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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.
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PMID:Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence. 283 Feb 48

Thrombomodulin, an endothelial cell protein, binds thrombin with high affinity and alters thrombin from a procoagulant to an anticoagulant molecule. In this study, chemical and/or proteolytic modification of thrombin was carried out to identify the essential components required for its interaction with thrombomodulin. Modification of thrombin at the catalytic site serine and histidine residues, with Diisopropylfluorophosphate and Tosyl-L-lysine chloromethyl ketone, resulted in loss of clotting and amidolytic activity. Both Diisopropyl phosphoryl-thrombin and Tosyl-L-chloromethyl ketone-thrombin inhibited native-thrombin: thrombomodulin catalyzed protein C activation with Ki values of 5 nM and 6 nM respectively indicating no loss of affinity for thrombomodulin. Oxidation of tryptophan residues with N-bromosuccinimide or iodination of tyrosine residues of thrombin led to reduced clotting and amidolytic activity as well as a reduced ability to interact with thrombomodulin. Modification of arginine residues with Phenylglyoxal and 2,3,Butanedione led to loss of thrombomodulin binding affinity. Limited proteolysis of thrombin by trypsin yielded the derivative beta-thrombin which had also lost its ability to interact with thrombomodulin. Deglycosylation of thrombin did not alter its binding affinity for thrombomodulin. These results indicate that one or more tryptophan, arginine and tyrosine residues are essential for the recognition of thrombin by thrombomodulin whilst the carbohydrate side chain and the active site residues of the thrombin molecule are not involved in thrombomodulin binding.
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PMID:Modification of human thrombin: effect on thrombomodulin binding. 284 49

Thrombomodulin is an endothelial cell surface protein which complexes with thrombin to accelerate protein C activation. To gain insight into the mechanisms of thrombomodulin-membrane association, limited proteolytic digestions of thrombomodulin with trypsin and elastase were performed. Trypsin digestion resulted in two major fragments (Mr = 54,000 and 27,000), both of which bound to phosphatidylcholine/phosphatidylserine vesicles. Elastase digestion also yielded two major fragments (Mr = 50,000 and 25,000), but only the smaller fragment bound to the phospholipid vesicles. The larger fragment obtained from both enzymatic digestions retained the ability to accelerate protein C activation. The Mr = 54,000 fragment from the trypsin digest retained a high affinity for thrombin (Kd less than or equal to 0.5 nM), a Km for protein C of approximately equal to 8 microM, and a half-maximal Ca2+ dependence of 0.3 mM. The Mr = 50,000 fragment from elastase digestion had a lower affinity for thrombin (Kd approximately equal to 6 nM) than intact thrombomodulin, and the Km for protein C was decreased to 0.3 microM in the presence of 0.3 mM Ca2+. The Ca2+ dependence of protein C activation with the Mr = 50,000 fragment was distinctly different from that of thrombomodulin or the active tryptic fragment. The active elastase fragment exhibited a Ca2+ optimum at 0.3 mM and activity rapidly decreased with further increases in Ca2+. At the Ca2+ optimum, the Km for protein C was similar to that observed on endothelial cell surfaces or with thrombomodulin reconstituted into liposomes. Our data demonstrate that thrombomodulin has one or more membrane-binding domains and that an active soluble form with catalytic activity can be generated by limited proteolytic digestion. Digestion with elastase appears to expose a site on thrombomodulin capable of recognizing the gamma-carboxyglutamic acid domain of protein C (residues 1-44 of the light chain). Whether this is the same site which is exposed on thrombomodulin upon incorporation into phospholipid vesicles (see accompanying manuscript) remains to be determined.
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PMID:Proteolytic formation and properties of functional domains of thrombomodulin. 302 70

In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.
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PMID:Antigenic, molecular and functional heterogeneity of Clara cell secretory proteins in the rat. 399 48

The complete covalent structure of protein C, a protein degraded during germination of Bacillus megaterium spores, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 30 and 31 to 71. The intact protein was also cleaved by cyanogen bromide into two peptides, residues 1 to 27 and 28 to 71. Cleavage of the larger cyanogen bromide peptide with trypsin allowed isolation of the COOH-terminal peptide, residues 59 to 71. Automated sequenator analysis of the intact protein and peptide fragments, together with previously published partial sequence data on this protein and carboxypeptidase A digestion of the intact protein provided data from which the following unique sequence was deduced: (formula: see text). The primary sequence of the C protein shows an extremely high degree of homology with that of the A protein--another protein degraded during germination of B. megaterium spores.
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PMID:Covalent structure of protein C. A second major low molecular weight protein degraded during germination of Bacillus megaterium spores. 677 41

Binding Ca2+ to a high affinity site in protein C and Gla-domainless protein C (protein C lacking residues 1-44) results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. Protein C modeling studies suggested the single high affinity Ca2+ binding-site might be present in a loop in the protease domain and involve Glu-70 and -80 (chymotrypsin numbering system). This loop, which is a known Ca(2+)-binding site in trypsin, is also conserved in other coagulation proteases, including factors VII, IX,and X. In thrombin, which does not bind Ca2+, Glu-70 is replaced by Lys, creating an internal salt bridge with Glu-80. We constructed and expressed a Gla-domainless protein C mutant in which Glu-80 is replaced with Lys. The activation of the resultant mutant is accelerated by thrombomodulin in a Ca(2+)-independent fashion. Unlike wild type Gla-domainless protein C, Ca2+ no longer inhibits activation of the mutant by free thrombin, and Ca2+ stimulation of chromogenic activity is also absent. The characteristic Ca(2+)-dependent quenching of Gla-domainless protein C intrinsic fluorescence is also absent in the mutant. We conclude that the high affinity Ca(2+)-binding site in protein C critical for zymogen activation involves Glu-80. The Glu-80 to Lys mutation probably results in a salt bridge with Glu-70 that stabilizes protein C zymogen in a conformation similar, if not identical, to the Ca(2+)-stabilized conformation favorable for rapid activation by the thrombin-thrombomodulin complex.
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PMID:Mutation of Glu-80-->Lys results in a protein C mutant that no longer requires Ca2+ for rapid activation by the thrombin-thrombomodulin complex. 790 67

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.
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PMID:Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen. 800 77

Human activated protein C (APC) has been shown to be physiologically susceptible to inhibition by the abundant serpin inhibitor alpha-1-anti-trypsin (AAT). Studies on the inactivation by AAT [Heeb, M. J., & Griffin, J. H. (1988) J. Biol. Chem. 263, 11613-11616] have shown that the calculated rate of this inactivation matches that of the observed half-life of APC in vivo [Wydro, R., Oppenheimer, C., Rodger, R., & Miemi, S. (1988) Clin. Res. 36, 329A] and complex formation therefore probably represents a physiologic regulation process for APC. In this study we observed that bovine APC, in contrast to human APC, is nearly completely resistant to inactivation by human AAT. An additional difference between human and bovine APC is that human APC is a potent anticoagulant in human plasma, whereas bovine APC is only minimally active in human plasma. These functional differences exist despite considerable structural similarity between the human and bovine molecules. In order to identify specific molecular regions responsible for function, a chimeric molecule consisting of the light chain of human protein C (PC) and the heavy chain of bovine PC was constructed, expressed, and characterized. The activated chimeric PC is similar to human APC in having potent anticoagulant activity in human plasma, but displays nearly identical resistance to AAT inhibition with the bovine molecule. The similarity between the chimeric and bovine molecules in resistance to AAT inhibition indicates that the structural determinants for inhibitor interactions reside within the heavy chain (serine protease) domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Resistance to inhibition by alpha-1-anti-trypsin and species specificity of a chimeric human/bovine protein C. 811 Jul 91

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
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PMID:Inhibition of coagulation factors by recombinant barley serpin BSZx. 884 56

The autolysis loop of thrombin comprises nine residues, from Glu146 to Lys149e, five of which (Ala149a-Lys149e) are inserted relative to trypsin and chymotrypsin. Deletion of the insertion Ala149a-Lys149e causes no significant change in the properties of the enzyme, except for a slight enhancement of protein C activation. Deletion of the entire Glu146-Lys149e loop, however, reduces fibrinogen clotting 240-fold, but decreases protein C activation only 2-fold. This loop-less mutant is de facto an exclusive activator of protein C, having lost the primary procoagulant function of thrombin. Because the autolysis loop affects fibrinogen binding, but not protein C activation, it provides a target for new drugs designed to suppress exclusively the procoagulant activity of thrombin.
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PMID:Selective loss of fibrinogen clotting in a loop-less thrombin. 924 18


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