The electrochemical proton gradient in Escherichia coli membrane vesicles

Abstract

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 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-muH+) 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 deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH 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 deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.

The Electrochemical Proton Gradient Génerated by Light in Membrane Vesicles and Chromatophores from Rhodopseudomonas sphaeroides

“…The membrane potentials determined from the accumulation of Ph3MePf in membrane vesicles are in good agreement with the values obtained in membrane vesicles from other bacteria [14,16,39]. The membrane potentials calculated from the SCN accumulation in chromatophores, are in good agreement with the membrane potentials obtained from the fluorescence enhancement of S-anilinonaphthalenel-sulphonic acid [36].…”

“…These data demonstrate that the pH gradient increases with increasing external pH. Contrary to the observations of Ramos and Kaback [14,16] the pH reached inter- …”

“…The electrical potential across the membrane has been estimated from the accumulation of lipophilic cations or anions [14,3 1, 34,351, the enhancement of the fluorescence of probes such as X-anilinonaphthalene-l-sulphonic acid [36], the quenching of the fluorescence of cyanine dyes [37] and, for membranes from photosynthetic organism, from the spectral shifts of the absorption bands of pigments [32]. Methods requiring measurement of the accumulation of highly membrane permeant solutes became available for studies in membrane preparations by application of the flow dialysis technique [14,16]. Several of the methods mentioned above have been applied in this investigation.…”

“…Uptake studies were performed by the flow-dialysis technique according to Ramos et al [14,15] at 30 "C. For anaerobic conditions the glass-covered upper chamber of the dialysis apparatus and the dialysis buffer were deoxygenated with oxygen-free nitrogen. The upper and lower chamber were separated by Vishking dialysis tubing (pore diameter 2.4 nm).…”

N‐ethylmaleimide induces K+−H+ antiport activity in Escherichia coli K‐12

“…Like in the presence of the ionophore nigericin [18,19], which catalyzes an electroneutral K'-H' exchange [20], the cells compensated the NEM-induced decrease in ApH with an increase in A$, with the result that the total protonmotive force remained constant ( fig.l,2 [ 141).…”