title: Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis.
abstract: ATP synthase was isolated from beef heart mitochondria by extraction with N,N-bis-(3-D-gluconamidopropyl)deoxycholamide or by traditional cholate extraction. The enzyme was purified subsequently by ion-exchange and gel-permeation chromatographies in the presence of glycerol and the protease inhibitor diisopropylfluorophosphate. The ATP synthase consisted of 12-14 subunits and contained three tightly bound nucleotides. The co-reconstitution of crude or purified ATP synthase with monomeric bacteriorhodopsin by the method of detergent incubation of liposomes yielded proteoliposomes capable of light-driven ATP synthesis, as detected with a luciferase system for at least 30 min. The reaction was suppressed by the inhibitors oligomycin (> 90%) and dicyclohexylcarbodiimide (85%) and by the uncoupler carbonylcyanide-p-trifluormethoxyphenylhydrazone (> 95%). The purified ATP synthase was apparently free of cytochrome impurities and of adenylate kinase activity, i.e. the enzyme exhibited light-driven ATP synthesis without the dark reaction. For the first time, this is demonstrated with purified ATP synthase from beef heart mitochondria.
authors.affiliation: Gustav-Embden-Zentrum der Biologischen Chemie, Klinikum der J. W. Goethe-Universität, Frankfurt/M., Germany.
authors.forename: B
authors.initials: B
authors.lastname: Deisinger
authors.forename: T
authors.initials: T
authors.lastname: Nawroth
authors.forename: K
authors.initials: K
authors.lastname: Zwicker
authors.forename: S
authors.initials: S
authors.lastname: Matuschka
authors.forename: G
authors.initials: G
authors.lastname: John
authors.forename: G
authors.initials: G
authors.lastname: Zimmer
authors.forename: H J
authors.initials: HJ
authors.lastname: Freisleben
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D008081:Liposomes; D001436:Bacteriorhodopsins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases