Molecular Physiological Analysis on the Accumulation and Reduction of a Metal Ion by Ascidians

海鞘对金属离子富集与还原的分子生理分析

• 批准号: 11440244
• 负责人: MICHIBATA Hitoshi
• 金额: $ 8.96万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11440244/
• 关键词: Ascidian; Vanadium; Accumulation; Reduction; cDNA cloning; Metal-binding; 金属濃縮; 酸化還元; 組み換えタンパク質; 遺伝子クローニング; 金属結合タンパク質

During 2 years supported by the research grant, we have obtained the following results. Full-length of cDNAs, encoding 12.5 kDa and 15 kDa vanadium-associated proteins (designated newly as Vanabin), respectively, which might have a clue to resolve the mechanism of vanadium accumulation, was analyzed. As a result, vanabin was disclosed to consist of about 120 amino acids in which the content of cysteine residues is very high and to be a novel protein having α-helix. Recombinant vanabin was found to bind about 20 atoms of vanadium and its binding constant is 10-7M.In vacuoles of the vanadocytes we previously pointed out a close energetic correlation between the concentrations of vanadium ions and protons accumulated by vacuolar-type H^+-ATPases (V-ATPases) expressed on the vacuole membrane. As the first step to elucidate the energetics, we isolated cDNA encoding subunit C of V-ATPase which plays the role of regulating ATPase activity and confirmed that the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued when the ascidian cDNA for subunit C was transformed. Furthermore, we obtained about 300 EST clones and extracted 50 proteins from the homogenate of vanadocytes using vanadium-affinity chromatography.

在研究资助的两年时间里，我们取得了以下成果。对编码12.5 kDa和15 kDa钒相关蛋白(新命名为Vanabin)的全长cDNA进行了分析，可能为解决钒的积累机制提供了线索。结果表明，VaBin由约120个氨基酸组成，其中半胱氨酸残基含量很高，是一种新的具有α-螺旋的蛋白质。重组钒与约20个钒原子结合，结合常数为10-7M。在钒细胞的液泡中，我们先前指出钒离子浓度与液泡膜上表达的液泡型H~(++)-ATPase(V-ATPase)积累的质子之间存在着密切的能量相关性。作为阐明能量学的第一步，我们分离了V-ATPase C亚基的编码基因，该基因具有调节ATPase活性的作用，并证实当C亚基的海鞘基因被转化时，萌芽酵母相应的vma5突变体的pH敏感表型被拯救。此外，我们还获得了约300个EST克隆，并用钒亲和层析从钒细胞匀浆中提取了50个蛋白质。

项目成果

Ueki,T.: "Exclusive expression of transketolase in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea."Biochim.Biophys.Acta. 1494. (2000)

Ueki,T.：“转酮醇酶在富含钒的海鞘（Ascidia sydneiensis Samea）的钒细胞中的独家表达。”Biochim.Biophys.Acta。

Kanamori, K.: "Direct reduction from vanadium(V) to vanadium(IV) by NADPH in the presence of EDTA. A consideration of the reduction and accumulation of vanadium in the ascidian blood cells."J. Inorg. Biochem.. 77. 157-161 (1999)

Kanamori, K.：“在 EDTA 存在下，NADPH 将钒 (V) 直接还原为钒 (IV)。考虑海鞘血细胞中钒的还原和积累。”J.

Ueki,T.: "Subunit C of the vacuolar-type ATPase from the vanadium-rich ascidians, Ascidia sydneiensis samea, rescued the pH sensitivity of yeast vma5 mutants."Marine Biotech.. (in press). (2001)

Ueki,T.：“来自富含钒的海鞘（Ascidia sydneiensis Samea）的液泡型 ATP 酶的 C 亚基，挽救了酵母 vma5 突变体的 pH 敏感性。”海洋生物技术..（出版中）。

Michibata, H.: "Physiological function of selective accumulation of vanadium in ascidian from sea water and its possible application"Nihon Kaisuigakkaishi. 53. 248-257 (1999)

Michibata, H.：“海水中海鞘选择性积累钒的生理功能及其可能的应用”Nihon Kaisuigakkaishi。

Michibata,H.: "Vanadocyte is a cell holding the key to resolve the highly selective accumulation and reduction of vanadium in ascidians." Microscop.Res.Technol.in press. (1999)

Michibata, H.：“钒细胞是解决海鞘中钒高度选择性积累和还原问题的关键细胞。”