Identification of the neuronal acceptor in bovine cortex for ammodytoxin C, a presynaptically neurotoxic phospholipase A2.
A specific, high-affinity binding site for ammodytoxin C in synaptic membranes from bovine cerebral cortex was detected and partially characterized. Equilibrium binding analysis revealed a single population of [125I]ammodytoxin C acceptors with the following binding parameters: Kd = 6.0 nM and Bmax = 5.7 pmol/mg membrane protein. Such binding was strongly inhibited by three ammodytoxins (A, B, and C) and by crotoxin B. Vipera berus berus phospholipase A2 was a weaker inhibitor; nontoxic phospholipase A2, ammodytin I2, and the myotoxic phospholipase A2 homologue, ammodytin L, both from Vipera ammodytes ammodytes venom, inhibited binding only at very high concentrations, whereas alpha-dendrotoxin, beta-bungarotoxin, and crotoxin A had no influence on the [125I]ammodytoxin C-specific binding. The ammodytoxin C neuronal binding site therefore overlaps, at least partially, with the neuronal acceptors for some of the related presynaptically neurotoxic phospholipases A2 (beta-neurotoxins). [125I]-Ammodytoxin C was covalently attached to its acceptor by chemical cross-linking. Subsequent SDS-PAGE analysis followed by autoradiography revealed saturably labeled membrane components with apparent M(r) values of 51,000 (weaker band) and 53,000-56,000 (stronger band). Pretreatment of synaptic membranes with Staphylococcus aureus V-8 proteinase and proteinase K, heat, or low pH decreased the [125I]ammodytoxin C-specific binding to various extents, but never abolished it completely. Membrane protein and certain phospholipids residing in its vicinity are therefore most likely involved in the binding of ammodytoxin C to bovine synaptic membranes.
Krizaj
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I
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia.
Dolly
J O
JO
Gubensek
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F
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D004120:Dimethyl Suberimidate; D054501:Group II Phospholipases A2; D066298:In Vitro Techniques; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism; D014757:Viper Venoms / Q000378:metabolism*; D017819:Viperidae
D009498:Neurotoxins; D014757:Viper Venoms; D004120:Dimethyl Suberimidate; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C060161:ammodytoxin C