Mass spectrometry analysis of the phospholipase A(2) activity of snake pre-synaptic neurotoxins in cultured neurons.
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.
Paoli
Marco
M
Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Padova, Italy.
Rigoni
Michela
M
Koster
Grielof
G
Rossetto
Ornella
O
Montecucco
Cesare
C
Postle
Anthony D
AD
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
D004546:Elapid Venoms; D008055:Lipids; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D012910:Snake Venoms; C005082:notexin; C483858:phosphatidylethanolamine; D054467:Phospholipases A2; D002118:Calcium