Navegando por Palavras-chave "primary sequence"

Agora exibindo 1 - 2 de 2
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    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.
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds
    (Elsevier B.V., 2006-03-01) Paiva, PMG; Oliva, MLV; Fritz, H.; Coelho, LCBB; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Pernambuco (UFPE); LMU Munchen
    Two Bowman-Birk type trypsin inhibitors (CmTI1 and CmTI2) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI1 and CmTI2, with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Legummosae. the putative reactive sites of CmTI1 are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI2: lysine at position 22 and leucine at position 49. the dissociation constant K-i of the complex with trypsin is 1.4 nM. the apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating. (c) 2005 Elsevier B.V. All rights reserved.