Cleavage of bovine mitochondrial ATPase inhibitor with endopeptidases, and binding of the resulting peptides to the interface between the alpha- and beta-subunits of F1ATPase.

Mitochondrial ATPase inhibitor binds to F1ATPase, forming an equimolar complex with the enzyme, and the binding site has been reported to be located in the beta-subunit [Klein, G. et al. (1980) Biochemistry 19, 2919-2925] or at the interface between the alpha- and beta-subunits of the enzyme [Mimura, H. et al. (1993) J. Biochem. 113, 350-354]. In the present study, bovine ATPase inhibitor as well as three peptide fragments of the inhibitor, Gly1-Asn51, Glu52-Asp84, and Lys46-Asp84, were used to examine the features of the binding of the inhibitor and F1ATPase. Only the amino terminal fragment, Gly1-Asn51, exhibited a similar level of inhibitory activity to that of the native ATPase inhibitor. Although the other two carboxyl terminal side fragments did not exhibit any inhibitory activity, they interfered with the action of the intact ATPase inhibitor when they were pre-loaded to F1ATPase. Since the two carboxyl fragments did not interfere with the inhibitory action of the amino fragment, it is inferred that the inhibitor interacts with F1ATPase at its carboxyl terminal region prior to binding at the amino terminal region of the enzyme. Cross-linking experiments revealed that ATPase inhibitor bound to the alpha- and beta-subunits of F1ATPase, and that the amino terminal peptide preferentially bound to the alpha-subunit and the carboxyl terminal peptides to the beta-subunit.