Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes.
The tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from electroplax  membranes of Electrophorus electricus has been purified. The toxin-binding site could be efficiently solubilized with Lubrol-PX, resulting in an extract of high initial specific activity. Purification was facilitated by the development of a rapid, quantitative binding assay. The binding component was stabilized during purification by the use of mixed lipid/detergent micelles of defined composition, and by the saturation of the site with tetrodotoxin. The purification was achieved by means of a highly selective adsorption of the toxin-binding component to DEASE-Sephadex A-25, followed by desorption at high ionic strength and chromatography over Sepharose 6B. Final peak specific activities were at least 50% of the specific activity expected for a pure, undenatured toxin-binding componenet of 230,000 molecular weight. The purified material exhibited a sedimentation coefficient of approximately 8 S and an unusual Stokes radius of 95 A. Purified material showed a relatively simple pattern on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, being comprised of only three polypeptides.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D003902:Detergents; D004557:Electric Organ / Q000032:analysis*; D004593:Electrophorus; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008823:Micelles; D010713:Phosphatidylcholines; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D012995:Solubility; D013779:Tetrodotoxin / Q000378:metabolism*
D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D013779:Tetrodotoxin; D012964:Sodium