| Project/Area Number |
17370026
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Animal physiology/Animal behavior
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| Research Institution | Hiroshima University |
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Principal Investigator |
MICHIBATA Hitoshi Graduate School of Acience, professor, 大学院理学研究科, 教授 (00111740)
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| Co-Investigator(Kenkyū-buntansha) |
UEKI Tatsuya Graduate School of Science, Associate Professor, 大学院理学研究科, 助教授 (10274705)
HIROTA Hiroshi RIKEN Yokohama Institute, Research fellow, 蛋白質構造・機能研究グループ, 副ディレクター(研究職) (00126153)
HAMADA Toshiyuki Kagoshima University, Faculty of Science, Associate professor, 理学部, 助教授 (40321799)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2006: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2005: ¥10,100,000 (Direct Cost: ¥10,100,000)
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| Keywords | ascidian / vanadium / metal binding proteins / three dimensional structure / cell culture / redox / SS bonds / 円二色性スペクトル |
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| Research Abstract |
In order to clarify sub-cellular localization and functions of vanadium-binding proteins in vanadium-rich ascidians and to establish a cell culture system, following subjects were done.
1. Vanabin2 mutants were constructed and their metal binding abilities were studied. Several amino acid residues were identified to be responsible for maximum metal binding number. When treated by several different types of reductant, Vanabin2 formed several types of intermediates. Mutant with cysteine substitutions were also constructed, and its structure-function relationship were studied. By gel filtration, metal transfer experiments were done for Vanabinl and Vanabin2.
2. Sub-cellular localization of Vanabins 1-4 were studied by specific monoclonal antibodies using Western blot and confocal fluorescent microscopy. Vanabin4 associated with the cytoplasmic membrane weakly, while Vanabins 1,2 and 3 were cytoplasmic.
3. VIP1, which interact with Vanabin2, was identified. VIP1 was localized in the cytoplasm and cytoplasmic membrane. By immunohistochemistry cytoplasm was strongly stained. Interactions among the five Vanabins and VIP1 was studied.
4. Homology modeling based on solution structure of Vanabin2 was done for five Vanabins from Ascidia sydneiensis samea and five from Ciona intestinalis. Crystallization of Vanabins 1, 2, and P was unsuccessful.
5. Construction of cell lines from adult mesenchyme and blood cells as well as from young juvenile was unsuccessful. Lipofection and polyamine methods lead to the introduction of plasmid DNA into primary culture of one type of adult blood cell.
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