title: Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.
abstract: The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.
authors.forename: T
authors.initials: T
authors.lastname: Kurokawa
authors.forename: L G
authors.initials: LG
authors.lastname: Tillotson
authors.forename: C C
authors.initials: CC
authors.lastname: Chen
authors.forename: K J
authors.initials: KJ
authors.lastname: Isselbacher
mesh_terms: D000345:Affinity Labels; D003571:Cytochalasin B; D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification*; D010777:Photochemistry; D012995:Solubility
chemical_list: D000345:Affinity Labels; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B