Nicholls P, Cooper C E, Freedman J A, Leece B D
Department of Biological Sciences, Brock University, St. Catharines, Ont., Canada.
Biochem Cell Biol. 1988 Nov;66(11):1218-25. doi: 10.1139/o88-139.
Antibodies have been raised in rabbits against whole beef heart cytochrome-c oxidase and purified subunit V. Antioxidase recognizes nearly all the enzyme subunits but reacts very strongly with subunits II and IV. Antisubunit V is quite specific against subunit V. Inhibition of enzyme activity by antioxidase is typically biphasic in time, indicating populations of both rapidly and slowly reacting molecules. Variation of cytochrome c concentration shows partially competitive kinetics, but the antibody also affects "internal" enzymatic events, including the catalytic turnover induced by N,N,N',N'-tetramethyl-p-phenylenediamine alone and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects can be seen however. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c, but not that with internally trapped cytochrome c. No functionally significant epitopes are detectable on the N side of the membrane in proteoliposomes, although some small effects can be seen with submitochondrial particles. Antisubunit V inhibits the isolated enzyme by at least 60%. The inhibition at high ionic strength induces a biphasic pattern with respect to cytochrome c concentration. Antisubunit V may thus slow the dissociation of cytochrome c from its complex with the enzyme. Antisubunit V has only small effects on the activities of proteoliposomal and submitochondrial particle oxidase in either orientation. On subunit V, some sites, the binding of which can give rise to inhibition, are thus not accessible to antisubunit V when the enzyme is embedded in a functional membrane system.
已在兔子体内产生了针对完整牛心细胞色素 c 氧化酶和纯化的亚基 V 的抗体。抗氧化酶能识别几乎所有的酶亚基，但与亚基 II 和 IV 的反应非常强烈。抗亚基 V 对亚基 V 具有相当的特异性。抗氧化酶对酶活性的抑制在时间上通常是双相的，这表明存在快速反应和缓慢反应的分子群体。细胞色素 c 浓度的变化呈现出部分竞争性动力学，但该抗体也会影响“内部”的酶促事件，包括仅由 N,N,N',N'-四甲基对苯二胺诱导的催化周转以及细胞色素 a 还原后细胞色素 a3 的自旋状态变化。然而，未观察到光谱效应。抗氧化酶也会抑制含有外部细胞色素 c 的蛋白脂质体呼吸，但不会抑制含有内部捕获的细胞色素 c 的蛋白脂质体呼吸。在蛋白脂质体的膜 N 侧未检测到功能上有显著意义的表位，尽管在线粒体亚颗粒上可以看到一些小的效应。抗亚基 V 至少能抑制分离出的酶 60%。在高离子强度下的抑制作用相对于细胞色素 c 浓度呈现出双相模式。因此，抗亚基 V 可能会减缓细胞色素 c 与其与酶的复合物的解离。抗亚基 V 对任一方向的蛋白脂质体和线粒体亚颗粒氧化酶的活性只有很小的影响。因此，当酶嵌入功能膜系统中时，抗亚基 V 无法接近亚基 V 上一些结合后会导致抑制作用的位点。
