Kuriyama K, Taguchi J
Department of Pharmacology, Kyoto Prefectural University of Medicine, Kamikyo-Ku, Kyoto 602, Japan.
Neurochem Int. 1987;10(3):253-63. doi: 10.1016/0197-0186(87)90098-2.
Purification of solubilized ?-aminobutyric acid (GABA) and benzodiazepine receptors by Nonidet P-40 from the bovine cerebral cortex was employed by the affinity column chromatography using a benzodiazepine, 1012-S, as immobilized ligand. Although this benzodiazepine affinity gel retained all of the solubilized benzodiazepine receptors from synaptic membranes applied to the column, 25.6% of the GABA receptor binding activity was recovered in the run-through fraction. After the washing with NaCl, the biospecific elution with 1 mM 1012-S resulted the elution of 31.3% of GABA receptor binding activity applied to the column. The highest purification fold thus obtained was 3789 (specific activity: 0.39 nmol/mg protein). Successive application of 1-2 mM NaSCN also resulted further elution of GABA receptor. Scatchard analysis of [(3)H]muscimol binding to these fractions indicated that the purified GABA receptor had high and low affinity binding sites as detected in solubilized and synaptic membrane fractions. In addition, it was found that the B(max) values for both binding sites in the 1012-S-eluted fraction increased significantly without changing the K(D) values. SDS-polyacrylamide gel electrophoretic profiles of the 1012-S-eluted fraction showed the existence of two major bands having the molecular weights of approx. 53,000 and 57,000, both of which were irreversibly photoaffinity-labeled with [(3)H]flunitrazepam. This result made a sharp contrast with the result obtained in the rat brain, in which the subunits of the purified receptor consisted of molecular weights of 48,500 and 54,500 and the former band was selectively photoaffinity-labeled with [(3)H]flunitrazepam. In contrast, the gel filtration using Sephadex G-200 of the purified GABA/benzodiazepine receptor complex from both animal species revealed the molecular weight of these receptor complexes was approx. 340,000. These results strongly suggest that the subunit structures of GABA/benzodiazepine receptors from the bovine and rat brains may be essentially different. On the other hand, the purification of GABA/benzodiazepine receptor complex and C1 channel solubilized by CHAPS, a non denaturating zwitterionic detergent, using the same benzodiazepine affinity column followed by DEAE-Sephacel ion exchange column chromatography indicated that the purified GABA receptor was functionally coupled with both benzodiazepine receptor and C1 channel. These results also indicate that GABA(A) receptor which is structurally coupled with benzodiazepine receptor and C1 channel is readily purified by the use of this benzodiazepine affinity gel, whereas GABA(B) receptor which is not associated with both components is recovered in its run-through fraction, respectively.
采用非离子去污剂NP-40从牛大脑皮层中增溶γ-氨基丁酸（GABA）和苯二氮䓬受体，并使用苯二氮䓬1012-S作为固定配体通过亲和柱色谱法进行纯化。尽管这种苯二氮䓬亲和凝胶保留了施加到柱上的突触膜中所有增溶的苯二氮䓬受体，但25.6%的GABA受体结合活性在穿透组分中被回收。用NaCl洗涤后，用1 mM 1012-S进行生物特异性洗脱，导致施加到柱上的31.3%的GABA受体结合活性被洗脱。由此获得的最高纯化倍数为3789（比活性：0.39 nmol/mg蛋白质）。连续施加1 - 2 mM NaSCN也导致GABA受体的进一步洗脱。对这些组分进行[(3)H]蝇蕈醇结合的Scatchard分析表明，纯化的GABA受体具有在增溶和突触膜组分中检测到的高亲和力和低亲和力结合位点。此外，发现1012-S洗脱组分中两个结合位点的B(max)值在不改变K(D)值的情况下显著增加。1012-S洗脱组分的SDS-聚丙烯酰胺凝胶电泳图谱显示存在两条主要条带，分子量约为53,000和57,000，两者均被[(3)H]氟硝西泮不可逆地光亲和标记。该结果与在大鼠脑中获得的结果形成鲜明对比，在大鼠脑中纯化受体的亚基由分子量为48,500和54,500组成，并且前一条带被[(3)H]氟硝西泮选择性地光亲和标记。相比之下，对来自两种动物的纯化的GABA/苯二氮䓬受体复合物使用Sephadex G-200进行凝胶过滤显示这些受体复合物的分子量约为340,000。这些结果强烈表明牛和大鼠脑内GABA/苯二氮䓬受体的亚基结构可能存在本质差异。另一方面，使用相同的苯二氮䓬亲和柱随后进行DEAE-琼脂糖离子交换柱色谱法对由非变性两性离子去污剂CHAPS增溶的GABA/苯二氮䓬受体复合物和Cl通道进行纯化，表明纯化的GABA受体在功能上与苯二氮䓬受体和Cl通道均偶联。这些结果还表明，与苯二氮䓬受体和Cl通道在结构上偶联的GABA(A)受体可通过使用这种苯二氮䓬亲和凝胶容易地纯化，而与这两种成分均不相关的GABA(B)受体则分别在其穿透组分中被回收。
