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

金属积累机制关键因素Vanabins的分子生理学研究

基本信息

批准号：
17370026
负责人：
MICHIBATA Hitoshi
金额：
$ 9.41万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370026/
关键词：
ascidian vanadium metal binding proteins three dimensional structure cell culture redox SS bonds 円二色性スペクトル

项目摘要

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.

为了明确富钒海鞘中钒结合蛋白的亚细胞定位和功能，建立富钒海鞘的细胞培养体系，进行了以下实验。构建了Vanabin2突变体并研究了其金属结合能力。确定了几个氨基酸残基与最大金属结合数有关。在几种不同类型的还原剂的作用下，Vanabin2形成了几种类型的中间体。构建了半胱氨酸取代突变体，并对其结构-功能关系进行了研究。通过凝胶过滤，对Vanabinl和Vanabin2.2进行了金属转移实验。采用Western blot和共聚焦荧光显微镜对Vanabins 1-4的亚细胞定位进行了特异性单克隆抗体的研究。Vanabin4与细胞质膜的相关性较弱，而vanabin1、2和3与细胞质膜的相关性较弱。VIP1与Vanabin2相互作用。VIP1定位于细胞质和细胞质膜。免疫组化染色细胞质呈强染色。研究了5种Vanabins与VIP1的相互作用。以Vanabin2的溶液结构为基础，对悉尼海鞘（Ascidia sidneiensis samea）中的5个Vanabin2和海鞘（Ciona肠子）中的5个Vanabin2进行了同源性建模。Vanabins 1、2和P的结晶不成功。从成体间充质细胞和血细胞以及幼体细胞中构建细胞系是不成功的。脂质体和多胺方法可将质粒DNA引入一种成人血细胞的原代培养中。