Myristoyl coenzyme a

For screening of NMT1 inhibitors or the measurement of IC₅₀, the 1× reaction buffer (50 mM HEPES and 0.5 mM EDTA), 140 nM of NMT1 purified enzyme (See the NMT1 purification Section, the enzyme stock was preserved in the buffer containing 1mM EDTA, 250 mM NaCl, and 20 mM Tris pH 8.5), 5 µM of peptide Gly-Ser-Asn-Lys-Ser-Lys-Pro-Lys (derived from the N-terminus of human pp60Src tyrosine kinase), and the inhibitor at 0, 10, 20, 40, 80, 120, 160, or 200 µM respectively were mixed in a 96-well plate. After incubation at 30 °C for 10 min, the reaction was started by adding 1 µM myristoyl coenzyme A (Avanti Polar Lipids). The total volume of the above mixture was set 80 µL/well. After incubation at 30 °C for 60 min, the released coenzyme A was detected by adding 80 µL of 30 µM of 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM) stock solution (Sigma Aldrich) to each well and incubated in the dark for 12 min. The fluorescence intensity was measured by a Flex Station 3, microplate reader (excitation at 390 nm; emission at 479 nm).