Phosphoenolpyruvic acid tris(cyclohexylammonium)salt

Phosphoenolpyruvic Acid Tri(Cyclohexylammonium)Salt for Luminescent PMT Activity Determination

Rui Wang, Mol. BioSyst., 2011,7, 2970-2981.

Phos(enol)pyruvate (PEP) is a highly functional small molecule. It plays an important role in metabolism. For example, phosphoenolpyruvic acid tri(cyclohexylammonium)salt can be used in luminescent PMT activity assays. The luminogenic PMT-activity assay was performed as below:
· Briefly, Incubate the peptide substrate, buffer solution, and PMT at ambient temperature for 12 hours.
· From the reaction mixture, 5 ml aliquot was transferred by a multiple-channel pipette into a 96-well plate containing 5 ml coupling buffer (100 mM Tris-acetate, pH = 7.7, 2 mM phosphoenolpyruvic acid tri(cyclohexylammonium)salt, 2 mM sodium pyrophosphate decahydrate, 2 mM 5-phospho-D-ribose1-diphosphate sodium salt, 15 mM NH4SO4, 15 mM (NH4)2MoO4, 10 mM MgSO4, 0.8 U ml1 PPDK, and 0.4 U ml1 APRT).
· After adding 50 ml of D-luciferin/luciferase assay buffer and shaking the mixture for 30 s in the dark, the luminescent signal was read on a luminescent microplate reader.