Mechanisms of Active Transport in Isolated Membrane Vesicles: II. THE MECHANISM OF ENERGY COUPLING BETWEEN d-LACTIC DEHYDROGENASE AND β-GALACTOSIDE TRANSPORT IN MEMBRANE PREPARATIONS FROM ESCHERICHIA COLI

Abstract

Abstract The results presented in this paper provide preliminary evidence for the concept that the of the d-lactic dehydrogenase-coupled transport systems in isolated membrane vesicles from Escherichia coli may be electron transfer intermediates. Initial rates of lactose transport and d-lactic dehydrogenase activity respond identically to temperature and both processes have the same activation energy of 8400 cal per mole. The steady state levels of lactose accumulation at a variety of temperatures represent equilibrium states in which there is a balance between influx and efflux. This balance can be easily influenced by raising or lowering the temperature. Temperature-induced efflux is a saturable process with an apparent affinity constant that is approximately 60 times higher than the affinity constant for influx determined under the same experimental conditions. The apparent maximum velocity of temperature-induced efflux, on the other hand, is the same as that of influx. Potassium cyanide also induces a saturable efflux phenomenon which has an apparent Km that is much higher than that of the influx process. p-Chloromercuribenzoate inhibits d-lactic dehydrogenase-coupled transport of lactose, galactose, arabinose, glucuronate, glucose-6-P, proline, glutamic acid, serine, alanine, tyrosine, lysine, and tryptophan, and inhibition of each system by p-chloromercuribenzoate is reversed by dithiothreitol. Furthermore, p-chloromercuribenzoate inhibits temperature-induced efflux of intramembranal lactose, exchange of external lactose with [14C]lactose in the intramembranal pool, and lactose efflux induced by 2,4-dinitrophenol. Inhibition of these experimental parameters and of d-lactic dehydrogenase by p-chloromercuribenzoate is reversed by dithiothreitol. Reduction of the respiratory chain between d-lactic dehydrogenase and cytochrome b1 is responsible for carrier-mediated efflux of lactose. Anaerobiosis, cyanide, and 2-heptyl-4-hydroxyquinoline-N-oxide, each of which inhibits electron transfer after cytochrome b1, cause marked efflux. Amytal causes slow efflux, and oxamate and p-chloromercuribenzoate do not cause efflux despite marked inhibition of d-lactic dehydrogenase and the initial rate of lactose transport. Addition of amino acids which are also transported by d-lactic dehydrogenase-coupled transport mechanisms results in little or no inhibition of lactose transport. These findings are discussed in terms of a conceptual working model in which the carriers are depicted as electron transfer intermediates between d-lactic dehydrogenase and cytochrome b1.