Derivativation of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin (IAPS-Fsk), has been synthesized, purified, and characterized. The K/sub i/ for inhibition of 3-0-methylglucose transport by TAPS-Fsk in human erythrocytes was found to be 0.1 uM. The carrier-free radioiodinated label has been shown to be a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes with 1-10 nM [I-125]IAPS-Fsk and analysis by SDS-PAGE showed specific derivatization of a broad band with an apparent molecular weight of 40-70 kDa. Photoincorporation using 2 nM [I-125]IAPS-Fsk was protected with D-glucose, cytochalasin B, and forskolin. No protection was observed with L-glucose. Endo-B-galactosidase digestion and trypsinization of [I-125]IAPS-Fsk labelled erythrocytes reduced the specifically radiolabelled transporter to 40 kDa and 18 kDa respectively. [I-125]-IAPS-Fsk will be used to study the structural aspects of the glucose transporter