Nakayama N, Kirley T L, Vaghy P L, McKenna E, Schwartz A
J Biol Chem. 1987 May 15;262(14):6572-6.
A putative Ca2+ channel protein was purified from rabbit skeletal muscle transverse tubules with the combined use of lectin affinity chromatography and ion-exchange chromatography, followed by sucrose density gradient centrifugation. The major component of the purified preparation detected by sodium dodecyl sulfate-gel electrophoresis was a protein of 150 kDa when reduced with 20 mM dithiothreitol and a 191-kDa protein when treated with 20 mM N-ethylmaleimide. Therefore, this protein appears to be identical with the alpha subunit previously described (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118). This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis, followed by electroelution and/or electroblotting, and its amino acid composition and NH2-terminal sequence were determined. The NH2-terminal sequence is: NH2-Glu-Pro-Phe-Pro-Ser-Ala-Val-X-Ile-Lys-Ser-X-Val-X-Lys-Met-Gln-.
通过结合使用凝集素亲和层析和离子交换层析，随后进行蔗糖密度梯度离心，从兔骨骼肌横管中纯化出一种假定的钙离子通道蛋白。用十二烷基硫酸钠 - 凝胶电泳检测纯化制剂的主要成分，当用20 mM二硫苏糖醇还原时是一种150 kDa的蛋白质，用20 mM N - 乙基马来酰亚胺处理时是一种191 kDa的蛋白质。因此，这种蛋白质似乎与先前描述的α亚基相同（柯蒂斯，B.M.，和卡特拉尔，W.A.（1984年）《生物化学》23，2113 - 2118）。通过制备性十二烷基硫酸钠 - 凝胶电泳纯化该蛋白质，随后进行电洗脱和/或电印迹，并测定其氨基酸组成和NH2末端序列。NH2末端序列为：NH2 - 谷氨酸 - 脯氨酸 - 苯丙氨酸 - 脯氨酸 - 丝氨酸 - 丙氨酸 - 缬氨酸 - X - 异亮氨酸 - 赖氨酸 - 丝氨酸 - X - 缬氨酸 - X - 赖氨酸 - 甲硫氨酸 - 谷氨酰胺 - 。
