Hekman C, Tomich J M, Hatefi Y
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Biol Chem. 1991 Jul 25;266(21):13564-71.
The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.
借助针对F0亚基b、d、6、F6、A6L、寡霉素敏感性赋予蛋白（OSCP）、N,N'-二环己基碳二亚胺（DCCD）结合蛋白脂质以及ATP酶抑制剂蛋白（IF1）产生的亚基特异性抗体作为内部对照，研究了牛线粒体内膜中ATP合酶复合体膜扇区F0亚基的拓扑结构。通过直接抗体结合以及这些亚基对各种蛋白酶降解的敏感性（通过膜消化产物的凝胶电泳和亚基特异性抗体免疫印迹监测），研究了F0亚基在翻转和外翻内膜中的暴露情况。结果表明，亚基b、d、F6、A6L（包括其C末端）和OSCP暴露于基质侧。这些亚基足以识别抗体或进行蛋白水解的质量未暴露于胞质侧。内膜两侧的亚基6和DCCD结合蛋白脂质也是如此。采用[125I]蛋白A结合的放射性进行定量免疫印迹，以估计样品中抗原的浓度，从而确定了几个F0亚基和IF1相对于F1-ATP酶的化学计量。结果表明，在牛心脏线粒体中，每摩尔F1含有1摩尔的d、OSCP和IF1，以及2摩尔的b和F6。亚基6和DCCD结合蛋白脂质无法定量，因为前者转移到硝酸纤维素膜的效果不佳，而后者的抗体不结合[125I]蛋白A。
