Arginine derivatives of dicarboxylic acids from the parotid gland secretions of common toad <Emphasis Type="Italic">Bufo bufo</Emphasis>—New agonists of ionotropic γ-aminobutyric acid receptors

Compounds activating γ-aminobutyric acid type A receptor were isolated from the toad Bufo bufo venom as a result of chromatographic separation. Analysis of the structure of these compounds by mass spectrometry and nuclear magnetic resonance showed that they are arginine derivatives of dicarboxylic acids and represent suberylarginine, pimeloylarginine, and adipoylarginine.     

New paradoxical three-finger toxin from the cobra <Emphasis Type="Italic">Naja kaouthia</Emphasis> venom: Isolation and characterization

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of “orphan” toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.     

Preparation and ESR study of spin-labeled derivatives of Naja naja oxiana neurotoxin II

After neurotoxin II Naja naja oxiana reaction with N-hydroxysuccinimidyl 2,2,6,6-tetramethyl-4-carboxymethylpiperidine-1-oxyl, six derivatives were isolated, each containing one spin label. Their analysis (reduction, carboxymethylation, tryptic hydrolysis, isolation and identification of the spin labeled peptide) allowed to localize the label position: the epsilon-amino groups of Lys15, Lys25, Lys26, Lys44, Lys48, and alpha-amino group of Leu1. The neurotoxin II reaction with N-hydroxysuccinimidyl 2,2,5,5-tetramethyl-3-carboxypyrrolin-1-oxyl followed by chromatography afforded 10 derivatives, each having two labeled lysine residues, wherein the position of the modified residues was determined. The reactivity and microenvironment of amino groups are discussed basing on the dependence between the reaction conditions and yields. For di-spin labeled derivatives of the pyrroline series, the inter-label distances were determined by EPR from the standard curve and used for refinement of the neurotoxin conformation in solution.     

Tachykinin receptors in the rat brain bind alpha-bungarotoxin

alpha-Bungarotoxin was found to inhibit effectively the binding of 125I-labelled substance P and eledoisin to membrane and to solubilize preparations of the rat brain. Other postsynaptic neurotoxins exerted similar but less pronounced influence on the interaction of tachykinins with their receptors. The obtained results suggest that some alpha-bungarotoxin-binding polypeptides in brain are components of tachykinin receptors.     

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.     

The First Recombinant Viper Three-Finger Toxins: Inhibition of Muscle and Neuronal Nicotinic Acetylcholine Receptors

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor'sα, β, γ, orδ subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Photoactivatable derivatives of peptide and protein ligands in the study of neuroreceptor structure

The application of photoactivatable derivatives for studies of different types of neuroreceptors belonging to two superfamilies, G-protein-dependent receptors and ligand-gated ionic channels, is discussed. Studies of the structure of voltage-gated and ion-gated channels with the use of specific photoactivatable derivatives of neurotoxins are also described. Possibilities and prospects for the application of photoactivatable ligands in studies of the spatial structure of neuroreceptors are reviewed.