Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Abstract

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstruction into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membrane did not mediate the transport inhibition. Reaction of [3H]N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethylmaleimide to cholate-solubilized membrane protein had no effect on transport activity in artificial phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner. consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.