alpha-chymotrypsin and chymostatin

Human chymase forms angiotenin (ANG) I to ANG II, whereas the roles of ANG II generated by chymase and the effects of chymase inhibitors have been unclear. On the other hand, rat chymase could not convert ANG I to ANG II. In isolated rat arteries, the ANG I-induced vascular contraction was completely suppressed by angiotensin-converting enzyme (ACE) inhibitor only. However, 30% of ANG I-induced vascular contraction in isolated human arteries was suppressed by an ACE inhibitor, but the remainder was blocked by chymostatin. In hamster hypertensive models, ANG II formation by ACE, but not by chymase, in vascular tissues plays an important role in maintaining hypertension. ANG II formation also induces vascular remodeling such as neointima formation. After balloon injury of vessels in dog, chymase and ACE activities were significantly increased in the injured vessels. In this model, an ANG II receptor antagonist was effective in preventing neointimal formation after balloon injury of vessels in dog, but an ACE inhibitor was ineffective. In dog grafted veins, the activities of chymase and ACEmin the grafted vein were significantly increased 15- and 2-fold, respectively, compared with those in the symmetrical veins. The intimal area of the grafted vein was reduced by a chymase inhibitor. Therefore, chymase-dependent ANG II formation plays an important role in the proliferative response, and chymase inhibitors may appear useful for preventing vascular proliferation.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Chymases; Chymotrypsin; Cricetinae; Dogs; Enzyme Inhibitors; Humans; Hypertension; Neovascularization, Physiologic; Oligopeptides; Rats; Serine Endopeptidases; Serine Proteinase Inhibitors; Species Specificity; Vasoconstriction

A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.

Topics: Amino Acid Sequence; Analysis of Variance; Animals; Base Sequence; Chromatography, Affinity; Chymotrypsin; Dimethyl Sulfoxide; DNA Primers; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Gastric Juice; Hydrogen-Ion Concentration; Kinetics; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Oligopeptides; Palinuridae; Phenylmethylsulfonyl Fluoride; Proteolysis; Sequence Analysis, DNA; Temperature; Toluene

In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug-target interactions. CE enables the separation of proteins and drug-enzyme complexes preserving their physiological activity in aqueous media. By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug-target complex after CID. In this study, alpha-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of alpha-CT and a chymotrypsin-chymostatin complex under MS-compatible conditions. The use of neutral-coated CE capillaries was mandatory to reduce analyte-wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID. Fragmentation of the drug-enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V(50) voltage that enables 50% complex fragmentation as a reference value for chymotrypsin-chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug-enzyme complexes during ionization-desolvation processes. With this optimized setup, different CT inhibitors could be investigated and compared.

Topics: Buffers; Chymotrypsin; Electrophoresis, Capillary; Hydrogen-Ion Concentration; Oligopeptides; Osmolar Concentration; Protein Interaction Mapping; Tandem Mass Spectrometry; Temperature

Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O(-)(2) by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O(-)(2), whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O(-)(2), whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the alpha-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions.

Topics: Bacterial Proteins; Cells, Cultured; Chymotrypsin; Complement C3; Enzyme-Linked Immunosorbent Assay; Gene Deletion; Humans; Immunoblotting; Luminescent Measurements; Matrix Metalloproteinase 9; Neutrophil Activation; Neutrophils; Oligopeptides; Peptide Hydrolases; Phagocytosis; Protease Inhibitors; Superoxides; Treponema denticola; Virulence Factors

Mass spectrometric techniques play a prominent role in the rapidly expanding field of high-throughput screening (HTS). In this paper, the authors present a novel qualitative approach for the screening of a small library of compounds using MALDI-TOF-MS and HPLC-ESI-MS/MS. Chymotrypsin (CT), a serine protease, was selected as the target protein. A well-known inhibitor of CT is chymostatin (CS), a naturally occurring peptide aldehyde, which is reported to be a mixture of 3 components-A, B, and C-differing only in one of the amino acids. The authors report that native CS mixture consists of 3 additional ring hydroxylated components and that each compound exists in 2 epimeric forms. In case of protein-binding compounds, only 1 of the epimers was found to be active. A unique feature of this study is the generation of a combinatorial library of CS derivatives applying a one-pot strategy followed by identification and structural elucidation of the library components. Analytical investigation of the library resulted in the identification of 22 compounds. After incubation with CT and centrifugal ultrafiltration, 10 compounds were detected as protein-binding ligands. Finally, the complementary potentials of MALDI-TOF-MS and HPLC-MS/MS in the screening of complex ligand mixtures have been discussed.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin; Combinatorial Chemistry Techniques; Drug Design; Molecular Structure; Oligopeptides; Peptide Library; Protein Binding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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 investigated the angiogenic effect of chymase, an alternative angiotensin II-generating enzyme, on angiogenesis using hamster sponge implant model. Exogenous administration of angiotensin II (Ang II) or angiotensin I (Ang I) directly into the sponges enhanced angiogenesis, as determined from the hemoglobin contents in the sponge granuloma tissues. Chymostatin, an inhibitor of chymase, inhibited angiogenesis induced by Ang I but not by Ang II, suggesting the presence of a chymase-like Ang II-generating activity in the sponge granuloma. TCV-116 (5 mg/kg p.o.), an antagonist of Ang II type 1 receptor, and chymostatin suppressed bFGF-induced angiogenesis, suggesting the significance of the endogenous angiotensin system. Chymase activity in the sponge granuloma increased in parallel with the rise in hemoglobin contents induced by bFGF. We also examined the effects of direct administration of human pro-chymase gene or purified hamster chymase, and demonstrated that in vivo human pro-chymase gene transfection and direct injection of purified chymase enhanced angiogenesis, which was 50% inhibited by TCV-116. Sponge granulomas treated with Ang II was supressed by vascular endothelial growth factor (VEGF) antisense. Our results suggest that chymase enhanced angiogenesis partly through the local production of Ang II, followed by up-regulation of VEGF.

Topics: Angiotensin I; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Benzimidazoles; Biphenyl Compounds; Chymases; Chymotrypsin; Cricetinae; Endothelial Growth Factors; Granuloma; Hemoglobins; Humans; Lymphokines; Male; Mesocricetus; Neovascularization, Physiologic; Oligopeptides; Protein Isoforms; Serine Endopeptidases; Serine Proteinase Inhibitors; Tetrazoles; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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 examined conversion of Big endothelin-1 (ET-1) to mature ET-1 and pressor action during perfusion of the isolated perfused rat lung with Big ET-1. Big ET-1 caused a concentration-related increase in perfusion pressure and the pressor molar potency of the peptide was fivefold less than that of ET-1. Pressor responses to Big ET-1 were accompanied by an increase in immunoreactive-ET (IR-ET) levels in the perfusate and in the lung tissues. Pretreatment with phosphoramidon (10(-4) M), a metalloproteinase inhibitor, markedly suppressed the pressor action and increment in IR-ET in the tissues. Unexpectedly, the amount of IR-ET in the perfusate during perfusion of Big ET-1 was not influenced by phosphoramidon treatment. On the other hand, chymostatin, an inhibitor of chymotrypsin-like enzymes, effectively suppressed IR-ET levels in the perfusate; however, this enzyme inhibitor was without effect on the pressor action of Big ET-1 or on the increase in IR-ET levels in lung tissues. We tentatively conclude that the phosphoramidon-sensitive conversion of Big ET-T to ET-1 is linked to the pressor action of Big ET-1 in the isolated perfused rat lung. In addition, it seems likely that chymostatin-sensitive conversion of Big ET-1 to ET-1 does not play a major role in the conversion of the precursor to the mature form. We propose that IR-ET present in the tissues rather than that in the perfusate is a better indicator of the functional conversion of Big ET-1 in the rat lung.

Topics: Animals; Aspartic Acid Endopeptidases; Chymotrypsin; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; In Vitro Techniques; Kinetics; Lung; Male; Metalloendopeptidases; Oligopeptides; Perfusion; Protein Precursors; Pulmonary Circulation; Rats; Rats, Sprague-Dawley

Invasion of human erythrocytes by Plasmodium falciparum is inhibited by chymostatin. This suggests that digestion of erythrocyte surface proteins by a protease with chymotrypsin-like activity may be involved in the invasion process. We find that treatment of intact erythrocytes with chymotrypsin cleaves the integral membrane protein, band 3, generating a major fragment with an apparent molecular weight of 58 kDa. We have used measurements of the rotational mobility of band 3, labelled with the phosphorescence probe, eosin-5-maleimide, as a monitor of the changes in the molecular organisation of the erythrocyte membrane which accompany band 3 cleavage. We report that the chymotrypsin treatment increases the rotational freedom of band 3, possibly due to conformational changes which disrupt its interaction with the underlying peripheral membrane proteins. We also show that chymotrypsin-treated erythrocytes undergo extensive endocytosis upon incorporation of exogenous fluorescently labelled phospholipid. We suggest that during the invasion process, digestion of band 3 by a chymotrypsin-like protease may induce a localised disruption of the erythrocyte membrane. This destabilised region of membrane may represent the site for the insertion of parasite-derived phospholipid, thus allowing the formation of the parasitophorous vacuole membrane.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Endocytosis; Erythrocyte Membrane; Humans; In Vitro Techniques; Oligopeptides; Plasmodium falciparum; Protein Conformation

We have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse alpha-amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse alpha-amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse alpha-amylase than the 28 kDa killer secretion signal. We detected a chymostatin-sensitive protease activity in the culture medium of S. pombe, which digests mouse alpha-amylase secreted into the culture medium. The addition of 5 micrograms/ml chymostatin was shown to protect mouse alpha-amylases from this degradation.

Topics: alpha-Amylases; Amino Acid Sequence; Animals; Chymotrypsin; Endopeptidases; Genetic Vectors; Mice; Molecular Sequence Data; Oligopeptides; Protease Inhibitors; Protein Sorting Signals; Recombinant Proteins; Schizosaccharomyces

A series of analogues of chymostatin, including Z-Arg-Leu-Phe-aldehyde (Z-Arg-Leu-Phe-H), have been synthesized. Analysis of the inhibitory potential of these analogues permits identification of residues and interactions that are important for inhibitory activity. Moreover, the structure-function relationship for Z-Arg-Leu-Phe-H and chymostatin inhibition of chymotrypsin and Streptomyces griseus proteinase A (SGPA) was probed further with the aid of molecular mechanics. This analysis identified interactions that provide an explanation for the enhanced activity of the natural product, chymostatin, over the synthetic analogues in the inhibition of chymotrypsin but not SGPA.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Cattle; Chymotrypsin; Kinetics; Molecular Sequence Data; Oligopeptides; Protein Conformation; Serine Endopeptidases; Serine Proteinase Inhibitors; Streptomyces griseus

We have recently reported that unipolar cell shedding from plantar stratum corneum incubated in vitro, and the associated degradation of the desmosomal protein desmoglein I, are dependent on the activity of a proteinase that can be inhibited by aprotinin, chymostatin and zinc ion. The aim of this work was to find a proteinase in plantar stratum corneum that fulfils the criteria for being the responsible enzyme. Dissociated plantar corneocytes were incubated with the chymotrypsin substrate 3-carbomethoxypropionyl-L-Arg-L-Pro-L-Tyr-p-nitroanilide hydrochloride (S-2586) and H-D-Ile-Pro-Arg-p-nitroanilide dihydrochloride (S-2288), a substrate for a wide range of serine proteinases with arginine specificity. There was a significant rate of hydrolysis of S-2586, but S-2288 was hydrolysed only very slowly. Extraction of dissociated corneocytes with buffers containing KCl or sodium dodecyl sulphate released one major proteinase that could be detected by electrophoresis in polyacrylamide gels with copolymerized casein and subsequent incubations of the gels. Both the caseinolytic activity and the S-2586-hydrolysing activities were inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. The S-2586-hydrolysing activity was also inhibited by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride. Both activities were optimal at pH 7-8 but were also significant at pH 5.5. On gel exclusion chromatography, the S-2586-hydrolysing and caseinolytic activities were eluted with an apparent molecular weight of around 18 kDa. When analyzed by electrophoresis in the presence of sodium dodecyl sulphate under non-reducing conditions the caseinolytic enzyme had an apparent molecular weight of around 25 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aprotinin; Cell Communication; Chromatography, Gel; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epidermis; Humans; Keratolytic Agents; Oligopeptides; Zinc

An alternative angiotensin II-forming system distinct from the vascular renin-angiotensin system was demonstrated using a rat hindlimb perfusion system and synthetic substrates. This pathway was resistant to captopril and aprotinin, but was highly sensitive to chymostatin. Moreover, angiotensin II formation was substrate-dependent, i.e. angiotensin II formation from tridecapeptide human renin substrate in the presence of captopril was more than twice than that from an equimolar amount of angiotensin I. Both pathways may play a role in regulating the peripheral circulation.

Topics: Angiotensin I; Angiotensin II; Animals; Aprotinin; Captopril; Chromatography, High Pressure Liquid; Chymotrypsin; Male; Oligopeptides; Radioimmunoassay; Rats; Rats, Inbred Strains; Renin; Substrate Specificity

When rat hepatocytes were cultured in the presence of various specific protease inhibitors, lysosomal acid phospholipase A1 activity decreased progressively. Exposure of the cultured cells to 0.1 micrograms/ml of pepstatin, E 64, leupeptin or chymostatin also reduced the catalytic activities of several lysosomal marker enzymes. Irrespective of the protease inhibitor type employed, acid phospholipase A1 activity reacted most sensitively, followed by acid phosphatase, acid beta-N-acetyl-D-hexosaminidase and acid beta-glucuronidase. Of the protease inhibitors studied, pepstatin appeared to be most potent in reducing lysosomal enzyme activities in cultured hepatocytes. These findings suggest that proteolytic processes at as yet unknown, possibly extralysosomal sites play an important role in the turnover rates of lysosomal enzymes.

Topics: Animals; Cells, Cultured; Chymotrypsin; Cysteine Proteinase Inhibitors; Leucine; Leupeptins; Liver; Lysosomes; Oligopeptides; Pepstatins; Phospholipases; Phospholipases A; Phospholipases A1; Protease Inhibitors; Rats; Time Factors

The reaction of chymase, a chymotryptic proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with time, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] much greater than [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kinetics. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants (k2) and dissociation constants (Ki) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation (k2/Ki), the first-order reactivation rate constant (k-2), and the dissociation constant of the covalent complex (Ki*) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.

Topics: Amino Acid Chloromethyl Ketones; Aprotinin; Binding Sites; Chymases; Chymotrypsin; Enzyme Reactivators; Humans; Kinetics; Mathematics; Oligopeptides; Serine Endopeptidases; Serine Proteinase Inhibitors; Skin; Trypsin Inhibitors

Sporothrix schenckii produces two extracellular proteinases in albumin- or collagen-supplemented unbuffered liquid medium. Proteinase I had an optimal pH of 6.0, and its activity was strongly inhibited by chymostatin. Proteinase II had an optimal pH of 3.5, and its activity was strongly inhibited by pepstatin. Speculating that these two proteinases are key enzymes for fungal growth, we investigated the influences of culture medium pH and either or both of the proteinase inhibitors pepstatin and chymostatin on the cell growth of S. schenckii. In buffered medium at a pH of 3.5, an optimal pH for proteinase II, only proteinase II activities were observed, while at pH 6.0, an optimal pH for proteinase I, only proteinase I activities were observed. However, there was no cell growth inhibition. These results suggested that the regulation of the production of the two proteinases is dependent on environmental pH. The addition of pepstatin or chymostatin to the culture medium did not inhibit the cell growth of S. schenckii, but the addition of both inhibitors at a concentration of 10 micrograms/ml strongly inhibited growth. These results suggested that at least one of the two proteinases was expressed to allow fungal growth in albumin-supplemented media. The indirect fungistatic action of the proteinase inhibitors, which inhibit proteolysis, may be applied to a topical therapeutic agent in vivo.

Topics: Chymotrypsin; Culture Media; Endopeptidases; Humans; Hydrogen-Ion Concentration; Oligopeptides; Pepstatins; Protease Inhibitors; Sporothrix

Cathepsin-D has been previously reported to cleave intact PTH into PTH-(1-34) and -(35-84) in membranous fractions of rat and bovine kidney. Whether PTH degradation occurs by intact kidney cells, however, has not been examined in detail. We have, therefore, examined this possibility using an opossum kidney (OK) cell line which possesses the characteristics of proximal renal tubules and responds to PTH. PTH radioimmunoreactivity recovered in trichloroacetic acid-soluble products and in fractions eluted from reverse phase HPLC was measured using an antibody directed to the midregion and C-terminus of PTH. In this study, intact OK cells, but not extracellular enzymes, cleaved human (h) PTH-(1-84) into three discrete fragments which were released into the medium in a time- and temperature-dependent fashion. Half-maximal velocity of PTH-degrading activity (PTHDA) was observed at 9 nM hPTH-(1-84). A 1000-fold molar excess of PTH antagonists [hPTH-(3-34) and [Tyr34]hPTH-(7-34)amide] markedly inhibited PTHDA, whereas ACTH, glucagon, or big gastrin did not suppress it, suggesting an involvement of the PTH receptor in PTHDA. This PTHDA was strongly inhibited by phenylmethylsulfonylfluoride and chymostatin, but not by trypsin inhibitor, elastatinal, or inhibitors of aspartic, cysteine, or metalloproteinases, suggesting that it is due to a seryl chymotrypsin-like endopeptidase. Analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by UV absorbance (210 nm), three of which were measurable by PTH RIA, and each corresponded to the three PTH fragments produced by OK cells. All three fragments were predominantly suppressed in the presence of chymostatin, suggesting that chymotrypsin-like activity is solely responsible for PTHDA in intact OK cells. To further explore the cleavage sites of PTH by chymotrypsin, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the conclusion that a chymotrypsin-like enzyme in OK cells cleaved the hormone between residues 23-24, and 34-35 to produce, at least, hPTH-(24-84) and -(35-84). Lysosomal blockers (chloroquine, ammonium chloride, or monensin) did not affect this PTHDA. Our present study indicates that chymotrypsin-like endopeptidase, but not other endopeptidase or lysosomal enzymes, is responsible for the limited hydrolysis of PTH by intact OK cells.

Topics: Amino Acids; Animals; Cell Line; Chromatography, High Pressure Liquid; Chymotrypsin; Endopeptidases; Kidney; Kidney Tubules, Proximal; Kinetics; Oligopeptides; Opossums; Parathyroid Hormone; Peptide Fragments; Phenylmethylsulfonyl Fluoride; Protease Inhibitors

The effect of the cathepsin B inhibitor chymostatin was studied in mice with an X chromosome-linked muscular dystrophy. Treatment for 7 weeks at a daily dose of 1 microgram/g body weight had no apparent beneficial effect: serum creatine kinase levels, histopathology and the activity of muscle cathepsin B were not significantly altered by the treatment.

Topics: Animals; Chymotrypsin; Female; Male; Mice; Mice, Inbred C57BL; Muscular Dystrophy, Animal; Oligopeptides

Lysosomotropic amino acid esters and amides kill Leishmania amazonensis amastigotes by a mechanism which probably involves enzymatic hydrolysis of the compounds and rapid accumulation of less permeant amino acid within the parasites. We show here that, in agreement with this model, the proteinase inhibitors antipain and chymostatin prevented the killing of intracellular and isolated parasites by L-leucine methyl ester (Leu-OMe). Survival of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by tetrazolium (MTT) reduction. Near maximal protection of intracellular parasites was obtained after 24 h incubation of macrophage cultures with 50 micrograms ml-1 antipain or chymostatin. Incubation for greater than 1 h with chymostatin or greater than 4 h with antipain alone resulted in loss of viability of the parasites. Protective activity was only slightly diminished by 20 h chase of isolated parasites in inhibitor-free medium. Two synthetic chymostatin analogues, Z-Val-Phe-Sc and Z-Ile-Phe-Sc, protected isolated amastigotes at 4 or 10 micrograms ml-1. With the exception of Trp-NH2, the toxicity of which was only minimally inhibited, antipain and chymostatin also prevented parasite destruction by other amino acid derivatives. Finally, in concentration-dependent fashion, the inhibitors reduced the accumulation of [3H]leucine in isolated amastigotes incubated with [3H]Leu-OMe. Since uptake of labelled ester was unaffected, we postulate that protection involves inhibition of the parasite enzymes which hydrolyse the amino acid derivatives.

Topics: Amino Acids; Animals; Antipain; Chymotrypsin; Esters; Female; Hydrogen-Ion Concentration; Hydrolysis; Leishmania; Leucine; Macrophages; Mice; Oligopeptides; Protease Inhibitors

The microbial, peptide-derived aldehyde chymostatin is a potent, competitive inhibitor of chymotrypsin and cathepsin G: Ki = 4 X 10(-10) and 1.5 X 10(-7) M, respectively. Et is "slow-binding inhibitor" of both proteases and, as such, allows determination of rate constants for its association with and dissociation from these proteases. Inhibition kinetics indicate second-order rate constants for the association of chymostatin with chymotrypsin and cathepsin G of 360,000 and 2000 M-1 S-1, respectively and a first-order rate constant for the dissociation of both protease-chymostatin complexes of approximately 0.0002 s-1. Thus, the extreme difference in potency of chymostatin as an inhibitor of chymotrypsin and cathepsin G originates entirely in Kon. Solvent deuterium isotope effects (SIE) were determined to probe the reaction step that rate limits Kon. For the reaction of chymotrypsin with chymostatin, the SIE for Kon is 1.6 +/- 0.1, while for the reaction of chymotrypsin with the peptide substrates Ala-Ala-Phe-pNA and Suc-Ala-Ala-Pro-Phe-pNA, the SIE's for Kc/Km are 2.8 +/- 0.2 and 1.9 +/- 0.1, respectively. These results suggest that Kon for the association of chymotrypsin with chymostatin is at least partially rate limited by a reaction step involving proton transfer. Combined with results for the inhibition of chymotrypsin by Bz-Phe-H [Kennedy, W.P., & Schultz, R. M. (1979) Biochemistry 18, 349-356], these data suggest a mechanism for inhibition by chymostatin involving the general-base-catalyzed formation of an enzyme-bound hemiacetal, followed by a conformational change of this intermediate that produces the final, stable complex of enzyme and inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cathepsin G; Cathepsins; Chymotrypsin; Kinetics; Molecular Weight; Oligopeptides; Protein Binding; Serine Endopeptidases

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

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

The low-molecular-weight inhibitor of chymase, chymostatin, and F(ab')2 fragments of anti-chymase markedly inhibited histamine release induced by anti-rat immunoglobulin E (IgE) but not that induced by compound 48/80. Inhibitors with molecular weights of more than 6,000, such as alpha 1-antichymotrypsin and aprotinin, and non-immunized F(ab')2 had no effect on histamine release. These results suggest that chymase in mast cell granules plays an essential role in the process of IgE-mediated degranulation. After degranulation, released chymase was associated with the cell surface while released tryptase was present in the extracellular milieu as a complex with a protein associated with tryptase (trypstatin).

Topics: alpha 1-Antichymotrypsin; Animals; Aprotinin; Chymotrypsin; Histamine Release; Immunoglobulin E; Immunoglobulin Fab Fragments; Leucine; Leupeptins; Mast Cells; Oligopeptides; p-Methoxy-N-methylphenethylamine; Rats; Rats, Inbred Strains

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Chemical Phenomena; Chemistry; Chymotrypsin; Computers; Crystallography; Endopeptidases; Histidine; Molecular Conformation; Oligopeptides; Protease Inhibitors; Serine Endopeptidases

A series of peptides based on the structure of the proteinase inhibitor chymostatin were tested for their toxicity and ability to suppress protein degradation in the isolated mouse diaphragm. The inhibitory activities of the analogues were very similar, in marked contrast to their disparate abilities as inhibitors of chymotrypsin. Toxicity was determined by measurement of the rates of protein synthesis and of leakage of lactate dehydrogenase into the incubation medium. No significant toxicity was measurable at concentrations of inhibitor that were effective at suppressing proteolysis. The structural features of the chymostatin molecule may be less than optimal for suppression of proteolysis in muscle.

Topics: Animals; Chymotrypsin; Female; Hydrolysis; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Male; Mice; Muscle Proteins; Oligopeptides

Procoat, the precursor form of the major coat protein of coliphage M13, assembles into the Escherichia coli inner membrane and is cleaved to mature coat protein by leader peptidase. This assembly process has previously been reconstituted using lipids and purified leader peptidase in a cell-free protein synthesis reaction (Watts, C., Silver, P., and Wickner, W. (1981) Cell 25, 347-353; Ohno-Iwashita, Y., and Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900). We now report that procoat can also cross a liposomal membrane composed of only purified phospholipids; leader peptidase is not needed to catalyze insertion. When procoat is synthesized in vitro in the presence of liposomes with encapsulated chymotrypsin, the procoat inserts spontaneously through the membrane and is degraded. The protease was shown by several criteria to be in the lumen of the liposomes. These results demonstrate that the precursor form of an E. coli integral membrane protein can cross a membrane without the aid of leader peptidase or any other membrane proteins.

Topics: Capsid; Capsid Proteins; Chymotrypsin; Coliphages; Endopeptidase K; Endopeptidases; Escherichia coli; Liposomes; Membrane Proteins; Oligopeptides; Phospholipids; Protease Inhibitors; Protein Precursors; Serine Endopeptidases

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.

Topics: alpha 1-Antichymotrypsin; Amino Acids; Binding, Competitive; Chymotrypsin; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Endopeptidases; Humans; Isoflurophate; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Oligopeptides; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Endopeptidases; Substrate Specificity

Analogues of the microbial proteinase inhibitor chymostatin have been synthesized. The two most promising analogues were tested on protein turnover in isolated rat hepatocytes. Their effect is much similar to the effect of chymostatin, but the analogues are even more powerful inhibitors, probably due to an increased effect on lysosomal thiol proteinases. The analogues blocked most of the lysosomal (i.e. methylamine-sensitive) degradation of endogenous protein and caused a 50% inhibition of the non-lysosomal degradation; the effect occurred rapidly and was reversed upon washing the cells. One of the analogues, Z-Arg-Leu-Phe(H), is the most potent inhibitor of hepatic protein degradation so far found.

Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Chymotrypsin; Fetuins; Liver; Male; Methylamines; Oligopeptides; Proteins; Rats; Rats, Inbred Strains; Time Factors

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

Topics: Animals; Antipain; Cathepsins; Cell Survival; Chymotrypsin; Endopeptidases; Heart; Leupeptins; Male; Myocardial Infarction; Myocardium; Oligopeptides; Pepstatins; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors; 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 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

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) exhibit increases in both a cell-associated and a secreted form of plasminogen activator (PA). The mechanism whereby the membrane-bound, cell-associated form of PA is processed to an extracellular, soluble form has been examined in cultures of chicken fibroblasts transformed by a temperature-sensitive mutant of RSV. We report that chymostatin, a protease inhibitor of limited specificity, inhibits the release of PA from tsRSVCEF while causing accumulation of cell-associated PA. Chymostatin's effect on PA release is specific, reversible and appears to be due to its anti-proteolytic capacity. Chymostatin does not inhibit cellular protein synthesis or interfere in the assay used to measure PA. A chymostatin-sensitive protease activity has been found in a membrane fraction isolated from tsRSVCEF.

Topics: Animals; Avian Sarcoma Viruses; Cell Adhesion; Cell Membrane; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Chymotrypsin; Oligopeptides; Plasminogen Activators; Protein Biosynthesis; Proteins; Secretory Rate; Solubility

A chymotrypsin-like enzyme (EC 3.4.21.20) was isolated from bovine granulocytes, and purified 14-fold by affinity chromatography on 4-phenylbutylamine Affi-gel. The molecular weights of the isoenzymes were estimated as 16 000, 19 300 and 24 000 by SDS-polyacrylamide gel electrophoresis. A Michaelis constant of 2 mM and a catalytic constant of 34.6 s-1 were determined with Bz-Tyr-OEt. The esterolytic activity of the enzyme could be inhibited both by PMSF and by TPCK. It was also inhibited by chymostatin (Ki = 0.11 microgram/ml) and by the cytosol inhibitor of the bovine granulocyte (K'i = 1 microM). The chymotrypsin-like enzyme of the bovine granulocyte shares a number of the kinetic properties common to the chymotrypsin-like enzyme of the human granulocyte. The two granulocytes showed nearly identical chymotrypsin-like enzyme activities per cell.

Topics: Animals; Cattle; Chromatography, Affinity; Chymotrypsin; Granulocytes; Isoenzymes; Molecular Weight; Oligopeptides; Protease Inhibitors