Costa B, Lucacchini A, Martini C
Dipartimento di Psichiatria, Farmacologia e Biotecnologie dell 'Università degli Studi di Pisa, Italy.
Neurochem Int. 1998 Aug;33(2):121-9. doi: 10.1016/s0197-0186(98)00019-9.
We report the characterization of A2a adenosine receptors (A2aARs) in porcine striatal membranes and their solubilization (25%) by the detergent digitonin. After solubilization, the drug specificity and equilibrium [3H]CGS-21680 ([3H]2-(4-(2-carboxyethyl)phenylethylamino)-5'-N-ethyl-carboxamido -adenosine) binding parameters were virtually identical to those obtained in intact membranes, indicating a conservation of the binding site after the removal of receptors from their lipid environment. Gel filtration on a calibrated Superdex 200 HR column revealed a main [3H]CGS-21680 binding peak with an apparent molecular weight of 171,000+/-9000 Da. In membranes, Scatchard analysis of saturation data carried out in a wide range of radioligand concentration (1-100 nM) resulted in a biphasic curve and, in accordance with the two binding sites model, yielded a Kd1 = 7.4+/-0.5 and Kd2 = 53.1+/-3.6 nM, a Bmax1 = 186+/-15 fmol/mg protein and a Bmax2 = 285+/-20 fmol/mg protein, respectively. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPgamma[S]) a shift from two affinity states to a single one was evidenced (Kd = 28.5+/-5.9 nM) and a Bmax value of 504+/-10 fmol/mg protein found. In the soluble extract, only one high-affinity state was detected (Kd = 19.3+/-1.1 nM and Bmax = 285+/-20 fmol/mg protein) and, in the presence of GTPgamma[S]), a two site model likewise provided a significantly (P < 0.01) better fit (Kd1 = 13.9+/-1.2 nM and Kd2 = 72.1+/-6.9 nM, Bmax1 = 125+/-10 fmol/mg protein and Bmax2 = 375+/-19 fmol/mg protein, respectively). These results suggest a close relation between the receptor and G protein solubilized as a functional unit and open the way to its purification.
我们报告了猪纹状体膜中A2a腺苷受体（A2aARs）的特性以及用去污剂洋地黄皂苷使其增溶（25%）的情况。增溶后，药物特异性和平衡态[3H]CGS - 21680（[3H]2 - (4 - (2 - 羧乙基)苯乙氨基)-5'-N - 乙基 - 羧酰胺基 - 腺苷）结合参数与完整膜中获得的参数几乎相同，这表明在将受体从其脂质环境中去除后结合位点得以保留。在校准的Superdex 200 HR柱上进行凝胶过滤显示，一个主要的[3H]CGS - 21680结合峰，其表观分子量为171,000±9000 Da。在膜中，在广泛的放射性配体浓度范围（1 - 100 nM）内对饱和数据进行Scatchard分析得到一条双相曲线，根据两个结合位点模型，分别得到Kd1 = 7.4±0.5和Kd2 = 53.1±3.6 nM，Bmax1 = 186±15 fmol/mg蛋白质和Bmax2 = 285±20 fmol/mg蛋白质。在存在鸟苷 - 5'-O - (3 - 硫代三磷酸)（GTPγ[S]）的情况下，证明从两个亲和状态转变为单一状态（Kd = 28.5±5.9 nM），并且发现Bmax值为504±10 fmol/mg蛋白质。在可溶性提取物中，仅检测到一种高亲和状态（Kd = 19.3±1.1 nM和Bmax = 285±20 fmol/mg蛋白质），并且在存在GTPγ[S]的情况下，双位点模型同样提供了显著更好的拟合（P < 0.01）（分别为Kd1 = 13.9±1.2 nM和Kd2 = 72.1±6.9 nM，Bmax1 = 125±10 fmol/mg蛋白质和Bmax2 = 375±19 fmol/mg蛋白质）。这些结果表明受体与作为功能单元增溶的G蛋白之间存在密切关系，并为其纯化开辟了道路。
