Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure

Abstract

Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 107 cells (10 mg protein) yielded about 12 μg of vesicular protein and 11 μg of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypetide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270000, 245000, 165000 and 115000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245000, 190000, 115000 and 25000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.