:: Asian Journal of Andrology ::

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

D. Anuradha, K. Veera Reddy, T. Charles Kumar, S. Neeraja, P.R.K. Reddy, P. Reddanna

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

Asian J Androl 2000 Dec; 2: 277-282

Keywords: testis; glutathione transferase; leukotrienes; prostaglandins

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 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 Y_b size (60%) with molecular weight of 27 kDa. The most predominant subunits, however, are GST Y_n2 (27%), followed by GST Y_c (24%) and GSTY_n1 (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 Y_b sized and are involved in the synthesis of eicosanoids like LTC₄ and PGD₂.

1 Introduction

Glutathione S-transferases (E.C. 2.5.1.18) are multigene, multifunctional, dimeric proteins involved in the detoxification of a range of xenobiotics^[1-3]. The reactions catalyzed by GSTs include conjugation, reduction, isomerization, peroxidation etc. Because of their wide range of biological activities in the cell, they protect cellular constituents from electrophilic and oxidative attack. There are at least seven classes of GSTs currently identified, five represent the cytosolic GSTs, alpha, mu, theta, pi, sigma and two membrane bound. Initially classification of GSTs was done by the Y configuration^[4]. Later in 1992, a consultant group had designed classification of human GSTs, but is generally applicable to other species^[5]. Listowisky recently reclassified mammalian GSTs that are primarily expressed in testis^[6] and compared it with other classification systems (Table 1). GST isoforms are expressed specifically in almost all the tissues, including the reproductive tissues like testes and ovaries in a discrete tissue-specific pattern^[7].

Table 1. Listowisky's subunit nomenclature for GSTs.

Listowiskys representation	Y system	Numerical system
GSTM1	Y_b1	3
GSTM2	Y_b2	4
GSTM3	Y_b3 or Y_n1	6
GSTM5	Y_n2	9
GSTM6	Y_o	11
GSTP1	Y_p	7
GSTA1	Y_a1	1
GSTA3	Y_c1	2
GSTA4	Y_k	8

Besides their role in xenobiotic metabolism, GSTs are also involved in various metabolic pathways including eicosanoid biosynthesis. Specific isoforms of GSTs are involved in the biosynthesis of eicosanoids by reducing hydroperoxides^[8], endoperoxides^[9], and formation of peptido leukotrienes^[10]. Eicosanoids, metabolites of arachidonic acid via lipoxygenase (LOX) and cyclooxygenase (COX) pathways, are extremely potent biologically active compounds with bewildering variety of actions on different processes, including reproduction. The fact that arachidonic acid is the predominant unsaturated fatty acid found in testis ^[11] and that essential fatty acid deficiency leads to male sterility^[12] suggests the importance of eicosanoids in reproduction. There is extensive literature on the metabolism, biochemistry and effects of prostaglandins on spermatogenesis and androgenesis^[13-15]. Recent studies on the isozymes of cyclooxygenase, the enzyme involved in the synthesis of prostaglandins revealed the expression of two isoforms COX-1 and COX-2. COX-1 is expressed ubiquitously where as COX-2 has a more restricted expression pattern. In contrast to the constitutive expression of COX-1, COX-2 is highly inducible by mitogens, cytokines, growth factors, lipopolysaccharides, etc.

Earlier studies have shown the PGF₂ is the major cyclooxygenase product in testis^[16] and the role of lipoxygenase products in vertebrate reproduction is reviewed^[17]. These arachidonic acid metabolites were shown to be intratesticular factors regulating LH-stimulated androgen production in rat testis^[18]. Recent studies from our laboratory have shown that COX-2 is constitutively present in testis and 12-HETE is the major LOX product formed in the seminiferous tubules and 5-HETE in Leydig cells^[19].

Since GSTs are involved in the metabolism of arachidonic acid to generate eicosanoids, that play an important role in testicular androgenesis, the present study was designed to isolate and identify different GSTs in rat testis, to separate various subunits on reverse-phase HPLC and to analyze their involvement in eicosanoid biosynthesis.

2 Materials and methods

2.1 Samples

Rat testes were isolated from healthy adult Wistar strain albino rats of 1005 days age group. They were isolated after perfusion with saline and stored at -80 for further use.

2.2 Purification of GSTs by affinity chromatography

Perfused rat testes were homogenized in 25 mmol/L Tris-HCl, pH 8.0 containing 0.25 mol/L sucrose and centrifuged at 10,000g for 30 min. The supernatant was centrifuged at 105,000g for 60 min and the resultant supernatant was referred to as the cytosolic fraction. The cytosolic fraction was passed through the S-hexylglutathione-linked epoxy-activated Sepharose 6B affinity chromatographic column^[8] which was previously equilibrated with 25 mmol/L Tris-HCl, pH 7.0 and then washed with the same buffer containing 0.2 mol/L potassium chloride. The affinity bound GSTs were eluted with the equilibration buffer containing 2.5 mmol/L GSH, 5 mmol/L S-hexylglutathione and 3 mL fractions were collected and the fractions with activity were pooled, dialyzed and concentrated by lyophilization.

2.3 Enzyme assays

GST activity was measured with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate and the thioether bond formed was measured at 340 nm and cumene hydroperoxide (CHP) was used as a conventional substrate for measuring the peroxidase activity, which was measured by decrease in absorption of NADPH at 340 nm^[8].

2.4 Electrophoresis and immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of affinity purified GSTs and HPLC separated subunits was performed on 12% polyacrilamide gels^[20]. The molecular weight of subunits was calculated using UVP-2000 gel documentation software program. Testicular affinity purified GSTs separated on SDS-PAGE were screened with polyclonal antibodies raised against rat hepatic cytosolic GSTs.

2.5 HPLC analysis

Affinity purified GSTs were separated by RP-HPLC into subunits using Waters ODS Bondapak TM C18 column (3.9 mm300 mm)^[21]. The mobile phase consisted of a complex step gradient of solvent A (0.1% trifluoroacetic acid in 35% acetonitrile) and solvent B (0.1% trifluoroacetic acid in 75% acetonitrile). Polypeptides eluted were detected at 214 nm.

2.6 GST catalyzed synthesis of prostaglandins

The affinity purified testicular GSTs (100 g) were incubated in a final volume of 4.5 mL of 100 mmol/L potassium phosphate buffer (pH 7.0) containing 5 mmol/L glutathione and 2 mmol/L EDTA, with 22 mol/L PGH₂ (containing 0.1 Ci [¹⁴C]AA) for 5 min^[9]. The reaction was terminated by adding 100 L 1 mol/L HCl and the products were extracted with chloroform. The concentrated samples were separated on silica gel coated TLC plates by using the solvent system consisting of ethylacetate : trimethylpentane: acetic acid: water (110:50:20:100, v:v:v:v). The TLC plates were sprayed with 50% sulfuric acid and baked at 120 for 10 min to view under UV light. Different prostaglandins were identified by their R-f values with reference to the standards. Quantification was done by scrapping the spots corresponding to those of the standards into the scintillation vials and measuring the radioactivity in a Beckman LS-1800 counter.

2.7 GST catalyzed synthesis of leukotrienes

In the reaction for LTC₄ synthase^[10]the typical assay mixture in a volume of 4.5 mL contained 75 mmol/L Tris-HCl (pH 8.0), 5 mmol/L GSH, affinity purified testicular GSTs (50 g) and 30 mol/L 5,6- LTA₄ methyl ester. The reaction was terminated after 10 min of incubation at 37 by adding 50 L 6 mol/L HCl. The reaction mixture was centrifuged at 10,000g and the resulting supernatant was passed through C₁₈ SEP PAK catridges that were previously equilibrated with methanol. The reaction products were eluted with 2 mL methanol, dried under argon and reconstituted in HPLC solvent. The products were separated on Shimadzu HPLC system using ODS C₁₈ column. The products were eluted with HPLC solvent consisting of methanol: water: acetic acid (65:35:0.1, v:v:v, pH 5.7) at a flow rate of 1 mL/min. Individual peaks collected were screened for characteristic UV/VIS spectrum of LTC₄. Quantification of the LTC₄ peak identified was carried out based on the HPLC integration data and by taking the molar extinction coefficient of LTC₄ at 280 nm as 410⁴.

3 Results

Testicular cytosolic GSTs from adult Wistar strain albino rats were purified by passing the cytosolic fraction through the affinity column. Table 2 shows typical purification profile of rat testicular cytosolic GSTs. As shown in the table, the affinity purified GSTs have specific activity of 59 units/mg protein with CDNB as the substrate, which was almost double the specific activity reported for rat liver affinity purified GSTs^[22]. The overall yield achieved was more than 75%. Testicular GSTs also showed non-selenium glutathione peroxidase activity (1.45 units/mg protein), the activity being much lesser than that reported for rat liver cytosolic GSTs (4.5 units/mg protein).

Table 2. Purification profile of rat testicular GSTs, monitored with 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. GST activity is represented as Units/min. One unit is defined as 1 mol of thioether formed/min.

Step	Total protein (mg)	Total activity (Units)	Specific activity (Units/mg protein)	Purification fold	Yield (%)
10,000g supernatant	1090	536	0.49	1	100
105,000g supernatant	781	469	0.6	1.22	87.5
Affinity Pooled	6.89	405	58.8	119	75.56

Testicular cytosolic GSTs when separated on SDS-PAGE (Figure 1A) showed predominantly Y_b sized subunits with molecular weight 27 kDa as against rat liver cytosolic GSTs where Ya (25.6 kDa) and Y_c (28 kDa) were known to be the major subunits^[21]. Western blot analysis of affinity purified GSTs separated on SDS-PAGE, probed with polyclonal antibodies raised against rat liver affinity purified GSTs (Figure 1B) also showed Y_b sized proteins as the major testicular GSTs followed by Y_c subunits.

Figure 1. (A) SDS-PAGE of affinity purified cytosolic GSTs.
(B) Western blot analysis of cytosolic GSTs probed with antibodies raised against hepatic cytosolic cytosolic GSTs.1. Hepatic GSTs. 2. Testicular cytosolic GSTs.

Further characterization of the various GST subunits was performed on RP-HPLC (Figure 2). Individual peaks obtained were identified based on their elution pattern and molecular weights^[23]. Y_n2 is the major subunit (27%) of GSTs found in testis followed by Y_c (24%), Y_n1 (20%), Y_b2 (8%), Y_a, Y_p, Y_b1, Y_o and Y_k (Table 3). The individual peaks were collected and further analyzed on SDS-PAGE (Figure 3). As shown in Table 3, Y_n1 and Y_n2 (50%) followed by Y_b (12%) which belong to the mu class GSTs formed the major class of GSTs present in the testis^[24]. Y_n1 and Y_n2 were found to be similar but not identical immunologically and differ only at two positions in the first 20 amino acid residues from the N-terminus^[25].

Table 3. Data represents the relative concentrations of individual subunits separated on RP-HPLC.

GST subunit	Relative abundance (% total)
Y_b1	4.53
Y_b2	8.13
Y_n2	27.15
Y_p	4.76
Y_c	24.14
Y_n1	20.34
Y_o	3.42
Y_a	4.92
Y_k	2.59

Figure 2. RP-HPLC separation of affinity purified testicular GST subunits.
Figure 3. SDS-PAGE of GST subunits separated on RP-HPLC.
1. Testicular GSTs
2. Peak 1 (Y_b1)
3. Peak 2 (Y_b2)
4. Peak 3 (Y_n2)
5. Peak 4 (Y_p)
6. Peak 5 (Y_c)
7. Peak 6 (Y_n1)
8. Peak 7 (Y_o)
9. Peak 8 (Y_a)
10. Peak 9 (Y_k)

To identify the role of testicular GSTs in leukotriene metabolism, affinity purified GSTs were incubated with LTA₄Me and products formed were separated on RP-HPLC. Testicular GSTs showed LTC₄ synthase activity (Figure 4), which was significantly higher than that of hepatic GSTs (Table 4). This might be due to the presence of Y_n1 subunit, which exhibits maximum LTC₄ synthase activity^[26].

Table 4. Production of prostaglandins and leukotrienes by testicular and hepatic affinity purified GSTs.

Product formed	Testicular affinity GSTs	Hepatic affinity GSTs
PGD₂	260005050b	56201770
PGE₂	120004200	210007520
PGF₂	2300950	62002140
LTC₄Me	48.209.3b	24.57.4

Data on prostaglandins is represented in CPM/mg protein for 5 min. Data on leukotrienes represented in nmoles of LTC₄ formed/(minmg protein). bdenotes significantly different from hepatic GSTs. Significance was set at P<0.05.

Figure 4. Separation of leukotrienes by RP-HPLC. Inset: UV/Vis absorption spectrum of 5,6-LTC₄ peak in methanol.

The involvement of GSTs in prostaglandin metabolism was analyzed by incubating testicular GSTs with PGH₂ and the products were separated on TLC and quantified by counting the radioactivity of the products separated on TLC. Affinity purified testicular GSTs showed maximum PGD synthase activity followed by PGE₂ and PGF₂ whereas hepatic GSTs showed PGE synthase activity (Table 4).

4 Discussion

Diverse subunit classes of GSTs were known to be expressed age, sex and tissue specifically. The presence of multiple but closely related gene products providea broad substrate specificity and there by allows detoxification of a wide variety of xenobiotics and endogenous toxicants. Four classes of GSTs alpha, mu, pi, theta are expressed in the somatic cells of the testis. The pi form is not expressed in normal spermatogonia but was over expressed in germ cell neoplasia and can be used as a marker of germ cell cancer^[27]. Mu3 is reported to be the major form of GST present in human testis^[6]. The mu class GST in the seminiferous tubules (STF) is secreted from Sertoli cells and is shown to be asteroid binding protein^[28]. The mu GST, present on the sperm was shown to be playing a role in acrosome reaction^[29]. Our study demonstrates that Y_n1 and Y_n2, which belong to the mu class GST are the predominant forms in rat testis cytosol and are of the molecular weight 27 kDa. GSTs in the testis are involved in the synthesis of eicosanoids (LTC₄ from LTA₄ and PGD₂ from PGH₂), which play a role in testicular steroidogenesis and spermatogenesis.

Acknowledgements

This work was supported by the grants from Council for Scientific and Industrial Research project 37(0987)/98/EMR-II and Department of Science and Technology (Grants SP/SO/D-33/88 and SP/SO/D-20/93), Government of India, New Delhi.

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Correspondence to: Dr P. Reddanna, School of Life Sciences, University of Hyderabad, Hyderabad-500 046, India.
Tel: +91-40-301 0745 Fax: +91-40-301 0120
e-mail: prsl@uohyd.ernet.in
Received 2000-08-15 Accepted 2000-11-06