کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533182 | 961855 | 2005 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A coupled fluorescent assay for histone methyltransferases
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
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 histone 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 86-92
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 86-92
نویسندگان
Evys Collazo, Jean-François Couture, Stacie Bulfer, Raymond C. Trievel,