Patel S D, Cleeter M W, Ragan C I
Department of Biochemistry, University of Southampton, U.K.
Biochem J. 1988 Dec 1;256(2):529-35. doi: 10.1042/bj2560529.
The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.
通过化学交联和用[125I]碘代重氮苯磺酸盐（IDABS）进行放射性标记，研究了牛心NADH脱氢酶在线粒体内膜中的组织方式。线粒体或亚线粒体颗粒用二硫代琥珀酰亚胺基酒石酸酯和亚胺基辛二酸二甲酯进行交联，并用亚基特异性抗血清通过蛋白质印迹法分析含有NADH脱氢酶亚基的二聚体产物。线粒体交联产生了（49 + 30）kDa和（49 + 19）kDa的二聚体以及一个含有30 kDa亚基的额外二聚体。亚线粒体颗粒交联产生了（75 + 51）kDa、（75 + 30）kDa和（49 + 13）kDa的二聚体以及另一个含有30 kDa亚基的二聚体。我们得出结论，49 kDa和30 kDa亚基是跨膜的，19 kDa亚基暴露于膜的细胞质面，而75、51和13 kDa亚基暴露于膜的基质面。分离的酶与IDABS反应导致75、49、42、33、30、13和10 kDa亚基被标记。在首先标记线粒体或亚线粒体颗粒然后通过免疫沉淀分离NADH脱氢酶的实验中，发现49 kDa亚基的标记主要发生在膜的细胞质侧。另一方面，75、13和10 kDa亚基的标记主要发生在膜的基质侧，而30和33 kDa亚基从两侧都被大量标记。这些发现与通过交联获得的结果一致。
