Binding Properties of 9K Protein to F1-ATPase: A Counterpart Ligand to the ATPase

Abstract

A regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with F_l-ATPase in the presence of ATP and Mg²⁺, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of F_l-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the F_l-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 × 10^-6 M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 × 10^-6 M). When added simultaneously the binding of the inhibitor and 9K protein to F_l-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to F_l-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound F_l-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectively