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Abstract

Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phospatidylserine 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 (K_i = 0.3 μM), and 55-pmol phlorizin-binding sites per mg of protein (K_D = 0.5 μM) 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.