本論文利用 Taiwan cobra PLA2 (NNA-PLA2) 及其氮端突變蛋白(M-PLA2, PLA2(N1M), PLA2(△N7)) 和具有與 NNA-PLA2 相同氮端區域序列的 Bungarus multicinctus PLA2 (BM-PLA2) 探討氮端區域參與磷脂酶 A2活性作用之分子機制。結果顯示氮端區域的突變對 Ca2+ 和 ANS 結合能力沒有明顯影響，但造成酵素活性急遽下降，對二級結構有顯著改變。相較於其他突變蛋白，移除氮端區域前七個胺基酸 (PLA2(△N7)) 造成Ca2+-binding site 細微結構的變化。由螢光分析、化學修飾及利用 detergent進行結構變性分析顯示移除氮端區域對 active site 和受質結合部位結構明顯改變，而以 Met 取代 Asn-1 (PLA2(N1M)) 對 NNA-PLA2 結構影響最小。以脂質體模擬細胞進行分析顯示，突變蛋白中以 PLA2(N1M) 破壞細胞膜能力最顯著約為NNA-PLA2 89%，而 PLA2(△N7) 則幾乎無破壞細胞膜能力，經化學修飾之 NNA-PLA2 破壞細胞膜能力與酵素活性間不具有相關性，但 Trp-18 、 Trp-61 、 Lys-65 、 Tyr-3和Tyr-63 與磷脂酶 A2 破壞細胞膜能力有關，而磷脂酶 A2 除直接水解磷脂質能力外，另有活性區域透過非磷脂酶 A2 活性方式導致細胞膜的破壞。綜合以上結果，本篇論文顯示氮端區域與維持磷脂酶 A2 酵素活性和非磷脂酶A2 酵素活性構形有關。

The goal of the present study is to explore the functional involvement of the N-terminal region in the biological activity of phospholipase A2 (PLA2) enzyme. Native PLA2 from the venoms of Naja naja atra and Bungarus multicinctus and N-terminally mutated N. naja atra PLA2, i.e. an additional Met before Asn-1(M-PLA2), substitution of Asn-1 with Met-1(PLA2(N1M)) and removal of N-terminal seven residues (PLA2(△N7)), were employed in this study. Mutations on the N-terminal region insignificantly perturbed the binding ability of PLA2 for Ca2+ and ANS, but the enzymatic activity of mutants drastically decreased. Moreover, an alteration in the secondary structure was observed as revealed by CD spectra. Compared to other mutants, the fine structure of Ca2+-binding site within PLA2(△N7)) changed. Additionally, removal of the N-terminal region caused significant alternation in the structures of active site and substrate-binding site as evidenced by the results of fluorescence measurement, chemical modification and denaturation with detergents. In all N-terminal mutants, substituting Ans-1 with Met-1 affected the NNA-PLA2 structure to a least extent. The membrane-damage activity of PLA2(N1M) and M-PLA2 was 89% and 34% that of NNA-PLA2, respectively. PLA2(△N7) did not exhibit the membrane-damage activity. Studies on the biological activities of chemically modified N. naja atra PLA2 reflected a dissociation of the enzymatic activity from membrane-damage activity, and suggested the involvement of Trp-18, Trp-61, Lys-65, Tyr-3 and Tyr-63 in membrane-damage activity. Collectively, our data indicate that the intact N-terminus was crucial for maintaining of the functional conformation of PLA2 in the manifestation of the enzymatic activity and membrane-damage activity, and the enzymatic activity of PLA2 is in aid of but not exclusively essential for the membrane-damage effect.

文摘要…………………………………………………………………i
英文摘要…………………………………………………………………ii
英文縮寫表……………………………………………………………...iv
序言………………………………………………………………………1
實驗材料…………………………………………………………………5
材料與方法………………………………………………………………7
結果……………………………………………………………………..25
討論……………………………………………………………………..39
參考文獻………………………………………………………………..44
表………………………………………………………………………..48
圖………………………………………………………………………..51
附錄……………………………………………………………………..66

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