A fluorescence-based assay for N-myristoyltransferase activity

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• 作者：Victor Goncalves^a ; James A. Brannigan^b ; Emmanuelle Thinon^a ; Tayo O. Olaleye^a ; Remigiusz Serwa^a ; Salvatore Lanzarone^a ; Anthony J. Wilkinson^b ; Edward W. Tate^a ; ^{e.tate@imperial.ac.uk} ; Robin J. Leatherbarrow^a ; ^{r.leatherbarrow@imperial.ac.uk}
• 关键词：N-Myristoyltransferase (NMT) ; Fluorescence ; 7-Diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin (CPM) ; Coenzyme A ; Screening
• 刊名：Analytical Biochemistry
• 出版年：2012
• 出版时间：1 February 2012
• 年：2012
• 卷：421
• 期：1
• 页码：342-344
• 全文大小：309 K

文摘

N-myristoylation is the irreversible attachment of a C₁₄ fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.