期刊
ANALYTICAL BIOCHEMISTRY
卷 421, 期 1, 页码 342-344出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.10.013
关键词
N-Myristoyltransferase (NMT); Fluorescence; 7-Diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin (CPM); Coenzyme A; Screening
资金
- Medical Research Council (MRC) [G0900278]
- Wellcome Trust [087792]
- Biotechnology and Biological Sciences Research Council [BB/D02014X/1]
- Cancer Research UK [C29637/A10711]
- Imperial College
- European Union [PIEF-GA-2010-273868]
- BBSRC [BB/D02014X/1] Funding Source: UKRI
- MRC [G0900278] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D02014X/1] Funding Source: researchfish
- Medical Research Council [G0900278] Funding Source: researchfish
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. (C) 2011 Elsevier Inc. All rights reserved.
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