Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.
Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.
D000596:Amino Acids / Q000378:metabolism*; D001693:Biological Transport, Active; D002414:Cations, Monovalent; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006096:Gramicidin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D013539:Simian virus 40; D012964:Sodium / Q000494:pharmacology; D013696:Temperature; D014634:Valinomycin / Q000494:pharmacology
D000596:Amino Acids; D002414:Cations, Monovalent; D006096:Gramicidin; D014634:Valinomycin; D010042:Ouabain; D012964:Sodium; D011188:Potassium