Search

Conotoxin ImI is a specific marker of α7 nicotinic acetylcholine receptors. To study the role of aromatic indole group of tryptophan 10 in biological activity of ImI, the analogue containing tyrosine at this position was synthesized by solid-phase peptide synthesis. The analogue obtained, as well as its iodinated derivatives, were shown to be active against rat brain α7 acetylcholine receptor expressed in Xenopus oocytes. Attachment of bulky aromatic p -benzoylbenzoyl group to N-terminal α-amino group of iodinated [Tyr 10 ]ImI only slightly affected the biological activity of the analogue. The data obtained suggest that indole ring of tryptophan 10 is not absolutely necessary for biological activity of conotoxin ImI, and that the N-terminus can accommodate a large aromatic group without loss of biological activity.

182 In the article by Yu.N. Utkin, Ch. Weise, et al., entitled “The New Peptide from the Fea’s Viper Azemiops feae Venom Interacts with Nicotinic Acetylcholine Receptors,” which was published in Dokl. Biochem. Biophys., 2012, vol. 442, there was a mistake in the state contract number. The correct number is 16.512.11.2207. Erratum: “The New Peptide from the Fea’s Viper Azemiops feae Venom Interacts with Nicotinic Acetylcholine Receptors” [Dokl. Biochem. Biophys., 2012, vol. 442, no. 1, pp. 33–35]

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

Snake venoms contain a vast array of toxic polypeptide components interacting with a variety of cell targets. Thus, Elapidae snake venoms contain α-neurotoxins with very high affinity for nicotinic acetylcholine receptors (nAChRs) and a few toxins able to suppress the activity of Ca2+ and K+ channels. Experimental evidence for the presence of nAChR antagonists and voltage-gated ionic channel blockers in venoms of Viperidae snakes is very scarce. In this study, effects of crude venoms of seven snake species (Vipera nikolskii, Echis multisquamatus, Gloydius saxatilis, Bitis arietans, Vipera renardi, Vipera lebetina, and Naja kaouthia) on nAChRs and voltage-gated Ca2+ channels were studied for the first time. The experiments were carried out on isolated identified neurons of the fresh-water mollusc Lymnaea stagnalis using voltage-clamp and intracellular perfusion techniques. All Viperidae snake venoms under study blocked nAChRs and voltage-gated Ca2+ channels. The potency of these venoms against nAChRs was significantly lower in comparison with N. kaouthia venom which is rich of α-neurotoxins; however, the extent of Ca2+ channel block by venoms of Viperidae snakes and N. kaouthia was similar. The data obtained suggest that Viperidae snake venoms tested in this study contain peptides with affinity both for nAChRs and for voltage-gated Ca2+ channels.

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.

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.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.

To determine whether the ability to induce neurite outgrowth in rat pheochromocytoma cell line PC12 is characteristic of phospholipases of different types, we have studied the influence of phospholipase A(2) (PLA2) from cobra Naja kaouthia venom and two PLA2s from viper Vipera nikolskii venom on PC12 cells. Phospholipases from the viper venom are heterodimers in which only one of the subunits is enzymatically active, while PLA2 from the cobra venom is a monomer. It was found that all three PLA2s induce neurite outgrowth in PC12. The PLA2 from cobra venom exhibits this effect at higher concentrations as compared to the viper enzymes. We have not observed such an activity for isolated subunits of viper PLA2s, since the enzymatically active subunits have very high cytotoxicity, while the other subunits are not active at all. However, co-incubation of active and inactive subunits before addition to the cells leads to a marked decrease in cytotoxicity and to restoration of the neurite-inducing activity. It has also been shown that all enzymatically active PLA2s are cytotoxic, the PLA2 from cobra venom being the least active. Thus, for the first time we have shown that PLA2s from snake venoms can induce neurite outgrowth in PC12 cells.

Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.

Polyclonal antibodies obtained by immunization of rabbits with native form of weak toxin (WTX) from cobra Naja kaouthia venom efficiently interacted with WTX and a weak toxin from Naja oxiana venom, but not so with their denaturated forms. These antibodies could also bind with lower affinity other groups of three-fingered toxins: long-chain alpha-neurotoxins, muscarinic toxins and cytotoxins, but practically did not bind short-chain alpha-neurotoxins. The efficiency of toxin-antibody interaction depends on the group (weak toxins, long or short alpha-neurotoxins, cytotoxins etc.) to which the toxin belongs, but not on species of snake from which the toxin originates. There is a correlation between the results obtained and phylogenetic analysis of the three-fingered toxins which revealed that WTX is very close to other weak toxins, relatively close to long alpha-neurotoxins, cytotoxins and muscarinic toxins, but is distant from the short alpha-neurotoxins.

Photoinduced cross-links between the iodinated Lys26-p-azidobenzoyl derivative of neurotoxin II from Naja naja oxiana cobra venom and nicotinic acetylcholine receptor from Torpedo californica (AChR) have been studied in the presence of alpha-conotoxin GI from the marine snail C. geographus. Preincubation of the AChR-enriched membranes with increasing concentrations of alpha-conotoxin GI protects first the gamma subunit from photolabelling and then the delta subunit, the IC50 values being 0.76 and 5.01 microM, respectively. The results obtained, in view of the relevant data in literature, demonstrate that the (alpha + gamma) site, which is the high affinity site for d-tubocurarine, has also a higher affinity for an alpha-conotoxin than the (alpha + delta) containing site. The latter has a somewhat higher affinity than the (alpha + gamma) site towards some naturally occurring snake venom alpha-neurotoxins or their derivatives.

Amino acid sequences of several fragments of the 25k 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 25k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.

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.

selected groupings gives crucial information on the conformation of proteins in solution and characterizes the topography of the sites responsible for biological activity, It also enables a comparison of the protein’s spatial structure in solution and in the crystal to be made on a quantitative basis, which is essential for understanding the dynamic features and conformational potentialities of the molecule. Evaluation of the intramolecular distances in proteins in solution can be done by optical spectroscopy, EPR and NMR techniques. For instance, detailed information can be obtained through the analysis of shift and relaxation effects of paramagnetic metal ions [l] or covalent spin labels (stable nitroxyl radicals) [2] in the NMR spectra of proteins. However, there arises a serious problem of signal assignment in overlapping proton spectra of proteins. In this respect it appears promising to use line-broadening evoked by spin labels in 19F NMR spectra of fluorine-containing protein derivatives. Such derivatives are obtained via chemical modification or biosynthetically, whereas their spectra usually consist of a limited number of signals [3,4 1. Feasibility of such an approach is demonstrated here with neurotoxin II (NT-II) isolated from the venom of Central Asian cobra (Naja naja oxiuna). A spin label was incorporated at the e-amino group of the Lys 27* residue. The amino groups of Leu 1, Lys 15, Lys 26, Lys 45 and Lys 47 residues were trifluoroacetylated. The distances between the spin label and the trifluoroacetyl groups were evaluated by r9F NMR. Comparison of the results on the spatial structure of neurotoxins in solution [2,6] and in the crystal [7] points to adequacy of the utilized approach.

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.

Fifteen Naja naja oxiana neurotoxin II derivatives mono and di spin-labeled on Lys-15, Lys-25, Lys-26, Lys-44 and Lys-46 have been prepared and the position of the label(s) identified. Spin label distances to a number of the side chains were determined by 1H NMR for the mono-labeled species and interlabel distances were determined by EPR for the di-labeled residues. Binding of the mono spin-labeled derivatives to solubilized acetylcholine receptor protein (AChR) from Torpedo marmorata was followed by EPR. By means of a spin probe technique, it was found that spin labels on Lys-15, Lys-25, Lys-46 and, especially Lys-26, are less accessible in the AChR complexes than in the free toxins. Involvement of the dansyl labels on Lys-26 and Lys-46 in the binding was demonstrated by fluorescence spectroscopy. The data obtained yielded evidence of a β-structure of the toxin and allowed preliminary characterization of the neurotoxin binding surface.

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).