N-myristoyl transferase assay using phosphocellulose paper binding

Abstract

N-myristoyl-CoA: protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH 2-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 pp60 src, 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 > spleen > heart > 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.