Assay of N-myristoyl transferase by selective adsorption of myristoyl-coenzyme A on acidic alumina

We have developed a simple and rapid method for detecting the enzyme myristoyl-CoA:protein N-myristoyl transferase. The enzyme catalyzes the transfer of the myristoyl moiety of myristoyl-CoA to the amino-terminal glycine residue of a peptide (protein). Incorporation of the 14C]myristate into the peptide is quantified after separation of the 14C]myristoyl-peptide from unreacted 14C]myristoyl-CoA by selective adsorption of 14C]myristoyl-CoA on acidic alumina. Optimal assay concentrations were 200 microM synthetic peptide, 1 microM 14C]myristoyl-CoA, 10 mM Tris-HCl/1 mM dithiothreitol/0.1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid/aprotinin (10 micrograms/ml) buffer, pH 7.4, and 1-10 micrograms protein.