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研究生:王聖宏
研究生(外文):Sheng-Hung Wang
論文名稱:磷脂水解酶A2作用在不同基質之選擇性探討
論文名稱(外文):The Study of Hydrolysis Activity of Phospholipase A2 at Different Lipid Substrates
指導教授:黃維寧
指導教授(外文):Wei-Ning Huang
學位類別:碩士
校院名稱:元培科學技術學院
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:96
語文別:中文
論文頁數:83
中文關鍵詞:磷脂水解酵素A2定點突變磷脂質
外文關鍵詞:Phospholipase A2Site-directed mutagenesisPhospholipids
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磷脂水解酵素A2 (phospholipase A2,PLA2,E.C. 3.1.1.4),催化水解磷脂質的sn-2位置,並且釋放脂肪酸與脫脂酸磷脂質。這些水解產物為重要的二級訊息傅遞者,且與免疫、發炎和生物毒性相關。在本論文中比較各種類的PLA2對於一系列不同的脂質來做酵素活性測定,我們也利用分子生物選殖技術人工重組Naja atra PLA2 (aPLA2)基因並且定點突變成D23A、D23F、D23K、D23N,利用大腸桿菌表現系統表現重組蛋白,再使用蛋白質工程技術,將形成包涵體的aPLA2,重新再折疊成構形正確的蛋白結構,使用逆相高效率色層分析純化重組蛋白,接著用圓二色旋光光譜測定其二級結構,最後使用呈色法來將實驗室中所有不同種類的PLA2去做酵素活性測定,來瞭解PLA2作用在不同基質時所扮演的選擇性角色為何。由過去其它實驗室的結果發現眼鏡蛇毒PLA2在水解PC類的磷脂質有最強的酵素活性,我們發現磷脂質若帶有三個甲基,會比頭部無甲基(PE)或只帶一個甲基、二個甲基的磷脂質酵素活性會強很多。aPLA2經定點突變後,其酵素活性明顯降低,但在PE以及PG卻無明顯改變,顯示aPLA2 D23這個位置一經突變,就會造成酵素功能在脂質選擇性上有很大的改變。至於人類Group IIa PLA2,其酵素活性在POPG很強,卻在DMPG上非常地微弱。綜合以上不同種類PLA2的酵素水解活性結果,我們對於PLA2蛋白質在基質的選擇性上有更深一層瞭解,日後可以讓我們進一步去探討更多酵素水解細胞膜的機制。
Phospholipase A2 (PLA2、E.C. 3.1.1.4) converts phospholipids into lysophospholipids and free fatty acids by hydrolyzing the ester bond of phospholipids at the sn-2 position. Some released fatty acids are precursors of eicosanoids or work as secondary messengers、which might be potentially involved in inflammation、immune response and signal transduction. In this thesis、we would like to determine whether enzyme activities of PLA2 from different species could be influenced as they reacted with different molecular species of phospholipids. By making use of techniques of genetic engineering and site-directed mutagenesis、we constructed Taiwanese cobra (Naja atra) PLA2 (aPLA2) gene and four mutated genes with a point mutation at D23A、D23F、D23K and D23N positions、respectively. Then、these recombinant aPLA2 proteins in inclusion bodies with the correct structure were over-expressed、collected and refolded by using E. coli expression system and protein engineering techniques. To purify aPLA2 and identify its secondary structures、we utilized combined techniques of reverse phase-high performance liquid chromatography (RP-HPLC) and Circular Dichroism spectra (CD spectra). Enzyme activities of these proteins were detected by colorimetric assay. Results in this study agreed with previous reports that PLA2 from cobra venom exerted the most powerful activity to hydrolyze phosphocholine (PC)、and also indicated that recombinant aPLA2 preferably hydrolyzed phospholipids with three methyl groups on the polar head、as compared to those containing no、one or two methyl groups. When enzyme activities of mutated aPLA2 were examined、hydrolytic activity on phosphotidylcholine (PC) was significantly reduced as phospholipids appeared、and the degree of hydrolysis was as same as that of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). These findings suggested that the position D23 of aPLA2 DNA sequence might be responsible for enzyme substrate specificity. Beside、we determined the other type of phospholipase A2、human Group IIa PLA2、and found the enzyme hydrolyzed 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (POPG) powerfully、however、no such effect was observed when worked with 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG). In conclusion、results from this thesis help us have a better understanding of the substrate specificity of phospholipase A2、and provide an approach to explore the possible mechanism of phospholipase-mediated hydrolysis occurs in the cell membrane.
誌謝 I
中文摘要 III
英文摘要 IV
目錄 VI
圖目錄 IX
表目錄 XI
第一章 緒論 1
1.1磷脂水解酶 1
1.1.1何謂磷脂水解酶 1
1.1.2 PLA2的生理功能 1
1.1.3 PLA2的分類 1
1.1.4 PLA2的作用機制 3
1.1.4.1鈣離子結合位置與膜結合角度 3
1.1.4.2 PLA2的之遲滯期現象 3
1.1.4.3 PLA2水解產物與膜幾何結構之關係 4
1.2生物細胞膜 4
1.2.1細胞膜的主要成份 4
1.3研究目的 4
第二章 材料與方法 6
2.1 實驗材料 6
2.2 實驗藥品 6
2.3 實驗儀器 11
2.4 實驗方法 12
2.4.1基因人工重組Naja atra PLA2 DNA片段重組之聚合酶鏈鎖反應 12
2.4.1.1全長PLA2基因之PCR引子片段 12
2.4.1.2含有Factor Xa之PCR引子 12
2.4.1.3定點突變之PCR引子 12
2.4.2 PCR產物膠體純化 13
2.4.3質體DNA之抽取 13
2.4.4限制酶酵素處理(Restriction enzyme digestion) 13
2.4.5質體之CIP處理 13
2.4.6接合作用(Ligation) 14
2.4.7 LB培養基之製備 14
2.4.8轉型作用(Transformation) 15
2.4.9重組蛋白的表現 15
2.4.10 SDS-聚丙烯醯胺板膠電泳法 16
2.4.11蛋白質電泳之操作方法 16
2.4.12蛋白質電泳膠片染色與褪色 17
2.4.13蛋白質再折疊 17
2.4.14利用Factor Xa酵素切除融合蛋白 17
2.4.15定點突變 17
2.4.16 RP-HPLC純化出重組以及定點突變之Naja atra PLA2蛋白 18
2.4.17旋光光譜儀量測Naja atra PLA2及其重組和定點突變之二級結構 18
2.4.18酵素活性螢光測定法 19
2.4.19酵素活性測定呈色法 19
第三章 結果與討論 20
3.1 Naja atra PLA2基因選殖 20
3.2 利用大腸桿菌表現aPLA2蛋白 20
3.3 Naja atra PLA2突變種之製備 21
3.4 PLA2s蛋白特性分析 21
第四章 結論 23
第五章 圖表 24
參考文獻 74
附件一 磷脂質相轉變點溫度 80
附件二 高壓細胞破碎機EF-C3操作說明 82

圖 目 錄
頁次
圖一 磷脂水解酵素依水解不同的區域命名之示意圖 24
圖二 脂質被磷脂水解酵素A2水解成脂肪酸鏈與脫脂酸磷脂質之示意圖 25
圖三 花生四烯酸藉由5-LO以及cyclooxygenase分解的代謝圖 26
圖四 三類分泌型磷脂水解酵素A2的結構圖 27
圖五 磷脂水解酵素A2之綵帶圖 28
圖六 磷脂水解酵素A2在與鈣離子相結合之結構圖 29
圖七 磷脂水解酵素A2 lag Time的測量圖 30
圖八 神經突觸中脂質膜的曲折度控制微粒的融合以及突觸的活動力之卡通圖 31
圖九 Naja atra PLA2在質體pET21b(+)基因選殖示意圖 32
圖十 經PCR放大的aPLA2 DNA電泳圖 33
圖十一 利用IPTG誘導大腸桿菌表現PLA2蛋白質SDS-PAGE電泳圖 34
圖十二 Naja atra PLA2在質體pAB3基因選殖示意圖 35
圖十三 構築Naja atra PLA2 Insert DNA選殖到pAB3質體PCR放大DNA電泳圖 36
圖十四 二次放大插入DNA電泳圖 37
圖十五 經BamHI和HindⅢ兩種限制酶反應切割的DNA電泳膠結果圖 38
圖十六 經BamHI和HindⅢ兩種限制酶切割pAB3質體DNA電泳圖 39
圖十七 經CIP酵素反應後的pAB3載體DNA電泳圖 40
圖十八 經轉型作用到JM109勝任細胞PCR反應電泳圖 41
圖十九 誘導aPLA2在SDS-PAGE上蛋白表現電泳圖 42
圖二十 在鈣離子存在下之aPLA2 3-D蛋白質結構圖 43
圖二十一 定點突變示意圖 44
圖二十二 定點突變電泳圖 45
圖二十三 誘導突變種aPLA2在SDS-PAGE上蛋白表現電泳圖 47
圖二十四 經Factor Xa酵素切割後的aPLA2蛋白質產物在SDS-PAGE電泳圖 48
圖二十五 使用RP-HPLC純化aPLA2和突變種 51
圖二十六 Native、WT和突變種的aPLA2圓二色光譜結果圖 52
圖二十七 螢光光譜儀測定Native以及人工重組和突變種的aPLA2水解NBD-PC的酵素活性分析結果圖 53
圖二十八 螢光光譜儀測定各種不同種類的PLA2在NBD-PC以及NBD-PG的酵素活性分析結果圖 54
圖二十九 測定酵素活性呈色法示意圖 55
圖三十 不同pH值溶液在吸收光譜558nm測定之結果圖 56
圖三十一 不同種類的PLA2對DLPC磷脂質水解活性 57
圖三十二 不同種類的PLA2胺基酸序列比對 58
圖三十三 不同種類的PLA2對各類磷脂質水解活性 60
圖三十四 不同種類的磷脂質對各類PLA2水解活性 62
圖三十五 PO種類的磷脂質對Native、WT和D23A PLA2水解活性 63
圖三十六 不同PE種類以及POPC的磷脂質對Native、WT和D23A PLA2水解活性 64
圖三十七 不同種類的磷脂質對人類IIa PLA2水解活性 65

表 目 錄
頁次
表一 分泌型磷脂水解酶A2家族 66
表二 胞內型磷脂水解酶A2家族 67
表三 非鈣離子依賴型磷脂水解酶A2家族 68
表四 血小板活化因子乙醯水解酶A2家族 69
表五 溶小體磷脂水解酶A2家族 70
表六 分泌型低分子量之磷脂水解酵素A2之比較列表 71
表七 引子核苷酸之設計 72
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