Madras B K, Seeman P
J Neurochem. 1985 Mar;44(3):856-61. doi: 10.1111/j.1471-4159.1985.tb12894.x.
To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin-solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9-11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.
为检测部分纯化的多巴胺受体对各种多巴胺能激动剂和拮抗剂的敏感性，通过等电聚焦法富集犬脑纹状体多巴胺受体。用洋地黄皂苷增溶的受体先用[³H]螺哌隆进行预标记，然后聚焦两个时间段。5小时后（不完全聚焦），在pH 6和9 - 11处检测到放射性峰。只有pH 6的峰显示出D2受体预期的药物敏感性。pH 6峰的受体回收率为79%，纯化倍数为7倍。过夜聚焦至平衡后，pH 6峰进一步分离为pH 4.6和6.8处的峰。仅在pH 4.6部分鉴定出该受体。pH 4.6峰中受体的回收率很低（10%），表明该受体几乎没有富集。抗精神病药物和多巴胺激动剂与纯化物质的结合顺序与原始可溶性受体制剂相似。除非将聚焦过程中使用的磷酸盐缓冲液替换为Tris缓冲液，多巴胺不会与纯化的pH 4.6部分结合。pH 6.8和pH 10部分虽然都含有预标记的[³H]螺哌隆，但没有受体，这表明在纯化的每个步骤对每个部分进行激动剂和拮抗剂测试的重要性。
