The adenosinetriphosphatase (ATPase) inhibitor peptide of rat liver mitochondria [Cintrón, N.M., & Pedersen, P.L. (1979) J. Biol. Chem. 254, 3439-3443] has been examined in detail with respect to its effect on the adenosine 5′-triphosphate (ATP) hydrolytic and functional activities catalyzed by the H+-ATPase of inverted inner membrane vesicles from the same source. The peptide inhibitor maximally inhibits the ATPase activity of the H+-ATPase when enzyme and inhibitor are incubated near pH 7.0 for ≥3 min prior to assay in the presence of the substrates ATP and Mg2+. Inosine 5′-triphosphate (ITP) and guanosine 5′-triphosphate (GTP), which are also effective substrates for the enzyme, will substitute for ATP in the preliminary incubation. When the ATP hydrolytic activity taking place during the preliminary incubation is prevented by addition of ethylenediaminetetraacetic acid (EDTA), the peptide inhibitor is rendered ineffective as an inhibitor of the H+-ATPase. Adenosine 5′-diphosphate (ADP) and Pi either separately or together are unable to replace ATP in the preliminary incubation. It is suggested that an intermediate H+-ATPase complex containing bound MgATP, ADP, and/or Pi interacts with the inhibitor peptide. Kinetic experiments show that in tris(hydroxymethyl)aminomethane (Tris)-HCO3 buffer velocity vs. [ATP] response patterns are hyperbolic (i.e., typical Michaelis-Menten) when no inhibitor is present, whereas when the ATPase inhibitor peptide is present, such patterns are biphasic. The inhibitor appears to unmask at least one ATP site on the rat liver H+-ATPase that is functionally silent in Tris-HCO3 buffer. Under conditions where the ATPase inhibitor peptide maximally inhibits ATPase activity of the H+-ATPase of inverted inner membrane vesicles, it maximally inhibits also the ATP-dependent NADH-NADP+ transhydrogenase and the ATP-dependent succinate-linked NAD+ reduction activities supported by the enzyme. Under these conditions, the ATP-Pi exchange reaction catalyzed by the enzyme is inhibited about 50%, and the ATP synthetic activity is unaffected. The ATP-Pi exchange reaction can be further inhibited (≥75%) by addition of higher concentrations of inhibitor peptide. Results reported here describe an unusual relationship between an enzyme inhibitor and its target enzyme in that the catalytic activity of the enzyme itself is required to “prime” formation of a nonproductive inhibitor-enzyme complex. They also supply new information about the mode of interaction between the inhibitor protein and the H+-ATPase. Finally, they show that the inhibitor peptide of rat liver is primarily a unidirectional inhibitor, which suppresses ATP-dependent processes while exerting little effect on the steady-state rate of ATP synthesis.
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