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Abstract

A simple procedure to reconstitute highly efficient Na⁺/D-glucose cotransport from solubilized brush-border membranes of proximal kidney tubules is described. Reconstitution of transport activity was possible with various phospholipid and cholesterol combinations; the presence, however, of cholesterol and at least one phospholipid was essential. When liposomes were synthesized from only one phospholipid and cholesterol, the highest uptake rats were observed with phosphatidylserine; phosphatidylcholine was less effective and phosphatidylethanolamine showed insignificant uptake of D-glucose in the presence of Na⁺. The rate at which an inward-directed Na⁺ gradient dissipated across the liposomal membranes was reduced if the cholesterol concentration of liposomes was increased. In the optimized system, proteoliposomes were formed from cholesterol and phosphatidylserine by a heat-sonication-freeze-thaw procedure. A Na⁺-gradient persisted for hours across these proteoliposomal membranes and a Na⁺/D-glucose cotransport with the following characteristics could be demonstrated: (1) dependency on the Na⁺ gradient; (2) a transient (3) rheogenicity; (4) stereospecificity; and (5) high-affinity phlorizin inhibition. Since the Na⁺-gradient-stimulated D-glucose uptake is linear for minutes, the initial uptake rates can be measured and the Na⁺/D-glucose cotransport activity of different protein fractions can be compared.