ATP synthetase (F1F0) of Escherichia coli K-12. High-yield preparation of functional F0 by hydrophobic affinity chromatography.
1. The purified ATP synthetase complex (F1F0) from Escherichia coli was adsorbed to immobilized poly-(L-lysine)-deoxycholic acid. About 0.7 mg F1F0 were bound per ml of settled gel. The hydrophilic F1 part was dissociated from the complex by treatment with 7 M urea. F0 was eluted in high yield either with deoxycholate (6 mM) or taurodeoxycholate (10 mM). About 14% of the total protein bound to the column was eluted as F0, which corresponds to 64% of the total F0 in the F1F0 complex. 2. The purified F0 preparation obtained was composed of three different kinds of subunits with apparent molecular weights of 24000 (a), 19000 (b) and 8300 (c), respectively as determined by sodium dodecyl sulfate gel electrophoresis. 3. After incorporation into liposomes and the generation of a potassium diffusion potential by valinomycin, the F0 preparation mediated H+ translocation. This H+ uptake is inhibited by either dicyclohexylcarbodiimide or purified F1 ATPase. 4. Incubation of F0-containing liposomes with F1 led to the reconstitution of an ATP-driven quenching of acridine-dye fluorescence. The quenching was abolished by uncoupler and prevented by dicyclohexylcarbodiimide.
D025181:ATP Synthetase Complexes; D000244:Adenosine Diphosphate / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D008081:Liposomes; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010770:Phosphotransferases / Q000302:isolation & purification*; D011522:Protons
D008081:Liposomes; D009097:Multienzyme Complexes; D011522:Protons; D000244:Adenosine Diphosphate; D010770:Phosphotransferases; D025181:ATP Synthetase Complexes