A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis.
Protein N-myristoylation is an important lipid modification that affects the activity and membrane-binding properties of crucial proteins belonging to signal transduction cascades. The aim of this work was to develop a rapid and easy diagnostic method to check for (i) effective N-myristoylation of any given protein and (ii) easy proteome annotation. The N-myristoylation reaction was coupled to that of pyruvate dehydrogenase, and NADH was continuously detected spectrophotometrically. This method was optimized for and applied to full-length Saccharomyces cerevisiae and Arabidopsis thaliana N-myristoyltransferases and two A. thaliana enzyme derivatives. The data were validated by comparison with a previously described discontinuous assay, modification of the chemical nature of the substrates, and use of specific inhibitors. The kinetics of N-myristoylation were determined in vitro with various compounds including a full-length polypeptide substrate, a small G protein of the RAB family already known to be N-myristoylated in vivo. This automated assay can be used for proteomic studies to determine the N-myristoylation state of any protein.
Boisson
Bertrand
B
Protein Maturation, ISV, UPR2355, Centre National de la Recherche Scientifique, Bâtiment 23, 1 avenue de la Terrasse, F-91198 Gif-sur-Yvette, France.
Meinnel
Thierry
T
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology; D002863:Chromogenic Compounds / Q000138:chemical synthesis; D003001:Cloning, Molecular; D007658:Ketone Oxidoreductases / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism; D009227:Myristic Acids / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015398:Signal Transduction; D013057:Spectrum Analysis
D000214:Acyl Coenzyme A; D002863:Chromogenic Compounds; D009227:Myristic Acids; D011994:Recombinant Proteins; D019814:Myristic Acid; C017418:alpha-hydroxymyristic acid; C040349:S-tetradecanoyl-coenzyme A; D009249:NADP; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase