Equilibrium between two forms of the lac carrier protein in energized and nonenergized membrane vesicles from Escherichia coli.
p-Nitrophenyl alpha-D-galactopyranoside is a competitive inhibitor of lactose transport in membrane vesicles prepared from  Escherichia coli ML 308-225 (Ki congruent to 6.6 muM) but is not accumulated by the vesicles. Binding of p-nitrophenyl alpha-D-[6-3H]galactopyranoside to membrane vesicles has been measured by flow dialysis. In the presence of D-lactate, ligand binds to the vesicles with a KD of about 6 muM, and a total of 2.3 nmol per mg of membrane protein is bound at saturation. In the absence of D-lactate, a small amount of binding can be detected (approximately 0.2 nmol per mg of membrane protein) with a similar affinity constant (KD congruent to 9 muM). Binding inthe 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. The results confirm previous observations that the bulk of the lac carrier protein is unable to bind ligand unless the membrane is energized. and they also corroborate observations 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 two forms: (i) a low affinity, cryptic form which predominates in the absence of energy coupling; and (ii) a high affinity form, accessible from the external surface of the membrane, which predominates in the presence of an electrochemical gradient of protons (interior negative and alkaline).
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004140:Dinitrophenols / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D009596:Nitrophenols / Q000378:metabolism
D001426:Bacterial Proteins; D004140:Dinitrophenols; D005697:Galactosides; D009596:Nitrophenols; D007785:Lactose