A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates
A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates
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DOI：
10.1016/j.ab.2009.09.005
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发表时间：
2010-03-15
影响因子：
2.9
通讯作者：
Hevel, Joan M.
中科院分区：
文献类型：
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作者：
Suh-Lailam, Brenda B.;Hevel, Joan M.
S-腺苷-L-甲硫氨酸 (AdoMet) 依赖性甲基转移酶对蛋白质残基的修饰会影响一系列细胞过程。在这里，我们描述了一种定量测量甲基转移速率的新方法，该方法与使用蛋白质底物兼容。该方法依赖于移液器吸头末端装填的反相树脂快速将未反应的 AdoMet 与放射性标记的蛋白质产物分离的能力。结合的放射性标记蛋白质产物直接洗脱到闪烁瓶中并进行计数。除了减少分析时间之外，该协议的灵敏度还允许确定初始速率数据。通过在 1 小时内通过人蛋白精氨酸甲基转移酶 1、变体 1 (hPRMT1v1) 生成异质核核糖核蛋白 K (hnRNP K) 蛋白的甲基化米氏曲线，显示了该协议的实用性。该测定的另一个优点是，与现有方案相比，放射性废物减少了 3000 多倍。 (C) 2009 Elsevier Inc. 保留所有权利。
Modification of protein residues by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols. (C) 2009 Elsevier Inc. All rights reserved.