Sodium-induced conformational changes in the glucose transporter of intestinal brush borders.
Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D007093:Imidazoles / Q000494:pharmacology; D007422:Intestines / Q000648:ultrastructure*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010695:Phlorhizin / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D013774:Tetranitromethane / Q000494:pharmacology; D013861:Thiocyanates / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D005452:Fluoresceins; D007093:Imidazoles; D009004:Monosaccharide Transport Proteins; D013861:Thiocyanates; D012964:Sodium; D010695:Phlorhizin; D016650:Fluorescein-5-isothiocyanate; D013774:Tetranitromethane; C031760:thiocyanate; C012160:N-acetylimidazole