Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography.
Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of D-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards D-glucose compared to other sugars tested was shown as well as the main features of D-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of D-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the D-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 A; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65,000. We conclude that this protein fraction is involved in D-glucose transport by renal brush borders.
J C
JC
Poirée
M
M
Starita-Geribaldi
P
P
Sudaka
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002458:Cell Fractionation / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010539:Permeability; D010695:Phlorhizin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D013552:Swine
D008081:Liposomes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose