Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.
Expression cloning of the cDNA for a polypeptide associated with rat hepatic sinusoidal reduced glutathione transport: characteristics and comparison with the canalicular transporter.
复制标题
与大鼠肝窦还原型谷胱甘肽转运相关的多肽的 cDNA 表达克隆：特征以及与小管转运蛋白的比较。
DOI：
10.1073/pnas.92.5.1495
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发表时间：
1995
影响因子：
11.1
通讯作者：
Kaplowitz,N
中科院分区：
文献类型：
--
作者：
Yi,JR;Lu,S;Fernández-Checa,J;Kaplowitz,N
利用非洲蟾卵母细胞表达系统，我们先前从大鼠肝脏中鉴定出大约4 kb的mRNA片段，表达巯基眼啡还原型谷胱甘肽s -共轭物（BSP-GSH）不敏感，以及大约2.5 kb的mRNA片段表达BSP-GSH敏感的还原型谷胱甘肽（GSH）转运。从前者中克隆了一个4.05 kb的cDNA，并被鉴定为推测的大鼠小管谷胱甘肽转运体。从大约2.5 kb片段构建的cDNA文库开始，我们现在已经分离出一个单克隆，该克隆导致BSP-GSH和半胱甘肽抑制GSH转运蛋白活性的表达，其正弦GSH转运蛋白具有大约3 mM的特征。大鼠正弦谷胱甘肽转运体相关多肽（RsGshT） cDNA全长2733个碱基，开放阅读框1059个核苷酸，编码353个氨基酸（39968 Da）的多肽，具有两个推测的跨膜结构域。在各种数据库中均未发现可识别的同源性。在RsGshT的cRNA的体外翻译中产生了大约40 kda的蛋白。Northern blot分析显示，在大鼠和人肝脏中有一个约2.8 kb的转录本，在其他器官中杂交信号可以忽略不计。苯巴比妥治疗后，RsGshT mRNA的丰度没有增加。BSP-GSH的顺式抑制和半胱甘氨酸的反式抑制以及苯巴比多的缺乏诱导是正弦GSH分泌的特征，因此表明RsGshT要么编码正弦GSH转运体本身，要么编码转运体的调控亚基，决定其肝脏特异性活性。
Using the Xenopus oocyte expression system, we previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein reduced glutathione S-conjugate (BSP-GSH)-insensitive and an approximately 2.5-kb fraction expressing BSP-GSH-sensitive reduced glutathione (GSH) transport. From the former, a 4.05-kb cDNA was cloned and characterized as the putative rat canalicular GSH transporter. Starting with a cDNA library constructed from the approximately 2.5-kb fraction, we have now isolated a single clone that leads to expression of a BSP-GSH- and cystathionine-inhibitable GSH transporter activity with Km approximately 3 mM characteristic of the sinusoidal GSH transporter. The cDNA for the rat sinusoidal GSH transporter-associated polypeptide (RsGshT) is 2733 bases with an open reading frame of 1059 nucleotides encoding a polypeptide of 353 amino acids (39,968 Da) with two putative membrane-spanning domains. No identifiable homologies were found in searching various data bases. An approximately 40-kDa protein is generated in in vitro translation of cRNA for RsGshT. Northern blot analysis revealed a single approximately 2.8-kb transcript in rat and human liver with negligible hybridization signal in other organs. The abundance of mRNA for RsGshT did not increase with phenobarbital treatment. Cis-inhibition by BSP-GSH and trans-inhibition by cystathionine and lack of induction by phenobarbital are characteristic of sinusoidal GSH secretion and thus indicate that RsGshT either encodes the sinusoidal GSH transporter itself or a regulatory subunit of the transporter that determines its liver-specific activity.