Abstract
6'-(N-Dansyl)aminohexyl-1-thio-beta-D-galactopyranoside binds specifically to the lac carrier protein in cytoplasmic membrane vesicles isolated from Escherichia coli. Binding can be induced by substrate oxidation (generation of an electrochemical gradient of protons), by potassium efflux in the presence of valinomycin (generation of a potassium diffusion potential), and by passive, carrier-mediated lactose efflux. We show that in all three cases the number of binding sites is temperature dependent. Binding is maximal and constant above 20 degrees ; it decreases between 20 degrees and 10 degrees . Oxidation of substrate (D-lactate) leads to the development of an electrochemical gradient of protons across the membrane (interior negative and alkaline), which is composed of interconvertible electrical and chemical gradients. We show that both the electrical potential across the membrane and the chemical difference in proton concentrations across the membrane are independent of temperature between 5 degrees and 25 degrees . We show that the number of binding sites induced by D-lactate oxidation depends on pH. At both 25 degrees and 5 degrees , the number of binding sites increases from pH 5 to pH 6.5, remains constant between pH 6.5 and 7, and decreases from pH 7 to pH 8. In contrast, the number of binding sites induced by passive, carrier-mediated lactose efflux is independent of pH between pH 5.5 and pH 8. From these findings, we conclude that the pH- and temperature-dependent effects on the number of 6'-(N-dansyl)aminohexyl-1-beta-thio-D-galactopyranoside binding sites have different origins. The pH dependence of binding is energy linked and reflects in part the pH dependence of the electrochemical gradient of protons across the membrane generated by substrate oxidation. The temperature dependence is not an energy-linked phenomenon. The decrease of the number of binding sites at low temperature probably reflects the aggregation of the lac carrier protein with other membrane proteins. This aggregation takes place as a consequence of the conformational disorder-to-order transition of the membrane lipids and the concomitant preferential segregation of the lac carrier protein in the membrane domains containing the disordered lipids.