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

为了阐明富钒海鞘中钒结合蛋白的亚细胞定位和功能，并建立细胞培养体系，进行了以下研究。构建了VaBin2突变体，并对其金属结合能力进行了研究。有几个氨基酸残基决定了最大的金属结合数。当用几种不同类型的还原剂处理时，Vanabin2形成了几种类型的中间体。构建了半胱氨酸取代突变体，并对其构效关系进行了研究。采用凝胶过滤的方法，对钒和钒2.2进行了金属迁移实验。利用免疫印迹和共聚焦荧光显微镜，用特异性单抗研究了Vanabins 1-4的亚细胞定位。花生素4与细胞膜结合较弱，花青素1、2、3为细胞质。鉴定出与Vanabin2相互作用的VIP1。VIP1定位于胞浆和胞膜。免疫组织化学染色显示胞浆呈强阳性。研究了五种哇那宾与VIP1的相互作用。根据Vanabin2的溶液结构，对来自海桑的5个Vanabins和来自沙棘的5个Vanabins进行了同源建模。钒类化合物1、2和P的结晶不成功。从成人间充质细胞和血细胞以及幼年幼年细胞构建细胞系失败。脂质体法和多胺法将质粒DNA导入到一种成人血细胞的原代培养中。

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.