Bharadwaj D, Roy M S, Bose D, Hati R N
Indian Institute of Chemical Biology, Jadavpur, Calcutta, West Bengal.
J Biol Chem. 1994 Jun 10;269(23):16229-35.
An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.
一种整合膜蛋白酶从大鼠下颌下腺线粒体中溶解并纯化至同质。纯化后的酶可使兔血浆凝固。在还原条件下，该酶在SDS - 聚丙烯酰胺凝胶电泳上的分子量为22 kDa，在Sephadex G - 100柱上进行凝胶过滤时分子量为24 kDa。其等电点为4.2 - 4.25。酶活性受到二异丙基氟磷酸酯、大豆胰蛋白酶抑制剂、苯甲脒、抑肽酶和抗蛋白酶的强烈抑制，表明该酶为丝氨酸蛋白酶。其活性的最适pH为8.5。Zn2 +具有强烈的抑制作用；在1 mM浓度下可产生72%的抑制率。该酶对在P1位置含有精氨酸且NH2末端封闭的不同合成底物（对硝基苯胺衍生物）具有活性。对于底物N - Bz - Pro - Arg - pNa（其中Bz为苯甲酰基，pNA为对硝基苯胺），Kcat / Km最高。纯化后的酶以剂量依赖方式使兔血浆凝固。在抑肽酶或大豆胰蛋白酶抑制剂存在下，该酶引起的血浆凝固完全被阻断，表明这种凝固反应需要蛋白酶活性。针对纯化酶产生的抗体可抑制该酶引发的血浆凝固。该酶可纠正X因子缺乏的人血浆延长的凝血时间，但无法将纯化的纤维蛋白原转化为纤维蛋白凝块，表明该酶具有Xa因子样活性。该酶具有激活凝血酶原的能力。该酶的几个特性使其与其他已报道的下颌下腺蛋白酶有所区别。
