The glucose transport system of muscle plasma membranes: Characterization by means of [ 3H]cytochalasin B binding

Abstract

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092–1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific d-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [ 3H]CB binding activity ( K d of 0.28 μ m). [ 3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by d-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [ 3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [ 3H]CB binding. Higher concentrations of trypsin abolished [ 3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 m m) and N-ethylmaleimide (15 m m) inhibited [ 3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the d-glucose-sensitive [ 3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of M r about 45K of muscle membranes which might represent the glucose carrier.