Identification of the Neuronal Acceptor in Bovine Cortex for Ammodytoxin C, a Presynaptically Neurotoxic Phospholipase A2

• Published in: Biochemistry (Easton) Vol. 33; no. 46; pp. 13938 - 13945
• Main Authors: Krizaj, Igor, Dolly, J. Oliver, Gubensek, Franc
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01-11-1994
• Subjects: Animals; Binding Sites; Cattle; Cerebral Cortex - cytology; Cerebral Cortex - metabolism; Dimethyl Suberimidate; Group II Phospholipases A2; In Vitro Techniques; Neurons - metabolism; Neurotoxins - metabolism; Phospholipases A - metabolism; Phospholipases A2; Synaptic Membranes - drug effects; Synaptic Membranes - metabolism; Viper Venoms - metabolism; Viperidae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00250a049

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.