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研究生(外文):Yi-Hsun Tsao
論文名稱(外文):The study of phospholipase A2 and inhibitors interaction
指導教授(外文):Wei-Ning Huang
中文關鍵詞:磷脂水解酶 A2人類非胰腺磷脂水解酶 A2酵素抑制劑對接分泌型
外文關鍵詞:phospholipase A2human non-pancreatic phospholipase A2enzyme inhibitorsdockingsecretory
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磷脂水解酶 A2 (phospholipase A2, PLA2, E.C.,主要水解磷脂質的sn-2位置,並且釋出游離脂肪酸 (free fatty acid) 與脫脂磷脂質 (lysophospholipid)。在生理上,PLA2 會活化免疫細胞內的訊息傳遞,啟動發炎機制。在本篇論文中利用 NBD 以及酚紅兩種指示劑做為酵素活性測定方法的基礎來比較各種抑制劑對不同類 PLA2 的作用進行研究。在此兩種測定方法中皆得到 MJ33 與 PK03 分別對蜂毒素 PLA2 及人類非胰腺 PLA2 具有最強的抑制能力,但是以此兩種方法測定 MJ33 對蛇毒素 PLA2 (aPLA2) 的抑制效力時卻得到不一致的結果。由於我們利用微脂粒包覆酚紅指示劑是新嘗試的酵素活性測定方法,因此結果的穩定性需更加確認。我們也利用 aPLA2 與 MJ33 的共晶體以 X-Ray 繞射法解析此複合體之三維結構,在此三維結構顯示鈣離子與 MJ33 磷酸氧原子的距離為 3.7 Å,較過去文獻之胰臟 PLA2 遠,因此推測 MJ33 對 aPLA2 的抑制作用不會受到鈣離子的加強,此結果可嘗試說明 MJ33 對 aPLA2 抑制能力為何較弱的原因。最後藉由電腦進行 PLA2 與抑制劑對接計算,經計算結果得知 PK03 尾端類似脂肪酸的結構會增強與 PLA2 間的疏水性交互作用,然而 aPLA2 與 MJ33 的計算結果卻是以 MJ33 尾端的長碳鏈進入 PLA2 的活性位置有最低的結合能,此計算結果雖與實際晶體結構不一致卻也提供了更多樣的可能結合態。
Phospholipase A2 (PLA2, E.C. converts phospholipids into free fatty acids and lysophospholipids by hydrolyzing the ester bond of phospholipids at sn-2 position. Physiologically, PLA2 activation in immune cells will pass the message to initiate the inflammation mechanism. In this study, we used NBD fluorescent agent and liposome coated phenol red as indicators to compare inhibitory mechanims of various inhibitors to different PLA2s. The two indicators showed that the inhibitor MJ33 strongly suppressed the activites of bee venom phospholipase A2 (bvPLA2) while the inhibitor PK03 strongly suppressed the activites of and human nonpancreatic phospholipase A2 (hnpPLA2); however, the two methods showed contrary results when MJ33 and PK03 were applied to Naja atra phospholipase A2 (aPLA2). As this was the first time to utilize liposome coated phenol red as the indicator for studying PLA2 activity assay, more studies would be needed to characterize properties of this method. We also used X-ray diffractions and the co-crystals of MJ33 and aPLA2 for the three-dimensional structural determination. We found that calcium ion was far away from MJ33 and the ability of inhibition of MJ33 might not be enhanced by calcium ion. Based on computational results on docking, the fatty-acid-like tail of PK03 would enhance hydrophobic interactions with PLA2. However, the profile of lowest binding energy of MJ33 and aPLA2 showed the carbon chain of MJ33 dock into the active site of aPLA2 and it was not matching the result of X-ray crystallgraphy. It may provide more possibility of interaction on PLA2/inhibitor.
誌謝 I
中文摘要 III
英文摘要 IV
總目錄 V
目錄 V
圖目錄 VII
表目錄 VIII
縮寫對照表 X
第一章 緒論 1
1.1 引言 1
1.2 磷脂水解酶 A2 的簡介 1
1.3 磷脂水解酶 A2 的分類 2
1.4 分泌型磷脂水解酶 A2 的生理特性 2
1.5 分泌型磷脂水解酶 A2 的結構 3
1.6 分泌型磷脂水解酶 A2 催化機制 4
1.7 磷脂水解酶 A2 抑制劑 4
1.8 研究目的 7
第二章 實驗原理 8
2.1 磷脂水解酶 A2 活性分析原理 8
2.2 磷脂水解酶 A2 晶體結構 9
1. 結構的決定 9
2. X 光繞射數據的收集 12
3. 晶體培養 13
4. 影響蛋白質晶體生長的過程 15
2.3 電腦計算分子對接 16
第三章 材料與方法 19
3.1 藥品 19
3.2 實驗方法 20
1. 蛋白質純化 20
2. 以 NBD 螢光劑測定酵素活性 22
3. 以酚紅指示劑測定酵素活性 24
4. aPLA2 與 MJ33 抑制劑晶體培養 25
5. 繞射數據收集 26
6. 繞射數據的處理 26
7. 結構的精調 27
8. 蛋白質與小分子對接 27
第四章 結果 29
4.1 以 NBD 螢光劑測定酵素活性 29
4.2 以酚紅指示劑測定酵素活性 29
4.3 晶體培養 29
4.4 晶體繞射數據分析 30
4.5 aPLA2 與 MJ33 複合體之結構 31
4.6 PLA2與小分子對接 (docking) 32
第五章 討論 34
5.1 酵素活性分析 34
5.2 晶體培養 35
5.3 aPLA2 與 MJ33 複合體之結構 35
5.4 PLA2 與小分子對接 36
第六章 結論 38
第七章 圖表 39
第八章 參考文獻 77

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