Navegando por Palavras-chave "blood clotting enzymes"

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    Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates
    (Elsevier B.V., 1999-12-01) Oliva, MLV; Mendes, C. R.; Juliano, M. A.; Chagas, JR; Rosa, J. C.; Greene, L. J.; Sampaio, M. U.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (K-i 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (K-i 2.0 nM) and Abz-FRSSRQ-EDDnp (K-i 2.5 nM). (C) 1999 Published by Elsevier Science B.V. All rights reserved.
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    Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds
    (Elsevier B.V., 1999-12-01) Oliva, MLV; Andrade, S.; Batista, IFC; Sampaio, M. U.; Juliano, M.; Fritz, H.; Auerswald, E. A.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Univ Munich
    Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with K-i values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (K-i = 80 nM). the comparison between BuXI and BvTI reactive site structure indicates differences at Met(59), Thr(66) and Met(67) residues. the hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (K-m = 0.68 mu M, k(cat)/K-m = 1.3 X 10(6) M-1 s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (K-m = 0.66 mu M, k(cat)/K-m = 2.2 X 10(3) M-1 s(-1)). the contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. the determined K-m and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes. (C) 1999 Elsevier Science B.V. All rights reserved.
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    Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds
    (Bentham Science Publ Ltd, 2003-07-01) Oliva, Maria Luiza Vilela [UNIFESP]; Andrade, S. A.; Juliano, Maria Aparecida [UNIFESP]; Sallai, Roberto C. [UNIFESP]; Torquato, R. J.; Sampaio, Misako Uemura [UNIFESP]; Pott, V. J.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
    The 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.