Hartshorne R P, Catterall W A
J Biol Chem. 1984 Feb 10;259(3):1667-75.
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.
本文描述了一种从大鼠脑中纯化钠通道的方法，纯化倍数达1380倍，达到基本均一性。该通道在Triton X-100中溶解，并通过添加磷脂酰胆碱和10 mM氯化钙来稳定。通过依次在DEAE-葡聚糖、羟基磷灰石和麦胚凝集素/琼脂糖上进行层析，然后通过蔗糖梯度沉降进行纯化。最终制剂每毫克蛋白质结合2910 pmol的石房蛤毒素（STX），或每摩尔Mr约为316,000的钠通道结合0.9摩尔STX。在纯蛋白质的十二烷基硫酸钠-聚丙烯酰胺凝胶上，三种多肽亚基占银染强度的90%，并在蔗糖梯度沉降中以与STX结合活性一致的化学计量复合物形式迁移：Mr约为260,000的α亚基、Mr约为39,000的β1亚基和Mr约为37,000的β2亚基。纯化的α亚基以及完整突触体中的α亚基在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳过程中表现异常，呈现出异常高的外推电泳自由迁移率。提出了α1(β1)1(β2)1的亚基化学计量关系。
