Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label. |
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| Authors: | B E Wadzinski M F Shanahan R B Clark and A E Ruoho |
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| Affiliation: | Department of Pharmacology, University of Wisconsin Medical School, Madison 53706. |
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| Abstract: | 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. |
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