AMMODYTOXIN-A ACCEPTOR IN BOVINE BRAIN SYNAPTIC-MEMBRANES

Ammodytoxin A, the presynaptic neurotoxin from Vipera ammodytes ammody tes venom, was found to bind specifically and with high affinity to bo vine cortex synaptic membrane preparation. The detected ammodytoxin A high-affinity binding was characterized by equilibrium binding analysi s which revealed a single high-affinity binding site with K-d 4.13 nM and B-max 6.67 pmoles/mg of membrane protein. I-125-ammodytoxin A was covalently cross-linked to its neuronal acceptor using a chemical cros s-linking technique. As revealed by subsequent SDS-PAGE analysis and a utoradiography, I-125-ammodytoxin A specifically attached to membrane components with apparent mol. wts 53,000-56,000. Besides by the native ammodytoxin A, the binding of radioiodinated ammodytoxin A to the neu ronal acceptor was highly attenuated, also by other two iso-neurotoxin s from V. a, ammodytes venom, ammodytoxins B and C, and neurotoxin cro toxin B from the venom of the South American rattlesnake (Crotalus dur issus terrificus). Vipera berus berus phospholipase A(2) was a weaker inhibitor, whereas nontoxic phospholipase A(2), ammodytoxin I-2 and my otoxic phospholipase A(2) homologue, ammodytin L, both from V. a. ammo dytes venom as well, were very weak inhibitors. No inhibitory effect o n I-125-ammodytoxin A specific binding at all was, however, obtained w ith alpha-dendrotoxin, beta-bungarotoxin and crotoxin A, respectively. Treatment of synaptic membranes with proteinase K and Staphylococcus aureus V-8 proteinase, a combination of PNGase F and neuroaminidase, h eat or acid lowered the I-125-ammodytoxin A specific binding to variou s extents but never completely abolished it. The ammodytoxin A binding site in bovine synaptic membranes is thus most likely a combination o f membrane glycoprotein acceptor and membrane phospholipids. As ammody toxin A reduced the second negative component of the perineural wavefo rm, measured on mouse triangularis sterni preparation, which is very l ikely a result of an inhibition of a fraction of the terminal K+ curre nts, the ammodytoxin A acceptor could well be connected with K+ channe ls.