title: Selective labeling of the erythrocyte hexose carrier with a maleimide derivative of glucosamine: relationship of an exofacial sulfhydryl to carrier conformation and structure.
abstract: Sulfhydryl-reactive derivatives of glucosamine were synthesized as potentially transportable affinity labels of the human erythrocyte hexose carrier. N-Maleoylglycyl derivatives of either 6- or 2-amino-2-deoxy-D-glucopyranose were the most potent inhibitors of 3-O-methylglucose uptake, with concentrations of half-maximal irreversible inhibition of about 1 mM. Surprisingly, these derivatives were very poorly transported into erythrocytes. They reacted rather with an exofacial sulfhydryl on the carrier following a reversible binding step, the latter possibly to the exofacial substrate binding site. However, their reactivity was determined primarily by access to the exofacial sulfhydryl, which, as predicted by the one-site model of transport, required a carrier conformation with the exofacial substrate binding site exposed. Once reacted, the carrier was "locked" in a conformation unable to reorient inwardly and bind cytochalasin B. In intact erythrocytes the N-maleoylglycyl derivative of 2-[3H]glucosamine labeled predominantly an Mr 45,000-66,000 protein on gel electrophoresis in a quantitative and cytochalasin B inhibitable fashion. By use of changes in carrier conformation induced by competitive transport inhibitors in a "double" differential labeling method, virtually complete selectivity of labeling of the carrier protein was achieved, the latter permitting localization of the reactive exofacial sulfhydryl to an Mr 18,000-20,000 tryptic fragment of the carrier.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2230.
authors.forename: J M
authors.initials: JM
authors.lastname: May
mesh_terms: D019325:3-O-Methylglucose; D001667:Binding, Competitive; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008757:Methylglucosides / Q000097:blood; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D010446:Peptide Fragments / Q000032:analysis; D010693:Phloretin / Q000494:pharmacology; D011487:Protein Conformation
chemical_list: D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; C059704:N-(N-maleoylglycyl)-6-amino-6-deoxyglucopyranose; C059706:N-(N-maleoylglycyl)-2-amino-2-deoxyglucopyranose; D019325:3-O-Methylglucose; D008320:Maltose; D004228:Dithionitrobenzoic Acid; D005944:Glucosamine; D010693:Phloretin; D005998:Glycine