The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein.
Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.
Villegas
R
R
Instituto Internacional de Estudios Avanzados (IDEA), Caracas (Venezuela).
Sorais-Landáez
F
F
Rodŕiguez-Grille
J M
JM
Villegas
G M
GM
D000327:Adsorption; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis; D002845:Chromatography; D003902:Detergents; D007473:Ion Channels / Q000032:analysis*; D008121:Nephropidae / Q000032:analysis*; D009420:Nervous System / Q000032:analysis*; D000077423:Polidocanol; D011092:Polyethylene Glycols; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels; D012995:Solubility; D013779:Tetrodotoxin / Q000378:metabolism
D002352:Carrier Proteins; D003902:Detergents; D007473:Ion Channels; D015222:Sodium Channels; C023790:tetrodotoxin-binding protein; D000077423:Polidocanol; D011092:Polyethylene Glycols; D013779:Tetrodotoxin; D012964:Sodium