大鼠睾丸谷胱甘肽S-转移酶的纯化与特性：在类花生酸合成中的作用

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

作者信息

Anuradha D, Reddy K V, Kumar T C, Neeraja S, Reddy P R, Reddanna P

机构信息

School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India.

出版信息

Asian J Androl. 2000 Dec;2(4):277-82.

PMID:11202416

Abstract

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. 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.

CONCLUSION

Majority of rat testicular GSTs are Yb sized and are involved in the synthesis of eicosanoids like LTC4 and PGD2.

摘要

目的

从大鼠睾丸中纯化谷胱甘肽S-转移酶（GSTs）；分离并鉴定各种亚基及其在类花生酸生物合成中的作用。

方法

采用S-己基谷胱甘肽连接的环氧活化琼脂糖凝胶6B柱通过亲和色谱法纯化大鼠睾丸GSTs，并通过反相高压液相色谱（RP-HPLC）分离各个亚基。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析对亲和纯化的GSTs进行表征。通过将GSTs与5,6-白三烯A4甲酯（LTA4Me）和前列腺素H2（PGH2）孵育并分析在HPLC/TLC上形成的产物来确定睾丸GSTs在类花生酸生物合成中的作用。

结果

本研究表明，大鼠睾丸GSTs的大多数为Yb大小（60%），分子量为27 kDa。然而，最主要的亚基是GST Yn2（27%），其次是GST Yc（24%）和GST Yn1（20%）。这些睾丸GSTs以5,6-白三烯A4甲酯（LTA4Me）为底物时表现出非常高的白三烯C4（LTC4）合酶活性，以前列腺素H2（PGH2）为底物时表现出前列腺素D（PGD）合酶活性。