Search PubMed⌕ Search

Biomedical subjects

V I Tsetlin

Publications and source records attributed to V I Tsetlin.

At least 73 records · Page 4Linked to original sources
PubMed1983

[Study of long-chain neurotoxins from Naja naja siamensis and Naja naja oxiana venoms by circular dichroism and fluorescence].

CD and fluorescence spectroscopy were used to study the conformational mobility of long-chain neurotoxins from the venom of cobra Naja naja siamensis and Naja naja oxiana (STX and NT-I, respectively). Microenvironment of tryptophan residues in these neurotoxins and their lysine-modified derivatives was also analyzed. Ionization of His22 (pK 5,4-5,6) in STX influences the invariant Trp29 microenvironment. No conformational change was observed on titration of single His residue situated in the C-terminal part of NT-I. Attachment of dansyl groups at Lys39 or Lys60 residues in NT-I is without effect on the native neurotoxin conformation. The distances from these labels to Trp29 or Trp33 indole rings are no shorter than 1,0 nm. The data obtained for STX and NT-I indicate that the STX X-ray structure may serve as a basis for studies on the solution conformation of the long-chain neurotoxins.

Amino Acid Sequence↗
PubMed1982

Interaction surfaces of neurotoxins and acetylcholine receptor.

Binding of neurotoxin II Naja naja oxiana derivatives containing one spin label at various positions (Leu 1, Glu 2, Lys 15, Lys 25, Lys 26, His 31, Lys 44 and Lys 46) to purified solubilized acetylcholine receptor protein (AchR) from Torpedo marmorata was studied by EPR techniques. AchR interaction with several dansylated neurotoxin II derivatives was followed by difference fluorescence spectroscopy. A series of neurotoxin II p-azidobenzoyl derivatives were prepared and in three of them modified lysine residues were identified. In combination, spectroscopic data and photolabeling implicate a considerable area of the neurotoxin in association with AchR. Rigidity of the neurotoxin II conformation allowed to regard its binding surface as a mould of the AchR corresponding site and to estimate the minimal size of the latter. Conformation of the long-chain neurotoxins and their binding to AchR are briefly discussed basing on the 1H and 19F NMR studies of neurotoxin I Naja naja oxiana, toxin 3 Naja naja siamensis and its acetylated or trifluoroacetylated derivatives, as well as on Achr interaction with the derivatives spin labeled at Lys 27 and His 71.

Acetylcholine↗
PubMed1979

Conformational studies of neurotoxin II from Naja naja oxiana. Selective N-acylation, circular dichroism and nuclear-magnetic-resonance study of acylation products.

After treatment of neurotoxin II, a component part of the venom of the Middle Asian cobra Naja naja oxiana, with acetoxysuccinimide all five possible epsilon-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacetyl)-neurotoxins II and the hexa(trifluoroacetyl)-neurotoxin II, which were studied by circular dichroism (CD), 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five epsilon-amino groups) in the hexa(trifluoroacetyl)-neurotoxin II NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the 19F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin II 19F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.

Acylation↗
PubMed1976

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.

Amino Acid Sequence↗
PubMedDate not supplied

[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. 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.

Amino Acid Sequence↗
PubMedDate not supplied

[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., 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.

Amino Acid Sequence↗
PubMedDate not supplied

[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. 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.

Animals↗
PubMedDate not supplied

[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. 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).

Anabasine↗
PubMedDate not supplied

[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.

Animals↗
PubMedDate not supplied

[Conformational aspects of peptide interaction with proteolytic enzymes. Effect of amino acid residue configuration on binding of stereoisomeric N-acetylleucyltyrosine and N-acetyltyrosylleucine methylamides with pepsin].

Ki, Km and kcat. constants which characterize the interaction with pepsin of a complete series of Ac-Leu-Tyr-NHMe and Ac-Tyr-Leu-NHMe stereoisomers are determined. In compounds, containing residues of different configuration, Ki was found to have higher values than Ki or Km in DD- and LL-dipeptides. The data obtained show that not only the rate of peptide bond hydrolysis but also the efficiency of the binding with the enzyme depend on the configuration of the amino acid residue. Differences in the efficiency of pepsin binding of diastereomers is a manifestation of differences in their conformational characteristics. Data on "secondary" binding sites of pepsin are used to account for a mixed type inhibition observed in some cases.

Binding Sites↗
PubMedDate not supplied

[Conformational aspects of peptide interaction with proteolytic enzymes. Pepsin-catalyzed hydrolysis of cyclic peptides, containing leucyl-tyrosine fragments].

Pepsin was found capable of splitting cycloota- and cyclodecapeptides cyclo (-L-leucyl-L-tyrosyl-glycln-), n=6 or 8. The 18-membered peptides cyclo(-L-leucyl-L-tyrosyl-glycyl4-) and cyclo(-L-leucyl-L-tyrosy-delta-aminovaleroyl2-) were found stable to the effect of pepsin. To study the kinectics of hydrolysis for tyrosine-containing substrates of pepsin, a method of isolation of the unsplit substrate using ion-exchange resins and quantitative spectrophotometric estimation by absortion of tyysis of cycoocta and cyclodecapeptides was characterized by Km and kcat values.

Hydrolysis↗
PubMedDate not supplied

[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. 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).

Amino Acid Sequence↗
PubMedDate not supplied

[5-fluoro-tryptophan-containing N-terminal domain of the alpha-subunit of the Torpedo californica acetylcholine receptor: preparation in E. coli and 19F NMR study].

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

Animals↗