Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli

Abstract

P-Nitrophenyl .alpha.-D-galactopyranoside is a competitive inhibitor of lactose transport in membrane vesicles prepared from E. coli ML 308-225 (Ki [inhibition constant] .simeq. 6.6 .mu.M) but is not accumulated by the vesicles. Binding of p-nitrophenyl .alpha.-D-[6-3H]galactopyranoside to membrane vesicles was measured by flow dialysis. In the presence of D-lactate, ligand binds to the vesicles with a KD [affinity constant] of .apprx. 6 .mu.M, and a total of 2.3 nmol/mg of membrane protein is bound at saturation. In the absence of D-lactate, a small amount of binding can be detected (.apprx. 0.2 nmol/mg of membrane protein) with a similar KD of .simeq. 9 .mu.M. Binding in the presence or absence of D-lactate is dependent upon a functional lac y gene product and upon the structural integrity of the vesicle membrane and is reversed by p-hydroxymercuribenzenesulfonate. Agents such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and valinomycin, alone or in combination, abolish D-lactate-dependent binding, but do not affect binding in the absence of electron donors. As previously suggested, the bulk of the lac carrier protein is apparently unable to bind ligand unless the membrane is energized, indicating that a small amount of binding occurs in the absence of energy coupling. The findings are discussed in terms of a model in which the lac carrier protein exists in a state of dynamic equilibrium between 2 forms: a low affinity, cryptic form which predominates in the absence of energy coupling and a high affinity form, accessible from the external surface of the membrane, which predominates in the presence of an electrochemical proton gradient (interior negative and alkaline).