Burchmore Richard J S, Wallace Lynsey J M, Candlish Denise, Al-Salabi Mohammed I, Beal Paul R, Barrett Michael P, Baldwin Stephen A, de Koning Harry P
Institute of Biomedical and Life Sciences, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
J Biol Chem. 2003 Jun 27;278(26):23502-7. doi: 10.1074/jbc.M301252200. Epub 2003 Apr 21.
While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.
近年来，虽然已从许多不同生物体中克隆出多种核苷转运体，其中一些还能转运核碱基，但后生动物、侧生动物或原生动物的高亲和力、核碱基选择性转运体的序列信息尚无报道。我们从布氏布氏锥虫中鉴定出一个基因TbNBT1，它编码一种属于平衡核苷转运体超家族的435个氨基酸残基的蛋白质。该基因在该生物体的前循环型和血流型中均有表达。在缺乏内源性嘌呤转运体的酿酒酵母菌株中表达TbNBT1，可使酵母利用腺嘌呤作为唯一嘌呤源生长，并赋予酵母对腺嘌呤和次黄嘌呤的高亲和力转运活性，其Km值分别为2.1±0.6和0.66±0.22微摩尔，对黄嘌呤、鸟嘌呤、鸟苷和别嘌呤醇也有高亲和力，对肌苷有中等亲和力。在布氏布氏锥虫前循环型中鉴定出一种动力学特征无法区分的转运体，并将其命名为H4。在前循环型中对TbNBT1进行RNA干扰可使相关mRNA水平降低约80%，H4转运活性降低约90%。在非洲爪蟾卵母细胞中表达TbNBT1进一步证实，该基因编码首个在分子水平上鉴定出的来自原生动物或动物的高亲和力核碱基转运体。
