Purification and reconstitution of the adipocyte plasma membrane D-glucose transport system.

Rat adipocyte plasma membranes have previously been shown to retain stereospecific transport activity for D-glucose following extraction of extrinsic proteins with dimethylmaleic anhydride (Shanahan, M. F., and Czech, M. P. (1977) J. Biol. Chem. 252, 6554-6561). When these extracted plasma membranes were incubated in 2% sodium cholate and centrifuged, the resultant supernatant contained only one major glycoprotein fraction of 94,000 daltons, as determined by dodecyl sulfate-polyacrylamide gel electrophoresis. This protein fraction was combined with cholate-dispersed, exogenous phospholipids. The detergent was removed by gel filtration and vesicles composed of phospholipid and membrane protein were formed which exhibited preferential, time-dependent uptake of D- versus L-glucose when measured by a rapid filtration method. In addition, D-glucose uptake was inhibited by cytochalasin B, phlorizin, phloretin, and dipyridamole. These results suggest the direct involvement of the 94,000-dalton glycoprotein fraction in fat cell hexose transport.