Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles

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Abstract
Membrane vesicles isolated from A. vinelandii strain O by lysis of spheroplasts in potassium or sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by 3 independent methods: fluorescence of 3,3''-dipropylthiodicarbocyanine and 3,3''-dihexyloxacarbocyanine; uptake of 86Rb+ in the presence of valinomycin; and uptake of [3H]triphenylmethyl phosphonium. In the 1st method the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxidation of electron donors. A linear relationship between this quenching and a K diffusion potential was employed to calibrate the probe response. In the 2nd method the steady-state concentration ratio of Rb across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In the 3rd method the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3''-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75-80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of H+ was observed during L-malate oxidation, and the amount of proton exodus was greater in K rather than the Na-containing vesicles. This indicates the presence of a Na-H+ antiport mechanism. D-Glucose uptake was observed during development of a K diffusion potential that was artificially imposed across the vesicle membrane. The presence of a glucose-proton symport mechanism in accordance with Mitchell''s principles is suggested.