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)

Human alpha thrombin acts as a mitogen for cultures of resting chick embryo fibroblasts (CEF) in serum free medium. The use of 125I-labeled thrombin shows that thrombin specifically binds to CEF and that after a lag of approximately 30 to 60 minutes it can not be removed by subsequent exposure to trypsin. The entry of 125I thrombin into the trypsin-insensitive domain is not inhibited to any great extent by excess unlabelled thrombin. The cell-associated thrombin retains its native molecular weight and its catalytic activity toward synthetic amide substrates. It appears to be located in the crude nuclear fraction of homogenized CEF cells. The association of thrombin with CEF is specific, since the non-mitogenic serine protease chymotrypsin is internalized to a much lesser extent than thrombin. The data are discussed in terms of a possible intracellular site for thrombin's mitogenic action.
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PMID:Binding and internalization of 125I thrombin in chick embryo fibroblasts: possible role in mitogenesis. 67 Mar 1

The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-Glu, were not susceptible to another serine protease from small intestine (SI-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the SI-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the SI-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the SI-protease.
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PMID:Selective cleavage of peptide bonds by a serine protease from rat skeletal muscle. 70 Dec 36

The one-electron reduction of chymotrypsin, trypsin, and their zymogens have been studied by pulse radiolysis. The optical spectra of the transient products from the two active enzymes display a pH-dependent band at 360 nm, associated with the histidine-electron adduct. The yield of the histidyl radical as a function of pH is consistent with a pK(a) less than 4.5, which suggests that the radical is located at the enzyme active site. The histidines of the proenzymes chymotrypsinogen and trypsinogen are unreactive towards the hydrated electron. We conclude that formation of the histidine-electron adduct at the serine protease active site is sensitive to the physical alterations which accompany protease activation.
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PMID:Application of pulse radiolysis to the study of proteins: chymotrypsin and trypsin. 70 36

Kinetic constants are reported for alpha-chymotrypsin- and Streptomyces griseus protease 3 (SGP3)-catalyzed amide hydrolysis of a number of peptide amides of varying substrate chain length. alpha-Chymotrypsin, but not SGP3, will hydrolyze rapidly specific acetyl amino acid amides. SGP3-catalyzed, but not alpha-chymotrypsin-catalyzed, hydrolysis is greatly stimulated by the presence of up to four amino acid residues N-terminal to the scissile bond of the substrate. The enzyme-substrate interactions utilized to promote hydrolysis, therefore, differ in these two enzymes, which, in other respects, show marked similarities. alpha-Chymotrypsin depends mainly on primary enzyme-substrate contacts, those with the amino acid residue (P1) whose carbonyl group forms part of the scissile bond, whereas SGP3 depends mainly on "secondary" enzyme-substrate contacts with amino acid residues (P2-P4) more remote from the scissile bond. A comparison with porcine elastase, a related serine protease, indicates that there is an inverse relation between the importance of primary and secondary enzyme substrate interactions in this family of enzymes. A rationale is proposed for this effect based on the observation that both types of enzyme-substrate interaction predominantly affect the rate constant for the acylation step of substrate hydrolysis.
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PMID:The active centers of Streptomyces griseus protease 3 and alpha-chymotrypsin: enzyme-substrate interactions remote from the scissile bond. 81 24

Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.
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PMID:Trypsin-like neutral protease associated with soluble elastin. 90 57

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes. 128 92

The presence of the enzymatically active allergens equivalent to Der p I (cysteine protease), Der p III (serine protease) and amylase in extracts of Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived from D. pteronyssinus and D. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared from D. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.
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PMID:A comparative study of allergenic and potentially allergenic enzymes from Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei. 128 68

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.
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PMID:Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis. 128 66

Buried water molecules in the structurally homologous family of eukaryotic serine proteases were examined to determine whether buried waters and their protein environments are conserved in these proteins. We found 16 equivalent water sites conserved in trypsin/ogen, chymotrypsin/ogen, elastase, kallikrein, thrombin, rat tonin and rat mast cell protease, and 5 additional water sites in enzymes which share the primary specificity of trypsin. Based on an alignment of 30 serine protease sequences, it appears that the protein environments of these 21 conserved buried waters are highly conserved. The protein environments of buried waters are comprised primarily of atoms from highly conserved residues or main chain atoms from nonconserved residues. In one instance, the protein environment of a water is conserved even in the presence of an unlikely Pro/Ala substitution. We also note 3 instances in which a histidine side chain substitutes for water, suggesting that the structural role of water at these sites is satisfied by the presence of an alternative hydrogen bonding partner. Buried waters appear to be integral structural components of these proteins and should be incorporated into protein structures predicted on the basis of sequence homology to this family, including the catalytic domains of coagulation proteases.
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PMID:Buried water in homologous serine proteases. 133 31

The serpin family of inhibitors have an important role in the control of coagulation and fibrinolysis. For a full understanding of how these pathways operate in vivo and correct measurement of enzyme and inhibitor activity, in vitro knowledge of the mechanism of action of serpins is essential. Using alpha 2-antiplasmin as a model inhibitor we find, in contrast to most previous reports, a reversible mechanism: E + I in equilibrium with EI in equilibrium with EI', where complex formation is two stepped, but both steps are reversible. Our work with plasmin in the presence of 50 mM aminohexanoic acid shows that binding of alpha 2-antiplasmin is very tight (but reversible) with an overall Ki (Ki final) = 4.0 pM. With chymotrypsin (a model serine protease) Ki final = 100 pM, so as expected binding of alpha 2-antiplasmin is weaker with chymotrypsin. However, analysis of the individual rate constants shows that the difference in strength of binding is accounted for by the dissociation rate constant for the second step (k-2) = 1.9 x 10(-6) s-1 for plasmin and 1.1 x 10(-4) s-1 for chymotrypsin. Thus k-2, the rate constant previously ignored, explains the different affinities of alpha 2-antiplasmin for these two enzymes. Furthermore, this model of two (or more) step, reversible binding is accepted for protease inhibitors of other families. With one of these, aprotinin (a Kunitz inhibitor) with plasmin we also obtain a two-stage reversible mechanism with a Ki final = 200 pM and the strength of inhibition is also largely determined by k-2 = 3.5 x 10(-5) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies on the mechanism of binding of serpins and serine proteases. 137 55


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