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±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.
Highlights
Out of the four genera of venomous snakes in Brazil (Bothrops, Crotalus, Lachesis and Micrurus), only Crotalus and Micrurus cause failure of the neuromuscular junction, producing peripheral muscular weakness
Bothrops neuwiedi pauloensis (BNP) venom was initially separated into 8 fractions by molecular exclusion (Figure 1A)
Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of ∼14 and ∼28kDa, respectively (Figure 1B, inset)
Summary
Out of the four genera of venomous snakes in Brazil (Bothrops, Crotalus, Lachesis and Micrurus), only Crotalus and Micrurus cause failure of the neuromuscular junction, producing peripheral muscular weakness. Cogo et al [9] reported the neurotoxicity of Bothrops insularis venom on mice and chicks and on mouse phrenic nerve-diaphragm (MPND) and chick biventer cervicis (CBC) isolated preparations. A neurotoxic presynaptic PLA2 fraction, which blocked indirectlyevoked twitches in CBC but did not affect the responses to acetylcholine (ACh) and KCl, was isolated from this venom [10]. Zamuner et al [53] reported that B. neuwiedi venom caused head-drop, loss of balance and respiratory failure in chicks, after an i.m. dose of 0.55 mg/kg. We have observed that the presynaptic neurotoxicity of BNP on MPND is Ca2+-dependent and accompanied by a pronounced increase in the frequency of mepps and the presence of giant mepps [12].
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