Purification of glutathione S‐transferase from lamb muscle and its effect on lipid peroxidation

Abstract

AbstractLamb muscle contained 0.4 mg glutathione S‐transferase (EC 2.5.1.18) per gram of tissue. On storage at 1°C, 20% of the activity was lost after 3 weeks. Glutathione S‐transferase was isolated from lamb muscle by affinity chromatography on S‐hexyglutathione Sepharose 6B. One major form of the enzyme was present in the purified preparation, a dimer with subunit molecular weight of 22 900 and isoelectric point of 6.7. This enzyme fits into the Pi class on the basis of substrate specificity. Linoleic acid hydroperoxides and trans, trans‐2,4‐decadienal, products of linoleate peroxidation, were both substrates (0.11 and 0.24 μmol min−1 mg−1 protein respectively). Glutathione S‐transferase inhibited copper‐stimulated peroxidation of arachidonate in the presence of glutathione. The inhibition was only slightly less efficient in the absence of glutathione. The latter effect probably arises from the presence of enzyme‐bound glutathione (4–5 mol mol−1 enzyme subunit) and also from a small but significant glutathione‐independent peroxidase activity. The latter was detected using an HPLC assay which monitors formation of product directly. Reduced glutathione per se showed only a slight inhibition of arachidonate peroxidation.