pepstatin and leupeptin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Topics: Agar; Blastomyces; Candida; Depression, Chemical; Endopeptidases; Geotrichum; Leupeptins; Mitosporic Fungi; Oligopeptides; Pepstatins; Species Specificity

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

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

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

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

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

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

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

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

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

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

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