Nakata H
Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892.
J Biol Chem. 1989 Oct 5;264(28):16545-51.
The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.
通过依次使用固定化黄嘌呤胺类似物 - 琼脂糖亲和色谱、羟基磷灰石色谱和再亲和色谱，大鼠脑膜中的A1腺苷受体已被纯化约50,000倍，达到明显的均一性。从脑膜开始的总产率约为4%。纯化制剂的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳通过银染或放射性碘化后的放射自显影片显示出一条明显分子量为34,000的宽单带。纯化的受体结合约24 nmol的8 - 环戊基 - 1,3 - [3H]二丙基黄嘌呤/ mg蛋白质，解离常数为1.4 nM。如果假设每个受体分子有一个配体结合位点，这个最大特异性结合值与分子量为34,000道尔顿的蛋白质的预期理论比活性（29.4 nmol/mg）一致。使用[3H]对苯二异硫氰酸酯 - 黄嘌呤胺类似物的亲和标记实验表明，分子量为34,000的蛋白带含有配体结合位点。纯化的受体具有典型的A1腺苷受体药理学特异性，类似于未纯化的受体制剂。
