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)

The clearance of thrombin-antithrombin (TAT) complexes from blood by the liver is through a receptor mediated pathway. We have used the established human hepatoma cell line, Hep G2, to determine if these hepatocytes have the capacity to bind this enzyme-inhibitor complex. The TAT complex was bound to the cells in a time and temperature dependent manner, reaching an apparent steady state at 90 minutes at both 4 and 37 degrees C. Binding at 4 degrees C was 5-7-fold less extensive than at 37 degrees C. The bound TAT was structurally similar to the added ligand. This interaction was specific, as it was inhibited by nonlabeled TAT but not by 50-fold molar excesses of unrelated proteins or by the individual constituents, thrombin or antithrombin. Factor Xa- antithrombin complex inhibited the binding reactions slightly. Specific binding isotherms at 37 degrees C were subjected to Scatchard plots. The apparent dissociation constant was 247 +/- 74 nM, and the number of TAT molecules bound per cell was 5.19 +/- 0.89 X 10(5). Bound TAT complexes did not undergo degradation at 4 or 37 degrees C for up to 2.5 hr, as greater than 85% of the bound ligand was acid precipitable during the time course of binding. Internalization of the TAT complex was compared with transferrin, a molecule known to be internalized by Hep G2 cells, by resistance of the cell-bound ligands to degradation by trypsin or pronase. In contrast to transferrin, most of the TAT complexes remained cell-surface associated for at least 2 hr at both 4 degrees and 37 degrees C, indicating that TAT was not substantially internalized by the Hep G2 cells.
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PMID:Specific association of thrombin-antithrombin complexes with a human hepatoma cell line. 300 66

Identification of lysyl residue(s) in human plasma antithrombin required for binding of heparin was approached using chemical modification with the amino-group reagent pyridoxal 5'-phosphate. Modification of antithrombin with limiting amounts of reagent yields an average incorporation of the phosphopyridoxyl label into 1 lysine/protein molecule (Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938). Fractionation of the labeled antithrombin by affinity chromatography on heparin-Sepharose separated a phosphopyridoxylated antithrombin species devoid of heparin binding from modified protein which retained affinity for heparin. To generate peptide maps of the two antithrombin species, the proteins were reductively denatured, S-carboxymethylated, and digested with trypsin. Fractionation of the tryptic digests by reverse-phase high performance liquid chromatography indicated one peak in the chromatogram of the non-heparin-binding species to be clearly different when compared to the chromatogram of the heparin-binding species. The sequence of the unique peptide, determined by automated Edman degradation, was Thr-Ser-Asp-Gln-Ile-His-Phe-Phe-Phe-Ala-Lys-Leu-Asn-Cys-Arg. This peptide corresponds to a tryptic fragment including residues 115-129 in the sequence of antithrombin, with the modified residue identified as Lys-125. Additionally, phosphopyridoxylation of antithrombin in the presence of added heparin indicated that several other lysyl residues were "protected" from modification. Identification of this critical lysine for heparin binding strongly supports previous data which indicate that the heparin-binding domain of antithrombin is located at the NH2 terminus within one of the disulfide cross-linked loops of the protein.
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PMID:Identification of a lysyl residue in antithrombin which is essential for heparin binding. 311 Jan 43

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).
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PMID:Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow. 316 33

Antithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; antithrombin Milano peak 1 of Mr approximately 60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr approximately 120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the approximately 120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr approximately 60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the approximately 120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxymethylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, Northwick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J. Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.
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PMID:Antithrombin Milano, single amino acid substitution at the reactive site, Arg393 to Cys. 323 50

Both normal antithrombin-III (AT-III alpha) and the high heparin affinity form (AT-III beta) were isolated from pooled human plasma. AT-III beta had a lower negative charge and lower molecular mass than AT-III alpha. Sialidase and endo-F digestion indicated that the inherent difference resided in the oligosaccharide component of the molecule. CNBr fragmentation showed there was an oligosaccharide sidechain missing between residues 104 and 251, subdigestion with trypsin indicated that Asn 135 was not glycosylated in AT-III beta. Chromatography of total tryptic digests on concanavalin A-Sepharose confirmed that the high heparin affinity form of antithrombin lacked an oligosaccharide moiety at Asn 135.
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PMID:Physiological variant of antithrombin-III lacks carbohydrate sidechain at Asn 135. 360 1

Antithrombin is a major proteinase inhibitor of the blood coagulation system. Its inherited deficiency or abnormality is often associated with thromboembolism. Antithrombin "Northwick Park," a functionally inactive variant antithrombin, has recently been shown by us (Lane, D.A., Flynn, A., Ireland, H., Erdjument, H., Samson, D., Howarth, D., and Thompson, E. (1987) Br. J. Haematol. 65,451-456) to be present in plasma, in part, as a high Mr (approximately 120,000) component which has a characteristic electrophoretic mobility in agarose gels in the absence of denaturing agents. In this communication, we present evidence that this Mr approximately 120,000 variant component is comprised of an antithrombin-albumin covalent disulfide-linked complex. This proposal is supported by results of: (a) fast atom bombardment mass spectrometry of the isolated reduced, S-carboxymethylated, trypsin-digested Mr approximately 120,000 complex; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this complex and its reduced and S-carboxymethylated constituents; (c) immunoblotting of these polyacrylamide gels with antisera specific for antithrombin and albumin; (d) NH2-terminal sequence analysis of one of the isolated, S-carboxymethylated proteins that comprise the Mr approximately 120,000 complex; and (e) fast atom bombardment mass spectrometry of its tryptic peptides.
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PMID:Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin "Northwick Park". 365 16

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.
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PMID:[Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis]. 367 59

Stereoisomeric oligopeptides were studied for their inhibitory effect on the hydrolysis of benzoyl-L-arginine methyl ester catalyzed by thrombin and trypsin, as well as on the thrombin-fibrinogen reaction. Comparison of the peptide structures, their conformational flexibility and inhibitory effects on thrombin and trypsin shows the availability of the essential differences in organization and functioning of the subsites S3, S2 and S'1 of these enzymes. In contrast to trypsin, thrombin is shown to be characterized by more pronounced secondary stereospecificity. This manifests in the more vigorous dropping of the catalytic constants of thrombin-catalyzed esterolysis than those of trypsin-catalyzed hydrolysis of the substrates, containing D-amino acids at the subsite P2. It is revealed that the peptide Tos-D-Val-D-Ala-D-Agr-D-Phe-OCH3 is the most powerful inhibitor among studied compounds. It is noteworthy that its antithrombin effect is almost an order of magnitude higher than its antitrypsin effect.
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PMID:[Inhibitory effect of methyl esters of arginine-containing oligopeptides on thrombin and trypsin]. 368 93

Human heparin cofactor II is a plasma protein that is known to inhibit thrombin. The rate of thrombin inhibition by heparin cofactor II is accelerated (greater than or equal to 1000-fold) in the presence of the glycosaminoglycans, heparin and dermatan sulfate. We have found that chymotrypsin A alpha is also inhibited by heparin cofactor II with a second-order rate constant value of 1.8 X 10(6) M-1 X min-1 at pH 8.0 and 25 degrees C. However, there was no measurable effect of heparin or dermatan sulfate on the rate of chymotrypsin inhibition. Arginine-modified heparin cofactor II showed a comparable percentage loss of both antichymotrypsin and antithrombin activities. Heparin cofactor II and chymotrypsin formed a stable complex with a Mr value near 90,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis; this suggests a 1:1 reaction stoichiometry. The chymotrypsin cleavage site in heparin cofactor II was the same as that for thrombin, and primary structure analysis of the inhibitor showed a P'1-P'8 sequence of Ser-Thr-Gln-Val-Arg-Phe-Thr-Val ... . The results indicate that, in contrast to alpha 1-antichymotrypsin, which does not inhibit trypsin-like enzymes, including thrombin, heparin cofactor II can effectively inhibit both thrombin and chymotrypsin.
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PMID:Inhibition of chymotrypsin by heparin cofactor II. 386 4

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.
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PMID:Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin. 395 55


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