Transport of adenosine by recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in Xenopus laevis oocytes.
Transport of adenosine by recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in Xenopus laevis oocytes.
复制标题
通过来自非洲爪蟾卵母细胞表达的大鼠空肠的重组嘌呤和嘧啶选择性钠/核苷协同转运蛋白转运腺苷。
作者：
S. Yao;A. Ng;M. Ritzel;W. P. Gati;C. Cass;J. Young
两个主要的Na+依赖性核苷转运蛋白亚型涉及在哺乳动物细胞中的腺苷转运功能上区分的基础上的底物特异性：一个是选择性的嘧啶核苷，但也结合腺苷，和其他嘌呤核苷的选择性，但也结合尿苷。通过测量[3 H]腺苷通量，已经建立了嘌呤选择性系统的腺苷转运能力，而腺苷渗透嘧啶选择性系统的结论是基于抑制试验。我们研究了腺苷转运介导的重组嘧啶选择性大鼠空肠/肾脏Na+/核苷协同转运蛋白（rCNT 1）在非洲爪蟾卵母细胞中表达，并将其与重组嘌呤选择性大鼠空肠/肝脏Na+/核苷协同转运蛋白（rCNT 2）介导的腺苷转运进行比较。rCNT 1介导的腺苷通量比rCNT 2介导的腺苷通量低1个数量级。在动力学研究中，rCNT 1转运腺苷的表观Km值（26 μ M）的流入类似于尿苷，但具有非常低的Vmax值，和Vmax/Km的比率分别为0.003和0.57腺苷和尿苷。重组rCNT 1介导[3 H]尿苷从预载卵母细胞中流出，证明了核苷渗透物双向转运的能力。尿苷外排刺激细胞外尿苷和细胞外腺苷抑制，这表明rCNT 1从其面向外的构象转换到其面向内的构象的速率增加时，转运蛋白与尿苷复合，并减少时，它与腺苷复合。因此，尽管rCNT 1以相似的亲和力结合腺苷和尿苷，但它在动力学上有利于尿苷的转运。
Two major Na+-dependent nucleoside transporter subtypes implicated in adenosine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: one is selective for pyrimidine nucleosides but also binds adenosine, and the other has selectivity for purine nucleosides but also binds uridine. Transportability of adenosine by the purine-selective system has been established by measurements of [3H]adenosine fluxes, whereas the conclusion that adenosine is permeant of the pyrimidine-selective system is based on inhibition assays. We investigated adenosine transport mediated by a recombinant pyrimidine-selective rat jejunal/kidney Na+/nucleoside cotransporter (rCNT1) expressed in Xenopus laevis oocytes and compared it with that mediated by a recombinant purine-selective rat jejunal/liver Na+/nucleoside cotransporter (rCNT2). Adenosine fluxes mediated by rCNT1 were 1 order of magnitude lower than those mediated by rCNT2. In kinetic studies, rCNT1 transported adenosine with an apparent Km value for influx (26 microM) similar to that for uridine but with a very much lower Vmax value, and the Vmax/Km ratios were 0.003 and 0.57 for adenosine and uridine, respectively. Recombinant rCNT1 mediated efflux of [3H]uridine from preloaded oocytes, demonstrating a capacity for bidirectional transport of nucleoside permeants. Uridine efflux was stimulated by extracellular uridine and inhibited by extracellular adenosine, suggesting that the rate of conversion of rCNT1 from its outward-facing conformation to its inward-facing conformation was increased when the transporter was complexed with uridine and decreased when it was complexed with adenosine. Thus, although rCNT1 binds adenosine and uridine with similar affinities, it kinetically favors transport of uridine.