Studies on the Activity Measurement for Blood Proteases using their Monoclonal Antibodies

组织因子增强了由因子催化的合成酯底物的水解。以N^< α >-苯氧羰基- l-精氨酸对硝基苄基酯（Z-Arg-ONb）为底物，建立了一种简便、灵敏的viia -组织因子复合物活性测定方法。这种底物以前已经合成过（Zur， M., and Nemerson， Y.(1978)）。化学，253,2203-2209)。新方法是在含0.1%三氟乙酸的45%乙腈中，采用3C_<18>的反相高效液相色谱法测定酶反应过程中对硝基苯醇的释放量。Z-Arg-ONb对血浆丝氨酸蛋白酶具有广泛的特异性，并且IXa因子可以水解该底物，表明该方法也可用于IXa因子活性的测定。用该方法考察了不同条件下组织因子对VIIa因子酯酶活性的影响，发现组织因子对VIIa因子酯酶活性的影响明显。人因子和牛因子VIIa对Z-Arg-ONb的水解均有显著影响。这些结果与以前的报告不一致。此外，即使在组织因子存在的情况下，因子viia介导的Z-Arg-ONb水解也不需要磷脂。我们还测定了在存在或不存在组织因子的情况下，VIIa因子对底物的动力学参数（Km和kcat）。单独使用VIIa因子的Km值比VIIa因子-组织因子复合物高6倍（3.2比0.54 mM），而kcat值比VIIa因子-组织因子复合物低12倍（14.3比173 s^<-1>）。这些结果表明，组织因子影响了VIIa因子的催化中心，增强了VIIa因子介导的酯底物水解。通过从VIIa中去除y-羧谷氨酸（Gla）结构域，组织因子的增强作用消失，而在没有组织因子的情况下，酯酶活性不受这种修饰的影响，这表明即使在反应混合物中不存在磷脂，Gla结构域也是VIIa因子与组织因子相互作用的有效决定因素。少

TISSUE FACTOR POTENTIATES THE HYDROLYSIS OF SYNTHETIC ESTER SUBSTRATE CATALYZED BY FACTOR VIIA.We established a simple and sensitive assay method for the activity of factor VIIa-tissue factor complex using N^<alpha>-benzyloxycarbonyl-L-arginine P-nitrobenzyl ester (Z-Arg-ONb) as a substrate. The substrate has been synthesized previously (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The new method was that the amount of P-nitrobenzyl alcohol released during the enzyme reaction is measured by reversed-phase HPLC, using a 3C_<18> column in 45% acetonitrile containing 0.1% trifluoroacetic acid with an isocratic elution. Z-Arg-ONb had a broad specificity for plasma serine proteases, and factor IXa could hydrolyze this substrate, indicating that the method is also useful for assay of the factor IXa activity. Using this assay method, we examined effect of tissue factor on the esterase activity of factor VIIa under various conditions, and found that tissue factor. . potenti … More ates greatly the hydrolysis of Z-Arg-ONb by both human and bovine factor VIIa's. These results did not agree with the previous report. Moreover, phospholipids were not required for the factor VIIa-mediated hydrolysis of Z-Arg-ONb even in the presence of tissue factor. We also determined the kinetic parameters (Km and kcat) of factor VIIa toward the substrate in the presence or absence of tissue factor. The Km value of factor VIIa alone was 6 times higher than that of factor VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of factor VIIa-tissue factor complex (14.3 ve rsus 173 s^<-1>). These results indicate that tissue factor affects the catalytic center of factor VIIa and enhances the factor VIIa-mediated hydrolysis of the ester substrate. The potentiating effect of tissue factor disappeared by removal of y-carboxyglutamic acid (Gla) -domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification, suggesting that the Gla-domain is required as the potent determinants on factor VIIa for the interaction with tissue factor, even if phospholipids are absent in the reaction mixture. Less