Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.
The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.
Wadzinski
B E
BE
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Shanahan
M F
MF
Clark
R B
RB
Ruoho
A E
AE
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000031:analogs & derivatives*; D004224:Diterpenes; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008223:Lymphoma / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D004224:Diterpenes; D007328:Insulin; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; C054433:3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin; D005576:Colforsin; D005947:Glucose