Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake

• Published in: The Journal of biological chemistry 1977-03, Vol.252 (6), p.1990-1997
• Main Author: Lever, J E
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Biological Transport, Active; Cations, Monovalent; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Transformation, Neoplastic; Endoplasmic Reticulum - metabolism; Fibroblasts - metabolism; Gramicidin - pharmacology; Hydrogen-Ion Concentration; Kinetics; Ouabain - pharmacology; Potassium - pharmacology; Simian virus 40; Sodium - pharmacology; Temperature; Valinomycin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)71854-1

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.