Purification and characterization of rat testicular glutathione S-transferases: role in the synthesis of eicosanoids

Aim: Purification of glutathione S-transferases (GSTs) from rat testis; sep aration and-identification of various subunits and their role in eicosanoid biosynthesis. Methods: Purification of mt testicular GSTs by affinity chro matography, employing S-hexylglutathione-linked epoxy-activated Sepharose 6 B column and separation of individual subunits by reverse phase-high pressu re liquid chromatography (RP-HPLC). Characterization of affinity purified G STs by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The role of testicular GSTs in eicosanoid biosy nthesis was determined by incubating GSTs with 5,6-Leukotriene A(4)Me (LTA( 4)Me) and prostaglandin H-2(PGH(2)) and analyzing the products formed on HP LC/TLC. Results: The present study reveals that majority of rat testicular GSTs are of Y-b size (60%) with molecular weight of 27 kDa. The most predom inant subunits, however, are GST Y-n2(27%), followed by GST Y-c(24%) and GS T Y-n1(20%). These testicular GSTs showed very high Leukotriene C-4 (LTC4) synthase activity with 5, 6-Leukotriene A(4)Me (LTA(4)Me) as the substrate and prostaglandin D (PGD) synthase activity with prostaglandin H-2(PGH(2)) as the substrate. Conclusion: Majority of rat testicular GSTs are Y-b sized and are involved in the synthesis of eicosanoids like LTC4 and PG(4).