| pubmed-article:9761456 | pubmed:abstractText | 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. | lld:pubmed |
