On the function of the natural ATPase inhibitor protein in intact mitochondria

The recently described methodology to extract the mitochondrial ATPase along with other mitochondrial proteins into organic solvents, and their subsequent incorporation into liposomes [Eur. J. Biochem. (1982) 121, 427-433] has been employed to estimate the number of ATPases that contain the natural ATPase inhibitor protein in its inhibitory site in intact mitochondria incubated in various metabolic states. It was found that in the presence of electrochemical gradients about 50% of the ATPases are without inhibitor protein in its inhibitory site (active ATPases). In the transition from state 4 to state 3 the percentage of active ATPases diminishes from about 50% to approximately 20%. This indicates that during steady-state phosphorylation only a limited number of ATPases are in the active catalytic state, and that not only during active hydrolysis does the inhibitor protein interact with its inhibiting site; rather the inhibitor seems to interact with an intermediate state of the enzyme that appears either during the synthetic or hydrolytic reactions. In addition it was found that ATP, with or without uncoupler, induces the interaction of the inhibitor protein in more than 80% the ATPases through an oligomycin-sensitive process. Thus, notwithstanding other factors the interaction may account for the low hydrolytic activity of mitochondria.