An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases
An enzyme-coupled ultrasensitive luminescence assay for protein methyltransferases
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DOI：
10.1016/j.ab.2010.03.010
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发表时间：
2010-06-15
影响因子：
2.9
通讯作者：
Luo, Minkui
中科院分区：
文献类型：
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作者：
Ibanez, Glorymar;McBean, Jamie L.;Luo, Minkui
通过蛋白质翻译后修饰的表观遗传调控在发育和疾病中是必不可少的。其中关键的化学修饰是由蛋白质甲基转移酶（pmt）进行的蛋白质甲基化。定量和灵敏的PMT活性分析可以为研究PMT功能提供有价值的工具。在这里，我们开发了一种基于s -腺苷- l-蛋氨酸（Ado-Met/SAM）的pmt的酶偶联发光检测方法。在这项实验中，pmt参与甲基化的副产物s -腺苷- l-同半胱氨酸（AdoHcy/SAH）分别通过5'-甲基硫代腺苷/AdoHcy核苷酶（MTAN）、腺嘌呤磷酸核糖基转移酶（APRT）和丙酮酸正磷酸二激酶（PPDK）依次转化为腺嘌呤、单磷酸腺苷和5'-三磷酸腺苷（ATP）。合成的ATP可以很容易地用荧光素/荧光素酶试剂盒定量。该方法对adhcy具有定量线性响应，对0.3 pmol adhcy具有超灵敏度。通过本实验，对SET7/9甲基化的动力学参数进行了表征，并明确支持以AdoMet结合为初始步骤，然后是底物结合和限速甲基化的有序机制。发光分析也有望普遍适用于许多其他adomet依赖性酶。此外，混合测量96 /384孔格式的分析使其适合自动化和高通量。因此，我们的酶偶联发光试验代表了一种方便和超灵敏的方法来检测甲基转移酶活性和鉴定甲基转移酶抑制剂。(C) 2010爱思唯尔公司版权所有。
Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-L-methionine (Ado-Met/SAM)-based PMTs. In this assay, S-adenosyl-L-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5'-triphosphate (ATP) by 5'-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3 pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors. (C) 2010 Elsevier Inc. All rights reserved.