Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom



Document title: Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom
Journal: The journal of venomous animals and toxins including tropical diseases
Database: PERIÓDICA
System number: 000299004
ISSN: 1678-9199
Authors: 1
2
3

4

5
Institutions: 1Universidade Estadual de Campinas, Faculdade de Ciencias Medicas, Campinas, Sao Paulo. Brasil
2Universidade Estadual de Campinas, Instituto de Biologia, Campinas, Sao Paulo. Brasil
3Universidade de Sao Paulo, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto, Sao Paulo. Brasil
4Universidade de Sao Paulo, Faculdade de Medicina de Ribeirao Preto, Ribeirao Preto, Sao Paulo. Brasil
5Universita di Ancona, Istituto di Scienza Sperimentale e Clinica, Ancona, Marche. Italia
Year:
Volumen: 13
Number: 1
Pages: 103-121
Country: Brasil
Language: Inglés
Document type: Artículo
Approach: Experimental
English abstract Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (µBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10µg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0&plusmn;8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2
Disciplines: Química,
Biología
Keyword: Bioquímica,
Reptiles,
Veneno de víbora,
Neurotoxinas,
Purificación,
Secuenciamiento,
Neuwieditoxina,
Unión neuromuscular,
Bothrops neuwiedi pauloensis,
Viperidae
Keyword: Chemistry,
Biology,
Biochemistry,
Reptiles,
Snake venom,
Neurotoxins,
Purification,
Sequencing,
Neuwieditoxin,
Neuromuscular junction,
Bothrops neuwiedi pauloensis,
Viperidae
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