Luedtke R R, Molinoff P B
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Biochem Pharmacol. 1987 Oct 1;36(19):3255-64. doi: 10.1016/0006-2952(87)90642-3.
Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.
设计实验以研究从犬尾状核中溶解出的[3H]螺哌啶醇（[3H]SPD）结合位点的生化特性，并确定去污剂对放射性配体结合的影响。发现用0.75 - 1.0%的洋地黄皂苷提取犬尾状核可产生[3H]SPD结合位点的最大产量，同时使膜蛋白的提取量最小化。虽然结合位点可用1.0%的洋地黄皂苷溶解，但为实现[3H]SPD的最大结合，去污剂浓度需降低10倍。激动剂和拮抗剂的亲和力排序与多巴胺受体D2亚型的药理特性一致。然而，发现拮抗剂的结合很复杂。对一些混合犬尾状核制剂的研究导致D2选择性拮抗剂多潘立酮和舒必利的竞争曲线最符合单一位点模型。其他制剂与这些拮抗剂呈现双相抑制曲线。对D2选择性拮抗剂具有低亲和力的[3H]SPD结合位点类别占结合位点的30 - 40%。通过尺寸排阻高效液相色谱，然后吸附到DEAE - 葡聚糖凝胶上并用离子强度增加的缓冲液洗脱，实现了[3H]SPD溶解结合位点的富集。结合位点的富集伴随着溶解位点对[3H]SPD亲和力的降低。
