权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

用单克隆抗体测定血液蛋白酶活性的研究

基本信息

批准号：
02557016
负责人：
IWANAGA Sadaaki
金额：
$ 6.02万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557016/
关键词：
Factor VII tissue factor Synthetic Peptide substrate Extrinsic coagulation gamma-Gla domain Blood protease Monoclonal antibody Esterase VII因子蛍光性ペプチド基質 IX因子 X因子

项目摘要

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

组织因子促进因子VIIA催化的合成酯底物的水解。我们建立了一种简便、灵敏的测定因子VIIa-组织因子复合体活性的方法，该方法以N^&lt；α&gt；-苄氧甲酰基-L-精氨酸对硝基苄酯(Z-Arg-ONB)为底物。底物以前已经合成过(Zur，M.和Nmerson，Y.(1978)J.Biol)。化学。253、2203-2209)。新的方法是用反相高效液相色谱法测定酶反应过程中释放的对硝基苯甲醇的量，使用3C；lt；18&gt；柱，在含有0.1%三氟乙酸乙腈的45%乙腈中进行等度洗脱。Z-Arg-onb对血浆丝氨酸蛋白酶具有广泛的特异性，并且因子IXa可以对该底物进行水解，这表明该方法也适用于因子IXa活性的测定。用该方法检测了不同条件下组织因子对因子VIIa酯酶活性的影响，发现组织因子对因子VIIa的酯酶活性影响较大。。Potenti…人和牛因子VIIa对Z-Arg-onb的水解率较高，这些结果与以前的报道不一致。此外，即使在组织因子存在的情况下，因子VIIa介导的Z-Arg-onb的水解也不需要磷脂。我们还测定了在存在或不存在组织因子的情况下，因子VIIa朝向底物的动力学参数(Km和kcat)。因子VIIa单独的Km值是因子VIIa-组织因子复合体的6倍(3.2vs0.54 mm)，而kcat值则比因子VIIa-组织因子复合体的Km值低12倍(14.3ve RSU 173 S)。这些结果表明，组织因子影响因子VIIa的催化中心，并促进因子VIIa介导的酯底物的水解。去除因子VIIa中的γ-羧基谷氨酸(GLA)结构域后，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，这表明即使反应混合物中没有磷脂，也需要GLA结构域作为因子VIIa与组织因子相互作用的决定因素。较少