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

为了明确钒结合蛋白在富钒海鞘中的亚细胞定位和功能，建立细胞培养体系，本研究进行了以下工作.构建了Vanabin 2突变体，并研究了它们的金属结合能力。几个氨基酸残基被确定为负责最大金属结合数。在几种不同类型的还原剂作用下，钒酸盐2生成了几种类型的中间产物。构建了半胱氨酸突变体，并对其结构与功能关系进行了研究。通过凝胶过滤，对Vanabin 1和Vanabin2.2进行金属转移实验。用特异性单克隆抗体对Vanabins 1-4进行了Western blot和共聚焦荧光显微镜的亚细胞定位研究。Vanabin 4与细胞质膜结合较弱，Vanabins 1、2和3则与细胞质结合. VIP 1与Vanabin 2相互作用。VIP 1定位于细胞质和细胞质膜。免疫组织化学染色细胞质强染。研究了五种钒酸盐与VIP 1之间的相互作用.基于Vanabin 2的溶液结构，对5个来自海鞘和5个来自玻璃海鞘的Vanabin 2进行了同源模建。钒酸盐1、2和P的结晶不成功.从成人间充质和血细胞以及从年轻的少年细胞系的建设是不成功的。脂质体转染和多胺方法导致质粒DNA引入到一种类型的成人血细胞的原代培养物中。

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.