[3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter.
Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.
D000345:Affinity Labels / Q000378:metabolism*; D005576:Colforsin / Q000378:metabolism*; D003571:Cytochalasin B / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006026:Glycoside Hydrolases; D006801:Humans; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010777:Photochemistry; D014316:Tritium; D001616:beta-Galactosidase / Q000378:metabolism
D000345:Affinity Labels; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D014316:Tritium; D005576:Colforsin; D003571:Cytochalasin B; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D005947:Glucose