Characterization and solubilization of the cytochalasin B binding component from human placental microsomes.
Human placental microsomes exhibit uptake of D-[3H]glucose which is sensitive to inhibition by cytochalasin B (apparent Ki = 0.78 microM). Characterization of [3H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by D-glucose with an ED50 = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a Kd = 0.15 microM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60-70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [3H]cytochalasin B to the soluble protein displays a pattern of inhibition by D-glucose similar to that observed for intact membranes, and the measurement of an ED50 = 37.5 mM D-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [3H]cytochalasin B incorporation into soluble protein (Mr range 42000-68000) is prevented by the presence of D-glucose. An identical photolabelling pattern is observed for incorporation of [3H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.
D000345:Affinity Labels / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008861:Microsomes / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D010777:Photochemistry; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding
D000345:Affinity Labels; D002352:Carrier Proteins; D003572:Cytochalasins; D009004:Monosaccharide Transport Proteins; C007215:cytochalasin E; D003571:Cytochalasin B; D005947:Glucose