Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl.
Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated with Mal-ANS suggested that the inhibition of transport was due to a partial channel-blocking effect, and not to competition for the substrate binding site or to hindrance of carrier conformational change. In purified glucose carrier prepared from cells labeled on the exofacial sulfhydryl with Mal-ANS, a blue shift in the peak of fluorescence indicated that the fluorophore was in a relatively hydrophobic environment. Mal-ANS fluorescence in such preparations was quenched by ligands with affinity for the outward-facing carrier (ethylidene glucose, D-glucose, and maltose), but not by inhibitors considered to bind to the inward-facing carrier conformation (cytochalasin B or phenyl beta-D-glucoside). The effect of ethylidene glucose appeared to be related to an interaction with the glucose carrier, since the concentration dependence of ethylidene glucose-induced quench correlated well with the ability of the sugar analog to inhibit cytochalasin B binding to intact cells. The hydrophilic quenchers iodide and acrylamide decreased carrier-bound Mal-ANS fluorescence, resulting in downward-curving Stern-Volmer plots. Whereas ethylidene glucose enhanced iodide-induced quench, it had no effect on that of acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)
May
J M
JM
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2230.
Beechem
J M
JM
D019325:3-O-Methylglucose; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D001667:Binding, Competitive; D003571:Cytochalasin B / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D019325:3-O-Methylglucose; D003571:Cytochalasin B; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid