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Biospecific sorbents for isolation of the latrotoxin receptor have been obtained and studied. Binding of the receptor components solubilized from the bovine cerebral cortex membrane to the immobilized toxin critically depends on the presence of calcium ions and the solution ionic strength. The procedure for semipreparative isolation of the highly active receptor preparation with Kd = 9 x 10(-10) M and Bmax = 0.9 nmol/mg has been developed. Binding activity of the isolated receptor is inhibited by heating as well as by proteases and denaturing agents. According to electrophoretic analysis in the presence of SDS the receptor complex contains protein components of molecular mass 200, 160, 79, 43 kDa, the former two being glycoproteins.

Iodine-125 labelled alpha-latrotoxin from the venom of Central Asia black widow spider Latrodectus mactans tredecimguttatus binds specifically to the bovine brain membrane receptor producing a stable slowly dissociating complex with Kd = 1.6 x 10(-10) M and Bmax = 0.5 pmol/mg protein. Treatment of the complex with alkaline high-salt buffer induces reversible dissociation of the bound toxin. The antitoxin polyclonal antibody does not increase the dissociation rate of the bound toxin. Wheat germ lectin as well as concanavalin A inhibit the toxin binding to the membrane receptor. The receptor is solubilized with ionic and non-ionic detergents, and methods of latrotoxin binding assay are developed. The solubilized receptor is shown to retain high affinity to toxin, its binding activity being stable but critically dependent on the presence of calcium ions. Chromatographic properties of the receptor suggest its glycoprotein nature.

A presynaptic neurotoxin isolated from the venom of the Central Asia spider karakurt (Black Widow Spider, Latrodectus mactans tredecimguttatus) is shown to consist of two identical subunits of mol. weight about 118 kDa. The iodinated neurotoxin binds to the rat brain synaptosomal plasma membranes with Kd 0.1 nM (Bmax 0.1 pmol/mg of protein) at 37 degrees C, and with Kd 0.35 nM (Bmax 0.2 pmol/mg of protein) at 5 degrees C. At intermediate temperatures both types of receptors are detectable. It is supposed that the dimeric form of the toxin interacts with a single class of receptors possessing lateral mobility in the membrane. By the use of different bifunctional reagents it is revealed that the neurotoxin interacts with a presynaptic membrane protein of mol. weight 95 kDa. A protein of the same size accompanied by a 71 kDa protein was isolated by the affinity chromatography of solubilized synaptosomal membranes on the absorbent, containing immobilized neurotoxin.

Homologous low molecular weight compounds blocking postsynaptic glutamate receptors were isolated from the Argiope lobata spider venom by ion-exchange chromatography and reverse-phase HPLC. Structures of nine different blocking agents were determined by NMR and mass spectroscopy. They can be divided into three groups: argiopin, argiopinins and pseudoargiopinines. The major principles of the structural organization of the novel class of antagonists of glutamate receptors were considered.

Several homologous low-molecular weight compounds were purified from venom of spider Argiope lobata. They selectively blocked ionic currents elicited by glutamate and its agonist kainate in the membrane of isolated hippocampal neurons of rat. Three groups of these compounds--argiopine, argiopinines and pseudoargiopinines, blocked glutamate- and kainate-activated ionic currents in a voltage-dependent manner, acting preferentially on agonist-activated ionic channels. These compounds did not change the Kd values for both agonists. The blocking action was partially reversible for argiopine and poorly reversible for other compounds. Rate constants for the interaction of toxins with membrane receptors were estimated from the dependences of two-component kinetics of the blocking action and its recovery on toxin concentrations. Argiopine, argiopinines and pseudoargiopinines may be useful tools for further electrophysiological and biochemical investigation of the mammalian CNS glutamate receptors.

Monospecific antibodies against neurotoxin from the black widow spider venom (alpha-latrotoxin) have been used to identify a neuronal protein (s) with latrotoxin-like epitopes. As determined by ELISA, the protein is localized in cytoplasmic fraction of bovine brain. It was partially purified by anion-exchange chromatography and preparative SDS-electrophoresis. Immunoblotting data indicate that this neuronal acidic protein has molecular mass of ca. 100 kDa.

Argiopine, a compound capable of blocking the glutamate-activated ion channels in experiments with glutamatergic synapses of blowfly larvae, was isolated from the venom of spider Argiope lobata. Argiopine-receptor complex has KD 6,7 X 10(-7) M. The venom solution was treated with 60% ethanol, centrifuged and supernatant was subjected to reversed-phase HPLC in the acetonitrile concentration gradient. Argiopine structure was established using 1H- and 13C-NMR spectroscopy, mass spectrometry, elemental and amino acid analyses. Argiopine (molecular mass 636) consists of arginine (free alpha-NH2) linke with polyamine--NH (CH2)3NH(CH2)3NH(CH2)5NH--through a peptide bond. The polyamine is connected to the alpha-carboxyl group of asparagine whose alpha-amino group is linked to 2,4-dihydroxyphenylacetic acid.

Black widow spider toxin (latrotoxin) was studied for its effect on bilayer phospholipid membrane conductance. Under the toxin action membranes became selective with respect to divalent cations. The selectivity decreased in the following order: Ba2+, Sr2+, Ca2+, Mg2+. The ion channels formed by toxin in a planar membrane were effectively blocked by Cd2+. Current-voltage characteristics of the membranes permit concluding that toxin molecules are incorporated into a plane bilayer in the oriented way and the formed channels are potential-dependent.

Homologous low molecular weight compounds, blocking postsynaptic glutamate receptors, were isolated from the venom of the spider Argiope lobata by ion exchange chromatography and reverse phase HPLC. Structures of nine blocking agents were determined by 1H-NMR and mass spectroscopy. Two-dimensional COSY spectra were used to identify spin systems of the protons coupled via two or three chemical bonds. The spin systems were combined into the sequence by means of the nuclear Overhauser effect between spatially close protons. Ionogenic groups were identified from pH dependences of the proton chemical shifts. Molecular structures thus obtained were checked against amino acid analysis and mass spectrometry data. The antagonists can be divided into three groups: argiopin, argiopinins and pseudoargiopinins depending on features of the chromophoric moiety (2,4-dihydroxyphenylacetic acid, 4-hydroxy-indole-3-acetic acid or indole-3-acetic acid).