Partial purification of the D-glucose transport system in rat adipocyte plasma membranes.

Abstract

Partially purified rat adipocyte plasma membranes were subjected to selective protein extractions using sodium hydroxide, sodium iodide, and dimethylmaleic anhydride in an effort to identify membrane components associated with glucose transport.Up to 80% of the membrane proteins were extracted in these procedures with the remaining proteins consisting almost entirely of two glycoprotein fractions, 78,000 and 94,000 daltons, as determined by dodecyl sulfate polyacrylamide gel electrophoresis.Sonic disruption of these extracted membrane residues resulted in the formation of vesicular structures as revealed by electron microscopy.These vesicles demonstrated high affinity binding of [3H]cytochalasin B in all three preparations.Vesicles from the dimethylmaleic anhydride-extracted membranes also exhibited a marked stereospecific uptake of o-glucose compared to L-glucose, as measured by a rapid filtration method.This uptake was markedly inhibited by cytochalasin B and this inhibition closely paralleled the high affinity binding of [3H]cytochalasin B to these vesicles.In addition, uptake of o-[3H]glucose was inhibited by phoretin, phlorizin, and dipyridamole, all potent inhibitors of n-glucose transport in the intact adipocyte.Competitive inhibitors of glucose transport such as 3-O-methylglucose and unlabeled