Stauber G B, Ransom R W, Dilber A I, Olsen R W
Eur J Biochem. 1987 Aug 17;167(1):125-33. doi: 10.1111/j.1432-1033.1987.tb13313.x.
The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.
大鼠脑内的γ-氨基丁酸受体蛋白复合物以高产率溶解，通过苯二氮䓬亲和层析纯化得到毫克量的产物，并用于制备高效价的兔抗血清。高浓度的Triton X-100去污剂加KCl可溶解约90%的膜结合γ-氨基丁酸受体（通过[³H]蝇蕈醇结合测定）和苯二氮䓬受体（通过[³H]氟硝西泮结合测定）活性。两种活性都保留在使用固定化苯二氮䓬配体的亲和柱上，并且大部分柱吸附的受体可用游离苯二氮䓬加4M尿素的溶液洗脱。纯化后的蛋白以受体样的药理特异性结合[³H]蝇蕈醇和[³H]氟硝西泮，两种配体的比活性分别约为1700 pmol和700 pmol/mg蛋白。这相当于纯化了600多倍且接近理论纯度，从100g脑中获得毫克量的产物。在十二烷基硫酸钠凝胶电泳上观察到四条肽带，分子量分别为31、47、52和57 kDa。后两条带染色最明显，通过用[³H]氟硝西泮（52 kDa）和[³H]蝇蕈醇（57 kDa）进行光标记，以及在Western印迹上与Schoch等人制备的针对该蛋白的单克隆抗体反应（[1985年，《自然》（伦敦）314, 168 - 171]）鉴定为受体亚基。用纯化后的蛋白制备兔抗血清，该抗血清在高稀释度下既能共沉淀可溶性γ-氨基丁酸/苯二氮䓬受体结合活性，又能在Western印迹上对受体亚基（主要是52 kDa条带）进行染色。
