The assay described for amino acid decarboxylase can be used to quantitate the substrates and products associated with the decarboxylation of arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine.
Kochhar et al. (1989) characterized an assay for glutamate decarboxylase activity. Glutamate and 4-aminobutyrate were separated on a Nucle-osil Cg column. The mobile phase was 13 mM trifluoroacetate and 1 mM 1-octanesulfonate. Detection was by postcolumn derivatization with o-phthaldialdehyde reagent (1 mL/min) mixed with the column eluate (also 1 mL/min). The Teflon reaction coil (3 m x 0.3 mm) was kept at room temperature. The o-phthaldialdehyde reagent was prepared by dissolving 800 mg of o-phthaldialdehyde in 20 mL of ethanol plus 2.5 mL of 2-mercaptoethanol and mixing with 980 mL of 0.4 M sodium borate (pH 9.7) and 3 mL Brij 35. The fluorometer was set to give excitation at 350 nm and emission was measured at 450 nm.
The reaction mixture contained 5 mM L-glutamate and 0.5 to 10 mU of enzyme in 100 mM sodium phosphate (pH 7.2) containing 0.1 mM pyridoxal 5'-phosphate and 1 mM S-2-aminoethylisothiouronium bromide. The reaction was started by adding 5 to 10 ju,L of enzyme solution to give a final volume of 50 JU.L. Aliquots of 10 ju,L were removed at 0,10, and 20 minutes and mixed with 10 ju.L of prechilled 0.2 M perchloric acid. After centrifugation, 10 ju,L of the supernate was mixed with 190 ju,L of the HPLC mobile phase; HPLC
analysis took 40 /xL. Assays were linear for 40 minutes and with protein added up to 200 /xg/mL.
The assay was used for both a partially purified preparation of glutamate decarboxylase from E. coli and rat brain homogenates. Chromatography conditions for the other substrate-product mixtures listed above were also described.
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