Graham D, Pfeiffer F, Simler R, Betz H
Biochemistry. 1985 Feb 12;24(4):990-4. doi: 10.1021/bi00325a027.
A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.
通过在氨基士的宁琼脂糖上进行亲和层析，开发了一种大规模纯化程序，用于分离猪脊髓中的甘氨酸受体。经过约10000倍的总体纯化后，甘氨酸受体制剂包含三种主要多肽，其分子量分别为48000、58000和93000。用[3H]士的宁进行光亲和标记表明，[3H]士的宁结合位点与分子量48000的多肽相关，与分子量58000的多肽的相关性较小。[3H]士的宁与纯化受体的结合表现出解离常数KD为13.8 nM，并受到激动剂甘氨酸、牛磺酸和β-丙氨酸的抑制。凝胶过滤和蔗糖梯度离心给出的斯托克斯半径为7.1 nm，表观沉降系数为9.6 S。对纯化的猪和大鼠甘氨酸受体制剂中[3H]士的宁标记的分子量48000多肽进行肽图谱分析表明，该受体亚基的士的宁结合区域在这些物种之间高度保守。此外，六种抗大鼠脊髓甘氨酸受体的单克隆抗体中有三种与猪甘氨酸受体的相应多肽发生了显著的交叉反应。这些结果表明，猪脊髓中的甘氨酸受体与已充分表征的大鼠受体蛋白非常相似，并且可以纯化到足以进行蛋白质化学分析的量。
