The mechanism of osmotic adaptation in marine invertebraes. -Structural and functional characterization of taurine transporter

本研究的目的是通过研究海洋无脊椎动物牛磺酸转运蛋白的功能来阐明海洋无脊椎动物的高渗透适应能力。第一年(1999)，我们调查了暴露于环境渗透压变化的牡蛎体内游离氨基酸的变化，表明牛磺酸是牡蛎体内最重要的渗透调节物质。因此，我们尝试从海洋无脊椎动物中克隆牛磺酸转运蛋白编码基因。因此，我们从海洋贻贝中克隆了与哺乳动物牛磺酸转运蛋白高度同源的部分基因片段，而从牡蛎中未能克隆到该片段。第二年(2000年)，我们获得了贻贝转运蛋白全长基因，与牛磺酸和甜菜碱/GABA转运蛋白具有很高的同源性。此外，Northern印迹分析结果表明，该基因的从头转录同时受到低渗透和高渗透两种环境的诱导。为了分析克隆转运蛋白的底物专一性，我们检测了在非洲爪哇卵母细胞中表达的转运蛋白对[~1H]氨基酸、牛磺酸和[^&lt；14&gt；C]甜菜碱的摄取活性。结果表明，克隆的转运蛋白表达的卵母细胞对牛磺酸具有摄取活性，表明克隆了编码牛磺酸转运蛋白的基因。本研究首次从海洋无脊椎动物中克隆了牛磺酸转运蛋白，并对其在渗透适应中的作用进行了研究。我们的结果是向阐明海洋无脊椎动物的高渗透适应能力迈出的第一步。

The aim of this study is to elucidate the high ability of osmo-adaptation of marine invertebrates by investigation into the function of taurine transporter of marine invertebrates.In the first year (1999), we investigated into the change in free amino acid in the oyster exposed to the change in the ambient osmolality, and indicated that taurine was most important as an osmdyte in the oyster. Therefore, we made an attempt to clone the gene coding taurine transporter from marine invertebrates. As a result, we cloned a partial cDNA fragment showing high homology with taurine transporter of mammals from the marine mussel, while we could not clone that from the oyster.In the second year (2000), we obtained the full length of cDNA of mussel transporter showing high homology with taurine and betaine/GABA transporter. In addition, as a result of northern blot analysis, it is indicated that de novo transcription of this gene is induced by both hypo- and hyper-osmolality.In the last year (2001), we made an attempt to make the substrate of cloned transporter clear. In order to analyze the substrate spedficity of cloned transporter, we examined uptake activity of [^3H]amino acids, taurine and [^<14>C]betaine in the transporter expressed in the Xenopus oocyte. As a result, cloned transporter expressed oocyte showed the uptake activity to taurine and it is indicated cloned cDNA codes taurine transporter. In this study, taurine transporter was cloned from marine invertebrate for the first time, and it's partidpate in osmo-adaptation wasindicated. Our result is the first step toward the elucidation of the high ability in osmo-adaptation of marine invertebrates.