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 ca tegorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or N BMPR-insensitive (ei). Inhibition of the es process arises from bindin g of NBMPR to a high-affinity site(s) on the es transporter that can b e identified by photoaffinity labeling with [H-3]NBMPR. This study exa mined the equilibrative nucleoside transport processes of cultured hum an 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 identificat ion of polypeptides by specific photolabeling of membranes with [H-3]N BMPR, indicated that K562 cells possess es nucleoside transporters (ca 500 000 copies/cell). The photolabeled polypeptides of K562 cells mig rated with lower relative mobility (peak M-r value, 63 000) than did t hose of human erythrocytes (peak M-r value, 53 000). This difference i n apparent M-r was abolished by prolonged treatment of membrane protei ns with N-glycosidase F, suggesting that equilibrative nucleoside tran sport in K562 cells and erythrocytes is mediated by the same, or a clo sely related, es isoform. A cDNA encoding the es nucleoside transporte r of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human eryth rocytes. hENT-like mRNA species were detected in K562 cells, as well a s in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysi s with a placental hENT1 probe. A cDNA that encoded a protein identica l to hENT1 was isolated by reverse transcriptase polymerase chain reac tion with primers specific for hENT1. NBMPR inhibited zero-trans influ x 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) tra nsport process, which accounted for 80-90% of total transport activity . The remaining component was identified as the equilibrative NBMPR-in sensitive (ei) transport process since it: (i) exhibited low (IC50 > 1 .0 mu M) sensitivity to NBMPR; (ii) was not concentrative; and (iii) w as unchanged by elimination of the sodium gradient. The kinetic parame ters (determined at 37 degrees C) for the es-and ei-mediated processes differed markedly. Values for transport of uridine by the es-and ei-m ediated processes were, respectively: K-m = 229 +/- 39 and 1077 +/- 22 0 mu M; V-max, 186 +/- 31 and 40 +/- 5 pmol/mu l cell water/sec. Value s 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 protec ted by NBMPR (10 mu M) from the cytotoxic effects of tubercidin (7-dea zaadenosine). (C) 1997 Elsevier Science Ltd.