Characterization of (3H)cytochalasin B binding to the fat cell plasma membrane.

Transport of D-[3H]deoxyglucose and D-[3H]glucose by purified fat cell plasma membranes was markedly inhibited by cytochalasin B,dipyridamole, or phlorizin. The rate of L-[3H]glucose uptake was significantly slower and was unaffected by cytochalasin B. Both the rates of association of [3H]cytochalasin B with the fat cell plasma membrane and its dissociation from the membrane was significantly enhanced by the presence of Ca2+ or Mg2+. Scatchard plot analysis of [3H]cytochalasin B binding to fat cell membranes indicated the presence of high affinity and low affinity sites which exhibited apparent dissociation constants of about 6 X 10(-7) M and 5 X 10(-6) M, respectively. The concentration of cytochalasin B which half-maximally inhibited D-glucose transport was about 5 X 10(-7) M, indicating the high affinity sites are involved in transport inhibition. D-Glucose at concentrations as high as 100 mM had no effect on the binding of [3H]cytochalasin B to these high affinity sites, while the transport inhibitors phlorizin, phloretin, and dipyridamole markedly inhibited this binding. The protein reagents N-ethylmaleimide, 2-hydroxy-5-nitrobenzylbromide, 1-fluoro-2,4-dinitrobenzene, dithiothreitol, and trypsin also reduced binding. Extracted fat cell plasma membrane lipids dispersed by sonication did not bind [3H]cytochalasin B nor take up glucose by a cytochalasin B-sensitive pathway. The results indicate that cytochalasin B inhibits hexose transport in fat cells secondary to its interaction with membrane protein sites distinct from those which bind D-glucose.