[Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

[New weak toxins from the cobra venom].

A protein with M 7485 Da containing five disulfide bonds was isolated from the venom of cobra Naja oxiana using various types of liquid chromatography. The complete amino acid sequence of the protein was determined by protein chemistry methods, which permitted us to assign it to the group of weak toxins. This is the first weak toxin isolated from the venom of N. oxiana. In a similar way, two new toxins with M 7628 and 7559 Da, which fall into the range of weak toxin masses, were isolated from the venom of the cobra N. kaouthia. The characterization of these proteins using Edman degradation and MALDI mass spectrometry has shown that one of these proteins is a novel weak toxin and the other is the known weak toxin WTX with an oxidized methionine residue in position 9. Such a modification was detected in weak toxins for the first time. A study of the biological activity of the toxin from N. oxiana showed that, like other weak toxins, it can be bound by muscle nicotinic cholinoreceptors and alpha7 nicotinic cholinoreceptors.

[Natural alpha-conotoxins and their synthetic analogues in studies of nicotinic acetylcholine receptors].

alpha-Conotoxins, peptide neurotoxins from poisonous marine snails of the genus Conus that highly specifically block nicotinic acetylcholine receptors (AChRs) of various types, are reviewed. Preliminarily, the structural organization of AChRs of the muscular and neuronal types, their involvement in physiological processes, and their role in various diseases are briefly discussed. In this connection, the necessity of quantitative determination of AChR subtypes using neurotoxins and other approaches is substantiated. The chemical structure, spatial organization, and specificity of alpha-conotoxins are mainly discussed, taking into consideration the recent results on the ability of alpha-conotoxins to interact with muscular or neuronal hetero- and homooligomeric AChRs exhibiting a high species specificity. Particular emphasis is placed upon a thorough characterization of the surfaces of interaction of alpha-conotoxins with AChRs using synthetic analogues of alpha-conotoxins, mutations in AChRs, and pairwise mutations in both alpha-conotoxins and AChRs. The discovery in 2001 of the acetylcholine-binding protein from the pond snail Lymnaea stagnalis and the determination of its crystalline structure led to rapid progress in understanding the structural organization of ligand-binding domains of AChRs with which alpha-conotoxins also interact. We discuss the interaction of various alpha-conotoxins with acetylcholine-binding proteins, the recently reported X-ray structure of the complex of the acetylcholine-binding protein from Aplysia californica with the alpha-conotoxin analogue PnIA, and the application of this structure to the modeling of complexes of alpha-conotoxins with various AChRs.

[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor]. / Chuvstvitel'nyi neradioaktivnyi metod skrininga soedinenii, vzaimodeistvuiushchikh s neironnym alpha7-kholinoretseptorom.

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).

[Photoactivatable analogues of alpha-conotoxins GI and MI and their interaction with nicotinic acetylcholine receptor]. / Fotoaktiviruemye proizvodnye alpha-konotoksinov GI i MI i ikh vzaimodeistvie s nikotinovym atsetilkholinovym retseptorom.

Two photoactivatable analogues of alpha-conotoxin GI with the benzoylphenylalanine residue (Bpa) substituted for His10 or Tyr11 were synthesized using the method of solid-phase peptide synthesis. In addition, alpha-conotoxin MI was chemically modified by placing an azidobenzoyl or a benzoylbenzoyl photo label at N alpha of Gly1 or N epsilon of Lys10. All the photoactivatable analogues were purified by HPLC, their structures were confirmed by MALDI MS, and the label positions in their molecules were localized by MS of their trypsinolysis fragments. All the analogues interacted with the nicotinic acetylcholine receptor (AChR) from Torpedo californica as efficiently as the native alpha-conotoxins, with the differences in the inhibition constants being within one order of magnitude under the same conditions. [125I]Derivatives prepared from all the analogues retained the ability to be bound by AChR and were used in the photoinduced AChR cross-linking. All the AChR subunits were found to be cross-linked to the photoactivatable analogues, with the linking depending on both the chemical nature of label and its position in the alpha-conotoxin molecule.

[Cobra venom contains a protein belongs to the CRISP family]. / Iad kobry soderzhit belok CRISP-tipa.

Amino acid sequences of several fragments of the 25 k protein (molecular mass 24,953 Da) previously isolated from cobra Naja kaouthia (Kukhtina et al. Bioorg. Khim., 2000, vol. 26, pp. 803-807) were determined. Their comparison with the primary structures of known proteins showed that the 25 k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.

[The weak neurotoxin from Naja kaouthia Cobra venom decreases the arterial blood pressure in rats]. / Slabyi neirotoksin iz iada kobry Naja kaouthia snizhaet arterial'noe davlenie u krys.

The weak neurotoxin from the Naja kaouthia cobra venom was found to reduce, under the intravenous administration to rats, the arterial blood pressure and increase the heart rate.

[ESR study of the interaction of cytotoxin II with model membranes]. / Izuchenie metodom EPR vzaimodeistviia tsitoksina II s model'nymi membranami.

Cytotoxin II from the venom of the Central-Asian cobra Naja oxiana spin-labeled at Lys35 (SLCT II) was studied by ESR spectroscopy in aqueous solution and upon interaction with phospholipid vesicles from egg phosphatidylcholine or its mixture with dimyristoylphosphatidylglycerol (molar ratio 9:1). The distribution of SLCT II between the aqueous and lipid phases depended on the toxin and lipid concentrations and on the solution ionic strength. It was analyzed using the modified Gouy-Chapman equation that takes into account different charges of the cytotoxin in solution and in membrane. The analysis revealed two states of the cytotoxin-lipid complex. The first state corresponds to monomeric SLCT II hydrophobically interacting with the lipid membrane [a binding constant of (8 +/- 3) x 10(3) M-1] and carrying the charge of 4.4 +/- 0.3. On the basis of these parameters and the spatial structure of cytotoxin II in dodecylphosphocholine micelles, we concluded that the cytotoxin is mainly incorporated into the region of polar groups of the lipid bilayer. The second state of SLCT II is realized at high cytotoxin concentrations in the membrane and corresponds to the formation of toxin-lipid complexes that destruct the membrane bilayer structure.

[Efficient synthesis of natural alpha-conotoxins and their analogs]. / Effektivnaia skhema sinteza prirodnykh al'fa-konotoksinov i ikh analogov.

An efficient scheme for the synthesis of alpha-conotoxins, containing 12-18 amino acid residues and two disulfide bridges, was proposed. Its advantages are: (1) the avoidance of orthogonal protections of Cys residues; (2) a lower number of stages in a cycle of the peptide chain elongation by the method of solid phase synthesis; (3) the linear product is sufficiently pure for being used at the next stage of the disulfide bond formation without additional purification; and (4) a substantially reduced time of oxidation to disulfides at pH 10, which led to the target product in a high yield. A number of natural alpha-conotoxins (GI, ImI, EI, MII, and SIA), affecting the muscle and neuronal nicotinic acetylcholine receptors of various types, and several new analogues of these conotoxins (in particular, [Tyr10]ImI, [Gln12]GI, and [Ser1]GI) were synthesized by this scheme. They were used for elucidating the spatial structure of alpha-conotoxins by 1H NMR spectroscopy and for studying the ligand-binding sites of their receptors.

[Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom]. / Struktura i konformatsionnaia geterogennost' slabogo toksina iz iada kobry Naja kaouthia.

Resonances in the two-dimensional 1H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthia for all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequentional assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthia venom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis-trans isomerization of the peptide bond Arg32-Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin-spin coupling of protons, and rates of exchange of amide protons by deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel beta-sheets, one of which consists of two strands (regions 1-5 and 13-17) and the other, of three strands (regions 23-28, 38-43, and 55-59).

[Photoactivated derivatives of peptide and protein ligands in the studies of neuroreceptors]. / Fotoaktiviruemye proizvodnye peptidno-belkovykh ligandov v issledovaniistruktury neiroretseptorov.

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.

[MALDI-mass spectrometry for identification of new proteins in snake venoms]. / MALDI-mass-spektrometriia dlia identifikatsii novykh belkov v iade zmei.

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7-25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.

[alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers]. / alpha-Neirotoksiny i alpha-konotoksiny--blokatory nikotinovykh kholinoretseptorov.

The review is devoted to the competitive blockers of different nicotinic acetylcholine receptors, alpha-neurotoxins from snake venoms, and alpha-conotoxins from marine snails of the Conidae family. The relationship between the structure and function of these toxins is discussed. Recent data on the mechanism of alpha-neurotoxin and alpha-conotoxin interaction with the nicotinic acetylcholine receptor are presented.

[Antagonists of the tachykinin receptors]. / Antagonisty takhikininovykh retseptorov.

Antagonists of the tachykinin receptors, which have been most widely used in scientific investigations during the past decade (up to 1995 inclusive), are reviewed. The structures and peculiar characteristics of peptide and nonpeptide compounds of this class are described. Applications of these antagonists in the search for new subtypes of tachykinin receptors and for structure-function studies are discussed.

[Secondary structure and conformational heterogeneity of Naja naja oxiana cytotoxin II]. / Vtorichnaia struktura i konformatsionnaia geterogennost' tsitotoksina II Naja naja oxiana.

The proton resonances of cytotoxin II from Naja naja oxiana were sequentially assigned in 2D 1H NMR spectra for all of its 60 amino acid residues of both major and minor components of the spectra. The presence of the minor component was shown to be due to a conformational heterogeneity of the cytotoxin. The proton-deuterium exchange rates of amide groups were measured in 2H2O at a pH of 5.0 and at 10 degrees C. Experimental data obtained (d-connectivities, H-NC alpha-H coupling constants and long-range NOEs) allowed for the determination of the secondary structure of the two cytotoxin conformers. Both conformer structures contain two antiparallel beta-sheets. The first beta-sheet involves two antiparallel beta-strands comprising the residues 2-5 and 10-13. The second sheet involves three antiparallel strands consisting of the residues 20-26, 35-39, and 49-55; its peripheral beta-strands are connected by cross-over. The most striking structural difference between these conformers is the nature of the beta-turn 6-9, which has trans- and cis-forms of the Val7-Pro8 peptide bond in the major and minor conformer, respectively. The structures of other beta-turns, some of which are ascribed to standard types, and the C-termini are almost the same for the both conformers.

[Effective binding of alpha-bungarotoxin with the solubilized alpha-subunit of Torpedo californica acetylcholine receptor]. / Effektivnoe sviazyvanie alpha-bungarotoksina soliubilizirovannoi alpha-sub''edinitsei atsetilkholinovogo retseptora Torpedo californica]

alpha-Subunit of the Torpedo californica nicotinic acetylcholine receptor was isolated by preparative SDS-PAGE followed by reversed-phase HPLC on a C4 column in an acetonitrile-isopropanol gradient in water. After removal of the organic solvents and solubilization in beta-octylglucoside, the purified alpha-subunit binds alpha-bungarotoxin with high affinity (Kd 28 nM).

[Fragments 183-198 and 125-145 of the alpha-subunits of the Torpedo californica nicotinic acetylcholinergic receptor binds alpha-bungarotoxin and neurotoxin II from Naja naja oxiana]. / Fragmenty 183-198 i 125-145 alpha-sub''edinitsy nikotinovogo atsetilkholinovogo retseptora Torpedo californica sviazyvaiut alph-bungarotoksin i neirotoksin II Naja naja oxiana.

Interaction of the mono[125I]iodinated alpha-bungarotoxin and neurotoxin II Naja naja oxiana with the synthetic peptides corresponding to the fragments of the alpha-subunit of nicotinic acetylcholine receptor from Torpedo californica was studied. It was found that both toxins bind to the fragments alpha 186-198, alpha 183-198 and alpha 125-145 adsorbed to the 96-well P.E.T.G. assay plates (COSTAR). Acm-groups on Cys residues did not prevent toxin binding by the peptides studied. Determination of the binding parameters showed that alpha-bungarotoxin interacts with fragment alpha 125-145 less effectively than neurotoxin II. The data obtained demonstrate the presence of different toxin-binding sites on alpha-subunit and confirm the model of multipoint neurotoxin-receptor interaction.

[Preparation and characteristics of a tritiated derivative of CP-96,345--a nonpeptide antagonist of the substance P receptor with a high specific radioactivity]. / Poluchenie i kharakteristika tritirovannogo proizvodnogo CP-96,345-- nepeptidnogo antagonista retseptora veshchestva P--s vysokoi udel'noi radioaktivnost'iu.

[3H]CP-96.345 with the specific radioactivity of 84.2 Ci/mmol has been prepared from CP-96.345 by the high-temperature solid state isotopic exchange. The tritiated compound was equipotent with the parent antagonist in inhibiting the iodinated substance P binding to brain membranes from various species. Direct binding of [3H]CP-96.345 was detected by radioligand analysis using the striatum-enriched membranes from the guinea-pig brain.

[Structure of peptide fragments of the complex (Lys(Abz)26) neurotoxin II from Naja naja oxiana cross-linked with the nicotinic acetylcholine receptor from Torpedo californica]. / Struktura peptidnykh fragmentov kross-sshitogo kompleksa (Lys(Abz)26)neurotoksina II Naja naja oxiana s nikotinovym atsetilkholinovym retseptorom iz Torpedo californica.

After irradiating the acetylcholine receptor complex with the title neurotoxin derivative, the labeled delta-subunit was separated by preparative SDS-PAGE, reduced-carboxymethylated and cleaved with LysC endoproteinase. One of the radioactive peptides isolated by HPLC was further purified by electrophoresis in a tricin gel. Edman degradation of the radioactive fractions yielded the sequence of the delta-subunit fragment starting from Phe148. The AspN-cleavage of the radioactive peptide from the LysC digest gave on HPLC a radioactive peak which eluted similarly to peptide 33-44 generated by LysC/AspN-cleavage of 125I-neurotoxin II. In model experiments, irradiation of the photoactivable derivative was found to produce a heterogeneous mixture of reaction products. Unusually low initial and repetitive yields were observed for neurotoxin II and its fragments containing the photolabeled and radiolabeled residues. These results might explain why the neurotoxin sequence was not detected on Edman degradation of the cross-linked products available at a low picomole level.