Subunit Ya-specific Glutathione Peroxidase Activity toward Cholesterol 7-Hydroperoxides of Glutathione S-Transferases in Cytosols from Rat Liver and Skin*

(Received for publication, July 9, 1996, and in revised form, October 18, 1996)

Akira Hiratsuka Dagger , Hidefumi Yamane Dagger , Shinji Yamazaki §, Naoki Ozawa § and Tadashi Watabe Dagger

From the Dagger  Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji-shi, Tokyo 192-03 and § Toxicology and Efficacy Research, Tsukuba Research Laboratories, Pharmacia & Upjohn, Tsukuba, Ibaraki 300-42, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Dermal 7alpha - and 7beta -hydroperoxycholest-5-en-3beta -ols (cholesterol 7alpha - and 7beta -hydroperoxides), regarded as good aging markers in the rat (Ozawa, N., Yamazaki, S., Chiba, K., Aoyama, H., Tomisawa, H., Tateishi, M., and Watabe, T. (1991) Biochem. Biophys. Res. Commun. 178, 242-247), were reduced in the presence of glutathione (GSH) with concomitant formation of GSSG by cytosol from rat liver in which no detectable level of the hydroperoxides had been demonstrated to occur. The GSH peroxidase (GSH Px) activity toward the toxic steroid hydroperoxides was exerted to almost the same extent by both Alpha-class GSH S-transferases (GSTs), Ya-Ya and Ya-Yc, and by selenium-containing GSH Px (Se-GSH Px) in rat liver cytosol. None of three Mu-class GSTs, Yb1-Yb1, Yb1-Yb2, and Yb2-Yb2, and a Theta-class GST, Yrs-Yrs, from rat liver and a Pi-class GST, Yp-Yp, from rat kidney showed any appreciable GSH Px activity toward the hydroperoxides. The subunit Ya-bearing GSTs and Se-GSH Px purified from rat liver cytosol showed marked differences in apparent specific activity toward the cholesterol hydroperoxides (GSTs Ya-Ya > Ya-Yc >>  Se-GSH Px). However, a kinetic study indicated that Se-GSH Px had a higher affinity for steroid hydroperoxides than did the GSTs, so that Se-GSH Px could catalyze the reduction of lower concentrations of cholesterol 7-hydroperoxides with approximately equal Vmax/Km values to those by the GSTs. Rat skin had no GST bearing the subunit Ya but contained only a very low concentration of Se-GSH Px, possibly resulting in the accumulation of cholesterol 7-hydroperoxides in the skin but not in the liver. From rat skin cytosol, GSTs Yc-Yc, Yb1-Yb1, Yb1-Yb2, Yb2-Yb2, and Yp-Yp were isolated, purified to homogeneity, and identified with the corresponding GSTs from liver and kidney. The GSTs accounted for 0.23% of total skin cytosolic protein, and the most abundant isoform of skin GSTs was Yb2-Yb2, followed by Yc-Yc, Yp-Yp, Yb1-Yb1, and Yb1-Yb2 in decreasing order.


INTRODUCTION

5alpha -Hydroperoxycholest-6-en-3beta -ol (cholesterol 5alpha -hydroperoxide)1 and 7alpha -hydroperoxycholest-5-en-3beta -ol (cholesterol 7alpha -hydroperoxide) are intrinsic mutagens to Salmonella typhimurium TA 1537 (1). Cholesterol 7alpha -hydroperoxide and its isomer, cholesterol 7beta -hydroperoxide, a cytotoxic steroid to human foreskin fibroblasts (2), are normal but very minor components of human serum lipoproteins (3) and increase upon oxidation of low density lipoprotein isolated from human sera (2, 4, 5).

Our previous study using a high-performance liquid chromatography (HPLC)-chemiluminescence detector system demonstrated that cholesterol 7alpha - and 7beta -hydroperoxides accumulate linearly with aging in rats from 1-45 weeks after birth, strongly suggesting the dermal steroid hydroperoxides to be good aging markers in these animals (6). However, rat livers contained no detectable levels of cholesterol 7-hydroperoxides even those from 45-week-old animals. In the present study on the mechanism of steroid hydroperoxide accumulation in the skin, we gave attention to glutathione (GSH)-dependent reduction of cholesterol 7-hydroperoxides by GSH S-transferases (GSTs) and Se-containing GSH peroxidase (Se-GSH Px) in the rat liver and skin.

A great deal of information has been accumulated on the GSH-dependent reduction of organic hydroperoxides to the corresponding alcohols with concomitant formation of GSH disulfide (GSSG) by homo- and heterodimeric GSTs as non-Se-GSH Pxes in the liver and various tissues (7-10) and by Se-GSH Px existing as a homotetramer in the liver and erythrocytes (11-13). However, little has been known of the GSH-dependent reduction of the toxic cholesterol hydroperoxides by GSTs as well as by Se-GSH Px. The only information available at present on the GSH-dependent reduction of steroid hydroperoxides is that Se-GSH Px from pig erythrocytes had little activity toward cholesterol 7beta -hydroperoxide compared with those toward fatty acid hydroperoxides and progesterone 17alpha -hydroperoxide (14).

Nothing is known of the molecular species of GSTs and Se-GSH Px in rat skin, although the existence of Alpha-, Mu-, and Pi-class GSTs has been demonstrated by immunoblotting (15-17). The present paper deals with 1) the GSH-dependent reduction of the toxic cholesterol hydroperoxides by GSTs and Se-GSH Px in the rat liver and skin cytosols, 2) a key role of Alpha-class GSTs bearing subunit Ya in the reduction of the steroid hydroperoxides, and 3) the existence in rat skin of GSTs Yc-Yc, Yb1-Yb1, Yb2-Yb2, Yb1-Yb2, and Yp-Yp and of a very low level of Se-GSH Px, and the absence of Alpha-class GSTs bearing the subunit Ya.


EXPERIMENTAL PROCEDURES

Materials

1-Chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 30% (w/w) hydrogen peroxide, yeast GSH reductase, and NADPH were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan) and cholesterol, ethacrynic acid, phenylmethylsulfonyl fluoride, cumene hydroperoxide, linoleic acid, linolenic acid, arachidonic acid, S-hexyl-GSH, and Mr marker proteins were from Sigma, DE52 from Whatman Ltd. (Maidstone, United Kingdom), and CM-Sephadex C-50, DEAE Sephadex A-50, Sephadex G-200, epoxy-activated Sepharose 6B, activated thiol-Sepharose 4B, PBE 94, PBE 118, blue Sepharose 6B, Pharmalyte 8-10.5, and Polybuffer 96 from Pharmacia Biotech Inc. (Uppsala, Sweden). Goat anti-rabbit IgG was purchased from Organo Teknika Co. (West Chester, PA), and peroxidase-conjugated rabbit antiperoxidase IgG from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). Nitrocellulose membranes were purchased from Bio-Rad, and Aquacide II from Calbiochem-Novabiochem Co. (La Jolla, CA). GSH with purity higher than 99.8% and free from its oxidized form, GSSG, was donated by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). Cholesterol 5alpha -hydroperoxide, cholesterol 7alpha -hydroperoxide, 7beta -hydroperoxycholest-5-en-3beta -ol (cholesterol 7beta -hydroperoxide) (18, 19), and sodium salt of 5-sulfoxymethylchrysene (SMCR) (20), and free forms of 13-hydroperoxy-9,11-octadienoic acid (linoleic acid 13-hydroperoxide), 13-hydroperoxy-9,11,15-octadecatrienoic acid (linolenic acid 13-hydroperoxide), and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (arachidonic acid 15-hydroperoxide) (21) were synthesized as reported previously. Other reagents used were of reagent grade.

Enzyme Assay

GSH-conjugating activities of GSTs toward SMCR (22) and other substrates (23) were determined as reported previously. GSH Px activities of GSTs and Se-GSH Px were measured by a modification of the coupled assay procedure of Lawrence and Burk (24). The reaction mixture contained, in a final volume of 1.5 ml, 50 mM sodium phosphate buffer, pH 7.4, 1 mM EDTA, 0.2 mM NADPH, 2 units of GSH reductase, 4 mM GSH, 100 µM cholesterol hydroperoxide (dissolved in 0.5% (w/v) Tween 80/methanol),100 µM fatty acid hydroperoxide (dissolved in ethanol), 1.5 mM cumene hydroperoxide, or 0.25 mM hydrogen peroxide, and the appropriate enzyme (Se-GSH Px, 0.01-6 µg; GSTs Ya-Ya and Ya-Yc, 0.2-3 µg; GSTs Yc-Yc and Yp-Yp, 2-20 µg; GSTs Yb1-Yb1, Yb1-Yb2, and Yb2-Yb2, 21 µg; or GST Yrs-Yrs, 0.5-20 µg of protein). Following preincubation of the mixture without the substrate at 37 °C for 5 min, the reaction was started by the addition of the substrate solution (50 µl). The initial rates of the enzymatic reactions were determined as µmole NADPH oxidized from the decrease in absorbance at 340 nm recorded as linear slopes for 3 min. Control runs were carried out under the same conditions as mentioned above using distilled water instead of the enzyme solutions. Data were expressed as means of at least three experiments.

Kinetic parameters, Km and Vmax, for the reduction of the cholesterol hydroperoxides by GSTs or Se-GSH Px were obtained by double-reciprocal plots at substrate concentrations of 5-100 µM versus apparent reaction rates. Data were expressed as means of at least three experiments.

Preparation of Cytosol

Male Sprague-Dawley rats, weighing 250-270 g (at 7 weeks), were used for preparation of liver, kidney, and skin cytosolic fractions after an overnight fast. The animals were anesthetized with ether and sacrificed. Their isolated livers were perfused with cold isotonic KCl through the portal vein. The kidney and the perfused liver were cut into pieces and rinsed with cold isotonic KCl before homogenization. The skin was removed from the back of each rat after the area was shaved. The skin was then chilled and washed in cold isotonic KCl to remove blood. The skin sample was then scraped with a scalpel blade to remove dermal fat and muscle and cut into small pieces with scissors. The resultant pieces of tissue were prehomogenized in 150 mM Tris-HCl buffer, pH 7.4, containing 5 mM EDTA, 193 mM KCl, 0.25 mM phenylmethylsulfonyl fluoride, and 6.25 mM dithiothreitol with a Polytron tissue homogenizer (Kinematica, Switzerland) and filtered through two layers of surgical gauze. The skin prehomogenate, liver and kidney isolated from the animals were homogenized in the same buffer with a Teflon-pestled Potter-Elvehjem homogenizer. Tissue cytosolic fractions were obtained by centrifugation of postmitochondrial fractions of 33% (w/v) homogenates at 105,000 × g for 60 min. The cytosolic fractions were dialyzed against 50 volumes. 10 mM Tris-HCl buffer, pH 7.8, containing 1 mM EDTA and 2 mM 2-mercaptoethanol.

Purification of GSTs

GSTs Ya-Ya, Ya-Yc, Yc-Yc, Yb1-Yb1, Yb1-Yb2, Yb2-Yb2 (25), and Yrs-Yrs (22) were purified from rat liver cytosol and GST Yp-Yp (26) from rat kidney cytosol as reported previously. Rat skin GSTs were purified from skin cytosol (3,666 mg of protein) by the same methods as used for purification of GSTs from liver and kidney (see legends for Fig. 4 and Table III in the text).


Fig. 4. Separation of rat skin cytosolic GSTs active toward CDNB by chromatofocusing after their retention on the S-hexyl-GSH-Sepharose 6B column. A pooled fraction (8.1 mg of protein) was obtained by elution with S-hexyl-GSH of the S-hexyl-GSH affinity column, to which a dialyzed cytosolic fraction (3,666 mg of protein) from rat skin was directly applied and subjected to chromatofocusing under the same conditions as described in Figs. 1 and 2. The chromatogram was monitored with CDNB.
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Table III.

Summary of purification of GSTs from rat skin cytosol

A cytosolic fraction from rat skin was dialyzed and applied to an S-hexyl-GSH-Sepharose 6B column. GSTs were eluted from the column with S-hexyl-GSH and separated by chromatofocusing into fractions I-IV. Fraction III consisting of GSTs Yp-Yp and Yb1-Yb2 was separated into fractions IIIa and IIIb by re-chromatography on the S-hexyl-GSH affinity column as described under "Experimental Procedures."
Purification step Total protein Total activity Specific activity Yield

mg µmol/min µmol/mg protein/min %
Cytosol 3,666 183 0.05 100
S-Hexyl-GSH 8.1 168 20.7 92
Chromatofocusing
Fraction I (Yc-Yc) 1.4 30.5 21.8 17
Fraction II (Yb1-Yb1) 1.0 23.6 23.6 13
Fraction III 1.9 33.5 17.6 18
  S-Hexyl-GSH
    Fraction IIIa (Yp-Yp) 1.2 15.0 12.5 8
    Fraction IIIb (Yb1-Yb2) 0.5 7.6 15.2 4
    Fraction IV (Yb2-Yb2) 2.3 29.3 12.7 16

Purification of Se-GSH Px from Rat Liver and Skin

Hepatic Se-GSH Px was purified as reported previously (27) from a pooled fraction passing through an S-hexyl-GSH affinity column to which dialyzed rat liver cytosol (4,778 mg of protein) was directly applied to obtain GSTs. Purification of cutaneous Se-GSH Px was performed by modifications of the previously reported methods (27, 28). The pooled fraction which had passed through (3,325 mg of protein) an S-hexyl-GSH affinity column (2.0 × 7 cm) used for obtaining skin GSTs was mixed with glycerol and EDTA to make final concentrations of 10% (v/v) and 1 mM, respectively, and the mixture was brought to 40% saturation with ammonium sulfate. The precipitate formed was removed by centrifugation at 10,000 × g for 30 min, and the resulting supernatant solution was brought to 60% saturation with ammonium sulfate. Precipitate collected by centrifugation was dissolved in 20 mM sodium phosphate buffer, pH 7.2, containing 1 mM EDTA and 0.7 mM 2-mercaptoethanol (buffer A) and then dialyzed overnight against 5,000 ml of buffer A. The dialyzed solution (1,144 mg of protein/15 ml) was applied to a Sephadex G-200 column (2.6 × 84 cm) equilibrated with buffer A and eluted with the same buffer. The column effluent was collected into 7-ml fractions. The chromatographic fractions active toward hydrogen peroxide were pooled and concentrated to 7 ml (542 mg of protein) against Aquacide II and dialyzed against 5,000 ml of 20 mM sodium phosphate buffer, pH 6.0, containing 0.7 mM 2-mercaptoethanol and 1 mM EDTA (buffer B) for 18 h, and then applied to a CM-Sephadex C-50 column (1.7 × 21 cm) equilibrated with buffer B. The column was eluted successively with buffer B (100 ml), a linear gradient of 0-0.4 M NaCl in buffer B (250 ml), and the same buffer (200 ml) containing 1 M NaCl. The column effluent was collected into 5-ml fractions. The chromatographic fractions active toward hydrogen peroxide were pooled and concentrated to 5 ml (108 mg of protein) against Aquacide II and dialyzed against 5,000 ml of 10 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA (buffer C) for 18 h and applied to an activated thiol-Sepharose 4B column (0.9 × 5 cm) which was pre-equilibrated with buffer C, containing 0.1 M NaCl. The enzyme adsorbed was eluted from the column with 0.3 mM cysteine in buffer C after the column was washed with buffer C (30 ml) containing 0.1 M NaCl. The column effluent was collected into 2-ml fractions.

Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (29). Proteins were stained with Coomassie Brilliant Blue R-250 or with silver nitrate.

Reverse Phase Partition HPLC

Separation, identification, and isolation of GST subunits were carried out on a µBondasphere C18-300 Å column (5 µm particle size, 3.9 × 300 mm, Nihon Waters Ltd., Tokyo) with an Atto model Constametric-II liquid chromatograph as reported previously (30). The column was eluted at a flow rate of 0.5 ml/min with a 22-55% (v/v) linear gradient of acetonitrile in water, both containing 0.1%(v/v) trifluoroacetic acid. Chromatograms were monitored by absorbance at 220 nm. GSTs (10-20 µg of protein) purified by chromatofocusing were used as samples.

Automated Amino Acid Sequence Analysis

N-terminal amino acid sequence analysis was carried out with an Applied Biosystems model 470A gas phase sequence analyzer (Foster, CA) and a Spectra Physics SP8700XR phenylthiohydantoin analyzer (San Jose, CA).

Immunoblotting

Polyclonal antibodies to rat GSTs Ya-Ya, Yb1-Yb1, Yp-Yp and Yrs-Yrs were prepared as reported previously (22). Western blot analysis was performed by the method of Towbin et al. (31).

Protein Determination

Protein concentration was determined by the method of Lowry et al. (32) with bovine serum albumin as a standard.


RESULTS

GSH-dependent Reduction of Cholesterol Hydroperoxides by GSTs and Se-GSH Px from Rat Liver Cytosol

A dialyzed cytosolic fraction from rat liver had a GSH Px activity of 1.9 nmol/mg of protein/min toward cholesterol 7alpha -hydroperoxide. Fifty-three percent of the GSH Px activity passed through an S-hexyl-GSH-Sepharose 6B affinity column to which the cytosolic fraction was directly applied, and the remainder was retained on and eluted from the affinity column with S-hexyl-GSH (Fig. 1). The retained fraction contained 92% of the cytosolic activity toward CDNB. All the GSH Px activity toward hydrogen peroxide passed through the affinity column. Both GSH Px activities toward cholesterol 7alpha -hydroperoxide and hydrogen peroxide in the passing-through fraction were inseparable and concentrated into a single protein throughout the purification steps most frequently used for hepatic Se-GSH Px. The purified enzyme protein showed a molecular mass of 80 kDa on a Sephadex G-200 gel filtration column and a subunit mass of 22 kDa on SDS-PAGE. The sequence of the first 28 N-terminal amino acids of the tetrameric protein was completely identical with that of known rat liver Se-GSH Px (33). No other protein with GSH Px activity toward cholesterol 7alpha -hydroperoxide was found in the passing-through fraction from the S-hexyl-GSH affinity column.


Fig. 1. Elution patterns of protein and activities of GSH Px toward cholesterol 7alpha -hydroperoxide and hydrogen peroxide and of GSTs toward CDNB from an S-hexyl-GSH-Sepharose 6B column. A rat liver 105,000 × g supernatant fraction (3,080 mg of protein) was dialyzed and applied to an S-hexyl-GSH-Sepharose 6B column (2.0 × 7.0 cm) equilibrated with 10 mM Tris-HCl buffer, pH 7.8, containing 5 mM EDTA and 2 mM mercaptoethanol. The column was washed with the same buffer containing 200 mM NaCl and eluted with the same buffer containing 5 mM S-hexyl-GSH and 200 mM NaCl. The column effluent was collected into 5-ml fractions. Protein concentrations were determined by absorbance at 280 nm. GSH Px activities were determined with cholesterol 7alpha -hydroperoxide and hydrogen peroxide and GST activities with CDNB as described under "Experimental Procedures."
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The GSH Px activity toward cholesterol 7alpha -hydroperoxide, which was retained on the S-hexyl-GSH affinity column and eluted with S-hexyl-GSH, was separated by chromatofocusing into peaks I and II, both of which showed GSH-conjugating activities toward CDNB (Fig. 2). The GSTs of peaks I and II were identified as Ya-Ya and Ya-Yc, respectively, by SDS-PAGE and Western blot analyses and by co-reverse phase partition HPLC on µBondasphere C18-300Å with the corresponding authentic specimens purified from rat liver cytosol in our laboratory as reported previously (22). The GSTs appeared as a single band at 25 kDa for Ya-Ya and as two bands at 25 and 28 kDa for Ya-Yc on SDS-PAGE and Western blot using anti-Ya-antiserum (Fig. 3). The chromatographic profiles of the GSTs identified as Ya-Ya and Ya-Yc were very similar to those reported previously (22).


Fig. 2. Separation and purification of rat liver cytosolic GSTs active toward cholesterol 7alpha -hydroperoxide and CDNB by chromatofocusing after their retention on the S-hexyl-GSH-Sepharose 6B column. A pooled fraction (82 mg of protein) eluted with S-hexyl-GSH from the S-hexyl-GSH affinity column as shown in Fig. 1 was dialyzed, concentrated, and applied to a chromatofocusing gel (PBE 118) column (1 × 36 cm) equilibrated with 25 mM triethylamine-HCl buffer, pH 11.4. The column was eluted at a flow rate of 30 ml/h with Pharmalyte 8-10.5-HCl, pH 8.0, diluted 80-fold with deaerated water. The column effluent was collected into 2.5-ml fractions. GSH Px and GST activities toward cholesterol 7alpha -hydroperoxide and CDNB were determined as described under "Experimental Procedures." The GST of each peak was assigned as described in the text.
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Fig. 3. SDS-PAGE (A) and Western blot (B) analyses of purified rat liver cytosolic GSTs active toward cholesterol 7alpha -hydroperoxide. Lanes 1 (5 µg of protein) and 2 (10 µg of protein) represent GSTs isolated as peaks I and II by chromatofocusing shown in Fig. 2, following S-hexyl-GSH affinity column chromatography of rat liver cytosol. Lane Mr is a mixture of Mr marker proteins (66,000, bovine albumin; 45,000, egg albumin; 36,000, rabbit muscle glyceraldehyde dehydrogenase; 29,000, bovine erythrocytes carbonic anhydrase; and 20,100, soybean trypsin inhibitor). Western blot analysis was performed using rabbit anti-Ya-antiserum. Rabbit anti-sera raised against GSTs Yb1-Yb1, Yp-Yp, and Yrs-Yrs showed no cross-reactivity with the GSTs of peaks I and II (data not shown).
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GSTs Ya-Ya and Ya-Yc showed much higher activities toward cholesterol hydroperoxides than did Se-GSH Px, whereas the activities of Se-GSH Px toward fatty acid hydroperoxides and cumene hydroperoxide were extremely high compared with the Alpha-class GSTs (Table I). Cholesterol 7beta -hydroperoxide was a 2.5-fold better substrate for GST Ya-Ya than the 7alpha -isomer. A kinetic study indicated that there existed a much smaller difference in GSH Px activity toward the cholesterol 7-hydroperoxides between Se-GSH Px and GST Ya-Ya because of their much smaller Km values for Se-GSH Px than for the GST (Table II).

Table I.

GSH Px activities of rat Se-GSH Px and GSTs toward cholesterol hydroperoxides and other organic and inorganic hydroperoxides


Substrate (concentration) Se-GSH Px GSTs
Alpha
Mu
Pi, Yp-Yp Theta, Yrs-Yrs
Ya-Ya Ya-Yc Yc-Yc Yb1-Yb1 Yb1-Yb2 Yb2-Yb2

µmol/mg protein/min µmol/mg protein/min µmol/mg protein/min µmol/mg protein/min
Cholesterol
  7alpha -hydroperoxide (0.1 mM) 0.18 0.75 0.5 NDa NDa NDa NDa NDa NDa
  7beta -hydroperoxide (0.1 mM) 0.10 1.80 0.83 NDa NDa NDa NDa NDa NDa
  5alpha -hydroperoxide (0.1 mM) 0.07 0.70 0.3 NDa NDa NDa NDa NDa NDa
Linoleic acid
  13-hydroperoxide (0.1 mM) 408 2.0 1.5 1.6 0.1 0.1 0.1 1.4 4.6
Linolenic acid
  13-hydroperoxide (0.1 mM) 402 2.7 1.9 1.5 0.2 0.2 0.2 1.6 9.7
Arachidonic acid
  15-hydroperoxide (0.1 mM) 511 1.6 1.4 1.2 0.1 0.1 0.1 1.3 13.6
Cumene hydroperoxide (1.5 mM) 472 1.4 2.5 3.3 0.1 0.3 0.5 0.01 2.0
Hydrogen peroxide (0.25 mM) 445 NDa NDa NDa NDa NDa NDa NDa NDa
CDNB (1 mM) NDb 33.0 24.4 17.7 39.2 31.4 13.6 14.1 NDb
DCNB (1 mM) NDa 0.14 0.1 0.1 7.4 3.9 0.56 0.08 NDa
SMCR (0.025 mM) NDc NDc NDc NDc NDc NDc NDc NDc 0.2

a  Less than 10 nmol/mg of protein/min.
b  Less than 100 nmol/mg of protein/min.
c  Less than 0.05 nmol/mg of protein/min.

Table II.

Kinetic parameters for GSH-dependent reduction of cholesterol hydroperoxides by Se-GSH Px and GST Ya-Ya purified from rat liver cytosol

Cholesterol hydroperoxides were incubated at 37 °C with Se-GSH Px (5.3 µg of protein) or GST Ya-Ya (1.6 µg of protein) in a final volume of 1.5 ml of 50 mM sodium phosphate buffer, pH 7.4, containing 4 mM GSH. The enzyme activities were determined by the coupled assay described under "Experimental Procedures."
Cholesterola Se-GSH Px
GST Ya-Ya
Km Vmax Vmax/Km Km Vmax Vmax/Km

µM nmol/mg protein/min µM nmol/mg protein/min
7alpha -Hydroperoxide 11 181 16 32 1,003 31
7beta -Hydroperoxide 3 105 39 56 2,083 37
5alpha -Hydroperoxide 4 65 16 11 798 72

a  The substrate concentration range was 5-100 µM. At higher concentrations than 100 µM, hydroperoxides were crystallized from the aqueous medium, containing Tween 80/methanol, used for enzyme assay.

Four other hepatic GSTs active toward CDNB were isolated as homogeneous proteins by chromatofocusing (Fig. 2) and identified by SDS-PAGE, Western blot, and reverse phase partition HPLC analyses with authentic specimens of GSTs Yc-Yc (Alpha), Yb1-Yb1 (Mu), Yb1-Yb2 (Mu), and Yb2-Yb2 (Mu) for peaks III, IV, V, and VI, respectively (data not shown). However, as readily predicted from the result of chromatofocusing, these purified Alpha- and Mu-class GSTs had no GSH Px activity toward cholesterol hydroperoxides, but showed activity toward fatty acid hydroperoxides and cumene hydroperoxide (Table I). Despite its highest activity toward the fatty acid hydroperoxides among the hepatic GSTs, Theta-class GST Yrs-Yrs isolated from the passing-through fraction from the S-hexyl-GSH affinity column had no activity toward the cholesterol 7-hydroperoxides. The Pi-class GST Yp-Yp isolated from rat kidney cytosol also had no activity toward the steroid hydroperoxides.

Absence of GSTs Bearing Subunit Ya and Presence of a Very Low Level of Se-GSH Px in Rat Skin Cytosol

A dialyzed rat skin cytosolic fraction had a very weak GSH Px activity of 0.05 nmol/mg of protein/min, 1/38 of the hepatic activity, toward cholesterol 7alpha -hydroperoxide. All the GSH Px activity passed through the S-hexyl-GSH affinity column together with the GSH Px activity toward hydrogen peroxide (0.12 µmol/mg of protein/min) on direct application of the cytosol to the affinity column.

However, 92% of the total skin cytosolic GST activity toward CDNB (183 µmol/min) was retained on and eluted with S-hexyl-GSH from the S-hexyl-GSH affinity column. The dermal GST activity eluted from the affinity column was separated into four major peaks, I-IV, and a minor peak eluted at pH 8.3 by chromatofocusing (Fig. 4). SDS-PAGE and Western blot analyses indicated peak III to be a mixture of a Mu- and a Pi-class GSTs. This mixture was separated into two single peaks, IIIa and IIIb, with activity toward CDNB on the S-hexyl-GSH affinity column from which they were eluted stepwise with 0.05 and 5 mM S-hexyl-GSH, respectively.

All the aforementioned five major skin GSTs showed single bands on SDS-PAGE at 28 (I), 26.5 (II), 24 (IIIa), 26.5 (IIIb), and 26.5 (IV) kDa (Fig. 5). Western blot analysis of the GSTs using anti-sera raised against rat GSTs Ya-Ya, Yb1-Yb1, Yp-Yp, and Yrs-Yrs indicated that the GSTs of peak I belonged to the Alpha-class, peaks II, IIIb, and IV to the Mu-class, and peak IIIa to the Pi-class (data not shown). Co-reverse phase partition HPLC with authentic rat liver GSTs indicated the Alpha-class GST of peak I to be Yc-Yc (single peak at 37.5 min), the Mu-class GSTs of peaks II, IIIb, and IV to be Yb1-Yb1 (single peak at 31.7 min), Yb1-Yb2 (double-peak at 31.7 and 33.6 min), and Yb2-Yb2 (single peak at 33.6 min), respectively, and the Pi-class GST of peak IIIa to be Yp-Yp (single peak at 35.1 min). N-terminal amino acid sequencing of these GST subunits eluted from the HPLC column showed that the GST subunits from skin samples were the same as those of rat liver (data not shown). Summary of the purification of the rat skin GSTs is shown in Table III.


Fig. 5. SDS-PAGE of GSTs purified from rat skin cytosol. GSTs were purified by chromatofocusing and chromatofocusing-S-hexyl-GSH affinity chromatography as described in the text. Purified skin GSTs (5 µg of protein each) were used for electrophoresis. Lanes 1-3, GSTs of peaks I, II, and IV in Fig. 4 and lanes 4 and 5, GSTs of peaks IIIa and IIIb as described in the text, respectively. Lane 6, mixture of rat liver GSTs Ya-Yc and Yb1-Yb2. Lane Mr, mixture of the same marker proteins as used in Fig. 3.
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The very weak skin cytosolic GSH Px activity toward cholesterol 7alpha -hydroperoxide existing in the passing-through fraction from the S-hexyl-GSH affinity column was inseparable from the GSH Px activity toward hydrogen peroxide throughout the purification steps used for Se-GSH Px (Table IV). A very small amount of homogeneous enzyme protein was isolated and identified with rat liver Se-GSH Px by gel filtration column chromatography and SDS-PAGE (Fig. 6). The purified skin Se-GSH Px showed substrate specificities very similar to those of the hepatic enzyme toward hydrogen peroxide, cholesterol 5alpha -, 7alpha -, and 7beta -hydroperoxides, and the monohydroperoxides of linoleic, linolenic, and arachidonic acids and had no activity toward CDNB as shown in Table I. No further characterization, including N-terminal amino acid sequencing, was made with the purified skin Se-GSH Px because of its very small quantity.

Table IV.

Isolation and purification of Se-GSH Px from rat skin cytosol

GSTs were removed from a dialyzed rat skin cytosol on an S-hexyl-GSH-Sepharose 6B column, and the passing-through fraction from the column was used for purification of Se-GSH Px as described under "Experimental Procedures." GSH Px activities were determined as described under "Experimental Procedures" except that 10 mM sodium azide (0.1 ml) was added to the reaction mixtures to inhibit catalase.
Purification step Total protein Specific activities
Purification folds
Yields
H2O2 Ch 7alpha -OOHa H2O2 Ch 7alpha -OOHa H2O2 Ch 7alpha -OOHa

mg µmol/mg protein/min %
Cytosol 3,666 0.11 0.05  × 10-3 1 1 100 100
S-Hexyl-GSH 3,325 0.12 0.05  × 10-3 1 1 99 95
(NH4)2SO4 precipitation 1,144 0.27 0.11  × 10-3 2 2 76 64
Sephadex G-200 542 0.39 0.21  × 10-3 4 4 52 58
CM-Sephadex C-50 108 0.87 0.44  × 10-3 8 8 23 24
Activated thiol 0.049 420 0.19 3,818 3,773 5 5

a  Ch 7alpha -OOH, cholesterol 7alpha -hydroperoxide.


Fig. 6. SDS-PAGE of Se-GSH Px purified from rat skin and liver cytosols. Lane Mr, mixture of Mr maker proteins (66,000, bovine albumin; 45,000, egg albumin; 36,000, rabbit muscle glyceraldehyde dehydrogenase; 24,000, bovine pancreas trypsinogen; and 20,100, soybean trypsin inhibitor); lane 1, Se-GSH Px (1 µg of protein) from skin cytosol; and lane 2, Se-GSH Px (1 µg of protein) from liver cytosol. Enzyme proteins were stained with a silver staining kit.
[View Larger Version of this Image (37K GIF file)]



DISCUSSION

The present study strongly suggests that the reason that the accumulation of cholesterol 7-hydroperoxides in skin is a good aging marker in rats may be in large part attributable to the absence of the Alpha-class GSTs bearing subunit Ya (Fig. 4) and to the presence of only a very low concentration of Se-GSH Px in dermal tissue (Table IV). As to GSTs, only Ya-Ya and Ya-Yc could catalyze the reduction of cholesterol 7-hydroperoxides (Table I). These Alpha-class GSTs have been demonstrated to be isoforms accounting for 9.6 and 10% of total GSTs for Ya-Ya and Ya-Yc, respectively, in rat liver which contains no detectable level of the steroid hydroperoxides (34).

In addition, rat liver had a considerable level of Se-GSH Px (1.0 µg/mg cytosolic protein) activity toward cholesterol 7-hydroperoxides. A kinetic study demonstrated that Vmax for the GSH-dependent reduction of the steroid peroxides was much higher by GST Ya-Ya than by Se-GSH Px (Table II). However, the seleno-enzyme may play a more important part than does GST Ya-Ya in scavenging lower concentrations of the hydroperoxides in cytosol, because the former showed much smaller Km than did the latter. The data on the apparent specific activity of GST Ya-Ya toward the 7-hydroperoxides in Table I might be underestimated because Km values for the 7alpha - and 7beta -hydroperoxides were only about one-third and one-half of the maximum substrate concentration (100 µM, solubility limit, see the legend in Table II) used for the enzymatic reactions.

To our knowledge, the present investigation provided the first evidence for the direct comparison of the Se-GSH Px activities toward a variety of organic hydroperoxides with the GSH-Px activities of all the known major GSTs from rat liver and kidney cytosols. As reported previously (12, 14), the activities of Se-GSH Px toward fatty acid hydroperoxides and cumene hydroperoxide were extremely high compared with those of the GSTs (Table I). The only enzyme catalyzing the GSH-dependent reduction of cholesterol 7-hydroperoxides in rat skin was Se-GSH Px, which was purified to homogeneity from skin cytosol and strongly suggested by co-SDS-PAGE to be identical with Se-GSH Px from rat liver cytosol (Fig. 6). No attempt has been made to identify the homotetrameric enzyme Se-GSH Px as a homogeneous protein from skin cytosol in mammalian species, including rats and humans.

Previous studies on skin GSTs in rats (15, 22) and mice (10, 15, 35) have been done only using polyclonal antibodies to the Alpha-, Mu-, Pi-, and Theta-class enzymes from rat or human liver cytosol. These immunochemical studies indicated rat skin cytosol to contain Alpha-, Mu-, and Pi-class GSTs. The present study provided the first direct evidence for the molecular species of GSTs in rat skin. N-terminal amino acid sequence analysis indicated all the skin GST subunits to be identical with those expressed in rat liver cytosol. Based on the specific activities toward CDNB of the skin GSTs separated by chromatofocusing, their sum total accounted for 0.23% of total cytosolic protein, and they existed in a molar ratio of 2.8 (Yc-Yc):2.0 (Yb1-Yb1):1.0 (Yb1-Yb2):4.6 (Yb2-Yb2):2.4 (Yp-Yp).

Rat skin has been demonstrated to have considerably high activity in synthesizing leukotriene (LT)C4 from LTA4. Using GSTs from rat liver, and based on immunochemical evidence (36), the LTC4 synthase activity was suggested to be attributable to GST Yb2-Yb2 if present in rat skin cytosol. The Mu-class GST Yb2-Yb2 had the highest LTC4 synthase activity of all the known rat liver GSTs (37-39). The present study demonstrating GST Yb2-Yb2 to exist at the highest level of all the GSTs found in rat skin provided the first direct evidence for the molecular species of the previously demonstrated Mu-class GST responsible for the high dermal LTC4 synthase activity. Skin GSTs in rodents have not been well studied; however, GSTs in human skin cytosol have been well studied by DelBoccio et al. (40) and Singhal et al. (41). Both have demonstrated the absence of any Mu-class GST and the presence of a Pi-class GST at a higher concentration than with all the other classes of GSTs and also the presence of at least two Alpha-class GSTs, including GST 9.9, as major isoforms in human skin cytosol. In addition, the presence of three additional minor unidentified GSTs were reported by DelBoccio et al. (40) with human skin cytosol. Human skin Pi-class GST was demonstrated to be identical with GST pi  from liver (40). Human skin GST 9.9 has been recognized as a homodimer expressed only in human skin, shares strong identity in N-terminal amino acid sequence with rat Yc rather than Ya, and has a GSH Px activity toward linoleic acid hydroperoxide (40).

It is of interest that cholesterol 7-hydroperoxides were reduced only by rat liver GSTs bearing subunit Ya, although cumene hydroperoxide and fatty acid hydroperoxides were substrates for all the hepatic GSTs examined in the present study. Despite its potent GSH Px activity toward the fatty acid hydroperoxides compared with the Alpha-class GSTs, rat liver Theta-class GST Yrs-Yrs had no activity toward steroid hydroperoxides (Table I). The Alpha-class GSTs Ya-Ya and Ya-Yc, previously called ligandins (42-45), are well known as binding proteins to endogenous acidic and basic compounds, such as bile acids (42, 44, 46), LTC4 (47), hematin (48), and bilirubin (42), and to exogenous basic compounds, such as carcinogenic aminoazo dyes (34, 42). Of rat GST subunits, Ya is most likely to have a specific affinity for oxygenated cholesterols, such as cholesterol hydroperoxides (Table I) and cholesterol 5,6alpha -epoxide (49). The cholesterol epoxide has been demonstrated to be a potential carcinogen markedly formed in rat skin irradiated by ultraviolet light (B-band, UVB) (50, 51), and its production from cholesterol has been shown by us to be initiated and accelerated by peroxidation of microsomal lipids (52-54). The steroid epoxide binds covalently to calf thymus DNA (55) and is specifically detoxified by the rat liver GSTs bearing Ya (49) to the GSH conjugate, S-(3beta , 5alpha -dihydroxycholestan-6beta -yl)-GSH (56). Our unpublished data indicated rat liver to contain no detectable level of cholesterol 5,6alpha -epoxide. The present study strongly suggests that the marked increase in the cholesterol 5,6alpha -epoxide level in rat skin by UVB irradiation may be attributable to the lack of the Alpha-class GSTs bearing the subunit Ya.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed.
1    The abbreviations used are: cholesterol 5alpha -hydroperoxide, 5alpha -hydroperoxycholest-6-en-3beta -ol; cholesterol 7alpha -hydroperoxide, 7alpha -hydroperoxycholest-5-en-3beta -ol; cholesterol 7beta -hydroperoxide, 7beta -hydroperoxycholest-5-en-3beta -ol; GSH, reduced glutathione; GST, glutathione S-transferase; Se-GSH Px, selenium glutathione peroxidase; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; SMCR, 5-sulfoxymethylchrysene; LT, leukotriene.

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