Curtis B M, Catterall W A
Biochemistry. 1984 May 8;23(10):2113-8. doi: 10.1021/bi00305a001.
[3H]Nitrendipine binds with high affinity to a calcium antagonist receptor preferentially localized to the transverse tubule membrane of skeletal muscle. Digitonin was used to solubilize a [3H]nitrendipine-receptor complex from transverse tubule membranes. The digitonin-[3H]nitrendipine-receptor complex was purified 330-fold by a combination of wheat germ agglutinin chromatography, ion-exchange chromatography, and sedimentation through sucrose gradients to yield a preparation estimated to be 41% homogeneous on the basis of specific activity. Analysis by sodium dodecyl sulfate gel electrophoresis demonstrated that three polypeptides termed alpha [molecular weight (Mr) 130 000], beta (Mr 50 000), and gamma (Mr 33 000) quantitatively comigrated with the [3H]nitrendipine-receptor complex on sucrose gradients and represented 62% of the total protein staining. These three polypeptides are associated noncovalently. However, the apparent molecular weight of the alpha polypeptide is reduced from 160 000 to 130 000 upon reduction, consistent with the presence of an internal disulfide bond. Our results suggest that these three polypeptides are the subunits of the calcium antagonist receptor and are major components of the transverse tubule voltage-sensitive calcium channel.
[3H]尼群地平以高亲和力与一种钙拮抗剂受体结合，该受体优先定位于骨骼肌的横管膜。用洋地黄皂苷从横管膜中溶解[3H]尼群地平 - 受体复合物。通过麦胚凝集素色谱法、离子交换色谱法和蔗糖梯度沉降相结合的方法，将洋地黄皂苷 - [3H]尼群地平 - 受体复合物纯化330倍，得到一种根据比活性估计为41%均一的制剂。十二烷基硫酸钠凝胶电泳分析表明，三种多肽，称为α（分子量130000）、β（分子量50000）和γ（分子量33000），在蔗糖梯度上与[3H]尼群地平 - 受体复合物定量共迁移，占总蛋白染色的62%。这三种多肽以非共价方式结合。然而，还原后α多肽的表观分子量从160000降至130000，这与存在一个内部二硫键一致。我们的结果表明，这三种多肽是钙拮抗剂受体的亚基，并且是横管电压敏感性钙通道的主要成分。
