Srivastava L K, Ross G M, Bajwa S B, Mishra R K
Department of Psychiatry, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada.
Neurochem Res. 1990 Jun;15(6):647-57. doi: 10.1007/BF00973757.
The bovine striatal dopamine D1 receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [3H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a Kd of 0.95 and 1.1 nM and a Bmax of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [3H]SCH-23390 binding exhibited a clear D1 dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent than cis-flupentixol in displacing [3H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[3H]SCH-23390 competition studies revealed the presence of a high-affinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40-80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obtained from the agonist-treated membrane. In both the preparations, 100 microM guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.
在用多巴胺能激动剂（SKF-82526-J）或拮抗剂（SCH-23390）处理膜之后，牛纹状体多巴胺D1受体在磷脂存在的情况下用胆酸钠和氯化钠的组合进行增溶。随后通过凝胶过滤将增溶的受体重构成脂质囊泡。配体结合特性的比较表明，增溶并重构的受体以可饱和、立体特异性和可逆的方式将[3H]SCH-23390结合到一个同质位点，对于激动剂预处理和拮抗剂预处理的制剂，其解离常数（Kd）分别为0.95和1.1 nM，最大结合量（Bmax）分别为918和885 fmol/mg蛋白质。这些值与膜结合受体获得的值非常相似。拮抗剂对[3H]SCH-23390结合的竞争在从激动剂或拮抗剂预处理膜获得的重构制剂中表现出明显的D1多巴胺能顺序，不同的是，与拮抗剂预处理膜相比，在从激动剂预处理膜获得的制剂中，（+）布他拉莫在取代[3H]SCH-23390结合方面的效力比顺式氟哌噻吨高约四倍。激动剂/[3H]SCH-23390竞争研究表明，在两种重构的受体制剂中均存在激动剂结合的高亲和力成分。然而，与从激动剂处理膜获得的制剂相比，从拮抗剂处理膜获得的重构制剂中高亲和力激动剂结合位点的数量高40-80%。在两种制剂中，100 microM鸟苷酰亚胺二磷酸（Gpp(NH)p）完全消除了激动剂结合的高亲和力成分，而在天然膜中只是部分消除，这表明G蛋白与增溶受体紧密相关。无论受体是在膜用激动剂还是拮抗剂预孵育后增溶，从空间排阻高效液相色谱柱洗脱的受体-去污剂复合物的表观分子大小为360,000。用Gpp(NH)p对增溶制剂进行预孵育对洗脱谱几乎没有影响，这表明G蛋白受体复合物缺乏鸟嘌呤核苷酸依赖性解离。
