Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro.
OBJECTIVE
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. In a coupled assay involving GSTs, arachidonic acid and cyclooxygenase-1, rat testicular macGSTs produced prostaglandin E2 and F2alfa, while the cGSTs caused the generation of prostaglandin D2, E2 and F2alfa. In vitro interaction studies on GSTs and COX at the protein level have shown dose-dependent inhibition of COX activity by macGSTs and vice versa. This effect, however, is not seen with cGSTs. The inhibitory effect of COX on macGST activity was relieved with increasing concentrations of reduced glutathione (GSH) but not with 1-chloro 2,4-dinitrobenzene (CDNB). The inhibition of COX by macGSTs, on the other hand, was potentiated by glutathione.


CONCLUSIONS
We isolated and purified macGSTs and cGSTs from rat testis and analyzed their involvement in prostaglandin biosynthesis. These studies reveal a reversible functional interaction between macGSTs and COX in vitro, with possible interactions between them at the GSH binding site of macGSTs.
Neeraja
S
S
Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Ramakrishna
B
B
Sreenath
A S
AS
Reddy
G V
GV
Reddy
P R K
PR
Reddanna
P
P
D000818:Animals; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology*; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism*; D008297:Male; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013737:Testis / Q000187:drug effects / Q000201:enzymology*
D004137:Dinitrochlorobenzene; D011451:Prostaglandin-Endoperoxide Synthases; D005982:Glutathione Transferase; D005978:Glutathione