(3.231.230.175) 您好!臺灣時間:2021/04/16 02:12
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:曹藝薰
研究生(外文):Yi-Hsun Tsao
論文名稱:磷脂水解酶A2與抑制劑之交互作用研究
論文名稱(外文):The study of phospholipase A2 and inhibitors interaction
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:元培科技大學
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
畢業學年度:99
語文別:中文
論文頁數:93
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
相關次數:
  • 被引用被引用:1
  • 點閱點閱:549
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:27
  • 收藏至我的研究室書目清單書目收藏:1
磷脂水解酶 A2 (phospholipase A2, PLA2, E.C.3.1.1.4),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. 3.1.1.4) 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
目錄
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


1. Araljio, A. L., & Radvanyi, F. (1987). Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator. Toxicon , 25, pp. 1181–1188.
2. Balsinde, J., & Dennis, E. A. (1996). Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. J. Biol. Chem. , 271, pp. 6758–6765.
3. Bartoli, F., Lin, H. K., Ghomashchi, F., Gelb, M. H., Jain, M. K., & Castro, A. R. (1994). Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombinstimulated human platelets. J. Biol. Chem. , 269, pp. 15625–15630.
4. Beers, S. A., Buckland, A. G., & Wilton, D. C. (2002). The antibacterial properties of secreted phospholipases A2: A major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall. J. Biol. Chem. , 277, pp. 1788–1793.
5. Berg, O. G., Gelb, M. H., Tsai, D. M., & Jain, M. K. (2001). Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm. Chem. Rev. , 101, pp. 2613–2653.
6. Bergfors, T. M. (1999). Protein Crystallization. Intl Univ Line.
7. Bianco ID, I. D., Kelley, M. J., Crowl, R. M., & Dennis, E. A. (1995). Identification of two specific lysines responsible for the inhibition of phospholipase A2 by manoalide. Biochim. Biophys. Acta , 1250, pp. 197–203.
8. Birts, C. N., Barton, C. H., & Wilton, D. C. (2009). Catalytic and non-catalytic functions of human IIA phospholipase A2. Trends Biochem Sci , 35, pp. 28–35.
9. Blow, D. (2002). Outline of Crystallography for Biologists . USA: Oxford University Press.
10. Bruno, L. D., & Jonathan, P. A. (2003). Phospholipase A2. Prostaglandins, Leukotrienes and Essential Fatty Acids , 69, pp. 87–97.
11. Chayen, N. E. (2005). Methods for separating nucleation and growth in protein crystallisation. Prog Biophys Mol Biol. , 88 (3), pp. 329-337.
12. Chayen, N. E., & Saridakis, E. (2008). Protein crystallization: from purified protein to diffraction-quality crystal. Nature methods , 5 (2), pp. 147–153.
13. Chayen, N. E., Boggon, T. J., Cassetta, A., Deacon, A., Gleichman, T., Habash, J., et al. (1996). Trends and challenges in experimental macromolecular crystallography. Q. Rev. Biophys. , 29, pp. 227–278.
14. Copeland, R. A. (2000). Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis (2 ed.). Wiley-VCH.
15. Dias, R., & Jr, d. A. (2008). Molecular Docking Algorithms. Current Drug Targets , 9, pp. 1040–1047.
16. Du, W., Li, L., Nichols, K. P., & Ismagilov, R. F. (2009). SlipChip. Lab Chip , 9, pp. 2286–2292.
17. Ducruix, A., & Giegé, R. (2000). Crystallization of Nucleic Acids and Proteins: A Practical Approach (Practical Approach Series) (2 ed.). USA: Oxford University Press.
18. Frieboes, K. C., Reynolds, L. J., Lio, C. Y., Hale, M. R., Wasserman, H. H., & Dennis, E. A. (1996). Activated ketones as inhibitors of intracellular Ca2+-dependent and Ca2+-independent phospholipase A2. J. Am. Chem. Soc. , 118, pp. 5519–5525.
19. Gelb, M. H., Jain, M. K., & Berg, O. G. (1994). Inhibition of phospholipase A2. The FASEB Journal , 8, pp. 916–924.
20. Hansen, C. L., Skordalakes, E., Berger, J. M., & Quake, S. R. (2002). A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. . Proc Natl Acad Sci U S A. , 99, pp. 16531–16536.
21. Heyes, S. J. (1999). Structures of Simple Inorganic Solids. Retrieved from http://www.chem.ox.ac.uk/icl/heyes/structure_of_solids/lecture1/lec1.html
22. Huang, K. C., Zhou, L., Liu, Y., & Lai, L. H. (2006). A continuous fluorescence assay for phospholipase A2 with nontagged lipid. . Anal. Biochem. , 351, pp. 11–17.
23. Huang, Z., Payette, P., Abdullah, K., Cromlish, W. A., & Kennedy, B. P. (1996). Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2. Biochemistry , 35, pp. 3712–3721.
24. Jain, M. K., & Berg, O. G. (2006). Coupling of the i-face and the active site of phospholipase A2 for interfacial activation. Curr. Opin. Chem. Biol. , 10, pp. 473–479.
25. Jain, M. K., Tao, W., Rogers, J., Arenson, C., Eibl, H., & Yu, B. Z. (1991). Active-Site-Directed Specific Competitive Inhibitors of Phospholipase A2: Novel Transition-State Analogues. Biochemistry , 30, pp. 10256–10268.
26. Kaapro, A., & Ojanen, J. (2002). Protein docking. pp. 1–18.
27. KiniRManjunatha. (1997). Venom phospholipase A2 enzymes: structure, function and mechanism. West Sussex, England: John Wiley & Sons Ltd.
28. Kudo, I., & Murakami, M. (2002). Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat. , 68-69, pp. 3–58.
29. Lambeau, G., & Gelb, M. H. (2008). Biochemistry and Physiology of Mammalian Secreted Phospholipases A2. Annu. Rev. Biochem. , 77, pp. 495–520.
30. Lee, H. C. (2010). 1-anilinonaphthalene-8-sulfonate (ANS); a versatile fluorescent probe from protein folding study to drug design. BioWave , 12 (6), pp. 1–12.
31. Li, L., & Ismagilov, R. F. (2010). Protein crystallization using microfluidic technologies based on valves, droplets, and SlipChip. . Annu. Rev. Biophys. , 39, pp. 139–158.
32. Lombardo, D., & Dennis, E. A. (1985). Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor. J. Biol. Chem , 260, pp. 7234–7240.
33. Lucas, K. K., & Dennis, E. A. (2005). Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors. Prostaglandins Other Lipid Mediat. , 77, pp. 235–248.
34. Macromolecular docking. 擷取自 維基百科: http://en.wikipedia.org/wiki/Macromolecular_docking
35. Mayer, M. R., & Marshall, L. A. (1993). New insights on mammalian phospholipase A2s: comparison of arachidonoyl-selective and -nonselective enzymes. FASEB J , 7, pp. 339–348.
36. Meyuhas, D., Yedgar, S., Rotenberg, M., Reisfeld, N., & Lichtenberg, D. (1992). The use of C6-NBD-PC for assaying phospholipase A2-activity: scope and limitations. Biochimica et Biophysics Acta. , 1124, pp. 223–232.
37. Morris, G. M., Goodsell, D. S., Pique, M. E., Lindstrom, W. L., Huey, R., Forli, S., et al. (2010). AutoDock Version 4.2: Automated Docking of Flexible Ligands to Flexible Receptors. Retrieved from AutoDock: http://autodock.scripps.edu/faqs-help/manual/autodock-4-2-user-guide/AutoDock4.2_UserGuide.pdf
38. Morris, G. M., Huey, R., & Olson, A. J. (2008). UNIT 8.14 Using AutoDock for Ligand-Receptor Docking. Curr Protoc Bioinformatics , pp. 1–40.
39. Pan, Y. H., & Bahnson, B. J. (2007). Structural Basis for Bile Salt Inhibition of Pancreatic Phospholipase A2. J Mol Biol. , 369 (2), pp. 439–450.
40. Pan, Y. H., Epstein, T. M., Jain, M. K., & Bahnson, B. J. (2001). Five coplanar anion binding sites on one face of phospholipase A(2). Relationship to interface binding. Biochemistry , 40, pp. 609–617.
41. Reynolds, L. J., Mihelich, E. D., & Dennis, E. A. (1991). Inhibition of venom phospholipases A2 by manoalide and manoalogue. Stoichiometry of incorporation. J. Biol. Chem. , 266, pp. 16512–16517.
42. Riendeau, D., Guay, J., Weech, P. K., Lalibert, F., Yergey, J., Li, C., et al. (1994). Arachidonyl trifluoromethyl ketone, a potent inhibitor of 85-kDa phospholipase A2, blocks production of arachidonate and 12-hydroxyeicosatetraenoic acid by calcium inopohore-challenged platelets. J. Biol. Chem. , 269, pp. 15619–15624.
43. Roberts, M. F., Deems, R. A., Mincey, T. C., & Dennis, E. A. (1977). Chemical modification of the histidine residue in phospholipaseA2 (Naja naja naja): a case of half-site reactivity. J. Biol. Chem. , 252, pp. 2405–2411.
44. Schevitz, R. W., Bach, N. J., Carlson, D. G., Chirgadze, N. Y., & Clawson, D. K. (1995). Structure-based design of the first potent and selective inhibitor of human nonpancreatic secretory phospholipase A2. Nat. Struct. Biol. , 2, pp. 458–465.
45. Scott, D. L., White, S. P., Browning, J. L., Rosa, J. J., & Gelb, M. H. (1991). Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate. Science , 254, pp. 1007–1010.
46. Sekar, K., Eswaramoorthy, S., Jain, M. K., & Sundaralingam, M. (1997). Crystal Structure of the Complex of Bovine Pancreatic Phospholipase A2 with the Inhibitor 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol. Biochemistry , 36, pp. 14186–14191.
47. Six, D. A., & Dennis, E. A. (2000). The expanding superfamily of phospholipase A2 enzymes: classication and characterization. Biochimica et Biophysica Acta , 1488, pp. 1–19.
48. Sousa, S. F., Fernandes, P. A., & Ramos, M. J. (2006). Protein–Ligand Docking: Current Status and Future Challenges. Proteins , 65, pp. 15–26.
49. Street, I. P., Lin, H. K., Laliberte, F., Ghomashchi, F., Wang, y. Z., Perrier, H., et al. (1993). Slow and tight binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry , 32, pp. 5935–5940.
50. Stroganov, O. V., Novikov, F. N., Stroylov, V. S., Kulkov, V., & Chilov, G. G. (2008). Lead Finder: An Approach To Improve Accuracy of Protein-Ligand Docking, Binding Energy Estimation, and Virtual Screening. J. Chem. Inf. Model. , 48, pp. 2371–2385.
51. Tsukanova, V., Grainger, D. W., & Salesse, C. (2002). Monolayer Behavior of NBD-Labeled Phospholipids at the Air/Water Interface. Langmuir , 18, pp. 5539–5550.
52. Verheij, H. M., Volwerk, J. J., Jansen, M. E., Puyk, C. W., Dijkstra, B. W., Drenth, J., et al. (1980). Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism. Biochemistry , 19, pp. 743–750.
53. Vinson, P. (2006). Automation of Protein Crystallography. Successful Identification of Structure Dependent on Producing High Quality Crystals. Genetic Engineering & Biotechnology News .
54. Wang, X. R., Liu, L., Lai, H. L., & Tang, Y. (1998). SCORE: A New Empirical Method for Estimating the Binding Affinity of a Protein-Ligand Complex. . J. Mol. Model. , 4, pp. 379–394.
55. Wingeta, J. M., Pana, Y. H., & Bahnson, B. J. (2006). The interfacial binding surface of phospholipase A2s. Biochimica et Biophysica Acta. , 1761 (11), pp. 1260–1269.
56. Zheng, B., Roach, S. L., & Ismagilov, R. F. (2003). Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets. . J. Am. Chem. Soc. , 125, pp. 11170–11171.
57. Zhou, L., Fang, C., Wei, P., Liu, S., Liu, Y., & Lai, L. (2008). Chemically Induced Dimerization of Human Nonpancreatic Secretory Phospholipase A2 by Bis-indole Derivatives. J. Med. Chem. , 51, pp. 3360–3366.
58. Zhou, X., Lau, L., Lam, W. W., Au, S. W., & Zheng, B. (2007). Nanoliter dispensing method by degassedpoly(dimethylsiloxane) microchannels and its application in protein crystallization. Anal. Chem. , 79, pp. 4924–4930.
59. 分子對接. 擷取自 維基百科: http://zh.wikipedia.org/wiki/%E5%88%86%E5%AD%90%E5%AF%B9%E6%8E%A5
60. 岳俊杰, 馮華, & 梁龍. (2010). 蛋白質結構預測實驗指南. 化學工業出版社.
61. 林榮信. 結構為基礎的虛擬篩選.
62. 盧光瑩, & 華子千. (2006). 生物大分子晶體學基礎. 北京大學出版社.
63. 鄭信民, & 林麗娟. (2002). X光繞射應用簡介. 工業材料雜誌 (181), 頁 100–108.
64. 閻隆飛, & 孫之榮. (1999). 蛋白質分子結構. 清華大學出版社.

連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔