King R G, Nielsen M, Stauber G B, Olsen R W
Department of Biochemistry, University of California, Riverside.
Eur J Biochem. 1987 Dec 15;169(3):555-62. doi: 10.1111/j.1432-1033.1987.tb13645.x.
Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.
用两性离子去污剂3-[(3-胆酰胺丙基)-二甲基铵基]丙烷磺酸盐（Chaps）溶解大鼠脑膜匀浆中惊厥剂t-丁基双环磷[35S]硫代酸盐结合活性，并在凝胶过滤和亲和色谱上显示其与苯二氮卓-γ-氨基丁酸（GABA）受体复合物共纯化。虽然在其他去污剂（如脱氧胆酸钠或Triton X-100）中溶解或向两性离子去污剂溶解的提取物中添加Triton X-100可消除惊厥剂结合活性，但不会消除GABA和苯二氮卓受体结合活性，惊厥剂活性并非不可逆转地丧失或选择性不稳定，而是可通过将蛋白质换回去污剂Chaps来恢复。单独用Chaps溶解的含有惊厥剂活性的GABA-苯二氮卓受体活性，以及在Chaps中添加Triton X-100且缺乏惊厥剂活性的样品，通过凝胶过滤柱色谱法或辐射失活靶标大小分析测量，其大小没有差异。这表明惊厥剂结合活性不需要任何额外的蛋白质亚基或其他大分子，相对于GABA和苯二氮卓受体结合也不需要任何独特的聚集状态，并且所有三种活性都存在于同一蛋白质复合物上。与完整大脑一样，惊厥剂结合活性的靶标大小是苯二氮卓结合活性的3至5倍，这表明惊厥剂活性需要受体复合物具有寡聚蛋白结构，且在天然膜环境中存在完整的强亚基相互作用，并且这种结构和其他特性在Chaps中比在其他去污剂中保留得更好。
