牛脑皮层膜中高亲和力[³H]5-羟色胺结合位点的增溶与表征

Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

作者信息

VandenBerg S R, Allgren R L, Todd R D, Ciaranello R D

出版信息

Proc Natl Acad Sci U S A. 1983 Jun;80(11):3508-12. doi: 10.1073/pnas.80.11.3508.

DOI:10.1073/pnas.80.11.3508

PMID:6574495

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC394074/

Abstract

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.

摘要

利用 Triton X - 100、吐温 - 80 和辛基 - β - D - 葡萄糖苷，已从牛脑皮质膜中溶解出高亲和力的[³H]5 - 羟色胺结合活性。这种去污剂混合物可溶解粗膜制剂中存在的高亲和力[³H]5 - 羟色胺结合活性，保留 75 - 90%的特异性结合。选择这种去污剂混合物是因为它可以通过透析和聚苯乙烯珠吸附轻易地从溶解部分中去除，从而允许进一步纯化和分离结合位点。饱和分析揭示了高亲和力[³H]5 - 羟色胺结合的多个成分。在粗牛皮质膜中，至少存在两个结合成分。根据曲线型 Scatchard 图定义，具有较高亲和力的结合成分对[³H]5 - 羟色胺的解离常数（Kd）为 1 - 3 nM，而较低亲和力成分的 Kd 为 10 - 20 nM。在溶解制剂中，仅出现一类结合位点，Kd 为 50 - 100 nM。通过透析和聚苯乙烯珠吸附去除去污剂后，会恢复曲线型 Scatchard 图，其表观 Kd 与在粗膜制剂中观察到的相似，且每个成分的最大结合量（Bmax）值增加。溶解制剂中的[³H]5 - 羟色胺结合活性对 Sephacryl S - 300 柱色谱和甘油梯度沉降稳定。对这两种方法的峰结合级分进行饱和分析，再次得到曲线型 Scatchard 图，表明多个高亲和力结合成分得以保留并一起迁移。已计算出结合位点的分子量、斯托克斯半径和摩擦系数。去除去污剂后，溶解的物质表现出许多通常归因于 S1 受体的特征，例如对[³H]5 - 羟色胺及其类似物具有高亲和力以及对 5 - 羟色胺拮抗剂具有低亲和力。