MERCURY INTERACTION WITH THE TAURINE TRANSPORT SYSTEM OF RED BLOOD CELLS

拟议研究的总体目标是确定和 表征分子靶点和相互作用机制 汞与膜转运蛋白。 我们将特别关注 汞与钠依赖的牛磺酸转运的相互作用 系统中的血红蛋白含有体腔细胞（红细胞，红细胞）的 海洋多毛类，二鳃囊海鞘。 该运输系统是 类似于哺乳动物组织如心脏中的牛磺酸转运系统， 肾脏和大脑。 在Glycera RBC中，牛磺酸维持在异常水平， 梯度（950：01; 190 mM细胞内牛磺酸：0.2 mM细胞外 牛磺酸）。 我们已经证明这种转运蛋白非常敏感 汞的抑制作用。 暴露在20 μ M的HgC 12中一分钟 抑制牛磺酸流入50%。 使用还原巯基试剂的研究 不同分子大小的反应位点表明， 转运蛋白似乎被膜部分封闭， 只有小分子才能获得完全逆转。 由于HgC 12在 高氯化物介质主要作为阴离子络合物存在， 是不渗透膜，我们提出的操作假设， 非离子HgC 12络合物是与牛磺酸反应的形式 巯基位于蛋白质的跨膜区域。 我们 我将通过以下具体目标来检验这一假设：（1） 从生理学上表征HgC 12的形式， 牛磺酸转运系统，并确定参与转运蛋白 对汞的相互作用敏感的细胞体积调节。 (2)从Glycera RBC中克隆Na依赖性牛磺酸转运蛋白。 （三） 表征汞与牛磺酸转运蛋白的相互作用 在非洲爪蟾卵母细胞中表达。 对完整RBC和 表达牛磺酸转运蛋白的卵母细胞将使用 放射性同位素法 分子程序将取决于反向 具有牛磺酸转运活性的poly（A）+RNA的转录和PCR 通过mRNA的牛磺酸转运活性的表达鉴定 部分显微注射到非洲爪蟾卵母细胞中。 生理学研究将 用各种技术测量溶质含量和净通量 包括离子选择电极、原子吸收光谱、HPLC 和放射性同位素方法。 这项研究的结果将提供 重要的基本信息的相互作用的分子机制 汞与牛磺酸转运体，有一个洞，在许多动物 组织，包括人的心脏、神经和肾组织。

The overall objective of the proposed research is to identify and characterize the molecular targets and mechanisms of interaction of mercury with membrane transport proteins. We will focus specifically on the interactions of mercury with the Na-dependent taurine transport system in the hemoglobin containing coelomocytes (red cells, RBCs) of the marine polychaete, Glycera dibranchiata. This transport system is similar to taurine transport systems in mammalian tissues such as heart, kidney and brain. In Glycera RBCs, taurine is maintained at exceptional gradients (950:01; 190 mM intracellular taurine: 0.2 mM extracellular taurine). We have shown that this transport protein is very sensitive to inhibition by mercurial. A one minute exposure to 20 muM HgC12 inhibits taurine influx 50%. Studies using reduced sulfhydryl reagents of different molecular size indicated that the reactive sites on the transport protein appear to be partially occluded by the membrane since complete reversal is obtained only with small molecules. Since HgC12 in high chloride media is present predominantly as anionic complexes that are impermeant to membranes, we propose the operational hypothesis that the nonionic HgC12 complex is the form that reacts with taurine sulfhydryl groups lying in membrane spanning regions of the protein. We will test this hypothesis by addressing the following specific aims: (1) Physiologically characterize the form of HgC12 that interacts with the taurine transport system and identify the transport proteins involved in cell volume regulation that are sensitive to interaction with mercury. (2) Clone the Na-dependent taurine transporter from Glycera RBCs. (3) Characterize the interaction of mercurial with the taurine transporter expressed in Xenopus oocytes. Flux measurements on intact RBCs and on oocytes expressing the taurine transport protein will be done using radioisotope methods. The molecular procedures will depend on reverse transcription and PCR of poly(A)+RNA with taurine transport activity identified by expression of taurine transport activity of the mRNA fractions microinjected into Xenopus oocytes. Physiological studies will measure solute contents and net fluxes with a variety of techniques including ion selective electrodes, atomic absorption spectroscopy, HPLC and radioisotopic methods. The results of this study will provide important basic information on the molecular mechanism of interaction of mercury with the taurine transporter that has a hole in many animals tissues including human cardiac, nerve and kidney tissue.