Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases.
Assays for S-adenosylmethionine (AdoMet/SAM)-dependent methyltransferases.
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S-腺苷甲硫氨酸 (AdoMet/SAM) 依赖性甲基转移酶的测定。
DOI：
10.1002/0471140856.tx0426s38
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Hevel,Joan
中科院分区：
文献类型：
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作者：
Wooderchak,WhitneyL;Zhou,ZhaohuiSunny;Hevel,Joan
甲基转移酶对小分子和蛋白质的修饰影响了广泛的生物过程。在这里，我们报告了两种方法来测量甲基转移酶的活性。首先，我们描述了一种用于定量表征S-腺苷-L-甲硫氨酸（SAM）依赖性甲基转移酶活性的酶偶联连续分光光度法。在本试验中，S-腺苷-L-高半胱氨酸（SAH或SAH），即腺苷蛋氨酸依赖性甲基转移酶的转甲基化产物，通过重组SAH-Hcy核苷酶水解为S-核糖高半胱氨酸和腺嘌呤。随后，由AHCY产生的腺嘌呤被重组腺嘌呤脱氨酶进一步水解为高黄嘌呤和氨。这种脱氨基作用与265 nm处吸光度的降低有关，可对其进行连续监测。其次，我们描述了一个不连续的测定，放射性标记纳入甲基受体。这两种检测的优点是通过β-Hcy核苷酶破坏β-Hcy，这增强了β-Hcy产物对S-腺苷甲硫氨酸依赖性甲基转移酶的反馈抑制。重要的是，这两种方法都是廉价的，鲁棒的，并且适合于高通量。协议毒理学. 38：4.26.1 - 4.26.12.© 2008年由约翰威利父子公司。
Modification of small molecules and proteins by methyltransferases impacts a wide range of biological processes. Here we report two methods for measuring methyltransferase activity. First we describe an enzyme‐coupled continuous spectrophotometric assay used to quantitatively characterizeS‐adenosyl‐L‐methionine (AdoMet or SAM)–dependent methyltransferase activity. In this assay,S‐adenosyl‐L‐homocysteine (AdoHcy or SAH), the transmethylation product of AdoMet‐dependent methyltransferase, is hydrolyzed toS‐ribohomocysteine and adenine by recombinant AdoHcy nucleosidase. Subsequently, the adenine generated from AdoHcy is further hydrolyzed to homoxanthine and ammonia by recombinant adenine deaminase. This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Secondly, we describe a discontinuous assay that follows radiolabel incorporation into the methyl receptor. An advantage of both assays is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition ofS‐adenosylmethionine‐dependent methyltransferases. Importantly both methods are inexpensive, robust, and amenable to high throughput.Curr. Protoc. Toxicol. 38:4.26.1‐4.26.12. © 2008 by John Wiley & Sons, Inc.