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A fluorescence-based assay for N-myristoyltransferase activity.

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.
AuthorsVictor Goncalves, James A Brannigan, Emmanuelle Thinon, Tayo O Olaleye, Remigiusz Serwa, Salvatore Lanzarone, Anthony J Wilkinson, Edward W Tate, Robin J Leatherbarrow
JournalAnalytical biochemistry (Anal Biochem) Vol. 421 Issue 1 Pg. 342-4 (Feb 01 2012) ISSN: 1096-0309 [Electronic] United States
PMID22051857 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)
CopyrightCopyright © 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
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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