IDENTIFICATION OF THE NEURONAL ACCEPTOR IN BOVINE CORTEX FOR AMMODYTOXIN-C, A PRESYNAPTICALLY NEUROTOXIC PHOSPHOLIPASE-A(2)

A specific, high-affinity binding site for ammodytoxin C in synaptic m embranes from bovine cerebral cortex was detected and partially charac terized. Equilibrium binding analysis revealed a single population of [I-125]ammodytoxin C accepters with the following binding parameters: K-d = 6.0 nM and B-max = 5.7 pmol/mg membrane protein. Such binding wa s strongly inhibited by three ammodytoxins (A, B, and C) and by crotox in B. Vipera berus berus phospholipase A(2) was a weaker inhibitor; no ntoxic phospholipase A(2), ammodytin I-2, and the myotoxic phospholipa se A(2) homologue, ammodytin L, both from Vipera ammodytes ammodytes v enom, inhibited binding only at very high concentrations, whereas alph a-dendrotoxin, beta-bungarotoxin, and crotoxin A had no influence on t he [I-125]ammodytoxin C-specific binding. The ammodytoxin C neuronal b inding site therefore overlaps, at least partially, with the neuronal accepters for some of the related presynaptically neurotoxic phospholi pases A(2) [beta-neurotoxins). [I-125]-Ammodytoxin C was covalently at tached to its acceptor by chemical cross-linking. Subsequent SDS-PAGE analysis followed by autoradiography revealed saturably labeled membra ne components with apparent M(r) values of 51 000 (weaker band) and 53 000-56 000 (stronger band). Pretreatment of synaptic membranes with S taphylococcus aureus V-8 proteinase and proteinase K, heat, or low pH decreased the [I-125]ammodytoxin C-specific binding to various extents , but never abolished it completely. Membrane protein and certain phos pholipids residing in its vicinity are therefore most likely involved in the binding of ammodytoxin C to bovine synaptic membranes.