N-myristoyl transferase assay using phosphocellulose paper binding.
N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.
King
M J
MJ
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Sharma
R K
RK
D000217:Acyltransferases / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives*; D002854:Chromatography, Paper / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions; D014018:Tissue Distribution
D010446:Peptide Fragments; D002482:Cellulose; C008881:phosphocellulose; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D011494:Protein Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)