Interaction of the mioflazine derivative R75231 with the nucleoside transporter: evidence for positive cooperativity

Abstract

This study investigated the interaction of the mioflazine derivative R75231 with the nucleoside transport system of rabbit cortical synaptosomes, and assessed the binding of [ 3H]R75231 to human erythrocyte ghost membranes. R75231 was a potent inhibitor of [ 3H]nitrobenzylthionosine binding and [ 3H]uridine uptkke in synaptosomes (K i < 10 nM). This inhibition was evident even after extensive washing of the synaptosomes, subsequent to exposure to R75231. In addition to its tight binding characteristics, R75231 was shown to be a ‘mixed’ type inhibitor of [ 3H]nitrobenzylthioinosine binding (increased K D, decreased B max). [ 3H]R75231 bound with high affinity (K D = 0.4 nM) to erythrocyte membranes with a B max of 44 pmol/mg protein, which is comparable to the number of [ 3H]nitrobenzylthioinosine binding sites in this preparation. Binding of [ 3H]R75231 to these membranes was reversible, but the rate of dissociation was dependent upon the displacer used. Nitrobenzylthioinosine and dipyridamole each induced a complete dissociation of site-bound [ 3H]R75231 at rates not significantly different from those observed using a protocol involving a 100-fold dilution with buffer (no displacer). However, R75231 and mioflazine slowed the rate of dissociation of [ 3H]R75231 and actually caused an initial increase in the amount of site-bound [ 3H]R75231. Adenosine, on the other hand, enhanced the rate of [ 3H]R75231 dissociation. These results indicate that R75231 binding to the nucleoside transporter is a complex reaction, which may involve multiple interacting sites demonstrating positive cooperativity.