Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli

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Abstract
Inverted membrane vesicles prepared from E. coli ML 308-225 generate a transmembrane electrochemical proton gradient (.DELTA..hivin..mu.H+; interior positive and acid) during oxidation of D-lactate, succinate, reduced phenazine methosulfate or NADH or hydrolysis of ATP. Using the distribution of the lipophilic anion thiocyanate to measure the membrane potential (.DELTA..PSI.) and the permeant weak base methylamine to measure the pH gradient (.DELTA.pH), maximal values for .DELTA..hivin..mu.H+ of approximately 160 mV are obtained. Many of the properties of .DELTA..hivin..mu.H+ in inverted vesicles are similar to those described previously in right-side-out vesicles. The magnitude of the .DELTA..PSI. (interior positive) generated in the presence of D-lactate or reduced phenazine methosulfate is similar to that observed in right-side-out vesicles but of opposite polarity and independent of pH from 5.5-8.0. Plots of .DELTA.pH vs. internal pH in inverted vesicles and external pH in the right-side-out vesicles are similar with D-lactate as the electron donor. As observed with right-side-out vesicles, dissipation of .DELTA..PSI. or .DELTA.pH leads to a concomitant increase in the other parameter without a change in the rate of respiration. Inverted vesicles catalyze Na+ accumulation, and it is apparent that the process can be driven by either .DELTA..PSI. (interior positive) or .DELTA.pH (interior acid).