The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes.
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 a Vmax of 1.28 mumol min-1 mg-1, whereas the Vmax of the complex without the inhibitor was 8.3 mumol min-1 mg-1. In contrast, the Km for Mg-ATP of F0F1I was 148 microM, comparable to the Km value of 142 microM of the F0F1 complex devoid of IF1. The hydrolytic activity of the F0F1I increased severalfold by incubation at 60 degrees C at pH 6.8, reaching a maximal ATPase activity of 9.5 mumol 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 uncoupler p-trifluoromethoxyphenylhydrazone.
Vázquez-Contreras
E
E
Departamento de Bioenergética, Universidad Nacional Autónoma de México, México, D.F.
Vázquez-Laslop
N
N
Dreyfus
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013367:Submitochondrial Particles / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
D000255:Adenosine Triphosphate; D008081:Liposomes; D046911:Macromolecular Substances; D011506:Proteins; D011510:Proteolipids; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein; C020485:proteoliposomes