Dills W L, Beavo J A, Bechtel P J, Myers K R, Sakai L J, Krebs E G
Biochemistry. 1976 Aug 24;15(17):3724-31. doi: 10.1021/bi00662a013.
Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.
合成了几种嘌呤环上连接有氨基烷基侧链的环核苷酸衍生物，并研究了它们在固定于溴化氰活化的琼脂糖4B之前和之后与3',5'-环磷酸腺苷（cAMP）依赖性蛋白激酶的相互作用。可溶性N6-取代衍生物在激活蛋白激酶方面与cAMP本身一样有效，并且比8-取代的cAMP衍生物更有效，而2-取代的cAMP衍生物和cGMP衍生物效果最差。所有测试的合成衍生物都是牛肉心磷酸二酯酶的不良底物，其水解速率低于cAMP本身的2%。利用开发的评估蛋白激酶对固定化环核苷酸亲和力的方法，发现所有固定化的环核苷酸都以生物特异性方式与蛋白激酶相互作用，依据以下标准判断：（1）固定化的环核苷酸与cAMP竞争蛋白激酶上的结合位点；（2）类似的间隔臂不竞争；（3）酶浓度、MgATP以及环磷酸环的裂解对蛋白激酶与固定化环核苷酸相互作用的影响与先前对游离cAMP所示的相同。此外，固定化配体的结合效果顺序与类似的可溶性配体相同。通过固定化过程，N6-H2N(CH2)2-cAMP激活0.005 μM蛋白激酶的观察到的Ka从0.23增加到3 μM。这种增加不受偶联密度和间隔臂长度的影响。0.10 μM蛋白激酶与固定化N6-H2N(CH2)2-cAMP结合的观察到的Kb随着所用基质的分子筛排阻极限降低而增加，这表明固定化后表观亲和力的至少部分降低是由于酶被排除在基质的一部分之外，因此也被排除在固定化配体分子之外。
