Reconstitution and partial purification of several Na+ cotransport systems from renal brush-border membranes. Properties of the L-glutamate transporter in proteoliposomes.
Brush-border membranes of renal proximal tubules were solubilized with deoxycholate and some proteins were separated and incorporated into proteoliposomes by a reconstitution procedure which was analyzed in detail. The proteoliposomes contained mainly polypeptides with molecular weights of 152,000, 94,000, and 52,000, each of which could be separated further into homologous polypeptides with different isoelectric points. In the proteoliposomes, Na+ cotransport systems for D-glucose, acidic and neutral amino acids, and mono- and dicarboxylic acids were demonstrated by showing that due to an inwardly directed Na+ gradient the substrate concentrations in the proteoliposomes increased significantly over their respective equilibrium values. Using inhibition experiments, selectivity of the different transporters could be demonstrated. Studying the reconstituted L-glutamate transporter in detail, countertransport of L-glutamate and K+ was shown (i) at Na+ equilibrium the intraliposomal L-glutamate concentration increased significantly over the equilibrium value if an outside-directed K+ gradient was applied; (ii) Rb+ influx was significantly stimulated by the outflux of L-glutamate. By applying a K+ diffusion potential across the liposomal membrane by addition of valinomycin it could be shown that during L-glutamate transport in the presence of Na+ and K+ positive charge is transferred together with L-glutamate and Na+. The apparent Km value of L-glutamate uptake driven by concentration differences of 89 mM Na+ (out greater than in) and 89 mM K+ (in greater than out) was 26.3 +/- 1.3 microM. The Vmax value of 70.2 +/- 2.3 pmol X mg of protein-1 X S-1 was half the value measured in intact membranes.
D027322:Amino Acid Transport System X-AG; D000596:Amino Acids / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002264:Carboxylic Acids / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates / Q000494:pharmacology; D007672:Kidney Cortex / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008854:Microscopy, Electron; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013552:Swine; D027981:Symporters
D027322:Amino Acid Transport System X-AG; D000596:Amino Acids; D002264:Carboxylic Acids; D002352:Carrier Proteins; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates; D008081:Liposomes; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D012964:Sodium; D011188:Potassium