Nakata H
Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jan 15;265(2):671-7.
Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.
利用新开发的亲和层析系统对大鼠睾丸的A1腺苷受体进行了纯化（中田浩，（1989年）《分子药理学》35卷，780 - 786页）。用洋地黄皂苷从大鼠睾丸膜中溶解A1腺苷受体，然后依次通过黄嘌呤胺类似物固定化琼脂糖亲和层析、羟基磷灰石层析、黄嘌呤胺类似物 - 琼脂糖再亲和层析，最后通过TSK - 3000SW凝胶渗透层析进行纯化，纯化倍数超过25000倍。最终制剂的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳经放射性碘化后，通过放射自显影显示出一条Mr为41000的单一宽带。这条Mr为41000的肽也被A1腺苷受体亲和标记试剂特异性标记。一种高亲和力的A1腺苷受体拮抗剂，8 - 环戊基 - 1,3 - [3H]二丙基黄嘌呤，以约1.4 nM的KD值与纯化的受体饱和结合。纯化的受体对腺苷激动剂和拮抗剂的特异性与未纯化的受体制剂基本相同，尽管纯化的腺苷受体对激动剂的亲和力与未纯化的受体制剂相比显著较低，这表明纯化的A1腺苷受体以低激动剂 - 高拮抗剂亲和力状态存在。用内切糖苷酶F对纯化的睾丸腺苷A1受体进行去糖基化处理后，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中受体蛋白的迁移率增加，表观Mr为30000，类似于大鼠脑膜去糖基化的A1腺苷受体。使用胰蛋白酶和V8蛋白酶对纯化的睾丸和脑A1腺苷受体进行肽图谱分析表明，这些受体显示出一些结构同源性。
