Adenosine 5'-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli

Abstract

ATP synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of E. coli was investigated. Membrane vesicles prepared in the presence of ADP were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2-0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone or azide to the assay buffer inhibited ATP synthesis. ADP and Mg2+ were required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-ATPase (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. The Mg2+-ATPase contained tightly bound ATP.