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KMID : 0358619910250020115
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Korean Journal of Physiology
1991 Volume.25 No. 2 p.115 ~ p.123
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Molecular Association of Glucose Transporter in the Plasma Membrane of Rat Adipocyte
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Hah Jong-Sik
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Abstract
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Molecular association of glucose transporters with the other proteins in the plasma membrane was assessed by gel electrophoresis and immunoblot techniques. Approximately of GLUT-4, of clathrin, 48.7% of total protein in the plasma membrane (PM) were found insoluble upon extraction with 1% Tx-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the Tx-100 insoluble PM fraction contained about 4 major polypeptides with apparent molecular weight of above 200, 100-120, 80 and 30-35 KDa that were readily removed upon wash with a high pH buffer which is known to remove clathrin and 0.5 M Tris-buffer which is known to remove assembly proteins (AP). Immunoblotting of GLUT4 and clathrin against specific antibodies showed that GLUT-4 and clathrin were co-solubilized up to 84.6% and 82.7% respectively by wash with a high pH buffer and 1% Tx-100. When the membrane was pre-washed with a high pH buffer and 0.5 M Tris solution, GLUT4 and clathrin were not solubilized further suggesting that GLUT4 molecules are in molecular association with clathrin, AP and/or other extrinsic membrane proteins in plasma membrane and the formation of clathrin-coated structures might be involved in insulin stimulated glucose transporter translocation mechanism.
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KEYWORD
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Glucose transporter, Glucose transporter translocation, GLUT4, AP, Immunoblotting, Silver stain
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