Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro.

Aim: To analyze the role of cytosolic glutathione S-transferases cGSTs and membrane-associated cytosolic GSTs macGSTs in prostaglandin biosynthesis and to evaluate the possible interaction between glutathione S-transferases GSTs and cyclooxygenase (COX) in vitro.

Methods: SDS-PAGE analysis was undertaken for characterization of GSTs, thin layer chromatography (TLC) to monitor the effect of GSTs on prostaglandin biosynthesis from arachidonic acid (AA) and spectrophotometric assays were done for measuring activity levels of COX and GSTs.

Results: SDS-PAGE analysis indicates that macGSTs have molecular weights in the range of 25-28 kDa.

Expression of cyclooxygenase-2 in rat testis.

Cyclooxygenase, the rate-limiting enzyme in the production of prostaglandins, exists in two isoforms, the constitutive cyclooxygenase 1 (Cox-1) and the inducible cyclooxygenase 2 (Cox-2). Cox-1 is involved in homeostatic functions while Cox-2 is implicated in various pathological processes such as inflammation and cancer. The present study describes the constitutive expression of Cox-2 in immature and mature rat testis, primarily localized in the spermatogonial cells.

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; separation and identification of various subunits 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.