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作者机构:Vanderbilt Univ Sch Med Dept Pathol Nashville TN 37232 USA
出 版 物:《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)
年 卷 期:2000年第275卷第22期
页 面:16428-16434页
核心收录:
学科分类:0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学]
基 金:NHLBI NIH HHS [HL07751 HL02832 HL38779] Funding Source: Medline
主 题:结合部位 结合 竞争性 催化作用 色谱法 亲和 酶激活 凝血酶原/代谢 硫酸盐类/代谢 凝血酶/代谢 动物 牛 人类
摘 要:Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin, Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO3-), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO3-) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 mu M and 25 +/- 2 nM, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO3-) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO3-) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a similar to 100-fold increase in affinity when thrombin is formed.