Silva W I, Andres A, Schook W, Puszkin S
J Biol Chem. 1986 Nov 5;261(31):14788-96.
Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).
从奎宁环基苯甲酸盐结合测定获得的证据表明，毒蕈碱型乙酰胆碱受体分子存在于纯化的牛脑包被小泡中。用抗网格蛋白的多克隆抗体从包被小泡形成的免疫沉淀物，结合在固定的金黄色葡萄球菌细胞表面，也显示出奎宁环基苯甲酸盐结合活性。通过生化标记物测定和电子显微镜确定了包被小泡的高纯度。奎宁环基苯甲酸盐与包被小泡结合的毒蕈碱受体位点的解离常数（Kd）为25皮摩尔，最大结合容量（Bmax）约为191飞摩尔/毫克蛋白质。使用阿托品、N-甲基东莨菪碱、氧化震颤素和氨甲酰胆碱进行的结合竞争实验证实了结合位点具有典型的毒蕈碱性质。受体的效价排序为：阿托品＞N-甲基东莨菪碱＞氧化震颤素＞卡巴胆碱。使用双位点模型对数据进行分析显示，氧化震颤素有13%的高亲和力位点，卡巴胆碱有66%的高亲和力位点，拮抗剂N-甲基东莨菪碱有62%的高亲和力位点。在包被小泡中检测到的毒蕈碱药物结合亲和力的异质性可能与脑组织中包被小泡亚群的存在有关，（科茨，D.S.，科茨，J.D.，舒克，W.J.，和普兹金，S.（1985年）《细胞生物学杂志》101，48a；菲弗，S.R.，和凯利，R.B.（1985年）《细胞》40，949 - 957）。
