Matsui H, Asakura M, Tsukamoto T, Imafuku J, Ino M, Saitoh N, Miyamura S, Hasegawa K
J Neurochem. 1985 May;44(5):1625-32. doi: 10.1111/j.1471-4159.1985.tb08805.x.
alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.
用[³H]可乐定（α₂激动剂）标记的α₂肾上腺素能受体，可以用两性离子去污剂3-[3-(胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐（CHAPS）从大鼠脑中以对鸟嘌呤核苷酸敏感的形式溶解出来。膜中约40%的原始[³H]可乐定结合位点用6 mM CHAPS溶解。使用聚乙烯亚胺处理过的GF/B滤器，通过真空过滤法分离可溶性[³H]可乐定结合复合物。溶解的[³H]可乐定结合位点保留了膜结合的α₂肾上腺素能受体的相同药理学特性。[³H]可乐定与溶解的α₂受体结合的Scatchard图呈曲线，表明存在两种不同的结合成分。溶解的受体从Bio-Gel A-1.5 m柱上以单峰形式洗脱，斯托克斯半径为6.6 nm。等电点为5.6 - 5.8。鸟嘌呤核苷酸、单价阳离子和巯基反应剂对受体结合的调节在溶解状态下保持完整，而二价阳离子的调节作用则丧失。受体和鸟嘌呤核苷酸结合调节成分在溶解状态下的明显保留，可能有助于在分子水平上研究脑α₂肾上腺素能受体系统的调节机制。
