EXPRESSION CLONING OF A RAT-LIVER NA+-INDEPENDENT ORGANIC ANION TRANSPORTER
EXPRESSION CLONING OF A RAT-LIVER NA+-INDEPENDENT ORGANIC ANION TRANSPORTER
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DOI：
10.1073/pnas.91.1.133
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发表时间：
1994-01-04
影响因子：
11.1
通讯作者：
MEIER, PJ
中科院分区：
文献类型：
--
作者：
JACQUEMIN, E;HAGENBUCH, B;MEIER, PJ
利用非洲爪蟾卵母细胞表达克隆技术，我们分离了一个大鼠肝脏有机阴离子转运多肽（oatp）的cDNA。克隆的oatp介导磺溴酞（BSP）的Na+非依赖性摄取，其在低BSP浓度（例如，2 μ M）。加入增加量的BSA对初始BSP摄取的最大速度没有影响，但它使K（m）值从1.5 μ M（无BSA）增加到24 μ M（BSA/BSP摩尔比，3.7）和35 μ M（BSA/BSP比，18.4）。除BSP外，克隆的oatp还介导结合（牛磺胆酸盐）和非结合（胆酸盐）胆汁酸的Na+非依赖性摄取。cDNA序列分析揭示了一个2010个核苷酸的开放阅读框架，编码670个氨基酸（计算分子量，74 kDa）的蛋白质，具有4个可能的N-连接糖基化位点和10个推定的跨膜结构域。体外翻译实验表明，该转运蛋白确实是糖基化的，其多肽骨架的表观分子量为59 kDa。北方印迹分析表明，与几种mRNA物种从大鼠肝，肾，脑，肺，骨骼肌，近端结肠，以及从小鼠和兔的肝组织，但不是冰鱼（Raja erinacea）和人类的交叉反应。
Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oatp). The cloned oatp mediated Na+-independent uptake of sulfobromophthalein (BSP) which was Cl--dependent in the presence of bovine serum albumin (BSA) at low BSP concentrations (e.g., 2 muM). Addition of increasing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the K(m) value from 1.5 muM (no BSA) to 24 muM (BSA/BSP molar ratio, 3.7) and 35 muM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na+-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Sequence analysis of the cDNA revealed an open reading frame of 2010 nucleotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indicated that the transporter was indeed glycosylated and that its polypeptide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several mRNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human.