Adenosine 5'-triphosphate synthesis driven by a protonmotive force in membrane vesicles of Escherichia coli

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Abstract
ATP synthesis energized by an artificially imposed protonmotive force (.DELTA.p) in ADP-loaded membrane vesicles of E. coli was investigated. The protonmotive force is composed of an artificially imposed pH gradient (.DELTA.pH) or membrane potential (.DELTA..psi.), or both. A .DELTA.pH was established by a rapid alteration of the pH of the assay medium. A .DELTA..psi. was created by the establishment of diffusion potential of K+ in the presence of valinomycin. The maximal amount of ATP synthesized was 0.4-0.5 nmol/mg of membrane protein when energized by a .DELTA.pH and 0.2-0.3 nmol/mg of membrane protein when a .DELTA..psi. was imposed. Simultaneous imposition of a .DELTA.pH and .DELTA..psi. resulted in the formation of greater amounts of ATP (0.8 nmol/mg of membrane protein) than with either alone. The amount of ATP synthesized was roughly proportional to the magnitude of the artificially imposed .DELTA.p. Although p-chloromercuribenzoate, 2-heptyl-4-hydroxyquinoline-N-oxide or NaCN each inhibits oxidation of D-lactate, and thus oxidative phosphorylation, none inhibited ATP synthesis driven by an artificially imposed .DELTA.p. Membrane vesicles prepared from uncA or uncB strains, which are defective in oxidative phosphorylation, likewise were unable to catalyze ATP synthesis when energy was supplied by an artificially imposed .DELTA.p.