Rauh J J, Lambert M P, Cho N J, Chin H, Klein W L
J Neurochem. 1986 Jan;46(1):23-32. doi: 10.1111/j.1471-4159.1986.tb12920.x.
Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin-solubilized receptors were representative of the entire membrane-bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin-agarose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N-acetylneuraminic acid residues reduced binding to wheat germ agglutinin-agarose by 40%; further treatment with endoglycosidases D and H, to remove all N-linked carbohydrate, decreased binding by a total of 67%. Removal of N-acetylneuraminic acid residues had no effect on agonist binding properties of the membrane-bound receptors. The carbohydrate-specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X-100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pI of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate-urea-polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N-linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.
将牛大脑皮层的毒蕈碱型乙酰胆碱受体用洋地黄皂苷溶解，以便随后测定其若干生化特性。就碳水化合物含量、等电点和分子量而言，用洋地黄皂苷溶解的受体代表了毒蕈碱受体整个膜结合群体。溶解受体的糖蛋白性质通过其与麦胚凝集素-琼脂糖的定量结合得以证明。结合拮抗剂的存在并不降低受体与这种凝集素的结合程度。用神经氨酸酶处理受体以去除N-乙酰神经氨酸残基，使与麦胚凝集素-琼脂糖的结合减少40%；再用内切糖苷酶D和H处理以去除所有N-连接的碳水化合物，使结合总共减少67%。去除N-乙酰神经氨酸残基对膜结合受体的激动剂结合特性没有影响。碳水化合物特异性酶还被用于评估碳水化合物对受体等电点和分子量的贡献。用洋地黄皂苷或 Triton X-100 溶解的毒蕈碱受体聚焦为一个主要物种，其 pI 为 4.3。神经氨酸酶处理导致受体的 pI 增加 0.17 个单位。用共价毒蕈碱拮抗剂丙基苯甲酰胆碱芥子碱标记的毒蕈碱受体在十二烷基硫酸钠-尿素-聚丙烯酰胺凝胶上作为一条单一的主要多肽迁移，分子量为 73,000。从这些凝胶中排除尿素会严重阻碍受体迁移，表明受体在 SDS 中有强烈的聚集倾向。用内切糖苷酶处理去除 N-连接的碳水化合物，使拮抗剂结合多肽的分子量降低不超过 5%。这些结果证明了哺乳动物大脑皮层毒蕈碱受体的糖蛋白性质，并为它们在碳水化合物含量方面的异质性提供了证据。
