Assay for GABA Transaminase Activity

The activity of GABA transaminase was measured as described (Marienhagen et al., 2005). Briefly, the assay buffer contained 200 mM Tris–HCl pH 8, 0.25 mM pyridoxal 5′-phosphate, 20 mM GABA, and 20 mM 2-oxoglutaric acid. To test if ammonium affects GABA transaminase activity, 30 mM (NH₄)₂SO₄ was added. The reaction mixture (initial volume 10 ml) was preincubated for 2 min at 30°C and started by the addition of purified protein at a final concentration of 3 μg mL^–1. Six 500 μl samples were collected over a period of 26 min and the reaction was immediately terminated by mixing each sample with 300 μL of 5% (v/v) perchloric acid and 38% (v/v) ethanol. After this, the sample was neutralized by addition of 200 μL of 20 mM Tris–HCl (pH 8) with 23 mM K₂CO₃. The precipitated salts were removed by centrifugation (10 min, 16,000g). Subsequently, the glutamate concentration in the sample was measured by HPLC (Agilent 1260 series) equipped with an Agilent Eclipse XDB-C18 column using a variable wavelength detector and a fluorescence detector. Elution was performed with a mixture of 43% (v/v) buffer A [10 mM Na₂HPO₄, 10 mM Na₂B₄O₂ (pH 8.2)] and 57% (v/v) methanol at a flow rate of 2 mL/min for 14 minutes. Before chromatographic separation, amino acids were derivatized with o-phthaldialdehyde (Lindroth and Mopper, 1979). 25–1000 μM sodium glutamate was used as standard.