Xin H B, Timerman A P, Onoue H, Wiederrecht G J, Fleischer S
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
Biochem Biophys Res Commun. 1995 Sep 5;214(1):263-70. doi: 10.1006/bbrc.1995.2283.
The ryanodine receptor (RyR)/calcium release channel isolated from skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) is tightly associated with FK506 binding protein of 12.0 kDa (FKBP12) (Jayaraman et al., (1992) J.Biol.Chem. 267, 9474-9477). In this study, we describe a new method of affinity chromatography for purifying the RyR from skeletal muscle SR based on: 1) its tight association with FKBP12; and 2) the finding that bound FKBP on the RyR can be exchanged with soluble FKBP12 (Timerman et al., (1995) J.Biol.Chem. 270, 2451-2459). Soluble glutathione S-transferase/FKBP12 (GST/FKBP12) fusion protein was first exchanged with bound FKBP12 on the RyR of TC. The TC were then solubilized with CHAPS and the complex of RyR.GST/FKBP12 was specifically adsorbed by glutathione Sepharose 4B and then eluted with glutathione. The RyR, purified by this method, has similar characteristics by SDS-PAGE, radioligand binding and immuno-reactivity as the RyR purified by multiple sequential column chromatography.
从骨骼肌肌浆网（SR）终末池（TC）分离出的兰尼碱受体（RyR）/钙释放通道与12.0 kDa的FK506结合蛋白（FKBP12）紧密相关（贾亚拉曼等人，（1992年）《生物化学杂志》267卷，9474 - 9477页）。在本研究中，我们描述了一种基于以下两点从骨骼肌SR中纯化RyR的亲和层析新方法：1）它与FKBP12的紧密结合；2）发现RyR上结合的FKBP可与可溶性FKBP12进行交换（蒂默曼等人，（1995年）《生物化学杂志》270卷，2451 - 2459页）。首先将可溶性谷胱甘肽S - 转移酶/FKBP12（GST/FKBP12）融合蛋白与TC的RyR上结合的FKBP12进行交换。然后用CHAPS使TC溶解，RyR.GST/FKBP12复合物被谷胱甘肽琼脂糖4B特异性吸附，随后用谷胱甘肽洗脱。通过该方法纯化的RyR，经SDS - PAGE、放射性配体结合和免疫反应性检测，与通过多次连续柱层析纯化的RyR具有相似的特性。
