentation to cloned T cells by protease inhibitors.
The effect of eight microbial protease inhibitors on Ag-presentation to six different Ag-specific T cell clones was investigated. We found that these protease inhibitors can inhibit Ag presentation in a highly selective manner. This selectivity was evident with T cell clones specific to different Ag as well as with T cells specific to the same Ag but differing in their H-2 restriction. The inhibition was to due to cytotoxicity or effects through the TCR because none of the eight inhibitors inhibited IL-2-induced T cell proliferation, and because they did not inhibit Ag presentation by fixed APC or synthetic polypeptide. The conclusion after these data suggests that each specific antigenic fragment is produced by a unique set of proteases. Topics: Animals; Antigen-Presenting Cells; Antipain; Clone Cells; Histocompatibility Antigens Class II; Immunosuppressive Agents; Leupeptins; Metalloendopeptidases; Mice; Oligopeptides; Protease Inhibitors; T-Lymphocytes | 1988 |
Processing requirements for T cell activation by Mycoplasma arthritidis-derived mitogen.
Mycoplasma arthritidis produces in culture a polyclonal mitogen which is active for murine and human T lymphocytes in the presence of accessory cells (AC). We studied the requirements for processing and presentation by AC of Mycoplasma arthritidis supernatant (MAS) mitogen to human T cells. As inhibitors of AC processing, several agents were used which inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing by presenting cells and washed out before T cells were added to culture, they inhibited lymphocyte activation and, therefore, we assume that they interfered with the presentation of the mitogen. Thus, if MAS requires a processing step, it appears to involve lysosomal proteolysis which can be blocked in vitro. Topics: Ammonium Chloride; Antigen-Presenting Cells; Chloroquine; Formaldehyde; Leupeptins; Lymphocyte Activation; Lysosomes; Macrophages; Mitogens; Monensin; Mycoplasma; Polymers; T-Lymphocytes | 1988 |
Thrombin-like inhibitory action of trypsin and trypsin-like proteases on human platelet adenylate cyclase.
The effects of trypsin, acrosin and a recently described trypsin-like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25 degrees C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50 approximately 0.1 microM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half-maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 micrograms/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N-ethylmaleimide at low concentrations and Mn2+ ions (greater than or equal to 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine-nucleotide-binding protein Gi, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin-like proteases have two opposite effects on the platelet adenylate cyclase system, the well-documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell-surface receptor sites in a manner similar to thrombin. Topics: Acrosin; Adenylyl Cyclase Inhibitors; Animals; Blood Platelets; Cattle; Epinephrine; Guanosine Triphosphate; Humans; Leupeptins; Male; Peptide Hydrolases; Spermatozoa; Thrombin; Trypsin | 1988 |
Role for intracellular proteases in the processing and transport of class II HLA antigens.
Human B-lymphoblastoid cell lines (B-LCL) incubated with the protease inhibitor leupeptin accumulate complexes of class II HLA antigens with a series of Mr 21,000-23,000 basic proteins termed leupeptin-induced proteins (LIP). The appearance of class II antigen-associated LIP coincides with the disappearance of class II antigen-associated invariant (I) chain. Glycopeptides generated by in vitro proteolysis of LIP and I chain using Staphylococcus aureus V8 protease are identical as determined by electrophoresis in sodium dodecyl sulfate. These results suggest that LIP is a proteolytic product derived from the I chain and are consistent with the view that further in vivo proteolysis of LIP by a leupeptin-sensitive enzyme normally facilitates its release from class II antigens. Incubation of B-LCL with monensin, which traps class II antigens and associated I chain in the Golgi apparatus, or chloroquine, which neutralizes intracellular acidic compartments and inhibits I-chain dissociation, blocks the leupeptin-induced appearance of LIP. Treatment of LIP with endoglycosidases F and H shows that both of its N-linked oligosaccharides are in the complex form, indicating that proteolysis of class II antigen-associated I chain to generate LIP occurs in a late-Golgi or post-Golgi compartment. The compartment in which these proteolytic events occur may be identical to the site in macrophages and B lymphocytes where foreign antigens are processed and interact with class II HLA molecules. Topics: Biological Transport; Chloroquine; HLA-D Antigens; Humans; Leupeptins; Molecular Weight; Monensin; Peptide Hydrolases; Protein Processing, Post-Translational | 1988 |
Evidence that cathepsin B contributes to skeletal muscle protein breakdown during sepsis.
The mechanisms of accelerated skeletal muscle protein degradation during sepsis have not been fully elucidated. Activity of the lysosomal protease cathepsin B is increased in skeletal muscle during various catabolic states other than sepsis. In the present study the protein degradation rate and cathepsin B activity were determined in extensor digitorum longus and soleus muscles from nonseptic and septic rats. The protein degradation rate during incubation in vitro with or without the cathepsin B inhibitor leupeptin was also determined. Both protein degradation and cathepsin B activity were increased in muscles from septic rats. Incubation with leupeptin reduced, but did not normalize, the protein degradation rate in both extensor digitorum longus and soleus muscles from septic animals. These studies suggest that increased cathepsin B activity contributes to the accelerated muscle proteolysis seen during sepsis and that proteases other than cathepsin B are also involved. Topics: Animals; Bacterial Infections; Cathepsin B; Leupeptins; Male; Muscle Proteins; Muscles; Rats; Rats, Inbred Strains | 1988 |
Degradation of the precursor of mitochondrial aspartate aminotransferase in chicken embryo fibroblasts.
The precursor of mitochondrial aspartate aminotransferase accumulates in the cytosol of cultured chicken embryo fibroblasts if its import into mitochondria is inhibited by an uncoupling agent. However, its accumulation is limited by degradation with a half-life of only approximately 5 min (Jaussi, R., Sonderegger, P., Flückiger, J., and Christen, P. (1982) J. Biol. Chem. 257, 13334-13340). The aim of the present study was the characterization of the proteolytic system(s) responsible for this very rapid intracellular degradation. On depleting chicken embryo fibroblasts of ATP, the rate of degradation of the precursor was lowered by approximately 70%. Chicken embryo fibroblasts depleted of divalent metal ions showed a degradative activity of 10% of the initial value. Reconstitution of these cells with Mg2+ and Ca2+ increased the degradative activity from 10 to 107 and 24%, respectively. Thiol reagents almost completely prevented the degradation, whereas specific peptide inhibitors of cysteine proteases or inhibitors of intralysosomal proteolysis decreased the rate of degradation by only approximately 30%. Inhibitors of serine proteases had little effect. No rapid degradation of the precursor was observed in crude extracts of chicken embryo fibroblasts. The data indicate that the bulk of the precursor accumulated under conditions of import block is degraded by one or several cytosolic proteases dependent on ATP, Mg2+, and thiol groups of unknown localization, conceivably by proteolytic enzymes identical with or similar to one of the high molecular weight cytosolic proteases (Waxman, L., Fagan, J.M., Tanaka, K., and Goldberg, A. L. (1985) J. Biol. Chem. 260, 11994-12000). The rest of the precursor appears to be degraded by lysosomes. Topics: Adenosine Triphosphate; Animals; Antipain; Aspartate Aminotransferases; Chick Embryo; Cytosol; Enzyme Precursors; Fibroblasts; Half-Life; Leupeptins; Methylamines; Mitochondria; Monensin | 1988 |
Degradation of endocytosed dermatan sulfate proteoglycan in human fibroblasts.
Endocytosis and subsequent degradation of iduronic acid-rich small dermatan sulfate proteoglycan from fibroblast secretions were studied in human fibroblasts. Upon endocytosis of [3H]leucine- and [35S]sulfate-labeled proteoglycan release of free leucine was 10 to 15 times more rapid than that of inorganic sulfate. Within approximately 3 h a steady state was approached between transport of proteoglycan to the compartment of core protein degradation and release of free leucine. No such steady state could be found with respect to the dermatan sulfate chains. In the presence of benzyloxycarbonyl-Phe-Ala-diazomethylketone or of other SH-protease inhibitors the degradation of the protein moiety of endocytosed proteoglycan was much less inhibited than the degradation of the polysaccharide chain. Benzyloxycarbonyl-Phe-Ala-diazomethylketone did not affect the degradation of dermatan sulfate chains taken up by fluid phase endocytosis and the activities of all known dermatan sulfate-degrading enzymes. Percoll gradient centrifugation indicated that also in the presence of the protease inhibitor the partially degraded proteoglycan accumulated in dense lysosomes. The isolation of intracellular dermatan sulfate peptides and molecular size determinations of endocytosed dermatan sulfate proteoglycan supported the conclusion that a critical proteolytic step is required before the dermatan sulfate chain becomes accessible to hydrolytic enzymes. Topics: Ammonium Chloride; Biological Transport; Cells, Cultured; Chondroitin; Chondroitin Sulfate Proteoglycans; Cycloheximide; Dermatan Sulfate; Diazomethane; Endocytosis; Fibroblasts; Half-Life; Humans; Kinetics; Leucine; Leupeptins; Lysosomes; Molecular Weight; Protease Inhibitors; Proteoglycans; Sulfates | 1988 |
Effects of indomethacin and leupeptin on muscle cathepsin B activity and protein degradation during sepsis.
The roles of prostaglandins and lysosomal proteases in accelerated skeletal muscle proteolysis during sepsis are not yet fully understood. In this study rats received intraperitoneal injections of the prostaglandin synthesis inhibitor indomethacin (IND, 5.0 mg/kg), the lysosomal cathepsin B inhibitor leupeptin (LEU, 2.5 mg/kg), or normal saline 2 hr before cecal ligation and puncture (a model of intraabdominal sepsis) or sham-operation. The injections were repeated every 6 hr for a total of four doses. Sixteen hours after operation, intact extensor digitorum longus (EDL) muscles were harvested and cathepsin B activity was measured in one muscle. The contralateral muscle was incubated in oxygenated Krebs-Henseleit bicarbonate buffer containing glucose (10 mM) and cycloheximide (0.5 mM), and protein degradation rate was determined as the release of tyrosine into the incubation medium. Both muscle cathepsin B activity and protein degradation rate were higher in septic than in sham-operated rats. Treatment with IND or LEU significantly reduced the elevated cathepsin B activity in septic muscles, but failed to significantly alter muscle proteolysis. In nonseptic muscle, both cathepsin B activity and protein degradation rate were unaffected by the different types of treatment. The results suggest that although prostaglandins may influence muscle lysosomal protease activity, neither prostaglandins nor the lysosomal protease cathepsin B appear to be major regulators of accelerated muscle protein breakdown during sepsis. Topics: Animals; Bacterial Infections; Cathepsin B; Indomethacin; Leupeptins; Muscle Proteins; Muscles; Oligopeptides; Reference Values | 1988 |
Thrombin inhibition with dipeptidyl argininals.
Human alpha- and gamma-thrombins (with high and essentially no fibrinogen clotting activities, respectively) were inhibited in chromogenic substrate assays by the dipeptidyl argininals: antipain less than leupeptin less than H-D-Phe-Pro-argininal approximately Boc-D-Phe-Pro-argininal. In clotting assays with alpha-thrombin, I50 values were slightly higher than Ki values from chromogenic substrate assays, except for a somewhat lower I50 for antipain. Our data cautions the use of argininal proteinase inhibitors in the assessment of thrombin functions, and the high potency of H-D-Phe-Pro-argininal and its derivative suggest pharmaceutical applications. Topics: Antipain; Blood Coagulation; Kinetics; Leupeptins; Oligopeptides; Thrombin | 1988 |
Perinatal leupeptin retards subsequent acquisition of a visual discrimination task in chicks.
Recent discoveries that calcium sensitive proteases (calpains) may play an important role in memory led to investigation of the effects of leupeptin, a calpain inhibitor, on learning. Intracerebral injections of either 0.9% saline, 80 micrograms aprotinin, 306 micrograms glutamate, or 8 or 80 micrograms leupeptin were administered to 95 two-day-old chicks. On Day 10 posthatch, animals were then tested on a task requiring discriminations between pebbles and food pellets. Chicks receiving 8 or 80 micrograms leupeptin or 306 micrograms glutamate were found to be deficient in acquiring the task. Aprotinin, a control serine protease inhibitor, did not retard discrimination learning. None of the drugs tested altered body weight or gross visual skills. These results suggest that retardation produced by leupeptin is not due to nonspecific changes in protease activity, visual competency, or growth. The reported results confirm earlier reports of leupeptin-induced memory impairments in rats. Topics: Animals; Aprotinin; Chickens; Discrimination Learning; Glutamates; Glutamic Acid; Leupeptins; Oligopeptides; Visual Perception; Vocalization, Animal | 1987 |
A G protein directly regulates mammalian cardiac calcium channels.
A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels. Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Colforsin; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guinea Pigs; Heart; Ion Channels; Isoproterenol; Leupeptins; Membrane Potentials; Phosphorylation; Thionucleotides; Ventricular Function | 1987 |
Potentiation of the cytotoxicity of peplomycin against Ehrlich ascites carcinoma by bleomycin hydrolase inhibitors.
Topics: Animals; Bleomycin; Carcinoma, Ehrlich Tumor; Cell Survival; Cysteine Endopeptidases; Drug Synergism; Female; Glycoside Hydrolases; Leucine; Leupeptins; Mice; Oligopeptides; Peplomycin | 1987 |
The diacylglycerol kinase inhibitor R59022 potentiates superoxide production but not secretion induced by fMet-Leu-Phe: effects of leupeptin and the protein kinase C inhibitor H-7.
The addition of low concentrations of the chemotactic factor fMet-Leu-Phe to rabbit neutrophils in the absence of cytochalasin B produces very little superoxide. This level of superoxide can be greatly increased in neutrophils pretreated for 30 min with 10 microM of the diacyl-glycerol kinase inhibitor R59022. This potentiation occurs also in the presence of cytochalasin B. In addition, while the small level of superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7), the increase by R59022 is completely abolished by this compound. In addition, this increase can be potentiated further by leupeptin. Unlike superoxide generation, the release of lysozyme or N-acetyl-beta-glucosaminidase produced by fMet-Leu-Phe is not stimulated by R59022. The results presented here suggest that stimulation of the oxidative burst requires the generation and the maintenance of a sufficient amount of diacylglycerol and/or the rearrangement of the cytoskeleton such as the inhibition of actin polymerization. Furthermore, the membrane-associated form of protein kinase C is the one responsible for the activation of the oxidative burst. The relationship between protein kinase C activation and the stimulated oxidative burst and the physiological role of chemotactic factors in the functions of the neutrophils are discussed. Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cytochalasin B; Diacylglycerol Kinase; Isoquinolines; Kinetics; Leupeptins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Phosphotransferases; Piperazines; Platelet Activating Factor; Protein Kinase C; Pyrimidinones; Rabbits; Superoxides; Thiazoles | 1987 |
Platelet factor XIII is activated by calpain.
The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII. Topics: Blood Platelets; Calcium; Calcium-Binding Proteins; Calpain; Enzyme Activation; Factor XIII; Humans; In Vitro Techniques; Leupeptins; Thrombin; Transglutaminases | 1987 |
Spatial learning in the rat: impairment induced by the thiol-proteinase inhibitor, leupeptin, and an analysis of [3H]glutamate receptor binding in relation to learning.
Rats were given continuous intraventricular infusion of saline or the thiol-proteinase inhibitor leupeptin, via subcutaneously implanted osmotic minipumps, while being trained on a spatial learning water task using spaced trials. Leupeptin caused overnight forgetting during training, but performance eventually reached asymptote in both groups. A retention test conducted 48 h later to assess spatial memory revealed no significant group differences, but did cause, in saline-treated rats only, a disruption of subsequent retraining back to the correct spatial location. The groups showed no differences in Cl-dependent [3H]glutamate receptor binding to hippocampal or entorhinal cortex membranes subsequent to training. In a second experiment, normal rats trained on the same task also showed no differences in Cl-dependent [3H]glutamate binding relative to rats exposed to the water task but given random spatial position training and handled controls. The results are discussed in relation to the hypothesis of Lynch and Baudry (Science (1984) 224, 1057-1063) that a calcium-dependent thiol proteinase is involved in memory formation through its ability to modify glutamate receptor distribution and dendritic spine shape. Topics: Animals; Brain; Discrimination Learning; Glutamates; Glutamic Acid; Hippocampus; Injections, Intraventricular; Leupeptins; Male; Memory; Mental Recall; Neural Pathways; Oligopeptides; Orientation; Rats; Receptors, Glutamate; Receptors, Neurotransmitter; Space Perception; Synaptic Membranes | 1987 |
The effect of analogues of chymostatin on lysosomal and non-lysosomal components of protein degradation in isolated hepatocytes.
Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo. Topics: Adenosine Triphosphate; Animals; Gluconeogenesis; In Vitro Techniques; Leupeptins; Liver; Lysosomes; Male; Oligopeptides; Protease Inhibitors; Rats; Structure-Activity Relationship; Tyrosine Transaminase | 1987 |
Evidence for two catabolic endoglycosidase activities in beta-mannosidase-deficient goat fibroblasts.
Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal beta-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Man beta 4GlcNAc beta 4GlcNAc and Man beta 4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [3H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Man beta 4GlcNAc beta 4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [3H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-beta-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus. Topics: Animals; beta-Mannosidase; Biological Transport; Carbohydrate Sequence; Carrier Proteins; Cells, Cultured; Fibroblasts; Glycoproteins; Glycoside Hydrolases; Goats; Leupeptins; Lysosomes; Mannosidases; Receptor, IGF Type 2; Structure-Activity Relationship | 1987 |
Antipain or leupeptin in combination with aspirin or indomethacin synergistically inhibit human platelet activation by thrombin and trypsin.
Topics: Antipain; Aspirin; Blood Platelets; Drug Synergism; Humans; In Vitro Techniques; Indomethacin; Leupeptins; Oligopeptides; Thrombin; Trypsin | 1987 |
Membrane glycoproteins are involved in the differentiation of the BC3H1 muscle cell line.
The nonfusing muscle cell line BC3H1 expresses a family of muscle-specific proteins when the fetal bovine serum (FBS) concentration is reduced from 20 to 1%. We have used a series of glycosylation inhibitors to assess the role played by glycoproteins in the initiation of differentiation in this cell line. Tunicamycin (TNM) and 2-deoxy-D-glucose, added to cells when the FBS concentration was reduced, blocked creatine phosphokinase (CPK) induction by 70-95%. These effects were dose dependent and reversible. TNM and 2-deoxy-D-glucose also reversed CPK induction in differentiated cells. Leupeptin and N-acetylglucosamine did not reverse these effects. 1-Deoxynojirimycin, 1-deoxymannojirimycin, and swainsonine have no effect on induced CPK expression, whereas castanospermine, a glucosidase I inhibitor, blocked its induction completely. As attempts to use conditioned medium from cells grown in 1 or 20% FBS have no effect on this differentiation process we conclude that high mannose structures, but not complex form glycoproteins, bound to the surface of BC3H1 cells play a role in transducing signals for differentiation and are probable mediators of cell/cell contact. Topics: 1-Deoxynojirimycin; Acetylglucosamine; Alkaloids; Animals; Cell Differentiation; Cell Line; Creatine Kinase; Deoxy Sugars; Deoxyglucose; Enzyme Induction; Glucosamine; Glycoproteins; Indolizines; Leupeptins; Membrane Glycoproteins; Membrane Proteins; Mice; Muscles; Oligosaccharides; Swainsonine; Tunicamycin | 1987 |
Analysis of proteolytic processing during specific antigen presentation.
In this report we have studied the effect of protease inhibitors on B-cell-antigen processing. As a source of antigen-presenting B cells we have utilized transformants transfected with a vector carrying immunoglobulin (Ig) genes specific for the hapten trinitrophenyl (TNP). B-cell-specific (TNP-proteins) and nonspecific antigen-presentation activities were blocked to the same extent upon addition of inhibitors for protease and endosomal function. Interestingly, the effect of leupeptin, a thiol protease inhibitor, varied depending on the antigen and helper T cells utilized. These results suggest that specific groups of proteases may be required for antigen processing so that discrete antigenic epitopes in association with major histocompatibility complex molecules can be recognized by interacting T cells. Topics: Animals; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Cells, Cultured; Chloroquine; Haptens; Leupeptins; Lymphocyte Cooperation; Mice; Peptide Hydrolases; Receptors, Antigen, T-Cell; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; Tosylphenylalanyl Chloromethyl Ketone; Trinitrobenzenes | 1987 |
Targeting of neoglycoprotein-drug conjugates to cultured human embryonal carcinoma cells.
Fluorescent neoglycoproteins were used to screen for the presence and sugar specificities of cell surface lectins in two human embryonal carcinoma cell lines. Efficient labeling correlated with extent of lectin-mediated uptake of neoglycoproteins, as measured by inhibition of DNA synthesis by drug-neoglycoprotein conjugates. These conjugates contain covalently linked carbohydrate moieties on the carriers to render them accessible to the membrane lectins, most effectively galactosides and alpha-glucosides. They furthermore contain chemically linked cytotoxic drugs (etoposide, cis-diamminedichloroplatinum II and methotrexate) which are intracellularly released after lysosomal breakdown of the carrier, as indicated by the effect of leupeptin. Sugars can confer a greater than 10-fold increase in cytotoxic capacity to the nonglycosylated carrier-drug conjugate, nearly reaching the level of toxicity of the freely diffusible drug. Two different neoglycoproteins, reacting with independently targeted membrane lectins, were shown to be useful in a model for combination chemotherapy. These results therefore suggest potential usefulness of custom-made glycosylated carriers in the targeting of therapeutic agents to human embryonal carcinoma cells. Topics: Antineoplastic Agents; Cell Line; Cell Survival; Cisplatin; Etoposide; Glycoproteins; Humans; Lectins; Leupeptins; Methotrexate; Pharmaceutical Vehicles; Receptors, Cell Surface; Teratoma | 1987 |
Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.
Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns. Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Brain; Carcinoma, Hepatocellular; Cattle; Cell Line; Endopeptidases; Fibroblast Growth Factors; Humans; Leupeptins; Liver Neoplasms; Molecular Weight; Protease Inhibitors | 1987 |
Sendai-viral HN and F glycoproteins as probes of plasma-membrane protein catabolism in HTC cells. Studies with fusogenic reconstituted Sendai-viral envelopes.
Reconstituted Sendai-viral envelopes (RSVE) were produced by the method of Vainstein, Hershkovitz, Israel & Loyter [(1984) Biochim. Biophys. Acta 773, 181-188]. RSVE are fusogenic unilamellar vesicles containing two transmembrane glycoproteins: the HN (haemagglutinin-neuraminidase) protein and the F (fusion) factor. The fate of the viral proteins after fusion-mediated transplantation of RSVE into hepatoma (HTC) cell plasma membranes was studied to probe plasma-membrane protein degradation. Both protein species are degraded at similar, relatively slow, rates (t1/2 = 67 h) in HTC cells fused with RSVE in suspension. Even slower degradation rates for HN and F proteins (t1/2 = 93 h) were measured when RSVE were fused with HTC cells in monolayer. Lysosomal degradation of the transplanted viral proteins is strongly implicated by the finding that degradation of HN and F proteins is sensitive to inhibition by 10 mM-NH4Cl (81%) and by 50 micrograms of leupeptin/ml (70%). Topics: Ammonium Chloride; Animals; Azides; Cell Line; Deoxyglucose; DNA, Viral; Hemagglutinins, Viral; HN Protein; Leupeptins; Liver Neoplasms, Experimental; Membrane Fusion; Membrane Proteins; Parainfluenza Virus 1, Human; Sodium Azide; Viral Envelope Proteins; Viral Fusion Proteins | 1987 |
Protein damage and degradation by oxygen radicals. I. general aspects.
Aggregation, fragmentation, amino acid modification, and proteolytic susceptibility have been studied following exposure of 17 proteins to oxygen radicals. The hydroxyl radical (.OH) produced covalently bound protein aggregates, but few or no fragmentation products. Extensive changes in net electrical charge (both + and -) were observed. Tryptophan was rapidly lost with .OH exposure, and significant production of bityrosine biphenol occurred. When incubated with cell-free extracts of human and rabbit erythrocytes, rabbit reticulocytes, or Escherichia coli, most .OH-modified proteins were proteolytically degraded up to 50 times faster than untreated proteins. The exceptions were alpha-casein and globin, which were rapidly degraded without .OH modification. ATP did not stimulate the degradation of .OH-modified proteins, but alpha-casein was more rapidly degraded. Leupeptin had little effect under any condition, and degradation was maximal at pH 7.8. The data indicate that proteins which have been denatured by .OH can be recognized and degraded rapidly and selectively by intracellular proteolytic systems. In both red blood cells and E. coli, the degradation appears to be conducted by soluble, ATP-independent (nonlysosomal) proteolytic enzymes. In contrast with the above results, superoxide (O2-) did not cause aggregation or fragmentation, tryptophan loss, or bityrosine production. The combination of .OH + O2- (+O2), which may mimic biological exposure to oxygen radicals, induced charge changes, tryptophan loss, and bityrosine production. The pattern of such changes was similar to that seen with .OH alone, although the extent was generally less severe. In contrast with .OH alone, however, .OH + O2- (+O2) caused extensive protein fragmentation and little or no aggregation. More than 98% of the protein fragments had molecular weights greater than 5000 and formed clusters of ionic and hydrophobic bonds which could be dispersed by denaturing agents. The results indicate a general sensitivity of proteins to oxygen radicals. Oxidative modification can involve direct fragmentation or may provide denatured substrates for intracellular proteolysis. Topics: Adenosine Triphosphate; Free Radicals; Hydroxides; Hydroxyl Radical; Leupeptins; Proteins; Superoxides | 1987 |
Effects of low calcium and inhibition of calcium-activated neutral protease (CANP) on mature nerve terminal structure in the rat sternocostalis muscle.
The possible role of calcium and a calcium-activated neutral protease (CANP) in the reorganisation of mature mammalian neuromuscular junctions was studied in the sternocostalis muscle in rats. After the well-documented loss of polyneuronal innervation has occurred, the remaining single mature nerve ending continues to change its terminal branching pattern by gradually becoming more complex. Reducing local calcium concentrations by the chelating agent BAPTA or inhibiting CANP by local application of an inhibitor, Leupeptin, resulted in the endings becoming more complex in appearance when examined after 6 or 7 days. It is concluded that calcium and CANP are important in the remodelling of mature neuromuscular junctions. Topics: Animals; Calcium; Calpain; Egtazic Acid; Leupeptins; Motor Neurons; Neuromuscular Junction; Neuronal Plasticity; Rats; Rats, Inbred Strains | 1987 |
Suppression of neurofilament degradation by protease inhibitors in experimental spinal cord injury.
Intraperitoneal administration of the neutral protease inhibitors leupeptin and E-64c substantially suppressed the degradation of neurofilament proteins (NFP) at the site of mechanical insult and secondary axonal degeneration, and facilitated the recovery of motor functions in acute spinal cord injury in rats. The drug effects were assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of NFP fractions from the injured tissue and by morphometry of degenerating axons revealed by the Fink-Heimer method in distal spinal cord segments with the aid of an automated image analyzer. The role of calcium-activated neutral proteases in acute central nervous tissue damage and potential use of protease inhibitors as therapeutic modalities are discussed. Topics: Animals; Electrophoresis, Polyacrylamide Gel; Intermediate Filament Proteins; Leucine; Leupeptins; Male; Nerve Tissue Proteins; Neurofilament Proteins; Oligopeptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries | 1987 |
Partial purification and characterization of cysteine proteinases in eccrine sweat.
Attempts were made to purify and characterize cysteine proteinases in human eccrine sweat and further clarify their origin. Benzoyl-DL-arginine-beta-naphthylamide (BANA) and L-leucine beta-naphthylamide (LeuNA) hydrolases in thermally induced sweat were sequentially purified by Sephacryl S-200 chromatography and chromatofocusing, which yielded two major peaks of BANA hydrolase activity, BANA-I and BANA-II. Both enzymes are cysteine proteinases as evidenced by stimulation of enzymic activity by dithiothreitol and ethylenediaminetetraacetic acid and its inhibition by iodoacetic acid, (PCMB), and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane (E-64). Unlike BANA-II, BANA-I showed an additional aminopeptidase activity, an affinity to concanavalin A-Sepharose but no affinity to organomercurial sepharose and failed to hydrolyze benzyloxycarbonyl-phenylalanyl-arginine 4-methyl 7-coumarylamide (Z-Phe-Arg-NMec), a specific substrate for cathepsin B, which is poorly sensitive to leupeptin [inhibitor constant (Ki) = 1 X 10(-5) M] and relatively heat resistant. These and other characteristics such as its isoelectric points (PI) (= 5.8) and the Km for Arg-NMec (0.1 mM) and BANA (0.71 mM) all support the possibility that BANA-I is closely related to cathepsin H. In contrast, BANA-II is sensitive to Zn2+, leupeptin (Ki = 5.5 X 10(-9) M), is not adsorbed by concanavalin A- (Con-A)Sepharose, but is bound to organomercurial sepharose. It has a specificity to Z-Phe-Arg-NMec but not to Arg-NMec, has the molecular weight of 27, PI of 5.2, the pH optima for BANA (6.0), and the Km for BANA of 3.3 mM and the Km for Z-Phe-Arg-NMec of 0.1 mM. These features resemble those of liver cathepsin B. Leupeptin-sensitive BANA hydrolase was observed in the glandular extract of isolated sweat glands, which was increased after stimulation with methacholine and isoproterenol in vitro. The data are consistent with the notion that cathepsins B- and H-like enzymes are present in eccrine sweat and the former may be derived from the sweat gland. Topics: Benzoylarginine-2-Naphthylamide; Chromatography, Affinity; Chromatography, Gel; Cysteine Endopeptidases; Dithiothreitol; Eccrine Glands; Edetic Acid; Endopeptidases; Hydrogen-Ion Concentration; Isoelectric Focusing; Isoenzymes; Leucine; Leupeptins; Molecular Weight; Sweat; Sweat Glands | 1987 |
Biosynthesis and maturation of lactase-phlorizin hydrolase in the human small intestinal epithelial cells.
The biosynthesis and maturation of the human intestinal lactase-phlorizin hydrolase (LPH; EC 3.2.1.23-3.2.1.62) has been studied in cultured intestinal biopsies and mucosal explants. Short time pulse labelling revealed on high mannose intermediate of Mr 215,000 which was converted upon endo-beta-N-acetylglucosaminidase H (endo-H) digestion to a polypeptide of Mr 200,000. The brush border form of LPH was revealed after longer pulse periods and has Mr 160,000. It possesses mainly complex oligosaccharide chains and, owing to its partial endo-H sensitivity, at least one chain of the high mannose type. Leupeptin partially inhibited the appearance of the Mr-160,000 polypeptide. Monensin treatment of biopsies resulted in the modification of the Mr-160,000 species to the Mr-140,000 molecule, which was endo-H sensitive. Pulse-chase analysis indicated a slow post-translational processing of the high mannose precursor (Mr 215,000) to yield the mature brush-border form (Mr 160,000) of LPH. Our results further indicate that LPH is synthesized as a single polypeptide precursor which is intracellularly cleaved to yield the mature brush border of LPH. The data presented suggest that this cleavage occurs during the translocation of the molecule across the Golgi complex. Topics: Antibodies, Monoclonal; beta-Galactosidase; Chemical Precipitation; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Epithelium; Galactosidases; Glucosidases; Humans; Intestine, Small; Isoenzymes; Lactase-Phlorizin Hydrolase; Leupeptins; Monensin; Multienzyme Complexes; Protein Biosynthesis; Tunicamycin | 1987 |
A possible role for dephosphorylation in glucocorticoid receptor transformation.
Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation. Topics: Alkaline Phosphatase; Animals; Cell Line; Cytosol; Dexamethasone; Edetic Acid; Leupeptins; Mice; Phosphorylation; Pituitary Neoplasms; Protease Inhibitors; Receptors, Glucocorticoid; Sodium Fluoride | 1987 |
Inhibitory effect of a protease inhibitor, leupeptin, on the development of influenza pneumonia, mediated by concomitant bacteria.
The protease inhibitor leupeptin prevented multiple step replication of an influenza virus (A/swine/1976/31, H1N1) mediated by staphylococcal proteases. It also suppressed virus replication and development of fatal pneumonia in mice co-infected with the virus and Staphylococcus aureus. Topics: Animals; Endopeptidases; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral; Influenza A virus; Leupeptins; Metalloendopeptidases; Mice; Oligopeptides; Orthomyxoviridae Infections; Pneumonia, Staphylococcal; Protease Inhibitors; Protein Processing, Post-Translational; Staphylococcus aureus; Virus Replication | 1987 |
Pharmacological protection against the toxicity of N-methyl-D-aspartate in immature rat cerebellar slices.
In order to delineate the pharmacological characteristics of the toxicity of N-methyl-D-aspartate (NMDA), slices of cerebellum from 7-day old rats were incubated with NMDA, together with various putative protective agents. These comprised three different groups: (i) a competitive receptor antagonist (kynurenic acid), (ii) direct (cobalt ions, flunarizine) and indirect (taurine) calcium entry blockers, (iii) cyclo-oxygenase inhibitors (indomethacin and acetylsalicylic acid) and a blocker of calcium-activated, neutral proteases (leupeptin). When the slices were incubated for 30 min in medium containing 100 microM NMDA, postmigratory granule cell nuclei were rounded and swollen. After 90 min of recovery in normal medium, the nuclei were pyknotic and the cells were irreversibly injured. As expected, these changes were completely blocked by kynurenate, indicating that NMDA receptors mediate the cell death. Cobalt ions abolished the acute toxicity of NMDA, but after recovery, some granule cell nuclei were swollen. This effect could be attributed to the toxicity of cobalt ions and not to delayed toxicity of NMDA. The other inhibitors of the uptake of calcium, flunarizine and taurine, did neither affect acute nor persistent toxicity of NMDA. These results support the previous finding that the toxicity of NMDA is calcium-dependent and that organic calcium channel blockers are ineffective against NMDA-induced uptake of calcium. Leupeptin had no effect on the toxicity of NMDA, suggesting that calcium-activated proteolysis was not the crucial event in excitotoxic necrosis. Indomethacin, but not acetylsalicylic acid, prevented neuronal degeneration provoked by NMDA, but only in very large concentrations (greater than or equal to 100 microM).(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Aspartic Acid; Aspirin; Calcium Channel Blockers; Cerebellum; Female; In Vitro Techniques; Indomethacin; Kynurenic Acid; Leupeptins; Male; N-Methylaspartate; Rats; Rats, Inbred Strains; Taurine | 1987 |
Treatment of experimental autoimmune myasthenia gravis in rabbits with leupeptin, a protease inhibitor.
We injected 12 New Zealand white rabbits intraperitoneally with 15 mg/kg Leupeptin on alternative days for about 4 months. After 1 week of Leupeptin treatment, they were challenged with purified acetylcholine receptor (AChR) from Torpedo californica in Freund's complete adjuvant. All control animals died within 60 days. Six animals treated with Leupeptin did not develop EAMG in spite of repeated AChR injections. Three animals developed clinical signs of EAMG after 65 days. The clinical course was short in the one that survived and prolonged in the 2 that finally died. All animals (Leupeptin-treated and controls) had circulating anti-AChR antibodies. Among the survivors, titers were slightly lower and EMG repetitive stimulation tests were normal. Leupeptin (0.02-200 mM) did not prevent curaremimetic [3H]toxin binding to AChR in membranes or in solution, nor dissociate AChR-toxin-antibody complexes. Immune response to antigens other than receptor remained intact in Leupeptin-treated animals. Leupeptin was not toxic at the doses given. The mechanism of this protection is not well understood. Leupeptin seems to decelerate the turnover rate of AChR induced by anti-AChR antibodies and/or to decrease the complement-mediated immune attack against the muscle end-plate. Topics: Animals; Antibodies; Autoimmune Diseases; Leupeptins; Male; Myasthenia Gravis; Oligopeptides; Rabbits; Receptors, Cholinergic | 1987 |
Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate.
Stimulation of intact human neutrophils with phorbol 12-myristate 13-acetate results in the selective phosphorylation of two cytoskeletal protein components with molecular masses of 20 and 48 kDa. After phosphorylation the 48-kDa protein is no longer recovered as a component of the cytoskeletal fraction but is present as a fully soluble phosphoprotein. Phosphorylation of the 20-kDa protein (probably myosin light chains) signals a proteolytic conversion, catalyzed by calpain, to a smaller species having a molecular mass of approximately 15 kDa. Phosphorylation of both the 48- and 20-kDa proteins is related to the conversion of protein kinase C, also catalyzed by calpain, to the soluble fully active form. Leupeptin, an inhibitor of calpain, blocks both the phosphorylation of the target proteins and the proteolytic modification of the 20-kDa polypeptide. Thus, phosphorylation of cytoskeletal proteins and signal-directed proteolysis appear to be related processes that follow stimulation of human neutrophils by phorbol esters. The resulting changes in cytoskeletal organization may be involved in the expression of some neutrophil functions, such as exocytosis of specific granules. Topics: Cytoskeletal Proteins; Humans; Leupeptins; Molecular Weight; Neutrophils; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate | 1987 |
Protein kinase C activity and hexamethylenebisacetamide-induced erythroleukemia cell differentiation.
Hexamethylenebisacetamide (HMBA) is a potent inducer of murine erythroleukemia (MEL) cell differentiation. The mechanism of action of HMBA is not known. In this study we provide evidence that protein kinase C has a role in inducer-mediated MEL cell differentiation: (i) HMBA induces the formation of a soluble, proteolytically activated form of protein kinase C that is catalytically active in the absence of Ca2+ and phospholipid; (ii) the protease inhibitor leupeptin blocks formation of this activated form of the kinase and inhibits HMBA-induced MEL cell hemoglobin accumulation; (iii) phorbol 12-myristate 13-acetate (PMA) inhibits HMBA-induced MEL differentiation and causes depletion of total protein kinase C activity; (iv) MEL cells depleted in protein kinase C activity by culture with PMA are resistant to induction by HMBA; (v) upon removal of PMA, restoration of MEL cell sensitivity to HMBA is correlated with reaccumulation of protein kinase C activity; and (vi) MEL cells grown to density arrest are both depleted of protein kinase C activity and resistant to HMBA. Together, these results suggest that HMBA-mediated MEL cell differentiation involves a protein kinase C-related mechanism and the proteolytically activated form of the kinase, which does not require Ca2+ or phospholipid for its catalytic activity. Topics: Acetamides; Animals; Calcium; Cell Differentiation; Cells, Cultured; Hemoglobins; Leukemia, Erythroblastic, Acute; Leupeptins; Mice; Phospholipids; Protein Kinase C; Tetradecanoylphorbol Acetate | 1987 |
Hepatic processing of transforming growth factor beta in the rat. Uptake, metabolism, and biliary excretion.
Transforming growth factor beta (TGF beta), a recently discovered polypeptide, modulates growth of normal and neoplastic cells. Since little is known concerning in vivo disposition of TGF beta, we performed studies to examine the hepatic processing of biologically active 125I-TGF beta in the rat. After intravenous injection, 125I-TGF beta disappeared from the plasma with an initial t1/2 of 2.2 min; partial hepatectomy delayed the plasma disappearance of 125I-TGF beta by 80%. 60 min after intrafemoral injection, 63% of the recovered label was present in liver and/or bile; by 90 min, most of the label removed by the liver (83%) had been slowly excreted into bile. Nearly all the label in bile (96%) was soluble in trichloracetic acid and not immunoprecipitable by specific antiserum. Colchicine and vinblastine inhibited cumulative biliary excretion of label by 28 and 37%, respectively; chloroquine and leupeptin each increased the amount of label in bile that was precipitable by trichloracetic acid and that coeluted with authentic 125I-TGF beta on molecular sieve chromatography. There was efficient first-pass hepatic extraction of 125I-TGF beta (36%) in the isolated perfused rat liver, which was inhibited by unlabeled TGF beta (but not by epidermal growth factor, EGF) and by lectins in a dose-dependent manner; prolonged fasting also decreased clearance (26%). After fractionation of liver by differential or isopycnic centrifugation, radiolabel codistributed with marker enzymes for lysosomes. The results indicate rapid, extensive, inhibitable, and organ-selective extraction of TGF beta by the liver. After extraction, TGF beta undergoes efficient transhepatic transport, extensive intracellular metabolism, and slow but complete biliary excretion of its metabolites. Liver fractionation studies and pharmacologic manipulations suggest that these processes are associated with organelles that include microtubules and lysosomes. The data suggest that the liver is a major target tissue or site of metabolism for biologically active TGF beta. Topics: Animals; Bile; Chloroquine; Leupeptins; Liver; Male; Organoids; Peptides; Rats; Transforming Growth Factors | 1987 |
Inhibition of H-ras oncogene transformation of NIH3T3 cells by protease inhibitors.
The protease inhibitors antipain, leupeptin, alpha 1-antitrypsin, and epsilon-aminocaproic acid were found to inhibit transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. Inhibition of focus formation by protease inhibitors was concentration dependent and maximal at 50% of control values. Transfection of a gene for neomycin resistance was not affected by protease inhibitors. Antipain was inactive if present only during the first 2 days of the gene transfer protocol or only during the final 10 days of the experiment. However, the full effect was observed when antipain was added at the subculture step on day 3 and during the subsequent cell proliferation. If cells were not subcultured, the yield of the foci per microgram of DNA was sharply reduced and addition of antipain did not further suppress the transformation rate. Subculture of NIH3T3 cells 3 days after transfection at lower cell densities resulted in higher transformation efficiency. The results suggest that transformation of NIH3T3 cells by a single mutated oncogene may involve multiple stages including cell proliferation and that part of this process is susceptible to inhibition by protease inhibitors. Topics: alpha 1-Antitrypsin; Aminocaproic Acid; Animals; Antipain; Cell Division; Cell Line; Drug Resistance; Leupeptins; Neomycin; Oncogenes; Plasmids; Protease Inhibitors; Transfection | 1987 |
Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody.
In studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of 131I-labeled aMM46 and aMM46-3H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action. Topics: Animals; Antibodies, Monoclonal; Cell Line; Immunotoxins; Leupeptins; Mammary Neoplasms, Experimental; Methotrexate; Mice; Mice, Inbred C3H | 1987 |
Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts.
The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase. Topics: Cell Line; Cross Reactions; Fibroblasts; Gaucher Disease; Glucosidases; Glucosylceramidase; Humans; Immunosorbent Techniques; Leupeptins; Methionine; Molecular Weight; Mutation | 1987 |
Leukocyte protease activities in myotonic dystrophy: studies on effects of protease inhibitors.
Neutral and acid protease activities inhibited by chymostatin, leupeptin, pepstatin and HgCl2 in mononuclear cells and granulocytes showed no significant differences between myotonic dystrophy patients and controls. These results suggest that chymotrypsin and cathepsin B and D activities are probably normal in leukocytes in myotonic dystrophy. Topics: Aspartic Acid Endopeptidases; Endopeptidases; Granulocytes; Humans; Leukocytes; Leupeptins; Mercuric Chloride; Monocytes; Myotonic Dystrophy; Neprilysin; Oligopeptides; Pepstatins; Protease Inhibitors | 1987 |
Significant effects of Z-Gln-Val-Val-OME, common sequences of thiol proteinase inhibitors on thiol proteinases.
The first studies on a series of the small synthetic thiol proteinase inhibitors, conservative common sequences in several thiol proteinase inhibitors, are described. Among the many interesting findings with synthetic thiol proteinase inhibitors was the observation that the most effective analogue, Z-Gln-Val-Val-Ala-Gly-OMe, whose amino and carboxyl groups were protected with Z and OMe, respectively, showed inhibitory activity on papain and cathepsin B and protected papain from egg cystatin, human low-molecular-weight kininogen and T-kininogen-induced inhibition but not from leupeptin-induced inhibition. Moreover, it was revealed that Z-Gln-Val-Val-OMe was the smallest peptide to exhibit a protective effect on papain. Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Cathepsin B; Cystatins; Kininogens; Leupeptins; Oligopeptides; Papain; Protease Inhibitors; Proteins; Structure-Activity Relationship | 1987 |
A tripeptide protease inhibitor attenuates conditioned avoidance behavior.
In a single 10-s training trial, hatchling chicks were conditioned to suppress their spontaneous peck response to a small spherical target by coating it with an aversive liquid. A 24-h test trial employing a dry target demonstrated a robust memory for the training manifested in passive avoidance behavior. Leupeptin, a low-molecular-weight antiprotease, injected i.c.v. 1 h or 15 min prior to training attenuated the long-term avoidance response but not the initial training-induced peck suppression. Leupeptin had no effect on memory if given posttraining. A dose response experiment revealed that a 100- or 200-micrograms dose of leupeptin reliably impaired conditioned avoidance, whereas a 25 or 50-micrograms dose was ineffective. A dipeptide leupeptin analog (L-leucyl-L-arginine) possessed one-half the potency of the tripeptide in the memory task, and a different protease inhibitor (aprotinin) failed to effect the conditioned avoidance behavior. The mechanisms by which leupeptin may exert its influence in this behavioral paradigm and others are discussed. Topics: Animals; Avoidance Learning; Conditioning, Classical; Depression, Chemical; Injections, Intraventricular; Leupeptins; Male; Memory; Oligopeptides; Protease Inhibitors; Time Factors | 1987 |
A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.
Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy. Topics: Autophagy; Binding Sites; Cell Fractionation; Cell Line; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Glyceraldehyde-3-Phosphate Dehydrogenases; Intermediate Filaments; L-Lactate Dehydrogenase; Leupeptins; Microinjections; Phagocytosis; Proteins; Pyruvate Kinase; Serum Albumin, Bovine | 1987 |
Mode of action of bestatin and leupeptin to induce the accumulation of acid soluble peptides in rat liver in vivo and the properties of the accumulated peptides. The important role of bestatin- and leupeptin-sensitive proteases in the protein degradation
The chemical properties of acid soluble peptides accumulated in liver or excreted into urine after administration of bestatin or leupeptin to rats were investigated extensively. At the same time, the effects of glucagon on the bestatin-induced accumulation of acid soluble peptides were studied. The results show the important role of bestatin- and leupeptin-sensitive proteases in the degradation pathway of intracellular proteins in vivo. Topics: Aminopeptidases; Animals; Glucagon; Kinetics; Leucine; Leupeptins; Liver; Male; Models, Biological; Oligopeptides; Peptide Mapping; Peptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Solubility | 1987 |
The role of microtubules in the phagocytosis of collagen by fibroblasts.
The effects of the anti-microtubular agents, colchicine and vinblastine, on the phagocytosis of collagen by fibroblasts were assessed quantitatively in cultured mouse bone explants. It was found that in the absence of the microtubular system the volume density of lysosomal vacuoles containing cross-banded collagen fibrils in periosteal cells did not differ from that seen in controls. In contrast, cytochalasin B which interferes with the microfilament system prevented the accumulation of collagen-containing vacuoles in the cytoplasm. The data indicate that the phagocytosis of collagen fibrils by fibroblasts does not depend on the integrity of the microtubular apparatus, but seems to require an intact microfilament system. Topics: Animals; Bone and Bones; Cells, Cultured; Colchicine; Collagen; Cytochalasin B; Cytoplasmic Granules; Fibroblasts; Leupeptins; Mice; Microscopy, Electron; Microtubules; Phagocytosis; Vacuoles; Vinblastine | 1987 |
[Effect of leupeptin on protein uptake and the degradation of the visceral yolk sac epithelium of the rat].
Topics: Animals; Epithelium; Female; Horseradish Peroxidase; Leupeptins; Oligopeptides; Pregnancy; Proteins; Rats; Rats, Inbred Strains; Yolk Sac | 1987 |
Sperm chymotrypsin-like enzymes of different inhibitor-susceptibility as lysins in ascidians.
Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors. Topics: Animals; Anti-Bacterial Agents; Chymotrypsin; Enzyme Inhibitors; Fertilization; Leupeptins; Male; Oligopeptides; Peptides; Spermatozoa; Urochordata | 1987 |
Enhancement of neuromuscular recovery after nerve repair in primates.
This investigation describes the use of the calcium-activated protease inhibitor, leupeptin, as an adjunctive therapy to the microsurgical repair of median nerves in a primate model. Our results indicate that leupeptin facilitates morphological recovery in denervated thenar muscles and in distal sensory and mixed motor-sensory nerve trunks and functional recovery measured by motor nerve conduction velocity. Toxicological testing of leupeptin showed that, when administered at a dose of 12 mg/kg, intramuscularly, once daily, haematological and clotting profiles were not adversely affected. Topics: Animals; Axons; Blood Coagulation; Cebus; Leupeptins; Median Nerve; Muscles; Myelin Sheath; Nerve Regeneration; Neural Conduction; Oligopeptides | 1987 |
Leupeptin inhibits the transformation of glucocorticoid receptor.
The effect of leupeptin upon the transformation of the glucocorticoid receptor was tested. When the labeled receptor was treated with heat or high salt in the presence of leupeptin, the binding to DNA-cellulose decreased in a dose-dependent manner. We observed 50% inhibition with about 40 mM leupeptin. The addition of leupeptin after the transformation procedures did not inhibit the binding to DNA-cellulose. In gradient centrifugation, 40 mM leupeptin retained approximately 10S, untransformed form. Elution profiles from DEAE-cellulose showed the preservation of the peak eluted with 0.2 M KCl, corresponding to the untransformed form. These results indicate that leupeptin might have the similar effects to molybdate in regard to blocking the transformation of rat liver glucocorticoid receptor, though the effects with leupeptin were not as great as those seen with molybdate. Topics: Adrenalectomy; Animals; Cellulose; Centrifugation, Density Gradient; Chromatography, Affinity; Cytosol; DNA; Kinetics; Leupeptins; Liver; Male; Molybdenum; Oligopeptides; Rats; Rats, Inbred Strains; Receptors, Glucocorticoid; Triamcinolone Acetonide | 1987 |
Ascorbic acid inhibits lysosomal autophagy of ferritin.
Ascorbic acid retards ferritin degradation in K562 erythroleukemia cells leading to an increase in the availability of cellular iron (Bridges, K. R., and Hoffman, K. E. (1986) J. Biol. Chem. 261, 14273-14277). To explore the mechanism of this effect, the influence of ascorbate on subcellular ferritin distribution was examined. Cellular ferritin was pulse-labeled with 59Fe for 2 h, after which the cells were hypotonically lysed and fractionated on an 8% Percoll density gradient. Immediately after the labeling, all of the ferritin was in the cytoplasmic fractions at the top of the gradient. When the labeling was followed by a 24-h period of growth, a portion of the ferritin shifted to the lysosome-associated fractions at the bottom of the gradient, consistent with lysosomal autophagy of cytoplasmic ferritin. When ascorbate was added to the culture medium during the 24-h incubation, the magnitude of the shift was reduced. This process was also examined by size-fractionation of the contents of labeled cells using a Sepharose CL-6B column. Immediately after labeling, ferritin emerged from the column in two peaks, indicating the existence of both ferritin monomer and aggregates within the cytoplasm. After a 24-h period of growth, the monomer peak disappeared, while a new ferritin peak coincident with lysosomes emerged again, indicative of lysosomal autophagy of ferritin. In cells cultured with ascorbate for 24-h, there was a marked attenuation of the shift of ferritin to the lysosomal fractions. The monomer peak disappeared, as in the controls, but there was instead, an accumulation of ferritin as cytoplasmic aggregates. The total ferritin content of the ascorbate-treated cells was increased by 4-fold over that of the control. These experiments indicate that ascorbate blocks the degradation of cytoplasmic ferritin by reducing lysosomal autophagy of the protein. The access to the cell of the potentially toxic iron stored within the ferritin molecule is thereby increased. Topics: Ascorbic Acid; Cell Line; Cytoplasm; Ferritins; Glucuronidase; Iron; Leupeptins; Lysosomes; Proteins | 1987 |
Effect of leupeptin on platelet aggregation, fibrin formation and amidolysis induced by thrombin.
Topics: Blood Platelets; Dipeptides; Fibrin; Humans; In Vitro Techniques; Leupeptins; Oligopeptides; Platelet Aggregation; Thrombin | 1987 |
Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity--formation of crinosomes.
The number of rat liver autophagic vacuoles (AVs) was increased by separate injection of three different inhibitors--vinblastine, leupeptin, and chloroquine--of lysosomal protein degradation. The different mechanisms of action of the agents correlated to the ultrastructure of the AVs. Accumulation of the base chloroquine with ensuing influx of water into AVs caused a significant swelling. The leupeptin-induced AVs were processed into residual-body-like structures within a few hours of exposure in line with the presence of a leupeptinase in liver tissue. Vinblastine was the most efficient agent in increasing the occurrence of AVs. The effect of vinblastine lasted for the entire study period (36 hr) with continuous formation of nascent AVs. In addition, vinblastine caused the appearance of a subpopulation of AVs laden with VLDL particles. The term crinosomes was suggested for these hybrid organelles, since they seemed to evolve by fusion between secretory granules and lysosomes. In addition to sequestered cell organelles, the AVs harbored cytosolic enzyme activities (LDH and aldolase). Leupeptin was the only agent that caused a decrease in cathepsin B and L activities. Similarly, leupeptin impeded protein breakdown in isolated AVs, whereas vinblastine and chloroquine evoked an increase. In vivo, chloroquine and vinblastine block protein degradation. The reason for this discrepancy is probably that during in vivo exposure the substrate (cytoplasmic proteins) is built up in the AVs because degradation is retarded. Upon isolation of the AVs the inhibitor block is released, and proteolysis proceeds at enhanced rates over control due to excess of substrates. Leupeptin, on the other hand, caused a substantial inhibition of thiol proteinases; this block remained in the isolated AVs. Accordingly, leupeptin-induced AVs displayed decreased protein degradation following shorter exposure times. Later, when leupeptin was metabolized, catch-up proteolysis was noted. The differing mechanisms of action of the inhibitors were also apparent as regards lipid contents and lipolysis. Whereas chloroquine and vinblastine increased the amounts of cholesterol and triglycerides parallel to proteins, leupeptin had no such effect. Lipolysis proceeded at normal rate following leupeptin administration, which was not the case after vinblastine and chloroquine exposure. Leupeptin has no effect on acid lipases; therefore lipids do not accumulate in AVs of hepatocytes that are exposed to le Topics: Animals; Autophagy; Chloroquine; Leupeptins; Lipid Metabolism; Liver; Lysosomes; Microscopy, Electron; Phagocytosis; Proteins; Rats; Vacuoles; Vinblastine | 1987 |
Influence of a calcium dependent protease inhibitor on platelet activation and secretion.
Continuous proteolysis resulting in consumption of major cytoskeletal proteins may be essential for platelet activation and aggregation. In this study we evaluated the effect of a known protease inhibitor, Leupeptin, on agonist induced platelet aggregation and secretion. Platelets exposed to 10 ugs/ml of Leupeptin did not aggregate in response to the action of thrombin (0.2 u/ml). However, a concentration of Leupeptin as high as 250 ugs/ml did not prevent arachidonate induced aggregation and secretion. Leupeptin (100 ugs/ml) effectively blocked thrombin (0.2 u/ml) induced elevation of cytosolic calcium, but did not affect arachidonate induced elevation of platelet intracellular calcium levels. At a concentration of 100 ug/ml, Leupeptin effectively blocked thrombin (0.5 u/ml) induced clot formation of platelet poor plasma, suggesting that it can exert its effect on thrombin by preventing fibrinogen degradation. Effective Ki for the competitive inhibition of thrombin induced hydrolysis of a chromogenic substrate, S2238, by Leupeptin was 2.4 uM. Leupeptin inhibition of platelet function was reversible by washing platelets free of the polypeptide. Results of our study demonstrate that Leupeptin inhibits thrombin induced platelet activation, probably by interfering with its proteolytic activity on the platelet surface membrane. However, inhibition of platelet surface membrane associated proteases did not prevent activation of platelets by other agonists. Topics: Blood Coagulation; Blood Platelets; Calcium; Cytosol; Dipeptides; Glycoproteins; Humans; Hydrolysis; Leupeptins; Oligopeptides; Platelet Aggregation; Serotonin; Therapeutic Irrigation; Thrombin | 1987 |
In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody.
In studies on antitumor antibody:drug conjugates as potential antitumor agents, methotrexate (MTX) was conjugated with a murine monoclonal antibody (aMM46) to an antigen on ascitic mouse mammary tumor MM46 cells (MM antigen) with human serum albumin (HSA) as an intermediary. MTX was linked to HSA which had been conditioned to have about 1 mol of thiol group per mol of HSA by dithiothreitol treatment followed by oxidation on standing at 4 degrees C. The MTX linking was performed, without protection of the thiol group of HSA, by using MTX N-succinimidyl ester prepared via MTX intramolecular anhydride. The resulting HSA:MTX was reacted with the immunoglobulin with the maleimide group introduced. The aMM46:HSA:MTX obtained retained both antibody binding and drug activities. The cytotoxicity of aMM46:HSA:MTX against MM antigen-positive MM46 cells was greater than that of control 96.5 (anti-human melanoma-associated antigen, p97):HSA:MTX and was inhibited by unconjugated aMM46. No different cytotoxicity of aMM46:HSA:MTX compared with that of 96.5:HSA:MTX was observed against MM antigen-negative mouse mammary tumor MM48 cells. The presence of ammonium chloride or leupeptin abrogated the selective cytotoxicity against MM46 cells of aMM46 conjugate but did not affect the nonspecific cytotoxicity of 96.5:HSA:MTX. These results support the idea that the selective cytotoxicity of aMM46:HSA:MTX is antibody directed and exhibited through lysosomal degradation of the conjugate. Topics: Ammonium Chloride; Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Line; Chemical Phenomena; Chemistry, Physical; Cytotoxicity, Immunologic; Leupeptins; Methotrexate; Mice; Mice, Inbred C3H; Serum Albumin | 1987 |
On the fates of receptor-bound ovine luteinizing hormone and human chorionic gonadotropin in cultured Leydig tumor cells. Demonstration of similar rates of internalization.
Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the fate of the receptor-bound ovine LH (oLH) and human CG (hCG) in cells incubated in the presence or absence of extracellular Na+. We have previously shown that Na+ does not affect the number of LH/CG receptors or the binding affinity of hCG, but it decreases the binding affinity of oLH. Thus, it was possible to compare the fate of these two hormones under conditions where their binding affinities differ by a factor of 8 (i.e. in the presence of Na+) or by a factor of less than 2 (i.e. in the absence of Na+). Moreover, since only the affinity of oLH is affected by Na+, we were able to distinguish between those effects mediated by a change in binding affinity from those effects that are more general in nature by comparing the behavior of hCG in cells incubated in the presence or absence of Na+. The results presented herein show that the rates of internalization of oLH and hCG are very similar regardless of the presence or absence of Na+; and the absence of Na+ leads to a 2- to 3-fold decrease in the rate of degradation of the internalized oLH and hCG. We have found, however, that the binding affinities of oLH and hCG have significant effects on the pathway of receptor-mediated endocytosis under conditions where there is no free hormone present in the medium. The results presented show that in the absence of free hormone in the medium, the rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment (i.e. hCG in the presence or absence of Na+, but oLH only in the absence of Na+). If the binding affinity of the hormone is low (i.e. oLH in the presence of Na+), then the rate of disappearance of the surface-bound hormone represents the sum of the rates of internalization and dissociation of the hormone and thus cannot be used to approximate the rate of hormone internalization. Topics: Ammonium Chloride; Animals; Biological Transport; Chorionic Gonadotropin; Endocytosis; Kinetics; Leupeptins; Leydig Cell Tumor; Luteinizing Hormone; Male; Mice; Receptors, LH; Sheep; Sodium | 1987 |
Degradation of short- and long-lived proteins in perfused liver and in isolated autophagic vacuoles--lysosomes.
The lysosomal contribution to breakdown of prelabeled short- and long-lived cell proteins in the perfused rat liver was monitored in two ways. In the first, either leupeptin or chloroquine was injected into rats as well as added to the perfusate. The extent of suppression of proteolysis compared to that of controls was equated with the lysosomal share of protein breakdown. Both compounds inhibited degradation of short-lived proteins by some 30% and long-lived proteins by some 60%. In the second approach, lysosomal-autophagic vacuolar (LAV) fractions were isolated from livers pretreated and perfused as above. The LAV fractions generated different amounts of degradation products during subsequent incubations. They were, in decreasing order of magnitude, that of the LAV fraction isolated from livers perfused with chloroquine, without inhibitor, and with leupeptin. The LAV fraction from control livers was considered to yield the most reliable estimate of the lysosomal share of proteolysis as recorded during liver perfusions. It was 54% for short-lived proteins and 75% for long-lived proteins. These figures are higher than those obtained in the perfusion experiments in the first approach. It is therefore suggested that quantitative assessment concerning the lysosomal share of overall proteolysis may be underestimated when based on inhibition experiments performed on intact cells. The ultrastructure of hepatocytes and LAV fractions was also investigated. Leupeptin-treated hepatocytes displayed more frequent and larger AVs than those of control livers. Cell vacuolation was an additional characteristic of chloroquine-exposed hepatocytes. The LAV fractions isolated from control and leupeptin-treated livers mirrored the observations made in liver tissue. The constituents of the LAV fraction isolated from chloroquine-treated tissue were, however, less swollen than in situ, probably due to the extraction of water during isolation. Topics: Animals; Autophagy; Chloroquine; Half-Life; Leupeptins; Liver; Lysosomes; Male; Phagocytosis; Proteins; Rats; Rats, Inbred Strains; Vacuoles | 1987 |
Leupeptin and chymostatin inhibit mammalian protein methylesterase activity.
Purified protein methylesterase (PME) from rat kidneys was incubated with ovalbumin-methyl esters and a series of protease inhibitors. All four inhibitors with C-terminal aldehyde, leupeptin, chymostatin, Boc-Gln-Leu-Lys-H and D-Phe-Pro-Arg-H completely blocked PME activity. Other inhibitors including, alpha-1 antitrypsin, soybean trypsin inhibitor, antithrombin III, phenylmethylsulfonylfluoride, aprotinin and lima bean trypsin inhibitor had no significant effect whereas pepstatin, at high concentration reduced the enzymatic activity by 25%. The most potent inhibitors, leupeptin and chymostatin, had a Ki of 3.5 X 10(-8) and 5.4 X 10(-7) M, respectively. These inhibitors provide two new tools to study PME function. Topics: Animals; Kinetics; Leupeptins; Oligopeptides; Protein Methyltransferases; Rats | 1987 |
Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin.
The inhibitory effect of leupeptin on [3H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [3H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [3H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S). Topics: Animals; Binding, Competitive; Centrifugation, Density Gradient; Dexamethasone; Female; Goats; Kinetics; Leupeptins; Mammary Glands, Animal; Oligopeptides; Receptors, Glucocorticoid | 1987 |
Calcium regulation of lysosomal proteolysis in skeletal muscle.
Topics: Animals; Calcimycin; Calcium; Calmodulin; Dopamine Antagonists; Female; Insulin; Ionophores; Leupeptins; Lysosomes; Methylamines; Muscle Proteins; Muscle, Skeletal; Protease Inhibitors; Rats; Trifluoperazine | 1986 |
Stabilization of insulin receptor subunit structure by glutathione-insulin transhydrogenase.
A partially purified insulin receptor preparation from rat liver was incubated at 37 degrees C with and without the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2/5.3.4.1). Insulin-binding activity was then assessed by crosslinking receptor-125I-insulin complexes and subjecting them to electrophoresis on SDS-polyacrylamide gels in the absence and presence of reductant followed by autoradiography. Prior incubation of the receptor at 37 degrees C in the absence of the enzyme markedly decreased the subsequent binding of 125I-insulin to the holoreceptor (Mr 350 000) and to its subunits (Mr 180 000 and 130 000), while addition of the enzyme to the preincubation medium served to substantially prevent this decrease. The loss in binding at 37 degrees C was not restored by subsequent addition of the enzyme, nor was the loss prevented by any of the several known inhibitors of proteolysis. The apparent stabilization of receptor by transhydrogenase, as evidenced by the increase in binding above control levels, was proportional to both the enzyme concentration and the duration of incubation. These effects seem to be specific for transhydrogenase, since several other disulfide-containing proteins were found to be ineffective. These data suggest that the stabilization of the subunit structure of the insulin receptor at physiological temperatures may take place via a disulfide interchange reaction catalyzed by glutathione-insulin transhydrogenase. Topics: Animals; Antipain; Aprotinin; Electrophoresis, Polyacrylamide Gel; Ethylmaleimide; Insulin; Isomerases; Leupeptins; Macromolecular Substances; Molecular Weight; Oxidoreductases; Protein Disulfide Reductase (Glutathione); Protein Disulfide-Isomerases; Rats; Receptor, Insulin | 1986 |
Degradation of [125I]iodoglucagon by normal rat plasma in radioimmunoassay mixture containing aprotinin and its prevention by p-chloromercuriphenyl sulfonate and leupeptin.
Degradation of [125I]iodoglucagon during RIA of glucagon would result in erroneously high values for immunoreactive glucagon (IRG). Normal rat plasma was found to have high activity for glucagon degradation that was not suppressed by the aprotinin and EDTA routinely added to the RIA system. About 30% of the added radioactive glucagon was degraded during RIA in assay mixture at pH 7.4 containing 0.2 ml normal rat plasma, and the IRG value was calculated to be 150-180 pg/ml plasma. The degradation was completely inhibited by addition of 2 mM p-chloromercuriphenyl sulfonate (PCMS) plus 0.25 mM leupeptin as protease inhibitors, and in their presence the IRG value was about 80 pg/ml. The glucagon-degrading activity was about half as much in assay mixture at pH 8.8 as in that at pH 7.4, but the degradation still affected the accuracy of IRG values. When rat plasma was incubated with [125I]iodoglucagon in the assay conditions used for RIA and then subjected to Bio-Gel P-6 filtration, three new peaks of radioactivity were found in low mol wt fractions, with decrease in the peak corresponding to [125I]iodoglucagon, whereas on similar treatment in the presence of PCMS and leupeptin all the radioactivity was recovered in the glucagon fraction. The average recoveries of authentic glucagon as IRG in the absence and presence of the inhibitors were less than 60% and more than 90%, respectively. These findings indicate that determination of plasma IRG in rats by RIA with assay mixture containing aprotinin gives spuriously high values owing to degradation of the radiotracer, and that PCMS and leupeptin should be added to the sample and assay mixture to prevent this degradation. Topics: 4-Chloromercuribenzenesulfonate; Animals; Aprotinin; Blood; Chromatography, Gel; Dose-Response Relationship, Drug; Glucagon; Immune Sera; Leupeptins; Male; Oligopeptides; Phenylmercury Compounds; Protease Inhibitors; Radioimmunoassay; Rats; Rats, Inbred Strains | 1986 |
Antigen-Ia interaction and the proteolytic processing of antigen: the structure of the antigen determines its restriction to the A or E molecule of the major histocompatibility complex.
The effect of a protease inhibitor, leupeptin, on the presentation of hen egg lysozyme (HEL) to cloned T cells was investigated. We found that leupeptin-sensitive thiol proteases are apparently less involved when HEL is presented by the I-Ad molecule, than when it is presented by the I-Ed molecule. This selectivity was more of a function of the antigen than that of the Ia molecule because presentation of denatured or fragmented HEL was not sensitive to leupeptin whereas antigen presentation to a number of I-A-restricted T cell clones specific to other antigens was sensitive to leupeptin. These data demonstrate that the particular combination of major histocompatibility complex/nominal antigen recognized by a certain T cell clone may require processing of the antigen molecule through a certain group of proteases and that other combinations are independent of that particular processing pathway. Furthermore, there is a preference for a certain type of processing depending on the Ia molecule involved. Topics: Animals; Antigen-Presenting Cells; Antigens; Clone Cells; Epitopes; H-2 Antigens; Histocompatibility Antigens Class II; Leupeptins; Major Histocompatibility Complex; Mice; Mice, Inbred Strains; Muramidase; Peptide Hydrolases; T-Lymphocytes | 1986 |
Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes.
Down regulation of phorbol diester receptors was studied with respect to proteolysis of protein kinase C, which is activated by Ca2+, phospholipids, and diacylglycerols and which binds to phorbol diesters. We used FRSK cells, a cell line derived from fetal rat skin keratinocytes, because in these cells specific binding of phorbol 12,13-dibutyrate decreased rapidly (50% decrease in 30 min). This decrease (down regulation) was inhibited by some protease inhibitors, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin, but not by inhibitors of lysosomal hydrolases. On treatment with 12-O-tetradecanoylphorbol 13-acetate, protein kinase C was rapidly translocated from the cytosol to the membranes and then decreased. This decrease in protein kinase C was also inhibited by TPCK, TLCK, and leupeptin. The decrease in membrane activity of protein kinase C was associated with increase in cytosolic activity of a protein kinase that was smaller in molecular weight (Mr 40,000-60,000) than protein kinase C, did not depend on Ca2+/phosphatidylserine/diacylglycerol, and did not bind to phorbol 12,13-dibutyrate. These results indicate that down regulation of phorbol diester receptors is probably caused by nonlysosomal proteolysis of protein kinase C. The kinase formed by cleavage may be an active catalytic site of protein kinase C. Topics: Animals; Caenorhabditis elegans Proteins; Calcium; Carrier Proteins; Cell Line; Chromatography, Gel; Cytosol; Diglycerides; Female; Keratins; Leupeptins; Molecular Weight; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphatidylserines; Pregnancy; Protein Kinase C; Rats; Receptors, Drug; Receptors, Immunologic; Skin; Tetradecanoylphorbol Acetate; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 1986 |
Effect of protease inhibitors on skeletal muscle lysosomal enzyme activity and resting membrane potential.
Inhibition of lysosomal enzyme activity by protease inhibitors in denervated muscle was studied in vitro by incubating muscle homogenates with increasing concentrations of inhibitor and determining acid protease activity. Pepstatin was the most potent inhibitor of protease activity, followed in order of inhibitor potency by chloroquine, aprotinin, and leupeptin. The in vivo uptake of chloroquine or leupeptin by denervated muscle was greater than that of innervated muscle, and repeated administration of the drug led to a decrease in muscle N-acetylglucosaminidase activity. Repeated administration of chloroquine had no effect on resting membrane potential of muscle fibers in the soleus or extensor digitorum longus measured in vivo. Chloroquine attenuated the marked decrease in the resting membrane potential of the extensor 2.5 days after denervation, but had no effect on that of the denervated soleus. Possible involvement of proteases in lowering the membrane potential of denervated muscle is discussed. Topics: Acetylglucosaminidase; Animals; Aprotinin; Chloroquine; Leupeptins; Lysosomes; Male; Membrane Potentials; Muscle Denervation; Muscles; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Rats; Rats, Inbred Strains | 1986 |
[Comparative studies of nafamostat mesilate and various serine protease inhibitors in vitro].
Inhibitory effects of nafamostat mesilate (nafamostat) on various enzymes were investigated, and they were compared with those of gabexate mesilate (gabexate), leupeptin, aprotinin and urinastatin in vitro. Nafamostat inhibited trypsin, plasmin, thrombin, pancreatic kallikrein, Clr and Cls more potently than gabexate and leupeptin. Gabexate and leupeptin did not inhibit pancreatic kallikrein and thrombin, respectively. Aprotinin inhibited trypsin, plasmin, pancreatic kallikrein and chymotrypsin. Urinastatin inhibited trypsin and chymotrypsin. Nafamostat inhibited the complement-mediated hemolysis in diluted serum more potently than gabexate and leupeptin, but aprotinin and urinastatin did not. Nafamostat, furthermore, inhibited the complement-mediated hemolysis in undiluted serum, but gabexate did not. Unlike aprotinin and urinastatin, nafamostat and gabexate inhibited alpha 2-macroglobulin bound trypsin as well as free trypsin to the same extent. The inhibitory effect of gabexate toward trypsin was reduced more markedly than that of nafamostat after incubation with plasma at 37 degrees C. These results show that nafamostat is more useful than other inhibitors such as gabexate, leupeptin, aprotinin and urinastatin. Topics: alpha-Macroglobulins; Aprotinin; Benzamidines; Complement Inactivator Proteins; Gabexate; Glycoproteins; Guanidines; Hemolysis; In Vitro Techniques; Leupeptins; Protease Inhibitors; Protein Binding | 1986 |
Enzymatic regulation of calcium current in dialyzed and intact molluscan neurons.
Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Animals; Calcium; Calmodulin-Binding Proteins; Egtazic Acid; Helix, Snails; In Vitro Techniques; Ion Channels; Leupeptins; Neurons; Phosphoprotein Phosphatases; Protein Kinases | 1986 |
Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells.
GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-E Topics: Ammonium Chloride; Animals; Cell Compartmentation; Cell Line; Cell Membrane; Chloroquine; Cytosol; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Leupeptins; Lysosomes; Pituitary Neoplasms; Protein Binding; Rats; Receptors, Cell Surface; Receptors, Somatostatin; Somatostatin | 1986 |
Targeting, internalization, and cytotoxicity of methotrexate-monoclonal anti-stage-specific embryonic antigen-1 antibody conjugates in cultured F-9 teratocarcinoma cells.
Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface. Topics: Animals; Antibodies, Monoclonal; Glycolipids; Immunoglobulin G; Leucovorin; Leupeptins; Lewis X Antigen; Lysosomes; Methotrexate; Mice; Mice, Inbred BALB C; Teratoma; Thiamine Pyrophosphate; Tritium | 1986 |
Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression.
Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20), castanospermine caused the formation of novel transient forms of higher Mr than corresponding controls, indicating a blocked removal of glucose residues. For the first three enzymes, the 'mature' (Golgi-processed) forms were similar in size to or slightly smaller than corresponding controls and were, as shown for aminopeptidase N, endoglycosidase-H-sensitive, evidence of a blocked attachment of complex sugars. Maltase-glucoamylase did not undergo conversion into a 'mature' form, suggesting that, unlike other microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane. Topics: Alkaloids; Aminopeptidases; Animals; Carbohydrate Metabolism; CD13 Antigens; Electrophoresis, Polyacrylamide Gel; Indolizines; Intestinal Mucosa; Leupeptins; Membrane Proteins; Microvilli; Organ Culture Techniques; Swine | 1986 |
Comparative studies on insulin-like growth factor II and insulin processing by vascular endothelial cells.
Processing of circulating polypeptide hormones by vascular endothelial cells may be critical to hormone transport from the vascular lumen to tissue sites of action. The comparative processing of insulin-like growth factor II (IGF II) and insulin by cultured bovine aortic endothelial cells was examined. These cells possess high-affinity receptors for IGF II and insulin, as shown by competitive-binding studies. At 37 degrees C, internalization determined by both resistance to an acid wash and electron microscopy was rapid with 50-70% of bound IGF II and insulin internalized at 60 min. Subsequently, between 70 and 75% of the internalized hormones were released from the cells within 60 min. Although most of both hormones were released intact, degradation of IGF II was greater than that of insulin by two- to threefold, as assessed by G-50 chromatography and trichloroacetic acid precipitability. Leupeptin, a specific lysosomal protease inhibitor, increased cell-associated 125I-labeled IGF II by 53.0 +/- 6.0% and decreased degradation by 55%; however, it was without effect on 125I-labeled insulin. Chloroquine and monensin, which act at the lysosomes and at other sites, increased both cell-associated IGF II and insulin and decreased the degradation of both hormones. The increases in cell-associated 125I-IGF II produced by chloroquine (42.0 +/- 7.4%) and monensin (78.3 +/- 8.5%) were quantitatively similar to the decreases in IGF II degradation caused by the agents; however, the increase in cell-associated insulin was approximately threefold greater than could be accounted for simply by decreased insulin degradation.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Aorta; Biological Transport; Cattle; Cells, Cultured; Chloroquine; Endothelium; Insulin; Insulin-Like Growth Factor II; Kinetics; Leupeptins; Models, Biological; Monensin; Receptor, Insulin; Receptors, Somatomedin; Somatomedins | 1986 |
Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway.
Substantial amounts of epidermal growth factor (EGF) are cleared from the circulation by hepatocytes via receptor-mediated endocytosis and subsequently degraded within lysosomes. We have used a combined biochemical and morphological approach to examine the fate of the receptor after exposure to EGF. Polyclonal antibodies were prepared against the purified receptor and their specificity established by immunoprecipitation and immunoblotting techniques. The EGF receptor was then localized by immunofluorescence and immunoperoxidase techniques and quantified on immunoblots. In untreated livers, EGF receptor was restricted to the sinusoidal and lateral surfaces of hepatocytes. 2-4 min after exposure of cells to EGF, the receptor was found in small vesicles (i.e., coated vesicles) as well as larger vesicles and tubules at the cell periphery. By 15 min the receptor was found in multivesicular endosomes located near bile canaliculi. Exposure of hepatocytes to EGF also resulted in a rapid loss of receptor protein from total liver homogenates and a decrease in its half-life from 8.7 h in control livers to 2.5 h. This EGF-induced loss of receptors was not observed when lysosomal proteinases were inhibited by leupeptin or when endosome/lysosome fusion was prevented by low temperature (16 degrees C). In the presence of leupeptin, receptor could be detected in structures identified as lysosomes using acid-phosphatase cytochemistry. All these results suggested rapid internalization of EGF receptors in response to ligand and degradation within lysosomes. However, four times more ligand was degraded at 8 h than the number of high-affinity (Kd of 8-15 nM) EGF-binding sites lost, suggesting either (a) high-affinity receptors were recycled, and/or (b) more than 300,000 receptors were available for EGF uptake. We identified and characterized a latent pool of approximately 300,000 low-affinity receptors (Kd approximately 200 nM) that could be separated on sucrose gradients from the plasma membrane pool of approximately 300,000 high-affinity receptors (Kd of 8-15 nM). Despite the differences in their binding affinities, the high- and low-affinity receptors appeared to be structurally identical and were both EGF-dependent protein kinases. In addition, the dynamics of the low-affinity receptors were consistent with a functional role in EGF uptake and delivery to lysosomes. Topics: Animals; Cell Compartmentation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Immunoenzyme Techniques; Leupeptins; Liver; Lysosomes; Molecular Weight; Phosphorylation; Protein Kinases; Rats; Receptors, Cell Surface; Temperature | 1986 |
Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor.
When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by greater than 50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferrin. Because the time involved in the transferrin cycle is shorter (12.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand. Topics: Antibodies, Monoclonal; Binding Sites, Antibody; Cell Line; Chloroquine; Endocytosis; Half-Life; Humans; Iron; Leukemia, Erythroblastic, Acute; Leupeptins; Receptors, Cell Surface; Receptors, Transferrin | 1986 |
Phosphorylation of bovine cardiac calcium-activated neutral protease by protein kinase-C.
Protein kinase C prepared from rat brain was used to phosphorylate a calcium-activated neutral protease, purified from bovine cardiac muscle. Attempts to phosphorylate the enzyme in the presence of calcium were unsuccessful, unless the protease inhibitor leupeptin was also present. Phosphorylation of the 74K subunit of the protease was completely inhibited in the absence of phosphatidylserine and diolein, indicating that phosphorylation of the enzyme was catalysed by the calcium and phospholipid-dependent protein kinase C. Topics: Animals; Calpain; Cattle; Enzyme Activation; Leupeptins; Macromolecular Substances; Molecular Weight; Myocardium; Phosphorylation; Phosphothreonine; Protein Kinase C | 1986 |
Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+-requiring proteinase.
Low concentrations of phorbol 12-myristate 13-acetate (PMA) elicit a specific response in human neutrophils, characterized by the production of oxygen radicals and the release into the medium of a membrane-bound serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G. and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 1685-1689). The following evidence indicates that this response is mediated by membrane-bound protein kinase C: 1) it is blocked by inhibitors of protein kinase C; and 2) it is enhanced in cells preloaded with leupeptin which prevents proteolysis of protein kinase C and its subsequent dissociation from the cell membrane. This response is not accompanied by significant exocytosis of granule enzymes. With higher concentrations of PMA, and more particularly on stimulation with formylmethionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B, a substantial exocytosis of constituents of both specific and azurophil granules is observed. With fMLP, exocytosis of granule enzymes is the predominant event, with little production of H2O2 and negligible release of membrane-bound serine proteinase. Exocytosis promoted either by a high concentration of PMA or by fMLP is inhibited by leupeptin, indicating that it is due to the action of an intracellular Ca2+-dependent thiol proteinase (calpain), either directly or by conversion by calpain of membrane-bound protein kinase C to the soluble Ca2+/phospholipid-independent form. Intracellular mobilization of Ca2+ is also observed following stimulation with either PMA or fMLP, but only the latter results in a net increase in the intracellular concentration of free Ca2+; under these conditions maximum exocytosis of granule contents is observed. Topics: Calpain; Cytochalasin B; Endopeptidases; Exocytosis; Humans; Hydrogen Peroxide; Leupeptins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Retinaldehyde; Serine Endopeptidases; Tetradecanoylphorbol Acetate; Trifluoperazine | 1986 |
Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein Ib.
Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin. Topics: Animals; Calcimycin; Calcium; Calpain; Chickens; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Isoelectric Point; Leucine; Leupeptins; Membrane Proteins; Molecular Weight; Phenotype; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Platelet Membrane Glycoproteins | 1986 |
Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases.
The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane. Topics: Anion Exchange Protein 1, Erythrocyte; Blood Proteins; Calcimycin; Calpain; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Leupeptins; Membrane Proteins; Mersalyl; Neuropeptides; Spectrin | 1986 |
Effect of low calcium and protease inhibitors on synapse elimination during postnatal development in the rat soleus muscle.
The mechanisms controlling the reorganisation of synaptic inputs to developing skeletal muscle fibres was studied using electrophysiological and histological methods. In the developing rat soleus muscle there is a rapid reduction of polyneuronal innervation between 9 and 12 days. Reducing the local concentration of calcium by applying chelating agents such as EGTA or BAPTA in vivo to 9-day-old rat soleus muscles over a period of 3 days slowed the rate of elimination of polyneuronal innervation. It was established that the reduction of calcium induced by EGTA or BAPTA was not sufficient to produce a detectable reduction in neuromuscular activity. The possibility that a calcium-dependent enzyme such as CANP may play a role in synapse reorganisation was therefore tested. Local application of inhibitors of calcium-activated neutral protease (CANP), leupeptin or E-64, to 9-day-old rat soleus muscles over 3 days had similar effects to those of EGTA or BAPTA, i.e. the elimination of polyneuronal innervation that usually takes place was much slower. Since the inhibition of thiol proteases had similar effects on synapse elimination as a reduction of calcium concentration, it is concluded that CANP is important in the reorganisation of the developing neuromuscular junction. Topics: Animals; Animals, Newborn; Calcium; Calpain; Egtazic Acid; Leupeptins; Mersalyl; Muscle Development; Neuromuscular Junction; Rats; Rats, Inbred Strains | 1986 |
Four Ca2+-dependent proteinase activities isolated from crustacean muscle differ in size, net charge, and sensitivity to Ca2+ and inhibitors.
Four Ca2+-dependent proteinase activities in lobster claw and abdominal muscle have been resolved by high-performance liquid chromatography on gel filtration and ion-exchange columns. These activities, which do not appear to be generated by autolytic or other degradative processes, differed from each other in molecular weight (peak I, Mr = 310,000; peak IIa, Mr = 125,000; peak IIb, Mr = 195,000; peak III, Mr = 59,000) and net charge, as indicated by elution from an ion-exchange column with a NaCl gradient. Although optimum activity occurred at 5-10 mM Ca2+ at pH 6.8, the enzymes differed in activation at lower Ca2+ concentrations. The concentrations required for half-maximal activation were 0.6 mM for peak III, 1 mM for peak I, 1.5 mM for peak IIa, and 2 mM for peak IIb. Only the peak III proteinase was active at 100 microM Ca2+; none were active at 10 microM and below. Although the lobster Ca2+-dependent proteinases were all inhibited, from 75 to 98%, by the cysteine proteinase inhibitors leupeptin, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine, and iodoacetamide, they showed differential responses to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor phenylmethanesulfonyl fluoride. Peak I was moderately (26%) inhibited by phenylmethanesulfonyl fluoride, whereas peaks IIb and III were inhibited 26 and 90%, respectively, by pepstatin. This is the first description of multiple forms of Ca2+-dependent proteinase that require Ca2+ at millimolar levels in any tissue, either vertebrate or invertebrate. Topics: Animals; Calcium; Calcium Channel Blockers; Calpain; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Iodoacetamide; Leupeptins; Molecular Weight; Muscles; Nephropidae; Pepstatins; Phenylmethylsulfonyl Fluoride | 1986 |
Calcium-induced degeneration of the cytoskeleton in monkey and human peripheral nerves.
Biopsy specimens of human and monkey peripheral nerves, when incubated in calcium containing media, showed a loss of neurofilaments and microtubules with replacement by granular debris. Cytoskeletal structures remained intact when incubated in calcium-free media. Disruption of neurofilaments and microtubules in calcium containing media was inhibited by the thiol protease inhibitor, leupeptin. Similar incubations of excised Pacinian corpuscles revealed evidence of early terminal axon degeneration in the presence of calcium. These data substantiate the hypothesis that neural cytoskeletal degradation in primates and in man is calcium-mediated. Topics: Animals; Calcium; Calpain; Cebus; Culture Media; Cytoskeleton; Humans; Intermediate Filaments; Leupeptins; Microtubules; Nerve Degeneration; Peripheral Nerves; Wallerian Degeneration | 1986 |
Ultrastructural study of the effect of calcium ionophore, A23187, on rat muscle.
The present study was undertaken to investigate changes in the muscle fiber when treated with calcium ionophore. Muscles treated with ionophore showed disruption of the plasma membrane of the muscle fiber, delta lesions, marked contraction of the myofibrills, and dissolution of Z lines and I bands. Black granules of calcium pyroantimonate were observed inside the plasma membrane in ionophore-treated muscle fibers without alteration of the other muscle organelles. The density of the intramembranous particles was less in muscle treated with calcium ionophore than in the control muscle. These results support the previous hypothesis that the increased concentration of intracellular calcium activates calcium-activated neutral protease and induces necrosis of the myofiber. The mechanism for the decrease in the density of intramembranous particles is unsolved. However, the disruption of the plasma membrane may not be a direct effect of calcium ionophore on it, but a secondary phenomenon which occurs after the calcium-induced necrosis of the muscle fibers. Topics: Animals; Calcimycin; Calcium; Cell Membrane; Creatine Kinase; Female; Freeze Fracturing; Leupeptins; Microscopy, Electron; Muscles; Muscular Dystrophies; Rats; Rats, Inbred Strains | 1986 |
Involvement of thiol proteases in galactosialidosis.
The activities of Z-Phe-Arg-NMec(ZPA) hydrolase, cathepsin B and cathepsin H and the concentration of endogenous thiol protease inhibitor in fibroblasts from patients with galactosialidosis were found not to be significantly different from those in control fibroblasts. Culture for 5 days with thiol protease inhibitors such as leupeptin, E-64 or Z-Phe-Phe-CHN2 partially restored the beta-galactosidase activity of fibroblasts from patients, but did not affect the beta-galactosidase activity of fibroblasts from control subjects. However, culture with leupeptin, but not other protease inhibitors, increased the ZPA hydrolase and cathepsin B activities of fibroblasts from both patients and controls 2- to 4-fold. Sephadex G-75 chromatography showed that the activity of high molecular weight ZPA hydrolase, which was initially predominant in fibroblasts, decreased markedly during their culture with leupeptin, while the activities of lower molecular weight ZPA hydrolase and cathepsin B increased about 5-fold. These results suggest that high molecular weight ZPA hydrolase, which is presumably cathepsin J, degrades beta-galactosidase, and that the defect in galactosialidosis is impaired protection of beta-galactosidase from degradation. Topics: beta-Galactosidase; Cathepsin B; Cathepsin H; Cathepsins; Cells, Cultured; Coumarins; Cysteine Endopeptidases; Diazomethane; Dipeptides; Endopeptidases; Fibroblasts; Galactosidases; Humans; Leucine; Leupeptins; Neuraminidase; Protease Inhibitors | 1986 |
Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts.
Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes. Topics: Antibodies; Antibodies, Monoclonal; beta-Galactosidase; Fibroblasts; G(M1) Ganglioside; Galactosidases; Gangliosidoses; Histocytochemistry; Humans; Immunochemistry; Leupeptins; Lysosomes; Metabolism, Inborn Errors; Microscopy, Electron; Neuraminidase; Subcellular Fractions | 1986 |
Selective inhibition of proteolytic enzymes in an in vivo mouse model for experimental metastasis.
Peptide aldehyde transition state analogue inhibitors of serine and cysteine proteases have been used to selectively inhibit proteases for which prior evidence supports a role in tumor cell metastasis. These enzymes include cathepsin B, urokinase plasminogen activator (PA), and thrombin. The inhibition constants of the peptidyl aldehyde inhibitors show that they are highly selective for a particular targeted serine or cysteine protease. The inhibitors are introduced by i.p. injection or by miniosmotic pumps into syngeneic C57BL/6 mice also given injections of B16-F10 melanoma cells, and the number of metastatic foci in the lung was determined. While the injection protocol gave an initially high but changing in vivo concentration of inhibitor over time, the minipump implant gave a constant steady state concentration of inhibitor over 5-7 days. Minipump infusion of leupeptin (acetylleucylleucylargininal), a strong inhibitor of cathepsin B at a steady state plasma concentration 1000-fold greater than its Ki(cathepsin B), gave no significant decrease in lung colonization by the B16 tumor cells. Ep475, a stoichiometric irreversible peptide inhibitor of cathepsin B-like proteases, also did not significantly inhibit metastatic foci formation. Introduction of selective inhibitors of urokinase PA, tert-butyloxycarbonylglutamylglycyl-argininal and H-glutamylglycylargininal at concentrations near its Ki, produced no significant decrease in mouse lung colonization. The selective thrombin inhibitor D-phenylalanylprolylargininal infused to a steady state concentration 100-fold greater than its Ki dramatically increased B16 melanoma colonization of mouse lung. The results indicate that neither secreted cathepsin B-like nor urokinase PA have roles in B16 colonization of mouse lung, while thrombin may have a role in preventing metastasis. These experiments do not eliminate roles for a cathepsin B-like enzyme or urokinase PA in the initial steps of the metastatic process. Topics: Animals; Cathepsin B; Cathepsins; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Endopeptidases; Female; Fibrinolysis; Leucine; Leupeptins; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Peptide Hydrolases; Plasminogen Activators; Plasminogen Inactivators; Protease Inhibitors; Serine Endopeptidases; Thrombin; Thrombosis | 1986 |
The activation of protein degradation in muscle by Ca2+ or muscle injury does not involve a lysosomal mechanism.
By use of different inhibitors, we distinguished three proteolytic processes in rat skeletal muscle. When soleus muscles maintained under tension were exposed to the calcium ionophore A23187 or were incubated under no tension in the presence of Ca2+, net protein breakdown increased by 50-80%. Although leupeptin and E-64 inhibit this acceleration of protein breakdown almost completely, other agents that prevent lysosomal function, such as methylamine or leucine methyl ester, did not inhibit this effect. A similar increase in net proteolysis occurred in muscle fibres injured by cutting, and this response was also inhibited by leupeptin, but not by methylamine. In contrast, all these inhibitors markedly decreased the 2-fold increase in protein breakdown induced by incubating muscles without insulin and leucine, isoleucine and valine. In addition, the low rate of proteolysis seen in muscles under passive tension in complete medium was not affected by any of these inhibitors. Thus the basal degradative process in muscle does not involve lysosomes or thiol proteinases, and muscle can enhance protein breakdown by two mechanisms: lack of insulin and nutrients enhances a lysosomal process in muscle, as in other cells, whereas Ca2+ and muscle injury activate a distinct pathway involving cytosolic thiol proteinase(s). Topics: Animals; Calcimycin; Calcium; In Vitro Techniques; Leupeptins; Lysosomes; Male; Methylamines; Muscle Contraction; Muscle Proteins; Muscles; Protease Inhibitors; Rats; Rats, Inbred Strains | 1986 |
Evidence against a possible involvement of the serine, and thiol proteases in the exocytotic mechanism of catecholamine secretion in cultured bovine adrenal medullary cells.
The effects of protease inhibitors on the secretion of catecholamines were studied in cultured bovine adrenal medullary cells. Although the inhibitors of serine proteases could inhibit the carbamylcholine-induced secretion, they failed to inhibit the secretion evoked by either high K+ or A23187. The thiol protease inhibitor had no effect on the secretion. These results therefore seem to indicate that the serine protease inhibitors may inhibit the receptor-mediated secretion probably through their effects on the plasma membrane, thus suggesting that a possible involvement of the serine, and thiol proteases in exocytosis may be unlikely. Topics: Adrenal Medulla; Animals; Calcimycin; Carbachol; Catecholamines; Cattle; Cells, Cultured; Cysteine Endopeptidases; Endopeptidases; Exocytosis; Kinetics; Leupeptins; Oligopeptides; Potassium; Serine Endopeptidases | 1986 |
Protease and cyclooxygenase inhibitors synergistically prevent activation of human platelets.
Thrombin induces platelet aggregation and formation of a fibrin clot in platelet-rich plasma; leupeptin, a protease inhibitor, partially inhibits platelet aggregation, but it does not inhibit fibrin clot formation. Indomethacin does not inhibit either thrombin-induced platelet aggregation or fibrin clot formation. However, when the two drugs are given together, a synergistic inhibition of thrombin-induced platelet aggregation occurs, while fibrin clot formation remains unaffected. Thrombin-induced stimulation of the release of serotonin in washed human platelets is also synergistically inhibited by the combined actions of leupeptin and indomethacin. Thrombin and collagen, added simultaneously, induce full platelet aggregation and release of serotonin. Neither leupeptin nor indomethacin inhibits platelet responses elicited by both agonists; however, when leupeptin and indomethacin are given together, a synergistic inhibition of thrombin- and collagen-induced response is observed. These findings might be relevant in prophylaxis and treatment of thromboembolic disease. Topics: Blood Platelets; Collagen; Humans; In Vitro Techniques; Indomethacin; Leupeptins; Oligopeptides; Platelet Aggregation; Secretory Rate; Serotonin; Thrombin | 1986 |
The effects of ascorbic acid on the intracellular metabolism of iron and ferritin.
An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron. Topics: Animals; Ascorbic Acid; Cell Line; Deferoxamine; Dose-Response Relationship, Drug; Ferritins; Iron; Leukemia, Erythroblastic, Acute; Leupeptins; Methionine | 1986 |
Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms.
We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0. Topics: Animals; Chromatography, Gel; Cysteine Endopeptidases; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Female; Helminth Proteins; Hemoglobins; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Leupeptins; Male; Molecular Weight; Peptides; Protease Inhibitors; Schistosoma mansoni; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 1986 |
Morphometric assessment of drug effects in experimental spinal cord injury.
The effect of large doses of methylprednisolone sodium succinate (MPSS) and two protease inhibitors, leupeptin and bestatin, on experimental acute spinal cord injury was evaluated by morphometric analysis of degenerating axons with the aid of an automated image analyzer. Spinal cord injury was produced by epidural compression with a surgical clip on the T-11 segment in rats. The extent of axonal damage was assessed in Rexed's lamina VIII in the L-6 segment by measuring the amount of silver grains, representing degenerating axons and their terminals, using the Fink-Heimer method. The severity of axonal damage was expressed as the degeneration index: that is, the amount of silver grains in experimental animals/the amount of silver grains in cord-transected animals. When examined on the 7th postoperative day, axonal degeneration in MPSS-treated rats was significantly decreased, with an average degeneration index difference of 6 (p less than 0.05). Increased preservation of axons was seen in the leupeptin-treated rats sacrificed 7, 10, and 14 days after trauma. The difference in the degeneration index between the leupeptin-treated and untreated groups was 16 on Day 7 (p less than 0.001), 12 on Day 10 (p less than 0.001), and 13 on Day 14 (p less than 0.01). Bestatin had no beneficial effect. The implications for the use of calcium-activated neutral protease inhibitors in acute spinal cord injury are discussed. Topics: Animals; Axons; Leucine; Leupeptins; Male; Methylprednisolone; Methylprednisolone Hemisuccinate; Rats; Rats, Inbred Strains; Spinal Cord Injuries | 1986 |
Pinocytosis and degradation of exogenous proteins by cystinotic fibroblasts.
Lysosomes of cystinotic human fibroblasts contain over 100-times the normal concentration of cystine. The high cystine concentration (probably in the millimolar range) might be expected to inhibit intralysosomal protein breakdown. A comparison of pinocytosis and degradation of five 125I-labelled proteins (bovine serum albumin, formaldehyde-denatured bovine serum albumin, bovine pancreatic ribonuclease A and porcine lactate dehydrogenase isoenzymes H4 and M4) by human fibroblasts has been made, using one cystinotic and two normal cell-lines. The proteins each entered fibroblasts by adsorptive pinocytosis and were then degraded within the lysosomes by enzymes susceptible to leupeptin, the thiol-proteinase inhibitor. Each protein was captured by the fibroblasts at a characteristic rate, which was not different in cystinotic cells. Normal and cystinotic fibroblasts did not differ in their proteolytic capacity, as measured in extracts of disrupted cells. In intact fibroblasts, four of the five proteins were rapidly and fully digested following pinocytosis, in both cystinotic and normal cells. However, with formaldehyde-denatured albumin, the most resistant to degradation of the proteins tested, or with some other proteins in the presence of leupeptin, when the proteolytic capacity of lysosomes is diminished, intralysosomal degradation of pinocytosed protein was incomplete. Moreover, under these conditions, cystinotic cells demonstrated a lower rate of protein digestion than normal cells. It is concluded that pinocytic capture, rather than intralysosomal proteolysis, is commonly the rate-limiting step in the overall process of uptake and degradation of proteins by fibroblasts, and that intralysosomal cystine inhibits digestion of pinocytosed protein only in circumstances when degradation becomes the rate-limiting step. Topics: Cells, Cultured; Cysteine Endopeptidases; Cystinosis; Endopeptidases; Humans; Leupeptins; Lysosomes; Pinocytosis; Proteins; Skin | 1986 |
Inhibition of proteolytic enzymes in the in vitro amnion model for basement membrane invasion.
The ability of B16-F10 mouse melanoma cells to cross an amnion basement membrane was determined in the presence of strong inhibitors of both serine and cysteine proteases. The concentrations of inhibitors were at orders of magnitude higher than their Ki values to serine and cysteine proteases implicated in metastasis, thus ensuring a complete inhibition for tumor secreted proteases such as cathepsin B-like proteases, plasminogen activators, and plasmin. Under these conditions of high serine and cysteine protease inhibitor concentrations, no significant decrease in B16-F10 melanoma cell invasion through the amnion was observed. Separate experiments showed that the inhibitors were neither toxic to the cells nor degraded. The results show that neither tumor cell secreted cathepsin B-like proteases nor plasminogen activator have a controlling role in basement membrane crossing in this metastatic model. A possible role for tumor cell membrane proteases in basement membrane invasion, in which the substrates of the protease bind to receptor sites near a membrane associated proteolytic activity, is not eliminated. Topics: Amnion; Animals; Basement Membrane; Blood Physiological Phenomena; Cathepsin B; Cathepsins; Cell Membrane; Cysteine Endopeptidases; Endopeptidases; Female; Humans; In Vitro Techniques; Leupeptins; Melanoma; Mice; Neoplasm Invasiveness; Plasminogen Activators; Pregnancy; Protease Inhibitors; Serine Endopeptidases; Urokinase-Type Plasminogen Activator | 1986 |
A protease acting on the estrogen receptor may modify its action in the adult rabbit epididymis.
We have previously shown that the cytosolic estrogen receptor in adult rabbit epididymides sediments as an congruent to 3 S species on sucrose gradients containing 0.01 M KCl while that from immature rabbit epididymides sediments at congruent to 9 S. This age-dependent decrease in sedimentation coefficient is attributable to the appearance of a leupeptin-sensitive protease as the animals mature. We now show that if adult epididymides are homogenized in buffer containing leupeptin, the 9 S receptor can be demonstrated, indicating inhibition of receptor degradation. In vitro nuclear uptake studies conducted in the absence of leupeptin indicated that the proteolyzed receptor was not an efficient nuclear binder. When leupeptin was present, nuclear uptake increased 6-fold and it was accompanied by depletion of receptor from the cytosol. Binding of the receptor to nuclei was specific since it could be inhibited by unlabeled estrogens but not by unlabeled 5 alpha-dihydrotestosterone or progesterone. In vitro mixing experiments indicated that the proteolytic activity was associated with the crude nuclear fraction since, in the absence of leupeptin, they had reduced ability to bind estrogen receptor present in immature epididymal cytosol. Specific in vivo binding of [3H]estradiol by adult and immature rabbit epididymides could be demonstrated. The time course of in vivo binding of [3H]estradiol by adult rabbit epididymal nuclei indicated maximum binding (70 fmol/g tissue) at 30 min following injection. By 60 min, the amount of binding had decreased to about 25 fmol. The accessory sex organs, which do not contain the protease, also exhibited maximum binding (150 fmol/g tissue) at 30 min. However, at the 60 min period binding was still about 140 fmol. Processing the tissues in buffers containing leupeptin had no effect on the results obtained. These results are interpreted to indicate that the presence of the protease decreases nuclear binding of the estrogen receptor and shortens nuclear occupancy. This combination of factors may be responsible for the decrease in estrogen action in the adult rabbit epididymis. Topics: Aging; Animals; Cell Nucleus; Centrifugation, Density Gradient; Epididymis; Estradiol; Estrogens; Genitalia, Male; In Vitro Techniques; Leupeptins; Male; Orchiectomy; Peptide Hydrolases; Rabbits; Receptors, Estrogen | 1986 |
Physiological role of thiol proteases in thyroid hormone secretion.
To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I- in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW less than 14K. Topics: Acid Phosphatase; Animals; Centrifugation, Density Gradient; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Leupeptins; Lysosomes; Male; Rats; Rats, Inbred Strains; Thyroid Gland; Thyroxine; Triiodothyronine | 1986 |
Induction and inhibition of cathepsin B and hemoglobin-hydrolase activity in murine B16 melanoma by thiol protease inhibitors.
The effects of potent thiol protease inhibitors in vitro (leupeptin, antipain, chymostatin and E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine) on intracellular cathepsin B and hemoglobin (Hb)-hydrolase from cultured B16 melanoma cells were studied. E-64 induced cultured B16 melanoma cells to decrease the activities of intracellular cathepsin B (EC 3.4.22.1.) but did not have this effect with Hb-hydrolase or acid phosphatase (EC 3.1.3.2). Leupeptin, antipain and chymostatin induced B16 melanoma cells to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. These results indicate that there are two kinds of thiol protease inhibitors, each with a varying reaction to cultured B16 melanoma--inhibition of intracellular cathepsin B, and conversely, inducement of both cathepsin B and Hb-hydrolase. Topics: Animals; Antipain; Cathepsin B; Kinetics; Leucine; Leupeptins; Melanoma, Experimental; Mice; Oligopeptides; Peptide Hydrolases; Protease Inhibitors | 1986 |
The bovine lens neutral proteinase comprises a family of cysteine-dependent proteolytic activities.
Inhibitor studies with peptide substrates demonstrate that bovine lens neutral proteinase comprises three distinct activities. Diisopropylfluorophosphate distinguishes the activity hydrolyzing carbobenzoxy-Gly-Gly-Leu-p-nitroanilide (inhibited) from that hydrolyzing carbobenzoxy-Leu-Leu-Glu-2-naphthylamide (not inhibited). Leupeptin inhibits hydrolysis of the substrate carbobenzoxy-Leu-Leu-Arg-2-naphthylamide, but not hydrolysis of carbobenzoxy-Gly-Gly-Leu-p-nitroanilide or carbobenzoxy-Leu-Leu-Glu-2-naphthylamide, demonstrating the presence of the third activity. Inhibition of the three activities by thiol reagents suggests that each activity may be dependent on an active-site cysteine residue. Topics: Animals; Cattle; Cysteine; Endopeptidases; Hydrolysis; Lens, Crystalline; Leupeptins; Neprilysin; Nitrobenzenes; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Substrate Specificity; Trypsin | 1986 |
Plasmodium falciparum: protease inhibitors and inhibition of erythrocyte invasion.
Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin. Topics: Animals; Chymotrypsin; Erythrocytes; Humans; In Vitro Techniques; Leupeptins; Oligopeptides; Plasmodium falciparum; Protease Inhibitors; Trypsin | 1986 |
Effect of leupeptin, a protease inhibitor, on dopa decarboxylase and protease activities in homogenates of submandibular salivary glands from young rats.
Dopa-decarboxylase activity was high at 2-3 weeks of age, low at 5-6 weeks, and very low in adult rats; protease activity increased with age. Trypsin-like proteases, which decrease dopa-decarboxylase activity, were detected in the gland and were inhibited by a protease inhibitor, leupeptin. High concentrations (20 mg/ml) of leupeptin increased dopa-decarboxylase activity in the glands of adult rats to the level found at 5-6 weeks of age. The low activity of dopa decarboxylase in adult gland may be due in part to endogenous proteases. Topics: Aging; Animals; Aromatic-L-Amino-Acid Decarboxylases; Dopa Decarboxylase; Female; Leupeptins; Male; Oligopeptides; Peptide Hydrolases; Rats; Rats, Inbred Strains; Submandibular Gland | 1986 |
Vascular permeability changes by proteinase inhibitors in carrageenin-induced inflammation in rats.
The effect of proteinase inhibitors such as TLCK, TPCK and leupeptin on vascular permeability was investigated in the carrageenin-air-pouch inflammation in rats. When each inhibitor was injected into the air-pouch immediately after carrageenin injection, TLCK, TPCK and leupeptin caused a rapid and significant increase in vascular permeability. The TLCK- and TPCK-induced increase declined gradually, whereas leupeptin inhibited the vascular permeability after the temporary increase. When the inhibitors were injected 5 h after carrageenin injection, TLCK and TPCK increased the vascular permeability, whereas leupeptin was without effect. Cyproheptadine, an anti-histamine and antiserotonin drug, inhibited the leupeptin-induced temporary increase, but failed to inhibit the TLCK- and TPCK-induced increase in vascular permeability. These results suggest that the leupeptin-induced increase in vascular permeability was mediated by histamine and serotonin, while TLCK and TPCK may increase vascular permeability as a result of a direct action on endothelial cells. Topics: Animals; Capillary Permeability; Carrageenan; Inflammation; Kinetics; Leupeptins; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone | 1986 |
Sustained proteolysis is required for human platelet activation by thrombin.
A maximally effective dose of indomethacin does not prevent serotonin release and aggregation in human platelets stimulated with thrombin. Thrombin induces rapid activation of inositol phospholipids-specific phospholipase C, which is reflected by the degradation of inositides and the phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Thrombin also activates protein kinase C and myosin light chain kinase as indicated by phosphorylation of the 40,000 and 20,000 dalton proteins, respectively. Leupeptin, a protease inhibitor that does not inhibit thrombin's proteolytic activity or its binding to platelet surface, is able to reverse platelet activation by thrombin when it is administered after the addition of the agonist and indomethacin. The results suggest a proteolytic-mediated pathway in transmembrane signalling involved in platelet activation by thrombin. Topics: Blood Platelets; Humans; Indomethacin; Inositol; Kinetics; Leupeptins; Platelet Aggregation; Serotonin; Thrombin | 1986 |
Evidence for a specific estradiol binding site on rat pituitary membranes.
Using adenohypophyses from normal female rats, we demonstrate that estradiol binds pituitary membranes to one homogeneous population of sites with high affinity [dissociation constant (Kd) = 0.041 +/- 0.014 nM; n = 6] and low capacity [maximum binding (Bmax) = 13.6 +/- 5.6 fmol/mg protein]. The binding is thermolabile. Association experiments show that the best experimental conditions are an overnight incubation at 0 C. When the amount of proteins is increased more than 0.3 mg/ml of membrane suspension, binding is rapidly nonlinear. The presence of 0.5 M leupeptin does not improve the binding. Extensive washing of the membranes does not decrease the amount of sites, indicating that the binding is not loosely attached to the membranes. Parenthetically, it should be noted that the membrane fraction was devoid of the cytosolic enzyme marker, lactate dehydrogenase. Binding is specific for estrogenic compounds. When 100% specific binding was determined in the presence of 10(-6) M diethylstilbestrol, 17 beta-estradiol, estrone, and estriol displaced total binding by 110, 80, and 75%, respectively. Neither 4-OH-tamoxifen nor dihydrotestosterone, progesterone, or cortisol displaced the binding. Taken together, these data argue in favor of the presence of specific membrane recognition sites for estradiol in the rat pituitary. Topics: Animals; Binding Sites; Binding, Competitive; Diethylstilbestrol; Dihydrotestosterone; Estradiol; Estriol; Estrone; Female; Hydrocortisone; Kinetics; Leupeptins; Pituitary Gland, Anterior; Progesterone; Rats; Rats, Inbred Strains; Temperature | 1986 |
Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm.
The effect of exposure to leupeptin (25 micrograms/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a double-labelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptin-treated yolk sacs were labelled with Con-A Fer at 4 degrees C and then incubated with HRP for 5, 15 or 60 min at 37 degrees C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptin-treated cells did not exhibit any labeling.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Cell Membrane; Cells, Cultured; Concanavalin A; Endocytosis; Endoderm; Horseradish Peroxidase; Intracellular Membranes; Leupeptins; Lysosomes; Oligopeptides; Rats; Yolk Sac | 1986 |
Influence of agents that alter lysosomal function on fetal mouse hearts recovering from anoxia and substrate depletion.
Recovery of fetal mouse heart myocytes from oxygen and substrate deprivation for 1 h is accompanied by complicated lysosomal and non-lysosomal vacuolar responses which can be subdivided temporally into four distinct phases that include production of lysosomal dense bodies; segregation of damaged subcellular organelles into vacuoles that initially lack lysosomal enzymes; delivery of lysosomal enzymes to these vacuoles through fusion with dense bodies, transforming them into lysosomal autophagic vacuoles and degradation of the sequestered organelles. These events are normally completed within 6 h of the resupply of oxygen and substrate. The progression of these events is influenced significantly by pharmacological interventions that alter lysosomal properties. Chloroquine inhibits all aspects of the lysosomally-related processes as well as the sequestration phase during recovery. Leupeptin delays the lysosomal degradation, presumably by slowing proteolysis. Hydrocortisone permits the engulfment phase and the appearance of lysosomal dense bodies but appears to prevent or postpone the delivery of lysosomal enzymes to many of the large vacuoles and to delay the degradation of sequestered organelles. These observations reveal that segregation of damaged organelles and lysosomally-mediated degradation of these subcellular structures are important events during recovery from ischemic-like injury, and that agents that interfere with normal lysosomal function can prevent or delay some or all of the lysosomal responses that are involved in the recovery process. Topics: Animals; Chloroquine; Female; Fetus; Hydrocortisone; Hypoxia; Kinetics; Leupeptins; Lysosomes; Mice; Microscopy, Electron; Myocardium; Oligopeptides; Organ Culture Techniques; Pregnancy; Vacuoles | 1986 |
Acidic lens-protein-degrading activity in aqueous humour.
Lens-protein-degrading activity in human aqueous humor was determined biochemically. Proteolytic activity was found in the pellet of aqueous humor from patients with Morgagnian cataract, traumatic cataract, after cataract, traumatic lens luxation, and phacolytic glaucoma. The activity was found at pH 3.8 and it was inhibited partly by pepstatin or leupeptin. Possibly, cathepsin D and lysosomal thiol proteinases in the macrophages play a major role in the degradation of escaping lens protein under pathologic conditions. Topics: Animals; Aqueous Humor; Cataract; Cattle; Crystallins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Leupeptins; Pepstatins; Protease Inhibitors | 1986 |
Preparation and purification of the proteinase inhibitor, leupeptin, from culture filtrates of Streptomyces lavendulae.
Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) was produced as an extracellular metabolite by Streptomyces lavendulae. Addition of lysine to the growth medium stimulated leupeptin production from 200 to 1400 micrograms/ml. Incubation of late exponential phase mycelium in a synthetic medium was used to prepare single labelled (3H) and double labelled (3H/14C) inhibitor for metabolic studies. An improved purification scheme that generates leupeptin of high purity with good recoveries is also reported. Topics: Carbon Radioisotopes; Culture Media; Kinetics; Leupeptins; Oligopeptides; Streptomyces; Tritium | 1986 |
Assay of plasma leupeptin using the reversible binding of leupeptin to bovine pancreatic trypsin.
A competitive binding radioassay for leupeptin has been developed utilizing the reversible binding of leupeptin to bovine pancreatic trypsin. An ethanol precipitation step was introduced to separate trypsin-bound leupeptin from its free form. Advantages of this method are simplicity of the procedure and avoidance of the preparation of antiserum. The possible metabolites of leupeptin exhibit no significant inhibitory effect on leupeptin-trypsin binding in this system. This method was applied to the determination of plasma leupeptin levels in dogs after oral administration of the peptide. Topics: Animals; Binding, Competitive; Cattle; Dogs; Kinetics; Leupeptins; Male; Oligopeptides; Pancreas; Protein Binding; Species Specificity; Trypsin | 1986 |
Secretion-coupled protein degradation: studies on mammary casein.
Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells. Topics: Adenine; Amino Acids; Animals; Biological Transport; Caseins; Colchicine; Cytochalasin B; Female; Leupeptins; Mammary Glands, Animal; Organ Culture Techniques; Rabbits; Tunicamycin | 1986 |
Processing and estrogen regulation of the 52-kilodalton protein inside MCF7 breast cancer cells.
A 52K glycoprotein is secreted by human breast cancer cells in culture after estrogen stimulation. Using monoclonal antibodies, we have quantitated and characterized the corresponding proteins of the cell compartment. Using pulse-chase experiments, we have shown that about 40% of the 52K protein is secreted, the majority being successively processed into a 48K and a 34K protein. This last protein is very stable. The processing is inhibited by lysosomotropic agents and leupeptin, suggesting that it occurs in acidic vesicles, such as lysosomes or endosomes. Estradiol increased the intracellular level of immunoreactive 52K related proteins by 4-fold. Its effect is, however, more obvious in the medium, since there is a constitutive level in the cell. The stimulatory effects of estradiol on [3H]mannose and [35S]methionine incorporation into these proteins were similar and the endoglycosydase H sensitivity of the proteins was not altered, suggesting that estradiol did not modulate the glycosylation step. Antiestrogens did not stimulate synthesis and glycosylation of the 52K related proteins. Estradiol also increased the stability of the 52K precursor as well as that of total proteins. We conclude that the secreted 52K protein is the precursor of two cellular proteins of 48K and 34K. Estradiol stimulates both the intracellular accumulation of these proteins and the secretion of the precursor. Topics: Ammonium Chloride; Antibodies, Monoclonal; Breast Neoplasms; Cell Compartmentation; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Glycosylation; Humans; Leupeptins; Molecular Weight; Monensin; Neoplasm Proteins; Protein Processing, Post-Translational | 1986 |
Leucine aminopeptidase and hatching of Schistosoma mansoni eggs.
Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed. Topics: Animals; Cold Temperature; Darkness; Leucine; Leucyl Aminopeptidase; Leupeptins; Ovum; Schistosoma mansoni; Sodium Chloride | 1986 |
Characterization of Ca2+-activated, phospholipid-dependent protein kinase C/protein kinase M in guinea pig peritoneal exudate macrophages.
Protein kinase C (C-kinase) is shown to be present in the cytosolic and particulate fractions of mineral oil induced peritoneal macrophages of guinea pigs. By omission or use of a high concentration of leupeptin, three forms of the enzyme were obtained: stimulant/Ca2+/phospholipid-dependent C-kinase, eluted from DEAE 52 cellulose at 0.08-0.16 M NaCl; stimulant/Ca2+/phospholipid-independent protein kinase M (M-kinase), and Ca2+-inhibited & stimulant/phospholipid-dependent form of protein kinase, both eluted from DEAE 52 cellulose at 0.18-0.22 M NaCl. Phorbol ester or 1,2-diacylglycerol were used as stimulants. It is suggested that Ca2+-inhibited & stimulant/phospholipid-dependent protein kinase represents the in vivo form of the M-kinase in intact cells. Topics: Animals; Calcium; Cell Compartmentation; Diglycerides; Enzyme Activation; Guinea Pigs; Kinetics; Leupeptins; Macrophages; Peptide Fragments; Peritoneal Cavity; Phorbol Esters; Phosphatidylserines; Phospholipids; Protein Kinase C | 1986 |
Regulation of Ca2+-dependent protein turnover in skeletal muscle by thyroxine.
Dantrolene, an agent that inhibits Ca2+ mobilization, improved protein balance in skeletal muscle, as thyroid status was increased, by altering rates of protein synthesis and degradation. Thyroxine (T4) caused increases in protein degradation that were blocked by leupeptin, a proteinase inhibitor previously shown to inhibit Ca2+-dependent non-lysosomal proteolysis in these muscles. In addition, T4 abolished sensitivity to the lysosomotropic agent methylamine and the autophagy inhibitor 3-methyladenine, suggesting that T4 inhibits autophagic/lysosomal proteolysis. Topics: Adenine; Animals; Calcium; Dantrolene; Female; Leupeptins; Methylamines; Muscle Proteins; Muscles; Potassium; Rats; Rats, Inbred Strains; Thyroxine | 1986 |
Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte.
Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment. Topics: Animals; Asialoglycoprotein Receptor; Bile; Biological Transport, Active; Cell Compartmentation; Galactose Oxidase; Humans; Immunoglobulin A; Immunoglobulin Fragments; Leupeptins; Ligands; Liver; Lysosomes; Male; Protein Binding; Rats; Rats, Inbred Strains; Receptors, Fc; Receptors, Immunologic; Secretory Component; Species Specificity; Time Factors | 1986 |
Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein.
Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out. Topics: 4-Chloromercuribenzenesulfonate; Cathepsin B; Cathepsins; Dithioerythritol; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Leucine; Leupeptins; Myelin Basic Protein; Protease Inhibitors | 1985 |
Effect of proteases on the beta-thromboglobulin radioimmunoassay.
Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of beta-thromboglobulin and platelet factor 4. The initial assays indicated that a beta-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the beta-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of alpha 1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. Topics: Animals; Aprotinin; Beta-Globulins; beta-Thromboglobulin; Histamine Release; Leupeptins; Mast Cells; p-Methoxy-N-methylphenethylamine; Pancreatic Elastase; Peptide Hydrolases; Platelet Factor 4; Radioimmunoassay; Rats; Rats, Inbred Strains | 1985 |
Various proteinase inhibitors decrease prolactin and growth hormone release by anterior pituitary cells.
Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated. Topics: Animals; Aprotinin; Cells, Cultured; Dose-Response Relationship, Drug; Female; Growth Hormone; Leupeptins; Oligopeptides; Phenylmethylsulfonyl Fluoride; Pituitary Gland, Anterior; Prolactin; Protease Inhibitors; Rats; Trypsin Inhibitor, Kunitz Soybean | 1985 |
Regulation of ppp(A2'p)nA-dependent RNase levels during interferon treatment and cell differentiation.
The intracellular effector oligonucleotides ppp(A2'p)nA (n = 2- greater than or equal to 4) regulate the breakdown of RNA by activating ppp(A2'p)nA-dependent RNase. Cellular levels of this RNase were demonstrated to be regulated during differentiation of murine embryonal carcinoma cells. An induction of this RNase by interferon was demonstrated in each of three differentiated cell types (F9 clone 9, PYS, and PSA 5E) by analyzing rRNA breakdown following the introduction of ppp(A2'p)nA into the intact cells. In contrast, in three undifferentiated embryonal carcinoma cell lines (F9, PC13 clone 5, and Nulli 2A) there was little if any ppp(A2'p)nA-dependent RNase either with or without interferon pretreatment. These results were confirmed by affinity labeling of the RNase in cell-free systems. Addition of the proteinase inhibitor, leupeptin, to the cell lysis buffer was necessary to stabilize the RNase against cleavage to discrete breakdown products. Moreover, during differentiation of PC13 clone 5 cells by retinoic acid and N6,O2'-dibutyryl-adenosine 3',5'-monophosphate there was a gradual induction of ppp(A2'p)nA-dependent RNase. The expression of this RNase is, therefore, greatly enhanced during cell differentiation. In addition, the double-stranded-RNA-dependent protein kinase was investigated and was found to be interferon-inducible in all of the cell lines regardless of the state of cell differentiation. Topics: Affinity Labels; Animals; Cell Differentiation; Cell Line; Endoribonucleases; Interferons; Leupeptins; Mice; Neoplasms, Germ Cell and Embryonal; Protein Kinases; Ribonucleases | 1985 |
Leupeptin as a tool for the detection of the sites of catabolism of rat high-density lipoprotein apolipoproteins A-I and E.
Leupeptin, an inhibitor of lysosomal cathepsin activity, was injected intravenously into male rats. Tissues obtained from leupeptin-treated animals showed a depressed cathepsin activity when compared with tissues from saline-treated control animals. Leupeptin treatment did not change the hepatic activities and subcellular distribution of marker enzymes for mitochondria, microsomes and plasma membranes. Hepatic lysosomal cathepsin activity was specifically inhibited, but the subcellular distribution of all lysosomal marker enzymes tested was changed, indicating the occurrence of enlarged lysosomes in the leupeptin-treated animals. No significant differences were observed in the serum concentrations of protein, cholesterol, cholesteryl esters, phospholipids and apolipoproteins A-I, A-IV and E between leupeptin-treated rats and control animals. When radioiodinated asialofetuin was injected intravenously, the radiolabel was retained for an extended period of time in the liver of leupeptin-treated animals, indicating diminished catabolism of this protein in the liver. When rat high-density lipoprotein, labelled specifically in the apolipoprotein A-I or E moiety was injected intravenously, only the kidneys and the liver showed a leupeptin-induced accumulation of radioactivity. These studies provide evidence for an important contribution of the kidneys and the liver to the in vivo catabolism of high-density lipoprotein apolipoproteins, using a method completely different from sugar-containing labelling compounds. Topics: alpha-Fetoproteins; Animals; Apolipoprotein A-I; Apolipoproteins A; Apolipoproteins E; Asialoglycoproteins; Cathepsins; Fetuins; Kidney; Leupeptins; Lipoproteins, HDL; Liver; Lysosomes; Male; Oligopeptides; Rats; Rats, Inbred Strains; Serum Albumin; Time Factors | 1985 |
Effects of lysosomal inhibitors on 125I-insulin and 125I-asialofetuin degradation by the isolated, perfused rat liver and isolated rat hepatocytes.
To further evaluate the role of the lysosomal system in insulin degradation, we have compared the effects of inhibitors of lysosomal function on the degradation of 125I-insulin with 125I-asialofetuin, a lysosomally targeted molecule, by the intact, perfused rat liver and the isolated rat hepatocyte. The inhibitors employed were chloroquine (125 microM), NH4Cl (10 mM), and leupeptin (50 micrograms/ml). In the intact, perfused liver the observed inhibition of 125I-asialofetuin degradation at 30 min was as follows: chloroquine, 38%; NH4Cl, 32%; and leupeptin, 86%. Chloroquine also inhibited 125I-insulin degradation in the intact, perfused liver (29%), but NH4Cl and leupeptin had no effect. Using the isolated hepatocyte, the observed values for inhibition of 125I-asialofetuin at 60 min were: chloroquine, 85%; NH4Cl, 76%; and leupeptin, 81%. Chloroquine produced a 28% inhibition of 125I-insulin degradation, while NH4Cl and leupeptin had no effect. Chloroquine and NH4Cl decreased cell-associated radioactivity when isolated hepatocytes were incubated with 125I-asialofetuin (leupeptin had no effect), whereas chloroquine caused a 107% increase in cell-associated radioactivity when 125I-insulin was added to the incubation media (NH4Cl and leupeptin had no effect). These results indicate that the effects of chloroquine on insulin degradation are an extralysosomal action and that lysosomes appear not to be involved in the physiologic degradation of the insulin molecule. Topics: alpha-Fetoproteins; Ammonium Chloride; Animals; Asialoglycoproteins; Chloroquine; Fetuins; In Vitro Techniques; Insulin; Iodine Radioisotopes; Leupeptins; Liver; Lysosomes; Male; Perfusion; Rats; Rats, Inbred Strains | 1985 |
The effects of cytotropic compounds on the resialylation of human asialotransferrin type 3 in the rat.
Effects of chloroquine, colchicine, leupeptin, taxol and vinblastine on the resialylation and degradation of human [125I]asialotransferrin type 3 were studied in rats. An improved experimental technique was applied that permitted the quantification of resialylated ligand produced by individual animals over 3 h by using deconvolution. All three microtubule inhibitors increased the proportion of the dose undergoing resialylation by 35-39%. In addition, colchicine, and, especially, vinblastine enhanced the overall recovery of the dose as protein-bound 125I. The dose recovery was also augmented by leupeptin without any concomitant change in resialylation. Chloroquine suppressed resialylation and this effect could only be partially lifted by the administration of colchicine. The blood of colchicine-treated rats possessed no resialylating activity toward the ligand even when supplemented with additional alkaloid in vitro. The observations support the view that the respective fractions of the ligand destined for resialylation and degradation can, to a certain extent, be varied independently of each other. The effects of short-term starvation (20 h) and refeeding (4 h) on these processes are also presented. Topics: Alkaloids; Animals; Asialoglycoproteins; Chloroquine; Colchicine; Female; Humans; Leupeptins; Liver; Lysine; Male; Microtubules; Paclitaxel; Protein Biosynthesis; Rats; Starvation; Transferrin; Vinblastine | 1985 |
Insulin processing and action in adipocytes: evidence for generation of insulin-containing vesicles by leupeptin and monensin.
Incubation of adipocytes with 125I-insulin plus leupeptin or monensin, but not chloroquine, resulted in the appearance of a novel peak of 125I-insulin (modal density about 1.20 g/ml) on density gradient centrifugation; the appearance of the peak depended on the presence of specific insulin receptors on the cell surface. The fractions comprising this peak contained vesicles, probably originating from the Golgi apparatus, and dit not appear to be contaminated with lysosomes, mitochondria or plasma membrane. Entrapment of insulin in these vesicles per se did not prevent the activation of glucose transport, acetyl-CoA carboxylase or pyruvate dehydrogenase by insulin. Topics: Acetyl-CoA Carboxylase; Adipose Tissue; Animals; Centrifugation, Density Gradient; Deoxyglucose; Enzyme Activation; Female; Furans; Insulin; Leupeptins; Microscopy, Electron; Monensin; Oligopeptides; Organoids; Pyruvate Dehydrogenase Complex; Rats; Rats, Inbred Strains | 1985 |
Inhibition of plant cell proteolytic activities that degrade tubulin.
The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity. Topics: Alkaloids; Electrophoresis, Polyacrylamide Gel; Immunosorbent Techniques; Leupeptins; Molecular Weight; Paclitaxel; Pepstatins; Plants; Protease Inhibitors; Tubulin | 1985 |
Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions.
When normal human fibroblasts are brought to a steady state with 125I-labeled epidermal growth factor (125I-EGF), greater than 90% of the radioactivity is intracellular. We investigated this material to determine whether the 125I-EGF is intact or degraded. Our results show that 125I-EGF is rapidly processed after internalization and can be resolved into four peaks by native gel electrophoresis. These different forms were isolated and tested for their ability to bind to cell-surface EGF receptors. The first processed form was fully capable of binding to EGF receptors, but the second processed form could not. The third form was a collection of small degradation products. We calculated that at steady state about 60% of internalized "125I-EGF" was in a form still able to bind to EGF receptors. We then investigated the ability of different reported inhibitors of EGF "degradation" to block the processing of EGF. Although inhibitors of cathepsin B (leupeptin, antipain, N alpha-p-tosyl-L-lysine chloromethyl ketone, and chymostatin) were able to inhibit the release of monoiodotyrosine from treated cells in a time- and concentration-dependent manner, they had little effect on the processing step that apparently inactivates 125I-EGF. In contrast, agents that raised intravesicular pH, such as methylamine and monensin, inhibited the initial steps in EGF processing as well as the later steps. Low temperatures inhibited the transfer of 125I-EGF to the lysosomes and inhibited the conversion of EGF to a nonbindable form, but had little effect on the initial processing. We conclude that the intracellular processing of EGF is a multistep process that is initiated prior to lysosomal fusion, involves cathepsin B activity, and requires an acidic pH. In addition, many of the protease inhibitors that have been utilized to investigate the role of EGF degradation in mitogenesis do not block the conversion of EGF to a form that is apparently unable to interact with its receptor. Topics: Animals; Cathepsin B; Cathepsins; Cell Line; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Hydrogen-Ion Concentration; Leupeptins; Methylamines; Mice; Models, Biological; Monensin; Receptors, Cell Surface | 1985 |
Leupeptin, a protease inhibitor, blocks insemination-induced flight muscle histolysis in the fire ant Solenopsis.
The effect of the protease inhibitor leupeptin on flight muscle histolysis in queen fire ants was studied by electron microscopy. In untreated animals artificially inseminated, muscle involution was apparent at 6 hr post-insemination and complete by 24 hr post-insemination. However, in animals pre-treated with leupeptin and subsequently artificially inseminated, no morphologic evidence of flight muscle breakdown was seen at any interval between 6 and 24 hr post-insemination. Such information appears to indicate that one or more proteases are involved in the process of insemination-induced muscle atrophy in fire ants. The most likely candidate is a soluble, calcium-activated myofilament-associated protease. Topics: Animals; Ants; Atrophy; Calpain; Cytoskeleton; Endopeptidases; Female; Insemination; Leupeptins; Muscle Proteins; Muscles; Oligopeptides; Protease Inhibitors; Wings, Animal | 1985 |
A human platelet angiotensin I-processing system. Identification of components and inhibition of angiotensin-converting enzyme by product.
Mechanisms controlling the local generation of angiotensin II by vascular tissue are incompletely understood. Human platelets were examined for their ability to metabolize angiotensin I. Platelet-dependent angiotensin I metabolism was detected by a high performance liquid chromatography assay which allowed quantitation of angiotensin I substrate utilized and products formed. The major product of platelet-dependent angiotensin I metabolism was identified as des-Leu10-angiotensin I. The platelet des-Leu10-angiotensin I-generating activity had a pH optimum of 6.0-6.5 and was inhibited 100% by mersalyl acid (10(-4) M), 86% by leupeptin (10(-4) M), and 95% by iodoacetamide (10(-2) M). The activity had an approximate Mr = 70,000 as determined by Sephacryl S-200 gel filtration. Intact human platelets stimulated with calcium ionophore (1-10 microM) released 13.7-30.8% of the des-Leu10-angiotensin I-generating activity. Des-Leu10-angiotensin I, the major product of platelet angiotensin I metabolism, inhibited human serum and purified rabbit lung angiotensin-converting enzymes with an I50 of 3.7 X 10(-6) and 2.0 X 10(-6) M, respectively. These results suggest that the platelet may control local angiotensin II formation at vascular sites both by metabolism of the precursor peptide angiotensin I and by generation of an endogenous angiotensin-converting enzyme inhibitor, des-Leu10-angiotensin I. This platelet-dependent pathway may contribute to the control of local levels of vasoactive peptides, such as bradykinin and angiotensin II, so as to alter local tissue blood flow. Topics: Angiotensin I; Angiotensin-Converting Enzyme Inhibitors; Angiotensins; Animals; Blood Platelets; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Hydrogen-Ion Concentration; Iodoacetamide; Leupeptins; Lung; Mersalyl; Molecular Weight; Rabbits | 1985 |
Inhibition of fibrinogen receptor expression and serotonin release by leupeptin and antipain.
Human platelet agonists such as thrombin, ADP, and collagen stimulate the rapid expression of fibrinogen receptors. In other cell types, calcium-activated proteases have been suggested to participate in the mechanism of expression of cell surface receptors (Lynch, G., and Baudry, M. (1984) Science 224, 1057-1063). In platelets the majority of the neutral protease activity is calcium-activated protease. We examined the effects of leupeptin and antipain, two calcium-activated protease inhibitors, on the expression of platelet fibrinogen receptors. These inhibitors abolished thrombin and ADP-induced fibrinogen binding. This inhibition required the addition of leupeptin or antipain prior to the agonist and was not due to displacement of fibrinogen from its receptor or inhibition of agonist binding to platelets. Leupeptin and antipain also inhibited fibrinogen-independent thrombin-stimulated release of serotonin. These results are discussed in relation to the involvement of calcium-activated protease in early events of platelet activation. Topics: Antipain; Binding, Competitive; Blood Platelets; Fibrinogen; Humans; Kinetics; Leupeptins; Oligopeptides; Platelet Membrane Glycoproteins; Receptors, Cell Surface; Serotonin; Thrombin | 1985 |
Activation of the PCSM-protein phosphatase by a Ca2+-dependent protease.
The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCSM) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca2+-dependent protease. The enzyme-directed protease effect to which the PCSH and PCSL phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase alpha activity ratio. The Ca2+-dependent protease is completely inhibited by EGTA or leupeptin. Topics: Animals; Calpain; Egtazic Acid; Enzyme Activation; Kinetics; Leupeptins; Muscles; Phosphoprotein Phosphatases; Rabbits | 1985 |
Effects of proteinase inhibitors on preimplantation embryos in the rat.
Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis. Topics: Animals; Anti-Bacterial Agents; Antipain; Blastocyst; Cystatins; Embryonic Development; Enzyme Inhibitors; Female; Glycopeptides; Leupeptins; Oligopeptides; Pepstatins; Peptides; Pregnancy; Protease Inhibitors; Rats; Rats, Inbred Strains | 1985 |
Pharmacological dissociation of memory: anisomycin, a protein synthesis inhibitor, and leupeptin, a protease inhibitor, block different learning tasks.
Inhibition of protein synthesis by anisomycin for a short duration impairs memory of a one-trial inhibitory avoidance task in rats. Memory of escape conditioning involving eight trials is disrupted only if the duration of protein synthesis is prolonged by repeated injections. In marked contrast, olfactory memory of rats trained on two odor discriminations is not affected by anisomycin even if the duration of inhibition is prolonged and the number of trials is reduced to a minimum. In previous work, leupeptin, a thiol proteinase inhibitor, was shown to impair olfactory discrimination learning, but left inhibitory and avoidance conditioning intact. Together, these results provide a pharmacological double dissociation of memory, and suggest that the same chemistries, or mixtures of chemistries, may not be involved in all types of memory. Topics: Animals; Anisomycin; Avoidance Learning; Discrimination Learning; Injections, Subcutaneous; Leupeptins; Male; Memory; Olfactory Pathways; Oligopeptides; Protease Inhibitors; Pyrrolidines; Rats; Rats, Inbred Strains | 1985 |
Regulation of protein breakdown by epidermal growth factor in A431 cells.
Addition of epidermal growth factor (EGF) to cultures of A431 human epidermoid carcinoma cells produces an increase in the rate of intracellular protein breakdown that cannot be accounted for by increased proteolysis in lysates from EGF-treated cells. In support of this observation, inhibition of protein synthesis with cycloheximide does not reduce the EGF response in cell monolayers. On the other hand, inhibitors of lysosomal proteolytic function such as leupeptin, vinblastine and especially the weak base, ammonia, are able to block the ability of EGF to increase protein breakdown. Additional results suggest that the EGF effect is mediated via a stimulation of autophagy. First, the autophagocytosis inhibitor, 3-methyladenine, reduces the EGF response, and second, the ability of insulin to inhibit protein breakdown by preventing the formation of autophagic vacuoles is overcome by EGF. Moreover, the actions of inhibitors and competing hormones are similar to those reported for glucagon, a hormone known to increase autophagy. The EGF response on protein breakdown persists for at least 6 h after thorough washing of the A431 monolayers. This result contrasts with the rapid reversal of EGF effects in other cell lines. Examination of the fate of bound EGF in cells washed and incubated for 2 h at 37 degrees C shows that some 500-fold more EGF per mg protein is retained on the surface of A431 cells compared to AG2804-transformed fibroblasts, a difference which probably explains the unusual persistence of the EGF effect on protein breakdown. Topics: Adenine; Ammonium Chloride; Autolysis; Carcinoma, Squamous Cell; Cell Line; Cycloheximide; Epidermal Growth Factor; Fibroblasts; Humans; Hydrogen-Ion Concentration; Insulin; Leupeptins; Lung; Proteins; Time Factors; Vinblastine | 1985 |
[Inhibition of extracellular yeast proteinases by pepstatin A and leupeptin in the plate test].
Topics: Agar; Blastomyces; Candida; Depression, Chemical; Endopeptidases; Geotrichum; Leupeptins; Mitosporic Fungi; Oligopeptides; Pepstatins; Species Specificity | 1985 |
Lysosomal digestion of thyroglobulin: role of cathepsin D and thiol proteases.
Purified hog thyroid lysosomes, prepared by a procedure previously developed in this laboratory, were used to study lysosomal digestion of [131I]thyroglobulin [131I]Tg). The lysosomal proteases were solubilized with 0.1% Triton X-100. Rates of proteolytic digestion, measured by the release of ethanol-ammonium acetate-extractable 131I, were greatly stimulated by thiol reagents. The pH optimum was also affected by the presence of thiols. In the absence of a thiol reagent, a broad pH optimum was observed, ranging from 3.5-4.5. However, in the presence of 1 mM mercaptoethanol, the maximum rate of digestion occurred at pH 5.0, very close to reported values for the internal pH of lysosomes. Pepstatin, an inhibitor of cathepsin D, markedly inhibited lysosomal digestion of [131I]Tg at concentrations as low as 0.01 micrograms/ml. Its inhibitory effect was greater at pH 3.5 (pH optimum of cathepsin D) than at pH 5.0. Leupeptin, an inhibitor of thiol proteases, was not as potent as pepstatin, but it was significantly inhibitory at a concentration of 1 microgram/ml. In contrast to pepstatin, leupeptin displayed a greater inhibitory effect at pH 5.0 than at pH 3.5. The pH optimum of hog thiol proteases has been reported to range from 5.5-6.5. The effects of the two inhibitors were additive at pH 5.0. We conclude from these results that both cathepsin D and thiol proteases play a role in lysosomal digestion of Tg. Cathepsin D appears to be quantitatively more important than thiol protease in the initial phase of the digestion. The stimulatory effect of thiols on lysosomal digestion of [131I]Tg probably involves two separate effects: 1) stimulation of thiol proteases, and 2) reduction of S-S bonds in Tg, making the protein more susceptible to attack by proteolytic enzymes. Poorly iodinated [131I]Tg was more rapidly hydrolyzed than well iodinated [131I]Tg, based on the release of ethanol-ammonium acetate-extractable 131I. However, there was little or no difference in the rate of total peptide bond cleavage between poorly iodinated and well iodinated Tg. These results suggest that the first sites of iodination of Tg are preferentially attacked by lysosomal proteases. Long term (24-h) digestion of [131I]Tg with solubilized thyroid lysosomes at pH 5.0 in the presence of thiol compounds was just as effective as digestion with pronase at pH 8.0 in liberating free 131I-labeled iodothyronines and 131I-labeled iodotyrosines. Thus, thyroid lysosomes contain the full complement of Topics: Animals; Cathepsin D; Cysteine Endopeptidases; Endopeptidases; Hydrogen-Ion Concentration; Iodine; Leupeptins; Lysosomes; Mercaptoethanol; Monoiodotyrosine; Pepstatins; Pronase; Protease Inhibitors; Swine; Thyroglobulin | 1985 |
Schistosoma japonicum: biochemistry and cytochemistry of dipeptidyl aminopeptidase-II-like activity in adults.
The biochemical characterization of dipeptidyl aminopeptidase II activity was investigated in the supernatant of centrifuged homogenates of adult Schistosoma japonicum using a lysine-alanine oligopeptide derivative of 4-methoxy-2-naphthylamide as a substrate. It was observed that the pH optimum of the enzyme is in the acid range, with an optimum at pH 6.3. Time and enzyme concentration studies, along with temperature studies, support the premise that the reaction is enzymatic. The Km was 3.3 X 10(-3) M, at pH 5.5 and 37 C. Tris and diisopropyl phosphofluoridate, when incorporated into the assay system at final concentrations of 500 and 2 mM, respectively, significantly inhibited the reaction by 70.9 and 75%, respectively. Leupeptin (5 X 10(-4) mM) had no effect. The results indicate that the enzyme under study in the present investigation strongly resembles mammalian dipeptidyl aminopeptidase II due to its affinity for substrate, sensitivity to Tris and diisopropyl phosphofluoridate inhibition, and pH optimum. Its inhibition by diisopropyl phosphofluoridate indicates that it may belong to the serine class of proteases. Cytochemical studies revealed reaction product in the lipid-like globules in the gastrodermis, adding further credence that these globules are lysosomal. Topics: Animals; Cecum; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Hot Temperature; Hydrogen-Ion Concentration; Isoflurophate; Kinetics; Leupeptins; Octoxynol; Polyethylene Glycols; Schistosoma japonicum | 1985 |
Isolation of two forms of the high-molecular-mass serine protease, ingensin, from porcine skeletal muscle.
Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin. Topics: Animals; Antipain; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Coumarins; Cysteine Endopeptidases; Endopeptidases; Isoenzymes; Leucine; Leupeptins; Multienzyme Complexes; Muscles; Oligopeptides; Pepstatins; Proteasome Endopeptidase Complex; Sodium Dodecyl Sulfate; Swine | 1985 |
Degradation of short- and long-time labelled proteins by rat liver lysosomes.
Topics: Animals; Carbon Radioisotopes; Fructose-Bisphosphate Aldolase; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Male; Microscopy, Electron; Peptide Hydrolases; Proteins; Radioisotope Dilution Technique; Rats | 1985 |
Comparison of proteolysis in perfused rat liver and in isolated autophagic vacuoles.
Topics: Animals; Autophagy; Chloroquine; Leupeptins; Liver; Lysosomes; Microscopy, Electron; Organoids; Peptide Hydrolases; Phagocytosis; Rats; Vacuoles | 1985 |
The role of lysosomes in the degradation of myofibrillar and non-myofibrillar proteins in heart.
Lysosomes are presumed to be involved in protein degradation in heart, but their exact role is poorly understood. Several interventions that are known to alter cardiac proteolysis (e.g., insulin) also produce lysosomal changes that might account for the observed changes in protein degradation; but many other interventions appear not to do so. Agents that interfere with lysosomal function (e.g., sucrose, chloroquine, methyladenine, leupeptin) cause a 25% reduction in the rate of degradation of total protein in fetal mouse hearts in organ culture; however, in the same hearts the rate of degradation of myosin and other myofibrillar proteins remains unchanged. Thus, it appears that lysosomes are involved in cardiac proteolysis, but may not play a rate-limiting or regulatory role in many circumstances. The regulation of proteolysis by insulin appears to involve non-lysosomal pathways in addition to any lysosomal alterations it may cause. Furthermore, the initial cleavage of myofibrillar proteins appears no to be dependent on normal lysosomal function. Topics: Adenine; Animals; Chloroquine; Fetus; Insulin; Kinetics; Leupeptins; Lysosomes; Mice; Myocardium; Myofibrils; Proteins | 1985 |
Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity.
The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation. Topics: 2,4-Dinitrophenol; Ammonium Chloride; Anti-Bacterial Agents; Biological Transport; Cells, Cultured; Dinitrophenols; Fibroblasts; Humans; Insulin; Iodine Radioisotopes; Kinetics; Leupeptins; Lysosomes; Pinocytosis; Povidone-Iodine; Skin; Sodium Fluoride | 1985 |
Biosynthesis and intracellular transport of alpha-glucosidase and cathepsin D in normal and mutant human fibroblasts.
In order to study the intracellular localization of the proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D in cultured human skin fibroblasts we have used incubation with glycyl-L-phenylalanine-beta-naphthylamide (Gly-Phe-NH-Nap) as described by Jadot et al. [Jadot, M., Colmant, C., Wattiaux-de Coninck, S. & Wattiaux, R. (1984) Biochem. J. 219,965-970] for the specific lysis of lysosomes. When a homogenate of fibroblasts was incubated for 20 min with 0.5 mM Gly-Phe-NH-Nap, a substrate for the lysosomal enzyme cathepsin C, the latency of the lysosomal enzymes alpha-glucosidase and beta-hexosaminidase decreased from 75% to 10% and their sedimentability from 75% to 20-30%. In contrast, treatment with Gly-Phe-NH-Nap had no significant effect on the latency of galactosyltransferase, a marker for the Golgi apparatus, and on the sedimentability of glutamate dehydrogenase and catalase, markers for mitochondria and peroxisomes, respectively. The maturation of alpha-glucosidase and cathepsin D in fibroblasts was studied by pulse-labelling with [35S]methionine, immunoprecipitation, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and fluorography. When homogenates of labelled fibroblasts were incubated with Gly-Phe-NH-Nap prior to immunoprecipitation, 70-80% of all proteolytically processed forms of metabolically labelled alpha-glucosidase and cathepsin D was recovered in the supernatant. The earliest proteolytic processing steps in the maturation of alpha-glucosidase and cathepsin D appeared to be coupled to their transport to the lysosomes. Although both enzymes are transported via the mannose-6-phosphate-specific transport system, the velocity with which they arrived in the lysosomes was consistently different. Whereas newly synthesized cathepsin D was found in the lysosomes 1 h after synthesis, alpha-glucosidase was detected only after 2-4 h. When a pulse-chase experiment was carried out in the presence of 10 mM NH4Cl there was a complete inhibition of the transport of cathepsin D and a partial inhibition of that of alpha-glucosidase to the lysosomes. Leupeptin, an inhibitor of lysosomal thiol proteinases, had no effect on the transport of labelled alpha-glucosidase to the lysosomes. However, the early processing steps in which the 110-kDa precursor is converted to the 95-kDa intermediate form of the enzyme were delayed, a transient 105-kDa form was observed and the conversion of the 95-kDa intermedia Topics: alpha-Glucosidases; Ammonium Chloride; Biological Transport; Cathepsin D; Cell Line; Centrifugation, Density Gradient; Dipeptides; Fibroblasts; Glucosidases; Humans; Immunochemistry; Leupeptins; Lysosomes; Mutation; Organoids; Skin | 1985 |
Acidic lens protein degrading activity in bovine ciliary body.
Biochemical studies of acidic lens protein degrading activity in the bovine ciliary body were performed. The activity showed two peaks, Fractions A and B, on Sephadex G-75 column chromatography. Fraction A contained cathepsin D and thiol proteinase activities and was inhibited by pepstatin and leupeptin. Fraction B contained thiol proteinase activity and was inhibited by leupeptin. The proportion of peptide released from the lens protein by Fractions A and B was higher than those from bovine serum albumin and casein. Lens protein may be a good assay substrate for cathepsin D and lysosomal thiol proteinase in the ciliary body. Topics: Animals; Cattle; Chromatography; Ciliary Body; Crystallins; Cysteine; Hydrogen-Ion Concentration; Leupeptins; Pepstatins; Protease Inhibitors | 1985 |
Diacylglycerol lipase activity in human platelet intracellular and surface membranes. Some kinetic properties and fatty acid specificity.
Diacyl glycerol lipase activity has been examined of intracellular and surface membranes isolated from human blood platelets by free flow electrophoresis. Enzyme activity is present on both membranes but is activated at different substrate concentrations (Km 14 microM and 140 microM for intracellular and surface membrane, respectively). Both enzyme activities are stimulated by EGTA and GSH, and inhibited by added Ca2+. The specificity of the intracellular membrane enzyme has been investigated using a range of diacylglycerol substrates differing only in their '2' position fatty acid. Arachidonic acid is clearly the preferred '2' position moiety with activities towards eicosatrienoic, linoleic, oleic and palmitic acid-containing substrates, all substantially lower. Topics: Blood Platelets; Cell Membrane; Centrifugation, Density Gradient; Egtazic Acid; Fatty Acids; Glutathione; Humans; Intracellular Membranes; Kinetics; Leupeptins; Lipoprotein Lipase; Substrate Specificity; Trifluoperazine | 1985 |
Platelet cytoskeleton alpha-actinin in normal and thrombasthenic platelets: distribution and immunologic characterization.
The subcellular localization of alpha-actinin (Mr 100,000) in human skeletal muscle is restricted to the Z line, in which it is believed to anchor actin filaments. Recently, this protein was identified in normal and thrombasthenic human platelets by its antigenic cross-reaction with antibodies to chicken gizzard alpha-actinin. In our study, the biochemical interaction between purified platelet alpha-actinin and striated muscle F-actin was examined by electron microscopy of negatively stained preparations. Like its muscle counterpart, platelet alpha-actinin promotes the cross-linking and bundling of actin filaments. Antibodies prepared to human platelet alpha-actinin cross-reacted with chicken gizzard alpha-actinin as shown by immunoelectrophoresis and the western blotting technique. Immunoblots prepared with normal and thrombasthenic platelets with antibodies to human platelet alpha-actinin revealed that this protein is susceptible to proteolysis. Extracts of freshly drawn platelets showed a protein band of 100 K. When the platelet extracts were incubated at 37 degrees C for various times, the immunoblots showed protein bands of 100 and 80 K. The proportion of the 80 K protein band increased with incubation time. This proteolysis can be prevented by chelating agents such as EDTA or the protease inhibitor leupeptin. Indirect immunofluorescent studies of human skin fibroblasts with antibodies to chicken gizzard actin and human skeletal muscle, chicken gizzard, and platelet alpha-actinin revealed the staining pattern characteristic of each protein. The distribution of alpha-actinin in normal and thrombasthenic platelets was assessed by ferritin-labeled immunoelectron microscopy. Ferritin particles were found in the cytoplasm immediately below the membrane and in some granules. There was no labeling associated with the mitochondria. Topics: Actinin; Animals; Blood Platelet Disorders; Blood Platelets; Cross Reactions; Edetic Acid; Fibroblasts; Humans; Immunoelectrophoresis; Leupeptins; Microscopy, Electron; Muscle, Smooth; Rabbits; Skin | 1985 |
A study of the low beta-galactosidase activity in cystinotic fibroblasts: effects of cysteamine.
beta-Galactosidase activity but not beta-glucuronidase, N-acetyl-beta-D-galactosaminidase or arylsulphatase A activity, is known to be significantly lower in cultured human skin fibroblasts from patients with cystinosis than in cells from control subjects. Incubation of cell homogenates with disulphide or thiol compounds did not affect beta-galactosidase activity, suggesting that decreased beta-galactosidase activity in affected cells was not caused by the presence of inhibiting substances or absence of activating substances. Incubating cells with 0.5 or 1.0 mmol/l cysteamine, a substance used in the clinical treatment of cystinosis because it depletes cells of excess cystine, greatly decreased beta-galactosidase activity in both cystinotic and normal cells. This effect is shown to result from enzyme instability in lysosomes with raised pH and increased thiol concentration. Thus, cysteamine, although effective in depleting cystinotic cells of excess cystine, may have the undesired side-effect of severely decreasing lysosomal beta-galactosidase. Topics: beta-Galactosidase; Cells, Cultured; Cysteamine; Cystinosis; Fibroblasts; Galactosidases; Humans; Hydrolysis; Leupeptins | 1985 |
Effect of inhibitors of proteolysis and arachidonic acid metabolism on burn-induced protein breakdown.
A rat model has been developed to study the local effects of burn injury on the underlying muscle tissue. Protein turnover was measured in soleus muscle incubated in vitro in which both tyrosine release and protein synthesis was measured. A scald injury (3 seconds) to a small area of one hindlimb produces an increase in muscle proteolysis and is without effect on the soleus muscle of the contralateral leg. A very high concentration of indomethacin (40 mumol/L) had no effect on proteolysis in the control muscle but specifically inhibited burn-induced protein breakdown. However, since other cyclooxygenase inhibitors (aspirin and ibuprofen), lipoxygenase inhibitors (ETYA, NDGA, and esculetin), and mepacrine (a phospholipase inhibitor) had no effect on protein breakdown, it is unlikely that a product of arachidonic acid metabolism maintains the increased proteolysis in vitro. In addition, endogenous production of prostaglandin E2 (PGE2) was not different in muscles from burned and control legs. Probes of the proteolytic pathway using inhibitors show that the burn-induced stimulation of proteolysis is consistent with the stimulation of lysosomal protease activity. These results are supported by the observation of increased acid protease activity in muscle homogenates from the burned leg. The best hypothesis that explains these data is that a lysosomal pathway of protein degradation may be enhanced by burn. Products of arachidonic acid metabolism do not appear to maintain burn-induced proteolysis in muscle, although their role in initiating the pathological changes in vivo cannot be excluded. Topics: Animals; Arachidonic Acid; Arachidonic Acids; Aspirin; Burns; Hindlimb; Ibuprofen; In Vitro Techniques; Indomethacin; Leupeptins; Lysosomes; Muscles; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Proteins; Quinacrine; Rats; Rats, Inbred Strains; Tyrosine | 1985 |
Evidence for two pathways for stimulation of collagenolysis in bone.
The effect of parathormone (PTH), lipopolysaccharide (LPS), or interleukin-1 (IL-1) on calcium release and collagen degradation in bone was examined in vitro using labeled neonatal calvaria of normal mice and also of osteopetrotic microphthalmic (mi/mi) mice that have defective osteoclasts. All three agents stimulated calcium release from normal bone but not from mi/mi bone. PTH stimulated the degradation of both noncalcified and calcified collagen in normal bone as well as the degradation of noncalcified collagen in mi/mi bone. However, LPS and IL-1 only stimulated the degradation of calcified collagen in normal bone. One-half maximal stimulation of noncalcified collagen degradation in normal or mi/mi bone was achieved by about 3 nM PTH compared with about 1 nM PTH for that of calcium release from normal bone. While calcitonin (CT) and leupeptin inhibited calcium release and thereby the degradation of calcified collagen, neither agent inhibited PTH-stimulated noncalcified collagen degradation in normal or mi/mi bone. The data indicate the existence of two pathways that lead to collagen degradation in bone. One is intimately connected with the resorptive process stimulated by a variety of agents, and is probably mediated by osteoclasts. A second mechanism is sensitive only to PTH and appears to be associated with nonosteoclastic cells since it can operate under conditions in which osteoclasts are thought to be inactive or are inhibited. Topics: Acetazolamide; Animals; Bone and Bones; Bone Resorption; Calcitonin; Calcium; Cartilage; In Vitro Techniques; Interleukin-1; Leupeptins; Lipopolysaccharides; Mice; Osteoclasts; Parathyroid Hormone; Proline | 1985 |
Biochemical evidence for the presence of an actin protein in Tetrahymena pyriformis.
A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine . HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8-12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts. Topics: Actins; Animals; Chromatography, Affinity; Deoxyribonuclease I; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Leupeptins; Microscopy, Electron; Molecular Weight; Protein Binding; Tetrahymena pyriformis | 1985 |
Regulation of myofibrillar accumulation in chick muscle cultures: evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins.
The effect of calcium on myofibrillar turnover in primary chick leg skeletal muscle cultures was examined. Addition of the calcium ionophore A23187 at subcontraction threshold levels (0.38 microM) increased significantly rates of efflux of preloaded 45Ca+2 but had no effect on total protein accumulation. However, A23187 as well as ionomycin caused decreased accumulation of the myofibrillar proteins, myosin heavy chain (MHC), myosin light chain 1f (LC1f), 2f (LC2f), alpha-actin (Ac), and tropomyosin (TM). A23187 increased the degradation rate of LC1f, LC2f, and TM after 24 h. In contrast, the calcium ionophore caused decreased degradation of Ac and troponin-C and had no effect on the degradation of MHC, troponin-T, troponin-I, or alpha, beta-desmin (Dm). In addition, A23187 did not alter degradation of total myotube protein. The ionophore had little or no effect on the synthesis of total myotube proteins, but caused a marked decrease in the synthesis of MHC, LC1f, LC2f, Ac, TM, and Dm after 48 h. The mechanisms involved in calcium-stimulated degradation of the myofibrillar proteins were also investigated. Increased proteolysis appeared to involve a lysosomal pathway, since the effect of the Ca++ ionophore could be blocked by the protease inhibitor leupeptin and the lysosomotropic agents methylamine and chloroquine. The effects of A23187 occur in the presence of serum, a condition in which no lysosomal component of overall protein degradation is detected. The differential effect of A23187 on the degradative rates of the myofibrillar proteins suggests a dynamic structure for the contractile apparatus. Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Chickens; Chloroquine; Contractile Proteins; Leupeptins; Lysosomes; Methylamines; Muscle Proteins; Muscles; Myosins; Tropomyosin; Troponin | 1985 |
Molecular forms of GM2-activator protein. A study on its biosynthesis in human skin fibroblasts.
The biosynthesis and secretion of lysosomal GM2-activator was studied in fibroblasts from controls and patients of GM2 gangliosidosis metabolically labelled with [3H]-leucine. Immunoprecipitation was performed with affinity-purified antibodies to human kidney GM2-activator protein. Normal fibroblasts and fibroblasts of variant B and O of GM2 gangliosidosis secrete GM2-activator protein as a 24-kDa polypeptide, which is able to stimulate degradation of ganglioside GM2 by beta-hexosaminidase A in the in vitro assay. In the presence of 10mM NH4Cl the rate of secretion is twice as high as in normal fibroblasts. Intracellularly, GM2-activator protein is represented in these cell lines by polypeptides with apparent molecular masses ranging from 21 kDa-22.5 kDa. Under the same labelling conditions, in two cell lines of patients with variant AB of infantile GM2 gangliosidosis intracellularly only traces of GM2-activator were detectable, whereas significant amounts of polypeptides with molecular masses between 25 and 26.5 kDa could be precipitated from the media of these fibroblasts. Topics: Cells, Cultured; Endocytosis; Enzyme-Linked Immunosorbent Assay; Fibroblasts; G(M2) Activator Protein; G(M2) Ganglioside; Gangliosides; Gangliosidoses; Humans; Kinetics; Leucine; Leupeptins; Lysosomes; Pepstatins; Phenylmethylsulfonyl Fluoride; Protein Biosynthesis; Skin; Tritium | 1985 |
Stimulation of rat Sertoli cell adenylate cyclase by germ cells in vitro.
The effect of germ cells or germ cell fractions on adenylate cyclase (AC) activity in membrane preparations from cultured rat Sertoli cells has been examined. Whole germ cells or 30,000 X g pellet or supernatant fractions of germ cells have the ability to stimulate Sertoli cell AC to levels comparable to those measured in follicle-stimulating hormone-stimulated Sertoli cell membranes. Treatment at 100 degrees C but not 60 degrees C for 1 min abolished the ability of germ cell preparations to stimulate Sertoli AC. Germ cell stimulation of Sertoli cell AC was not calcium dependent, was not blocked by propranolol, and was observed to be dose dependent. Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cells, Cultured; Enzyme Activation; Follicle Stimulating Hormone; Germ Cells; In Vitro Techniques; Indomethacin; Isoproterenol; Leupeptins; Male; Propranolol; Rats; Sertoli Cells | 1985 |
Muscle damage induced by the ionophore A23187 can be prevented by prostaglandin inhibitors and leupeptin.
Frog skeletal muscle incubated in vitro with the ionophore A23187 shows extensive morphological alterations. Myofilament disruption, presumably mediated by excess intracellular calcium, can be partially prevented by preincubating the muscle with inhibitors of prostaglandin synthesis and the lysosomal thiol protease inhibitor leupeptin. Topics: Actin Cytoskeleton; Animals; Calcimycin; Calcium; Leupeptins; Muscle Contraction; Muscles; Oligopeptides; Prostaglandin Antagonists; Rana pipiens | 1985 |
Discrepancies in the catabolic pathways of human and rat high-density lipoprotein apolipoprotein A-I in the rat.
The in vivo metabolism in the rat of radioiodinated human and rat high-density lipoprotein was compared with a double-label procedure using 125I and 131I. While rat high-density lipoprotein showed a biphasic serum decay, human high-density lipoprotein was characterized by a monoexponential serum decay. No differences were observed between the serum decay of human high-density lipoprotein-2 and -3 subfractions, isolated by rate zonal ultracentrifugation. The catabolic sites of human and rat high-density lipoprotein were analysed using the lysosomal cathepsin inhibitor leupeptin. Radioiodinated rat high-density lipoprotein was catabolized by the kidneys and by the liver. In contrast, radioiodinated human high-density lipoprotein was catabolized almost exclusively in the liver. No difference in the catabolic sites of human high-density lipoprotein-2 and -3 subfractions was observed. The catabolic sites of human high-density lipoprotein apolipoprotein A-I in the rat were further analysed using the O-(4-diazo-3-[125I]iodobenzoyl) sucrose label. Compared with rat high-density lipoprotein apolipoprotein A-I, the kidneys played a minor role in the catabolism of human high-density lipoprotein apolipoprotein A-I. It is concluded that in the rat the catabolic pathways of the apolipoprotein A-I moieties of rat and human high-density lipoproteins are different, indicating that homologous high-density lipoproteins should be used for the investigation of in vivo metabolism. Topics: Animals; Apolipoprotein A-I; Apolipoproteins A; Humans; Kidney; Kinetics; Leupeptins; Lipoproteins, HDL; Liver; Male; Rats; Rats, Inbred Strains; Species Specificity; Tissue Distribution | 1985 |
Inhibition of the growth of human coronavirus 229E by leupeptin.
The protease inhibitor leupeptin prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells. The IC50 of leupeptin in plaque reduction tests was 0.4 micrograms/ml, whereas growth of host cells was unaffected by leupeptin at 50 micrograms/ml. Inhibition of plaque formation could be prevented by the addition of proteases to the overlay medium. In single-cycle growth experiments, leupeptin reduced virus yield only if added within 2 h of infection, indicating its action on an early stage of virus replication. Topics: Cell Line; Coronaviridae; Humans; Leupeptins; Oligopeptides; Protease Inhibitors; Viral Plaque Assay; Virus Replication | 1985 |
Effect of amino acids and cycloheximide on changes caused by vinblastine, leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo.
The number of autophagic vacuoles in hepatocytes of 24 h fasted mice in vivo increased manyfold following the administration of vinblastine, leupeptin and methylamine. The effect of each chemical is characterized by the predominance of a certain kind of vacuole. Vinblastine treatment is accompanied by a large proportion of vacuoles containing morphologically unaltered organelles, leupeptin causes preferential accumulation of dense and complex vacuoles, methylamine administration produces mostly large, electron-lucent, swollen vacuoles. The amounts of segregated and accumulated cytoplasmic material, expressed as percentage cytoplasm per hour, were 0.84%, 2.08% and 0.74% following vinblastine, leupeptin and methylamine treatment respectively. The actual rate of segregation was probably higher than this. Inhibition of degradation of the sequestered cytoplasmic material is proposed to be a main factor in the increase in the size of the autophagic/lysosomal compartment. Treatment with cycloheximide or exogenously added mixture of amino acids cut down the size of the autophagic/lysosomal system in control cells and strongly inhibited the accumulation caused by vinblastine, leupeptin and methylamine. Topics: Amino Acids; Animals; Autophagy; Cycloheximide; Leupeptins; Liver; Lysosomes; Male; Methylamines; Mice; Organoids; Phagocytosis; Proteins; Vacuoles; Vinblastine | 1985 |
Formation of a 55 000-weight cross-linked beta crystallin dimer in the Ca2+-treated lens. A model for cataract.
Incubation of lens in Ca2+-containing media, considered by several investigators to be a useful model of cataract formation, gave rise to significant alterations in the covalent structures of various proteins. In rabbit lens, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used after reduction of disulfides in urea, the most readily observable changes were (i) disappearance of 210K, 95K, and 60K proteins, (ii) modifications of alpha crystallin subunits, (iii) alterations of beta H crystallins, and (iv) de novo production of 55K and higher molecular weight polymers. The addition of leupeptin inhibited the disappearances of 210K, 95K, and 60K proteins and the alteration of alpha crystallins, suggesting that all these were caused by a Ca2+-activated protease. The proteolytically sensitive 60K species was identified as vimentin, a component of intermediate filaments. Formation of the 55K material and of higher molecular weight polymers during Ca2+ treatment of the lens could be prevented by histamine, a compound known to inhibit the transglutaminase-mediated cross-linking of proteins by epsilon-(gamma-glutamyl)lysine peptide bonds in other biological systems. It could also be shown by immunoblotting that an antibody raised against the 55K material reacted selectively with beta crystallins of normal lens. This indicates that the 55K product is in all likelihood an essential intermediate toward higher polymers and that the 55K product is a cross-linked dimer of certain polypeptides of beta crystallin.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Calcium; Cataract; Cross Reactions; Crystallins; Electrophoresis, Polyacrylamide Gel; Lens, Crystalline; Leupeptins; Macromolecular Substances; Molecular Weight; Rabbits; Vimentin | 1985 |
Participation of lysosomes in basal proteolysis in perfused rat liver. Discrepancy between leupeptin-induced lysosomal enlargement and inhibition of proteolysis.
Livers of non-starved rats were perfused cyclically for 2 h in the presence of either 4 x the normal concentration of amino acids (known to suppress proteolysis to basal level), or the same medium together with leupeptin (an inhibitor of cathepsin B, H and L). Stereologic analysis revealed that the drug elicited a linear increase in the fractional cytoplasmic volume of the hepatocytic autophagic vacuolar compartment amounting to 1.34%/h. Measurements of proteolysis under the same experimental conditions showed that addition of leupeptin to the perfusate reduced proteolysis from 1.74%/h to 1.34%/h, i.e. an inhibition of 21.6% was observed. Thus, although proteolysis was only little inhibited by leupeptin, cytoplasm was sequestered at a rate that almost fully accounted for overall protein breakdown during basal state. The reasons for this discrepancy, such as subtotal inhibition of proteinases, increase of lysosomal contents and compensatory increased non-lysosomal degradation are discussed. Topics: Amino Acids; Animals; Cytoplasmic Granules; Leupeptins; Liver; Lysosomes; Oligopeptides; Proteins; Rats | 1985 |
Studies in vivo and in vitro on the uptake and degradation of soluble collagen alpha 1(I) chains in rat liver endothelial and Kupffer cells.
Intravenously administered 125I-labelled monomeric alpha 1 chains (125I-alpha 1) of collagen type I were rapidly cleared and degraded by the liver of rats. Isolation of the liver cells after injection of the label revealed that the uptake per liver endothelial cell equalled the uptake per Kupffer cell, whereas the amount taken up per hepatocyte was negligible. The uptake of 125I-alpha 1 in cultured cells was 10 times higher per liver endothelial cell than per Kupffer cell. The ligand was efficiently degraded by cultures of both cell types. However, spent medium from cultures of Kupffer cells, unlike that from cultures of other cells, contained gelatinolytic activity which degraded 125I-alpha 1. The presence of hyaluronic acid, chondroitin sulphate or mannose/N-acetylglucosamine-terminal glycoproteins, which are endocytosed by the liver endothelial cells via specific receptors, did not interfere with binding, uptake or degradation of 125I-alpha 1 by these cells. Unlabelled alpha 1 and heat-denatured collagen inhibited the binding to a much greater extent than did native collagen. The presence of fibronectin or F(ab')2 fragments of anti-fibronectin antibodies did not affect the interaction of the liver endothelial cells, or of other types of liver cells, with 125I-alpha 1. The accumulation of fluorescein-labelled heat-denatured collagen in vesicles of cultured liver endothelial cells is evidence that the protein is internalized. Moreover, chloroquine, 5-dimethylaminonaphthalene-1-sulphonylcadaverine (dansylcadaverine), monensin and cytochalasin B, which impede one or more steps of the endocytic process, inhibited the uptake of 125I-alpha 1 by the liver endothelial cells. Leupeptin, an inhibitor of cathepsin B and 'collagenolytic cathepsins', inhibited the intralysosomal degradation of 125I-alpha 1, but had no effect on the rate of uptake of the ligand. The current data are interpreted as follows. (1) The ability of the liver endothelial cells and the Kupffer cells to sequester circulating 125I-alpha 1 efficiently may indicate a physiological pathway for the breakdown of connective-tissue collagen. (2) The liver endothelial cells express receptors that specifically recognize and mediate the endocytosis of collagen alpha 1(I) monomers. (3) The receptors also recognize denatured collagen (gelatin). (4) Fibronectin is not involved in the binding of alpha 1 to the receptors. (5) Degradation occurs intralysosomally by leupeptin-inhibitable cathepsins. Topics: Animals; Antimetabolites; Cells, Cultured; Collagen; Endothelium; Iodine Radioisotopes; Kinetics; Kupffer Cells; Leupeptins; Liver; Male; Rats; Rats, Inbred Strains | 1985 |
Metabolism of [3H]leupeptin by rat liver.
Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 microM) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 microM the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (less than 21 degrees C). [3H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [3H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period. Topics: Animals; Binding, Competitive; Biological Transport, Active; Chloroquine; Leupeptins; Liver; Lysosomes; Male; Oligopeptides; Perfusion; Rats; Rats, Inbred Strains; Temperature | 1985 |
Olfactory discrimination learning is blocked by leupeptin, a thiol protease inhibitor.
Rats were trained on successive two-odor discriminations with the cues randomly located in an 8-arm radial maze. After several days of training using different odor pairs, the thiol protease inhibitor leupeptin was infused into the ventricles and testing continued. Leupeptin caused a pronounced, dose-dependent and reversible deficit in performance in this task. Previous studies have shown that these drug concentrations do not influence spontaneous activity, feeding and drinking, or the acquisition and retention of avoidance conditioning. The results are interpreted as supporting the hypothesis that a calcium-sensitive proteinase is involved in certain forms of memory that require modification of telencephalic circuitries. Topics: Animals; Brain; Calcium; Depression, Chemical; Discrimination Learning; Hippocampus; Injections, Intraventricular; Leupeptins; Male; Memory; Olfactory Pathways; Oligopeptides; Rats; Smell | 1985 |
Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal.
Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M. Topics: Chemical Phenomena; Chemistry; Chromatography, Affinity; Humans; Leupeptins; Ligands; Oligopeptides; Urokinase-Type Plasminogen Activator | 1985 |
Inhibition of intracellular degradation of proteoglycans by leupeptin in rat ovarian granulosa cells.
Previous work (Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10270-10283) has indicated that heparan sulfate (HS) proteoglycans in rat ovarian granulosa cells are degraded by two kinetically distinct pathways. Pathway 1 degrades proteoglycans rapidly with a t 1/2 approximately 25 min without generating appreciable degradative intermediates. Pathway 2 degrades proteoglycans more slowly with a t 1/2 approximately 4 h, generating distinct degradative intermediates: single HS chains of Mr = approximately 10,000 and approximately 5,000. Effects of leupeptin, an inhibitor of thiol proteases, on the intracellular degradation of proteoglycans in the rat ovarian granulosa cell culture were examined using various chase protocols after labeling cells with [35S]sulfate. The presence of leupeptin at 100 micrograms/ml in the culture medium inhibited the intracellular degradation of proteoglycans by approximately 80% during a 7-h chase period after a 20-h labeling. Leupeptin affected neither the cellular content nor the in vitro activities of beta-hexosaminidase and arylsulfatase. Structural analyses of heparan sulfate species in leupeptin-treated cells demonstrated that the drug inhibited the degradation of HS proteoglycans at two distinct points. First, degradation of the core protein was partially inhibited and delayed before the start of glycosaminoglycan degradation. This resulted in the accumulation of degradative intermediates with partially degraded core proteins bearing intact glycosaminoglycan chains. This establishes the initial sequence for HS proteoglycan degradation, with proteolysis preceding endoglycosidase digestion, and suggests that these two degradation steps may occur in physically separate compartments. Second, the final depolymerization of HS fragments through pathway 2 was totally inhibited, resulting in the continuous accumulation of Mr = 5,000 HS chains. This is not due to the direct inhibition of the lysosomal exoglycosidase and sulfatase enzymes responsible for the complete depolymerization of HS chains, since pathway 1, while slowed, continued to completely depolymerize the HS chains in the presence of leupeptin. The results suggest that the intracellular compartment which completely degrades heparan sulfate chains is separate from those containing partially, endoglycosidically processed heparan sulfate chains and that leupeptin interfered with the translocation of glycosaminoglycans to the final degradation site. Topics: Animals; Female; Gonadotropins, Equine; Granulosa Cells; Kinetics; Leupeptins; Oligopeptides; Proteoglycans; Rats; Sulfur Radioisotopes | 1985 |
Comparative effects of cathepsin inhibitors on rat embryonic development in vitro. Evidence that cathepsin D is unimportant in the proteolytic function of yolk sac.
The effects of two proteinase inhibitors, leupeptin and pepstatin on the development of 9.5-day rat conceptuses in vitro has been studied. All cultures were of 48 h duration and the inhibitors were present throughout the entire period. When pepstatin was added to the culture medium (5-25 micrograms/ml) conceptuses developed and grew to an extent that did not differ from untreated controls. However, leupeptin (1-4 micrograms/ml) caused severe growth retardation and abnormal development of conceptuses. The effects of the two inhibitors on the hydrolysis of 125I-labelled BSA and haemoglobin by homogenates of 10.5-day yolk sac indicated the biochemical basis for the differential toxic effects of the two inhibitors on development. Leupeptin was highly inhibitory of the degration of both substrates whereas pepstatin caused no inhibition of 125I-labelled BSA hydrolysis, and only a slight inhibition of haemoglobin hydrolysis. These observations demonstrate that cathepsin D, a lysosomal aspartic proteinase that is specifically inhibited by pepstatin is not involved in yolk-sac-mediated protein utilization by early organogenesis-phase conceptuses and that lysosomal cysteine proteinases, specifically inhibited by leupeptin, are of paramount importance in this yolk sac function. Topics: Animals; Cathepsin D; Cathepsins; Cells, Cultured; Embryonic and Fetal Development; Hemoglobins; Hydrogen-Ion Concentration; Leupeptins; Pepstatins; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Time Factors; Yolk Sac | 1985 |
Is a nicotinic influence involved in denervation-induced depolarization of muscle?
The neurotrophic role of acetylcholine (ACh) in the denervation-dependent decline of muscle resting membrane potential (RMP) was evaluated. Freshly dissected rat hemidiaphragms with short or long (2 cm) nerve stumps attached ("-N" and "+N" preparations, respectively) were incubated in organ culture in the presence or absence of the nicotinic blockers, alpha-bungarotoxin (alpha-BTX) or d-tubocurarine (curare). Subsequently, RMPs and miniature endplate potentials (MEPPs) of the junctional region were measured. Spontaneous MEPPs disappeared with a half-life of 12 and 20 hr in -N and +N preparations, respectively. A 10- to 15-mV depolarization of RMP was observed between 15 and 20 hr in -N muscles and between 24 and 28 hr in +N muscles. This time course of disappearance of spontaneous potentials and of membrane depolarization agrees well with that observed in vivo. Although nicotinic transmission was blocked from the initiation of the incubation period in alpha-BTX- or curare-treated muscles, no acceleration of RMP decline in -N muscles in vitro was observed. Moreover, in +N preparations the effect of the nerve stump in delaying RMP depolarization persisted despite the continuous presence of alpha-BTX or curare. Since excess ACh triggers a lysosomal proteolytic response at the nerve-muscle junction and since this may occur early in denervation, the possible role of a nicotinic-induced proteolytic mechanism was tested in vitro with the potent protease inhibitor leupeptin. This inhibitor did not delay or prevent the denervation-dependent alterations.(ABSTRACT TRUNCATED AT 250 WORDS) Topics: Animals; Electrophysiology; Leupeptins; Male; Membrane Potentials; Motor Endplate; Muscle Denervation; Muscles; Nicotine; Organ Culture Techniques; Parasympatholytics; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors | 1985 |
Rat mammary gland in culture secretes a stable high molecular weight form of cathepsin L.
Culture medium from rat mammary gland explants was analyzed for the presence of cysteine proteinases. In addition to a putative precursor of the lysosomal enzyme cathepsin B, a cysteine proteinase with enzymatic properties similar to those reported for cathepsin L was found. Further evidence of the cathepsin L-like nature of this activity was provided by its high sensitivity towards the diazomethane inhibitors Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2 and towards leupeptin. The secreted form of cathepsin L is distinguished from the lysosomal form by its increased stability at alkaline pH and by its larger molecular size. It may thus represent an incompletely processed precursor form of the lysosomal enzyme. Topics: Animals; Cathepsin B; Cathepsin L; Cathepsins; Culture Techniques; Cysteine Endopeptidases; Diazomethane; Endopeptidases; Female; Hydrogen-Ion Concentration; Lactation; Leupeptins; Mammary Glands, Animal; Molecular Weight; Pregnancy; Rats; Rats, Inbred Strains; Substrate Specificity | 1985 |
Specific targeting of high density lipoproteins to liver hepatocytes by incorporation of a tris-galactoside-terminated cholesterol derivative.
A triantennary galactose-terminated cholesterol derivative, N-(tris(beta-D-galactopyranosyloxymethyl) methyl)-N alpha-(4(5-cholesten-3 beta-yloxy)succinyl)glycinamide (Tris-Gal-Chol), which dissolves easily in water, was added to human apolipoprotein E-free high density lipoproteins (HDL) in varying quantities. Incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein) stimulates the liver association of the HDL apoprotein radioactivity 24- and 55-fold, respectively, at 10 min after intravenous injection into rats. The increased interaction of Tris-Gal-Chol HDL with the liver is blocked by preinjection of asialofetuin or N-acetylgalactosamine but not influenced by N-acetylglucosamine. The parenchymal liver cell uptake of HDL is stimulated 42- or 105-fold, respectively, by incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein), while the association with nonparenchymal cells is stimulated only 1.7- or 5-fold. It can be calculated that 98.0% of the Tris-Gal-Chol HDL is associated with parenchymal cells. In contrast, incorporation of 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) leads to a selective association of LDL with nonparenchymal cells (92.3% of the total liver uptake). It is concluded that Tris-Gal-Chol incorporation into HDL leads to a specific interaction of HDL with the asialoglycoprotein (galactose) receptor on parenchymal cells whereas Tris-Gal-Chol incorporation into LDL leads mainly to an interaction with a galactose receptor from Kupffer cells. Probably this highly selective cellular targeting of LDL and HDL by Tris-Gal-Chol is caused by the difference in size between these lipoproteins. The increased interaction of HDL with the parenchymal cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein in the lysosomes. It is concluded that Tris-Gal-Chol incorporation into LDL or HDL leads to a markedly increased catabolism of LDL by way of the Kupffer cells and HDL by parenchymal cells which might be used for lowering serum cholesterol levels. The use of Tris-Gal-Chol might also find application for targeting drugs or other compounds of interest to either Kupffer or parenchymal liver cells. Topics: Animals; Chloroquine; Cholesterol Esters; Endothelium; Humans; In Vitro Techniques; Kinetics; Kupffer Cells; Leupeptins; Lipoproteins, HDL; Lipoproteins, LDL; Liver; Male; Rats; Rats, Inbred Strains; Receptors, LDL | 1985 |
Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C.
Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed. Topics: Adult; Blood Platelets; Enzyme Activation; Humans; In Vitro Techniques; Inositol Phosphates; Leupeptins; Middle Aged; Oligopeptides; Phosphatidic Acids; Phosphorylation; Platelet Aggregation; Serotonin; Thrombin; Trypsin; Trypsin Inhibitors; Type C Phospholipases | 1985 |
Alleviation of axonal damage in acute spinal cord injury by a protease inhibitor: automated morphometric analysis of drug-effects.
The degenerating axons and axon terminals developed in Rexed's lamina VIII in the anterior horn of the L6 segment after acute spinal cord compression at Th11 level in rats were visualized by the method of Fink-Heimer and the extent of axonal damage was quantitatively assayed with the aid of an automated image analyzer. Leupeptin, a potent protease inhibitor, substantially reduced the extent of the axonal damage (17% on average). Topics: Animals; Leupeptins; Male; Oligopeptides; Rats; Rats, Inbred Strains; Spinal Cord; Spinal Cord Injuries; Wounds, Nonpenetrating | 1985 |
The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64.
Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen fibrils (CCV) in periosteal fibroblasts. After an incubation period of 48 hours, leupeptin (in a concentration of 65 microM) caused a thirty-fold increase in the volume fraction of CCV. This effect proved to be reversible following upon the withdrawal of the drug. Since the leupeptin-related accumulation of intracellular collagen fibrils was not significantly inhibited by alpha, alpha dipyridyl (a drug that interferes with collagen fibril formation), it is thought unlikely that the fibrils represented newly synthesized collagen. This view is further substantiated by data obtained from explants incubated in the presence of the phagocytosis-inhibiting agent cytochalasin B. This compound completely inhibited the leupeptin-related accumulation of CCV. The data strongly suggest that collagen fibrils found in cytoplasmic vacuoles of periosteal fibroblasts represent collagen taken up by phagocytosis, the integrity of cytoplasmic actin filament systems is a prerequisite for phagocytosis of collagen to occur, and thiol-proteinases, such as cathepsin B, L, and/or N, play an essential role in the digestion of internalized collagen. Topics: 2,2'-Dipyridyl; Animals; Ascorbic Acid; Bone and Bones; Collagen; Culture Techniques; Cycloheximide; Cytochalasin B; Fibroblasts; Leucine; Leupeptins; Mice; Microscopy, Electron; Oligopeptides; Phagocytosis; Vacuoles | 1985 |
Reversibility of the stabilization effect of sodium molybdate on uterine estrogen and progesterone receptors of the vervet monkey.
Sodium molybdate affected the stability of vervet monkey (Cercopithecus aethiops pygerythrus) uterine estrogen (ER) and progesterone (PR) receptors. Yields of receptors were invariably higher (20-40%) when cytosols were prepared in the presence of 10mM sodium molybdate. No changes were observed in the binding affinities for the natural ligands as reflected in dissociation constants. Receptor-ligand association at 0 degrees C and 20 degrees C was not affected in the presence or absence of molybdate. Stability studies at 37 degrees C indicated both receptors to be more resistant to inactivation in the presence of molybdate. Dissociation of ER and PR was biphasic, indicating the existence of slow (SDC), as well as fast dissociating (FDC) complexes. Rate constants of dissociation were significantly affected by the presence of sodium molybdate. Although no significant changes in the sedimentation coefficients were observed, marked differences in the actual gradient profiles could be illustrated in the presence or absence of sodium molybdate. Observed effects could only be partially reversed in sedimentation dialysis experiments. Proteolytic inhibitors phenylmethylsulfonylfluoride (PMSF) and leupeptin had no inhibitive effect on the molybdate stabilization of ER and PR. Topics: Animals; Chlorocebus aethiops; Cytosol; Female; In Vitro Techniques; Kinetics; Leupeptins; Molybdenum; Phenylmethylsulfonyl Fluoride; Receptors, Estrogen; Receptors, Progesterone; Uterus | 1985 |
The fate of the surface protein gp70 during entry of retrovirus into mouse fibroblasts.
The kinetics of the viral surface protein gp70 and the viral core proteins p30 and p15C were followed during retrovirus entry into mouse fibroblasts. All three proteins were internalized, but whereas essentially all the gp70 was degraded, approximately one-third of the core proteins remained stable in the cells. These diverging routes of the different proteins are in agreement with the proposed route, that retrovirus enters the cells by endocytosis followed by a membrane fusion between the virus membrane and the vesicle membrane. Topics: Ammonium Chloride; Animals; Cell Line; Chloroquine; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Glucosamine; Kinetics; Leucine; Leupeptins; Mice; Retroviridae; Retroviridae Proteins; Tritium; Viral Envelope Proteins | 1985 |
Intracellular transport of asialoglycoproteins in rat hepatocytes. Evidence for two subpopulations of lysosomes.
The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles. Topics: Animals; Asialoglycoproteins; Cell Fractionation; Cellobiose; Cells, Cultured; Colchicine; Depression, Chemical; Leupeptins; Liver; Lysosomes; Male; Monensin; Orosomucoid; Rats; Rats, Inbred Strains; Tyramine | 1985 |
Trypsin-like enzyme from eggs of the ascidian (protochordate), Halocynthia roretzi. Purification, properties, and physiological role.
A trypsin-like enzyme has been purified to apparent homogeneity from eggs of the ascidian, Halocynthia roretzi, by a procedure including column chromatography on diethylaminoethyl-cellulose, phenyl-Sepharose, and soybean trypsin inhibitor-immobilized Sepharose 4B. The molecular weight of the enzyme was estimated to be 31,000 and 33,000 by gel electrophoresis in sodium dodecyl sulfate under the reducing and the nonreducing conditions, respectively. The isoelectric point of the enzyme was 4.8. The pH optimum of the activity was 8.4. The enzyme was stable between pH 6 and 9 in the presence of 0.005% Brij 35 as a stabilizer. Substrate specificity of the purified enzyme was broad toward various peptidyl-arginine (or -lysine) 4-methylcoumaryl-7-amides and was similar to that of a trypsin-like enzyme found in the fertilization product. The purified enzyme was inhibited by diisopropyl fluorophosphate and a variety of trypsin inhibitors including leupeptin, but not, or scarcely, inhibited by p-chloromercuribenzoic acid, pepstatin, chymostatin, bestatin, elastatinal, and tosyl-phenylalanyl-chloromethane. The rankings in the potencies of leupeptin and its six analogs as the inhibitors of the purified enzyme were well correlated with those found in their inhibitory effects on the expansion of perivitelline space. Thus, the trypsin-like enzyme possibly present in the fertilization product participates in the expansion of perivitelline space of the egg during fertilization of the ascidian. Topics: Animals; Fertilization; Isoelectric Point; Leupeptins; Molecular Weight; Ovum; Protease Inhibitors; Substrate Specificity; Trypsin; Urochordata | 1985 |
High-performance liquid chromatographic method for monitoring leupeptin in mouse serum and muscle by pre-column fluorescence derivatization with benzoin.
A high-performance liquid chromatographic method is described for the determination of leupeptin, a possible therapeutic drug for muscular dystrophy, in mouse serum and muscle. Leupeptin is reduced with sodium borohydride to leupeptinol, and then converted to a fluorescent derivative with benzoin. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) with isocratic elution and determined with fluorescence detection. The detection limits of leupeptin in serum and muscle are 250 pmol/ml (107 ng/ml) and 500 pmol/g (214 ng/g), respectively, corresponding to approximately 150 fmol each in a 100-microliters injection volume. This method is simple and sensitive enough to permit the quantification of leupeptin in biological samples from mice dosed with leupeptin. Topics: Animals; Benzoin; Chromatography, High Pressure Liquid; Leupeptins; Mice; Mice, Inbred C57BL; Muscles; Oligopeptides; Spectrometry, Fluorescence | 1985 |
Quantitative assessment of the contribution of autophagy to intracellular protein breakdown.
Topics: Animals; Autolysis; Cathepsins; Cycloheximide; Cytoplasm; Drug Administration Schedule; Fasting; Homeostasis; Leupeptins; Liver; Lysosomes; Microtubules; Protein Conformation; Proteins; Rats | 1985 |
Protein degradation in the lysosome.
Topics: Animals; Autolysis; Hydrogen-Ion Concentration; Leupeptins; Liver; Lysosomes; Male; Phagocytosis; Proteins; Rats; Subcellular Fractions | 1985 |
Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody.
Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I. Topics: Antibodies, Monoclonal; Calcium; Calcium-Binding Proteins; Calpain; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Erythrocyte Membrane; Humans; Immunoelectrophoresis; Immunoglobulin G; Leupeptins; Protease Inhibitors | 1984 |
The biochemistry of memory: a new and specific hypothesis.
Recent studies have uncovered a synaptic process with properties required for an intermediate step in memory storage. Calcium rapidly and irreversibly increases the number of receptors for glutamate (a probable neurotransmitter) in forebrain synaptic membranes by activating a proteinase (calpain) that degrades fodrin, a spectrin-like protein. This process provides a means through which physiological activity could produce long-lasting changes in synaptic chemistry and ultrastructure. Since the process is only poorly represented in the brain stem, it is hypothesized to be responsible for those forms of memory localized in the telencephalon. Topics: Animals; Calcium; Calpain; Carrier Proteins; Cerebral Cortex; Endopeptidases; Glutamates; Glutamic Acid; Hippocampus; Humans; Learning; Leupeptins; Memory; Microfilament Proteins; Neuronal Plasticity; Rabbits; Rats; Receptors, Cell Surface; Receptors, Glutamate; Receptors, Neurotransmitter; Synapses; Synaptic Membranes; Telencephalon | 1984 |
Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation.
Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation. Topics: Animals; Asialoglycoprotein Receptor; Asialoglycoproteins; Cells, Cultured; Chloroquine; Colchicine; Cold Temperature; Endocytosis; Endosomes; Exocytosis; Kinetics; Leupeptins; Liver; Lysosomes; Monensin; Rats; Receptors, Immunologic | 1984 |
Comparative studies on the calcium paradox in cardiac muscle: the effect of temperature on the different phases.
By measuring characteristic ultrastructural damage, the Ca2+-paradox has been demonstrated in isolated frog hearts, and in frog and mouse ventricle strips. During Ca2+-free perfusion (phase I), PCa of the sarcolemma is increased; 4 degrees C provides complete protection against these molecular changes in frog and a partial protection in mouse tissue. Re-introduction of extracellular Ca2+ (phase II) now causes typical Ca2+-triggered damage which is markedly reduced at 4 degrees C in both species but is not inhibited by leupeptin. Major sarcolemma damage occurs at this point. Topics: Animals; Calcium; Leupeptins; Mice; Mice, Inbred BALB C; Microscopy, Electron; Myocardial Contraction; Myocardium; Perfusion; Rana temporaria; Temperature | 1984 |
Acid proteolytic activities in mouse liver and muscle tissues after treatment with protease inhibitor leupeptin.
The activities of acid proteolytic enzymes were assayed in the liver and muscular tissues of mice (Mus musculus) 1, 6 and 24 hr after the administration of a protease inhibitor leupeptin (i.p., 15.5 mg/kg body wt). Leupeptin administration induced a strong inhibition of cathepsin B and a moderate inhibition of cathepsin C and acid autolytic rate in mouse liver 1 hr after injection. Thereafter the inhibition reduced and disappeared during 24 hr. The activity of cathepsin D was increased in liver 6 and 24 hr after injection. The activity of beta-glucuronidase was not affected by the leupeptin treatment. The administration of leupeptin did not affect the rate of acid autolysis and the activities of cathepsin C and D in cardiac and skeletal muscles. A slight increase in cathepsin B activity was observed 1 hr after leupeptin treatment in calf muscles. The cause of both tissue and enzyme specific changes after leupeptin treatment is discussed. Topics: Animals; Cathepsin B; Cathepsin C; Cathepsin D; Cathepsins; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Glucuronidase; Leupeptins; Liver; Male; Mice; Muscles; Oligopeptides; Peptide Hydrolases; Time Factors | 1984 |
Comparative studies on the role of calcium in triggering subcellular damage in cardiac muscle.
Isolated cardiac muscle strips from amphibians and mammals, together with isolated frog hearts, have been used as model systems for studying the action of elevated [Ca2+]i in promoting severe damage. A23187 and caffeine are believed to cause a rise in [Ca2+]i. Elevated [Ca2+]i causes characteristic damage which has been categorized and includes hypercontraction, Z-line damage and myofilament dissolution. The damage closely resembles that described in the isolated mammalian heart and in skeletal muscle preparations when [Ca2+]i is raised dramatically. Damage can therefore be triggered by releasing Ca2+ from intracellular sites, as distinct from increasing Ca2+ entry (as in the Ca2+-paradox). DNP and ruthenium red also cause identical damage and the results suggest that whilst the fall in pHi associated with ischaemia is probably the consequence of Ca2+/2H+ exchange at the mitochondria, coupled with ATP hydrolysis, lowered pHi by mitochondrial action is probably not the only cause of myofilament dissolution. Damage is not prevented by pretreatment with leupeptin, an inhibitor of Ca2+-activated neutral proteases, and it is concluded that the latter are probably not implicated in rapid and dramatic damage. The possible involvement of lysosomal enzymes in damage triggered by high [Ca2+]i is discussed. Topics: Animals; Caffeine; Calcimycin; Calcium; Dinitrophenols; Heart; Leupeptins; Mice; Mice, Inbred Strains; Mitochondria, Heart; Myocardial Contraction; Myocardium; Rana temporaria; Ruthenium Red; Subcellular Fractions | 1984 |
Leupeptin's effect on organ weight, RNA, DNA, and protein content after long bone fracture in the rat.
Long bone fracture in the rat is accompanied by enhanced urinary nitrogen loss reflecting changes in protein synthesis and breakdown. The effects of leupeptin, an inhibitor of lysosomal proteases, were assessed on organ weights, RNA, DNA, and protein content after injury in the rat. Two groups of 8-week-old rats were studied: The first group received left femoral fracture. Half of these received leupeptin (25 mumole ip/day), and the remainder received saline. The second group served as uninjured pair-fed controls, with half receiving leupeptin and half receiving saline. On Days 0, 1, 2, 4, and 7 after injury, animals were sacrificed and organs were removed for determination of weight, RNA, DNA, and protein content. All injured rats lost weight, with maximum loss occurring on Day 4. Food intake was also reduced. Pair-fed rats lost the same amount of weight as injured ones, and leupeptin could not prevent whole body weight loss. Expressed as percentage of total body weight, livers from leupeptin-treated injured rats weighed 10% greater than saline-treated ones on Days 2, 4, and 7 after injury (P less than 0.05). No differences occurred in RNA, DNA, or protein contents. Diaphragms similarly weighed 10, 20, and 30% greater on Days 2, 4, and 7 after injury, respectively, in leupeptin-treated rats (P less than 0.05). In addition, the RNA and DNA contents of diaphragms were 96 and 88% greater, respectively, in treated rats than in controls (P less than 0.05) on Day 4. It is concluded that leupeptin causes a relative increase in the weights of livers and diaphragms after injury, and causes a marked increase in the RNA and DNA contents of diaphragms. Topics: Animals; Body Weight; Diaphragm; DNA; Eating; Femoral Fractures; Leupeptins; Liver; Male; Oligopeptides; Organ Size; Proteins; Rats; Rats, Inbred Strains; RNA | 1984 |
125I-Insulin internalization by perfused rat liver: comparison of its subcellular distribution with that of a lysosomally targeted molecule, 125I-asialofetuin.
The subcellular distribution of 125I-insulin in the perfused rat liver was compared with the subcellular distribution of the lysosomally targeted asialoglycoprotein, 125I-asialofetuin. The use of Percoll density gradient medium provided excellent separation of lysosomes from the subcellular membrane fractions. Following perfusion with 125I-asialofetuin, a distinct peak of TCA-precipitable radioactivity could be observed in the lysosomal region of the gradient. In contrast, the gradient distribution of TCA-precipitable radioactivity following perfusion with physiological concentrations of 125I-insulin was unimodal, the observed peak corresponding to the distribution of intracellular membrane marker enzymes. Leupeptin, an inhibitor of lysosomal proteolysis, inhibited the degradation of 125I-asialofetuin but had no effect on 125I-insulin degradation. In addition, leupeptin produced a marked increase in TCA-precipitable radioactivity in the lysosome rich region of gradients prepared from livers perfused with 125I-asialofetuin. No such effect was observed following perfusion with 125I-insulin. These findings are consistent with an initial localization of the internalized insulin molecule with the membraneous system of the liver cell rather than the lysosomal system. Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Fetuins; Insulin; Intracellular Membranes; Leupeptins; Liver; Lysosomes; Male; Perfusion; Rats; Rats, Inbred Strains; Subcellular Fractions | 1984 |
Glycoprotein Ib in the Triton-insoluble (cytoskeletal) fraction of blood platelets.
Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl2, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with 32P as sodium phosphate led to incorporation of phosphatase-sensitive 32P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This supports the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ib alpha-chain exposed at the outer surface. Topics: Blood Platelets; Cytoskeleton; Edetic Acid; Glycoproteins; Humans; Immunoelectrophoresis, Two-Dimensional; Leupeptins; Membrane Proteins; Molecular Weight; Octoxynol; Platelet Membrane Glycoproteins; Polyethylene Glycols; Solubility; Thrombin | 1984 |
High-performance affinity chromatography of plasmin and plasminogen on a hydrophilic vinyl-polymer gel coupled with p-aminobenzamidine.
p-Aminobenzamidine was covalently attached via a spacer moiety to a microparticulate hydrophilic vinyl-polymer gel (Toyopearl HW65S) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. Toyopearl HW65S was alkylated with chloroacetylglycylglycine in dimethyl sulphoxide using methylsulphinyl carbanion as a catalyst, then p-aminobenzamidine was coupled to the carboxyl group of glycylglycine to form an acid amide bond. A column packed with the adsorbent retained both plasmin and plasminogen. Plasminogen was eluted with 6-aminohexanoic acid, a haptenic compound for the lysine-binding sites of plasminogen. For the elution of plasmin, the coexistence of 6-aminohexanoic acid and leupeptin (a competitive inhibitor for plasmin) was necessary. The results indicate a two-site interaction of plasmin with the immobilized ligand, i.e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of plasmin activity using a fluorogenic substrate, peptidylmethylcoumarylamide, revealed that effective chromatographic separation of the enzyme could be achieved with high sensitivity (10 micrograms) within 1 h. Topics: Amidines; Benzamidines; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Leupeptins; Plasminogen; Spectrophotometry, Ultraviolet; Trypsin Inhibitors | 1984 |
The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB15).
Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum-free medium alone, so serum-free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phosphodiesterase inhibitor R020-1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed. Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Animals; Antipain; Cell Differentiation; Cyclic AMP; Isoflurophate; Leupeptins; Mice; Neuroblastoma; Trypsin Inhibitor, Kunitz Soybean | 1984 |
Structural comparisons of wild-type and nuclear transport-defective simian virus 40 large tumor antigens.
PARA(nT) is a defective SV40-adenovirus 7 hybrid virus which contains the entire early region of the SV40 genome and codes for the synthesis of SV40 large tumor antigen (T-ag). A transport-defective variant of this hybrid, PARA(cT), encodes T-ag that is not transported to the nucleus, but accumulates in the cytoplasm. The structures of T-ags extracted from wild-type (WT) SV40-, PARA(nT)-, and PARA(cT)-infected cells were compared by peptide mapping. All three types of T-ag underwent considerable degradation when extracted using Tris-buffered Nonidet P-40 at pH 8.0. The addition of 200 microM leupeptin to the extraction buffer significantly inhibited this degradation. Comparison of methionine-containing tryptic peptides revealed no differences among the T-ags, suggesting that their primary structures are similar or identical. Phosphopeptide mapping revealed no differences between SV40- and PARA(nT)-encoded T-ags. In contrast, PARA(cT)-encoded T-ag lacked a prominent phosphopeptide that was present in both of the others. The possible relevance of this difference in phosphorylation to the transport defect is discussed. Topics: Adenoviridae; Amino Acid Sequence; Antigens, Viral, Tumor; Biological Transport; Cell Nucleus; Hybridization, Genetic; Leupeptins; Methionine; Mutation; Octoxynol; Peptides; Phosphopeptides; Phosphorylation; Polyethylene Glycols; Simian virus 40; Viral Proteins | 1984 |
Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics.
We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes. Topics: Animals; Cycloheximide; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Horseradish Peroxidase; In Vitro Techniques; Kinetics; Leupeptins; Ligands; Liver; Male; Microscopy, Electron; Rats; Receptors, Cell Surface; Subcellular Fractions | 1984 |
Leupeptin, a thiol proteinase inhibitor, causes a selective impairment of spatial maze performance in rats.
The effects of chronic intraventricular infusion of leupeptin, a potent inhibitor of thiol proteinases, were tested on ingestive behaviors, escape and avoidance conditioning, and spatial memory in rats. The drug did not detectably influence feeding, drinking, body temperature, or the latency to escape from a mild footshock or inhibitory avoidance behavior. However, rats treated with leupeptin made numerous errors ( reentries ) in an eight-arm spatial maze. These results are interpreted as supporting the hypothesis that calcium-activated thiol proteinases are involved in the formation of certain types of memory. Topics: Animals; Avoidance Learning; Brain; Cysteine Endopeptidases; Discrimination Learning; Dose-Response Relationship, Drug; Leupeptins; Male; Mental Recall; Oligopeptides; Orientation; Protease Inhibitors; Rats; Rats, Inbred Strains; Space Perception; Synaptic Transmission | 1984 |
Antigen conformation determines processing requirements for T-cell activation.
We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. Topics: Ammonium Chloride; Animals; Antigens; Chloroquine; Clone Cells; Leupeptins; Lymphocyte Activation; Lysosomes; Mice; Monensin; Myoglobin; Peptide Fragments; Protein Conformation; Protein Denaturation; Structure-Activity Relationship; T-Lymphocytes | 1984 |
Partial characterization of protease(s) in human breast cancer cytosols that can degrade estrogen and progesterone receptors selectively.
Proteolytic activity in human breast cancer cytosols was studied using hormone receptors from rats as the substrates. Under the conditions tested, limited proteolysis of both the estrogen and the progesterone receptors in uterine cytosol was observed, but not proteolysis of the glucocorticoid or androgen receptors in liver or prostate cytosols, respectively. Although both the nonactivated and activated uterine estrogen receptors were attacked by the enzyme(s), molybdate-stabilized receptors were resistant to proteolysis. The product of estrogen receptor cleavage sedimented at approximately 4S in low-salt gradients and at 3 to 4S in high-salt gradients. This fragment retained both the steroid-binding and DNA-binding domains. The marked decrease in its DNA-binding ability, compared with the salt-dissociated but non-proteolyzed receptors, may be attributable to interactions of the fragment with dialyzable modulator(s) in cytosol. The proteolytic activity in tumor cytosol was leupeptin sensitive and was precipitated by (NH4)2SO4 at 30 to 60% saturation. Its sedimentation coefficient was 4 to 5S. The proteolytic activity was identified in 70% of estrogen receptor-negative tumors but in only 40% of estrogen receptor-positive tumors. Topics: Breast Neoplasms; Cytosol; Estradiol; Female; Humans; Kinetics; Leupeptins; Molybdenum; Peptide Hydrolases; Receptors, Estradiol; Receptors, Estrogen; Receptors, Progesterone; Substrate Specificity; Uterus | 1984 |
Biosynthesis of intestinal microvillar proteins. Further characterization of the intracellular processing and transport.
The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvillar enzymes was greatly reduced by tunicamycin but could be partially restored by leupeptin, suggesting the existence of a mechanism whereby newly synthesized, malprocessed enzymes are recognized and degraded. In the presence of tunicamycin, polypeptides likely to represent non-glycosylated forms of the enzymes persisted in the Mg2+-precipitated membrane fraction, indicating that high mannose glycosylation is essential for transport to the microvillar membrane. Treatment of aminopeptidase N and sucrase-isomaltase with endo F reduced the size of the high mannose forms approximately to those seen in the presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N-linked carbohydrate. Topics: alpha-Glucosidases; Aminopeptidases; Animals; Biological Transport; Glucosamine; Glucosidases; Glycoproteins; Intestinal Mucosa; Leupeptins; Microvilli; Molecular Weight; Multienzyme Complexes; Sucrase-Isomaltase Complex; Swine; Tunicamycin | 1984 |
Regulatory proteolysis during corticosteroid hormone action.
Topics: Adrenal Cortex Hormones; Animals; Humans; Kidney; Leupeptins; Liver; Peptide Hydrolases; Protein Conformation; Receptors, Androgen; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone; Trypsin Inhibitors | 1984 |
Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver.
Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver. Topics: Animals; Cysteine Endopeptidases; Endopeptidases; Enzyme Induction; Hemoglobins; Hydrolysis; Kinetics; Leupeptins; Liver; Male; Microscopy, Electron; Oligopeptides; Rats; Rats, Inbred Strains; Subcellular Fractions | 1984 |
Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts.
Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation. Topics: Cells, Cultured; Collagen; Endopeptidases; Female; Fibroblasts; Humans; Leupeptins; Lung; Lysosomes; Pepstatins; Pregnancy; Procollagen; Tosyllysine Chloromethyl Ketone | 1984 |
Effects of the protease inhibitor leupeptin on proteolytic activities and regeneration of mouse skeletal muscles after exercise injuries.
Leupeptin, a nontoxic thiol protease inhibitor, has been proposed to have therapeutic use in hereditary muscular dystrophies. The purpose of this study was to characterize the in vivo changes in proteolytic activity of skeletal muscles induced by the repeated administration of leupeptin. Further, whether the modulation of proteolytic capacity by leupeptin affects the repair process of muscle injuries caused by heavy exercise was studied. Leupeptin was administered in mice intraperitoneally at a dose level of 15.5 mg/kg twice a day for 9 days. Leupeptin, known to be an inhibitor of cathepsin B both in vitro and after a single injection in vivo, paradoxically induced an increase of cathepsin B activity in mouse skeletal muscles after repeated administration. In addition, leupeptin administration for 9 days increased the activities of cathepsins C and D, as well as the rate of acid autolysis. The activity of beta-glucuronidase also increased, while those of arylsulfatase, ribonuclease, and alkaline protease were unaffected. No histopathologic changes were observed. At the low dosage used, leupeptin had no effect on the repair process of skeletal muscle after exercise injuries, although several proteolytic processes occur during the regeneration. It is suggested that the increase of acid protease activities in skeletal muscles is an adaptive response to the administration of the proteolytic inhibitor leupeptin and that leupeptin can be administered without prevention or delay of regenerative processes after the onset of myopathic changes. Topics: Animals; Cathepsin B; Cathepsin D; Cathepsins; Glucuronidase; Leupeptins; Male; Mice; Muscles; Oligopeptides; Peptide Hydrolases; Physical Exertion; Regeneration; Stimulation, Chemical; Time Factors | 1984 |
Inhibition of in-vitro fertilization of hamster oocytes by a thiol proteinase inhibitor.
Amongst the proteinase inhibitors tested, thiolstatin, a specific inhibitor for the thiol proteinases, leupeptin and antipain, both specific inhibitors of serine- and thiol-proteinases, strongly reduced fertilization of hamster oocytes in vitro. These results suggest the possible involvement of thiol proteinase(s), as well as acrosin, in the fertilization process. A possible role for thiol proteinase in sperm adhesion to the zona pellucida is proposed. Topics: Animals; Antipain; Cricetinae; Culture Media; Cystatins; Cysteine Endopeptidases; Enzyme Inhibitors; Female; Fertilization in Vitro; Leupeptins; Male; Oocytes; Protease Inhibitors; Sperm Motility; Time Factors | 1984 |
Mechanism of autodegradation of cell-surface macromolecules shed by human melanoma cells.
The mechanism of autodegradation of cell-surface macromolecules shed by human melanoma cells was studied by incubating radio-iodinated shed macromolecules with unlabeled sister cells and measuring the appearance of acid-soluble radioactivity. After a preliminary latent period of 1-3 h, degradation continually increased up to 24 h and was concentration-dependent. By contrast, binding to cells was very rapid reaching half-maximal value within 15 min. Autodegradation was markedly reduced (44-82%) by pharmacological agents which interfere with endocytosis or lysosomal enzyme activity, including drugs which inhibit receptor migration into coated pits (dansylcadaverine), endocytosis and intracellular transport (colchicine, cytochalasin B, and monensin), and the activity of lysosomal enzymes (chloroquine, ammonium chloride, leupeptin). Degradation was almost totally suppressed (95%) at 4 degrees C. These data suggest that surface macromolecules shed by melanoma cells are autodegraded in part by re-uptake into melanoma cells followed by degradation in lysosomes. Topics: Ammonium Chloride; Biological Transport; Cadaverine; Cell Line; Chloroquine; Coated Pits, Cell-Membrane; Colchicine; Cytochalasin B; Endocytosis; Humans; Leupeptins; Lysosomes; Melanoma; Membrane Proteins; Monensin; Neoplasm Proteins; Time Factors; Tunicamycin | 1984 |
An abnormality in intracellular protein degradation in fibroblasts from patients with I-cell disease.
Degradation of endogenous proteins in five normal and five I-cell (mucolipidosis II) storage disease human fibroblasts was compared. In growing cultures (low density) long half-life proteins were degraded normally in each group of cells. However, the enhancement of proteolysis when confluence is reached, which we have characterised previously as being a lysosomal function, was less in the I-cell fibroblasts. The lysosomotropic agents ammonium chloride, pepstatin and Z-Phe-Ala-diazomethylketone, inhibited similarly proteolysis in growing and confluent cultures of both cell types. Leupeptin depressed proteolysis in both growth states for both cell types, but, whereas it failed to abolish the enhancement of degradation in confluent normal cells, surprisingly it depressed degradation in confluent I-cell fibroblasts to a lower rate than in growing I-cell fibroblasts. In spite of this, the inhibitory effects of ammonium chloride and leupeptin were not additive in either normal or I-cell fibroblasts, indicating that they act upon the same proteolytic mechanism(s). Non-lysosomal mechanisms may degrade short half-life proteins, and it seemed that turnover of such proteins was slower in I-cell fibroblasts. It is suggested that mutual regulation of participating proteolytic pathways may be responsible for the dysfunction of intracellular protein degradation in I-cell fibroblasts. Topics: Ammonium Chloride; Cytosol; Fibroblasts; Half-Life; Humans; Leupeptins; Lysosomes; Mucolipidoses; Pepstatins; Proteins | 1984 |
Effects of inhibitors of protein degradation on the rate of protein synthesis in Chinese hamster ovary cells.
In the absence of serum and amino acids, cultured Chinese Hamster Ovary cells released to the medium two thirds of the leucine produced by protein degradation. Because protein synthesis requires all the amino acids, the loss of leucine implies incomplete reincorporation of the other amino acids as well. Leupeptin (0.45 mg/ml) and chloroquine (up to 40 microM) inhibited protein breakdown by 21 and up to 41%, respectively, and resulted in proportional decreases in protein synthesis. Chloroquine abolished the stimulation of protein breakdown by amino acid deprivation. From the values of protein synthesis and leucine output with and without chloroquine, it is estimated that the stimulation of protein degradation not only permitted continuing protein synthesis but also increased amino acid output. In the presence of serum or amino acids protein breakdown was slower than in their absence and less sensitive to inhibition by chloroquine, but proportional effects on synthesis and degradation were still observed. It is suggested that protein degradation may be necessary for the maintenance of optimum intracellular concentrations of amino acids even in the presence of extracellular amino acids. Topics: Amino Acids; Animals; Cell Line; Chloroquine; Cricetinae; Cricetulus; Female; Kinetics; Leupeptins; Oligopeptides; Ovary; Protein Biosynthesis; Proteins | 1984 |
The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture.
The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts. Topics: Amino Acid Chloromethyl Ketones; Animals; Chick Embryo; Collagen; Fibroblasts; In Vitro Techniques; Leupeptins; Oligopeptides; Pepstatins; Tendons; Tosyllysine Chloromethyl Ketone | 1984 |
Heterogeneity of free valine pools for protein synthesis on free and membrane-bound polysomes in rat liver.
When rat liver was pulse-labeled with [3H]valine in vivo, the nascent peptide on membrane-bound polysomes was found to be more highly labeled than that on free polysomes. Nascent peptides were purified from both classes of polysomes and, after hydrolysis, the amino acids were reacted with 14C-labeled 1-fluoro-2,4-dinitrobenzene. The specific activity of [3H]valine was determined from the [14C]-dinitrophenyl-[3H]valine after purification by two-dimensional thin layer chromatography. With this approach we found that the specific activity of [3H]valine in the nascent peptide of membrane-bound polysomes was more than twice that of free polysomes. Moreover, when rats were pretreated with a lysosomal protease inhibitor, the differences between the specific activities of valine in nascent peptides of the two classes of polysomes tended to decrease. Our results indicate the existence of two distinct pools for valine used for protein synthesis in liver cells; one serves as a precursor for the synthesis of secretory proteins on membrane-bound polysomes and the other as a precursor for the synthesis of intracellular proteins on free polysomes. Topics: Animals; Kinetics; Leupeptins; Liver; Molecular Weight; Polyribosomes; Protein Biosynthesis; Proteins; Rats; Rats, Inbred Strains; Tritium; Valine | 1984 |
Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain.
Injections of leupeptin (a thiol proteinase inhibitor) or chloroquine (a general lysosomal enzyme inhibitor) into the brains of young rats induced the formation of lysosome-associated granular aggregates (dense bodies) which closely resembled the ceroid-lipofuscin that accumulates in certain disease states and during aging. The dense material increased in a dose- and time-dependent fashion and was differentially distributed across brain regions and cell types. These observations provide clues to the origins of ceroid-lipofuscin and suggest means for studying the consequences of its accumulation. Topics: Animals; Brain; Chloroquine; Leupeptins; Lysosomes; Microscopy, Electron; Oligopeptides; Rats; Rats, Inbred Strains | 1984 |
Suppression of experimental allergic encephalomyelitis (EAE) with liposome-encapsulated protease inhibitor: therapy through the blood-brain barrier.
Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoallergic demyelinating disease which is caused by immunization with a particular neuroantigen, such as myelin basic protein (MBP). Results have suggested that protease inhibitors might be useful therapeutically. Leupeptin (acetyl-L-leucyl-L-leucyl-argininal), a protease inhibitor of tripeptide nature, was effective in suppressing EAE in guinea pigs, when administered in a form of liposomes consisting of egg lecithin, cholesterol and sulfatide. The drug seemed to be transported into the central nervous system (CNS) tissues across the blood-brain barrier with the aid of a particular type of liposomes as vehicle. Some outbred Hartley guinea pigs completely recovered from distinct symptoms of EAE, such as loss of weight, paralysis, incontinence and/or diarrhea, when treated i.p. every day with lecithin-cholesterol-sulfatide (molar ratio, 4:5:1) reverse-phase evaporation vesicles-encapsulated leupeptin (REV-Leu) from day 6 after sesitization with 30 micrograms of bovine MBP. Scarcely any typical histopathological changes of EAE were found in the CNS of most survivors treated with REV-Leu. Topics: Animals; Blood-Brain Barrier; Cholesterol; Encephalomyelitis, Autoimmune, Experimental; Guinea Pigs; Leupeptins; Liposomes; Oligopeptides; Phosphatidylcholines; Sulfoglycosphingolipids | 1984 |
Similar effects of various low-molecular-weight enzyme inhibitors on enzyme networks in dystrophic mice.
We compared the therapeutic effects of various low-molecular-weight enzyme inhibitors on dystrophic mice. Leupeptin, bestatin, forphenicinol and forphenicine significantly affected the enzymatic activities in the dystrophic muscles. The pattern of enzymatic changes in the muscles of forelimb and hindlimb caused by these inhibitors were similar in spite of the variety of their inhibitory spectra in vitro. However, comparing the pattern of enzymatic changes in spleen, forphenicinol differed from the other inhibitors tested. This may be related to the peculiar effects of this inhibitor on immunologically responsive cells. Topics: Alkaline Phosphatase; Aminopeptidases; Animals; Esterases; Forelimb; Glutamyl Aminopeptidase; Glycine; Hindlimb; Leucine; Leucyl Aminopeptidase; Leupeptins; Male; Mice; Mice, Mutant Strains; Molecular Weight; Muscles; Muscular Dystrophy, Animal; Oligopeptides; Spleen | 1984 |
Elevated ammonia release from dystrophic chicken muscle cell and fibroblast cultures.
The activity of adenosine monophosphate (AMP)-aminohydrolase, the major NH3-producing enzyme in skeletal muscle, was approximately 35% lower in 7-day dystrophic muscle cell cultures than in normal muscle cell cultures. However, the release rate of NH3 from dystrophic muscle cells was 45% higher than that from normal muscle cells. The reasons for this apparent discrepancy are not clear. To determine indirectly if deamination of amino acids from protein degradation contributed to NH3 release, cells were incubated with 100 micrograms/ml of the protease inhibitor, leupeptin. Leupeptin reduced the rate of NH3 release by only 18.8% in normal muscle cells and 16% in dystrophic muscle cells. The release of NH3 was also higher from dystrophic chicken fibroblast cultures. Topics: Adenosine Deaminase; Ammonia; Animals; Cells, Cultured; Chick Embryo; Fibroblasts; Leupeptins; Muscular Dystrophy, Animal; Protein Denaturation | 1984 |
Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells.
The synthesis of preribosomal RNA is inhibited "in vivo" and "in vitro" by the protease inhibitor leupeptin. "In vivo" leupeptin decreases by 74% the incorporation of labeled uridine into 45S pre rRNA while the synthesis of other RNA species is only slightly decreased. "In vitro", the elongation of already initiated pre rRNA chains that is achieved by incubation of isolated nucleoli is blocked by leupeptin. On the other hand, "in vitro" leupeptin has no direct effect on RNA polymerase I, tested in a nonspecific transcriptional system with Calf thymus DNA as template and in run off experiments with a cloned DNA containing the initiation site of the rDNA gene. A 100 kDa nucleolar protein which has been shown to be endoproteolytic cleaved "in vivo" (1) acts as an inhibitor of rDNA transcription in presence of leupeptin but produces little effect on the nonspecific transcription. In absence of the drug, the 100 kDa protein is processed in specific peptides which appeared to be similar to the "in vivo" maturation products. The possible role of the 100 kDa maturation process in the regulation of rDNA transcription is discussed. Topics: Animals; Cell Line; Cell Nucleolus; Cloning, Molecular; Cricetinae; Cricetulus; DNA; DNA, Ribosomal; Female; Leupeptins; Molecular Weight; Nucleic Acid Precursors; Nucleoproteins; Oligopeptides; Ovary; Plasmids; RNA Precursors; RNA, Ribosomal; Transcription, Genetic | 1984 |
Endocytosis of formaldehyde-denatured serum albumin by nonparenchymal liver cells in vitro.
The uptake and degradation of 125I-labeled formaldehyde-denatured serum albumin in nonparenchymal rat liver cells were studied in vitro. Nonparenchymal cells bound formaldehyde-denatured serum albumin at two types of binding site, one with a high affinity and one a low affinity. The number of high affinity binding sites was approx. 10(5) per cell and the association constant, Ka 10(8) M-1. Inhibition of protein synthesis with cycloheximide did not affect the uptake and degradation of formaldehyde-denatured serum albumin suggesting reutilization of the binding sites. The presence of monensin-reduced uptake and degradation to less than 10% of control values. Pronase treatment of nonparenchymal liver cells completely abolished the uptake and degradation of the ligand. The uptake mechanism was not specific for formaldehyde-denatured serum albumin. Unlabeled acetylated, as well as malondialdehyde treated, serum albumin reduced the uptake of 125I-labeled formaldehyde-denatured serum albumin as effectively as unlabeled formaldehyde-denatured serum albumin itself. Topics: Animals; Endocytosis; Formaldehyde; Leupeptins; Liver; Lysosomes; Monensin; Protein Conformation; Protein Denaturation; Rats; Serum Albumin; Structure-Activity Relationship | 1984 |
Uptake and degradation of 125I-labeled rat asialoorosomucoid by the perfused rat liver.
The uptake and degradation of a homologous rat serum asialoglycoprotein, 125I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. 125I-Asialoorosomucoid was rapidly taken up by the liver (t1/2 = 5.7 min) and acid-soluble degradation products began to appear in the circulating perfusate medium after 20-30 min. These products accounted for 60-65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of 125I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of 125I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathepsin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat 125I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occurred in the native molecular weight of the starting glycoprotein. Topics: Animals; Asialoglycoproteins; Biological Transport; In Vitro Techniques; Iodine Radioisotopes; Leupeptins; Liver; Male; Orosomucoid; Perfusion; Rats; Rats, Inbred Strains | 1984 |
Inhibition of fusion of skeletal myoblasts by tunicamycin and its reversal by N-acetylglucosamine.
The differentiation of a permanent line of rat skeletal myoblasts is inhibited by a low concentration of tunicamycin in the growth medium. At a level of 0.9 micrograms/mL, mannose incorporation in trichloroacetic-acid-insoluble material is inhibited to the extent of about 50% by the antibiotic. Blotting of glycoproteins of the cytoplasmic membrane of myoblasts separated by gel electrophoresis by radioiodinated concanavalin A revealed that four major glycoproteins of 230,0000, 145,000, 119,000, and 46,000 daltons were present in lower relative amounts in the plasma membrane following tunicamycin treatment. The 119,000 dalton glycoprotein was a major radioiodinated protein in intact cells and was presumably localized on the periphery of the membrane. The effect of tunicamycin on both fusion and glycosylation of membrane proteins could be reversed by N-acetylglucosamine, but not by the protease inhibitors leupeptin and pepstatin. Topics: Acetylglucosamine; Animals; Cell Division; Cell Fusion; Cell Line; Cell Membrane; Glucosamine; Glycoproteins; Kinetics; Leupeptins; Membrane Proteins; Molecular Weight; Muscles; Rats; Tunicamycin | 1984 |
Inhibition of Leishmania amastigote growth by antipain and leupeptin.
Antipain and leupeptin were found to be potent inhibitors of the growth of Leishmania mexicana mexicana amastigotes in explanted mouse unstimulated peritoneal macrophages. Antipain at 100 mg l-1 reduced eight-fold the percentage of macrophages infected and to 5% of the control the number of amastigotes present after seven days incubation. At 1 mg l-1, antipain and leupeptin were as effective antileishmanial agents as pentamidine isethionate, all three stopping parasite multiplication over the seven-day incubation. Topics: Animals; Antipain; Antiprotozoal Agents; Leishmania; Leupeptins; Macrophages; Mice; Microbial Sensitivity Tests; Oligopeptides; Pentamidine | 1984 |
Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.
Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover. Topics: Animals; Cathepsin D; Cathepsins; Cysteine; Glutathione; Hydrogen-Ion Concentration; In Vitro Techniques; Iodoacetamide; Kidney; Leupeptins; Liver; Lysosomes; Mice; Pepstatins; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Sulfhydryl Compounds | 1984 |
Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.
N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis. Topics: 2,4-Dinitrophenol; Acrylamides; Animals; Chromatography, Gel; Dinitrophenols; Female; Kinetics; Leupeptins; Pinocytosis; Polymers; Rats; Tyrosine; Yolk Sac | 1984 |
Characterization of subfractions of autolysosomes isolated from rat liver after leupeptin treatment.
Autolysosomes were isolated from leupeptin-treated rat liver (Furuno, K., Ishikawa, T., & Kato, K. (1982) J. Biochem. 91, 1943-1950). They were disrupted by hypotonic treatment and subfractionated by centrifugation in a discontinuous sucrose gradient into three distinct parts: the membranes, the soluble contents, and the insoluble remnants. The content fraction contained the bulk of the activities of lysosomal enzymes with a protein yield of about 36%. The membrane fraction, representing about 5% of protein of the autolysosomes, had a high specific activity of acid phosphatase. In SDS-polyacrylamide gel electrophoretic analysis, the autolysosomal membranes showed protein profiles similar to those of normal lysosomal membranes. The aggregates of partially digested cellular components, including organelles, in the autolysosomes were recovered as granular materials with a very high density (designated as the remnant fraction). This fraction accounted for more than half of protein of the autolysosomes but contained little of the activities of lysosomal enzymes. Lipid analyses revealed that the autolysosomes were poor in lipids because the lipid content of the insoluble remnants was very low. Measurements of the rate of protein degradation in vitro in the crude lysosomal fraction and the isolated autolysosomes from leupeptin-treated rat liver showed that proteolysis was suppressed within the autolysosomes. It was suggested that lipids of sequestered cellular components were preferentially digested within the autolysosomes due to the inhibition of proteolytic activity by leupeptin, and the resulting massive accumulation of proteins was responsible for the enhanced autolysosomal density. Topics: Animals; Electrophoresis, Polyacrylamide Gel; Intracellular Membranes; Leupeptins; Lipids; Liver; Lysosomes; Male; Membrane Proteins; Oligopeptides; Rats; Rats, Inbred Strains; Solubility | 1984 |
Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.
The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process. Topics: Ammonium Chloride; Animals; Cell Line; Cricetinae; Cycloheximide; Half-Life; Humans; Intracellular Membranes; Leucine; Leupeptins; Liver Neoplasms, Experimental; Membrane Proteins; Mice; Microscopy, Fluorescence; Mitochondria, Liver; Rats | 1984 |
Kinetics of cytotoxicity induced by immunotoxins. Enhancement by lysosomotropic amines and carboxylic ionophores.
The kinetics of cytotoxicity induced by ricin and a series of immunotoxins consisting of ricin A-chain coupled to antibodies against cell-surface antigens has been studied. The inhibition of protein synthesis in cells treated with immunotoxins or ricin occurs after a lag period. The rate of protein synthesis decreases according to a mono-exponential function, indicating a first-order process. With increasing concentration of immunotoxin, a maximal rate of inhibition is reached. The inactivation rate induced by immunotoxins was much slower than that achieved with ricin, even when products were compared on a basis of an identical number of molecules bound per cell, demonstrating the real higher efficacy of ricin. The time required to reduce protein synthesis by 90%, denoted T10, was 1.4-1.6 h with ricin, 60 h with anti-T65 immunotoxin on CEM human T leukemia cells (T65 positive), 65 h with anti-p97 immunotoxin on SK-MEL 28 human melanoma cells (p97 positive), and 20 h with an IgM anti-Thy 1.2 immunotoxin on WEHI-7 mouse T leukemia cells (Thy 1.2 positive). In this latter case, when the IgM antibody was replaced by an IgG anti-Thy 1.2, a 5-fold increase in the inactivation rate was obtained, demonstrating the importance of the binding moiety for the immunotoxins. Lysosomotropic amines such as ammonium chloride, chloroquine, and methylamine and carboxylic ionophores such as monensin, which are known to interfere with the uptake of certain macromolecules, strongly increased the rate of protein synthesis inhibition by all immunotoxins tested and increased 4-50,000-fold the sensitivity of cells to the immunotoxin. Enhancement in the inactivation rate was as much as 7-10-fold when either of these compounds was added, generating T10 values comparable to those of ricin. Topics: Amines; Ammonium Chloride; Animals; Antigens, Surface; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Humans; Ionophores; Isoantibodies; Kinetics; Leukemia, Experimental; Leupeptins; Mice; Monensin; Nigericin; Pepstatins; Protein Biosynthesis; Ricin | 1984 |
Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin.
Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later. Topics: Acid Phosphatase; Aminopeptidases; Animals; Autophagy; Cathepsin D; Cathepsin H; Cathepsins; Centrifugation, Density Gradient; Cycloheximide; Cysteine Endopeptidases; Cytoplasm; Dexamethasone; Fructose-Bisphosphate Aldolase; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Male; Oligopeptides; Organoids; Phagocytosis; Rats; Rats, Inbred Strains; Tyrosine Transaminase; Vacuoles | 1983 |
Neurofilament accumulation induced in synapses by leupeptin.
The hypothesis that the usual absence of neurofilaments in synaptic terminals is due to their degradation by the calcium-activated protease present in axoplasm was tested by injecting leupeptin, which inhibits the protease, into the optic tectum of goldfish kept at 15 degrees and at 25 degrees C. The resulting accumulation of neurofilaments in synaptic terminals provides in vivo evidence in support of the hypothesis. The significance of these results and the potential uses of this pharmacological tool are discussed. Topics: Animals; Axonal Transport; Calpain; Cytoskeleton; Fishes; Leupeptins; Nerve Endings; Neuronal Plasticity; Oligopeptides; Protease Inhibitors; Synapses; Synaptic Transmission | 1983 |
Factors affecting the reproducibility of androgen receptor determinations in human prostate.
Several factors affecting the reproducibility of androgen receptor assays in the human prostate gland have been identified. Experiments in the presence of proteolytic inhibitors such as PMSF, leupeptin and aprotinin resulted in no change in the estimates of androgen receptors. The androgen binding was also unaffected by the concentrations of endogenous steroids in the tissue. Although the receptor assay was reliable down to protein concentrations of about 5 g/l, at lower concentrations the reliability of the method was significantly reduced. Similarly, tissue storage at -25 degrees C drastically lowered the binding capacity of the prostatic tissue extracts to 50% of initial receptor content over a period of 3 months. The high degree of correlation between receptor levels in the stroma and epithelium suggests that the binding in the total gland will not be drastically affected by the proportion of stroma and epithelium it contains. Following the widely practised 2-h extraction of the nucleus with 0.6 mol/l KCl buffer, a highly significant non-extractable binding component remained. Analysis of data by the single-point assay produced results comparable to those obtained by standard Scatchard analysis and seemed reliable so long as careful quality control was employed. Topics: Aprotinin; Dihydrotestosterone; Estrenes; Humans; Leupeptins; Male; Methods; Metribolone; NADP; Prostate; Receptors, Androgen; Receptors, Steroid | 1983 |
Proteolysis of the epidermal growth factor receptor by endogenous calcium-activated neutral protease from rat liver.
In A-431 membranes but not in rat liver membranes, the epidermal growth factor (EGF) receptor was converted from a Mr=180,000 to a Mr=160,000 form by a protease activated when cells were broken in the presence of calcium. Calcium-activated neutral protease (CANP) activity in rat liver cytosol was separated from its protein inhibitor by DEAE-cellulose chromatography. When fractions containing this protease activity were incubated with rat liver membranes in the presence of calcium, the Mr=180,000 form of the receptor was converted to the Mr=160,000 form. This conversion was blocked both by the separated endogenous inhibitor and by leupeptin. Apparently CANP is a highly regulated endogenous protease which could degrade the EGF receptor-kinase in most tissues. Topics: Animals; Calpain; Endopeptidases; ErbB Receptors; Leupeptins; Liver; Male; Molecular Weight; Phosphorylation; Rats; Rats, Inbred Strains; Receptors, Cell Surface | 1983 |
Adhesion of cells to polystyrene surfaces.
The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin. Topics: Animals; Bacteriological Techniques; Cell Adhesion; Cricetinae; Culture Media; Cycloheximide; Kidney; Leupeptins; Oxidation-Reduction; Polystyrenes; Trypsin | 1983 |
Cytochemical demonstrations of protease in human peripheral blood cells by use of new alpha-naphthyl ester substrates.
The proteases of human leukocytes were cytochemically studied by use of new alpha-naphthyl esters, tosyl-L-lysine-alpha-naphthyl ester (TLNE) and acetyl-L-tyrosine-alpha-naphthyl ester (ATNE). The hydrolytic activities were strong only in neutrophils, with both substrates. They were inhibited completely by DFP and chymostatin, but not by leupeptin and iodoacetate. These results indicate that chymotrypsin-like enzyme(s), capable of hydrolyzing both substrates, exist in neutrophils. Topics: Chymotrypsin; Histocytochemistry; Humans; Iodoacetates; Iodoacetic Acid; Isoflurophate; Leukocytes; Leupeptins; Lysine; Male; Naphthols; Neutrophils; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Substrate Specificity; Tyrosine | 1983 |
Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin.
The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation. Topics: Aspartic Acid Endopeptidases; Cell Adhesion; Dictyostelium; Edetic Acid; Endopeptidases; Glucosamine; Leucine; Leupeptins; Oligopeptides; Tunicamycin | 1983 |
The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.
When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes. Topics: 2,4-Dinitrophenol; Ammonium Chloride; Animals; Dinitrophenols; Female; Immunoglobulin G; In Vitro Techniques; Iodine Radioisotopes; Leupeptins; Povidone; Pregnancy; Rats; Rotenone; Serum Albumin, Bovine; Yolk Sac | 1983 |
Binding and degradation of doubly radioiodinated luteinizing hormone by mouse Leydig cells.
Doubly radioiodinated LH (**LH) was used to examine the fate of both subunits during interaction with freshly isolated Leydig cells. Cells that had bound **LH and were then resuspended in **LH-free medium released radioactivity continuously from both subunits in acid-soluble form, but to only a limited extent in acid-precipitable form. With cells from BALB/c mice, the initial rate of release of acid-soluble radioactivity was substantially greater from alpha-subunit than from beta-subunit; this difference was not apparent with cells from Swiss-Webster mice. Appearance of acid-soluble radioactivity was inhibited by leupeptin; testosterone production was not affected. Cell-associated radioactivity declined when the resuspension medium contained unlabeled LH, but assumed a steady state when cells were incubated continuously in **LH. Thus, upon binding of LH to receptor, both subunits are internalized and degraded within the lysosome. Binding and degradation can proceed simultaneously, yet independently. LH degradation has no role in acute testosterone production. Topics: Animals; Leupeptins; Leydig Cells; Luteinizing Hormone; Male; Mice; Testosterone; Time Factors | 1983 |
Inhibition of proteolysis in rat yolk sac as a cause of teratogenesis. Effects of leupeptin in vitro and in vivo.
Conceptuses from 9.5-day pregnant rats were cultured for 48 h in heat-inactivated homologous serum to which leupeptin, a specific inhibitor of the lysosomal cysteine-proteinases, was added for the final or the penultimate 6 h. The presence of leupeptin (25 micrograms/ml or above) increased the protein content of yolk sacs at harvesting to approximately twice the control value. The protein content of the embryo at harvesting was lower than that of controls. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. The presence of leupeptin did not affect the rate of uptake of the radiolabelled macromolecule by the yolk sac, nor facilitate its entry into the embryo. When formaldehyde-denatured 125I-labelled bovine serum albumin was added to the culture medium for the final 6 h of culture, little radioactivity was found in the yolk sac at harvesting, and barely any was found in the embryo. Trichloroacetic acid-soluble radioactivity was found in the culture serum. The presence of leupeptin sharply increased the levels of radioactivity in the yolk sac (but not the embryo) and sharply decreased the acid-soluble radioactivity of the culture medium. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in both yolk sac and embryo at harvesting. The presence of leupeptin increased the amount found in the yolk sac and decreased that found in the embryo. These results are interpreted as follows. Leupeptin enters the lysosomes of the yolk sac, inhibiting their cysteine proteinases. The digestion of proteins pinocytosed by the yolk sac is consequently inhibited, resulting in the accumulation of protein by the yolk sac and a decreased flow of amino acids to the embryo. Leupeptin (50 mg/kg), injected into pregnant rats at either 8.5 days or 9.5 days of gestation, induced congenital malformation in the offspring. It is proposed that leupeptin exerts its teratogenic action by inhibiting proteolysis in the lysosomes of the yolk sac, and so depriving the developing embryo of its supply of amino acids at a critical stage of development. Topics: Abnormalities, Drug-Induced; Animals; Embryo, Mammalian; Female; Leucine; Leupeptins; Oligopeptides; Pinocytosis; Povidone; Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Yolk Sac | 1983 |
Degradation of [6-3H]- and [1-14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver.
We studied the degradation and metabolism of radioactive glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver. Removal of 130 micrograms of the protein from the perfusate occurred with a T 1/2 of 10.3 min. Radioactivity associated initially with the microsomal fraction of the tissue homogenate and was then transferred to the lysosomes where hydrolysis of N-acetylglucosamine residues took place. Radioactivity left the lysosomal-rich fraction of the homogenate with a T 1/2 of 50 min. Final accumulation of radioactivity was in the supernatant and greater than 80% of this material was found to be UDP-N-acetylhexosamines. In those studies, 10 mumol of unlabeled glucosamine had been added to the perfusate to expand the intracellular pool of UDP-GlcNAc and prevent further metabolism of any radioactive N-acetylglucosamine released by the lysosomes. When this procedure was not done, greater than one-third of the lysosomally derived N-acetylglucosamine was reused by the liver to form new radioactive glycoproteins secreted into the perfusate. Overall hydrolysis of N-acetylglucosamine from the glycoprotein substrate was greater than 80% during 2 h of perfusion. However, leupeptin, a thiol cathepsin inhibitor, decreased the release of amino sugar by 50%, even though this compound had no effect on the activity of any lysosomal glycosidase in vitro. Large glycopeptides of molecular weight 35,000 accumulated in the lysosomes due to leupeptin. From these studies, we conclude that efficient digestion of the carbohydrate component of this glycoprotein in situ requires the concerted activity of both lysosomal glycosidases and proteases. Topics: Animals; Asialoglycoproteins; Biological Transport; Carbon Radioisotopes; Glucosamine; Kinetics; Leupeptins; Liver; Male; Orosomucoid; Perfusion; Rats; Rats, Inbred Strains; Tritium | 1983 |
Paradoxical effect of leupeptin in vivo on cathepsin B activity.
Leupeptin is a potent inhibitor of cathepsin B in vitro and is presumed to act in a similar manner in vivo. It is currently being used in several laboratories to examine the role of lysosomal proteinases such as cathepsin B in mouse models of muscular dystrophy. This report clearly demonstrates that leupeptin in adequate concentrations in vivo, is a potent stimulator of cathepsin B activity in striated muscle, heart, liver and kidney of the mouse. This paradoxical effect indicates that care is required in the interpretation of the results of the use of leupeptin as a cathepsin B inhibitor in vivo and that its use as an antiprotease for therapeutic purposes may be limited. Studies on CBZ-Phe-Ala-CHN2 demonstrated that this agent, when administered in vivo, inhibited Cathepsin B in the tissues assayed. Topics: Animals; Cathepsin B; Cathepsins; Kidney; Kinetics; Leupeptins; Liver; Lysosomes; Male; Mice; Mice, Inbred DBA; Muscles; Myocardium; Oligopeptides | 1983 |
Ultrastructural studies on autolysosomes in rat hepatocytes after leupeptin treatment.
We have studied the morphological alterations of the lysosomal compartment in rat hepatocytes following intraperitoneal administration of leupeptin, using electron microscopy and cytochemical techniques. At 30 min after the injection, autophagic vacuoles (autophagosomes and autolysosomes), containing cytoplasmic organelles, increased in number in the vicinity of bile canaliculi and also near the Golgi apparatus. At 1 h, most of the autophagic vacuoles were autolysosomes, single membrane-limited bodies positive for acid phosphatase activity. Development of the autolysosomes was accompanied by the reciprocal disappearance of pre-existing secondary lysosomes. From 1 to 8 h, the autolysosomes varied to a great extent in both size and shape as a result of coalescence. Segregated organelles within the autolysosomes were gradually degraded into electron-lucent unidentifiable debris. At later, residual bodies were abundant in the cytoplasm, and occasionally, their contents were discharged into the space of Disse. From 9 to 12 h, the autolysosomes decreased in the volume and number and secondary lysosomes of normal shape and size appeared. The autolysosomes seem to persist for long periods because of a retarded degradation of sequestered materials in leupeptin-treated hepatocytes. Topics: Acid Phosphatase; Animals; Autophagy; Leupeptins; Liver; Lysosomes; Male; Microbodies; Microscopy, Electron; Oligopeptides; Organoids; Rats; Rats, Inbred Strains; Time Factors | 1983 |
Degradation of acetylcholine receptors in muscle cells: effect of leupeptin on turnover rate, intracellular pool sizes, and receptor properties.
The cellular mechanisms of degradation of a transmembrane protein, the acetylcholine receptor (AChR), have been examined in a mouse muscle cell line, BC3H-1. The halftime of degradation of cell surface receptors labeled with [125I] alpha-Bungarotoxin ([125I] alpha-BuTx) is 11-16 h. Leupeptin, a lysosomal protease inhibitor, slows the degradation rate two- to sixfold, depending on the concentration of inhibitor used. The inhibition is reversible since the normal degradation rate is regained within 20 h after removal of the inhibitor. Cells incubated with leupeptin accumulate AChR. Little change in the number of surface AChR occurs but the amount of intracellular AChR increases two- to threefold. Accumulated AChR are unable to bind [125I] alpha-BuTx if excess, unlabeled alpha-BuTx is present in the culture medium during leupeptin treatment. Thus, leupeptin causes the accumulation of a surface-derived receptor population not previously described in these cells. Subcellular fractionation studies utilizing Percoll and metrizamide gradient centrifugation in addition to molecular exclusion chromatography suggest that the accumulated AChR reside in a compartment with lysosomal characteristics. In contrast, the subcellular component containing another intracellular pool of AChR not derived from the surface is clearly separated from lysosomes on Percoll gradients. The sedimentation properties of AChR solubilized from the plasma membrane and the lysosomal fraction have been compared. The plasma membrane AChR exhibits a sedimentation coefficient of 9S in sucrose gradients containing Triton, whereas the AChR derived from the lysosomal fraction exists in part in a high molecular weight form. The large aggregate and the organelle in which it resides may represent important intermediates in the degradative pathway of this membrane protein. Topics: Animals; Bungarotoxins; Cell Line; Centrifugation, Density Gradient; Leupeptins; Mice; Muscles; Oligopeptides; Receptors, Cholinergic | 1983 |
A comparison of lysosomal involvements in the degradation of normal and abnormal endogenous proteins of differing half-lives in MRC5 cells.
Protein degradation by diploid human-embryo lung fibroblasts (MRC5 cells) in monolayer culture was studied. 1. Varying the labelling period of proteins was found to alter the half-lives of labelled abnormal canavanine-containing proteins to an extent very similar to that obtained with normal proteins. 2. By manipulating the times of labelling it was possible to generate a species of abnormal protein with a greater half-life than that of a species of normal protein. A comparison of the lysosomal involvement in their degradation as determined both by inhibition by methylamine, a lysosomotropic agent, and by the degree of increase in protein degradation in step-down conditions, indicated that the degree of lysosomal involvement was not entirely dependent upon the half-life of the protein, but that abnormal proteins are preferentially degraded non-lysosomally. 3. The microtubule inhibitors colchicine and vinblastine were found to stimulate statistically basal protein degradation of normal long-labelled protein, whereas they had less effect upon the basal degradation of the other species of proteins studied and very little effect upon step-down degradation of all proteins studied. The stimulation in protein degradation found did not seem to involve the acid proteinases of lysosomes. Topics: Amino Acids; Cell Line; Colchicine; DNA; Fibroblasts; Half-Life; Humans; Leupeptins; Lysosomes; Methylamines; Proteins; Vinblastine | 1983 |
Effects of protein-degradation inhibitors on the inactivation of tyrosine aminotransferase, tryptophan oxygenase and benzopyrene hydroxylase in isolated rat hepatocytes.
The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism. Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzopyrene Hydroxylase; Cell Separation; Cycloheximide; In Vitro Techniques; Indoleamine-Pyrrole 2,3,-Dioxygenase; Leupeptins; Liver; Male; Methylamines; Oligopeptides; Rats; Rats, Inbred Strains; Tryptophan Oxygenase; Tyrosine Transaminase | 1982 |
Antiproteinase chemotherapy of acute experimental pancreatitis using the low molecular weight oligopeptide aldehyde leupeptin.
Continuous intravenous infusion of the low molecular weight trypsin inhibitor leupeptin prolonged the survival of rats with acute haemorrhagic pancreatitis (p less than 0.001) compared with controls receiving saline alone. Rats receiving high dose intravenous Trasylol (aprotinin) survived no longer than saline-only controls. Combination therapy of leupeptin with Trasylol conferred no additional benefit over animals treated with leupeptin alone. The nature of the infusion was selected blind after the induction of pancreatitis and survival was quantified by recording of body temperature. These preliminary results suggest that sterically favourable molecules which can complete the inhibiton of alpha 2-macroglobulin bound proteinases should contribute to the effective specific chemotherapy of the disease. Topics: Acute Disease; Animals; Aprotinin; Chromatography, Gel; Infusions, Parenteral; Leupeptins; Male; Oligopeptides; Pancreatitis; Rats; Rats, Inbred Strains | 1982 |
Receptor properties of corticosteroid binder IB.
Topics: Animals; Antipain; Cell Nucleus; Deoxyribonuclease I; Endodeoxyribonucleases; Kidney; Leupeptins; Liver; Male; Micrococcal Nuclease; Oligodeoxyribonucleotides; Pyridoxal Phosphate; Rats; Rats, Inbred Strains; Receptors, Steroid; Spermidine | 1982 |
Calcium facilitates endogenous proteolysis of the EGF receptor-kinase.
Topics: Animals; Calcium; Carcinoma, Squamous Cell; Cell Line; Electrophoresis, Polyacrylamide Gel; ErbB Receptors; Humans; Leupeptins; Mice; Molecular Weight; Peptide Hydrolases; Phosphorylation; Protein Kinases; Receptors, Cell Surface | 1982 |
On the mechanisms of inhibition of insulin action by small-molecular-weight trypsin inhibitors.
Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable. Topics: Adipose Tissue; Animals; Benzamidines; Glucose; Guanidines; In Vitro Techniques; Insulin; Insulin Antagonists; Isoflurophate; Leupeptins; Molecular Weight; Rats; Tosylarginine Methyl Ester; Trypsin Inhibitors | 1982 |
Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man.
In normal human fibroblasts, an enzymically active 85,000-dalton precursor form of beta-galactosidase is processed, via a number of intermediates, into a mature 64,000-dalton form. In addition there is an enzymically inactive 32,000-dalton component and its 54,000-dalton precursor. In fibroblasts from patients with a combined deficiency of beta-galactosidase and neuraminidase these last two components are absent and hardly any mature beta-galactosidase can be demonstrated. Nevertheless, in the mutant fibroblasts, precursor beta-galactosidase is synthesized and processed normally. The excessive intralysosomal degradation that is responsible for the deficiency of mature beta-galactosidase can be partially corrected by addition of the protease inhibitor leupeptin, which results in the accumulation of 85,000-dalton precursor beta-galactosidase and of a partially processed 66,000-dalton form. When mutant cells were grown in the presence of a "corrective factor" purified from the medium of NH4Cl-stimulated cell cultures, both beta-galactosidase and neuraminidase activities were restored to low control levels. The immunoprecipitation pattern was completely normal after addition of the corrective factor, and mature 64,000-dalton beta-galactosidase accumulated in the mutant fibroblasts. We propose that the combined beta-galactosidase/neuraminidase deficiency is caused by a defective 32,000-dalton glycoprotein which is normally required to protect beta-galactosidase and neuraminidase against excessive intralysosomal degradation and to give these enzymes their full hydrolytic activity. Topics: beta-Galactosidase; Cells, Cultured; Enzyme Precursors; Fibroblasts; Humans; Lactose Intolerance; Leupeptins; Lysosomes; Molecular Weight; Neuraminidase | 1982 |
Interaction of methotrexate poly(L-lysine) with transformed hepatic cells in culture.
Topics: Animals; Biological Transport; Cell Survival; Kinetics; Leucovorin; Leupeptins; Liver Neoplasms, Experimental; Methotrexate; Peptides; Polylysine; Rats | 1982 |
The degradation of endogenous and exogenous proteins in cultured smooth muscle cells.
The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards 125I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [3H]phenylalanine, was reduced by only 10-17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (Ea = 18 kcal/mol) and short-lived proteins (Ea = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37 degrees C in contrast to the breakdown of LDL which ceased below 20 degrees C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes. Topics: Animals; Aorta, Thoracic; Cattle; Cell Line; Chloroquine; Chymotrypsin; Embryo, Mammalian; Humans; Kinetics; Leupeptins; Lipoproteins, LDL; Lysosomes; Muscle, Smooth, Vascular; Oligopeptides; Proteins | 1982 |
Intracellular transport and degradation of 125I-Labelled denatured serum albumin in isolated nonparenchymal rat liver cells.
The intracellular movement, following uptake of 125I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker beta-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker beta-acetylglucosaminidase, suggesting lysosomal degradation. Topics: Animals; Biological Transport, Active; In Vitro Techniques; Leupeptins; Liver; Lysosomes; Male; Protein Denaturation; Rats; Rats, Inbred Strains; Serum Albumin; Subcellular Fractions | 1982 |
The regulation of proteolysis in normal fibroblasts as they approach confluence. Evidence for the participation of the lysosomal system.
The effect of the lysosomotropic agent NH4Cl and the proteinase inhibitors leupeptin, Z-Phe-Ala-CHN2 (benzyloxycarbonylphenylalanylalanyldiazomethane) and pepstatin on the degradation of intracellular proteins in Swiss 3T3 mouse and normal human fibroblasts in both the exponential and stationary (confluent) growth phases in nutritionally complete conditions was investigated. Inhibitory effects of all four agents on degradation in both growth states were detected. The increase in proteolysis normally occurring as cells approach confluence could be completely blocked by NH4Cl, by Z-Phe-Ala-CHN2, or by pepstatin in the presence of leupeptin. These results suggest that the lysosomal system is responsible for the regulation of proteolysis at confluence and further confirm its role in 'basal' proteolysis in growing cells. Topics: Ammonium Chloride; Animals; Cells, Cultured; Diazomethane; Fibroblasts; Humans; Leupeptins; Lysosomes; Mice; Pepstatins; Proteins | 1982 |
Evidence for exchange of inhibitors which bind to the active site of trypsin. Displacement of one inhibitor with a competitive inhibitor.
Two classes of inhibitors of trypsin (ED 3.4.21.4) have been studied, viz. active site-directed agents such as ovomucoid and active site titrants such as 4-methylumbelliferyl-4-guanidinobenzoate. The kinetics of beta-naphthyl-amidase inhibition by an active site-directed agent were markedly different from simultaneous assays of the availability of the active site towards active site titrants in the presence of the active site-directed agents. Analysis of these data indicated an exchange of active site-directed agent by subsequent addition of active site titrant. One class of trypsin inhibitor could be displaced by another from the trypsin active centre. Competitive chase experiments were designed to measure this exchange in which the active site-directed agent was first equilibrated with trypsin, then partially displaced by incremental additions of an active site titrant; the degree of active sites occupied by these two agents was then determined by active site titration with a second reagent. Topics: Aprotinin; Binding Sites; Binding, Competitive; Kinetics; Leupeptins; Ovomucin; Protein Binding; Trypsin; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitors | 1981 |
Autolysis of calcium-activated neutral protease of chicken skeletal muscle.
The conditions and process of autolysis of calcium-activated neutral protease (CANP) were examined. Optimal conditions for autolysis were the same as those required for expression of activity of CANP. The autolysis proceeded rapidly by a strictly limited proteolysis. During autolysis the molecular weight of CANP changed from 82 K (native CANP or mCANP) to 79 K and then 60 K. The 79 K and 60 K molecular species were both active at microM order of Ca2+ (muCANP), whereas the native CANP is active at mM order of Ca2+ (mCANP). Various proteases examined did not produce muCANP from mcanp under the conditions tested. Furthermore, muCANP did not yield muCANP from mCANP at lower Ca2+ concentrations where only muCANP was active. Therefore, muCANP is produced from mCANP only by the specific autolysis of mCANP. Topics: Animals; Calcium; Calpain; Chickens; Edetic Acid; Endopeptidases; Hydrogen-Ion Concentration; Kinetics; Leupeptins; Molecular Weight; Muscles; Peptide Hydrolases; Protease Inhibitors; Temperature | 1981 |
Easily releasable myofilaments from skeletal and cardiac muscles maintained in vitro. Role in myofibrillar assembly and turnover.
Gentle treatment with an ATP-containing relaxing solution of isolated myofibrils from rat diaphragm, soleus, extensor digitorum longus, and left atria maintained in vitro releases a small amount of myofilaments constituting less than 5% of total myofibrillar protein. Successive extraction of myofibrils produced little further filament release. Releasable myofilaments lack alpha-actinin (Mr = 95,000), certain very high molecular weight proteins (greater than 200,000), and possibly M-line protein but contain other myofibrillar proteins. After pulse-labeling with [3H]leucine for 8 min, specific activity of the myosin heavy chain in the easily releasable myofilaments is 3-6 times higher than the specific activity of myosin heavy chain in the residual myofibrils, although 85-90% of total label is in the myofibrillar myosin. In the absence of protein synthesis, releasable filament specific activity decreases, with a half-time of 60-90 min, to that of the myofibrillar myosin. This labeling pattern appears inconsistent with a simple precursor-product relationship between releasable filaments and myofibrils suggesting that the filaments originate largely from myofibrils. Preincubation of muscles with several factors known to decrease proteolysis, i.e. passive stretch, leupeptin, colchicine, and cycloheximide, reduced the size of the releasable filament fraction. Treatment of muscles with the calcium ionophore A23187, which accelerates proteolysis, and pretreatment of myofibrils with either trypsin or calcium-dependent protease increased filament release. Therefore, the releasable filament fraction may contain intermediates in the breakdown of myofibrils. The labeling kinetics may indicate a mixing of myofilaments within myofibrils which functions in the movement of contractile protein to its possible site of degradation, i.e. the myofibrillar surface. Topics: Animals; Calcimycin; Cell Fractionation; Female; Leupeptins; Molecular Weight; Muscle Proteins; Muscles; Myocardium; Myofibrils; Myosins; Rats; Rats, Inbred Strains; Trypsin | 1981 |
The effects of low temperature and chloroquine on 125I-insulin degradation by the perfused rat liver.
Topics: Animals; Biological Transport; Chloroquine; Cold Temperature; Insulin; Kinetics; Leupeptins; Liver; Perfusion; Rats; Subcellular Fractions | 1981 |
Purification and properties of an enzyme reducing leupeptin acid to leupeptin.
An enzyme catalyzing the reduction of leupeptin acid to leupeptin was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The enzyme was tentatively named leupeptin acid reductase. The molecular weight was estimated to be 320,000 by chromatography on Sepharose 6B. The reductase eluted with leupeptin acid synthetase both in molecular sieve chromatography and in affinity chromatography. The main properties of the reductase were: (1) ATP and NADPH were required for activity. ATP could not be replaced by GTP, ADP or AMP. NADPH could not be replaced by NADH. (2) Michaelis constants for ATP and NaDPH were 4.2 . 10(-5) M and 1.3 10(-6) M, respectively. (3) The enzyme was inhibited by leupeptin, the reaction product, and antipain. Both inhibitors have an L-argininal residue at the C-terminal structure. (4) The enzyme did not catalyze the conversion of leupeptin to leupeptin acid. Leupeptin acid reductase and leupeptin acid synthetase were found in the 10,000 x g pellet of the cell homogenate. The reductase was not released as readily from the pellet as the synthetase either by washing or by repeated freeze-thawing. Synthesis of leupeptin from acetyl-CoA, L-lucine and L-arginine in vitro was accomplished by combining leucine acyltransferase and the enzyme complex consisting of leupeptin acid synthetase an leupeptin acid reductase. Topics: Adenosine Triphosphate; Aldehyde Oxidoreductases; Cell Membrane; Drug Stability; Leupeptins; Molecular Weight; NADP; Oligopeptides; Oxidation-Reduction; Peptide Synthases; Streptomyces | 1981 |
Induced cell injury and cell death as a cause of congenital malformations in rats.
Embryopathy has been produced by inhibition of histiotrophic nutrition in the rat using a number of agents which prevent this process. The experiments were carried out in vitro at various stages of development before the inception of a chorio allantoic placenta. Dose-dependent effects on the embryo were demonstrated using the acid bisazo dye Trypan Blue, which inhibits endocytosis, and an enzyme inhibitor of bacterial origin known as leupeptin which inhibits cathepsin B, H and L. Homogenates of rat kidney and placenta also produced congenital defects; the concommitant electron microscopical changes in the yolk sac suggest that these effects too are due to alterations in the availability or quality of histiotroph. Topics: Abnormalities, Drug-Induced; Animals; Cathepsins; Cell Survival; Congenital Abnormalities; Dose-Response Relationship, Drug; Endocytosis; Female; Kidney; Leupeptins; Microscopy, Electron; Oligopeptides; Placenta; Pregnancy; Rats; Trypan Blue; Yolk Sac | 1981 |