MECHANISM OF LACTOSE TRANSLOCATION IN MEMBRANE-VESICLES FROM ESCHERICHIA-COLI .2. EFFECT OF IMPOSED DELTA-PSI, DELTA-PH, AND DELTA-MU-H+

Imposition of a membrane potential (ΔΨ, interior negative) or a pH gradient (ApH, interior alkaline) across the membrane of Escherichia coli ML 308-225 vesicles leads to a marked, transient increase in the fluorescence of 6'-(N-dansyl)aminohexyl l-thio-β-D-galactopyranoside. The maximum increase in fluorescence appears to be a linear function of the magnitude of the imposed ΔΨ or ApH, and the effect of each parameter is additive. Imposition of ΔΨ or ApH also alters the rate of carrier-mediated lactose efflux from the intravesicular pool, and the effects are dependent upon the polarity of the imposed ΔΨ or ApH. The rate of efflux is diminished with ΔΨ (interior negative) or ApH (interior alkaline) and enhanced with ΔΨ (interior positive) or ApH (interior acid). These effects are also additive, and importantly kinetic experiments demonstrate that ΔΨ and ApH alter the maximum velocity of efflux without a significant effect on the apparent Km of the process. Strikingly, moreover, imposition of ΔΨ, ApH, or AμH+ of either polarity has no effect whatsoever on the rate of exchange. The data provide support for the suggestion [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry (preceding paper in this issue)] that the rate-limiting step for carrier-mediated lactose efflux down a concentration gradient involves a step that is associated with the return of the carrier to the inner surface of the membrane. In addition, the results are consistent with the notion that the loaded carrier (i.e., the ternary complex between the carrier, protons, and lactose) is neutral while the unloaded carrier is negatively charged. Finally, comparative studies of the effects of ΔΨ and ApH on influx and efflux demonstrate that the translocation reactions catalyzed by the lac carrier are ki-netically asymmetrical. © 1979, American Chemical Society. All rights reserved.