Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Abstract

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ( Δ \\ ̄ gm H + ), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H + symport, to an observable decrease of Δ \\ ̄ gm H + , and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H + symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.