NEUROTOXIC PHOSPHOLIPASES A(2) AMMODYTOXIN AND CROTOXIN BIND TO DISTINCT HIGH-AFFINITY PROTEIN ACCEPTORS IN TORPEDO-MARMORATA ELECTRIC ORGAN

We studied the binding of radioiodinated ammodytoxin C, a monomeric ph ospholipase A(2) neurotoxin from Vipera ammodytes, and of radioiodinat ed crotoxin, a dimeric phospholipase A(2) neurotoxin from Crotalus dur issus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific b inding sites were identified and characterized. The high-affinity bind ing sites for both toxins have been shown to be proteins. The low-affi nity binding sites were not affected by proteinases or heat, suggestin g the involvement of certain lipid structures in this type of binding. By affinity-labeling, [I-125]ammodytoxin C was shown to be associated predominantly with membrane proteins of apparent molecular masses of 70 000 and 20 000 Da and to a lesser extent with several proteins of a pparent molecular masses ranging between 39 000 and 57 000 Da. [I-125] crotoxin, on the other hand bound primarily to a 48 000 Da membrane pr otein. All phospholipases A(2) tested, except beta-bungarotoxin, inhib ited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases Az prevented the high-affinity binding and t he cross-linking of ammodytoxin C and crotoxin. The inhibition profile s of high-affinity binding for [I-125]crotoxin and for [I-125]ammodyto xin C were quite different. Ammodytoxin C and crotoxin did not inhibit each other on their respective high-affinity binding sites. These obs ervations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with crotoxin, the isolated basi c subunit CB of crotoxin was able to completely inhibit the high-affin ity binding of [I-125]ammodytoxin C. Therefore, the acidic subunit CA of crotoxin does not simply act as a chaperone for CB subunit, but it also confers a distinct binding specificity to the crotoxin.