Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University, Germany.
BMC Biochem. 2010 May 20;11:20. doi: 10.1186/1471-2091-11-20.
Although numerous non-radioactive methods are in use to measure the catalytic activity of protein kinases, most require specialized equipment and reagents and are not sufficiently sensitive for the detection of endogenous kinase activity in biological samples. Kinases of the DYRK family have important functions in developmental and pathophysiological processes in eukaryotic organisms including mammals. We aimed to develop a highly sensitive, low-tech assay suitable to determine the activity of DYRK family kinases in tissues or cells from diverse sources.
Phosphorylation-site specific antibodies can be used to monitor the accumulation of the phosphorylated product in kinase assays. We present a modified configuration of an enzyme-linked immunosorbent assay (ELISA)-based kinase assay by using the phosphospecific antibody as the capture antibody. This assay format allowed the detection of small amounts of phosphopeptide in mixtures with an excess of the unphosphorylated substrate peptide (10 fmol phosphorylated peptide over a background of 50 pmol unphosphorylated peptide). Consequently, low substrate turnover rates can be determined. We applied this method to the measurement of endogenous DYRK1A activity in mouse heart tissue by immunocomplex kinase assay. Furthermore, we detected DYRK1-like kinase activity in Xenopus laevis oocytes and identified this kinase as a DYRK1 isoform distinct from the Xenopus DYRK1A ortholog.
We present a non-radioactive and highly sensitive method for the measurement of endogenous activities of DYRKs in biological samples. Xenopus laevis oocytes contain an active DYRK1-related protein kinase more similar to mammalian DYRK1B than DYRK1A.
尽管有许多非放射性方法可用于测量蛋白激酶的催化活性，但大多数方法需要专门的设备和试剂，并且对于检测生物样品中的内源性激酶活性不够敏感。DYRK 家族的激酶在真核生物（包括哺乳动物）的发育和病理生理过程中具有重要功能。我们旨在开发一种高度敏感、低技术的测定法，适用于测定来自不同来源的组织或细胞中的 DYRK 家族激酶活性。
磷酸化位点特异性抗体可用于监测激酶测定中磷酸化产物的积累。我们提出了一种基于酶联免疫吸附测定（ELISA）的激酶测定的改良构型，该测定将磷酸化特异性抗体用作捕获抗体。该测定格式允许在与未磷酸化底物肽过量的混合物中检测少量磷酸肽（50 pmol 未磷酸化肽背景下有 10 fmol 磷酸化肽）。因此，可以确定低的底物周转率。我们应用该方法通过免疫复合物激酶测定法测量了小鼠心脏组织中的内源性 DYRK1A 活性。此外，我们在非洲爪蟾卵母细胞中检测到 DYRK1 样激酶活性，并将该激酶鉴定为与非洲爪蟾 DYRK1A 同源物不同的 DYRK1 同工型。
我们提出了一种非放射性和高度敏感的方法，用于测量生物样品中内源性 DYRKs 的活性。非洲爪蟾卵母细胞中含有一种活性 DYRK1 相关蛋白激酶，与哺乳动物 DYRK1B 更相似，而与 DYRK1A 不同。
