An Assay for Myristoyl-CoA:Protein N-Myristoyltransferase Activity Based on Ion-Exchange Exclusion of [3H] Myristoyl Peptide

✍ Scribed by S.A. French; H. Christakis; R.R. Oneill; S.P.F. Miller

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 552 KB
• Volume: 220
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1306

✦ Synopsis

We developed an assay to test for inhibition of myristoyl-CoA:protein (\mathbf{N})-myristoyltransferase (NMT) activity since this enzyme is important in viral replication and cellular biochemistry. Saccharomyces cerevisae NMT was harvested from Escherichia coli carrying a plasmid vector containing the yeast NMT cDNA. Following the enzyme-catalyzed reaction of (\left[{ }^{3} \mathrm{H}\right]) myristoylCoA and an octapeptide substrate (GlyAsnAla (\mathbf{A r g}{2}) (\mathbf{N H}{2}) ), the assay mixture was loaded on AG1-8X anion-exchange resin which bound negatively charged reactants and by-products and left a doubly positively charged and nonbinding (\left[{ }^{3} \mathrm{H}\right]) myristoyl-peptide product in the supernatant. Optimum conditions for separating reactants and by-products from myristoyl-peptide in a (100-) pmol reaction were (450 \mathrm{mg}) resin and (25 %) methanol at pH 5.8. Under these conditions (97 %) of myristic acid and (98 %) of myristoyl-CoA bound to the resin, whereas (99 %) of myristoyl peptide remained in the supernatant. The potent inhibitor (S)-(2-oxopentadecyl)CoA was tested in our assay system. In addition, highspecific-activity (\left[{ }^{3} \mathrm{H}\right]) myristoyl-CoA, synthesized using acyl-CoA synthetase, was purified on a (200-\mu \mathrm{Ci}) scale (60 nmol) using a reverse-phase (\mathrm{C}-18) silica gel cartridge. Impurities, including free CoA, were washed from the column using (10 %) acetonitrile in (10 \mathrm{~mm}) potassium phosphate buffer, pH 7.5, while purified ( (95 %) by radiochemical scan) myristoyl-CoA was eluted from the column using 1:1 acetonitrile:phosphate buffer. & 1994 Academic Press, Inc.