Molecular Physiology of the Highly-Selective Bioaccumulation of Metal Ions

海鞘选择性地积累极高浓度的钒离子。其在蓄积池中的浓度达到350 mm，相当于海水中浓度的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盐离子。这一异常现象可作为研究金属离子选择性积累的模型系统。(1)揭示了银杏叶海桑VaBin-1和VaBin-2的金属离子结合能力，即与每个蛋白质结合的最大金属离子数和解离常数。通过电子自旋共振(ESR)分析，发现VaBin2中的胺氮和氧原子与钒离子发生配位作用，而硫原子则不与之配位。(2)从洋黄瓜的cDNA文库中鉴定出5个与VaBins同源的基因。(3)表达序列标签(EST)分析和固定化钒亲和层析发现了三个新的钒结合蛋白：Vanabin3、Vanabin4和VanabinP。通过核磁共振分析，基本确定了Vanabin2的三维结构。(5)克隆了属于Nramp家族、金属ATPase家族、氯通道家族和硫酸盐转运家族的膜转运蛋白和通道。将每个基因在哺乳动物或酵母细胞中进行外源表达。

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.