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研究生:謝瑀
研究生(外文):Yu Hsieh
論文名稱:發展針對絲胺酸水解酶活性偵測之化學探針
論文名稱(外文):Developing chemical probe for activity profile of serine hydrolases
指導教授:林照雄林照雄引用關係
指導教授(外文):Chao-Hsiung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:54
中文關鍵詞:絲胺酸水解酶化學探針化學蛋白質體學有機磷類化合物
外文關鍵詞:serine hydrolaseschemical probechemical proteomicsorganophosphorus compoundsfluorophosphonatefluorophosphatePhaZFP-peg-biotin
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絲胺酸水解酶(serine hydrolases)家族包含了各種蛋白酶、脂肪酶、酯酶,以及醯胺酶等,是數量最為龐大且最具多樣性的一個酵素族類。而絲胺酸水解酶在生物體內扮演著許多重要的角色,同時亦參與了許多重要的功能,並且有許多人類疾病皆與絲胺酸水解酶或其調控蛋白質的功能異常有關。為了有系統地瞭解各種絲胺酸水解酶的作用與功能,必須建立一個研究方法以系統地偵測或純化絲胺酸水解酶家族,並進一步探討其生物意義。
Fluorophosphonate/fluorophosphate(簡稱FP)是目前已知的絲胺酸水解酶酵素活性抑制物,其化學活性能與絲胺酸水解酶催化部位內之絲胺酸產生不可逆的共價鍵結,並進而抑制其酵素活性。因此我們與其他實驗室合作製備了一個帶有生物素標記之FP(簡稱FP-peg-biotin)作為針對絲胺酸水解酶的化學探針以標記所偵測到的酵素,之後再以一維凝膠電泳、西方墨點法以及NeutrAvidin beads去分析實驗條件以純化、確認及實際分離出具有FP-peg-biotin標記之絲胺酸水解酶,並期望利用此新化學探針來建立一具有高反應性及專一性的絲胺酸水解酶研究方式。
Serine hydrolases represent one of the largest and diverse families of enzymes comprising numerous proteases, lipases, esterases, and amidases. Serine hydrolases play important roles and also regulate numerous important functions in many organisms. Disorder of such serine hydrolases may cause different kinds of human diseases. To understand the functions of diverse serine hydrolases, it is necessary to develop a method to systematically detect or enrich serine hydrolases and thus the following studies of their biological significances.
Fluorophosphonate/fluorophosphates (FP) is a well-known enzyme inhibitors of serine hydrolases. It has been demonstrated to covalently react with the catalytic serine residue of serine hydrolases and thus irreversibly inhibit the enzymatic activity. We collaborated with other laboratory to prepare an FP analogue with the function of biotin and polyethylene glycol (FP-peg-biotin) as a chemical probe to identify serine hydrolases. The following verification used SDS-PAGE, Western blot, and NeutrAvidin beads to separate, detect and enrich serine hydrolases. In summary, we anticipate this probe will help us to develop a high efficient and specific toll for studies of diverse serine hydrolases.
摘要 .......................... 2

英文摘要 ...................... 3

第一章 序論 .................. 4

第二章 材料及實驗方法 ........ 13

第三章 實驗結果 .............. 19

第四章 討論 .................. 26

第五章 參考文獻 .............. 33

第六章 圖表 .................. 41
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