Abstract
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.
Highlights
(Iao10 p ~ )N.o protectionwas observedwith L-glucose iodo-azido derivatives of glucose (17, 1a8s)well as a phenyl (600 mM)
We report the synthesis of a derivative of forskolin which promises to be an extremely useful probe of the mammalian D-glUCOSe transporter
The apparIeWnftor the inhibition shows any photoincorporationof radiolabel. of 3-0-methylglucose transportby forskolin was found to be Enzymatic Digestion of ['25Z]IAPS-forskolin-labeled Memapproximately 20 times greater than thaotbserved for IAPS- branes-It has beenpreviously reported that treatmenotf the forskolin
Summary
The product could be visualized lin in ethanol (1pl, final concentration 1.0-2.0nM)was added and as a UV-positive spot (RF0.4) on thin layer chromatography using the membranes incubated for an additional 30 min on ice in the dark. The membranes were centrifuged a t 30,000 X g for purified by preparative thick layer (1mm) chromatography on silica 30 min a t 4 "C, and the pellets were resuspended in 125 pl of the gel plates developed with to1uene:ethyl acetate (3:2). VI-The the purified transporter preparation (20 pg, 0.13 mg/ml) was quickly ['2SI]IAPA diluted with 5 ml of ice-cold 5P8 buffer containing the same concenwas dried under nitrogen, and 50 plof the N-hydroxysuccinimidy1-7- tration of D-gluCOse, L-glucose,cytochalasin B, and forskolin used in.
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