Hoppe J, Freist W
Eur J Biochem. 1979 Jan 2;93(1):141-6. doi: 10.1111/j.1432-1033.1979.tb12804.x.
8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.
8-叠氮腺苷5'-三磷酸（n8(3)ATP）似乎是一种适用于依赖3':5'-环磷酸腺苷（cAMP）的蛋白激酶的光亲和标记物。它在激酶的未解离形式以及催化亚基催化的磷酸转移反应中，与ATP竞争高亲和力ATP位点。此外，它在磷酸转移反应中被接受为底物。掺入全酶中的n8(3)ATP经辐照后会共价结合。用ATP进行的保护实验表明，这种共价结合发生在酶的高亲和力ATP位点。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳显示，n8(3)ATP与催化亚基结合。辐照后，酶被cAMP解离。发现磷酸转移酶活性的损失与掺入的[γ-32P]n8(3)ATP成比例。这些结果支持了我们的模型，即两个ATP位点在腺嘌呤结合部分是重合的。因此，调节亚基与催化亚基的结合会将低亲和力的催化活性ATP位点转变为高亲和力的无活性位点。
