ATP synthesis by purified ATP-synthase from beef heart mitochondria after coreconstitution with bacteriorhodopsin.
An ATP-synthase complex active in ATP synthesis was isolated from beef heart mitochondria by solubilization of submitochondrial particles with dodecyl-beta-D-maltoside and purified in a one-step procedure by subsequent ion-exchange chromatography. The electrophoretic analysis resulted in 14 subunits for the F0 F1 complex. ATP hydrolysis activity of the purified enzyme was 25 mumol ATP min-1 mg-1F0F1. ATP hydrolysis could be stimulated by addition of lipid vesicles. Further stimulation was observed in the presence of uncoupler. The inhibitors dicyclohexylcarbodiimide and oligomycin reduced hydrolytic activity to 70 and 40%, respectively. The preservation of ATP synthesis capability was demonstrated by reconstitution of the purified enzyme together with the light-driven proton pump bacteriorhodopsin. Upon illumination of ATP-synthase/bacteriorhodopsin proteoliposomes ATP synthesis activity was detectable for at least 7 min. At reduced temperature this time could be increased to 20 min. The maximum synthesis rate of 58 nmol ATP min-1 mg-1 F0F1 was obtained after reconstitution into liposomes made from crude soy bean lecithin by a detergent dialysis procedure using octyl-beta-D-glucopyranoside and monomeric bacteriorhodopsin. ATP synthesis was partially inhibited by oligomycin or dicyclohexylcarbodiimide and was completely abolished in the presence of uncoupler. The ability of the purified enzyme to synthesize ATP shows that the described isolation procedure results in an ATP-synthase complex which is intact in structure and function.
Matuschka
S
S
Universitätsklinik, Gustav-Embden-Zentrum der Biologischen Chemie, Frankfurt, Germany.
Zwicker
K
K
Nawroth
T
T
Zimmer
G
G
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D001436:Bacteriorhodopsins / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008929:Mitochondria, Heart / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology
D009840:Oligomycins; D011510:Proteolipids; C020485:proteoliposomes; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases