Pharmacological characterization of mikatoxin, an α-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka
Introduction
The New-Guinean small-eyed snake (Micropechis ikaheka; Elapidae), which is widely distributed throughout Papua New Guinea, Irian Jaya and adjacent Indonesian islands, has been implicated in many cases of severe and fatal envenoming (Warrell et al., 1996). The predominant clinical features of envenoming by M. ikaheka include peripheral neurotoxic symptoms, generalized myalgia, spontaneous systemic bleeding, incoagulable blood and passage of very dark urine (Hudson and Pomat, 1988, Warrell et al., 1996). However, despite its clinical importance, the venom of this elapid has not been characterized in detail. Preliminary in vitro studies have found the venom of M. ikaheka to show high phospholipase A2 (PLA2) enzyme activity, which could probably account for the myotoxic and anticoagulant effects that followed envenomation by this snake (Kamigutti et al., 1995, Geh et al., 1997). Myoglobinuria, suspected to be the cause of the dark urine following envenomation, was also attributed to the PLA2 activity of the venom (Warrell et al., 1996). Recently, Gao et al. (1999) from our laboratory, purified to homogeneity, a novel PLA2 (MiPLA-1) from the venom of M. ikaheka which produced dark urine in mice, attributable to haemoglobin and not myoglobin. This was the first report of a haemoglobinuria-inducing toxin from snake venom and probably contributes to the dark urine seen in patients after envenomation. Subsequently, Gao et al. (2001) isolated three new PLA2 isoenzymes (MiPLA-2, MiPLA-3 and MiPLA-4) from this snake venom that produced rapid concentration-dependent myonecrosis in mice.
The myotoxic effects of PLA2 enzymes can also contribute significantly to producing neuromuscular block (Rowan et al., 1989, Geh et al., 1992). Furthermore, snake venom PLA2 enzymes are also known to produce neuromuscular block as a consequence of a pre-synaptic mechanism (Strong et al., 1976). However, Geh et al. (1997) have suggested that the main effect of the crude venom of M. ikaheka on neuromuscular transmission in humans is likely to be on postsynaptic acetylcholine receptors. We were, therefore, interested in identifying the component(s) in this snake venom responsible for mediating its effects on neuromuscular transmission and the mechanism by which they produce neurotoxicity. Here, we report the purification and pharmacological characterization of a new neurotoxin, called mikatoxin, from M. ikaheka venom (MiV) that produces irreversible, postsynaptic neuromuscular blockade in vitro, in the absence of myotoxicity associated with phospholipase A2 activity. Preliminary data of part of this work was presented as an abstract at the 13th World Congress on Animal, Plant and Microbial Toxins, Paris, September 2000.
Section snippets
Materials
Lyophilised MiV was obtained from Venom Supplies, (Tanunda, SA, Australia). The pre-packed columns, Superdex 30 and Sephasil C8 were purchased from Pharmacia Biotech (Sweden). The following chemicals were purchased from the sources indicated: reagents for N-terminal sequencing (Applied Biosystem, Foster City, CA, USA), acetonitrile (Fisher Scientific, Fair Lawn, NJ, USA), 4-vinlypyridine (Sigma, St Louis, MO, USA) and trifluoroacetic acid (Fluka Chemika-Biochemika, Switzerland). All other
Preliminary studies on M. ikaheka venom
Preliminary pharmacological studies (n=3) showed that MiV (3 μg/ml) produced complete neuromuscular block in the CBCM that was accompanied by a progressive increase in the tone of the muscle. There was also a complete abolition of responses of the CBCM to exogenous ACh and CCh and a significant reduction (75±5.1%) in the contracture produced by KCl. MiV also showed high PLA2 activity in biochemical assays (∼680 μmol/min/mg).
Purification and isolation of mikatoxin
Mikatoxin was purified to homogeneity by a combination of gel filtration
Discussion
Snake venoms are complex mixtures of pharmacologically active protein and polypeptide toxins. A large number of these toxins can interfere with cholinergic transmission at postsynaptic or pre-synaptic sites of the neuromuscular junction (Karlsson, 1979, Yang, 1994). Postsynaptic neurotoxins (i.e. α-bungarotoxin, erabutoxin-b) bind to postsynaptic nicotinic acetylcholine receptors (nAChR) and interfere with neuromuscular transmission. These curaremimetic α-neurotoxins, are found in the venoms of
Acknowledgements
S. Nirthanan and Rong Gao are recipients of Graduate Research Scholarships from the National University of Singapore.
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