chymostatin and leupeptin

A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

Topics: Antipain; Diffusion; Dose-Response Relationship, Drug; Gelatin; Kinetics; Leupeptins; Models, Biological; Oligopeptides; Particle Size; Peptide Hydrolases; Serine Proteinase Inhibitors; Spectrophotometry; Structure-Activity Relationship; Surface Properties

How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. In these studies, we investigated whether this occurs in other cell types and whether lysosomes are required. Release of ferritin-Fe was induced with desferoxamine (DFO) in (59)Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. The half-lives for ferritin-(59)Fe and protein were parallel (23, 16, and 11 h for the hepatic, Caco2, and K562 cells, respectively). Co-treatment with 180 microM Fe, leupeptin, chymostatin, or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except for a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis.

Topics: Animals; Caco-2 Cells; Chloroquine; Deferoxamine; Enterocytes; Erythroid Cells; Ferritins; Hepatocytes; Humans; Immunoelectrophoresis; Iron; K562 Cells; Leupeptins; Lysosomes; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Rats

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

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

Topics: Adult; Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Calpain; Captopril; Cathepsins; Chymotrypsin; Cysteine Proteinase Inhibitors; Humans; Leucine; Leupeptins; Lisinopril; Lymphocyte Activation; Male; Oligopeptides; Peptidyl-Dipeptidase A; Protease Inhibitors; T-Lymphocytes

We previously reported that desmosomes play a key role in the adhesion of corneocytes, and their digestion by two types of serine proteases leads to desquamation. Patients with recessive X-linked ichthyosis show hyperkeratosis attributable to desmosomes, associated with an increased content of cholesterol sulfate (CS) and an increased thickness of stratum corneum. In this study, therefore, we examined the possibility that CS provokes the abnormal desquamation, acting as a protease inhibitor. Scaling was induced on mice after topical application of chymostatin and leupeptin. Visible scale was also observed on mice after topical application of CS. We found that the stratum corneum thickness of CS-treated mice was increased in comparison with that of vehicle-treated mice. The thickness of the epidermis and the labeling index with proliferating cell nuclear antigen from CS-treated mice was almost the same as that from vehicle-treated mice. Moreover, in the stratum corneum of CS-treated mice, the content of desmosomes was higher than that in vehicle-treated mice. CS also inhibited the protease-induced cell dissociation of human stratum corneum sheets. In vitro, CS competitively inhibited both types of serine protease: the Ki for trypsin was 5.5 x 10(-6) M and that for chymotrypsin was 2.1 x 10(-6) M. These results indicate that CS retards desquamation by acting as a protease inhibitor. Thus, accumulation of stratum corneum in recessive X-linked ichthyosis may be a result of the inhibition by excessive CS of proteases involved in the dissolution of desmosomes, required for desquamation of the stratum corneum.

Topics: Animals; Cholesterol Esters; Epidermis; Humans; Ichthyosis, X-Linked; Leupeptins; Male; Mice; Mice, Hairless; Oligopeptides; Serine Proteinase Inhibitors

Ferritin (Ft) plays an important role in cellular iron metabolism. It can store substantial amounts of iron in a nontoxic soluble form. However, its ability to donate iron for cellular needs, in particular for hemoglobin (Hb) synthesis in human erythroid cells, is still controversial. We studied the role of intracellular Ft-iron in Hb synthesis and the involvement of lysosomal proteolysis in iron release from Ft. Ft-iron release and its subsequent incorporation into heme was investigated in normal human erythroid precursors developing in culture. Dual staining flow cytometry with antibody (Ab)-specific for Ft and for Hb showed a decrease in cellular Ft content in erythroid cells during their maturation. Cellular Ft-iron participation in heme synthesis was studied by labeling cells with 59Fe. Cells were incubated with 59Fe-labeled human diferric transferrin (Tf), then chased, and intracellular radioiron distribution between Ft and Hb was determined on subsequent days by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or Ft immunoprecipitation and heme extraction. On day 6, most of the 59Fe accumulated in Ft. Thereafter, a progressive decrease of radioiron in Ft and a corresponding increase of the label in Hb was observed. Inhibition of heme synthesis with succinylacetone caused radioiron to remain in Ft and prevented its redistribution. Addition of unlabeled diferric Tf to the culture medium did not prevent radioiron from appearing in Hb. Chloroquine repression of lysosomal function prevented radio-iron redistribution between Ft and Hb. Inhibition of proteolysis by chymostatin and/or leupeptin led to Ft-protein accumulation in the cells and also prevented radioiron transfer from Ft to Hb. The results of the present study suggest that intracellular Ft donates iron for heme synthesis and that proteolytic Ft degradation in a lysosomal-like compartment is necessary for iron release and its transfer to heme.

Topics: Autoradiography; Cells, Cultured; Chloroquine; Enzyme-Linked Immunosorbent Assay; Erythroblasts; Erythroid Precursor Cells; Ferritins; Flow Cytometry; Heme; Hemoglobins; Humans; Iron; Iron Radioisotopes; Kinetics; Leupeptins; Oligopeptides; Protease Inhibitors

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

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.

Topics: Amino Acids, Branched-Chain; Animals; Caseins; Cations; Endopeptidases; Hot Temperature; Leupeptins; Multienzyme Complexes; Muscle Proteins; Muscles; Myofibrils; Nephropidae; Oligopeptides; Peptide Fragments; Protease Inhibitors; Substrate Specificity; Troponin

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

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 peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.

Topics: Amino Acid Sequence; Animals; Antipain; Cattle; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Erythrocytes; Humans; Insulin; Kinetics; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Oxidation-Reduction; Pentamidine; Proteasome Endopeptidase Complex

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

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

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.

Topics: Animals; Cell Division; Cell Line; Chromatography, Ion Exchange; Cricetinae; Cricetulus; Dipeptides; gamma-Glutamylcyclotransferase; In Vitro Techniques; Leupeptins; Methylamines; Oligopeptides; Proteins; Transglutaminases

The effect of eight microbial protease inhibitors on Ag-presentation to six different Ag-specific T cell clones was investigated. We found that these protease inhibitors can inhibit Ag presentation in a highly selective manner. This selectivity was evident with T cell clones specific to different Ag as well as with T cells specific to the same Ag but differing in their H-2 restriction. The inhibition was to due to cytotoxicity or effects through the TCR because none of the eight inhibitors inhibited IL-2-induced T cell proliferation, and because they did not inhibit Ag presentation by fixed APC or synthetic polypeptide. The conclusion after these data suggests that each specific antigenic fragment is produced by a unique set of proteases.

Topics: Animals; Antigen-Presenting Cells; Antipain; Clone Cells; Histocompatibility Antigens Class II; Immunosuppressive Agents; Leupeptins; Metalloendopeptidases; Mice; Oligopeptides; Protease Inhibitors; T-Lymphocytes

Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo.

Topics: Adenosine Triphosphate; Animals; Gluconeogenesis; In Vitro Techniques; Leupeptins; Liver; Lysosomes; Male; Oligopeptides; Protease Inhibitors; Rats; Structure-Activity Relationship; Tyrosine Transaminase

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

Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors.

Topics: Animals; Anti-Bacterial Agents; Chymotrypsin; Enzyme Inhibitors; Fertilization; Leupeptins; Male; Oligopeptides; Peptides; Spermatozoa; Urochordata

Purified protein methylesterase (PME) from rat kidneys was incubated with ovalbumin-methyl esters and a series of protease inhibitors. All four inhibitors with C-terminal aldehyde, leupeptin, chymostatin, Boc-Gln-Leu-Lys-H and D-Phe-Pro-Arg-H completely blocked PME activity. Other inhibitors including, alpha-1 antitrypsin, soybean trypsin inhibitor, antithrombin III, phenylmethylsulfonylfluoride, aprotinin and lima bean trypsin inhibitor had no significant effect whereas pepstatin, at high concentration reduced the enzymatic activity by 25%. The most potent inhibitors, leupeptin and chymostatin, had a Ki of 3.5 X 10(-8) and 5.4 X 10(-7) M, respectively. These inhibitors provide two new tools to study PME function.

Topics: Animals; Kinetics; Leupeptins; Oligopeptides; Protein Methyltransferases; Rats

The effects of protease inhibitors on the secretion of catecholamines were studied in cultured bovine adrenal medullary cells. Although the inhibitors of serine proteases could inhibit the carbamylcholine-induced secretion, they failed to inhibit the secretion evoked by either high K+ or A23187. The thiol protease inhibitor had no effect on the secretion. These results therefore seem to indicate that the serine protease inhibitors may inhibit the receptor-mediated secretion probably through their effects on the plasma membrane, thus suggesting that a possible involvement of the serine, and thiol proteases in exocytosis may be unlikely.

Topics: Adrenal Medulla; Animals; Calcimycin; Carbachol; Catecholamines; Cattle; Cells, Cultured; Cysteine Endopeptidases; Endopeptidases; Exocytosis; Kinetics; Leupeptins; Oligopeptides; Potassium; Serine Endopeptidases

The effects of potent thiol protease inhibitors in vitro (leupeptin, antipain, chymostatin and E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine) on intracellular cathepsin B and hemoglobin (Hb)-hydrolase from cultured B16 melanoma cells were studied. E-64 induced cultured B16 melanoma cells to decrease the activities of intracellular cathepsin B (EC 3.4.22.1.) but did not have this effect with Hb-hydrolase or acid phosphatase (EC 3.1.3.2). Leupeptin, antipain and chymostatin induced B16 melanoma cells to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. These results indicate that there are two kinds of thiol protease inhibitors, each with a varying reaction to cultured B16 melanoma--inhibition of intracellular cathepsin B, and conversely, inducement of both cathepsin B and Hb-hydrolase.

Topics: Animals; Antipain; Cathepsin B; Kinetics; Leucine; Leupeptins; Melanoma, Experimental; Mice; Oligopeptides; Peptide Hydrolases; Protease Inhibitors

Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin.

Topics: Animals; Chymotrypsin; Erythrocytes; Humans; In Vitro Techniques; Leupeptins; Oligopeptides; Plasmodium falciparum; Protease Inhibitors; Trypsin

Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated.

Topics: Animals; Aprotinin; Cells, Cultured; Dose-Response Relationship, Drug; Female; Growth Hormone; Leupeptins; Oligopeptides; Phenylmethylsulfonyl Fluoride; Pituitary Gland, Anterior; Prolactin; Protease Inhibitors; Rats; Trypsin Inhibitor, Kunitz Soybean

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.

Topics: Animals; Anti-Bacterial Agents; Antipain; Blastocyst; Cystatins; Embryonic Development; Enzyme Inhibitors; Female; Glycopeptides; Leupeptins; Oligopeptides; Pepstatins; Peptides; Pregnancy; Protease Inhibitors; Rats; Rats, Inbred Strains

A rat model has been developed to study the local effects of burn injury on the underlying muscle tissue. Protein turnover was measured in soleus muscle incubated in vitro in which both tyrosine release and protein synthesis was measured. A scald injury (3 seconds) to a small area of one hindlimb produces an increase in muscle proteolysis and is without effect on the soleus muscle of the contralateral leg. A very high concentration of indomethacin (40 mumol/L) had no effect on proteolysis in the control muscle but specifically inhibited burn-induced protein breakdown. However, since other cyclooxygenase inhibitors (aspirin and ibuprofen), lipoxygenase inhibitors (ETYA, NDGA, and esculetin), and mepacrine (a phospholipase inhibitor) had no effect on protein breakdown, it is unlikely that a product of arachidonic acid metabolism maintains the increased proteolysis in vitro. In addition, endogenous production of prostaglandin E2 (PGE2) was not different in muscles from burned and control legs. Probes of the proteolytic pathway using inhibitors show that the burn-induced stimulation of proteolysis is consistent with the stimulation of lysosomal protease activity. These results are supported by the observation of increased acid protease activity in muscle homogenates from the burned leg. The best hypothesis that explains these data is that a lysosomal pathway of protein degradation may be enhanced by burn. Products of arachidonic acid metabolism do not appear to maintain burn-induced proteolysis in muscle, although their role in initiating the pathological changes in vivo cannot be excluded.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Aspirin; Burns; Hindlimb; Ibuprofen; In Vitro Techniques; Indomethacin; Leupeptins; Lysosomes; Muscles; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Proteins; Quinacrine; Rats; Rats, Inbred Strains; Tyrosine

The proteases of human leukocytes were cytochemically studied by use of new alpha-naphthyl esters, tosyl-L-lysine-alpha-naphthyl ester (TLNE) and acetyl-L-tyrosine-alpha-naphthyl ester (ATNE). The hydrolytic activities were strong only in neutrophils, with both substrates. They were inhibited completely by DFP and chymostatin, but not by leupeptin and iodoacetate. These results indicate that chymotrypsin-like enzyme(s), capable of hydrolyzing both substrates, exist in neutrophils.

Topics: Chymotrypsin; Histocytochemistry; Humans; Iodoacetates; Iodoacetic Acid; Isoflurophate; Leukocytes; Leupeptins; Lysine; Male; Naphthols; Neutrophils; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Substrate Specificity; Tyrosine

The following three potent inhibitors of hepatocytic proteolysis were investigated to see if they would inhibit the intracellular inactivation of enzymes: chymostatin and leupeptin (proteinase inhibitors) and methylamine (a lysosomotropic weak base). Chymostatin inhibited the inactivation of two of the three enzymes tested: tyrosine aminotransferase (EC 2.6.1.5) and tryptophan oxygenase (tryptophan 2,3-dioxygenase, EC 1.13.11.11). Leupeptin had no effect on any of the enzymes, whereas methylamine had only a weak inhibitory effect on tyrosine aminotransferase inactivation. Apparently proteolytic cleavage (probably by a non-lysosomal proteinase, since only chymostatin is effective) is involved in the inactivation of tyrosine aminotransferase and tryptophan oxygenase. The third enzyme, benzopyrene hydroxylase (flavoprotein-linked mono-oxygenase, EC 1.14.14.1), is probably inactivated by a non-proteolytic mechanism.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzopyrene Hydroxylase; Cell Separation; Cycloheximide; In Vitro Techniques; Indoleamine-Pyrrole 2,3,-Dioxygenase; Leupeptins; Liver; Male; Methylamines; Oligopeptides; Rats; Rats, Inbred Strains; Tryptophan Oxygenase; Tyrosine Transaminase

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards 125I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [3H]phenylalanine, was reduced by only 10-17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (Ea = 18 kcal/mol) and short-lived proteins (Ea = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37 degrees C in contrast to the breakdown of LDL which ceased below 20 degrees C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.

Topics: Animals; Aorta, Thoracic; Cattle; Cell Line; Chloroquine; Chymotrypsin; Embryo, Mammalian; Humans; Kinetics; Leupeptins; Lipoproteins, LDL; Lysosomes; Muscle, Smooth, Vascular; Oligopeptides; Proteins