Akita Shoji, Umezawa Naoki, Kato Nobuki, Higuchi Tsunehiko
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
Bioorg Med Chem. 2008 Aug 15;16(16):7788-94. doi: 10.1016/j.bmc.2008.07.007. Epub 2008 Jul 8.
We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.
我们在此报告一种利用肽阵列对丝氨酸/苏氨酸激酶进行高效荧光检测的方法。我们的方法基于磷酸丝氨酸和磷酸苏氨酸残基特有的化学反应，即碱介导的磷酸基团β-消除以及随后含硫醇荧光试剂的迈克尔加成反应。该过程能够将荧光部分共价引入磷酸化肽中。为此设计并合成了新型荧光试剂。使用这些试剂，可轻松检测蛋白激酶A（PKA）和Akt-1的活性。我们的方法还可用于测量激酶抑制剂的活性。预计该检测方法将在激酶研究中得到广泛应用。
