Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds

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dc.contributor.authorOliva, Maria Luiza Vilela [UNIFESP]
dc.contributor.authorAndrade, S. A.
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorSallai, Roberto C. [UNIFESP]
dc.contributor.authorTorquato, R. J.
dc.contributor.authorSampaio, Misako Uemura [UNIFESP]
dc.contributor.authorPott, V. J.
dc.contributor.authorSampaio, CAM
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionEmpresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
dc.date.accessioned2016-01-24T12:33:56Z
dc.date.available2016-01-24T12:33:56Z
dc.date.issued2003-07-01
dc.description.abstractThe specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K-i 14 nM).BuXI and BvTI are highly homologous (70%). the major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition.Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. the catalytic efficiency k(cat)/K-m 4.3 x 10(7) M(-1)sec(-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate.Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P-1') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa.Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.en
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.affiliationEMBRAPA, Campo Grande, MS USA
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent1085-1093
dc.identifierhttp://dx.doi.org/10.2174/0929867033457548
dc.identifier.citationCurrent Medicinal Chemistry. Hilversum: Bentham Science Publ Ltd, v. 10, n. 13, p. 1085-1093, 2003.
dc.identifier.doi10.2174/0929867033457548
dc.identifier.issn0929-8673
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/27321
dc.identifier.wosWOS:000183379100001
dc.language.isoeng
dc.publisherBentham Science Publ Ltd
dc.relation.ispartofCurrent Medicinal Chemistry
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.subjectblood clotting enzymesen
dc.subjectfactor Xaen
dc.subjectfluorescent substrateen
dc.subjectplant Kunitz inhibitoren
dc.subjectpeptide substrateen
dc.subjectplasma kallikrein inhibitoren
dc.subjectserine proteinase inhibitoren
dc.subjectprimary sequenceen
dc.titleKinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seedsen
dc.typeinfo:eu-repo/semantics/article
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