[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.

[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.

[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.

[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor].

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.

[Photoactivatable analogues of alpha-conotoxins GI and MI and their interaction with nicotinic acetylcholine receptor].

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.

[MALDI-mass spectrometry for identification of new proteins in snake venoms].

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%.

[Cobra venom contains a protein belongs to the CRISP family].

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.

[The weak neurotoxin from Naja kaouthia Cobra venom decreases the arterial blood pressure in rats].

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.

[Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom].

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.

[Efficient synthesis of natural alpha-conotoxins and their analogs].

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.

[ESR study of the interaction of cytotoxin II with model membranes].

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.

[Photoactivated derivatives of peptide and protein ligands in the studies of neuroreceptors].

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.

[alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers].

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].

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.

[Effective binding of alpha-bungarotoxin with the solubilized alpha-subunit of Torpedo californica acetylcholine receptor].

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).

[Secondary structure and conformational heterogeneity of Naja naja oxiana cytotoxin II].

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.

[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].

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.

[Preparation and characteristics of a tritiated derivative of CP-96,345--a nonpeptide antagonist of the substance P receptor with a high specific radioactivity].

[3H]CP-96.345 with the specific radioactivity of 84.2 Ci/mmol has been prepared from CP-96.

[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].

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.

[Interaction of substance P receptor from rat brain with antibodies to its synthetic fragments and to substance P].

The peptides which correspond to the fragments 55-64, 182-192, 225-236 and 236-245 (P1-P4, respectively) of the rat brain substance P receptor were synthesized. Antibodies (Ab1-Ab4, respectively) against the KLH-conjugates of these peptides were raised and purified by affinity chromatography. None of the antibodies inhibited the Bolton-Hunter labelled substance P, [125I]BH-SP, binding to the rat brain membranes.

[Expression of a gene fragment from the alpha-subunit of the acetylcholine receptor from Torpedo californica in Saccharomyces cerevisiae].

A fragment of the AChR alpha-subunit gene from Torpedo californica (about 800 bp) was joined in frame with a synthetic duplex, coding for a changed leader peptide of the Kluyveromyces lactis killer toxin under the control of the reconstructed glyceraldehyde-3-phosphate dehydrogenase gene promoter in the pUC8 vector. Translation termination codons in all frames were inserted as a part of a self-complementary oligodeoxynucleotide to the Eco47III site (807 bp from the start of the gene). Vector pJDAch was constructed by cloning the expression cassette between the BamHI and HindIII sites in the multicopy yeast plasmid pJDB207.

[Conserved and variable segments of amino acid sequences in alpha-subunits of nicotinic acetylcholine receptors and alpha-bungarotoxin-binding proteins].

A comparative study has been carried out of homologous amino acid sequences of alpha-subunits of acetylcholine receptors (AChR) and related proteins classified into three groups: (i) alpha-bungarotoxin-binding alpha-subunits of nicotine AChR from vertebrate muscles and electrical organ of the skate; (ii) alpha-bungarotoxin-binding alpha-subunits of neuronal AChR from chicken and rat brain and (iii) alpha-bungarotoxin-binding alpha-subunit of chicken brain proteins. The experimental results were plotted as intergroup variability profiles obtained by comparison of all the sequences within one group with each of the sequences in the other group. All of the local variability profiles appeared to be similar and contained both highly conservative and highly variable sites.

[A new immunochemical method for detecting substance P receptors based on the biotin-streptavidin system].

A novel method for detecting the membrane receptors of Substance P (SP) has been developed. The method does not require radioactive derivatives of SP and is based on quantitation of specifically bound biotinylated SP (Bt-SP), the analysis being performed after destruction of the ligand-receptor complex in acidic conditions and Bt-SP extraction into solution. The acidic extract from the Bt-SP-membrane complex after neutralization is added to immulon microELISA plate coated with affinity purified anti-SP-antibodies.

[Effect of toxic components of snake venoms on binding of substance P with rat brain membranes].

Ion-exchange HPLC is used for purification of the snake venom alpha-neurotoxins, chi-bungarotoxin, cytotoxins, and phospholipases A2. Among these purified polypeptides, phospholipases A2 are found to be the most potent in inhibiting the substance P binding to rat brain membranes, Ki approximately 10(-8) M. Other toxins are weak inhibitors (Ki greater than or equal to 10(-4)-10(-5) M), earlier data on the inhibiting activity of alpha-bungarotoxin being caused by the commercial preparations' contamination with phospholipase A2.