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)

Crotalocytin, a platelet activating protein from timber rattlesnake venom, was studied to characterize its nature and to investigate its action on platelets. It exhibited proteolytic activity on the substrate azocoll and amidolytic activity on several peptide p-nitroanilides. The platelet activating and amidolytic activity of Crotalocytin was inhibited by diisopropylfluorophosphate. In addition, phenylmethylsulfonyl fluoride inhibited Crotalocytin's ability to stimulate platelets. Active site titration with p-nitrophenyl guanidobenzoate indicated that 52% of Crotalocytin's molecules were active and that the enzyme could also hydrolyze the titrant. These studies showed that Crotalocytin is a serine protease. Like thrombin and collagen, Crotalocytin induced simultaneous platelet aggregation and adenosine triphosphate (ATP) secretion. EDTA and prostaglandin E (PGE1) blocked Crotalocytin's ability to activate platelets; hirudin and antithrombin III did not. Crotalocytin stimulated the secretion of serotonin from dense granules and low affinity platelet factor 4 and fibrinogen from alpha-granules. Crotalocytin did not cause platelet lactic dehydrogenase loss or agglutinate formalin-fixed platelets, but it did aggregate chymotrypsin-treated platelets. Studies with antimycin A and 2 deoxy- D-glucose showed that Crotalocytin-induced platelet secretion was dependent on metabolic energy. Furthermore, Crotalocytin's induction of platelet secretion was prevented by eliminating exogenous ADP and blocking activation of the arachidonate pathway. Timber rattlesnake venom contains a serine protease that is unique, potent platelet activator.
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PMID:Crotalocytin: characterization of the timber rattlesnake platelet activating protein. 625 83

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.
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PMID:Cleavage and activation of human factor IX by serine proteases. 638 97

An acid-stable protease inhibitor (AS-PI) has been previously demonstrated in ascitic fluid from patients with ovarian carcinoma. In this study, the AS-PI was further purified using DEAE-cellulose and isoelectric focusing (IEF), and a partial characterization was undertaken. On DEAE-cellulose ion-exchange column chromatography, AS-PI activity was observed in both adsorbed and non-adsorbed fractions. The former represented the main AS-PI peak. By IEF, the respective pI values were 1.6 and 4.5. By gel filtration, the molecular weight of the main (adsorbed fraction) AS-PI was 78 000. This AS-PI strongly inhibited trypsin and to a lesser extent chymotrypsin, but exerted no inhibitory effect on plasmin. It slightly inhibited SH proteases such as papain and ficin. Immunologically, AS-PI was distinct from alpha 1-antitrypsin, alpha 1-antichymotrypsin, inter-alpha-trypsin inhibitor, antithrombin III, C1-inactivator, alpha 2-macroglobulin and alpha 2-plasmin inhibitor. The main AS-PI reacted with and was neutralized by antiurinary trypsin inhibitor serum, and on immunoelectrophoresis, had a mobility slightly cathodal to serum albumin.
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PMID:Further purification and characterization of acid-stable protease inhibitor from ascites of an ovarian carcinoma patient. 643 8

The reactive site of human alpha 1-antichymotrypsin has been identified as encompassing a leucyl-seryl bond at the apparent P1 and P'1 positions. This has been determined by dissociation of complexes of the inhibitor with bovine alpha-chymotrypsin, followed by identification of new NH2-terminal sequences, as well as by proteolytic inactivation by porcine pancreatic elastase. The latter results in peptide bond cleavage between the apparent P5 and P4 positions of the inhibitor, yielding a fragment whose sequence overlaps with that obtained through complex dissociation. Some homology with the sequence obtained and that already reported for both antithrombin III and alpha 1-proteinase inhibitor can be noted.
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PMID:Amino acid sequence at the reactive site of human alpha 1-antichymotrypsin. 655 93

Human alpha 2-antiplasmin was digested with trypsin and with chymotrypsin and about 70 percent of the amino acids were sequenced and aligned in peptides ranging from 2 to 33 residues. Here we report five sequences of 21 to 33 residues. When these were compared with the primary structures of antithrombin III, alpha 1-antitrypsin and ovalbumin, which belong to the same protein superfamily (Hunt and Dayhoff [1980] Biochem Biophys Res Commun 95: 864-871), three peptides showed clear homologies with these proteins, indicating that alpha 2-antiplasmin also belongs to that superfamily. In addition, alpha 2-antiplasmin appeared to contain at least one internal homology.
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PMID:Partial primary structure of human alpha 2-antiplasmin-homology with other plasma protease inhibitors. 698 43

Protease inhibitors and protease (caseinolytic, elastinolytic and esterolytic) activity were analysed in 190 milk samples from 94 mothers from day 1 to day 160 after delivery. The main protease inhibitors in human milk are alpha 1-antichymotrypsin and alpha 1-antitrypsin. As measured by electroimmunoassay, the level of alpha 1-antichymotrypsin in day 1 colostrum was higher than that in normal serum. Trace amounts of inter-alpha-trypsin inhibitor, alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, or antileukoprotease could be demonstrated. According to their protease inhibiting activity, the 53 milk samples from day 1-3 could be divided into two groups. (1) Presence of protease inhibiting activity (n = 35). Both alpha 1-antitrypsin and alpha 1-antichymotrypsin appeared intact and were able to form complexes with added trypsin or chymotrypsin although the major part of alpha 1-antichymotrypsin showed a retarded electrophoretic mobility. The proteolytic inhibiting activity, in spite of the presence of immunoreactive inhibitors (n = 18). alpha 1-antichymotrypsin had a precipitate pattern similar to group 1, whereas alpha 1-antitrypsin had a major fraction with slightly retarded mobility and two minor peaks in the alpha 1-and beta-regions. These precipitate patterns were unchanged on addition of human trypsin or chymotrypsin compatible with the presence of nonreactive inhibitor only. These samples had a caseinolytic and esterolytic activity with an electrophoretic mobility in the beta-region. All samples from day 4 and later had a demonstrable protease inhibiting activity.
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PMID:Protease inhibitors and their relation to protease activity in human milk. 704 31

Human alpha 2-antiplasmin was digested with CNBr and the partial NH2-terminal amino acid sequences of nine purified fragments were determined. One of these sequences (12 residues) revealed homologies with that of residues 158 to 168 and with that of residues 316-327 in the antithrombin III sequence and with the corresponding sequences in alpha 1-antitrypsin and ovalbumin. One CNBr-fragment of 69 residues with blocked NH2-terminus was subdigested with trypsin and chymotrypsin and most of its sequence aligned. This peptide probably represents the major part of the previously reported COOH-terminal fragment of alpha 2-antiplasmin with Mr 8,000 (1). In this sequence no clear homology with antithrombin III, alpha 1-antitrypsin or ovalbumin could be detected.
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PMID:Purification and partial primary structure of cyanogen bromide fragments from human alpha 2-antiplasmin. 712 13

Heparin and heparan sulfate, exhibiting wide biological interactions, are constituted of block structures. A defined pentasaccharide motif was found responsible for the enhancement of the rate of inactivation of factor Xa by antithrombin III. Heparin also interacts with other serine proteinase inhibitors as protease nexin I, and thus possibly modulates extracellular matrix proteolysis by serine proteinases in the pericellular environment. Human neutrophil elastase (HNE) activity is inhibited by heparin with Ki = 75 pM. This strong interaction is electrostatic, involving HNE/arginine residues disposed in a "cluster shoe" arrangement on the surface of the molecule and mainly OSO3- groups of heparin. HNE-heparin interactions also interfere with HNE associations with its natural inhibitors: it decreases the rate of association of HNE with alpha 1 proteinase inhibitor (alpha 1 P(i)) by 3 orders of magnitude, while increasing kass between HNE and mucus bronchial inhibitor (MBI) by > 10 fold. In vivo experiments demonstrated that heparin fragments lacking anticoagulant activity were able to nearly completely abolish emphysematous lesions induced in mice by a single intratracheal administration of 200 micrograms HNE. Long chain unsaturated fatty acids peptide conjugates were described as competitive HNE inhibitors (Hornebeck W. et al. 1985). We synthesized N-oleoyl heparin derivative (3 oleoyl groups/one molecule of heparin); such a lipophilic glycosaminoglycan (LipoGAG), although acting as an elastin protecting agent, possessed lower HNE inhibitory capacity as compared with heparin. In contrast, however, it was able to inhibit other serine proteinases such as urokinase, plasmin, porcine pancreatic apha-chymotrypsin and elastase. Such Lipo GAG's can be therefore useful to control matrix metalloproteinases (MMPs) during tissue remodeling or tumor invasion.
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PMID:Heparin and its derivatives modulate serine proteinases (SERPS) serine proteinase inhibitors (SERPINS) balance. Physiopathological relevance. 789 38

Protein C is a vitamin K-dependent serine protease zymogen that upon activation inhibits the coagulation cascade by inactivating factors Va and VIIIa. In an attempt to improve the anticoagulant activity of activated protein C (APC), we have prepared a mutant of protein C in mammalian cells in which Glu at position 192 (chymotrypsin numbering system) has been replaced with Gln (PC E192Q). Our strategy is based on the observation that the same substitution in thrombin improves the catalytic activity toward natural and synthetic substrates that contain Asp residues at P3 and P3'. Since factor Va also has an Asp at position P3 in the APC cleavage site of the factor Va heavy chain, we hypothesized that APC E192Q would inactivate factor Va more rapidly than wild type APC. The mutant inactivated factor Va approximately 2-3-fold faster than wild type. In plasma the mutant exhibited slightly less anticoagulant activity than wild type enzyme. Further characterization revealed that APC E192Q is inhibited 280 times faster than APC by alpha 1-antitrypsin (K2 = 2.8 x 10(3) M-1S-1 versus 10 M-1 S-1), and unlike APC, APC E192Q is inhibited by antithrombin III in the presence of heparin (K2 = 1.17 x 10(3) M-1 S-1) M-1 S-1) and absence of heparin (K2 = 57 M-1 S-1). Ca2+ increased K2 more than 4-fold with or without heparin. Unlike wild type APC, APC E192Q was effectively inhibited by pancreatic trypsin inhibitor (Ki = 10.6 +/- 0.26 nM) and tissue factor pathway inhibitor (58 +/- 5 nM). Like factor Xa, APC E192Q rapidly processed factor IX to factor IX alpha. These observations suggest that even though Glu at position 192 is not an optimal residue for catalyzing factor Va inactivation, it is an evolutionary adaptation to slow inhibition by plasma protease inhibitors.
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PMID:Conversion of glutamic acid 192 to glutamine in activated protein C changes the substrate specificity and increases reactivity toward macromolecular inhibitors. 810 82

Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as alpha-thrombin or collagenase induce a continuous, dose-dependent enhancement of the levels of cell-secreted NGF. Incubation of astrocytes for a 24-h period with 300 ng/ml of alpha-thrombin (approximately 9 nM, or 1 U/ml) results in an increase of the levels of cell-secreted NGF by a factor of three- to fourfold, and at doses 10 times higher, stimulation by a factor of up to four- to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP-1 family, such as c-fos, c-jun or junB. Trypsin, plasmin, alpha-chymotrypsin, or elastase also enhanced, to different extents, the levels of cell-secreted NGF. However, unlike alpha-thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose-response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell-substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of alpha-thrombin indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of alpha-thrombin with D-phenyl-alanyl-L-propyl-L-arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of alpha-thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of alpha-thrombin require binding to the cell surface. We found that gamma-thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as alpha-thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Enhancement of the synthesis and secretion of nerve growth factor in primary cultures of glial cells by proteases: a possible involvement of thrombin. 843 76


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