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)

The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.
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PMID:Purification and characterization of the mouse mammary tumor virus protease expressed in Escherichia coli. 133 Nov 10

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the first epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X (Lin, S.-W., Smith, K. J., Welsch, D., and Stafford, D. W. (1990) J. Biol. Chem. 265, 144-150). The chimeric protein, factor IX(Xegf1), as well as the wild-type, factor IXwt, produced in embryo kidney cells, and factor IX isolated from human plasma were radiolabeled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXa(Xegf1) (566 sites/platelet, Kd = 0.69 nM) and factor IXawt (590 sites/platelet, Kd = 0.61 nM) in the presence of factor VIIIa (5 units/ml) and factor X (1.5 microM) compared to factor IXaN (558 sites/platelet, Kd = 0.67 nM). The concentration of factor IXaN, factor IXawt, and factor IXa(Xegf1) required for half-maximal rates of factor Xa formation were 0.63, 0.7, and 0.83 nM, indicating that the Kdapp for binding of factor IXa(Xegf1) to the factor X activating complex on activated platelets is normal. These studies suggest either that the EGF-1 domain of factor IXa is not involved in factor IXa binding to platelets or that the EGF-1 domain from factor X when inserted into factor IXa, suffices to promote normal factor IXa binding.
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PMID:The role of the first growth factor domain of human factor IXa in binding to platelets and in factor X activation. 156 3

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.
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PMID:Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding. 163 19

Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.
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PMID:A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity. 166 May 25

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd,app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.
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PMID:The role of the second growth-factor domain of human factor IXa in binding to platelets and in factor-X activation. 765 78

To identify the amino acids in the Gla domain that mediate factor IXa binding to human platelets, we have used chimeric molecules and point mutations in the Gla domain of recombinant factor IX, based on molecular modeling using the coordinates of the Gla domain of bovine prothrombin, which reveals two surface structures whose sequences differ among factor IX, factor X, and factor VII. Binding to thrombin-activated platelets of factor IXa in the presence of factor VIIIa (2 units/mL) and factor X (1.5 microM) revealed a stoichiometry of approximately 550 sites per platelet with a Kd of approximately 0.65 nM compared with a Kd of approximately 2.5 nM in the absence of factor VIIIa and factor X. In contrast, mutations of factor IX to factor X residues at positions 4 and 5 or at positions 9, 10, and 11 results in decreases in the number of sites and affinity of factor IXa binding in the presence or absence of factor VIIIa and factor X. A chimera consisting of the Gla domain of factor VII with factor IX residues at positions 33, 34, 35, 39, and 40 displayed abnormal factor IXa binding and a decreased Vmax and a normal Km for factor X activation, and the replacement of amino acid residues 3-10 with those of factor IX restored normal binding and factor X activation kinetics to this chimeric protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High-affinity, specific factor IXa binding to platelets is mediated in part by residues 3-11. 791 24

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.
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PMID:Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity. 813 16

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.
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PMID:Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity. 819 58

The cDNA encoding a chimeric human protein C (PC), in which its epidermal growth factor-(EGF) like regions have been replaced with equivalent structures from human factor IX (fIX), was constructed and the gene product was expressed in human 293 cells. A molecular subpopulation of the recombinant chimeric protein (r-[PC/delta EGF-1,2/delta fIXEGF-1,2]) was purified that contained the full complement (9 residues/mol) of gamma-carboxyglutamic acid (Gla). After conversion by thrombin to its activated form (r-[APC/delta EGF-1,2/delta fIXEGF-1,2]), this latter enzyme was found to possess approximately 10% of the activity of wild-type recombinant APC (wtr-APC) in an APTT assay. In assay systems employing purified components, the activity of the mutant enzyme toward prothrombinase cofactor Va (fVa) and tenase cofactor VIII (fVIII) was approximately 30% and < 10%, respectively, of that of wtr-APC. The chimeric protein displayed full reactivity with a Ca(2+)-dependent monoclonal antibody to the Gla domain of PC, yielding a C50 for Ca2+ that was very similar to that obtained with wtr-PC (ca. 3.7 mM). Titrations of the dependency on Ca2+ of the intrinsic fluorescence of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] allowed calculation of a C50 value of 0.34 mM, again very similar to that of wtr-PC. As with wtr-PC, Ca2+ inhibited the thrombin-catalyzed activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] with aKi of 148 microM, as compared to a Ki of 125 microM for wtr-PC. At a saturating level of Ca2+, activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2/] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at approximately 70% of the rate of that of wtr-PC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Construction, expression, and properties of a recombinant chimeric human protein C with replacement of its growth factor-like domains by those of human coagulation factor IX. 829 11

The Moloney murine leukemia virus (Mo-MuLV) protease has been cloned into the prokaryotic expression vector pGEX-2T, expressed in fusion with the glutathione S-transferase from Schistosoma japonicum, and purified to apparent homogeneity after thrombin cleavage of the chimeric protein. The purified protease showed maximum activity at pH 6.0 and was inhibited by several aspartyl protease inhibitors, found to be active toward the human immunodeficiency virus-1 (HIV-1) protease. Peptides representing maturation cleavage sites in Gag and Gag-Pol polyproteins were accurately cleaved by the recombinant protease, and kinetic parameters have been determined. In addition, oligopeptides mimicking the cleavage site found in the transmembrane protein and leading to the formation of p15E and p2E were also hydrolyzed at the expected position. The Mo-MuLV protease appears to be more closely related to the HIV-1 protease than to the mouse mammary tumor virus enzyme, based on its substrate specificity and sensitivity to aspartyl protease inhibitors.
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PMID:Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme. 837 34


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