Purification and characterization of rat testicular glutathione S-transferases: role in the synthesis of eicosanoids.
OBJECTIVE
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. The role of testicular GSTs in eicosanoid biosynthesis was determine 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 subunits, however, are GST Yn2(27%), followed by GST Yc(24%) and GST Yn1(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.


CONCLUSIONS
Majority of rat testicular GSTs are Yb sized and are involved in the synthesis of eicosanoids like LTC4 and PGD2.
Anuradha
D
D
School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India.
Reddy
K V
KV
Kumar
T C
TC
Neeraja
S
S
Reddy
P R
PR
Reddanna
P
P
D000728:Androgens / Q000096:biosynthesis; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015777:Eicosanoids / Q000096:biosynthesis*; D005982:Glutathione Transferase / Q000378:metabolism*; D015289:Leukotrienes / Q000096:biosynthesis; D008297:Male; D011453:Prostaglandins / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013737:Testis / Q000166:cytology / Q000201:enzymology*
D000728:Androgens; D015777:Eicosanoids; D015289:Leukotrienes; D011453:Prostaglandins; D016718:Arachidonic Acid; D005982:Glutathione Transferase