Model for the interaction of crotoxin, a phospholipase A2 neurotoxin, with presynaptic membranes.
Crotoxin is a phospholipase A2 neurotoxin that impairs the release of acetylcholine at neuromuscular junctions, primarily at the presynaptic level. It associates a phospholipase A2 subunit, CB, with a chaperon subunit, CA. We have shown elsewhere that the purely cholinergic synaptosomes from the Torpedo electric organ provided a convenient model to study the pharmacology of crotoxin and other related neurotoxins [Délot, E., & Bon, C. (1992) J. Neurochem. 58, 311-319]. In the present experiments, we labeled crotoxin with 125I and demonstrated saturable binding to Torpedo presynaptic membranes. In the range of concentrations that was effective on synaptosomes, crotoxin bound to a single class of sites without cooperativity. The binding was affected by divalent cations, and its kinetics was rather complex. We observed a competition between crotoxin and related neurotoxins, but not CB. Although CA could not bind, it could compete efficiently with crotoxin. We compare our results with those previously obtained by others on guinea pig brain membranes. On Torpedo membranes, as on all models tested, CB was the major species bound to the membrane, while CA remained in solution. However, the mechanism underlying this phenomenon had never been clarified. The major question is the time scale of the events, i.e., does CB bind before or after dissociating from CA? Our results indicate that the predominant pathway involves the formation of a ternary complex between crotoxin's subunits and the acceptor site preceding the release of CA.
Délot
E
E
Unité des Venins, Institut Pasteur, Paris, France.
Bon
C
C
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D017609:Barium Compounds / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002458:Cell Fractionation; D002712:Chlorides / Q000494:pharmacology; D017839:Crotalus; D003439:Crotoxin / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity*; D004557:Electric Organ / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008954:Models, Biological; D009498:Neurotoxins / Q000378:metabolism* / Q000633:toxicity*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D014101:Torpedo; D017967:Zinc Compounds / Q000494:pharmacology
D017609:Barium Compounds; D002413:Cations, Divalent; D002712:Chlorides; D046911:Macromolecular Substances; D009498:Neurotoxins; D017967:Zinc Compounds; C024986:barium chloride; C016837:zinc chloride; D003439:Crotoxin; D010741:Phospholipases A; D054467:Phospholipases A2; D002122:Calcium Chloride; D000109:Acetylcholine