Yao S Y, Ng A M, Muzyka W R, Griffiths M, Cass C E, Baldwin S A, Young J D
Membrane Transport Research Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1997 Nov 7;272(45):28423-30. doi: 10.1074/jbc.272.45.28423.
Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.
哺乳动物细胞中的平衡核苷转运过程要么对硝基苄硫基肌苷（NBMPR）敏感（es），要么对NBMPR不敏感（ei）。此前，我们从人胎盘中分离出一种编码456个残基糖蛋白hENT1的cDNA。当在非洲爪蟾卵母细胞中表达时，hENT1介导es型转运活性，并受到可能与核苷和NBMPR竞争结合底物结合位点的冠状血管活性药物（双嘧达莫和地拉齐普）的抑制。我们现在报告从大鼠组织中克隆hENT1的es和ei同源物并进行功能表达；rENT1（457个残基）在氨基酸序列上与hENT1有78%的同一性，rENT2（456个残基）与rENT1/hENT1有49 - 50%的同一性，对应于延迟早期增殖反应基因产物HNP36的全长形式，HNP36是一种功能未知的蛋白质，之前以截短形式被克隆。rENT1受到NBMPR的抑制（在10 microM尿苷存在时IC50 = 4.6 nM），而rENT2对NBMPR不敏感（IC50 > 1 microM）。两种蛋白质都介导可饱和的尿苷内流（Km分别为0.15和0.30 mM），对嘌呤和嘧啶核苷具有广泛的选择性，包括腺苷，并且对双嘧达莫和地拉齐普的抑制相对不敏感（IC50 > 1 microM）。这些观察结果表明，es和ei核苷转运活性由不同但同源的蛋白质介导，并确定了HNP36基因产物的功能。
