JBC, Vol. 252, Issue 23, 8341-8343, Dec, 1977
Purification and reconstitution of the adipocyte plasma membrane D-glucose transport system
M. F. Shanahan and M. P. Czech
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.
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