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.2012 Feb 1;421(1):342-4.
doi: 10.1016/j.ab.2011.10.013. Epub 2011 Oct 14.

A fluorescence-based assay for N-myristoyltransferase activity

Affiliations

A fluorescence-based assay for N-myristoyltransferase activity

Victor Goncalves et al. Anal Biochem..

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.

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Figures

Fig. 1
Fig. 1
(A) NMT-catalysedN-myristoylation of target proteins. (B) Changes in fluorescence emission spectra upon formation of the CPM-coenzyme A adduct in assay buffer at 25 °C with excitation at 390 nm (RFU: relative fluorescence units).
Fig. 2
Fig. 2
(A) Chemical structure of inhibitors1 and2. (B) Inhibition assay: IC50 values were determined in the presence of 4 μM Hs pp60src(2-9) and 4 μM myristoyl-CoA. Data points represent means of duplicates.
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References

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