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.
D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000494:pharmacology; D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003571:Cytochalasin B / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D004176:Dipyridamole / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000378:metabolism; D050356:Lipid Metabolism; D008274:Magnesium / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D014316:Tritium
D014316:Tritium; D003571:Cytochalasin B; D013395:Sucrose; D004176:Dipyridamole; D015077:2-Hydroxy-5-nitrobenzyl Bromide; D003847:Deoxyglucose; D010695:Phlorhizin; D004139:Dinitrofluorobenzene; D008274:Magnesium; D005947:Glucose; D002118:Calcium