F0F1-ATP synthase of potato tuber mitochondria Structural and functional characterization by resolution and reconstitution studies

Abstract

ATP hydrolase activity and oligomycin-sensitive proton conductivity have been measured in sonic potato submitochondrial particles with various degrees of resolution of the F1-ATPase. In F1-containing particles, the ATP hydrolase activity was oligomycin-sensitive both in particles prepared by ultrasonic exposure in the presence of EDTA or in the presence of Mg2+. Removal of F1 by urea treatment resulted in almost complete removal of the ATP hydrolase activity of the particles in both cases. Addition of purified F1 to the urea-treated particles restored the membrane bound ATP hydrolase activity. The activity was oligomycin-sensitive when the reconstitution was carried out with urea-treated EDTA submitochondrial particles, but oligomycin insensitive when the reconstitution was carried out with urea-treated Mg2+ particles. Comparison of polyacrylamide gel electrophoresis patterns of Mg2+ particles and of the urea-treated Mg2+ particles showed, in the latter, that the decrease of the amount of the F1α and F1β subunits was accompanied by the decrease of the amount of a protein of apparent molecular mass of 27 kDa. Addition of the purified 27 kDa protein upon reconstitution of the urea-treated Mg2+ particles with F1, restored the oligomycin sensitivity of the membrane bound ATP hydrolase activity. In F1-containing particles the oligomycin-sensitive proton conductivity was measured by kinetic analysis of anaerobic release of transmembrane proton gradient set up by respiration. Extrapolation of the initial slope of oligomycin titration of this process showed high affinity binding site at 0.4 μg oligomycin per mg particle protein, i.e., stoichiometry of 1 mol oligomycin per mol F0F1-ATP synthase. In F1-depleted particles, the oligomycin sensitive proton conductivity was measured as proton release induced by a diffusion potential (positive inside) imposed by valinomycin mediated potassium influx. In these particles, also, the initial phase of oligomycin inhibition indicated a high-affinity binding site at 0.4 μg oligomycin per mg particle protein.