A coupled fluorescent assay for histone methyltransferases
A coupled fluorescent assay for histone methyltransferases
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DOI：
10.1016/j.ab.2005.04.007
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发表时间：
2005-07-01
影响因子：
2.9
通讯作者：
Trievel, RC
中科院分区：
文献类型：
--
作者：
Collazo, E;Couture, JF;Trievel, RC
组蛋白甲基转移酶（hmt）催化核小体核心组蛋白H3和H4以及历史性连接体H1b中赖氨酸和精氨酸的s -腺苷蛋氨酸（AdoMet）依赖甲基化。这些残基的甲基化调节转录激活或沉默，这取决于修饰的残基及其甲基化程度。尽管人们对阐明hmt在转录调控中的功能有浓厚的兴趣，但这些酶的定量分析仍然具有挑战性。为了表征hmt的底物特异性，我们开发了一种基于偶联荧光的adomet依赖性甲基转移酶检测方法。该实验利用s -腺苷型同型半胱氨酸水解酶（SAHH）将甲基转移产物s -腺苷型同型半胱氨酸（AdoHcy）水解为同型半胱氨酸（Hcy）和腺苷（Ado）。然后通过将其游离巯基部分偶联到巯基敏感荧光团来测定Hcy浓度。利用这一实验，我们确定了一种H3 lys -9特异性甲基转移酶Schizosaccharomyces pombe CLR4对合成组蛋白H3肽（对应于天然蛋白残基1-15）甲基化的动力学参数。荧光sahh偶联分析可以快速简便地测定HMT动力学，并可用于测量各种adomet依赖性甲基转移酶的酶活性。(c) 2005爱思唯尔公司版权所有。
Histone methyltransferases (HMTs) catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of lysines and arginines in the nucleosomal core histones H3 and H4 and the linker historic H1b. Methylation of these residues regulates either transcriptional activation or silencing, depending on the residue modified and its degree of methylation. Despite an intense interest in elucidating the functions of HMTs in transcriptional regulation, these enzymes have remained challenging to quantitatively assay. To characterize the substrate specificity of HMTs, we have developed a coupled-fluorescence-based assay for AdoMet-dependent methyltransferases. This assay utilizes S-adenosylhomocysteine hydrolase (SAHH) to hydrolyze the methyltransfer product S-adenosylhomocysteine (AdoHcy) to homocysteine (Hcy) and adenosine (Ado). The Hcy concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore. Using this assay, we have determined the kinetic parameters for the methylation of a synthetic histone H3 peptide (corresponding to residues 1-15 of the native protein) by Schizosaccharomyces pombe CLR4, an H3 Lys-9-specific methyltransferase. The fluorescent SAHH-coupled assay allows rapid and facile determination of HMT kinetics and can be adapted to measure the enzymatic activity of a wide variety of AdoMet-dependent methyltransferases. (c) 2005 Elsevier Inc. All rights reserved.