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.
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Authors | Victor Goncalves, James A Brannigan, Emmanuelle Thinon, Tayo O Olaleye, Remigiusz Serwa, Salvatore Lanzarone, Anthony J Wilkinson, Edward W Tate, Robin J Leatherbarrow |
Journal | Analytical biochemistry
(Anal Biochem)
Vol. 421
Issue 1
Pg. 342-4
(Feb 01 2012)
ISSN: 1096-0309 [Electronic] United States |
PMID | 22051857
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)
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Copyright | Copyright © 2011 Elsevier Inc. All rights reserved. |
Chemical References |
- Coumarins
- Fluorescent Dyes
- Myristic Acids
- N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide
- Acyltransferases
- glycylpeptide N-tetradecanoyltransferase
- Coenzyme A
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Topics |
- Acyltransferases
(analysis, antagonists & inhibitors, genetics, metabolism)
- Coenzyme A
- Coumarins
- Fluorescence
- Fluorescent Dyes
- Humans
- Kinetics
- Myristic Acids
(metabolism)
- Protein Processing, Post-Translational
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