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標題: | 穀胱甘肽與金屬蛋白活化銅離子鑲嵌至阿拉伯芥銅鋅超氧岐化酶之研究 Study of Glutathione and Metalloprotein Cooperation in Copper Ion Incorporation into Arabidopsis Copper/Zinc-Superoxide Dismutase |
作者: | Ching-Chih Lin 林敬智 |
指導教授: | 靳宗洛(Tsung-Luo Jinn) |
關鍵字: | 榖胱甘?,銅鋅超氧歧化?,金屬鑲嵌蛋白, glutathione,copper/zinc superoxide dismutase,metalloprotein., |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 超氧歧化酶(Superoxide Dismutase;SOD),可將超氧分子(O2.-)轉變為過氧化氫(H2O2)及氧分子(O2),具有解除氧化逆境的功能。對於銅鋅超氧歧化酶(CuZnSOD或CSD)的活化機制,目前已知有兩條路徑,一者是藉由銅鑲嵌輔助蛋白(Copper Chaperone of SOD1;CCS)的幫助,達到銅離子鑲嵌與形成內生性雙硫鍵的活化型態; 另一者在沒有CCS情況下,CSD仍然具有少量的活性,且榖胱甘肽(Glutathione)參與在非CCS的活化CuZnSOD的路徑上,但其活化途徑與機制仍未明朗。本篇論文中,利用巰基(R-SH)阻斷劑處理,進一步證實Glutathione作為氧化還原緩衝劑,透過對於金屬鑲嵌蛋白(Metalloprotein)上巰基氧化的轉譯後修飾機制,參與在非CCS的活化路徑中。此外,亦利用硫酸銨沉澱、逆向色層分析法配合巰基差異性標定與液態層析質譜儀,篩選出金屬硫蛋白(Metallothionein)可做為CCS活化過程中的銅離子來源,並且延伸探討Glutathione催化氧化還原調控在Metallothionein和金屬伴護蛋白(Metallochaperone)的金屬結合當量化學。綜合本研究之結果,說明了當金屬誘發氧化逆境發生時,細胞內銅離子相關的抗氧化逆境機制如何複雜且精密的運作,並且對於銅離子分布提供重要的金屬傳遞機制,進一步去闡述CuZnSOD如何活化於CCS或非CCS相關的路徑。 Superoxide dismutases (SODs, EC 1.15.1.1) are the enzymes that scavenge superoxide radicals(O2.-) to protect cells from oxidative damage. The major pathway for copper/zinc superoxide dismutase (CuZnSOD or CSD) activation involves the function of a copper chaperone for SOD (CCS). Besides, the minor CCS-independent pathway remains unclear mechanism. Glutathione had been reported cooperatively assisting for CSD activation and reconstitution but the mechanism is still unclear. In this study, we aimed to elucidate how the glutathione and the metalloproteins cooperate on the activation for enzyme, especially on Metal Incorporation into Superoxide Dismutase System (MISS). Through thiol block agent treatment, we elucidate that glutathione would involve CCS-independent activation pathway as a redox buffer via sulfur-based oxidative post-translational modification. Moreover, reverse-phase high performance liquid chromatography (HPLC) and LC-MS/MS analyses were performed to identify MT2A, MT2B as a candidate. The reconstitution assay showed MT2A and MT2B providing copper storage as a source for CCS-dependent activation. The glutathione-catalyzed redox regulation experiment fine-tunes the mechanism for glutathione-mediated oxidative switch of copper binding stoichiometry. In summary, our results elucidate the copper-dependent antioxidation process on CSD activation while copper toxicity stress occurring. The findings provide important information for how copper ion trafficking in metalloproteins and potential mechanism for CCS-dependent and CCS-independent activation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67770 |
DOI: | 10.6342/NTU201701982 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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