The sodium channel from rat brain. Purification and subunit composition.
A procedure is described for purification of the sodium channel 1380-fold from rat brain to essential homogeneity. The channel is solubilized in Triton X-100 and stabilized by addition of phosphatidylcholine and 10 mM CaCl2. It is purified by sequential chromatography on DEAE-Sephadex, hydroxylapatite, and wheat germ agglutinin/Sepharose followed by sedimentation through sucrose gradients. The final preparation binds 2910 pmol of saxitoxin (STX)/mg of protein or 0.9 mol of STX/mol of sodium channel of Mr approximately 316,000. Three polypeptide subunits comprise 90% of the silver stain intensity on sodium dodecyl sulfatepolyacrylamide gels of the pure protein and migrate as a stoichiometric complex coincident with STX-binding activity in sucrose gradient sedimentation: alpha with Mr approximately 260,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. The alpha subunit, both purified and in intact synaptosomes, is shown to behave anomalously during sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting an unusually high extrapolated electrophoretic free mobility. A subunit stoichiometry of alpha 1(beta 1)1(beta 2)1 is proposed.
D029845:Amphibian Proteins; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D046911:Macromolecular Substances; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D012964:Sodium