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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha-thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha-thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393-Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha-thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.
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PMID:Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin. 220 28

alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.
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PMID:Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358. 229 53

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.
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PMID:Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI. 233 43

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region. 239 12

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.
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PMID:Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins. 240 7

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S-methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.
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PMID:Biology of human megakaryocyte factor V. 242 59

Human alpha 2-antiplasmin (alpha 2AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r alpha 2AP) is immunologically identical with natural alpha 2AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k1) for the interaction of alpha 2AP and r alpha 2AP with plasmin are both (1-2) X 10(7) M-1 s-1. In order to examine the effects of alterations within the reactive site of alpha 2AP, deletions of the P1 residue Arg-364 (r alpha 2AP-delta Arg364) or the P'1 residue Met-365 (r alpha 2AP-delta Met365) were introduced by in vitro site-directed mutagenesis. r alpha 2AP-delta Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that alpha 2AP has no absolute requirement for Met in the P'1 position. Unexpectedly, no increase in antithrombin activity was observed. r alpha 2AP-delta Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor (k1 = 1.5 X 10(5) M-1 s-1).
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PMID:Characterization of recombinant human alpha 2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site. 244 52

Oxidative inactivation of alpha-1-proteinase inhibitor (A1-P1) and plasminogen activator inhibitor-1 (PAI-1), both members of the serine protease inhibitor (serpin) superfamily, using mild oxidation conditions has been already demonstrated. The oxidation mechanism has been shown to involve conversion of methionine to methionine sulfoxide in the reactive center of the inhibitors. In this study evidence is presented that alpha-2-antiplasmin (A2-PI) and antithrombin III (AT III) can also be inactivated by means of oxidation. For total inactivation of 50 pM A1-PI about 10 nM chloramine T (CT) and for the same molar concentration of A2-PI and AT III about 250 nM CT were found necessary. C1-inhibitor (C1-INH) showed some resistance to oxidation that could be overcome only by increasing CT to an amount (greater than 2000 nM) already beginning to inactivate the corresponding C1-esterase. As target enzymes for A2-PI, AT III, and A1-PI plasmin, thrombin and elastase, respectively, were used. Their activity was not impaired by the oxidation conditions applied. As there is no methionine in the reactive center of AT III an additional mechanism for oxidative inactivation of serpins has to be taken into consideration. Oxidation seems to be a general mechanism for altering the balances between serine proteases and their inhibitors in favour of the protease.
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PMID:Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor. 245 61

High molecular weight kininogen (HMWK) functions as a cofactor for activation of plasma serine zymogens and as an inhibitor of tissue cysteine proteases. Cell surfaces to which HMWK binds may provide sites for regulation of these systems. Localization of these HMWK-dependent processes at sites of vascular injury may depend on its binding to specific receptors on endothelial cells. In culture, passaged human umbilical vein endothelial cells (HUVEC) bind anti-HMWK antibody to the cell surface and contain 171 +/- 75 ng of HMWK/10(8) cells. [35S]Methionine-labeled HUVEC in culture synthesize a 120-kDa protein immunoisolated using an anti-kininogen antibody, and a 3500-nucleotide message for human HMWK was detected by Northern blot in RNA extracted from HUVEC. HUVEC also express unoccupied binding sites for HMWK on their surface. 125I-HMWK specifically binds to HUVEC in a reaction requiring Zn2+. 125I-HMWK binding to HUVEC is saturable at 4 degrees C but not at 23 degrees C. 125I-HMWK binds to HUVEC with equal affinity as unlabeled HMWK. Kallikrein, factor XII, fibrinogen, fibronectin, and thrombin do not inhibit 125I-HMWK binding to HUVEC. 125I-HMWK-HUVEC binding remains fully reversible at 60 min following the addition of a 50-fold molar excess HMWK. HUVEC express 9.3 +/- 2.0 X 10(5) (mean +/- S.E.) HMWK binding sites/cell (Kd = 52 +/- 13 nM). Both added and cell-bound 125I-HMWK migrate at 120 kDa on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein remains uncleaved upon binding to the HUVEC surface. These studies indicate that HUVEC synthesize HMWK and the HUVEC surface has a site for its expression. By synthesizing and localizing HMWK to the cell surface, endothelial cells may contribute to the activation of plasma's contact serine zymogens and regulation of tissue cysteine proteases.
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PMID:The expression of high molecular weight kininogen on human umbilical vein endothelial cells. 246 Apr 46

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.
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PMID:Sulfation of two tyrosine-residues in human complement S-protein (vitronectin). 247 56


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