A decrease in the rate of
ATP hydrolysis was observed after preincubation of intact mitochondria from
hepatoma 22a with an uncoupler. This effect is due both to a decrease in the rate of
ATP transport and to an inactivation of the F0F1-ATPase. The former effect is shown to result from an uncoupler-induced
ADP efflux. In de-energized mitochondria from
hepatoma (but not from mice liver), the concentration of
adenine nucleotides in the matrix equilibrates with the medium concentration via a
carboxyatractyloside (CATR)-insensitive transport system. CATR-insensitive accumulation of medium
ADP and stoichiometric exchange of added
ATP are observed in energized
hepatoma mitochondria. The dependence of the uncoupler-induced inactivation of
ATPase activity on delta mu H+, pH, and
ATP is consistent with the effect being caused by the natural
protein inhibitor (IF1) of F0F1.
ATP- and pH-dependent inactivation of the
enzyme is also observed after disruption of mitochondria with the
detergent Lubrol-WX. Almost all F0F1 in
hepatoma mitochondria have IF1 bound in a noninhibitory manner. In the presence of uncoupler, this complex converts, via a reversible pH-dependent and an irreversible
ATP-dependent process, to an inhibitory complex. The pH-dependent step can be blocked by Zn2+ and Cd2+
ions which probably bind to negatively charged residues on IF1, thereby preventing their protonation and conversion of the
protein to an inhibitory conformation.