title: Characterisation of the heterotrimeric presynaptic phospholipase A(2) neurotoxin complex from the venom of the common death adder (Acanthophis antarcticus).
abstract: While Australo-Papuan death adder neurotoxicity is generally considered to be due to the actions of reversible competitive postsynaptic alpha-neurotoxins, the neurotoxic effects are often poorly reversed by antivenom or anticholinesterases. This suggests that the venom may contain a snake presynaptic phospholipase A(2) (PLA(2)) neurotoxin (SPAN) that binds irreversibly to motor nerve terminals to inhibit neurotransmitter release. Using size-exclusion liquid chromatography under non-reducing conditions, we report the isolation and characterisation of a high molecular mass SPAN complex, P-elapitoxin-Aa1a (P-EPTX-Aa1a), from the venom of the common death adder Acanthophis antarcticus. Using the chick biventer-cervicis nerve-muscle preparation, P-EPTX-Aa1a (44,698Da) caused inhibition of nerve-evoked twitch contractions while responses to cholinergic agonists and KCl remained unaffected. P-EPTX-Aa1a also produced significant fade in tetanic contractions and a triphasic timecourse of neuromuscular blockade. These actions are consistent with other SPANs that inhibit acetylcholine release. P-EPTX-Aa1a was found to be a heterotrimeric complex composed of alpha, beta and gamma-subunits in a 1:1:1 stoichiometry with each subunit showing significant N-terminal sequence homology to the subunits of taipoxin, a SPAN from Oxyuranus s. scutellatus. Like taipoxin, only the alpha-chain produced any signs of neurotoxicity or displayed significant PLA(2) enzymatic activity. Preincubation with monovalent death adder antivenom or suramin, or inhibition of PLA(2) activity by incubation with 4-bromophenacyl bromide, either prevented or significantly delayed the onset of toxicity by P-EPTX-Aa1a. However, antivenom failed to reverse neurotoxicity. Early intervention with antivenom may therefore be important in severe cases of envenomation by A. antarcticus, given the presence of potent irreversible presynaptic neurotoxins.
authors.affiliation: Department of Medical & Molecular Biosciences, University of Technology, Sydney, NSW, Australia.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Blacklow
authors.forename: Pierre
authors.initials: P
authors.lastname: Escoubas
authors.forename: Graham M
authors.initials: GM
authors.lastname: Nicholson
mesh_terms: D000818:Animals; D001681:Biological Assay; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000737:chemistry* / Q000633:toxicity; D017815:Elapidae / Q000502:physiology*; D009119:Muscle Contraction / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects; D009498:Neurotoxins / Q000737:chemistry*; D054467:Phospholipases A2 / Q000737:chemistry* / Q000633:toxicity; D017729:Presynaptic Terminals / Q000187:drug effects; D017661:Receptors, Presynaptic / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004546:Elapid Venoms; D009498:Neurotoxins; D017661:Receptors, Presynaptic; D054467:Phospholipases A2