title: Solubilization and physical characterization of acceptors for dendrotoxin and beta-bungarotoxin from synaptic membranes of rat brain.
abstract: Dendrotoxin (DTX), an Mr 7000 convulsant polypeptide from the venom of Dendroaspis angusticeps, or its facilitatory homologues act through blockade of certain voltage-sensitive K+ currents in a variety of neurons. High-affinity acceptors for DTX have been demonstrated in synaptic plasma membranes of rat or chick brain, and a fraction of these avidly bind beta-bungarotoxin (beta-BuTX), a presynaptically active protein whose lighter B polypeptide is homologous to this toxin. Extraction of rat synaptic plasma membranes using Triton X-100 in K+-containing buffer yielded binding sites with KD values of approximately 0.5 and 0.7 nM for 125I-labeled DTX and beta-BuTX, respectively. The content of high-affinity sites obtained for beta-BuTX, including the contribution of a lower affinity component, approximates to the Bmax (approximately 1.3 pmol/mg of protein) obtained for the apparent single set of DTX acceptors. On solubilization, the pharmacological specificity of the acceptor for neurotoxic DTX congeners was retained. 125I-beta-BuTX binding (2.1 nM) was blocked efficaciously by DTX (IC50 = 1.6 nM) while the binding of 2.1 nM 125I-DTX was inhibited completely by beta-BuTX (IC50 = 25 nM); the lower potency of the latter could relate to the noncompetitive nature of the mutual competition and to the presence of high- and low-affinity sites for beta-BuTX. On gel filtration, or sedimentation analysis in H2O/sucrose and 2H2O/sucrose gradients, one peak of DTX binding activity was observed, and this was inhibitable by beta-BuTX. From the hydrodynamic properties of the acceptor/detergent/lipid complex (s20,w = 13.2 S; Stokes radius = 8.6 nm), a molecular weight of 405,000-465,000 was estimated.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Imperial College, London, U.K.
authors.forename: A R
authors.initials: AR
authors.lastname: Black
authors.forename: C M
authors.initials: CM
authors.lastname: Donegan
authors.forename: B J
authors.initials: BJ
authors.lastname: Denny
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002038:Bungarotoxins / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D004546:Elapid Venoms / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D015221:Potassium Channels / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013570:Synaptic Membranes / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D002352:Carrier Proteins; D004546:Elapid Venoms; D015221:Potassium Channels; C020273:dendrotoxin