Isolation, amino acid sequence and functional assays of SGTx1. The first toxin purified from the venom of the spider scodra griseipes.
A new toxin (SGTx1) was purified from the venom of the spider Scodra griseipes by a combination of gel filtration and reverse-phase chromatography. The complete amino acid sequence of SGTx1, TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF, was established by direct automated Edman degradation, and is in perfect agreement with the molecular mass of 3775 Da found by mass spectrometry. The primary structure of SGTx1 exhibited sequence identity with other spider toxins such as hanatoxin (76%), TxP5 toxin (32%) and huwentoxin (26%). The six cysteines in the sequence suggested three disulfide bridges, the presence of which was demonstrated by mass spectrometry after dithiothreitol reduction. Analysis of secondary structure using circular dichroism spectrometry yielded more than 50% beta-sheet and about 15-20% beta-turn. The extent of the beta-content and the presence of disulfide bridges suggest a structure of interconnected beta-strands. In addition, a study of membrane/toxin interactions was carried out by reconstitution in planar lipid bilayers and by antibacterial assays. SGTx1 displays moderate pore-forming ability (conductance of about 100 pS in 1 M NaCl), but antibacterial activity was not observed against Gram-positive or Gram-negative strains. As a preliminary assay, the activity of SGTx1 was investigating using electrophysiological measurements. At 0.15 microM, SGTx1 reversibly inhibits more than 40% of outward potassium currents in rat cerebellum granular cells. This result is reminiscent with the effect described for hanatoxin extracted from the venom of Grammostola spatulata.
Marvin
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Spectrométrie de Masse Bio-oRGANIQUE cnrs-upresa 6014, Mont Saint Aignan, France.
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Cosette
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Gagnon
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Molle
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Lange
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000187:drug effects; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D008051:Lipid Bilayers / Q000737:chemistry; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013111:Spider Venoms / Q000737:chemistry* / Q000494:pharmacology
D008051:Lipid Bilayers; D009498:Neurotoxins; D015221:Potassium Channels; C400227:SGTx1 toxin; D013111:Spider Venoms