Metal binding ability of vanabins and the redox reaction of vanadium

本研究的目的是揭示钒结合蛋白Vanabin2的金属结合结构域，揭示二硫键结构与金属结合的关系，揭示钒在海鞘中的氧化还原反应和生物学功能。构建了几个突变的钒结合蛋白，并对它们的金属结合能力进行了测定。位于第64位组氨酸附近的赖氨酸残基对于钒(IV)的高亲和力结合是重要的。半胱氨酸的丝氨酸取代导致突变蛋白的结构不稳定和降解。尤其是第五个二硫键对结构稳定性最重要，这些突变并不影响钒的结合。测定谷胱甘肽转移酶(AsGST)的金属选择性。在pH值为4.5时，AsGST与铁(III)、铜(II)和钒(IV)特异结合。当在体外用几种还原剂处理VaBin2时，观察到中间结构。在体外研究了钒(V)的还原与二硫键氧化还原的关系。提出了氧化还原中间体结构。利用基因芯片技术，研究了添加钒(IV)和钒(V)离子对番茄基因表达的影响。除磷酸戊糖途径外，基本糖代谢途径不受影响。此外，谷胱甘肽代谢中的酶也受到影响。

The purpose of this study is to reveal the metal binding domains of a vanadium-binding protein Vanabin2, to reveal the relationship between disulfide banding structures and metal binding, and to reveal the redox reaction and biological function of vanadium in ascidians.1. Several mutant vanabins were constructed and their metal binding ablitily was assayed. lysine residues located near the 64th histidine are important for high affinity binding of vanadium(IV).2. Serine substitutions of cysteines caused structural instability and degradation of the mutant proteins. Especially fifth disulfide bonding was most important for structural stability These mutation did not affect the binding of vanadium.3. Metal selectivity of glutathione transferase (AsGST) was assayed. AtpH4.5, AsGST bound specifically to iron (III), copper(II) and vanadium (IV).4. When Vanabin2 was treated with several reductants in vitro, intermediate structures were observed. Relationship between the reduction of vanadium(V) and the redox of disulfide bonds in Vanabin2 was assessed in vitro. Redox intermediate structure of Vanabin2 was suggested.5. By micro array analysis on Ciona intestinalis, genes whose expression was affected by the addition of vanadium(IV) and vanadium(V) ions were explored. Basic sugar metabolism pathways were not affected, except for pentose phosphate pathway. In addition, enzymes in glutathione metabolism were affected.