Tokumaru H, Anzai K, Abe T, Kirino Y
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Eur J Pharmacol. 1992 Dec 1;227(4):363-70. doi: 10.1016/0922-4106(92)90152-l.
The 1,4-dihydropyridine receptor associated with L-type Ca2+ channels was purified about 1700-fold from porcine cardiac sarcolemmal membranes using a simple and rapid (ca. 8 h) two-step procedure: wheat germ agglutinin affinity chromatography followed by immunoaffinity chromatography with a monoclonal antibody (MCC-1) against the alpha 2 delta subunit of the skeletal muscle Ca2+ channel with a glycine elution buffer (pH 3). Gel electrophoresis of this purified sample under non-reducing conditions revealed a major polypeptide band with molecular weight of 190 kDa, which was separated under reducing conditions to a 155 kDa band and 2-3 bands with M(r) about 20 kDa, corresponding to alpha 2 and delta subunits, respectively. The peptide band corresponding to the alpha 1 subunit was not detected in this gel electrophoresis. However, the alpha 1 subunit without bound alpha 2 delta was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the alpha 1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of alpha 1 was smaller than that of the alpha 2 subunit in the purified sample obtained here.
利用一种简单快速（约8小时）的两步法，从猪心肌肌膜中纯化出与L型钙离子通道相关的1,4 - 二氢吡啶受体，纯化倍数约为1700倍：第一步为麦胚凝集素亲和层析，第二步为用针对骨骼肌钙离子通道α2δ亚基的单克隆抗体（MCC - 1）进行免疫亲和层析，洗脱缓冲液为甘氨酸（pH 3）。在此纯化样品的非还原条件下进行凝胶电泳，显示出一条分子量为190 kDa的主要多肽带，在还原条件下该带分离为一条155 kDa的带和2 - 3条分子量约为20 kDa的带，分别对应α2和δ亚基。在该凝胶电泳中未检测到对应α1亚基的肽带。然而，未结合α2δ的α1亚基用1% Triton X - 100从MCC - 1琼脂糖凝胶中选择性洗脱。通过荧光显影和银染法，在用Triton X - 100洗脱的组分中可见一条对应α1亚基的190 kDa带。从电泳结果来看，在此获得的纯化样品中α1的量比α2亚基的量少。
