title: Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids.
abstract: We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.
authors.affiliation: Laboratoire des Venins, Unité associée Pasteur, Institut Pasteur, Paris, France.
authors.forename: F
authors.initials: F
authors.lastname: Radvanyi
authors.forename: A
authors.initials: A
authors.lastname: Keil
authors.forename: B
authors.initials: B
authors.lastname: Saliou
authors.forename: M P
authors.initials: MP
authors.lastname: Lembezat
authors.forename: C
authors.initials: C
authors.lastname: Bon
mesh_terms: D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D003435:Crotalid Venoms / Q000378:metabolism*; D003439:Crotoxin / Q000378:metabolism*; D004789:Enzyme Activation; D007811:Lanthanum / Q000378:metabolism* / Q000494:pharmacology; D008823:Micelles; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D003435:Crotalid Venoms; D008823:Micelles; D010743:Phospholipids; C028521:lanthanum chloride; D007811:Lanthanum; D003439:Crotoxin; D010740:Phospholipases; D010741:Phospholipases A