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Titolo:
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
Autore:
BOLETI H; COE IR; BALDWIN SA; YOUNG JD; CASS CE;
Indirizzi:
UNIV ALBERTA,DEPT BIOCHEM EDMONTON AB T6G 2H7 CANADA UNIV ALBERTA,DEPT BIOCHEM EDMONTON AB T6G 2H7 CANADA UNIV ALBERTA,MOL BIOL MEMBRANE PROT GRP EDMONTON AB T6G 2H7 CANADA UNIV ALBERTA,MEMBRANE TRANSPORT GRP EDMONTON AB T6G 2H7 CANADA UNIV ALBERTA,DEPT ONCOL,DIV EXPT ONCOL EDMONTON AB T6G 2H7 CANADA UNIV LEEDS,DEPT BIOCHEM & MOL BIOL LEEDS LS2 9JT W YORKSHIRE ENGLAND UNIV ALBERTA,DEPT PHYSIOL EDMONTON AB T6G 2H7 CANADA
Titolo Testata:
Neuropharmacology
fascicolo: 9, volume: 36, anno: 1997,
pagine: 1167 - 1179
SICI:
0028-3908(1997)36:9<1167:MOTEN(>2.0.ZU;2-K
Fonte:
ISI
Lingua:
ENG
Soggetto:
ASCITES TUMOR-CELLS; LEUKEMIA-CELLS; ERYTHROCYTE NUCLEOSIDE; NITROBENZYLTHIOINOSINE BINDING; ADENOSINE TRANSPORT; RAT; KINETICS; PROTEINS; INHIBITION; DIFFERENTIATION;
Keywords:
NUCLEOSIDE TRANSPORT; NITROBENZYLTHIOINOSINE; ADENOSINE; URIDINE; K562 ERYTHROLEUKEMIA; TRANSPORT KINETICS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
39
Recensione:
Indirizzi per estratti:
Citazione:
H. Boleti et al., "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", Neuropharmacology, 36(9), 1997, pp. 1167-1179

Abstract

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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/01/21 alle ore 15:53:33