Kinetics of nitrobenzylthioinosine binding to the human erythrocyte nucleoside transporter.
The kinetics of [3H]nitrobenzylthioinosine binding to human erythrocyte membranes was studied. The pseudo-first-order association was linear and consistent with a simple bimolecular reaction mechanism between nitrobenzylthioinosine and the nucleoside-transport mechanism. Dissociation of the [3H]nitrobenzylthioinosine complex at 22 degrees C was also linear (apparent k-1 congruent to 0.20 min-1). Adenosine was a competitive inhibitor of equilibrium high-affinity [3H]nitrobenzylthioinosine-binding activity (apparent Ki 0.1 mM). Dissociation of the [3H]nitrobenzylthioinosine-membrane complex was faster in the presence of adenosine and uridine, and this effect was proportional to the nucleoside concentration. Nucleoside concentrations less than 1 mM had no significant effect on the dissociation rate constant. In contrast, dissociation was slower in the presence of high concentrations (micromolar) of dipyridamole. Low concentrations of dipyridamole (2-200 nM) and nitrobenzylthioinosine concentrations as high as 2.5 microM had no effect on the rate of [3H]nitrobenzylthioinosine dissociation. These results are discussed in terms of possible distinct inhibitor and permeation sites, and are suggested to be consistent with both a single-site model for the binding of nitrobenzylthioinosine and permeant to the same site, or an allosteric-site model in which permeant and inhibitor bind to different sites.
D000241:Adenosine / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood*; D004176:Dipyridamole / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007288:Inosine / Q000031:analogs & derivatives*; D007700:Kinetics; D008954:Models, Biological; D009705:Nucleosides / Q000097:blood*; D011485:Protein Binding; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014529:Uridine / Q000494:pharmacology
D002352:Carrier Proteins; D009705:Nucleosides; D013868:Thioinosine; D007288:Inosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine; D000241:Adenosine; D014529:Uridine