Corbin J D, Beebe S J, Blackmore P F
J Biol Chem. 1985 Jul 25;260(15):8731-5.
Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.
通过用各种环磷酸腺苷（cAMP）类似物孵育大鼠肝细胞，激活了大鼠肝细胞蛋白激酶。然后制备煮沸提取物，并进行葡聚糖凝胶G - 25柱色谱分析。由于该树脂具有以不同亲和力结合环核苷酸的意外特性，G - 25柱色谱法将这些类似物与cAMP分离开来。进行分离是必要的，因为否则这些类似物会干扰为检测cAMP而开发的灵敏的蛋白激酶激活方法。这些cAMP类似物，而非5'-AMP，可使基础cAMP降低50 - 70%。这种作用迅速，呈类似物浓度依赖性，且与磷酸化酶激活同时发生，表明cAMP类似物通过激活依赖cAMP的蛋白激酶发挥作用。一种cAMP类似物完全阻断了相对低浓度胰高血糖素所引起的cAMP升高，但未阻断磷酸化酶反应，这表明cAMP类似物可替代cAMP作为蛋白激酶的细胞内激活剂。这些结果的一个含义是，激素引起的cAMP升高和蛋白激酶活性升高对细胞cAMP水平具有负反馈作用。
