The electrochemical proton gradient in Escherichia coli membrane vesicles

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Abstract
Membrane vesicles isolated from E. coli grown under various conditions generate a transmembrane pH gradient (.DELTA.pH) of .apprx. 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure .DELTA.pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (.DELTA..PSI.) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (.DELTA..hivin..mu.H+) of almost -200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a .DELTA.pH of .apprx. -120 mV. As external pH is increased, .DELTA.pH decreases, reaching 0 at .gtorsim. pH 7.5, while .DELTA..PSI. remains at .apprx. -75 mV and internal pH remains at pH 7.5-7.8. The variations in .DELTA.pH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for .DELTA.pH. Finally, .DELTA.pH and .DELTA..PSI. can be varied reciprocally in the presence of valinomycin and nigericin with little change in .DELTA..hivin..mu.H+ and no change in respiratory activity. These and other data show the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.