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.

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