Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label.
An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
B E
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Wadzinski
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Shanahan
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Ruoho
D019325:3-O-Methylglucose; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D005576:Colforsin / Q000031:analogs & derivatives* / Q000378:metabolism; D004224:Diterpenes; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D008757:Methylglucosides / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000032:analysis*; D010782:Photolysis; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; C054433:3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin; D019325:3-O-Methylglucose; D005576:Colforsin; D014357:Trypsin