Molecular mechanisms underlying the pharmacological activities of naja sputatrix venom

MOLECULAR MECHANISMS UNDERLYING THE BIOCHEMICAL ACTIVITIES OF NAJA SPUTATRIX VENOM CHARMIAN CHER DYI NI NATIONAL UNIVERSITY OF SINGAPORE 2005 MOLECULAR MECHANISMS UNDERLYING THE BIOCHEMICAL ACTIVITIES OF NAJA SPUTATRIX VENOM CHARMIAN CHER DYI NI B.Sc (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I really need to thank my supervisor, Professor Kandiah Jeyaseelan, for so many years of guidance, teaching, mentorship and support I have learnt so much working with him and am very grateful that he had allowed me to mature and come into my own as a researcher, though at times, I surely brought him to the brink of exasperation Thank you so much, Prof, for not giving up even when I almost did! I am also very grateful to Dr Arunmozhiarasi Armugam for so willingly imparting her technical expertise and for being so patient in teaching me I truly appreciate the fact that she shared her ideas with me and was always ready to hear mine She had helped me in so many other ways and her dedication and generosity are an inspiration My sincerest thanks to Prof Marelyn and Dr Zhu for allowing me to spend time in their labs and for being ever so willing to teach me and answer my many questions Their expert advice and support were invaluable I also want to thank Dr Ram for teaching me all that I know about microarray analysis I could not have come this far without the constant support and encouragement of all my labmates, in particular Siaw Ching, Dawn, Joyce and Yimin In the battle that is ‘PhD’, they shared all my victories and defeats They are the greatest bunch of colleagues and have helped me maintain my sanity till today! Special thanks to Siaw Ching for helping me so much and never complaining, even though she is also up to her neck with work I am eternally thankful to Daddy and Che for the many years of love and support! Thank you so much for having been so selfless in accommodating me Thank you too, Pris, for simply being the best friend anyone could have! Thank you for all your help and for always being there for me I really owe you one! The completion of this thesis is really the culmination of years of sacrifice, unconditional love, quiet support and constant prayer I dedicate it to my mother, who has so patiently waited for me to complete my studies and has given up so much in order that I can pursue my dreams I’ve finally finished! Most importantly, “ in every victory, let it be said of me…that my source of strength, my source of hope…is Christ alone.” Contents Publications i Summary ii Abbreviations iv Chapter 1: Introduction 1.1 Venomous snakes 1.1.1 Snake venoms 1.1.2 Snakebites and treatment 1.2 The elapids 1.3 The cobras 1.4 Naja sputatrix 1.5 Cardiotoxins 10 1.5.1 The classification of cardiotoxins 12 1.5.2 The cloning of cardiotoxin 13 1.5.3 The structure of cardiotoxin 13 1.5.4 Mode of action of cardiotoxin 15 1.5.4.1 Cardiotoxin-membrane interactions 15 1.5.4.2 Mechanisms of cardiotoxin-induced cell lysis 19 1.5.4.3 Cardiotoxin-induced apoptosis 23 1.5.4.4 Modulation of calcium ions by cardiotoxins 24 1.5.5 Putative functional sites in cardiotoxins 26 1.6 Phospholipase A2 29 1.6.1 Catalysis by phospholipase A2 30 1.6.2 Classification and diversity of phospholipase A2 31 1.6.3 The cloning of phospholipase A2 34 1.6.4 Structure of phospholipase A2 35 1.6.4.1 Primary structure 35 1.6.4.2 Secondary and tertiary structures 36 1.6.4.3 Quarternary structure 37 1.6.5 Snake venom phospholipase A2 38 1.6.5.1 Pharmacological properties of snake venom phospholipase A2 40 1.6.5.1.1 Myotoxicity 40 1.6.5.1.2 Inflammatory and edematogenic effects 42 1.6.5.1.3 Presynaptic neurotoxicity 44 1.6.5.1.4 Anti-coagulant activity 45 1.6.5.2 Pharmacological sites on phospholipase A2 46 1.6.5.2.1 Myotoxicity site 47 1.6.5.2.2 Presynaptic neurotoxicity site 48 1.6.5.2.3 Anti-coagulant site 48 1.7 Snake venom and their components as therapeutic agents 49 Fibrinogenolytic and fibrinolytic activity of snake venom 50 1.7.2 Anti-neoplastic activity of snake venom 51 1.7.3 Anti-inflammatory activity of snake venom 51 1.7.4 Other therapeutic activities of snake venom 52 1.8 The study of gene expression 53 1.8.1 Microarrays 54 1.8.1.1 Principles of method 55 1.8.1.2 Data-mining 56 1.8.2 Real-time polymerase chain reaction 57 1.8.2.1 Detection methods 57 1.8.3 Applications of gene expression studies 58 1.8.4 Snake venoms, toxins and gene expression studies 60 1.9 Aims of this study 62 1.7.1 Chapter 2: Materials and Methods 2.1 Materials 64 2.1.1 Animals and anaesthesia 64 2.1.2 Cell lines and culture medium 64 2.1.3 Naja sputatrix venom 65 2.1.4 Buffers and solutions 65 2.1.4.1 Phosphate buffer (0.1M) 65 2.1.4.2 Phosphate-buffered saline (1X) 65 2.1.4.3 SSPE buffer (20X) 66 2.1.4.4 MES stock buffer (12X) 66 2.1.4.5 Tetrazolium-Blue stock solution 66 2.1.4.6 Sodium acetate (3M) 66 2.1.5 Reagents for the isolation of DNA and RNA 66 2.1.6 Reagents for the electrophoresis of DNA 67 2.1.7 Reagents for the electrophoresis of RNA 67 2.1.8 Reagents for oligonucleotide microarray 69 2.1.9 Reagents for real-time PCR 71 2.1.10 Reagents for the isolation of proteins 71 2.1.11 Reagents for Tris-tricine SDS-PAGE 72 2.1.12 Reagents for Western blotting 73 2.1.13 Reagents for histological analysis and immunohistochemistry 74 2.1.14 Reagents for MTT cell viability assay 74 2.1.15 Reagents for propidium iodide/Hoechst nuclear staining 75 2.1.16 Reagents for caspase-3 activity assay 75 2.1.17 Reagents for TUNEL assay 76 2.1.18 Reagents for histological analysis of cerebral ischemia 76 Methods 77 2.2 2.2.1 Purification of phospholipase A2, cardiotoxin and neurotoxin 77 2.2.2 Gene and protein expression studies 77 2.2.2.1 Isolation of total cellular RNA 77 2.2.2.2 Formaldehyde agarose gel electrophoresis of RNA 78 2.2.2.3 Quantitative real-time PCR 78 2.2.2.4 Oligonucleotide microarray 80 2.2.2.5 Protein isolation 88 2.2.2.6 Protein determination using the method of Bradford 89 2.2.2.7 Tris-tricine SDS-PAGE 89 2.2.2.8 Western blotting 90 2.2.2.9 Immunohistochemistry 91 2.2.3 Histological analysis 94 2.2.3.1 Preparation of tissue sections 94 2.2.3.2 Haematoxylin and eosin staining 94 2.2.4 Biochemical assays 94 2.2.4.1 Detection of DNA fragmentation 94 2.2.4.2 MTT cell viability assay 96 2.2.4.3 Propidium iodide/Hoechst nuclear staining 96 2.2.4.4 Assay for the detection of Annexin V 97 2.2.4.5 Assay for the determination of caspase-3 activity 97 2.2.4.6 Assay for the detection of tumor necrosis factor-α 98 2.2.4.7 TUNEL assay 99 2.2.5 Surgical manipulations and physiological measurements 101 2.2.5.1 Tracheostomy 101 2.2.5.2 Permanent middle cerebral artery occlusion 101 2.2.5.3 Transient middle cerebral artery occlusion 102 2.2.5.4 Histological analysis of cerebral ischemia 103 2.2.5.5 Intravenous administration of PLA2 and tPA 104 2.2.5.6 Laser-Doppler flowmetery 105 2.2.5.7 Determination of serum troponin T 105 2.2.5.8 Blood gas analysis 106 2.2.5.9 Surface electrocardiogram (ECG) measurements 106 2.2.6 Statistical analysis 107 2.3 Chemicals and reagents 111 Chapter 3: The molecular basis of cardiotoxicity upon cobra envenomation 3.1 Introduction 114 3.2 Purification of N sputatrix crude venom 116 3.2.1 Gel filtration 116 3.2.2 RP-HPLC 117 3.3 Hierarchical clustering and principle component analysis of expression data 117 3.4 Functional clustering of genes in the heart 120 3.5 Real-time quantitative PCR 121 3.6 Electrocardiographic measurements 122 3.7 Measurement of cardiac troponin T in NS venom-treated mice 123 3.8 Histological analysis of envenomed cardiac tissue 124 3.9 Microarray analysis after PLA2, CTX and NTX treatment 125 Chapter 4: The mode of cardiotoxin-induced cell death in heart cells 4.1 Introduction 139 4.2 Cell viability upon CTX treatment 140 4.3 Cellular morphology after CTX and staurosporin treatment 141 4.4 Detection of oligonucleosomal DNA fragmentation 142 4.5 Analysis of phosphatidylserine externalization 142 4.6 Analysis of cell death by fluorescence microscopy 143 4.7 Assessment of caspase-3 activation 144 Chapter 5: Pulmonary inflammation and edema induced by phospholipase A2 5.1 Introduction 155 5.2 Lung morphology 156 5.3 Histological analysis of pulmonary inflammation and edema 157 5.4 Determination of lung fluid accumulation 158 5.5 Microarray analysis of pulmonary gene expression 158 5.6 Expression of Na+/K+ -ATPase 160 5.7 Expression of AQPs and 161 5.8 Immunohistochemistry 162 5.9 Summary of pathways involved 163 Chapter 6: The neuroprotective effect of phospholipase A2 in a rat model of focal ischemia 6.1 Introduction 175 6.2 Laser-Doppler flowmetry 180 6.3 Intravenous administration of PLA2 confers protection during transient ischemia 181 PLA2 does not confer protection in a permanent model of focal ischemia 182 6.5 MK801, but not tPA, confers neuroprotection 183 6.6 Administration of PLA2 protects neurons from ischemic cell death 184 Gene expression analysis of ischemia and PLA2induced neuroprotection 186 6.8 Real-time quantitation of gene expression 189 6.9 PLA2 does not cause systemic inflammation 190 6.4 6.7 Chapter 7: Discussion 7.1 Snake venom and its constituents 205 7.2 Cobra envenomation induces major gene expression changes in the heart 205 7.3 The cardiotoxicity of cobra venom 212 7.4 Cardiotoxin-induced cell death in cardiomyocytes 217 7.4.1 Classification of the mode of cell death 218 7.4.2 Cardiotoxin induces necrosis in cardiomyocytes 219 7.5 Effects of phospholipase A2 on the lung 223 7.6 Gene expression analysis of pulmonary inflammation and edema 224 7.7 Neuroprotective activity of phospholipase A2 231 7.7.1 Gene expression analysis of focal ischemia and phospholipase A2-mediated neuroprotection 231 Possible mechanisms of phospholipase A2mediated neuroprotection 238 Conclusions 242 7.7.2 7.8 References 244 References Ratcliffe, P.J., O’Rourke, J.F., Maxwell, P.H and Pugh, C.W (1998) Oxygensensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression J Exp Biol 201,1153-1162 Read, S.J., Parsons, A.A., Harrison, D.C., Philpott, K., Kabnick, K., O’Brien, S., Clark, S., Brawner, M., Bates, S., Gloger, I., Legos, J.J and Barone, F.C (2001) Stroke genomics: Approaches to identify, validate, and understand ischemic stroke gene expression J Cereb Blood Flow Metab 21, 755-778 Rees, B., Samama, J.P., Thierry, J.C., Gilibert, M., Fischer, J., Scheweitz, H., Lazdunski, M and Moras, D (1987) Crystal structure of a snake venom cardiotoxin Proc Natl Acad Sci USA 84, 3132-3136 Reid, A H (1964) Cobra-bites Brit Med J 2, 540-545 Reid, A H (1968) Symptomatology, pathology and treatment of land snake bite in India and South East Asia In: Venomous animals and their venoms Bϋcherl, W and Buckley, E.E (eds) Volume I Venomous vertebrates Academic Press, New York, pp 611-642 Reid, H A (1975) Epidemiology and clinical aspects of sea snake bites In: The Biology of Sea Snakes Dunson, W.A (ed) University Park Press, Baltimore pp 417-462 Renetseder, R., Brunie, S., Dijkstra, B.W., Drenth, J and Sigler, P.B (1985) A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom J Biol Chem 260, 11627-11634 Rioux, V., Gerbod, M.C., Bouet, F., Menez, A and Galat, A (1998) Divergent and common groups of proteins in glands of venomous snakes Electrophoresis 19, 788-796 Rodrigues, O.G., Scannone, H.R and Parra, N.D (1974) Enzymatic activities and other characteristics of Crotalus durissus cumaneusis venom Toxicon 12, 297-302 Roelofsen, B (1982) Phospholipases as tools to study the localization of phospholipids in biological membranes J Toxicol Toxin Rev 1, 87-197 Roest, M and Banga, J.D (2003) Editorial comment – genetic make-up for increased PAI-1 expression protects against stroke Stroke 34(12), 2828-2829 Romanic, A.M., White, R.F., Arleth, A.J., Ohlstein, E.H and Barone, F.C (1998) Matrix metalloproteinase expression increases after cerebral focal ischemia in rats Inhibition of matrix metalloproteinase-9 reduces infarct size Stroke 29, 1020-1030 276 References Rosenberg, G.A (1995) Matrix metalloproteinases in brain injury J Neurotrauma 12, 833-842 Rosenberg, G.A., Navratil, M., Barone, F.C., Feuerstein, G.Z (1996) Proteolytic cascade enzymes increase in focal cerebral ischemia in rat J Cereb Blood Flow Metab 16, 360-366 Rosenberg, P (1997) Lethal potency of snake venom phospholipase A2 enzymes In: Venom phospholipase A2 enzymes-Structure, function and mechanism Kini, R.M (ed) John Wiley and Sons Ltd, England pp 129-155 Rothwell, N.J and Hopkins, S.J (1995) Cytokines and the nervous system II: Actions and mechanisms of action Trends Neurosci 18, 130-136 Russel, F.E (1980) Chemistry and pharmacology of snake venom In: Snake venom poisoning Scholium International, New York pp 139-234 Russell, F.E and Dart, R.C (1992) Toxic effects of animal toxins In: Casarett and Doull’s Toxicology, 4th edition Amdur, M.O., Doull, J and Klaassen, C.D (eds) Pergamon Press, Oxford pp 753-803 Sarkar, B.B., Maitra, S.R and Roy, P.K (1946) Ann Biochem Exptl Med (Calcutta) 6, 87 Sarkar, N K (1947) Isolation of cardiotoxin from cobra venom (Naja tripudians, monocellate variant) J Indian Chem Soc 24, 227-232 Sarkar, N K (1951) Action mechanism of cobra venom, cardiotoxin and allied substances on muscle contraction Proc Soc Exp Biol Med 78, 469-471 Saunders, J.J.W (1966) Death in embryonic systems Science 154, 604-612 Schabitz, W.R., Sommer, C., Zoder, W., Kiessling, M., Schwaninger, M and Schwab, S (2000) Intravenous brain-derive neurotrophic factor reduces infarct size and counterregulates Bax and Bcl-2 expression after temporary focal cerebral ischemia Stroke 31, 2212-2217 Schagger, H and von Jagow, G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from to 100kDa Anal Biochem 166, 368-379 Schaller, B and Graf, R (2004) Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy J Cereb Blood Flow Metab 24, 351-371 277 References Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A and Reulen, H.J (1998) A critical re-evaluation of the intraluminal thread model of focal cerebral ischemia Evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by Laser-Doppler flowmetry Stroke 29, 2162-2170 Schuab C.D., Fankersley C., Schwartz A.R., Smith P.L., Robotham J.L and O’Donnell C.P (1998) Effect of sleep/wake state on arterial blood pressure in genetically identical mice J Appl Physiol 85(1), 366-371 Schulze, A and Downward, J (2001) Navigating gene expression using microarrays – a technology review Nature Cell Biol 3, E190-195 Scott, D.L (1997) Phospholipase A2: Structure and catalytic properties In: Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism Kini, R.M (ed) John Wiley & Sons Ltd., UK pp 97-128 Scott, D.L., Achari, A., Vidal, J.C and Sigler, P.B (1992) Crystallographical and biochemical studies of the (inactive) Lys49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus J Biol Chem 267, 22645-22657 Scott, D.L., Otwinowski, Z., Gelg, M.H and Sigler, P.B (1990) Crystal structure of bee-venom phospholipase A2 Science 250, 1563-1566 Scremin, O.U and Jenden, D.J (1996) Cholinergic control of cerebral blood flow in stroke, trauma and aging Life Sci 58(22), 2011-2018 Seilhamer, J.J., Randall, T.L., Johnson, L.K., Heinzmann, C., Klisak, I., Sparkes, R.S and Lusis, A.J (1989) Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes J Cell Biochem 39, 327-337 Seilhamer, J.J., Randall, T.L., Yamanaka, M and Johnson, L.K (1986) Pancreatic phospholipases A2: Isolation of the human gene and cDNAs from porcine pancreas and human lung DNA 5(6), 519-527 Senior, K (1999) Taking the bite out of snake venom Lancet 353, 1946 Serra, P., Amrani, A., Han, B., Yamanouchi, J., Thiersen, S.J and Santamaria, P (2002) RAG-dependent peripheral T cell receptor diversification in CD8+ T lymphocytes Proc Natl Acad Sci USA 99, 15566-15571 Sharma, J.N (2003) Does the kinin system mediate in cardiovascular abnormalities? An overview J Clin Pharmacol 43, 1187-1195 278 References Sharp, J.D., White, D.L Chiou, X.G (1991) Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2 J Biol Chem 266, 1485014853 Sherman, A.M (1990) Neurotoxic snake envenoming Sem in Neurol 10(1), 52-61 Sherman, D.G., Atkinson, R.P., Chippendale, T., Levin, K.A., Ng, K., Futrell, N., Hsu, C.Y and Levy, D.E (2000) Intravenous Ancrod for treatment of acute ischemic stroke: the STAT study: a randomized controlled trial Stroke Treatment with Ancrod Trial JAMA 283, 2395-2403 Shiels, A and Bassnet, S (1996) Mutations in the founder of the MIP gene family underlie cataract development in the mouse Nature Genet 12, 212-215 Shimizu, T., Hansson, G.C and Strandvik, B (1994) Effects of pancreatic and snake venom phospholipase A2 on the generation of leukotriene B4 and C4 by human leucocytes in vitro Pancreas 9(1), 37-41 Shimkets, R.A., Lowe, D.G., Tai, J.T.N., Sehl, P., Jin, H., Yang, R., Predki, P.F., Rothberg, B.E.G., Murtha, M.T., Roth, M.E., Shenoy, S.G., Windemuth, A., Simpson, J.W., Simons, J.F., Daley, M.P., Gold, S.A., McKenna, M.P., Hillan, K., Went, J.T., and Rothberg, J.M (1999) Gene expression analysis by transcript profiling coupled to a gene database query Nature Biotechnol 17, 798-803 Shu, J.S and Trai, M.Y (1999) The dynamic responses of pro-inflammatory and anti-inflammatory cytokines of human mononuclear cells induced by uromodulin Life Sci 65, 2581-2590 Siaghy, E.M., Deraux, Y., Sfaksi, N., Carteaux, J.P., Ungureanu-Longrois, D., Zannad, F., Villemot, J,P,, Burlet, C and Mertes, P.M (2000) Consequences of inspired oxygen fraction manipulation on myocardial oxygen pressure, adenosine and lactate concentrations: a combined myocardial microdialysis and sensitive oxygen electrode study in pigs Mol Cell Cardiol 32(3), 493-504 Siigur, J and Siigur, E (1992) The direct acting fibrin(ogen)olytic enzymes from snake venoms J Toxicol Toxin Review 11, 91 Sim, F.J., Zhao, C., Li, W.W., Lakatos, A and Franklin, J.M (2002) Expression of the POU-domain trabscription factors SCIP/Oct-6 and Brn-2 is associated with Scwann cell but not oligodendrocyte remyelination of the CNS Mol Cell Neurosci 20, 669-682 Singh, G.S and Sanyal, R.K (1967) Cardiovascular and respiratory systems in Elapidae envenomation Ind J Med Res 55(10), 1092-1098 279 References Six, D.A and Dennis, E.A (2000) The expending superfamily of phospholipase A2 enzymes: classification and characterization Biochim Biophys Acta 1488, 1-19 Smith, C.A and Hinman, C.L (2004a) A cyclic peptide, L1AD3, induces early signs of apoptosis in human leukemic T-cell lines J Biochem Mol Toxicology 18(4), 204-220 Smith, C.A and Hinman, C.L (2004b) Evidence that L1AD3, an apoptosisinducing cyclic peptide, binds a leukemic T-cell membrane protein receptor Arch Biochem Biophys 432, 88-101 Smith, L.A and Schmidt, J.J (1990) Cloning and nucleotide sequences of crotaline genes Toxicon 28, 575-585 Smith, W.S (2004) Pathophysiology of focal cerebral ischemia: a therapeutic perspective J Vasc Interv Radiol 15, S3-12 Smrcka, M., Otevrel, F., Kuthtickova, S., Horky, M., Juran, V., Duba, M and Graterol, I (2001) Experimental model of reversible focal ischaemia in the rat Scripta Medica (BRNO) 74(6), 391-398 Soares, A.M., Marcussi, Stabeli, R.G., Franca, S.C., Giglio, J.R., Ward, R.J and Arantes, E.C (2003) Structural and functional analysis of Bmj-MIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake venom Biochem Biophys Res Commun 302, 193-200 Sommers, C.D., Bobbitt, J.L., Bemis, K.G and Snyder, D.W (1992) Porcine pancreatic phospholipase A2-induced contractions of guinea pig lung pleural strips Eur J Pharmacol 216, 87-96 Song, Y., Jayaraman, S., Yang, B., Matthay, M.A., & Verkman, A.S (2001) Role of aquaporin water channels in airway fluid transport, humidification and surface liquid hydration J Gen Physiol 117, 573-582 Spellman, P.T., Sherlock, G., Zhang, M.Q., Iyer, V.R., Ander,s K., Eisen, M.B., Brown, P.O., Botstein, D and Futcher, B (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization Mol Biol Cell 9(12), 3273-97 Sprivulis P and Jelinek G (2000) Toxicology In: Snakebite in Textbook of Adult Emergency Medicine Cameron P, Jelinek G, Kelly AM, Murray L and Heyworth J (ed) Edinburgh, Churchill Livingstone, pp 649-652 280 References Sribar, J., Copic, A., Paris, A., Sherman, N.E., Gubensek, F., Fox, J.W and Krizaj, I (2001) A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin J Biol Chem 276(16), 12493-12496 Steinfeld, S Cogan, E., King, L.S., Agre, P., Kiss, R., Delporte, C (2001) Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sjogren’s syndrome patients Lab Invest 81, 143-148 Steinmetz, W.E., Bougis, P.E., Rochat, H Redwine, O.D., Braun, W and Wuthrich, K (1988) 1H nuclear-magnetic-resonance studies of the threedimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structure of homologous toxins Eur J Biochem 172, 101-116 Stenzel-Poore, M.P., Stevens, S.L., Xiong, Z., Lessov, N.S., Harrington, C.A., Mori, M., Meller, R., Rosenzweig, H.L., Tobar, E., Shaw, T.E., Chu, X and Simon, R.P (2003) Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation nad hypoxiatolerant states Lancet 362, 1028-1037 Stephens, L.L., Walker, J.L and Myers, W (1898) J Pathol Bacteriol 5, 279-301 Stevens-Truss, R and Hinman, C.L (1996) Chemical modification of methionines in a cobra venom cytotoxin differentiates between lytic and binding sites Toxicol Appl Pharmacol 139, 234-242 Stevens-Truss, R and Hinman, C.L (1997) Activities of cobra venom cytotoxins toward heart and leukemic T cells depend on localized amino acid differences Toxicon 35(5), 659-669 Stiles, B.G and Choumet, V (1997) Antibody studies with venom phospholipase s In: Venom Phospholipases A2 enzymes: Structure, Function and Mechanism Kini, R.M (ed) Wiley & Sons Ltd., pp 223-224 Stocker, K (1999) Snake venoms affecting hemostasis and fibrinolysis In: Medical use of snake venom proteins Stocker, K (ed) CRC Press, Boca Raton pp 97 Stocker, K.F (1990) Composition of snake venoms In: Medical Use of Snake Venom Proteins Stocker, K.W (ed) CRC Press, Boca Raton, pp 33-56 Stueven, H., Aprahamian, C Thompson, B., Horwitz, L and Darin, J (1983) Cobra envenomation: An uncommon emergency Ann Emerg Med 12, 636-638 Sturn A., Quackenbush J and Trajanoski Z (2002) Genesis: Cluster analysis of microarray data Bioinformatics 18, 207-208 281 References Su, C., Chang, C and Lee, C.Y (1967) Pharmacological properties of the neurotoxin of cobra venom In: Animal toxins Russell, F.E and Saunders, P.R (eds) Pergamon Press, Oxford pp 259-267 Su, S.H., Su, S.J., Lin, S.R and Chang, K.L (2003) Cardiotoxin-III slectively enhances activation-induced apoptosis of human CD8+ T lymphocytes Toxicol Appl Pharmacol 193, 97-105 Sue, C.S., Jarrell, H.C., Brisson, J.R and Wu, W.G (2001) Dynamic characterization of the water binding loop in the P-type cardiotoxin: Implicatin for the role of the bound water molecule Biochemistry 40, 12782-12794 Sue, S., Rajan, P.K., Chen, T.S., Hsieh, C.H and Wu, W (1997) Action of Taiwan cobra cardiotoxin on membrane: binding modes of a β-sheet polypeptide with phosphatidylcholine bilayers Biochemistry 36, 9826-9836 Sue, S.C., Chien, K.Y., Huang, W.N., Abraham, J.K., Chen, K.M and Wu, W.G (2002) Heparin binding stabilizes the membrane-bound form of cobra cardiotoxin J Biol Chem 277(4), 2666-2673 Sun, J.J and Walker, J.A (1986) Actions of cardiotoxins from Southern Chinese cobra (Naja naja atra) on rat cardiac tissue Toxicon 24, 233-245 Sun, Y.J., Wu, W.G., Chiang, C.M., Hsin, A.Y and Hsiao, C.D (1997) Crystal structure of cardiotoxin V from Taiwan cobra venom: pH-dependent conformational change and a novel membrane-binding motif identified in the threefinger loops of P-type cardiotoxin Biochemistry 36, 2403-2413 Sundell, I.B., Khalil, A.A., Zuzel, M and Theakston, R.D.G (2003) The role of phospholipase A2 in th stimulation of neutrophil motility by cobra venoms Toxicon 41, 459-468 Svanik, N., Westman, G., Wang, D and Kubista, M (2000) Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution Anal Biochem 281, 26-35 Sznajder, J.I., Factor, P and Ingbar, D.H (2002) Lung edema clearance: role of Na+-K+ -ATPase J Appl Physiol 93, 1860-1866 Szold O., Ben-Abraham R., Frolkis I., Sorkine M and Sorkine P (2003) Tumour necrosis factor as a mediator of cardiac toxicity following snake envenomation Crit Care Med 31, 1449-1453 Tamura A., Graham DI, McCulloch J and Teasdale GM (1981) Focal ischaemia in the rat 1: description of technique and early neuropathological consequences following middle cerebral artery occlusion J Cereb Blood Flow Metab 1, 53-60 282 References Tan, N H and Armugam, A (1989a) Isolation and characterization of a lethal acidic phospholipase A2 from Malayan cobra (Naja naja sputatrix) Biochem Int 18, 785792 Tan, N H and Armugam, A (1989b) The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes Biochem Int 19, 803-810 Tan, N H and Armugam, A (1990) In vivo interactions between neurotoxin, cardiotoxin and phospholipases A2 isolated from Malayan cobra (Naja sputatrix) venom Toxicon 28 (10), 1193-1198 Tan, N.H (1982a) Cardiotoxins from the venom of Malayan cobra (Naja naja sputatrix) Arch Biochem Biophy 218, 51-58 Tan, N.H (1982b) Isolation and preliminary characterization of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix) Biochim Biophy Acta 719, 599-605 Tan, N.H (1983a) Improvement of Malayan cobra antivenom Toxicon 21, 75-79 Tan, N.H (1983b) Isolation and characterization of two toxins from the venom of the Malayan cobra (Naja naja sputatrix) Toxicon 21, 201-207 Tan, N.H (1991) The biochemistry of venoms of some venomous snakes of Malaysia- A review Tropical Biomedicine 8, 91-103 Tang, Y., Lu, A., Aronow, B.J., Wagner, K.R and Sharp, F.R (2002) Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainite seizures, hypoglycemia and hypoxia Eur J Neurosci 15, 1937-1952 The National Institute of Neurological Disorders and Stroke rtPA Stroke Study Group (1995) Tissue plasminogen activator for acute ischemic stroke N Engl J Med 333, 1581-1587 Thiagarajan, P and Tait, J.F (1990) Binding of annexinV/placental anticoagulant protein I to platelets Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets J Biol Chem 265, 17420-17423 Thunnissen, M.M., Elso, A.B., Kalk, K.H Drenth, J., Dijkstra, B.W., Kuipers, O.P., Dijkman, R., de Haas, G.H and Verheij, H.M (1990) X-ray structure of phospholipase A2 complexed with a substrate-derived inhibitor Nature 347, 689691 283 References Tilg, H., Vannier, E., Vachino, G., Dinarello, C.A and Mier, J.W (1993) Antiinflammatory properties of hepatic acute phase proteins: Preferential induction of interleukin 1(IL-1) receptor antagonists over IL-1 beta synthesis by human peripheral blood mononuclear cells J Exp Med 178, 1629-1636 Tithof, P.K., Peters-Golden, M and Ganey, P.E (1998) Distinct phospholipase A2 regulates the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils J Immuno 160, 953-960 Tonsing, L., Potgieter, D.J.J., Louw, A.L and Visser, L (1983) The binding of snake venom cardiotoxins to heart cell membranes Biochim Biophys Acta 732, 282-288 Torras, J., Bordalba, J.R., Seron, D., Moliner, R., Carrera, M., Valles, J., MartinezCastelao, A., Alsina, J and Grino, J.M (1993) Protective effect of the PAF antagonist BN52021 in an experimental renal warm ischemia model Tranpl Int 6:236-238 Torres, P.S., Zapata, D.A., Pacheo-Castro, A., Rodriguez-Fernandez, J.L., Cabanas, C and Regueiro, J.R (2002) Contribution of CD3γ to TCR regulation and signaling in human mature T lymphocytes Int Immunol 14, 1357-1367 Towne, J.E., Harrod, K.S., Krane, C.M., & Menon, A.G (2000) Decreased expression of aquaporin (AQP) and AQP5 in mouse lung after acute viral infection Am J Respir Cell Mol Biol 22, 34-44 Towne, J.E., Krane, C.M., Bachurski, C J., & Menon, A.G (2001) Tumour necrosis factor-α inhibits Aquaporin expression in mouse lung epithelial cells J Biol Chem 276(22), 18657-18664 Townson, J.R and Nibbs, R.J.B (2002) Characterization of mouse CCX-CKR, a receptor for the lymphocyte-attacting chemokines TECK/mCCL25, SLC/mCCL21 and MIP-3â/mCCL19 Eur J Immunol 32, 1230-1241 Trumble, W.R, Huang, J.L., Trumble, M.J., Wesson, C., Huang, M and He, Z (1992) Cobra cardiotoxin mediates calcium release from muscle sarcoplasmic reticulum vesicles and binds to specific membrane proteins In: Recent advances in toxinology research, Vol Gopalakrishnakone, P and Tan, C.K (eds) Venom & Toxin Research Group, National University of Singapore, Singapore Trump, B.F., Berezesky, I.K and Cowley, R.A (1982) The cellular and subcellular characteristics of acute and chronic injury with emphasis on the role of calcium In: Pathophysiology of Shock, Anoxia and Ischemia Cowley, R.A and Trump, B.F (ed) Williams and Wilkins, Baltimore pp 6-46 284 References Tsirka, S.E., Rogove, A.D., Bugge, T.H., Degen, J.L and Strickland, S (1997) An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus J Neurosci 17, 543-552 Tsubota, K., Hirai, S., King, L.S., Agre, P and Ishida, N (2001) Defective cellular trafficking of lacrimal gland aquaporin-5 in Sjogren’s syndrome Lancet 357, 688689 Tu, A.T (1977) Venoms: Chemistry and molecular biology John Wiley & Sons, Inc, New York Tweedie, M.W.F (1983) The snakes of Malaya Singapore National Printers, Singapore Tzeng, W.F and Chen, Y.H (1988) Supression of snake-venom cardiotoxininduced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases Biochem J 256, 89-95 Uemura, Y., Kowall, N.W and Moskowitz, M.A (1991) Focal ischemia in rats causes time-dependent expression of c-fos protein immunoreactivity in widespread regions of ipsilateral cortex Brain Res 552, 99-105 Vadas, P and Pruzanski, W (1986) Role of secretory phospholipases A2 in the pathobiology of disease Lab Invest 55, 391-404 Vadas, P., Browning, J., Edelson, J and Pruzanski, W (1993) Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states J Lipid Mediators 8, 1-30 Vadas, P., Pruzanski, W., Kim, J and Fornasier, V (1989) The proinflammatory effect of intra-articular injection of soluble human and venom phospholipase A2 Am J Pathol 134, 807-811 Valentin, E and Lambeau, G (2000) What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2? Biochimie 82, 815-831 Van Hoogevest, P Du Maine, A.P., de Kruiff, B and De Gier, J (1984) The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles Biochim Biophys Acta 777, 241-252 Vanden Heuvel, J.P., Tyson, F.L and Bell, D.A (1993) Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR Biotechniques 14, 395-398 285 References Velculescu, V.E., Zhang, L., Vogelstein, B and Kinzler, K.W (1995) Serial analysis of gene expression Science 270, 484-487 Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Yandell, M., Evans, C.A., Holt, R.A., Gocayne, J.D., Amanatides, P., Ballew, R.M et al (2001) The sequence of the human genome Science 291, 13031351 Verger, R., Mieras, M.E.C and de Haas, G.H (1973) Action of phospholipase A2 at interfaces J Biol Chem 248, 4023-4034 Verheij H.M (1996) Structure and mechanism of pancreatic phospholipase A2 – A molecular biology approach In: Phospholipases in Clinical Inflammation Glaser, K.B and Vadas, P (ed) CRC Press, Boca Raton, pp 3-24 Verheij, H.M., Slotboom, A.J and de Haas, G.H (1981) Structure and function of phospholipase A2 Rev Physiol Biochem Pharmacol 91, 91-203 Verkleij, A.J., Zwaal, R.F., Roelofsen, B., Comfurius, P., Kastelijn, D and van Deenen, L.L (1973) The asymmetric distribution of phospholipids in the human red cell membrane A combined study using phospholipases and freeze-ethch electron microscopy Biochim Biophys Acta 323, 178-193 Vincent, J.P., Balerna, M and Lazdunski, M (1978) Properties of association of cardiotoxin with lipid vesicles and natural membranes A fluorescence study FEBS Lett 85, 103-108 Viravan, C., Veeravat, U., Warrell, M.J., Theakston, R.D.G and Warrell, D.A (1986) ELISA confirmation of acute and past envenoming by the monocellate Thai cobra (Naja kaouthia) Am J Trop Med Hyg 35, 173-181 Vyas, A.A., Pan, J.J., Patel, H.V., Vyas, K.A., Chiang, C.M., Sheu, Y.C., Hwang, J.K and Wu, W.G (1997) Analysis of binding of cobra cardiotoxins to heparin reveals a new β-Sheet heparin-binding structural motif J Biol Chem 272, 96619670 Wade, D Andreu, D., Mitchell, S.A., Silveira, A.M.V., Boman, A., Boman, H.G and Merrifield, R.B (1992) Antibacterial peptides designed as analogs or hybrids of cecropins and melittin Int J peptide Protein Res 40, 429-436 Wahlgren, N.G and Ahmed, N (2004) Neuroprotection in cerebral ischaemia: facts and fancies – the need for new approaches Cerebrovasc Dis 17(Suppl 1), 153166 Waite, M (1987) The Phospholipases Plenum Press, New York 286 References Walsh, S., Anderson, M and Cartinhour, S.W (1998) ABCDE: a database for genome information Methods Biochem Anal 39, 299-318 Walther, T., Olah, L., Harms, C., Maul, B., Bader, M., Hortnagl, H., Schultheiss, H.P and Mies, G (2002) Ischemic injury in experimental stroke depends on angiotensin II FASEB J 16, 169-176 Wang, H., Zhan, Y., Xu, L., Feuerstein, G.Z and Wang, X (2001) Use of suppression subtractive hybridization for differential gene expression in stroke: discovery of CD44 gene expression and localization in permanent focal stroke in rats Stroke 32(4), 1020-1027 Wang, H.X., Lau, S.Y., Huang, S.J., Kwan, C.Y and Wong, T.M (1997) Cobra venom cardiotoxin induces perturbations of cytosolic calcium homeostasis and hypercontracture in adult rat ventricular myocytes J Mol Cell Cardiol 29, 27592770 Wang, X.K., Louden, C., Yue, T.L., Ellison, J.A., Barone, F.C., Solleveld, H.A and Feuerstein, G.Z (1998a) Delayed expression of osteopontin after focal stroke in the rat J Neurosci 18(6), 2075-2083 Wang, X.K., Barone, F.C., White, R.F and Feuerstein, G.Z (1998b) Subtractive cloning identifies tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) increased gene expression following focal stroke Stroke 29, 516-520 Wang, X.K., Xu, L., Wang, H., Zhan, Y.T., Pure, E and Feuerstein, G.Z (2002) CD44 deficiency in mice protects brain from cerebral ischemia injury J Neurochem 83, 1172-1179 Wang, X.K., Yue, T.L., Young, P.R., Barone, F.C and Feuerstein, G.Z (1995a) Expression of interleukin-6, c-fos and zif268 mRNA in rat ischemic cortex J Cereb Blood Flow Metab 15, 166-171 Wang, X.K., Yue, T.L., Barone, F.C and Feuerstein, G.Z (1995b) Monocyte chemoattractant protein-1 (MCP-1) mRNA expression in rat ischemic cortex Stroke 26, 661-666 Wang, Y.F., Tsirka, S.E., Strickland, S., Stieg, P.E., Soriano, S.G and Lipton, S.A (1998) Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischemia in wild-type and t-PA-deficient mice Nat Med 4, 228-231 Warrell D.A (1986) Tropical snakebites: clinical studies in Southeast Asia In: Natural Toxins Harris J.B (ed) Clarendon Press, Oxford pp 25-45 287 References Warrell, D.A (1996) Clinical features of envenoming from snake bites In: Envenomings and Their Treatments Bon, C and Goyffon, M (ed) Fondation Marcel Merieux, Lyon pp 63-76 Weeraratna, A.T., Nagel, J.E., Mello-Coelho, V.D and Taub, D.D (2004) Geneexpression profiling: From microarrays to medicine J Clin Immunol 24(3), 213224 Wei, J., Walton, E.A., Millici, A and Buccafusco, J.J (1994) m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC J Neurochem 63, 815821 Wei, M., Wang, X., Asahi, M., Kano, T., Asahi, K., Ackerman, R.H and Lo, E.H (1999) Effects of tissue-type plasminogen activator in embolic versus mechanical models of focal cerebral ischemia in rats J Cereb Blood Flow Metab 19, 13161321 Weil, M., Jacobson, M.D., Coles, H.S.R., Davies, T.J., Gardner, R.L., Raff, K.D and Raff, M.C (1996) Constitutive expression of the machinery for programmed cell death J Cell Biol 133, 1053-1059 Weisbrot-Lefkowitz, M., Reuhl, K., Perry, B., Chan, P.H., Inouye, M and Mirochnitchenko, O (1998) Overexpression of human glutathione peroxidase protects transgenic mice against focal cerebral ischemia/reperfusion damage Mol Brain Res 53, 333-338 Wetzel, W.W and Christy, N.P (1989) A king cobra bite in New York City Toxicon 27, 393-395 Whitcombe, D., Theaker, J., Guy, S.P., Brown, T and Little, S (1999) Detection of PCR products using self-probing amplicons and fluorescence Nat Biotechnol 17, 804-807 White, J (1995) Clinical toxicology of snakebite in Australia and New Guinea, In: Handbook of Clinical Toxicology of Animal Venoms and Poisons Meier, J and White, J (ed) CRC Press, Florida pp 595-617 Wilhelm, J and Pingoud, A (2003) Real-time Polymerase Chain Reaction ChemBioChem 4, 1120-1128 Willingham, M.C (1999) Cytochemical methods for the detection of apoptosis J Histochem Cytochem 47(9), 1101-1110 Winzeler, E., Shoemaker, D,D,, Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J.D., Bussey, H., Chu, A.M et al (1999) Functional characterization of the Saccharomyces cerevisiae genome by precise 288 References deletion and parallel analysis Science 287, 873-880 Wolfsberg, T.G (1999) Candidate regulatory sequence elements for cell cycledependent transcription in Saccharomyces cerevisiae Genome Res 9(8), 775-792 Wu, W.G (1998) Cobra cardiotoxin and phospholipase A2 as GAG-binding toxins On the pathway from structure to cardiotoxicity and inflammation Trends Cardiovasc Med 8, 270-278 Wuster, W and Thorpe, R.S (1987) In: Proc 4th Ordinary General Meeting of the Societas Europea herpetological Van Gelder, J.J., Strijbosch, H and Bergers, P.J.M (ed) Faculty of Science, Nijmegen pp 449-464 Wyllie, A.H (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation Nature 284, 555-556 Wyllie, A.H., Kerr, J.F.R., Currie, A.R (1980) Cell death: the significance of apoptosis Int Rev Cytol 68, 251-306 Yamada, M., Lamping, K.G., Duttaroy, A., Zhang, W., Cui, Y., Bymaster F.P., McKinzie D.L., Felder, C.C., Deng, C.X., Faraci, F.M and Wess, J (2001) Cholinergic dilation of cerebral blood vessels is abolished in M5 muscarinic acetylcholine receptor knockout mice Proc Natl Acad Sci USA 98(24), 1409614101 Yang, C.C (1994) Structure-function relationship of phospholipase A2 from snake venoms J Toxicol Toxin Rev 13, 125-177 Yang, C.C (1997) Chemical modification and functional sites of phospholipase A2 In: Venom Phospholipases A2 enzymes: Structure, Function and Mechanism Kini, R.M (ed) Wiley & Sons Ltd., UK pp 185-204 Yang, G., Chan, P.H., Chen, S.F., Babuna, O.A., Simon, R.P and Weinstein, P.R (1994) Reduction of vasogenic edma and infarction by MK-801 in rats after temporary focal cerebral ischemia Neurosurgery 34(2), 339-345 Ye, Z.R., Liu, K.F and Garcia, J.H (2001) Mechanisms of neuronal cell death after ischemic brain injury In: Current Review of Cerebrovascular Disease Fisher, M and Bogousslavsky, J (ed) Current Medicine, Philadelphia pp 15-24 Yedgar, S., Lichtenberg, D., & Schnitzer E (2000) Inhibition of phospholipase A2 as a therapeutic target Biochim Biophy Acta 1488, 182-187 Yeo, M.S., Jeyaseelan, K., Chung, M.C., Gopalakrishnakone, P., Tan, C.H and Wong, H.A (1993) Molecular cloning of a cardiotoxin structural gene from 289 References Malayan spitting cobra (Naja naja sputatrix) Toxicon 31(1), 53-60 Yuan, J., Shaham, S., Ledoux, S Ellis, H.M and Horvitz, H.R (1993) The C elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin1b-converting enzyme Cell 75, 641-652 Zamorano, P.L., Mahesh, V.B and Brann, D.W (1996) Quantitative RT-PCR for neuroendocrine studies Neuroendocrinology 63, 397-407 Zhang, C and Gopalakrishnakone, P (1999) Histopathological studies of the acute inflammation in synovial tissue of rat knee joint following intra-articular injection of PLA2 from Chinese cobra (Naja naja atra) venom Toxicon 37, 783-799 Zhang, F., Xu, S and Iadecola, C (1995) Role of nitric oxide and acetylcholine in neocortical hyperemia elicited by basal forebrain stimulation: evidence for an involvement of endothelial nitric oxide Neuroscience 69, 1195-1204 Zhang, H., Hoang, T., Saeed, B and Ng, S.C (1996) Induction of apoptosis in prostatic tumor cell line DU14S by staurosporine, a potent inhibitor of protein kinases Prostate 26, 69-76 Zhao, Q et al (1994) Acta Physiol Scand 152, 349-350 Zhao, S., Du, X.Y., Chai, M Q., Chen, J.S., Zhou, Y.C and Song, J.G (2002) Secretory phospholipase A2 induces apoptosis via a mechanism involving ceramide generation Biochim Biophy Acta 1581, 75-88 Zheng, W., Aschner, M and Ghersi-Egea, J.F (2003) Brain barrier systems : A new frontier in metal neurotoxicological research Toxicol Appl Pharmacol 192, 1-11 Zivin, J.A., Fisher, M., DeGirolami, U., Hemenway, C.C and Stashak, J.A (1985) Tissue plasminogen activator reduces neurological damage after cerebral embolism Science 230, 1289-1292 Zivin, J.A., Lyden, P.D., DeGirolami, U., Kochhar, A., Mazzarella, V., Hemenway, C.C and Johnston, P (1988) Tissue plasminogen activator: reduction of neurologic damage after experimental embolic stroke Arch Neurol 45, 387-391 290 ... symptoms of envenomation and the ultimate severity of a bite depend on the size and species of the snake, the amount and degree of toxicity of the venom injected, Introduction the location of the. . .MOLECULAR MECHANISMS UNDERLYING THE BIOCHEMICAL ACTIVITIES OF NAJA SPUTATRIX VENOM CHARMIAN CHER DYI NI B.Sc (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF. .. depends on a number of factors such as the curvature of the layer, the physical state of the lipids and the presence of other molecules A site independent of the catalytic site, the interfacial recognition