Molecular identification of the equilibrative NBMPR-sensitive (es) nucleoside transporter and demonstration of an equilibrative NBMPR-insensitive (ei) transport activity in human erythroleukemia (K562) cells

Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [H-3]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 +/- 0.9 x 10(5)/cell, K-d = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [H-3]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500 000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak M-r value, 63 000) than did those of human erythrocytes (peak M-r value, 53 000). This difference in apparent M-r was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like mRNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a protein identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of H-3-labeled adenosine, uridine and thymidine by 50% (IC50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which accounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (IC50 > 1.0 mu M) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37 degrees C) for the es-and ei-mediated processes differed markedly. Values for transport of uridine by the es-and ei-mediated processes were, respectively: K-m = 229 +/- 39 and 1077 +/- 220 mu M; V-max, 186 +/- 31 and 40 +/- 5 pmol/mu l cell water/sec. Values for transport of adenosine by the es and ei-mediated processes were, respectively, 61 +/- 9 and 133 +/- 17 mu M; V-max; 70 +/- 5 and 23 +/- 8 pmol/mu l cell water/sec. The ei-mediated process, although small, was of pharmacologic: importance since K562 cells could not be protected by NBMPR (10 mu M) from the cytotoxic effects of tubercidin (7-deazaadenosine). (C) 1997 Elsevier Science Ltd.