(Received for publication, July 9, 1996, and in revised form, October 18, 1996)
From the Dermal 7 5 Our previous study using a high-performance liquid chromatography
(HPLC)-chemiluminescence detector system demonstrated that cholesterol
7 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 7 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.
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 5 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.
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.
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).
Summary of purification of GSTs from rat skin cytosol
Department of Drug Metabolism and Molecular
Toxicology,
- and
7
-hydroperoxycholest-5-en-3
-ols (cholesterol 7
- and
7
-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.
-Hydroperoxycholest-6-en-3
-ol (cholesterol
5
-hydroperoxide)1 and
7
-hydroperoxycholest-5-en-3
-ol (cholesterol 7
-hydroperoxide) are intrinsic mutagens to Salmonella typhimurium TA 1537 (1). Cholesterol 7
-hydroperoxide and its isomer, cholesterol
7
-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).
- and 7
-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.
-hydroperoxide compared with
those toward fatty acid hydroperoxides and progesterone
17
-hydroperoxide (14).
Materials
-hydroperoxide, cholesterol 7
-hydroperoxide, 7
-hydroperoxycholest-5-en-3
-ol (cholesterol 7
-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.
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.
[View Larger Version of this Image (24K GIF file)]
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
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.
ElectrophoresisSodium 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 HPLCSeparation, 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 AnalysisN-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).
ImmunoblottingPolyclonal 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 DeterminationProtein concentration was determined by the method of Lowry et al. (32) with bovine serum albumin as a standard.
A dialyzed cytosolic
fraction from rat liver had a GSH Px activity of 1.9 nmol/mg of
protein/min toward cholesterol 7-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 7
-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 7
-hydroperoxide was found in the
passing-through fraction from the S-hexyl-GSH affinity
column.
The GSH Px activity toward cholesterol 7-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).
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 7-hydroperoxide was a 2.5-fold better
substrate for GST Ya-Ya than the 7
-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).
|
|
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 CytosolA 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
7-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.
The very weak skin cytosolic GSH Px activity toward cholesterol
7-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 5
-, 7
-, and 7
-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.
|
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
7- and 7
-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 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 5,6-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-(3
,
5
-dihydroxycholestan-6
-yl)-GSH (56). Our unpublished data
indicated rat liver to contain no detectable level of cholesterol
5,6
-epoxide. The present study strongly suggests that the marked
increase in the cholesterol 5,6
-epoxide level in rat skin by UVB
irradiation may be attributable to the lack of the Alpha-class GSTs
bearing the subunit Ya.
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