Determination of glutamate decarboxylase by high-performance liquid chromatography

An improved method for the determination of glutamate decarboxylase (GAD) activity is described. The enzyme was evaluated by incubation with glutamic acid (L-Glu) in the presence of pyridoxal 5'-phosphate (PLP): the gamma-aminobutyric acid (GABA) formed was derivatized to PTC-GABA; the latter was subsequently separated and assayed by isocratic HPLC (LiChrospher RP-18 column; isocratic elution with pH 5.8 acetate buffer in acetonitrile-water) with UV absorbance detection at 254 nm. The method described is a sensitive, reproducible and specific assay useful for following variations of GAD activity in vitro; this assay was subsequently used for the evaluation of GAD activity variations after irradiation with low doses of He-Ne laser radiation.