Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of a histidine residue(s)

Abstract

Addition of lactose to E. coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced E. coli ML 30. The magnitude of the phenomenon is enhanced about 3-fold in vesicles from E. coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined from lactose-facilitated diffusion and quantitative comparison of the initial rates of the 2 fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.