Genetic Analysis of the 6-Thiobenzylpurine Binding Site of the Nucleoside Transporter in Mouse Lymphoblasts

From a mutagenized population of wild-type S49 T lymphoblasts, cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 μM methotrexate, 30 μM thymidine, 30 μM deoxycytidine, and 30 μM p-nitrobenzyl-6-thioinosine (NBMPR), a potent inhibitor of nucleoside transport. Unlike wild-type parental cells, two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild-type cells, KAB1, and KAB5 cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that nucleoside incorporation was much less sensitive to inhibition by NBMPR in the mutant cells. Rapid transport studies indicated that the mutant cell lines, unlike the wild-type parent, had acquired an NBMPR-insensitive nucleoside transport component which was similar to the NBMPR-sensitive wild-type transporter with respect to affinities for nucleosides and sensitivities toward N-ethylmaleimide and dipyridamole. Binding studies with [³H]NBMPR indicated that KAB5 cells were 70–75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild-type complement of NBMPR binding sites with wild-type binding characteristics. These data suggest that the NBMPR binding site in wild-type S49 cells is genetically distinguishable from the nucleoside carrier site and that the former may be a regulatory site.