Amino Acid Decarboxylase Kochhar et al 1989

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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