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V I Tsetlin

Publications and source records attributed to V I Tsetlin.

At least 55 records · Page 3Linked to original sources
PubMed1994

[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. Yeast cells harbouring the pJDAch vector produced mainly an N-glycosylated fragment of the acetylcholine receptor alpha-subunit, detected by means of [125I]-alpha-bungarotoxin. The fragment's location in the membrane fraction after disruption of the yeast cells simplified its isolation.

Amino Acid Sequence↗
PubMed1994

[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. To the former one may relate the transmembrane segments M1, M3 and, particularly, M2 as well as the 81-100 domain comprising several links pertaining to the ligand-binding domain in AChR. Domains 153-171 and 177-197 comprising most of the identified links involved in the binding of low molecular weight agonists and of alpha-bungarotoxin appeared to be highly variable ones.

Amino Acid Sequence↗
PubMed1993

Two-dimensional 1H-NMR study of the spatial structure of neurotoxin II from Naja naja oxiana.

The spatial structure of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana was determined by two-dimensional 1H-NMR techniques and computational analysis. Nearly complete proton resonance assignments for 61 amino acid residues have been made using two-dimensional (2D) homonuclear total correlated spectroscopy, 2D homonuclear double-quantum-filtered correlated spectroscopy and 2D homonuclear NOE spectroscopy (NOESY) experiments. The cross-peak volumes in NOESY spectra spin-spin coupling constants of vicinal protons NH-C alpha H and C alpha H-C beta H and the observation of slow deuterium exchange of amide protons were used to define local structure and a set of constraints for distance geometry program DIANA. The average root-mean-square deviations are 53 pm for backbone heavy atoms and 118 pm for all heavy atoms of 19 final neurotoxin II conformations. The spatial structure is characterized by a short double-stranded (residues 1-5 and 13-17) and a triple-stranded (residues 22-30, 33-41 and 50-54) antiparallel beta-sheets.

Amino Acid Sequence↗
PubMed1993

[Neuroactive complexed zinc compounds from bovine brain].

The fraction obtained from acidic extract of bovine brain homogenate after several steps of chromatographic purification provokes spontaneous aggressive encounters in rats upon intracerebroventricular injection. The simultaneous long-term raising of electric shock-induced aggression with the suppressing of muricidal and intraspecies aggressive behaviour has been observed. Intravenous and intraperitoneal injections of this fraction induce no behavioural changes in rats. It has been determined that the fraction consists of complex compounds of zinc with various aliphatic amines. A similar or higher behavioural activity has been discovered in series of synthetic complexes of zinc with different ligands, that are suggested for use in modelling any nervous and psychiatric disorders connected with an increased aggression level.

Aggression↗
PubMed1992

Investigation of ligand-binding sites of the acetylcholine receptor using photoactivatable derivatives of neurotoxin II from Naja naja oxiana.

Several photoaffinity derivatives of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana have been prepared. After reaction of the 125I-labeled derivatives with the nicotinic acetylcholine receptor from electric organ, the alpha-subunit of the nAChR is almost exclusively labeled by the derivative carrying the photoactivatable group in position Lys46. In contrast to this, a reactive group at Lys26 predominantly labels the gamma- and delta-subunits, while the alpha- and beta-subunits incorporate much less radioactivity. Competition experiments with d-tubocurarine show that the gamma-subunit is labeled when this derivative occupies the high affinity d-tubocurarine-binding site, while the delta-subunit is labeled by the toxin bound at the low-affinity d-tubocurarine site. A model is discussed for the orientation of different loops of the toxin molecules in the binding site for agonists and competitive antagonists.

Animals↗
PubMed1992

[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. The sensitivity of subsequent detection of Bt-SP enhances by using a new type of the streptavidin conjugate with a polymeric form of horseradish peroxidase. Enzyme immunoassay conditions were optimized using [125I]-labelled SP derivative. The developed method allows the determination of femtomoles of SP in a sample and has a sensitivity comparable to that of radioligand analysis.

Amino Acid Sequence↗
PubMed1992

[Identification of a segment of the gene for the substance B receptor in the human DNA genome using the polymerase chain reaction].

Polymerase chain reaction was applied to human genomic DNA using primers corresponding to the rat substance P receptor cDNA. As a result, a fragment of 94 b.p. was isolated identical to the fragment 771-864 of the above-mentioned cDNA, with the exception of the G796----A substitution (Val----Ile in the amino acid sequence). A comparison of the established sequence with the published structures of tachykinin receptors of NK-1, NK-2 and NK-3 types allows its assignment to the substance P receptor (NK-1 tachykinin receptor) gene detected in the human genome.

Amino Acid Sequence↗
PubMed1992

[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.

Animals↗
PubMed1989

alpha-Bungarotoxin interacts with the rat brain tachykinin receptors.

alpha-Bungarotoxin (alpha Bgt) was shown to inhibit the binding of the 125I-labeled substance P (SP) and eledoisin (EL) to the rat brain membranes with Kl values of 8.0 +/- 5.0 x 10(-8) and 1.1 +/- 0.5 x 10(-6) M, respectively. Lower inhibitory activity was manifested by several other postsynaptically acting snake venom neurotoxins. The alpha Bgt inhibition of SP binding with a Kl value of 8.5 +/- 5.5 x 10(-8) M to solubilized preparations of the rat brain membranes was demonstrated. The capacity to displace SP was found for d-tubocurarine and phencyclidine, although at concentrations considerably higher than those affecting the nicotinic acetylcholine receptors (AChRs). The results obtained suggest that some of the alpha Bgt-binding polypeptides, distinct from neuronal AChRs, may be functionally associated with the tachykinin receptors (TchR).

Animals↗
PubMed1989

Muramyl peptides bind specifically to rat brain membranes.

The capability of immunoactive muramyl peptides to bind specifically to rat brain membranes has been discovered. The reaction of an N-acetylglucosaminylmuramyl dipeptide analog having an additional C-terminal lysine residue (GMDP-Lys) with Bolton-Hunter reagent or N-hydroxysuccinimidyl-4-azidosalicylate afforded two acylated derivatives, GMDP-Lys-(Hp) and GMDP-Lys(Azs). Their iodination resulted in radioactive derivatives (spec. act. approximately 2000 Ci/mmol) whose binding to rat brain membranes is characterized by Kd approximately 3 nM and Bmax approximately 10 fmol/mg membrane protein. Binding could be inhibited by muramyl dipeptide (MDP), GMDP-Lys, and GMDP, while fragments of the latter, N-acetylglucosamine dipeptide and disaccharide, were ineffective.

Acetylation↗
PubMed1989

[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.

Amino Acid Sequence↗
PubMed1988

Tritium thermal activation study of bacteriorhodopsin topography.

The action of thermally activated tritium on the purple membrane and delipidated bacteriorhodopsin fragments has been studied, tritium incorporation into specified amino acid residues being quantified by Edman degradation. The membrane environment was found to affect the accessibility of amino acid residues for tritium. Bacteriorhodopsin fragments 14-31, 45-63, 81-89, 171-179, and 210-225 were localized to the membrane interior while fragments 4-12, 32-44, 64-65, 73-80, and 156-170 should lie outside or close to membrane surface. It was demonstrated that the peptide fragments joining transmembrane rods are not fully exposed to the solution.

Amino Acids↗
PubMed1987

[Specific binding of muramyl peptides with rat brain membranes].

A tripeptide analogue of N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl dipeptide (GMDP) which contains C-terminal Lys residue (GMDP-Lys) was prepared. Its reaction with N-hydroxysuccinimidyl 3-(4-hydroxyphenyl)propionate (BH) followed by iodination gave the 125I-labelled derivative with specific activity ca. 2000 Ci/mmol. This compound was shown to bind specifically with rat brain membranes, dissociation constant Kd = 3.1 +/- 0.9 nM, binding capacity Bmax = 11.0 +/- 12 fmol/mg protein. Binding was inhibited by the non-radioactive iodinated derivative, unmodified GMDP-Lys and GMDP. Thus, the specific binding of immunoactive myramyl peptides with brain has been demonstrated for the first time.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1986

[Effect of modification of Naja naja oxiana neurotoxin II on the stoichiometry of its complexes with acetylcholine receptor].

It was discovered that illumination of the complex formed by the solubilized acetylcholine receptor from Torpedo marmorata and Lys25-p-azidobenzoyl derivative of neurotoxin II results in the appearance on the receptor of up to 4 additional binding sites. Acetylcholine and neurotoxin II, but not the long-chain neurotoxins bind specifically to these sites. The additional binding sites could be also detected after illuminating the receptor complex with other photoactivable derivatives, provided the latter were displaced from one of the two main binding sites by hexa(trifluoroacetyl)neurotoxin II. A similar, but less pronounced effect, was observed on binding Lys25 (Ac) derivative of neurotoxin II. The formation of the additional binding sites was found to depend on the activity of the receptor preparations as well as on the mutual influence of the two main toxin-binding sites.

Amino Acid Sequence↗
PubMed1984

[Relative localization of the bound acetylcholine receptor subunits and neurotoxin].

A series of neurotoxin II (Naja naja oxiana) derivatives, each containing one p-azido-[14C]benzoyl group, have been prepared. Those labeled at Leu1, Lys15, Lys25, Lys26, or Lys46 associate specifically with the acetylcholine receptor from the Torpedo marmorata electric organs and form the crosslinks with it as a result of irradiation. Electrophoresis in polyacrylamide gel and gel chromatography revealed the contacts between the neurotoxins and alpha, beta, gamma and delta subunits of the receptor, modification of a particular subunit being governed by the photoactivable group position in the neurotoxin molecule. The differences of the two neurotoxin binding sites in the receptor were demonstrated by analysis of the photoinduced crosslinks under the conditions of one site being blocked by hexa (trifluoroacetyl) neurotoxin II. The mutual arrangement of the two bound neurotoxin molecules was established. On the basis of data obtained, two models for the acetylcholine receptor subunit topography were proposed.

Animals↗
PubMed1984

[2-D-NMR study of the conformation characteristics of toxin 3 from Naja naja siamensis venom].

The spatial structure of "long" toxin 3 Naja naja siamensis in solution has been studied by methods of two-dimensional (2D) 1H NMR spectroscopy. The individual signal assignments for 67 out of 71 residues and analysis of nuclear Overhauser effects between distinct protons of the molecule allowed the comparison of the toxin 3 conformations at different pH values and temperatures. It was shown that the deprotonated imidazole ring of His22 residue (at pH greater than or equal to 7,5) is surrounded by the side chains of Cys17, Pro18, Val23, Cys24, Cys45, Ala46 and Thr48 residues. On the contrary, the protonated imidazole ring of His22 (at pH less than 4,0) is exposed into solvent. Ionization of His22 is accompanied by a change in the Tyr25 aromatic ring orientation and affects the conformational mobility of the Cys17, His22, Cys45 and Ala47 side chains. The revealed conformational features of toxin 3 in solution are discussed in connection with the differences between "long" and "short" neurotoxins in the kinetics of their binding to acetylcholine receptor.

Amino Acids↗