Ligand interactions with the solubilized porcine atrial muscarinic receptor.
Ligand interactions with the solubilized porcine atrial muscarinic receptor.
复制标题
配体与溶解的猪心房毒蕈碱受体的相互作用。
DOI：
10.1021/bi00532a016
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Schimerlik,MI
中科院分区：
文献类型：
--
作者：
Herron,GS;Miller,S;Manley,WL;Schimerlik,MI
材料和方法根据Cremo et al.（1981）的描述，将猪心房微体中的mAcChR溶于10 mM磷酸钠、1 mM EDTA、1 mM PMSF、0.4% w/v洋地黄苷和0.08% w/v胆酸缓冲液中，pH为7.4。通过使用Schmidt & Raftery（1973）开发的DEAE滤片法，根据QNB结合位点对溶解受体进行定量（Yamamura & Snyder, 1974），并在本实验室进行了改进，用于上述缓冲系统（Cremo et al., 1981）。蛋白质浓度测定采用Lowry等人（1951）（经peterson（1977）改进）的方法，以结晶牛血清白蛋白为标准。可溶性提取物的比活性在每g蛋白1 ~ 5 nmol的QNB位点之间变化，心房微粒体的总QNB位点的回收率在85% ~ 98%之间。溶解后的mAcChR要么立即使用，要么在0℃下保存，至少稳定一周（在0℃下8天后，大约20%的QNB位点丢失）。
Materials and MethodsThe mAcChR was solubilized from porcine atrial micro-somes in 10 mM sodium phosphate, 1 mM EDTA, 1 mM PMSF, 0.4% w/v digitonin, and 0.08% w/v cholate buffer, pH 7.4, as described by Cremo et al.(1981). The solubilized receptor was quantitated in terms of QNB binding sites (Yamamura & Snyder, 1974) by using the DEAE filter disc assay developed by Schmidt & Raftery (1973) and modified in this laboratory for use in the above-mentioned buffer system (Cremo et al., 1981). Protein concentration was determined by the method of Lowry et al.(1951) as modified byPeterson (1977) with crystalline bovine serum albumin as the standard. Specific activities of the solubilized extract varied between 1 and 5 nmol of QNB sites per g of protein, and recoveries of total QNB sites from the atrial microsomes were between 85 and 98%. The solubilized mAcChR was either used immediately or stored at 0 C where it appeared to be stable for at least a week (about 20% of the total QNB sites were lost after 8 days at 0 C).