5'-Atp → Tetradecanoyl-coa

Participants

Reaction Steps

The compounds are then tested on the supernatant in competition with the peptide substrate Gly-Asn-(Ala)4 -(Arg)2 ("G8R") according to the conditions which follow, as described by TOWLER et al. (PNAS, (1987), 84, 2708-2711). Myristoylcoenzyme A is synthesized enzymatically immediately before use by incubation of myristate with ATP, Coenzyme A (lithium salt) and Pseudomonas Acyl-Coa synthetase (20 min at 30° C.). The peptide substrate and the biological source as obtained above are then added. The mixture is incubated for 10 min at 37° C.; the reaction is then stopped with 110 μl of methanol and 10 μl of trichloroacetic acid. The medium is left at 4° C. for 10 min and then centrifuged at 10,000 g for 5 min. An aliquot (30 μl) of the final supernatant is injected into an HPLC system equipped with a μBondapak column (Waters) and developed with a linear gradient as described by Towler et al. (PNAS, (1987), 84, 2708-11). Detection of the acylated peptide is performed using a radioactive line detector (Berthold) by means of the addition of 5 ml/min of scintillation fluid (Zinsser). The acylated peptide is eluted at 85% acetonitrile-TFA/15% H20-TFA.

Provenance & Evidence