ISOLATION BY PREPARATIVE FREE-FLOW ELECTROPHORESIS AND AQUEOUS 2-PHASE PARTITION FROM RAT ADIPOCYTES OF AN INSULIN-RESPONSIVE SMALL VESICLEFRACTION WITH GLUCOSE-TRANSPORT, ACTIVITY

Preparative free-flow electrophoresis and aqueous two-phase polymer pa rtition were used to obtain a plasma membrane-enriched fraction of adi pocytes isolated from epididymal fat pads of the rat together with a f raction enriched in small vesicles with plasma membrane characteristic s (thick membranes, clear dark-light-dark pattern). The electrophoreti c mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged mo lecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fa t cells treated or not treated with 100 mu U/ml insulin and the reside nt proteins of the two fractions analyzed by SDS-PAGE, the two fractio ns exhibited characteristic responses involving specific protein bands , Insulin treatment for 2 min resulted in the loss of a 90 kDa band fr om the plasma membrane. At the same time, a ca. 55-kDa peptide band th at was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 gluc ose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation b y treatment of adipocytes with insulin.