The native F₀F₁-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

Abstract

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28Μmol min⁻¹ mg⁻¹, whereas theV _max of the complex without the inhibitor was 8.3Μmol min⁻¹ mg⁻¹. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148ΜM, comparable to theK _m value of 142ΜM of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60‡C at pH 6.8, reaching a maximal ATPase activity of 9.5Μmol min⁻¹ mg⁻¹; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.