title: Mass spectrometry analysis of the phospholipase A(2) activity of snake pre-synaptic neurotoxins in cultured neurons.
abstract: Snake pre-synaptic phospholipase A(2) neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time-course of the phospholipase A(2) activity (PLA(2)) hydrolysis of notexin, beta-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro, these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA(2) hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA(2)s appear to contribute little, if any, to the phospholipid hydrolysis measured here.
authors.affiliation: Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Paoli
authors.forename: Michela
authors.initials: M
authors.lastname: Rigoni
authors.forename: Grielof
authors.initials: G
authors.lastname: Koster
authors.forename: Ornella
authors.initials: O
authors.lastname: Rossetto
authors.forename: Cesare
authors.initials: C
authors.lastname: Montecucco
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Postle
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004546:Elapid Venoms / Q000145:classification / Q000494:pharmacology*; D008055:Lipids / Q000032:analysis; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D054467:Phospholipases A2 / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D012910:Snake Venoms / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: D004546:Elapid Venoms; D008055:Lipids; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D012910:Snake Venoms; C005082:notexin; C483858:phosphatidylethanolamine; D054467:Phospholipases A2; D002118:Calcium