| Abstract: | A cDNA clone for glutathione S-transferaseA (GSTA) from plaice (Pleuronectes platessa) was expressed in Eschericia coli (E. coli) and purified to homogeneity by S-hexylglutathione affinity chromatography. When compared to literature values for a variety of purified mammalian GSTs, the heterologously expressed purified plaice enzyme had moderate activity towards the model substrate 1,2-chloro-2,4-dinitrobenzene (CDNB) and exhibited a Km of 2.5 ± 2 mM and Vmax of 30.9 ± 2.3 μmol min−1 mg−1. It had little or no activity towards several other model GST substrates including 1,2-dinitrochloro-4-benzene (DCNB), ethacrynic acid (EA), and p-nitrobenzylchloride (NBC). However plaice GSTA was a relatively efficient catalyst for the conjugation of a series of alk-2-enals and alk-2,4-dienals and also 4-hydroxynonenal. The highest activity observed with this series of substrates was with trans-non-2-enal with a Km of 17.9 ± 2.2 μM and a Vmax of 3.01 ± 0.57 μmol min−1 mg−1. These unsaturated alkenals have been identified in cells and cell extracts as highly toxic products arising from peroxidation of unsaturated fatty acids particularly during periods of oxidative stress. Fish are relatively rich in polyunsaturated fatty acids and thus GSTA mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products. |
