1. A fluorescence-based assay for N-myristoyltransferase activity
- Author
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Goncalves, James A. Brannigan, Emmanuelle Thinon, T.O. Olaleye, Remigiusz A. Serwa, Edward W. Tate, S Lanzarone, Robin J. Leatherbarrow, and Anthony J. Wilkinson
- Subjects
Biochemistry & Molecular Biology ,N-Myristoyltransferase (NMT) ,Coenzyme A ,Biophysics ,Myristic acid ,COA ,Biochemistry ,Article ,Biochemical Research Methods ,Fluorescence ,chemistry.chemical_compound ,Coumarins ,MYRISTOYL TRANSFERASE ,Peptide bond ,Humans ,Molecular Biology ,IN-VIVO ,Fluorescent Dyes ,chemistry.chemical_classification ,GAG ,Science & Technology ,Chemistry, Analytical ,NMT2 ,Fatty acid ,Cell Biology ,Molecular biology ,Small molecule ,Kinetics ,Chemistry ,Enzyme ,TARGET ,chemistry ,Glycine ,Physical Sciences ,Screening ,lipids (amino acids, peptides, and proteins) ,MEMBRANE ,INHIBITORS ,Myristic Acids ,Protein Processing, Post-Translational ,Life Sciences & Biomedicine ,Acyltransferases ,7-Diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin (CPM) - 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.
- Published
- 2011