Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells - ENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine
Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells - ENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine
复制标题
DOI：
10.1074/jbc.275.12.8375
复制
发表时间：
2000-03-24
影响因子：
4.8
通讯作者：
Tse, CM
中科院分区：
文献类型：
--
作者：
Ward, JL;Sherali, A;Tse, CM
我们将克隆的人平衡核苷转运蛋白 1 和 2（hENT1 和 hENT2）稳定转染到核苷转运蛋白缺陷的 PK15NTD 细胞中。尽管 hENT1 和 hENT2 预计为 50 kDa 蛋白质，但在 SDS 聚丙烯酰胺凝胶电泳上，hENT1 运行为 40 kDa，hENT2 迁移为 50 和 47 kDa。肽 N-糖苷酶 F 和糖苷内切酶 H 将 hENT1 去糖基至 37 kDa，将 hENT2 去糖基至 45 kDa，hENT1 更敏感，对硝基苄基硫代肌苷 (NBMPR) 的敏感性分别有 7000 倍和 71 倍的差异（IC50，0.4 +/- 0.1 nM 与 2.8 +/- 0.3 mu M）双嘧达莫（IC50，5.0 +/- 0.9 nM 对比 356 +/- 13 nM）。 [H-3]NBMPR 以 0.377 +/- 0.098 nM 的高亲和力 K-d 与 ENT1 细胞结合，每个 ENT1 细胞具有 34,000 个转运蛋白，尿苷周转数为 46 分子/秒。虽然两种转运蛋白都具有广泛的选择性，但 hENT2 通常是一种低亲和力核苷转运蛋白，具有 2.6-、2.8-、7.7- 和对胸苷、腺苷、胞苷和鸟苷的亲和力分别比 hENT1 低 19.3 倍。相比之下，hENT2对肌苷的亲和力比hENT1高4倍。核碱基次黄嘌呤抑制hENT2对[H-3]尿苷的摄取，但对hENT1的影响很小。综上所述，这些结果表明hENT2可能在ENT2主要表达的骨骼肌等组织中转运腺苷及其代谢物（肌苷和次黄嘌呤）方面发挥重要作用。
We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa, With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC50, 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 mu M) and dipyridamole (IC50, 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [H-3]NBMPR binds to ENT1 cells with a high affinity K-d of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine, Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7.7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1, The nucleobase hypoxanthine inhibits [H-3]uridine uptake by hENT2 but has minimal effect on hENT1, Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.