Molecular physiological study on Vanabins that are key factors for metal accumulation mechanisms

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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.