Frey T G, Chan S H, Schatz G
J Biol Chem. 1978 Jun 25;253(12):4389-95.
The structure and the orientation of cytochrome c oxidase molecules in crystalline cytochrome c oxidase membranes (Vanderkooi, G., Senior, A.E., Capaldi, R.A., and Hayashi, H. (1972) Biochim. Biophys. Acta 274, 38-48) were studied by image analysis of electron micrographs and by reacting the crystalline preparations with immune gamma-globulins against individual cytochrome c oxidase subunits. Binding of gamma-globulins to the membranes was detected by the following two methods: (a) electrophoretic identification of gamma-globulin polypeptides in the washed membranes; (b) electron microscopic examination of the negatively stained membranes. The membranes bound immune gamma-globulins against subunit IV (which faces the matrix side in intact mitochondria) but failed to bind immune gamma-globulins against subunits II + III (which face the outer side of the inner membrane in intact mitochondria). In contrast, solubilized cytochrome c oxidase bound either of the two immune gamma-globulins. All cytochrome c oxidase molecules in the crystalline membranes are thus asymmetrically arranged so that subunit IV faces outward and subunits II + III face toward the interior. This orientation is opposite to that found with intact mitochondria. The data also suggest that the crystalline membranes form closed vesicles which are impermeable to externally added gamma-globulins.
通过对电子显微镜照片进行图像分析，并使结晶制剂与针对细胞色素c氧化酶各个亚基的免疫球蛋白反应，研究了结晶细胞色素c氧化酶膜（范德库伊，G.，西尼尔，A.E.，卡帕尔迪，R.A.，和林，H.（1972年）《生物化学与生物物理学报》274卷，38 - 48页）中细胞色素c氧化酶分子的结构和取向。通过以下两种方法检测免疫球蛋白与膜的结合：（a）对洗涤后的膜中的免疫球蛋白多肽进行电泳鉴定；（b）对经负染色的膜进行电子显微镜检查。这些膜结合了针对亚基IV（在完整线粒体中面向基质侧）的免疫球蛋白，但未能结合针对亚基II + III（在完整线粒体中面向内膜外侧）的免疫球蛋白。相比之下，溶解的细胞色素c氧化酶结合了这两种免疫球蛋白中的任何一种。因此，结晶膜中的所有细胞色素c氧化酶分子都是不对称排列的，使得亚基IV面向外侧，亚基II + III面向内部。这种取向与在完整线粒体中发现的取向相反。数据还表明，结晶膜形成封闭的囊泡，对外加的免疫球蛋白是不可渗透的。
