臺灣博碩士論文加值系統

鈣調素 (calmodulin, CaM) 是真核細胞中普遍存在的泛蛋白。與四個鈣離子鍵結後可負責調節三十幾種標的酵素以利各種生理反應的進行，像是肌球蛋白輕鏈磷酸□(myosin light chain kinase；MLCK)，鈣調素依賴蛋白質磷酸□ (calmodulin-dependent protein kinase)，蛋白質去磷酸□2B (protein phosphatase 2B；calcineurin)，以及一氧化氮合成□ (nitric-oxide synthase)…等。在本篇研究嘗試由牛腦組織中萃取與純化蛋白質去磷酸□2B，並利用遺傳工程技術將原生種與變異種的鈣調素合成出來。建立鈣調素與標的酵素的活性測試，研究鈣調素不同功能區 (domain) 對於蛋白質去磷酸□2B與肌球蛋白輕鏈磷酸□活化能力的影響。

Calmodulin (CaM) is a ubiquitous regulatory protein that is found in all eukaryotic cells. It transmits the Ca2+ signal and activates many enzymes including protein phosphatase 2B (calcineurin), myosin light chain kinase, protein kinase II etc.. The structural nature underling the activation of CaM with it’s target enzymes are important for the understanding of the diverse roles of CaM.In this study, we have overexpressed and purified the recombinant wild type and N-terminal domain of CaM. We have also tried to purify one of the CaM-dependent enzymes, protein phosphatase 2B. An fluorometric activity assay of protein phosphatase 2B has been established based on a published method. The roles of the two domains of CaM in the activation of its target enzyme are compared

目錄

謝誌 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 V
表目錄 VI
第一章、緒論 1
第二章、實驗方法與材料 7
第三章、實驗結果與討論 20
文獻參考 24




圖目錄
圖 1 鈣調素在細胞內負責的生理機制……………………….2
圖 2 蛋白質去磷酸□2B與鈣調素、Ca2+以及免疫抑制劑/免疫親和蛋白複合物的機制………………………………………………………5
圖 3 肌肉的收縮與舒張的作用機制……………………………..6
圖4 蛋白質去磷酸□2B以親和性層析管柱純化示意圖...................17
圖5 鈣調素原生種及變異種經SDS-PAGE跑片結果………………27
圖6 鈣調素N端蛋白質經DEAE層析管柱純化結果........................28
圖7 鈣調素N端蛋白質流經分子篩層析管柱純化結果......................29
圖8 牛腦組織流經affi-gel blue層析管柱利用免疫染色法及跑片後的呈色結果………………………………………………………………..30
圖9蛋白質去磷酸□2B型經鈣調素親和性管柱純化結果………….31
圖10蛋白質去磷酸□ 2B流經Mono-Q 管柱純化結果……………32
圖11蛋白質去磷酸□2B型以15％SDS-PAGE鑑定純度……………33
圖12 4-MU螢光強度的標準校正直線………………………………..34
圖13 在鈣調素作用下4-MUP被蛋白質去磷酸□2B水解成4-MU的螢光放射光譜圖………………………………………………………..35
圖14無鈣調素作用4-MUP被蛋白質去磷酸□2B水解成4-MU的螢光放射光譜圖…………………………………………………………..36
圖15鈣調素對於蛋白質去磷酸□2B活化能力的螢光放射光譜比較圖………………………………………………………………………..37
圖16 鈣調素變異種利用urea-gel 跑片結果…………………………38
圖17 鈣調素不同變異種對於肌球蛋白輕鏈磷酸□的活性測試……39






























表目錄

表一 原生種鈣調素以及變異種鈣調素的胺基酸序列………………40
表二 鈣調素N端蛋白質的純化流程...................................................41
表三 蛋白質去磷酸□2B萃取與純化流程………………………….42

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