The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reduc tase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cy tochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determine d by applying three novel and independent approaches that gave consistent r esults: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and c atalytic activities in the samples before and after ultracentrifugation wer e used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electropho resis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamid e gel electrophoresis) and Coomassie blue-staining indices of subunits of c omplexes was used for determining the ratios of complexes I, III, IV, and V , and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of compl exes I and III. For complexes I, II, III, IV, and V in bovine heart mitocho ndria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3: 6.7 +/- 0.8:3.5 +/- 0.2 was deter mined.