Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence- and stereospecificity of the S1- and S' i-subsites (i = 1-3) of bovine alpha-chymotrypsin and trypsin, proteinase K and penicillin amidase from E. coli and A. viscosus has been determined by hydrolysis and kinetically controlled peptide synthesis using different substrates. The data are compared with results for other serine proteases and the thiol protease papain. The stereospecificities differ by orders of magnitude, decreased when the enzyme was immobilized and were influenced when organic solvent molecules were bound to the enzyme.
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PMID:Stereo- and sequence specificity of serine proteases in peptide synthesis. 182 58

The alpha-glyceryl esters of Z-Gly, Z-Phe and Z-Tyr were synthesized and their use for protease catalyzed peptide synthesis was studied. Three enzymes isolated from crude papain were compared in their catalytic potency. Syntheses with alpha-chymotrypsin were performed in a biphasic system.
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PMID:Use of Z-amino acid-glyceryl esters in protease catalyzed peptide synthesis. 182 68

The tripeptide Z-Cys(Bzl)-Tyr-Ile-OtBu (I) has been synthesized by papain, chymotrypsin and thermolysin catalysis using two different strategies: a) starting from the C-terminal amino acid and b) starting from the N-terminal amino acid. The optimum reaction conditions for obtaining the peptides Z-Tyr-Ile-OtBu (II), Z-Cys(Bzl)-Tyr-OtBu (III) and Z-Cys(Bzl)-Tyr-Ile-OtBu (I) were established after analyses of the effects of pH, reaction time, concentrations of buffer, enzyme and substrates, relative proportions of the carboxyl to amine components and nature of the organic solvent on the coupling yield. The highest yields obtained for II and I using chymotrypsin and papain as catalysts were 74% and 45%, respectively. Starting from the N-terminal amino acid, and using papain and thermolysin as catalysts, the yields obtained were 91% for III and 92% for I. The effects of amine- and carboxyl-protecting groups on the extent of dipeptide synthesis catalyzed by chymotrypsin were also studied.
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PMID:A methodological study of the enzymatic synthesis of the tripeptide Z-Cys(Bzl)-Tyr-Ile-OtBu. 182 Nov 69

Fifteen 1-peptidyl-2-haloacetyl hydrazines, which can be considered halometanes of azapeptides containing Phe in P2 and alpha-aza-Ala or alpha-aza-Gly in P1, were synthesized and tested as models of cysteine-proteases inhibitors. By use of kinetic methods, they proved to irreversibly inactivate papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants of inactivation in the range 26-23000 M-1s-1 were observed for papain and 2000-39600 M-1s-1 for cathepsin B. KI for the reversible EI adducts ranged from 230 to 0.16 microM for papain and from 11 to 0.37 microM for cathepsin B. Structure of possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. Title compounds proved to be selective for cysteine-proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration, after 6 h incubation. Relatively low aspecific alkylating properties were also verified in tests using glutathione as the nucleophile.
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PMID:1-Peptidyl-2-haloacetyl hydrazines as active site directed inhibitors of papain and cathepsin B. 182 30

1. Methacholine relaxed phenylephrine-contracted aorta of the rat with the endothelium intact. This effect was inhibited by haemoglobin, methylene blue, gossypol, phenidone and L-NG-nitroarginine methyl ester (L-NAME). Rat aorta denuded of endothelium failed to relax in response to methacholine, histamine and the peptidoleukotrienes C4, D4 and E4. 2. Methacholine and histamine but not leukotrienes C4, D4 and E4 relaxed phenylephrine-contracted rat aorta without endothelium when surrounded by rabbit epithelium-intact bronchus. The muscarinic antagonist atropine antagonized the methacholine-induced relaxation. 3. Removal of the epithelium either mechanically or chemically, abolished methacholine-induced relaxation of rat aorta in the co-axial bioassay. These data indicate that the epithelium is responsible for the observed relaxant effect to methacholine and histamine. 4. The cyclo-oxygenase inhibitor, indomethacin, the phospholipase A2 inhibitor, mepacrine and the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), failed to inhibit methacholine-induced relaxation of rat aorta in the co-axial bioassay. This indicates that the epithelium-derived inhibitory factor (EpDIF) is not a product of the cyclo-oxygenase or lipoxygenase pathway or a product derived from activation of phospholipase A2. 5. Haemoglobin, methylene blue, phenidone, gossypol and L-NAME failed to inhibit the relaxation of rat aorta in the co-axial bioassay. These results demonstrate that EpDIF detected in the co-axial bioassay is not endothelium-derived relaxing factor (EDRF) or nitric oxide. Similarly, catalase was without effect. 6. EpDIF is unlikely to be a peptide since papain and alpha-chymotrypsin failed to alter the methacholine-induced relaxation of rat aorta in the co-axial bioassay. Furthermore, thiorphan, captopril and aprotinin were also without effect, suggesting that EpDIF is not a substrate for airway peptidases. 7. The results presented in this paper demonstrate the release of a vasoactive epithelium-derived inhibitory factor (EpDIF) from rabbit intrapulmonary bronchi by use of a co-axial bioassay preparation.
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PMID:The release of a non-prostanoid inhibitory factor from rabbit bronchus detected by co-axial bioassay. 185 18

A new approach to the assay of proteinases is described. The method relies on water-insoluble protein substrates, such as gluten and fibrin, which form expanded gels in the presence of sodium dodecyl sulfate (SDS) reagent. Powdered substrate is dispersed in buffer and aliquots are pipetted into long, narrow, 400-microliters tubes made of clear polypropylene. After the addition of enzyme and a period of incubation, a SDS reagent is added, the tubes are centrifuged, and the height of the SDS-protein gel is measured. Reduction of gel height gives a direct measure of enzyme activity. Salt concentration, pH, and incubation times must be consistent for both test and control reactions in order to obtain reproducible results. Examples of proteinases measured by this method are trypsin, chymotrypsin, elastase, pronase, papain, pepsin, an insect (Nysius huttoni) salivary proteinase, and wheat proteinase. The assay could detect enzyme in crude extracts or in purified form. In 1-h incubations, 10 ng of pepsin and elastase or 20 ng of purified insect proteinase could be detected. The assay was simple, fast, economical, and sensitive.
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PMID:General proteinase assay by formation of SDS-protein gels of proteolyzed substrate proteins. 195 67

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.
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PMID:Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor. 196 31

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.
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PMID:A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus. 198 74

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex.
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PMID:Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting. 199 68

Bacterial cell wall peptidoglycan (PGN) and lipopolysaccharide (LPS), which are both macrophage activators and polyclonal B cell mitogens, were shown to bind to the same dominant 70-kDa 6.5 pI protein on the surface of mouse B lymphocytes. This conclusion was supported by the following results: (a) the PGN- and LPS-binding proteins co-migrated following photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis; (b) cross-linking of PGN to this 70-kDa protein was competitively inhibited by LPS (IC50 = 7.3 microM), LPS from a deep rough mutant (IC50 = 6.9 microM), and lipid A (IC50 = 18-72 microM); (c) cross-linking of LPS to this 70-kDa protein was competitively inhibited by polymeric soluble PGN (IC50 = 0.09 microM) and sonicated high Mr PGN (IC50 = 0.6 microM); (d) cross-linking of both PGN and LPS to this 70-kDa protein was also competitively inhibited by dextran sulfate (IC50 = 115-124 microM); (e) cross-linking of both PGN and LPS to this 70-kDa protein was inhibited by a (GlcNAc)2-specific lectin; and (f) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, staphylococcal protease V, or papain were identical for PGN- and LPS-binding proteins and unique for each enzyme. Based on competitive inhibition experiments, binding of PGN to the 70-kDa protein was 20-1200 times stronger than the binding of LPS or lipid A on a per mol basis. However, when aggregated micellar structures of LPS or lipid A were considered, the avidities of LPS and PGN binding were similar. These results demonstrate binding of PGN and LPS to the same 70-kDa protein on lymphocytes and suggest that the binding is specific for the (GlcNAc-MurNAc)n backbone of PGN and the (GlcNAc)2 part of lipid A.
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PMID:Peptidoglycan and lipopolysaccharide bind to the same binding site on lymphocytes. 200 21


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