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Molecular Physiology of the Highly-Selective Bioaccumulation of Metal Ions

金属离子高选择性生物富集的分子生理学

基本信息

批准号：
14340264
负责人：
MICHIBATA Hitoshi
金额：
$ 9.54万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Ascidians accumulate extremely high concentration of vanadium ions selectively. Its concentration in the accumulating cells reaches 350 mM, which corresponds to 10 million times higher than that in sea water. The vanadium ions are stored in the vacuole of the vanadocytes at +3 oxidation state, which is rarely found in living cells.The pH of the vacuole is very low, and the vacuole contains high concentration of sulfate ions. This unusual phenomenon can be a model system for the study of selective accumulation of metal ions.(1) The metal binding ability, namely maximum number of metal ions that bind to each protein and the dissociation constants, of Vanabin 1 and Vanabin 2 of Ascidia sydneiensis samea were revealed. By electron spin resonance (ESR), it was found that amine nitrogens and oxygen atoms of Vanabin2 coordinate to vanadyl ions, and sulfur atoms do not.(2) From cDNA libraries of Ciona intestinalis, five genes homologous to Vanabins were identified. Their gene structure, expression profile, and metal binding activity was examined.(3) Expression sequence tags (EST) analysis and immobilized vanadium affinity chromatography revealed three novel vanabins, Vanabin3, Vanabin4 and VanabinP. Their expression pattern and metal binding ability was examined.(4) By nuclear magnetic resonance (NMR) analysis, 3D structure of Vanabin2 was almost determined.(5) Membrane transporters and channels belonging to Nramp family, metal ATPase family, chloride channel family and sulfate transporter family was cloned from vanadocytes of Ascidia sydneiensis samea. Exogenous expression of each gene in mammalian or yeast cells was performed.

海鞘对钒离子的选择性富集浓度极高。其在积累细胞中的浓度达到350 mM，相当于海水中的1000万倍。钒离子以+3氧化态储存在钒细胞的液泡中，这在活细胞中是很少见的，液泡的pH值很低，液泡中含有高浓度的硫酸根离子。这种异常现象可以作为研究金属离子选择性富集的模型体系。(1)揭示了悉尼海鞘Vanabin 1和Vanabin 2的金属结合能力，即与每个蛋白质结合的最大金属离子数量和解离常数。通过电子自旋共振（ESR）研究发现，Vanabin 2中的胺氮和氧原子与钒氧离子配位，而硫原子不与钒氧离子配位。(2)从玻璃海鞘cDNA文库中筛选到5个与Vanabins同源的基因。他们的基因结构，表达谱，和金属结合活性进行了检查。(3)表达序列标签（EST）分析和固定化钒亲和层析鉴定了3个新的钒素：Vanabin3、Vanabin4和VanabinP。检测它们的表达模式和金属结合能力。(4)通过核磁共振（NMR）分析，Vanabin 2的3D结构基本确定。(5)从悉尼海鞘（Ascidiasydneiensissamea）的钒细胞中克隆了Nramp家族、金属ATP酶家族、氯离子通道家族和硫酸盐转运蛋白家族的膜转运蛋白和通道。在哺乳动物或酵母细胞中进行每个基因的外源表达。