ATP synthase from bovine heart mitochondria: reconstitution into unilamellar phospholipid vesicles of the pure enzyme in a functional state.
A highly purified and monodisperse preparation of proton-translocating F1F0-ATPase from bovine heart mitochondria is an assembly of 16 unlike polypeptides. This preparation has been reconstituted in the presence of various detergents into unilamellar phospholipid vesicles. Incorporation of the enzyme into vesicles increases the ATP hydrolase activity of the enzyme by 10-20-fold, depending on the detergent, and the highest activities of ATP hydrolysis, 70 units/mg, were obtained by reconstitution from dodecylmaltoside or CHAPS. This activity is mostly sensitive to inhibitors that act on the F0 membrane sector of the complex. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-methoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co-reconstitution of the enzyme with bacteriorhodopsin, it was demonstrated that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteristic biological functions of the F1F0-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its physical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.
Groth
G
G
Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
Walker
J E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000609:Aminoacridines / Q000378:metabolism; D000818:Animals; D001436:Bacteriorhodopsins / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D003902:Detergents; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D008081:Liposomes / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009550:Nigericin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D000609:Aminoacridines; D003902:Detergents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008081:Liposomes; D009840:Oligomycins; D011510:Proteolipids; C020485:proteoliposomes; C010847:9-amino-6-chloro-2-methoxyacridine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009550:Nigericin