title: A new, robust, and nonradioactive approach for exploring N-myristoylation.
abstract: Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.
authors: Department of Cellular and Molecular Pathology,  German Cancer Research Center, Heidelberg,  Germany.
authors: Francesca
authors: F
authors: Rampoldi
authors: Roger
authors: R
authors: Sandhoff
authors: Robert W
authors: RW
authors: Owen
authors: Hermann-Josef
authors: HJ
authors: Gröne
authors: Stefan
authors: S
authors: Porubsky
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000368:Aged; D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009227:Myristic Acids / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D009227:Myristic Acids; C040349:S-tetradecanoyl-coenzyme A; D000217:Acyltransferases; C527098:N-myristoyltransferase, mouse; C050480:glycylpeptide N-tetradecanoyltransferase