Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration-and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (AiH+, interior negative and alkaline) was generated across the vesicle membrane. Although P-D-galactopyranosyl 1-thio-t-D-galactopyranoside blocked DEPC The chemosmotic hypothesis of Mitchell (1-3) proposes that energy derived from respiration, photochemical reactions, or ATP hydrolysis can be transformed into a transmembrane electrochemical gradient of protons (AOH+) that is the immediate driving force for many active transport systems in bacterial cells. More specifically, the hypothesis states that substrate-specific membrane proteins (i.e., porters, carriers, or permeases) mediate the coupling between AH+ and substrate accumulation by catalyzing the translocation of substrate with protons, the substrate moving against and the protons with their respective electrochemical gradients (i.e., symport).Cytoplasmic membrane vesicles prepared from Escherichia coli, which retain the same polarity and configuration as the membrane in the intact cell (4-6) as well as the capacity to convert respiratory energy into a 4AH+ (interior negative and alkaline) (7), catalyze the active transport of many substrates, including 3-galactosides (8). Moreover, studies with intact cells (9) and this in vitro system (7,(10)(11)(12)(13)(14) provide virtually unequivocal evidence for the central obligatory role of chemosmotic phenomena in active transport. Recent evidence with isolated membrane vesicles (15, 16) indicates that carriermediated lactose efflux down a concentration gradient is an ordered mechanism in which lactose is released first from the carrier, followed by loss of a proton, and that the unloaded carrier may be negatively charged. In
MATERIALS AND METHODSGrowth of Cells and Preparation of Membrane Vesicles. E. coli ML 308-225 (lac-, lacZ-, lacY+, lacA +) and ML 30 (lad +, lacZ +, lacY +, lacA +) were grown on minimal medium A containing 1.0% disodium succinate (hexahydrate), and membrane vesicles were prepared as described (19,20).Treatment with DEPC. Vesicles suspended in 100 mM potassium phosphate (pH 6.6) were titrated to pH 6.0 with 100 mM monobasic potassium phosphate and adjusted to a final concentration of about 2.0 mg of protein per ml by adding appropriate volumes of 100 mM potassium phosphate (pH 6.0). Small portions of 2.0 M DEPC (freshly prepared in absolute ethanol) were added to the membrane suspensions such that the final concentration of ethanol was never more than 0.5% (at concentrations up to 1.0%, ethanol has no significant effect on any of the activities studied). Immediately after addition of DEPC, the suspensions were vigorously agitated to achieve complete mixing and incubated at room temperature on a magnetic stirrer for given periods of time. Reactions were terminated by 1:5 dilution ...