Neurotoxic phospholipases A2 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 phospholipase A2 neurotoxin from Vipera ammodytes, and of radioiodinated crotoxin, a dimeric phospholipase A2 neurotoxin from Crotalus durissus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific binding sites were identified and characterized. The high-affinity binding sites for both toxins have been shown to be proteins. The low-affinity binding sites were not affected by proteinases or heat, suggesting the involvement of certain lipid structures in this type of binding. By affinity-labeling, [125I]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 apparent molecular masses ranging between 39,000 and 57,000 Da. [125I]crotoxin, on the other hand bound primarily to a 48,000 Da membrane protein. All phospholipases A2 tested, except beta-bungarotoxin, inhibited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases A2 prevented the high-affinity binding and the cross-linking of ammodytoxin C and crotoxin. The inhibition profiles of high-affinity binding for [125I]crotoxin and for [125I]ammodytoxin C were quite different. Ammodytoxin C and crotoxin did not inhibit each other on their respective high-affinity binding sites. These observations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with crotoxin, the isolated basic subunit CB of crotoxin was able to completely inhibit the high-affinity binding of [125I]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.
Krizaj
I
I
Unite des Venins, Institut Pasteur, Paris, France.
Faure
G
G
Gubensek
F
F
Bon
C
C
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D003432:Cross-Linking Reagents / Q000378:metabolism; D003439:Crotoxin / Q000737:chemistry / Q000378:metabolism*; D004557:Electric Organ / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D054501:Group II Phospholipases A2; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014757:Viper Venoms / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D009498:Neurotoxins; D011956:Receptors, Cell Surface; D014757:Viper Venoms; D003439:Crotoxin; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C060161:ammodytoxin C; D014357:Trypsin