title: An unusual phospholipase A₂ from puff adder Bitis arietans venom--a novel blocker of nicotinic acetylcholine receptors.
abstract: The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity.
authors.affiliation: Institute of Cell Biophysics, Russian Academy of Sciences, Ul. Institutskaya 3, Pushchino, Moscow Region 142290, Russia. vulfius@gmail.com
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Vulfius
authors.forename: Elena V
authors.initials: EV
authors.lastname: Gorbacheva
authors.forename: Vladislav G
authors.initials: VG
authors.lastname: Starkov
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Osipov
authors.forename: Igor E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Andreeva
authors.forename: Maxim E
authors.initials: ME
authors.lastname: Astashev
authors.forename: Victor I
authors.initials: VI
authors.lastname: Tsetlin
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Utkin
mesh_terms: D000818:Animals; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D013058:Mass Spectrometry; D018733:Nicotinic Antagonists / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2 / Q000235:genetics* / Q000302:isolation & purification*; D011978:Receptors, Nicotinic / Q000378:metabolism; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
chemical_list: D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D014757:Viper Venoms; D054467:Phospholipases A2