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

在研究补助金支持的2年期间，我们取得了以下成果。分析了分别编码12.5 kDa和15 kDa钒相关蛋白（新命名为Vanabin）的全长cDNA，这可能为阐明钒积累机制提供线索。其结果是，公开了由约120个氨基酸组成，其中半胱氨酸残基的含量非常高，并且是具有α-螺旋的新型蛋白质。重组钒酸结合蛋白能与约20个钒原子结合，结合常数为10- 7 M。我们曾指出，在钒细胞的液泡中，液泡膜上表达的液泡型H^+-ATP酶（V-ATP酶）积累的质子与钒离子浓度有密切的能量相关性。作为阐明能量学的第一步，我们分离了编码V-ATP酶C亚基的cDNA，该亚基起着调节ATP酶活性的作用，并证实了当转化C亚基的海鞘cDNA时，芽殖酵母的相应vma 5突变体的pH敏感表型被拯救。此外，我们获得了约300个EST克隆，并利用钒亲和层析从钒细胞匀浆中提取了50个蛋白。

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.