Shen Congcong, Xia Xiaodong, Hu Shengqiang, Yang Minghui, Wang Jianxiu
College of Chemistry and Chemical Engineering, Central South University , Changsha 410083, China.
Anal Chem. 2015 Jan 6;87(1):693-8. doi: 10.1021/ac503492k. Epub 2014 Dec 17.
A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5'-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.
利用聚胞嘧啶寡核苷酸（dC12）模板化的银纳米簇（Ag NCs），开发了一种用于监测蛋白激酶（PKA）活性和抑制作用的简单灵敏的荧光方法。发现三磷酸腺苷（ATP）可增强Ag NCs的荧光，而PKA将ATP水解为二磷酸腺苷（ADP）会降低Ag NCs的荧光。与现有的激酶活性检测方法相比，所开发的方法不涉及底物肽的磷酸化，这显著简化了检测程序。该方法对PKA检测具有高灵敏度、良好的选择性和宽线性范围。还研究了激酶抑制剂H-89对PKA活性的抑制作用。该传感方案也应用于HeLa细胞裂解物中药物刺激的PKA激活检测。
