A coupled fluorescent assay for histone methyltransferases

被引：98

作者：

Collazo, E ^{[1
]}

Couture, JF ^{[1
]}

Bulfer, S ^{[1
]}

Trievel, RC ^{[1
]}

机构：

[1] Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2005年 / 342卷 / 01期

关键词：

histone methylation; microplate fluorometry; enzyme-coupled assay; S-adenosylhomocysteine hydrolase; AdoMet-dependent methyl-transferases;

D O I：

10.1016/j.ab.2005.04.007

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：86 / 92

页数：7

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