RESUMEN
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.
Asunto(s)
Venenos Elapídicos , Glicoproteínas , Secuencia de Aminoácidos , Animales , Cisteína , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Venenos Elapídicos/farmacología , Elapidae , Animales , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , TriptófanoRESUMEN
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).
Asunto(s)
Venenos Elapídicos/química , Neurotoxinas/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas de ReptilesRESUMEN
Nerve growth factor (NGF) from the venom of cobra Naja kaouthia is highly homologous to mouse NGF. However, the differences between these two factors include the sequence regions determining the specificity of NGF interaction with Trk A or p75 receptors. To test if these variations can bring about dissimilarity in biological activity between these two NGFs, we have studied the effect of cobra factor on the survival of the primed PC12 cells after serum withdrawal. It was found that in a serum-free medium, cobra NGF prevented the death of PC12 cells with efficacy comparable to that of NGF from mouse submaxillary glands. In the course of purification two forms of cobra NGF were observed, both acting as a survival and a differentiation factor for PC12 cells in a serum-free medium. The form, eluting later from a reversed-phase column, displays survival effect at lower concentrations than the earlier eluting one.
Asunto(s)
Proteínas Neurotóxicas de Elápidos/farmacología , Factor de Crecimiento Nervioso/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Ratones , Células PC12 , Ratas , Glándula SubmandibularRESUMEN
A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.
Asunto(s)
Venenos Elapídicos/farmacología , Músculo Esquelético/fisiología , Receptores Nicotínicos/fisiología , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bungarotoxinas/farmacocinética , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Clonación Molecular , Proteínas Neurotóxicas de Elápidos/farmacología , Venenos Elapídicos/química , Elapidae , Escherichia coli , Femenino , Humanos , Técnicas In Vitro , Modelos Moleculares , Neurotoxinas/farmacología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Conformación Proteica , Ratas , Receptores Colinérgicos/efectos de los fármacos , Receptores Colinérgicos/fisiología , Receptores Nicotínicos/efectos de los fármacos , Proteínas Recombinantes/farmacocinética , Torpedo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.
Asunto(s)
Venenos Elapídicos/química , Neurotoxinas/química , Triptófano/química , Secuencia de Aminoácidos , Animales , Venenos Elapídicos/toxicidad , Hidrólisis , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Neurotoxinas/aislamiento & purificación , Neurotoxinas/toxicidad , Receptores Nicotínicos/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Torpedo , TripsinaRESUMEN
Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.
Asunto(s)
Colinérgicos/química , Venenos Elapídicos/química , Secuencia de Aminoácidos , Animales , Células CHO , Cromatografía por Intercambio Iónico , Quimotripsina/farmacología , Cricetinae , ADN Complementario/metabolismo , Disulfuros , Elapidae , Concentración 50 Inhibidora , Cinética , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Receptores Muscarínicos/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
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%.