Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: VIII. THE TRANSPORT OF AMINO ACIDS BY MEMBRANES PREPARED FROM ESCHERICHIA COLI

Abstract

Abstract The studies presented in this paper demonstrate that the transport of 16 amino acids by membrane vesicles prepared from Escherichia coli ML 308–225 exhibits properties similar to those found previously for respiration-linked β-galactoside transport. Concentrative uptake of all 16 amino acids is stimulated maximally by d-lactate and by the artificial electron donor system ascorbate-phenazine methosulfate, and to a lesser extent by succinate, l-lactate, dl-α-hydroxybutyrate, and NADH. Competitive uptake and displacement studies indicate that the vesicles possess at least nine distinct amino acid uptake systems with the following substrate specificities: proline, lysine, glycine-alanine, serine-threonine, glutamic acid-aspartic acid, phenylalanine-tyrosine-tryptophan, histidine, leucine-isoleucine-valine, cysteine. In the presence of ascorbate-phenazine methosulfate, the initial rates of lysine, alanine, serine, glutamic acid, tyrosine, leucine, cysteine, and lactose transport by membrane vesicles are 42 to 102% of those exhibited by appropriately treated whole cells. Initial rates of d-lactate-dependent transport as a function of external amino acid concentration yield simple hyperbolic kinetics for all but four amino acids: histidine, leucine, isoleucine, and valine yield biphasic velocity curves. Apparent Km values determined for the amino acids are in general agreement with results reported for intact cells. Steady state levels of d-lactate-dependent amino acid accumulation vary with temperature. These levels represent a balance between rates of influx and efflux, which can be shifted readily from one steady state level to another by raising or lowering the temperature. Different amino acid transport systems exhibit different steady state temperature optima, ranging from 20°–35°. In contrast, temperature optima for the initial rate of uptake occur at the same temperature (45°) with nearly all of the transport systems, and are similar to the temperature optimum for d-lactic dehydrogenase. There is no relationship between rates of oxidation of electron donors by the membranes (NADH g d-lactate g succinate g dl-α-hydroxybutyrate g l-lactate) and the ability of these compounds to stimulate amino acid uptake. These results indicate that the site of energy coupling between respiration and amino acid transport lies between the primary dehydrogenase and cytochrome b1. Furthermore, the relative effectiveness of these electron donors in supporting uptake varies widely among the various transport systems. The d-lactate-coupled concentrative uptake of proline, lysine, alanine, threonine, aspartic acid, tyrosine, histidine, leucine, and cysteine is inhibited by anaerobiosis, by the electron transfer inhibitors cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, amytal, and oxamate, and by p-chloromercuribenzoate; inhibition by the latter compound is reversed by dithiothreitol. Furthermore, anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, and amytal cause rapid efflux of the same amino acids from the intravesicular pool. Oxamate and p-chloromercuribenzoate, however, produce little or no efflux of accumulated amino acids. Moreover, p-chloromercuribenzoate blocks tyrosine exchange and cyanide-induced tyrosine efflux; in each case inhibition is reversed by dithiothreitol. Finally, cyanide-induced efflux of proline exhibits a Km for intramembranal proline which is 350 times higher than the Km determined for d-lactate-dependent uptake of external proline, whereas the Vmax values are the same for both processes. The findings presented are consistent with the conceptual model suggested for d-lactic dehydrogenase-coupled β-galactoside transport.