An assay for myristoyl-CoA: protein N-myristoyltransferase activity based on ion-exchange exclusion of [3H]myristoyl peptide.
We developed an assay to test for inhibition of myristoyl-CoA:protein 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 [3H]myristoyl-CoA and an octapeptide substrate (GlyAsnAla4Arg2-NH2), 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 [3H]myristoyl-peptide product in the supernatant. Optimum conditions for separating reactants and by-products from myristoyl-peptide in a 100-pmol reaction were 450 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, high-specific-activity [3H]myristoyl-CoA, synthesized using acyl-CoA synthetase, was purified on a 200-microCi scale (60 nmol) using a reverse-phase C-18 silica gel cartridge. Impurities, including free CoA, were washed from the column using 10% acetonitrile in 10 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.
Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland 20892.
S A
SA
French
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H
Christakis
R R
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O'Neill
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Miller
D000217:Acyltransferases / Q000032:analysis*; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D014316:Tritium
D010455:Peptides; D014316:Tritium; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase