| Co-Investigator(Kenkyū-buntansha) |
HIROTA Hiroshi RIKEN Yokohama Center, Sub Project Director (researcher), タンパク質構造機能研究グループ, プロジェクト副ディレクター(研究職) (00126153)
KANAMORI Kan Toyama University, Faculty of Science, Professor, 理学研究科, 教授 (00019001)
UEKI Tatsuya Hiroshima University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (10274705)
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| Research Abstract |
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.
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