Analytical Biochemistry 2012-02-01

A fluorescence-based assay for N-myristoyltransferase activity.

Victor Goncalves, James A Brannigan, Emmanuelle Thinon, Tayo O Olaleye, Remigiusz Serwa, Salvatore Lanzarone, Anthony J Wilkinson, Edward W Tate, Robin J Leatherbarrow

Index: Anal. Biochem. 421(1) , 342-4, (2012)

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Abstract

N-myristoylation is the irreversible attachment of a C(14) 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.Copyright © 2011 Elsevier Inc. All rights reserved.