Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium.
Vesicles were reconstituted from a purified dicyclohexyl-carbodiimide-sensitive ATPase complex (TF0-F1) and phospholipids of a thermophilic bacterium PS3. These vesicles synthesized ATP from ADP and Pi with energy from an electrochemical proton gradient (delta-micronH+) formed by a pH gradient and an electrical potential across their membranes. Maximal ATP synthesis was achieved by incubating the vesicles in malonate at pH 5.5 with valinomycin, and then rapidly transferring them to a solution of pH 8.4 and 150 mM K+. Under these conditons ATP synthesis continued at a decreasing rate for 30 s at 40 degrees. Appreciable formation of ATP (40 to 150 nmol/mg of TF0-F1) occurred at an initial delta-micronH+ above 205 mV and moderate formation at an initial value above 180 mV. ATP hydrolysis by the vesicles produced a delta-micronH+, and the additions of 32Pi and hexokinase to them resulted in 32Pi esterification. Analysis of the time courses of 32Pi esterification and decays of the pH difference and membrane potential, followed using 9-aminoacridine and 8-anilinonaphthalene-1-sulfonate, respectively, as probes, showed a relationship between delta-micronH+ and the rate of ATP synthesis. These results demonstrate that purified TF0-F1 is itself a reversible H+-translocating ATPase of oxidative phosphorylation.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D001419:Bacteria; D004024:Dicyclohexylcarbodiimide; D006593:Hexokinase / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D010085:Oxidative Phosphorylation; D010710:Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011522:Protons; D013025:Glycine max
D008081:Liposomes; D010710:Phosphates; D010743:Phospholipids; D011522:Protons; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006593:Hexokinase; D000251:Adenosine Triphosphatases