FIGURE 3.

Substrate specificities of recombinant HuGADL1. Chromatograms A, B, and C show the accumulation of hypotaurine in a HuGADL1 and cysteine sulfinic acid reaction mixture, the accumulation of taurine in a HuGADL1 and cysteic acid reaction mixture, and the accumulation of β-alanine in a HuGADL1 and aspartate reaction mixture, respectively. Chromatogram D shows the absence of GABA in the HuGADL1 and glutamate reaction mixture. Chromatogram E shows a comparative study of the substrate preference of HuGADL1 under different concentrations of substrates. Chromatogram F illustrates the retention time of hypotaurine, taurine, β-alanine, and GABA standards under the same assay conditions. The reaction was initiated by the addition of purified HuGADL1 into each substrate preparation. An equal volume of 100% ethanol was added to each reaction mixture at 10 min after incubation at 25 °C, followed by the addition of two volumes of OPT agent. The mixture was incubated for 3 min. Determination of the products was based on the detection of OPT derivatives by reverse phase liquid chromatography with electrochemical detection. A C18 reverse phase column (4.6 mm × 100 mm, 5-μm spherical particle) was used for substrate and product separation. The mobile phase consisted of 50 mm phosphate buffer (pH 3.5) containing 25% acetonitrile with a flow rate of 0.5 ml/min. The working electrode was maintained at +0.75 V versus an Ag/AgCl reference electrode.