牛脑纹状体多巴胺D_2受体的纯化

Purification of Dopamine D_2 Receptor from Bovine Striatal

目的:纯化牛脑纹状体多巴胺Ｄ２受体。方法:以胆酸和氯化钠增溶纹状体膜制剂,以氟哌啶醇偶联的Ｓｅｐｈａｒｏｓｅ ６Ｂ为亲和基质,用螺哌隆洗脱纯化Ｄ２受体。通过[３Ｈ]螺哌隆结合饱和实验和竞争实验测定其平衡解离常数(Ｋｄ)及配基结合特征。结果:经一次亲和层析,Ｄ２受体被纯化约１９００倍,回收率约８．７%,比活性为１６８．７ｐｍｏｌ·ｍｇ－１,相对分子质量约９４ ０００。通过Ｓｃａｔｃｈａｒｄ作图分析,纯化的受体Ｋｃ为０．２７ｎｍｏｌ·Ｌ－１,并保持与Ｄ２受体的配基结合特征。结论:以氟哌啶醇偶联的Ｓｅｐｈａｒｏｓｅ６Ｂ为亲和基质是纯化Ｄ２受体的有效方法。

Aim:To purify bovine striatal dopamine D2 receptor.Methods:The solution of cholic acid and sodium chloride was used to soluble dopamine D2 receptor from prepared bovine striatal membrane, and then the receptor was purified by the ligand affinity matrix of haloperidol-linked Sepharose 6B. The equilibrium dissociation constant( Kd) and the characteristics of the ligand-binding receptor were determined by the method of [3H] spiroperidol saturation and competition test. Results:Dopamine D2 receptor had been purified 1900-fold by one cycle affinity chromatography, and resulted in a recovery of 8. 7% of the original membrane-bound dopamine D2 receptor with a specific activity of 168. 7 pmol · mg-1. The molecular weight of protein obtained by affinity chromatography was about 94 000. Scatchard analysis revealed a Kd of 0.27 nmol · L-1 in affinity-purified receptor. Purified receptor maintained the ligand-binding characteristics of a dopamine D2 receptor. Conclusion: The affinity matrix, haloperidol-linked Sepharose 6B is an efficient method for purification of the dopamine D2 receptor.