Kidric M, Petrović J, Soskić V, Trajković D
Br J Pharmacol. 1984 Nov;83(3):687-95. doi: 10.1111/j.1476-5381.1984.tb16222.x.
Dopamine D2-receptors were solubilized from synaptosomal membranes of the bovine caudate nucleus using different detergents. They were labelled with [3H]-spiperone and assayed by polyethylene glycol precipitation. CHAPS was found to be the best solubilizing agent among all detergents used. Optimal conditions for solubilization were: 0.25% CHAPS, 3.5 mg ml-1 protein, 25 min, 4 degrees C and the yield of D2-receptors was 18.6%. Addition of some sulphobetain detergents increased the extent of solubilization, 125 mM NaCl and 0.25 M sucrose decreased it, while SH-group protecting agents (2 mM dithiothreitol and 6 mM beta-mercaptoethanol), as well as MEGA-9 and MEGA-12 were almost ineffective. -log IC50 values for solubilized dopamine D2-receptors are in linear correlation with the corresponding values for membrane-bound receptors (r = 0.962, slope factor 0.96) and Kd value of solubilized receptors was 3.61 +/- 0.94 nM, while that of membrane-bound receptors was 1.25 +/- 0.10 nM. Specific binding of [3H]-spiperone to the solubilized receptors resolved by linear sucrose density gradient centrifugation shows two maxima, one in the first several fractions from the bottom and the other with an apparent S value of 7.3.
使用不同的去污剂从牛尾状核的突触体膜中溶解多巴胺D2受体。用[3H] - 螺哌隆标记它们，并通过聚乙二醇沉淀法进行测定。发现在所有使用的去污剂中，CHAPS是最佳的增溶剂。溶解的最佳条件为：0.25% CHAPS、3.5 mg/ml蛋白质、25分钟、4℃，D2受体的产量为18.6%。添加一些磺基甜菜碱去污剂可增加溶解程度，125 mM NaCl和0.25 M蔗糖则会降低溶解程度，而SH基团保护剂（2 mM二硫苏糖醇和6 mMβ-巯基乙醇）以及MEGA - 9和MEGA - 12几乎无效。溶解的多巴胺D2受体的-log IC50值与膜结合受体的相应值呈线性相关（r = 0.962，斜率因子0.96），溶解受体的Kd值为3.61±0.94 nM，而膜结合受体的Kd值为1.25±0.10 nM。通过线性蔗糖密度梯度离心法分离的[3H] - 螺哌隆与溶解受体的特异性结合显示出两个最大值，一个在底部的前几个组分中，另一个的表观S值为7.3。
