title: Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.
abstract: Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.
authors.affiliation: Venom & Toxin Research Group, Department of Anatomy, Faculty of Medicine, National University of Singapore, Republic of Singapore.
authors.lastname: Maung-Maung-Thwin
authors.forename: P
authors.initials: P
authors.lastname: Gopalakrishnakone
authors.forename: R
authors.initials: R
authors.lastname: Yuen
authors.forename: C H
authors.initials: CH
authors.lastname: Tan
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002413:Cations, Divalent / Q000494:pharmacology; D007106:Immune Sera / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D009498:Neurotoxins / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism* / Q000648:ultrastructure; D013997:Time Factors; D014757:Viper Venoms / Q000378:metabolism*; D017819:Viperidae
chemical_list: D002413:Cations, Divalent; D007106:Immune Sera; D009498:Neurotoxins; D018377:Neurotransmitter Agents; D011506:Proteins; D014757:Viper Venoms; C094035:daboiatoxin; D010741:Phospholipases A; D054467:Phospholipases A2