Resolution and Reconstitution of H+-ATPase Complex from Beef Heart Mitochondria

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Abstract
Mitochondrial H+-ATPase complex, purified by the lysolecithin extraction procedure, was resolved into a membrane (NaBr-Fo) and a soluble fraction by treatment with 3.5 M NaBr. The NaBr-Fo fraction is completely devoid of .beta., .delta. and .epsilon. subunits of the F, ATPase and largely devoid of .alpha. and .gamma. subunits of F1, where Fo is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 .mu.g .cntdot. mg-1 Fo) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to inhibitors as submitochondrial particles. The present communication describes resolution of this F1-Fo preparation using NaBr and reconstitution of ATPase and Pi-ATP exchange activities. The NaBr-Fo prepared from this preparation shows no dependence on lipids, and the same or increased sensitivity to energy transfer inhibitors when reconstituted with F1-ATPase. F1 ATPase activity does not decrease on binding of F1 to NaBr-Fo, even though the reconstituted ATPase activity is 99% sensitive to oligomycin and dicyclohexylcarbodiimide. These properties are in contrast to the protperties of Fo reported by other workers.

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