A Fluorescence Lifetime-Based Assay for Abelson Kinase
A Fluorescence Lifetime-Based Assay for Abelson Kinase
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基于荧光寿命的 Abelson 激酶测定
DOI：
10.1177/1087057110385817
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发表时间：
2011
影响因子：
--
通讯作者：
U. Hassiepen
中科院分区：
文献类型：
--
作者：
S. Pritz;Gabriele Meder;K. Doering;P. Drueckes;Julian Woelcke;L. Mayr;U. Hassiepen
我们提出了一种新的同质体外分析格式，并将其应用于酪氨酸激酶酶活性的定量测定。该试验采用含有单个酪氨酸和通过半胱氨酸侧链连接的单个探针的短肽底物。肽的结构灵活性允许探针被非磷酸化酪氨酸侧链动态猝灭。探针响应其荧光寿命的变化取决于酪氨酸的磷酸化状态。我们利用这种效应直接跟踪底物的酶磷酸化，而不必求助于额外的检测成分，如针对磷酸酪氨酸的抗体。作为应用该分析原理的一个例子，我们介绍了用于化合物分析的Abelson激酶（c-Abl）分析的发展结果。在肽序列的调整将使这种分析格式适用于各种各样的其他酪氨酸激酶。
We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.
影响因子：
56.9
作者：
Schindler, T;Bornmann, W;Kuriyan, J
通讯作者：
Kuriyan, J
c-Abl 激酶结构域与小分子抑制剂 PD173955 和伊马替尼 (STI-571) 复合物的晶体结构。
影响因子：
11.2
作者：
Nagar,Bhushan;Bornmann,WilliamG;Pellicena,Patricia;Schindler,Thomas;Veach,DarrenR;Miller,WTodd;Clarkson,Bayard;Kuriyan,John
通讯作者：
Kuriyan,John