Partial purification and reconstitution of the Na+-D-glucose cotransport protein from pig renal proximal tubules.
Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phosphatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 microM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 microM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed.
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008871:Microvilli / Q000378:metabolism; D010695:Phlorhizin / Q000378:metabolism; D011510:Proteolipids; D012964:Sodium / Q000378:metabolism; D013552:Swine
D002352:Carrier Proteins; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose