Purification of a new acidic glutathione S‐transferase, GST‐Yn1 Yn1, with a high leukotriene‐C4 synthase activity from rat brain

Abstract

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20–25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (M_T or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the M_T in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn₁) of two spots (Yn₁ and Yn₂) of the testis GST-M_T. Therefore, the brain acidic form is a homodimer, and named GST-Yn₁ Yn₁. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C₄ synthase. This form (GST-Yn₁Yn₁) showed the highest leukotriene-C₄ synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The K_m values for leukotriene A₄ and glutathione were 26 μM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7–7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C₄ synthase activity, 67.2 nmol/mg protein in 5 min, but the K_m for leukotriene A₄ was 100 μM, fourfold higher than that of GST-Yn₁ Yn₁. These results suggest that mainly GST-Yn₁ Yn₁ may be involved in leukotriene-C₄ synthesis in rat brain.