Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: pharmacological characterization of allosteric interactions with the [3H]nitrobenzylthioinosine binding site.
Kinetic analysis of the binding of [3H]nitrobenzylthioinosine ([3H] NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of [3H] NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the [3H]NBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of [3H]NBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of [3H]NBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of [3H]NBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of [3H]NBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both [3H]NBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of [3H]NBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of [3H]NBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of [3H]NBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of [3H]NBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam. Comparable effects of adenosine and dipyridamole on [3H]NBMPR dissociation rate were obtained upon solubilization of the membranes with octylglucoside, suggesting that this phenomenon was not due to changes in membrane fluidity. These results are compatible with the existence of specific ligand recognition sites on the nucleoside transport complex of Ehrlich cells that are pharmacologically distinct from, but allosterically linked to, the high affinity binding sites for [3H]NBMPR. The marked effects on [3H]NBMPR binding kinetics that result from ligand interactions with these sites must be considered in the design and analysis of all studies involving the use of [3H]NBMPR as a high affinity probe for the nucleoside transport system.
Hammond
J R
JR
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
D000345:Affinity Labels / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism*; D002286:Carcinoma, Ehrlich Tumor; D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D033703:Nucleoside Transport Proteins; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001798:Blood Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D014316:Tritium; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine