Phospholipase A2(PLA2)廣泛的存在於各種蛇毒中，而其酵素活性對細胞膜的破壞所扮演的角色及其重要性一直備受爭議。 因此本論文以脂質體模擬細胞膜，排除接受器(receptor)及膜蛋白的影響，討論酵素活性是否對細胞膜產生破壞及其可能的機制。 結果顯示，雖然在有酵素活性的狀態下Naja naja atra phospholipase A2 (NNA-PLA2)及Notexin 破壞脂質體的能力較高，但在以Sr+2、Ba+2 抑制酵素活性的狀態下，NNA-PLA2 及Notexin依然具有破壞脂質體的能力，此外Notexin 的Lys-82 和Lys-115 經化學修飾後，其酵素活性不受影響，然而破壞脂質體的能力卻明顯下降。Circular Dichroism、acrylamide quenching 及Tryptophan fluorescence lifetime assay 實驗中亦發現Notexin 的二級結構會受脂質體誘導產生變化且NNA-PLA2 及Notexin的 tryptophan residues 所處微環境亦產生改變。 在Rhodamine self-quenching 的實驗結果顯示NNA-PLA2 及Notexin 均會形成oligomer，而以ρ-bromophenacyl bromide 作化學修飾得到的BPB-PLA2 僅以monomer 存在，並且亦無法破壞脂質體。 根據上述結果可以得知，NNA-PLA2 與Notexin 的酵素活性並不是造成細胞膜破壞的決定因素，且破膜能力可能與oligomer 之形成有關。

Phospholipase A2 (PLA2) extensively exists in various snake venom. Till now, a controversy remained to elucidate whether the PLA2 activity exclusively associates with the manifestation of the pharmacological activities. In the present study, we used liposome to imitate cell membrane for excluding the effects of receptor and membrane proteins, and estimating the molecular mechanism of snake venom phospholipase A2 on damaging liposome. Although a greater membrane damaging activity of Naja naja atra phospholipase A2 (NNA-PLA2) and notexin was noted in the presence of Ca2+, inhibitions of PLA2 activity by Sr2+ and Ba+2 were unable to abolish the membrane damaging effect. In addition, modification of Lys-82 and Lys-115 of notexin retained the full PLA2 activity, but the membrane damaging activity notably decreased. Fluorescence quenching studies, CD measurement, and tryptophan fluorescence lifetime assay indicated that liposome induced the α-helix conformation change and the tryptophan residues microenviroment change with the addition of Ca2+, Sr2+ or EDTA. Rhodamine quenching assay revealed that NNA-PLA2 and notexin formed oligomers when they bound with liposome. Besides, the modified PLA2 (BPB-PLA2) only formed monomer when it bound with liposome and lost the membrane damaging activity. Taken together, these results indicate that the membrane damaging effects of NNA-PLA2 and notexin are not critically caused by their enzymatic activitys and are probably associated with oligomerization.

目錄.......................................................... 1
摘要.......................................................... 2
Abstract .................................................. 3
英文縮寫.................................................. 4
前言.......................................................... 5
實驗材料................................................ 10
材料與方法............................................ 11
結果........................................................ 18
討論........................................................ 27
參考文獻................................................ 32
圖............................................................ 36
附錄........................................................ 53

Banerjee, J., Singh, J., Joshi, M.C., Ghosh, S., and Banerjee, N. (2006) The cytotoxic fimbrial structural subunit of Xenorhabdus nemtophila is a pore-forming toxin. J. Bacteriol., 188, 7957-7962
Bell, J.D., Sanchez, S.A., and Hazlett T.L. (2003) Liposomes in the study of phospholipase A2 activity, Methods in enzymology, 19-39, Elsevier Inc.
Bortoleto-Bugs, R.K., Neto, A.A., and Ward, R.J. (2004) Activation of Ca+2-independent membrane-damaging activity in Lys49-phospholipase A2 promoted by amphiphilic molecules. Biochem. Biophys. Res. Commun., 322, 364-372
Chang, L.S., Kuo, K.W., and Chang, C.C. (1993) Identification of tryptophan residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.Biochim. Biophys. Acta., 1202, 216-220
Chang, L.S., Wen, E.Y., and Chang, C.C. (1996a) The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2. J. Protein Chem., 15,255-260
Chang, L.S., Lin, S.R., and Chang, C.C. (1996b) The essentiality of calcium ion in the emzymatic activity of Taiwan cobra phospholipase A2. J. Protein Chem., 15, 701-707
Chioato, L., Aragao, E.A., Ferreira, T.L., Ivo de Medeiros, A., Faccioli, L.H., and Ward, R.J. (2007) Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis. Biochem. Biophys. Acta, 1768, 1247-1257
Cumming, B.S., Mchowat, J., and Schnellmann R.G. (2000) Phospholipase A2s in cell injury and death. J. Pharmacol. Exp. Ther., 294, 793-399
Dennis, E.A. (1994) Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem., 269, 13057-13060
Ellens, H., Bentz, J., and Szoka, F.C. (1985) H+- and Ca+2-induced fusion and destabilization of liposomes. Biochemistry, 24, 3099-3106
Fenard, D., Lambeau, G., Valentin, E., Lefebvre, J.C., Lazdunski, M., and Doglio A. (1999) Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells. J. Clin. Invest., 104, 611-618
Forouhar, F., Huang, W.N., Liu, J.H., Chien, K.Y., Wu, W.G., and Hsiao, C.D. (2003) Structural basis of membrane-induced cardiotoxin A3 oligomerization. J. Biol. Chem., 278, 21980-21988
Ghomashchi, F., Lin, Y., Hixon, M.S., Yu, B.Z., Annand, R., Jain, M.K., and Gelb, M.H. (1998) Interfacial recognition by bee venom phospholipase A2 : insights into nonelectrostatic molecular determinants by charge reversal mutagenesis. Biochemistry, 37, 6697-6710
Golding, C., Senior, S., Wilson, M.T., and O’Shea, P. (1996) Time resolution of binding and membrane insertion of a mitochondrial signal peptide: correlation with structural changes and Evidence for cooperativity. Biochemistry, 35, 10931-10937
Gorbenko, G.P., Ioffe, V.M., and Kinnunen P.K.J. (2007) Binding of lysozyme to phospholipids bilayers:evidence for protein aggregation upon membrane association. Biophys. J., 93, 140-153
Graddis, T.J., Myszka, D.G.., Chaiken, I.M. (1993) Controlled formation of model homo- and heterodimer coiled coil polypeptides. Biochemistry, 32, 12664-12671
Kini, R.M. (1997) Venom phospholipase A2 enzymes: structure, function and mechanism., John Wiley & Sons Ltd, West Sussex, England
Kroes, S.J., Canters, G.W., Gilardi, G., Hoek, A., and Visser, A.J.W.G. (1998) Time-resolved fluorescence study of Azurin variants: conformational hererogeneity and tryptophan mobility. Biophys. J., 75, 2441-2450
Ladokhin, A.S., Selsted, M.E., and White, S.H. (1997) Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by Melittin. Biophys. J., 72, 1762-1766
Maria, L., Vind , J., Oxenboll, K.M., Svendsen, A., and Patkar, S. (2007) Phospholiase and their industrial applications. Appl. Microbiol. Biotechnol., 74, 190-300
Mora, R., Valverde, B., Diaz, C., Lomonte, B., and Gutierrez, J.M. (2005) Calcium plays a key role in the effects induced by a snake venom Lys49 phospholipase A2 homologue on a lymphoblastoid cell line. Toxicon, 47, 75-86
Martinez, D., Otero, A., Alvarez C., Pazos, F., Tejuca, M., Lanio, M.E., Ion, G.A., Barlic, A., Iloro, I., Arrondo, J.L., Juan Manuel, G.M., and Lissi, E. (2007) Effect of sphingomyelin and cholesterol on the interaction of St II with lipidic interfaces. Toxicon, 49, 68-81
Nemec, K.N., Pande, A.H., Qin, S., Bieber Urbauer, R.J., Tan, S., Moe, D., and Tatulian S.A. (2006) Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2. Biochemistry, 45, 12448-12460
Nicol, F., Nir, S., and Szoka, F.C. (1996) Effect of Cholesterol and charge on pore formation in bilayer vesicles by a pH-sensitive peptide. Biophys. J., 71, 3288-3301
Qin, S., Pande, A.H., Nemec, K.N., and Tatulian S.A. (2004) The N-terminal α-Helix of pancreatic phospholipase A2 determines productive-mode orientation of the enzyme at the membrane surface. J. Mol. Biol., 344, 71-89
Qin, S., Pande, A.H., Nemec, K.N., and Tatulian S.A. (2005) Evidence for the regulatory role of the N-terminal helix of secretory phospholipase A2 from studies on native and chimeric proteins. J. Biol. Chem., 280, 36773-36783
Rigoni, M., Schiavo, G., Weston, A.E., Caccin, P., Allegrini, F., Pennuto, M., Valtorta, F., Montecucco, C., and Rossetto, O. (2004) Snake presynaptic neurotoxins with phospholipase A2 activity induce punctuate swellings of neuritis and exocytosis of synaptic vesicles. J. Cell Sci., 117, 3561-3570
Rufini, S., Pedersen, J.Z., Desideri A., and Luly, P. (1990) β-Bungarotoxin-mediated liposome fusion: spectroscopic characterization by fluorescence and ESR. Biochemistry, 29, 9644-9651
SÁ, J.M., Chioato, L., Ferreira, T.L., De Oliveira, A.H., Ruller, R., Rosa, J.C., Greene, L.J., and Ward, R.J. (2004) Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity. Biochem. J., 382, 191-198
Sharpe, J.C., and London, E. (1999) Diphtheria toxin forms pores of different size depending on its concentration in membranes: probable relationship to oligomerization. J. Membrane Biol., 171, 209-221
She, M., Dong, W.J., Umeda, P.K., and Cheung H.C. (1997) Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle Troponin C. Biophys. J., 73, 1042-1055
Six, D.A., and Dennis, E.A. (2000) The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim. Biophys. Acta., 1488, 1-19
Tatulian, S.A., Steczko, J., and Minor W. (1998) Uncovering a calcium-regulated membrane binding mechanism for soybean lipoxygenase-1. Biochemistry, 37, 15481-15490
Tatulian, S.A. (2001) Toward understanding interfacial activation of secretory phospholipase A2 (PLA2): membrane surface properties and membrane-induced structural changes in the enzyme contribute synergistically to PLA2 activation. Biophys. J., 84, 1773-1783
Tatulian, S.A. (2003) Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites. Biophys. J., 84, 1773-1783
Tatulian, S.A., Qin, S., Pande, A.H., and He, X. (2005) Positioning membrane proteins by novel protein engineering and biophysical approaches. J. Mol. Biol., 351, 939-947
Welches, W., Reardon, I., Heinrikson, R.L. (1993) An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom of Agkistrodon p. piscivorus. J. Protein Chem., 12, 187-193.
Yang, C.C., and Chang L.S. (1988) Role of the N-terminal region in phospholipases A2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra)venoms. Toxicon, 26, 721-731
Yang, C.C., and Chang, L.C. (1989) Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom. Biochem. J., 262, 855-860