A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Analytical Biochemistry Année : 2003

A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis.

Résumé

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.

Dates et versions

hal-00134350 , version 1 (01-03-2007)

Identifiants

Citer

Bertrand Boisson, Thierry Meinnel. A continuous assay of myristoyl-CoA:protein N-myristoyltransferase for proteomic analysis.. Analytical Biochemistry, 2003, 322 (1), pp.116-23. ⟨10.1016/j.ab.2003.07.007⟩. ⟨hal-00134350⟩
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