RESUMEN
A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Caracoles/química , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Conotoxinas/aislamiento & purificación , Electrofisiología , India , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.
Asunto(s)
Bloqueadores de los Canales de Potasio/aislamiento & purificación , Canales de Potasio con Entrada de Voltaje , Canales de Potasio , Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Canal de Potasio Kv.1.1 , Datos de Secuencia Molecular , Escorpiones , Homología de Secuencia de AminoácidoRESUMEN
A short chain peptide has been isolated from the venom of a red scorpion of Indian origin, Buthus tamulus. This peptide was purified using ion exchange and reverse phase chromatography and was characterized by molecular weight determination and amino acid sequence. The primary structure analysis shows that BtITx3 is a short peptide of 35 amino acid residues having a molecular weight of 3796 Da. The toxin shows toxicity towards the Lepidopteran species of insect Helicoverpa armigera causing flaccid paralysis and even death within 24 h. It shows more than 50% homology with the short insectotoxins having four disulfide bridges, which suggests that the toxin belongs to the class of short chain toxins blocking the chloride ion channels. This sequence homology study has also helped to bring out the structure-function relationship between the various short toxins. Homology modeling done by using template structure of a known toxin indicated that this toxin consists of a similar alpha/beta scaffold, as present in other scorpion toxins.
Asunto(s)
Venenos de Escorpión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Lepidópteros , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Venenos de Escorpión/química , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/química , Escorpiones/genética , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
This report summarizes our efforts towards depth-dependent analysis of membranes by design of suitable fluorescent and photoactivable lipid probes, which can be incorporated into membranes. The objective of depth-dependent analysis has been two fold, one to obtain information on lipid domains and other on transmembrane domains of membrane-bound proteins. In view of increasing importance of lipid rafts and other localized domain and limited success in case of structure determination of membrane-bound proteins vis-à-vis their soluble counterparts, it is tempting to rapidly attach fluorescent or photoactivable probes to lipids to get a probes where relatively little attention is paid to design of such probes. We have shown here how careful design of such probes is required to immobilize such probes in membranes for effective depth-dependent analysis of membranes. An effective design has become important when identification of putative transmembrane domains predicted primarily from the genome data based on hydropathy plots, often needs confirmation by contemporary methodology.