Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis.
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.
Deisinger
B
B
Gustav-Embden-Zentrum der Biologischen Chemie, Klinikum der J. W. Goethe-Universität, Frankfurt/M., Germany.
Nawroth
T
T
Zwicker
K
K
Matuschka
S
S
John
G
G
Zimmer
G
G
Freisleben
H J
HJ
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
D008081:Liposomes; D001436:Bacteriorhodopsins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases