Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase
Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase
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DOI：
10.1016/j.ab.2005.04.028
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发表时间：
2005-07-15
影响因子：
2.9
通讯作者：
Jeltsch, A
中科院分区：
文献类型：
--
作者：
Gowher, H;Zhang, X;Jeltsch, A
蛋白质甲基转移酶对蛋白质的修饰具有几个重要的生物学功能。在此，我们研究了Dim-5历史赖氨酸甲基转移酶对组蛋白H3尾部Lys9位点的甲基化，该酶参与表观遗传信号传导和基因沉默，并触发粗神经孢子虫的DNA甲基化。我们开发了一种新的检测方法，使用生物素化的合成肽底物和放射性标记的辅酶来检测蛋白质甲基化。我们表明，该分析是线性的时间和酶浓度（在多个周转条件下），其背景是非常低的。数据点在3%以内可重复。至少200 pmol的生物素化肽完全结合到微孔板上。我们利用该分析系统确定了Dim-5酶的k - M和k（cat）值分别为7.4 μ M和2.3 min（-1）。此外，我们确定了四种Dim-5变体的活性，范围从完全活性到少于1%的剩余活性。本研究开发的微孔板生物素/亲和素肽甲基化测定方法方便、准确、可重复性好、价格低廉。因为它产生定量结果，它可以用来表征历史赖氨酸甲基转移酶和其他蛋白质甲基转移酶的酶性质。该分析也非常适合高通量应用。(c) 2005爱思唯尔公司版权所有。
Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 historic lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K-m and k(cat) values of the Dim-5 enzyme for the methylation or a 20mer peptide to be 7.4 mu M and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of historic lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications. (c) 2005 Elsevier Inc. All rights reserved.