Vesicular preparation of a highly coupled ATPase-ATP synthase complex from pig heart mitochondria.

1. A method is described to prepare an ATPase-ATP synthase complex from pig heart mitochondria exhibiting a very high ATP-32Pi exchange activity (1.6 mumol/min per mag protein in optimal conditions). 2. The preparation is virtually devoid of nucleoside diphosphokinase and adenylate kinase activities. 3. Freeze-fracture studies show that the ATPase-ATP synthase complex is integrated in lipid vesicles of 400-600 A in diameter. 4. It contains the endogenous natural proteic inhibitor which seems to behave as a coupling factor. 5. The rate of ATP hydrolysis catalyzed by the ATPase-ATP synthase complex is competitively inhibited by ADP, while the presence of ADP increases the initial rate of 32Pi incorporation into ATP. 6. The 32Pi incorporation into ATP can occur at a rate almost equal to that of nucleoside triphosphate (NTP) hydrolysis provided that the rate of NTP hydrolysis is kept low and that the ADP concentration is high enough. In these conditions, a very high coupling between NTP hydrolysis and ATP synthesis can be demonstrated.