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

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。

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.