Accessibility of sulfhydryl residues induced by cytochalasin B binding and conformational dynamics in the human erythrocyte glucose transporter.
Studies with intact cells have implicated essential sulfhydryl groups in the carrier-mediated glucose transport of human erythrocytes. In an attempt to identify and characterize such essential sulfhydryl residues we have studied the interaction of p-chloromercuribenzoate (PCMB) with a purified glucose transporter preparation (band 4.5) from human erythrocytes, in the presence and absence of its ligands, and the effects of this interaction on the binding of cytochalasin B (CB) to the transporter. At least 3 mol of PCMB reacted per mol of this preparation. A portion of the reaction was significantly enhanced in the presence of cytochalasin B. This enhancement was a saturable function of CB concentration, and was half-maximal at a CB concentration equal to the dissociation constant for the CB binding to the preparation. This CB-sensitive, PCMB reaction product comigrated with the band 4.5 on lithium dodecyl sulfate-polyacrylamide gel electrophoresis. An excess of D-glucose did not affect the PCMB reaction by itself in the absence of CB, but totally abolished the CB-induced enhancement of the PCMB reaction. PCMB inhibited the CB binding activity of the transporter preparation, and this inhibition was also enhanced in the presence of CB. These results suggest that CB binding perturbs the conformational dynamics of the glucose transporter resulting in an exposure of at least two sulfhydryl residues to PCMB reaction, and that some of these CB-sensitive sulfhydryl groups are essential for CB binding to the transporter.
D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins / Q000097:blood*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003571:Cytochalasin B / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000097:blood*; D020245:p-Chloromercuribenzoic Acid
D002352:Carrier Proteins; D002729:Chloromercuribenzoates; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D003571:Cytochalasin B; D020245:p-Chloromercuribenzoic Acid