Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents
✍ Scribed by Czech, Michael P. ;Pillion, Dennis J. ;Shanahan, Michael F.
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1978
- Tongue
- English
- Weight
- 565 KB
- Volume
- 9
- Category
- Article
- ISSN
- 0091-7419
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✦ Synopsis
Sensitivity of the adipocyte D-glucose transport system in intact plasma menibranes or following solubilization and reconstitution into phospholipid vesicles t o several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleiniide (20 niM) o r 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 membranes did not mediate the transport inhibition. Reaction of [ HI N-ethylinaleirtiide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethylmaleimide t o cholate-solubilized membrane protein had no effect o n transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylinaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol o r glutathione ( 1 5 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport systeni differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal o r amino groups by the reagents studied.