Identification of the monkey lens glucose transporter by photoaffinity labelling with cytochalasin B.
Polypeptide constituents of the lens glucose transporter have been identified by photoaffinity labelling with cytochalasin B. The urea-insoluble fraction of monkey lens was irradiated at 280 nm for 30 min in the presence of 5 X 10(-7) M 3H-cytochalasin B. After extensive washing, the membranes were solubilized and their polypeptide composition determined by SDS-PAGE. Radioactivity was extracted from gel slices to determine the position of photoincorporated label. 3H-cytochalasin B was irreversibly incorporated into a broad molecular weight region from Mr greater than 94,000 to 43,000 with the peak of activity occurring at Mr 53,000. Photoincorporation was inhibited by D-glucose (500 mM) and phloretin (1 X 10(-5] but was unaffected by L-glucose (500 mM), cytochalasin E (1 X 10(-5) and phloridzin (1 X 10(-5) M). Cortex and nucleus membrane preparations contained the same range of labelled polypeptides after photoaffinity labelling but nuclear membranes contained approximately twice the activity of cortical membranes indicating an enrichment of glucose transporters in the nucleus. Treatment of labelled membranes with endoglycosidase F converted the broad band of labelling to a sharp band of Mr 45,000. The lens glucose transporter is therefore a glycoprotein and the broadness of the photaffinity labelled peak is due to heterogeneous N-linked glycosylation of a core polypeptide. From these studies it appears that the monkey lens glucose transporter closely resembles that of the human erythrocyte.
D000345:Affinity Labels; D000818:Animals; D003571:Cytochalasin B; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D007904:Lens Cortex, Crystalline / Q000378:metabolism; D007907:Lens Nucleus, Crystalline / Q000378:metabolism; D007908:Lens, Crystalline / Q000032:analysis*; D008027:Light; D008253:Macaca mulatta; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009004:Monosaccharide Transport Proteins / Q000032:analysis*
D000345:Affinity Labels; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase