Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.
Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.
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细胞色素 C 氧化酶的免疫学研究：溶解和膜结合酶中蛋白质亚基的排列。
DOI：
10.1111/j.1432-1033.1978.tb12564.x
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发表时间：
1978
期刊：
影响因子：
--
通讯作者：
Russell P. Tracy
中科院分区：
文献类型：
--
作者：
Samuel H. P. Chan;Russell P. Tracy
用十二烷基硫酸钠（SDS）和盐酸胍（V、VI、VII）作凝胶过滤剂，用6 M盐酸胍解离细胞色素c氧化酶，再用6 M尿素作离子交换层析剂，从牛心脏中分离出7个蛋白质亚基。当在尿素存在下通过高度交联的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳分析时，表观分子量为= I，36700; II，24300; III，20400; IV，17300; V，12300; VI，8700;和VII，5100。单特异性兔抗血清获得了对亚基I，IV，V，VI和VII和亚基II和III的混合物。这些亚基特异性抗血清与抗-I血清的例外都与洗涤剂溶解的天然氧化酶交叉反应。对纯化的氧化酶的酶促研究表明，针对亚基II + III、IV、V、VI和VII的免疫球蛋白分别引起25%、65%、20%、30%和25%的抑制，而抗I免疫球蛋白不抑制活性。亚单位特异性抗血清用于检查膜中亚单位的排列。用牛心线粒体和大鼠肝线粒体毛地黄皂苷颗粒进行的酶促研究表明，抗（II + III）血清、抗V血清和抗VII血清均抑制氧化酶活性，而其他抗血清则不抑制。另一方面，使用125I标记的免疫球蛋白的结果显示，抗IV、抗V和抗VII血清结合到倒置囊泡的表面（基质侧），而所有其它抗血清不结合。这些结果表明，细胞色素氧化酶亚基II和III位于外表面上，和亚基IV是专门的基质表面上，而亚基V和VII暴露在线粒体膜的两个表面上。亚基I和VI埋在膜内，两侧都不暴露。
Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.