Interaction of the mioflazine derivative R75231 with the nucleoside transporter: evidence for positive cooperativity.
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]nitrobenzylthioinosine binding and [3H]uridine uptake in synaptosomes (Ki < 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 KD, decreased Bmax). [3H]R75231 bound with high affinity (KD = 0.4 nM) to erythrocyte membranes with a Bmax 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.
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
K W
KW
Jones
J R
JR
Hammond
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D010879:Piperazines / Q000494:pharmacology*; D011817:Rabbits; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D010879:Piperazines; C072040:draflazine; D013868:Thioinosine; C041518:mioflazine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine; D014529:Uridine