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Initial Rate Kinetics and Evidence for Duality of Mediated Transport of Adenosine, Related Purine Nucleosides, and Nucleoside Analogues in L1210 Cells¹

Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [P. L. C., F. M. S., D. M. D., C-H. Y.], and Southern Research Institute, Birmingham, Alabama 35255 [J. A. M.]

In studies using a rapid kinetic technique, evidence was derived for multiplicity of systems mediating [³H]adenosine transport in L1210 cells. A variety of approaches were used in discriminating between transport and kinase-mediated phosphorylation. Under these conditions, two systems mediating influx were delineated which exhibited high-affinity [K_m = 13.9 ± 2 (S.E.) µM or low-affinity [K_m = 199 ± 27 (S.E.) µM for [³H]-adenosine. Both systems exhibited high capacities, but that associated with the low-affinity system (V = 263 ± 43 nmol/sec/g, dry weight) was 2- to 3-fold greater than that for the high-affinity system (V = 99.6 ± 12 nmol/sec/g dry weight). The relative difference in affinity of these two systems during influx was also reflected in the values for influx K_i obtained with other nucleosides and nucleoside analogues. Influx of [³H]-adenosine by each mediated system was inhibited by 6-(2-hydroxy-5-nitrobenzyl)thioguanosine, a specific transport inhibitor, and by 9-ß-D-arabinofuranosylpurine-6(1H)thione which is not phosphorylated in L1210 cells. Influx kinetics were the same in L1210 cells, in adenosine triphosphate-depleted L1210 cells, in cells (L1210/ara-C/MMPR) which have substantially reduced ability for [³H]adenosine phosphorylation, and in the presence of 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase. The same multiplicity in mediated influx of [³H]adenosine was shown at 0° when transport became rate limiting to total uptake. The high-affinity system mediating [³H]adenosine influx was also elucidated in L1210 cell plasma membrane vesicles in the presence or absence of 2'-deoxycoformycin. Almost all of the natural nucleosides examined competed less effectively with [³H]adenosine for influx by the high-affinity system than by the low-affinity system. These results are discussed with respect to possible pharmacological implications.

¹ Supported in part by Grants CA 08748, CA 24153, and CA 26318 from the National Cancer Institute, Department of Health and Human Services; Grant CH-26 from the American Cancer Society; and Elsa U. Pardee Foundation.

² To whom requests for reprints should be addressed, at Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, N. Y. 10021.

³ Special fellow of the Leukemia Society of America during the course of these studies.

Received 4/29/82. Accepted 10/ 5/82.