A simple liposomal system to reconstitute and assay highly efficient Na+/D-glucose cotransport from kidney brush-border membranes.
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.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis; D005947:Glucose / Q000378:metabolism; D007668:Kidney / Q000648:ultrastructure*; D008081:Liposomes / Q000032:analysis / Q000378:metabolism; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin / Q000494:pharmacology; D013552:Swine
D002352:Carrier Proteins; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D002784:Cholesterol; D010695:Phlorhizin; D005947:Glucose