Nicholls P, Cooper C E, Leece B, Freedman J A, Chan S H
Dept. of Biol. Sciences, Brock Univ., St. Catharines, Ont., Canada.
Prog Clin Biol Res. 1988;274:637-51.
Antibodies have been raised in rabbits against whole beef heart cytochrome oxidase and against purified subunit V. Western blot analysis showed that antioxidase was largely composed of anti-II and anti-IV antibodies but some anti-I and antibodies against small subunits were elicited. Similar analysis of anti-V showed it to be relatively specific against subunit V. Three types of anti-V were identified by ELISA with intact enzyme: (i) weak binding and redox independent, (ii) stronger binding, redox-dependent (binding only to reduced or partially reduced enzyme), and membrane-independent, and (iii) moderate-binding, redox-dependent & membrane-dependent. Inhibition of enzyme activity by anti-oxidase was biphasic in time, indicating populations of rapidly- and slowly- reacting molecules. Variation of cytochrome c concentration showed partially competitive kinetics, but the antibody also affected 'internal' enzymatic events including the turnover rate with TMPD and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects could however be seen. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c but not that with internally-trapped cytochrome c. No functionally significant epitopes occur on the N (matrix) side of the membrane. The more strongly binding anti-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. Anti-V may thus slow the dissociation of cytochrome c from its complex with the enzyme. The redox-dependent, membrane-dependent anti-V (reported previously) gave only about 20% inhibition of the enzyme. The effective anti-V antibody had little if any influence on the activity of proteoliposomal oxidase in either orientation. Some sites on subunit V whose binding can give rise to inhibition are not accessible to anti-V when the enzyme is embedded in a functional membrane system.
已在兔体内产生了针对全牛肉心脏细胞色素氧化酶和纯化的亚基V的抗体。蛋白质印迹分析表明，抗氧化酶主要由抗II和抗IV抗体组成，但也引发了一些抗I抗体和针对小亚基的抗体。对抗V的类似分析表明，它对亚基V具有相对特异性。通过用完整酶进行酶联免疫吸附测定（ELISA）鉴定出三种类型的抗V：（i）弱结合且与氧化还原无关，（ii）较强结合、氧化还原依赖性（仅与还原或部分还原的酶结合）且与膜无关，以及（iii）中等结合、氧化还原依赖性且与膜依赖性。抗氧化酶对酶活性的抑制在时间上呈双相性，表明存在快速反应和缓慢反应的分子群体。细胞色素c浓度的变化显示出部分竞争性动力学，但该抗体也影响“内部”酶促事件，包括与四甲基对苯二胺（TMPD）的周转速率以及细胞色素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。抗氧化酶也抑制含有外部细胞色素c的蛋白脂质体呼吸，但不抑制含有内部捕获的细胞色素c的蛋白脂质体呼吸。在膜的N（基质）侧未出现功能上重要的表位。结合更强的抗V对分离的酶的抑制作用至少为60%。在高离子强度下的抑制作用相对于细胞色素c浓度呈现双相模式。因此，抗V可能会减缓细胞色素c与其与酶的复合物的解离。氧化还原依赖性、膜依赖性抗V（先前已报道）对该酶的抑制作用仅约为20%。有效的抗V抗体对两种取向的蛋白脂质体氧化酶的活性几乎没有影响。当酶嵌入功能性膜系统中时，亚基V上一些其结合可导致抑制的位点对抗V不可及。
