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

Abstract

Aim: Purification of glutathione S-transferases (GSTs) from rat testis; separation and identification of various sub-units and their role in eicosanoid biosynthesis. Methods; Purification of rat testicular GSTs by affinity chromatography, employing S-hexylglutathione-linked epoxy-activated Sepharose 6B column and separation of individual subunits by reverse phase-high pressure liquid chromatography (RP-HPLC). Characterization of affinity purified GSTs by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The role of testicular GSTs in eicosanoid biosynthesis was determined by incubating GSTs with 5, 6-Leukotriene A4Me (LTA4Me) and prostaglandin H2(PGH2) and analyzing the products formed on HPLC/TLC. Results; The present study reveals that majority of rat testicular GSTs are of Yb size (60%) with molecular weight of 27 kDa. The most predominant sub-units, however, are GST Yn2 (27%), followed by GST Yc (24%) and GST Ynl (20%). These testicular GSTs showed very high Leukotriene C4(LTC4.) synthase activity with 5, 6-Leukotriene A4Me (LTA4Me) as the substrate and prostaglandin D (PGD) synthase activity with prostaglandin H2(PGH2) as the substrate. Conclusion; Majority of rat testicular GSTs are Yb sized and are involved in the synthesis of eicosanoids like LTC4 and PGD2. © 2000, Asian Journal of Andrology.