Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial, the brown antechinus (Antechinus stuartii)

Article abstract of DOI:10.1080/004982597240352

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight a

Full text of DOI:10.1080/004982597240352

xenobiotica , 1997, vol. 27, no. 6, 573±586
Puri®cation and characterization of hepatic
glutathione transferases from an insectivorous
m arsupial, the brown antechinus
stuartii)
(Antechinus
R. M. BOLTON and J. T. AHOKAS*
Key Centre for Applied and Nutritional Toxicology, RMIT-University,
GPO Box 2476V, Melbourne, Victoria 3001, Australia
Received 16 September 1996
1. Five unique glutathione transferase isoenzymes were puri®ed from the hepatic
cytosol of an insectivorous marsupial, the brown antechinus. The puri®ed GSTs were
characterized by structural and catalytic properties including apparent molecular weight
and isoelectric point, speci®city towards model substrates, kinetic parameters, sensitivity to
inhibitors and cross-reactivity with antisera raised against human GSTs.
2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol,
representing 71% of the total GST puri®ed. The substrate speci®city of Antechinus GST
1-1 was similar to that of other alpha class GSTs, particularly with respect to its high
activity with cumene hydroperoxide. The mu class was represented by three GST
isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented
8, 2 and 10 % of the total GST puri®ed respectively. A single GST, Antechinus GST 22,
belonged to the pi class of GSTs and represented 12 % of the total GST puri®ed. The
hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar
to that observed in the human than in rat.
3. A previous study investigating a herbivorous marsupial, the brushtail possum
(Trichosurus vulpecula) also identi®ed a predominant hepatic GST belonging to the alpha
class and displaying peroxidase activity. The evolutionary conservation of a similar
predominant GST isoenzyme in these marsupials suggests that they play an important role
in the detoxication metabolism of these unique mammals.
Introduction
The glutathione transferases (GSTs‹, EC 2\5\1\18) are a multifunctional
family of enzymes that by both catalytic activity and direct binding functions make
a signi®cant contribution towards cellular protection against the toxic challenges of
endogenous compounds and a variety of xenobiotics (Daniel 1993). The main
function of GSTs involves the catalysis of the conjugation of electrophilic,
hydrophobic compounds with cellular glutathione, increasing aqueous solubility
and thus facilitating excretion of the compound. This pathway results in the
detoxication of many endogenous electrophiles as well as exogenously derived
environmental chemicals (Motoyama and Dauterman 1980, Ketterer and Meyer
1989). GSTs may further function in a protective role by covalently binding
electrophilic compounds, resulting in their inactivation in the cell (Mannervik and
Danielson 1988). GSTs also non-covalently bind numerous non-substrate ligands
* Author for correspondence.
‹
GST, glutathione transferase ; GSH, glutathione ; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB,
1,2-dichloro-4-nitrobenzene ; SBP, sulphobromophthalein ; EPNPP, 1,2-epoxy-3-(p-nitrophenoxy)-
propane; EA, ethacrynic acid ; Sbf-Cl, ammonium 4-chloro-7-sulphobenzofurazan ; tPBO, trans-4-
phenyl-3-buten-2-one ; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
±
’
0049±8254}97 $12 00
1997 Taylor & Francis Ltd

574
R. M. Bolton and J. T. Ahokas
and may thus serve as intracellular binding, storage and transport proteins (Litwack
et al. 1971, Listowsky et al. 1988). Mammalian cytosolic GSTs are presently
classi®ed into four multigene classes (alpha, mu, pi and theta), each of which consist
of two or more genes that encode diåerent subunit types. The evolutionary events
which have led to the present-day diversity of GSTs have still to be clearly de®ned,
however the cytosolic GSTs are thought to have diverged from a common ancestral
gene belonging to the theta class (Buetler and Eaton 1992, Taylor et al. 1993).
Diversi®cation into distinct classes is thought to have occurred prior to the
divergence of mammals (Mannervik and Danielson 1988).
Investigation of glutathione transferases in the brushtail possum revealed
signi®cant diåerences in the expression of this detoxication enzyme in comparison
to eutherian mammals (Bolton and Ahokas 1997). Of particular signi®cance was the
®nding that the brushtail possum expressed a single predominant hepatic GST
isoenzyme belonging to the alpha class of GSTs (Possum GST 1-1). The peroxidase
activity shown by this isoenzyme suggested that it might play a signi®cant role in the
detoxication of allelochemicals present in the eucalypt diet of the brushtail possum.
However, the lack of GST isoenzymes from other GST classes may result in
susceptibility to particular types of environmental chemicals in this herbivorous
marsupial.
If the major role of possum GST 1-1 is to contribute to the detoxication of
eucalypt allelochemicals, then this may be considered an adaptation to the species’
¯oral environment, and thus diet. The evolutionary signi®cance of this ®nding, to all
Australian marsupials, is unknown at present. In light of the possible disadvantages
of the expression of a predominant GST isoenzyme, it is important to determine if
the pattern observed in the brushtail possum represents an evolutionary divergence
within the marsupial sub-class. Investigations of GST expression in a wider range
of marsupial species will therefore contribute to determining the potential eåects of
environmental chemicals on Australian marsupials. Thus, puri®cation of the hepatic
glutathione transferases of an insectivorous marsupial, the brown antechinus, was
undertaken. The brown antechinus is a small dasyurid marsupial which is common
to the forests of south-eastern Australia and characterised by an unusual life history
strategy in which all males of the species die at the end of the annual breeding season
(Wood 1970).
M aterials and m ethods
Materials
CDNB, EA, SBP, EPNPP, Sbf-Cl, cumene hydroperoxide, glutathione, S-hexyl glutathione,
dithiothreitol, EDTA, BSA, haematin, cibacron blue and Coomassie brilliant blue R were obtained from
Sigma Chemical Co. (USA). DCNB, tPBO, tributyltin acetate, triethyltin bromide and triphenyltin
chloride were obtained from Aldrich Chemical Co. (USA). Sepharose 6B, polybuåers, pharmalytes and
the chromatofocusing resin PBE-118 were from Pharmacia (Sweden). Hydrogen peroxide was obtained
from BDH Merck Chemicals (Australia), and tri¯uoroacetic acid from Applied Biosystems (USA).
Animals
Brown antechinus (Antechinus stuartii) were trapped in an area of sclerophyll forest near Powelltown,
70 km east of Melbourne, Victoria (Department of Conservation and Natural Resources permit RP-91-
046). Elliot traps, baited with a mixture of oats and peanut butter, and containing shredded paper for
nesting, were set at dusk and cleared at dawn. Ten adult male brown antechinus were trapped in mid±late
June 1991, and nine adult female brown antechinus were trapped between late June and early August
1991 (winter). When in the ®eld, animals were kept in the traps and food and bedding changed daily.
Upon transportation to Melbourne, animals were housed in rat cages, with shredded paper bedding, and

Hepatic glutathione transferases
575
provided with water and food daily. The food consisted of ground beef heart and liver, dry pet food and
dried fruit as recommended by Woolley (1982). Animals were killed by cervical dislocation a maximum
of 4 days after capture.
Preparation of cytosolic fraction
All procedures were carried out at 0±4 C unless otherwise stated. Livers were perfused in situ with
°
ice-cold 10 m potassium phosphate buåer, pH 7 0, containing 1 m EDTA, 2 m dithiothreitol and
±
0 15 KCl, then excised. Homogenization of the liver (10 % w v in the perfusion buåer) was performed
m
m
m
±
m
}
using three passes on an Ultra-Turrax T25 homogeniser (Janke and Kunkel GMBH and Co., Germany).
The homogenate was centrifuged at 10 000 g for 20 min to remove cell debris, nuclei and mitochondria.
The resulting supernatant was further centrifuged at 100000 g for 60 min. The cytosolic fraction used in
°
this study was the resulting 100000 g supernatant. Cytosol was stored at ®80 C until used. This is a
standard way of storing cytosol and no evidence of loss of activity was observed.
Enzyme assays
Glutathione transferase activity with the substrates CDNB, DCNB, EA, tPBO, EPNPP, and SBP
was measured spectrophotometrically according to the method of Habig et al. (1974). Glutathione
peroxidase activity towards cumene hydroperoxide and hydrogen peroxide was determined as described
by Lawrence and Burk (1976). GST activity with the ¯uorescent substrate Sbf-Cl was determined as
°
previously described (Bolton et al. 1994). All assays were carried out at 37 C and speci®c activity
expressed as mol product formed min mg protein. Protein concentration of cytosols and pure
l
}
}
isoenzyme samples were determined according to the methods of Lowry et al. (1951) and Bradford (1976)
respectively. BSA was used as the standard.
Puri®cation of glutathione transferases
All procedures were performed at 0±4 C unless otherwise stated. Pooled samples of either male or
°
±
m
female cytosols were diluted with 10 m Tris-HCl, pH 7 8 and applied to an S-hexyl glutathione column
in tandem with a glutathione-linked aænity column as described previously (Bolton and Ahokas 1997).
Elution of glutathione transferases from the S-hexyl glutathione column was achieved using two elution
±
buåers; 50 m Tris-HCl containing 10 m GSH, pH 7 0 followed by 50 m Tris-HCl containing
m
m
m
±
10 m GSH, pH 9 6 (Ooi et al. 1993). Two fractions of GSTs were therefore eluted, one containing
acidic and near-neutral GSTs and the other containing basic GSTs. The glutathione column was washed
m
±
with 22 m phosphate buåer, pH 7 0 and the GSTs eluted using 50 m Tris-HCl containing 5 m
GSH, pH 9 6. Puri®ed GST samples were concentrated using an Amicon 8010 ultra®ltration unit with
m
m
m
±
a YM 10 membrane (10 000 molecular weight cut-oå).
Aænity puri®ed GSTs were further fractionated by chromatofocusing using an LKB 2150 hplc
system (Sweden) ®tted with a Mono P HR 5 20 column (5¬200 mm, Pharmacia, Sweden). The column
}
±
was pre-equilibrated at room temperature with 25 m diethanolamine, pH 9 6. The ®rst elution buåer,
m
±
1 ml Pharmalyte 8±10.5 and 5 2 ml Polybuåer 96 diluted to 200 ml with water and adjusted to pH 8 with
HCl, was run on the column (5 ml) prior to sample application. The sample was diluted in the elution
m
1
buåer prior to injection. The second elution buåer, Polybuåer 74 diluted 20 times with water and
m
adjusted to pH 4 with 1 HCl, was run onto the column after the pH of the eluant was stable at pH 8.
±
The ¯ow rate during elution was 0 5 ml min. Column eçuent was continuously monitored at 280 nm
}
using a Spectro¯ow 757 Absorbance Detector (Kratos Analytical Instruments, USA) and pH was
continuously monitored using a glass combination electrode. Each fraction was measured for GST
°
m
m
activity at 25 C using substrate concentrations of 1 m CDNB and 1 m GSH. Ampholyte removal and
concentration of pooled fractions was achieved using an Amicon 8010 ultra®ltration unit with a YM 30
membrane (30 000 molecular weight cut-oå) (Ooi et al. 1993). Each fraction was washed ®ve times with
°
22 m phosphate buåer containing 1 m dithiothreitol, 1 m EDTA and 20 % glycerol, pH 7 0 (4 C).
±
m
m
m
°
Pure isoenzymes were stored at ®80 C until used.
Hplc analysis of GST subunits
Hplc analysis of brown antechinus GST subunits was performed according to the method of Ostlund
È
±
l
Farrants et al. (1987) using a Brownlee Aquapore 7 m, C reverse-phase column (220 mm¬4 6 mm,
")
Applied Biosystems, Australia). The mobile phase linear gradient (35±55% B over 60 min) of 0 05 %
±
±
(v v) tri¯uoroacetic acid in water (solvent A) and 0 05 % tri¯uoroacetic acid in acetonitrile (solvent B) was
}
employed at a ¯ow rate of 1 ml min.
}
Inhibition studies
Investigation of the inhibitory eåect of cibacron blue, SBP, S-hexyl glutathione, haematin, tributyltin
acetate, triphenyltin chloride and triethyltin bromide on Antechinus GST 1-1 was performed essentially
°
as described by Tahir et al. (1985). Assays were carried out at 37 C using substrate concentrations of
m
6 m glutathione (saturating concentration) and 1 m CDNB.
m

576
R. M. Bolton and J. T. Ahokas
Kinetic constants
GST assays were performed in quadruplicate and at 37 C with varying concentrations of CDNB
°
±
±
±
(0 1±3 0 m ) and glutathione (0 1±7 5 m ).
±
m
m
SDS-polyacrylamide gel electrophoresis
The apparent molecular weight of the puri®ed Antechinus GST isoenzyme subunits was determined
±
according to the procedure of Laemmli (1970) using SDS-PAGE in 12 % acrylamide 2 6 % bis-
}
acrylamide. After electrophoresis, protein bands were visualized using Coomassie blue staining. The
±
protein molecular weight standards used were 14 4 kDa (hen egg white lysozyme), 21 5 kDa (soyabean
±
±
trypsin inhibitor), 31 kDa (bovine carbonic anhydrase), 45 kDa (hen egg white ovalbumin), 66 2 kDa
(bovine serum albumin) and 97 4 kDa (rabbit muscle phosphorylase B) (Biorad Laboratories, USA).
±
Immunochemical methods
Antisera raised in rabbit to human alpha (GST A1-1), mu (GST M1-1) and pi (GST P1-1) GST were
a kind gift from Professor Bengt Mannervik, Department of Biochemistry, Uppsala University. Brown
antechinus GSTs were electrophoretically transferred onto nitrocellulose membrane according to the
method of Towbin et al. (1979). Upon complete protein transfer (30 V for 16 h), the membrane was
washed, blocked and the colour developed using a Biorad Immun-blot assay kit. All steps were performed
according to the manufacturer’s instructions. The nitrocellulose membrane was incubated for 1 h with
human GST antisera (diluted 2000 times). The secondary antibody employed was goat anti-rabbit IgG
alkaline phosphatase conjugate (Biorad).
Results
Puri®cation of brown antechinus glutathione transferases
Aænity chromatography yielded two fractions containing partially puri®ed
glutathione transferases. Both fractions were obtained from the S-hexyl glutathione
aænity column. Although only 81 % of the starting CDNB activity was recovered
from this column (table 1), the use of a glutathione aænity column loaded with the
wash from the S-hexyl glutathione aænity column yielded no further glutathione
transferases (measured via CDNB activity). The use of two elution buåers of
diåering pH allowed initial fractionation of GSTs into a basic and an acidic near-
}
neutral fraction. This method was particularly useful since the basic fraction was
shown to contain a single isoenzyme, termed Antechinus GST 1-1. This fraction
represented 70 % of the total cytosolic GST puri®ed (table 1) and hplc analysis
showed a single subunit peak with a retention time of 37 min.
Hplc analysis of the acidic near-neutral fraction showed the presence of four
}
subunit peaks, with retention times of 25, 26, 32 and 37 min (®gure 1). This fraction
was further puri®ed using chromatofocusing (®gure 2) to yield ®ve pure isoenzyme
peaks. These isoenzymes are named in order of their puri®cation. The ®rst peak
yielded a further small amount of Antechinus GST 1-1 and eluted in the void
volume. Antechinus GST 2-2 yielded a single subunit peak at 25 min upon hplc
analysis, and Antechinus GST 3-3 consisted of a single subunit peak at 26 min. Hplc
analysis showed that the following protein peak consisted of two subunit peaks with
retention times of 26 and 32 min. This isoenzyme is termed Antechinus GST 3-4,
and was the only heterodimer puri®ed. The ®nal isoenzyme puri®ed, Antechinus
GST 4-4, consisted of a single subunit peak at 32 min when analysed by hplc.
A summary of the puri®cation of brown antechinus glutathione transferases is
shown in table 1. Estimation of the proportion of total GST puri®ed contributed by
each isoenzyme shows that 71 % of the GST puri®ed was Antechinus GST 1-1.
Antechinus GST 2-2 and Antechinus GST 4-4 represented 12 and 10 % of the total
GST protein, with Antechinus GST 3-3 and Antechinus GST 3-4 contributing
lesser proportions of the total soluble hepatic GST content of this marsupial species.

Hepatic glutathione transferases
577

578
R. M. Bolton and J. T. Ahokas
Figure 1. Analysis of brown antechinus glutathione transferase subunits by reverse-phase hplc. An
l
aliquot (100 l) of the acidic fraction obtained after aænity chromatography of pooled male cytosol
was injected onto the hplc column (7 m, C reverse-phase) under the conditions described in the
l
")
Materials and methods section. When similar samples obtained from female animals were
analysed, a clear sex-diåerence in the GST isoenzyme pro®le was not observed.
Molecular properties
The apparent isoelectric point of individual brown antechinus GSTs was
determined as the pH of elution from the chromatofocusing column. Antechinus
GST 1-1 eluted in the void volume, indicating that its pI was greater than the pH
±
of the starting buåer (pH 9 6). A sample of this isoenzyme was chromatofocused
±
between pH 11 and 8 using PBE-118 and eluted at pH 9 7. The remaining brown
antechinus GSTs displayed a range of isoelectric points, from the basic Antechinus
±
GST 2-2 (pI 8 7), to the near-neutral Antechinus 3-3 (pI 6 9) and acidic Antechinus
±
±
3-4 and 4-4 (pI 5 7 and 5 6 respectively).
±
The subunit molecular weights of brown antechinus GSTs were determined by
SDS-PAGE (®gure 3). Antechinus GSTs 1-1, 2-2, 3-3 and 4-4 were homodimers of
subunits with M ¯ 24 800, 25 700, 25 700 and 27 200 respectively. Although there
was insuæcient Antechinus GST 3-4 protein available to run on the gel, it is
r
assumed based on hplc analysis that this isoenzyme consists of a heterodimer of two
subunits with M ¯ 25 700 and 27 200.
r
Substrate speci®cities and kinetic constants
Five male and ®ve female cytosols were randomly chosen and cytosolic substrate
activities measured (table 2). Signi®cant sex diåerences were observed in both 1-
"
±
chloro-2,4-dinitrobenzene (male female, p % 0 007) and ethacrynic acid (female
"
±
male, p % 0 004) activity. The limited sample available for study necessitated the
pooling of individual samples into sex-speci®c groups for the puri®cation of

Hepatic glutathione transferases
579
Figure 2. Separation of brown antechinus hepatic glutathione transferases using chromatofocusing.
Preliminary runs showed that the ®nal three peaks could be more easily visualized if the sensitivity
of the absorbance detector was increased after the elution of the second peak. Thus, these two parts
of the trace are shown on diåerent axes. The acidic near-neutral fraction of aænity puri®ed GSTs
±
}
m
was applied to a Mono P HR 5 20 column equilibrated with 25 m diethanolamine, pH 9 6.
}
Elution was performed using ampholine buåers at pH 8 and 4 (see experimental procedures). The
absorbance of the eluant was monitored continuously at 280 nm, fractions collected and their
CDNB activity measured. Individual GST isoenzymes were named according to their order of
puri®cation. Antechinus GST 3-4 was identi®ed as a heterodimer using hplc analysis of the
relevant fractions.
cytosolic GSTs. Hplc analysis of the pooled male and pooled female aænity puri®ed
GSTs did not indicate a clear sex diåerence in GST isoenzyme pro®le.
The substrate speci®cities of the pure antechinus GSTs are shown in table 3.
CDNB displayed broad speci®city for antechinus GSTs, although the greatest
activity was observed with Antechinus GST 1-1. EA was also a substrate for all of
the antechinus GSTs, with Antechinus GST 2-2 displaying the greatest speci®city
towards this substrate. Antechinus GST 1-1 was the only isoenzyme that displayed
activity with the substrates DCNB and cumene hydroperoxide. Sbf-Cl activity was
shown only by Antechinus GST 2-2.
Kinetic constants were determined for the three major brown antechinus GST
isoenzymes using bisubstrate (CDNB and GSH) kinetic analysis (table 4.) Similarly
to Possum GST 1-1 (Bolton and Ahokas 1997), the K for CDNB shown by
m
Antechinus GSTs 1-1 and 4-4 was greater than this substrate’s aqueous solubility,
thus speci®c activities determined under standard conditions (1 mm CDNB and
1 mm GSH) are considerably below the V . The K for glutathione shown by
max
m
Antechinus GST 4-4 was 10-fold lower than that of Antechinus GSTs 1-1 and 2-2

580
R. M. Bolton and J. T. Ahokas
Figure 3. SDS polyacrylamide-gel electrophoresis of brown antechinus glutathione transferases.
}
Antechinus GST were analysed on a 12 % SDS polyacrylamide gel and proteins were visualized
}
with Coomassie blue staining. Lane 1 contained protein molecular weight standards with M ¯
97400 (rabbit muscle phosphorylase B), 66 200 (bovine serum albumin), 45 000 (hen egg white
ovalbumin), 31 000 (bovine carbonic anhydrase), 21 500 (soyabean trypsin inhibitor) and 14 400
r
±
(hen egg white lysozyme). Lanes 2±5 contained approximately 0 1 g Antechinus GSTs 1-1, 2-2,
l
3-3 and 4-4 respectively.
Table 2. Hepatic cytosolic glutathione transferase activity in the brown antechinus.
a
Speci®c activity
l
( mol min mg protein)
}
}
Substrate
Male (n ¯ 5) Female (n ¯ 5)
b
±
±
±
±
± ±
0 84³0 09
1-Chloro-2,4-dinitrobenzene
1,2-Dichloro-4-nitrobenzene
Ethacrynic acid
1 32³0 10
± ±
0 02³0 00
0 02³0 00
c
±
±
±
±
0 03³0 00
0 06³0 01
trans-4-Phenyl-3-buten-2-one
Sulphobromophthalein
Ammonium 4-chloro-7-sulfobenzofurazan
Cumene hydroperoxide
Hydrogen peroxide
nd
nd
nd
nd
nd
nd
±
±
±
±
± ±
0 13³0 01
± ±
0 07³0 00
0 15³0 01
0 07³0 00
a
b
c
Values are shown as mean³SE.
Males signi®cantly greater than females, p % 0 007.
Females signi®cantly greater than males, p % 0 004.
±
±
nd, Activity not detected.
indicating diåerences in the catalytic eæciency and GSH-binding site between these
%
brown antechinus GST isoenzymes. The ratio of k to K was in the order of 10
cat
m
for all of the antechinus GSTs investigated, indicating that these isoenzymes are
eæcient in their catalysis of this substrate.
Inhibitor sensitivity
The sensitivity of Antechinus GST 1-1 to a range of inhibitors is shown in table
5. Activity of Antechinus GST 1-1 toward the substrate CDNB was extensively
inhibited by tributyltin acetate, haematin and triphenyltin chloride. Intermediate
inhibition was observed with triethyltin-bromide and cibacron blue whereas the
binding aænity of this GST for SBP and S-hexyl glutathione was low.

Hepatic glutathione transferases
581

582
R. M. Bolton and J. T. Ahokas
Table 5. Inhibition characteristics of brown antechinus glutathione transferase 1-1.
a
lm
)
Inhibitor
I
(
&!
±
2 10
Cibacron blue
±
0 22
Tributyltin acetate
Triethyltin bromide
Triphenyltin chloride
Sulphobromophthalein
Haematin
±
1 23
±
0 80
±
7 93
±
0 77
±
19 30
S-hexylglutathione
a
The I is the concentration of inhibitor giving 50 % inhibition of the enzyme activity assayed at
°
&!
pH 6 5 and 37 C, with 1 m CDNB and 6 m glutathione as substrates.
±
m
m
Figure 4. Immunological relationships of brown antechinus glutathione transferases. Antechinus
GSTs 1-1, 2-2 and 4-4 were investigated for immuno-cross reactivity with antisera raised against
human alpha, mu and pi GSTs using Western blotting. Lanes 1±3, 4±6 and 7±9 were treated with
antisera raised against human pi, mu and alpha GST respectively. Lanes 1, 4 and 7 contained
±
±
0 1 g Antechinus GST 1-1. Lanes 2, 5 and 8 contained 0 1 g Antechinus GST 2-2. Lanes 3, 6
l
l
±
and 9 contained 0 1 g Antechinus GST 4-4. Lane 10 contained prestained molecular weight
standards (Novex).
l
Immunochemical analysis
Immuno-cross reactivity of Antechinus GSTs 1-1, 2-2 and 4-4 with antisera
raised against human alpha, mu and pi GST was investigated using Western
blotting techniques (®gure 4). Antechinus GST 1-1 showed immuno-cross
reactivity with antisera raised against human alpha GST, whereas Antechinus GST
2-2 and 4-4 displayed immuno-cross reactivity with antisera raised against human pi
and mu GSTs, respectively.
Discussion
Five GST isoenzymes were detected in hepatic cytosol from adult male and
female brown antechinus. All forms were puri®ed to apparent homogeneity using a
combination of aænity chromatography and chromatofocusing.
Antechinus GST 1-1 represented 71 % of the total GST protein puri®ed (table
1) and was characterized by a combination of catalytic and structural properties.

Hepatic glutathione transferases
583
Comparison of these characteristics with those of GSTs of known class puri®ed
from other mammalian species indicates that Antechinus GST 1-1 belongs to the
alpha GST family. Similarly to Possum GST 1-1 (Bolton and Ahokas 1997) and
other members of the alpha class of GSTs, Antechinus GST 1-1 displayed a highly
basic apparent isoelectric point, an intermediate molecular weight, substrate
speci®city towards an organic hydroperoxide (cumene hydroperoxide) (table 3),
high aænity towards haematin and low aænity toward cibacron blue (table 5)
(Mannervik et al. 1985, Mannervik and Danielson 1988).
The classi®cation of Antechinus GST 1-1 as an alpha class GST was con®rmed
by investigation of structural characteristics of the enzyme. Antechinus GST 1-1
cross-reacted with antisera raised against human alpha GSTs and showed no
reaction with antisera raised against human mu and pi GSTs (®gure 4).
Four minor GST isoenzymes were puri®ed from brown antechinus hepatic
cytosol (table 1). These isoenzymes were characterized by catalytic and structural
properties and their classi®cation was achieved using immunological cross-reactivity
with antisera raised against human alpha, mu and pi GST’s. Antechinus GST 2-2
displayed cross-reactivity with antisera raised against human pi GST, whereas
Antechinus GST 4-4 showed an immunological cross-reaction with antisera raised
against human mu GST (®gure 4). Insuæcient samples were available to investigate
the immunological properties of Antechinus GST 3-3 and 3-4. Dimeric structures
with combinations of subunits from diåerent GST families have not been observed
in other mammalian species (Mannervik and Danielson 1988). Since Antechinus
GST 4-4 is a mu class GST, and Antechinus subunit 4 forms a heterodimer with
Antechinus subunit 3, it is assumed that both Antechinus GST 3-3 and 3-4 also
belong to the mu GST family. Thus, brown antechinus alpha class GSTs were
represented by a single isoenzyme that contributed 71 % of the total hepatic soluble
GST content. Mu class GSTs were represented by three isoenzymes and
contributed a total of 20 % of the GSTs puri®ed. Pi class GSTs were represented by
one isoenzyme that constituted 12 % of the total hepatic soluble GST content in the
brown antechinus. Clearly, the liver of the brown antechinus contains three distinct
GST classes, formed by at least four unique subunits. Assuming similarity to
®ndings in other mammals (Mannervik et al. 1985, Board et al. 1990), the distinct
proteins belonging to each GST class are most likely the products of at least three
separate genes, a speculation that remains to be con®rmed.
Like the brushtail possum (Bolton and Ahokas 1997), the brown antechinus
expresses an alpha class GST as the predominant hepatic isoenzyme. In contrast to
the possum, the brown antechinus also expresses isoenzymes belonging to both the
mu and pi GST class, although these isoenzymes are relatively minor in their
contribution to the overall soluble hepatic GST content. In comparison with the
eutherian mammals, the marsupials studied clearly express a diåerent GST
isoenzyme pro®le, in which a single (alpha) GST isoenzyme predominates.
However, when the isoenzyme pattern is compared in terms of GST classes, a
similar pro®le is observed in man and the brown antechinus, in which alpha GSTs
predominate (71 and 75 % for antechinus and man respectively), mu GSTs are
intermediate (20 and 30 % respectively) and pi GSTs are a minor contributor (12
and 4 % respectively) (van Ommen et al. 1990). In contrast, the rat pro®le shows that
alpha and mu GSTs are both major representatives (37 and 54 % of total hepatic
GST respectively) (Jernstrom et al. 1988).
The evolutionary conservation of an isoenzyme pattern in which a dominant

584
R. M. Bolton and J. T. Ahokas
hepatic alpha GST isoenzyme is expressed suggests that alpha GSTs have a
functional role important to the successful survival of these marsupials. Both
Antechinus GST 1-1 and Possum GST 1-1 display speci®city towards organic
hydroperoxide and it has been suggested that this activity is important for the
detoxication of plant allelochemicals and their oxidised metabolites (Bolton and
Ahokas 1997). Antechinus GST 1-1 lacks the inorganic hydroperoxide activity
displayed by Possum GST 1-1, a ®nding that may result from the diåerent diets of
these marsupials. The brown antechinus does not feed directly on Eucalyptus foliage,
but on insects which may have ingested eucalypt leaves (Fox and Archer 1984).
Therefore in the brown antechinus, the need for detoxication of plant allelochemicals
is concomitant to the need to detoxify xenobiotics found in the insect diet, and may
thus result in expression of members of the mu and pi GST families. Alternatively,
the expression of GST isoenzymes in these species may be in¯uenced by presently
unknown endogenous functions of these enzymes. Wildlife permit restrictions
resulted in an unavoidable period (up to 4 days) when the animals were fed an
unnatural diet and this may also have aåected the GST expression pro®le in this
species.
Previously, it had been postulated that the expression of a single predominant
hepatic GST by the brushtail possum may result in a limited ability to detoxify a
wide range of xenobiotics. The brown antechinus relies on its predominant hepatic
GST to a lesser extent and expresses isoenzymes from three diåerent GST classes.
Thus, these ®ndings indicate that the brown antechinus may have a greater adaptive
ability for metabolism of environmental chemicals in comparison to the brushtail
possum. Further investigation of detoxication enzymes in marsupials will thus
contribute to environmental management in Australia.
Sex diåerences in GST isoenzyme pro®le were indicated by signi®cant
diåerences in cytosolic substrate activities for CDNB and ethacrynic acid (table 2).
Since all brown antechinus GST isoenzymes displayed signi®cant but varying
speci®city towards CDNB, the greater cytosolic activity with this substrate observed
in male brown antechinus may represent an increased expression of one or more
GST isoenzymes. These animals were trapped at around the time of the start of the
annual breeding season. In males of the species, this period coincides with an
increase in free corticosteroid concentration in the plasma (Lee et al. 1977).
Glucocorticoids have been shown to transcriptionally up-regulate GST expression
in the mouse (Rushmore et al. 1993, Rushmore and Pickett 1993). Sex-dependent
regulation of GST isoenzyme expression has been observed in several mammalian
species (Igarashi and Satoh 1989) and is thought to be in¯uenced by a range of
hormones (Srivastava and Waxman 1993). The mechanisms that regulate GST
isoenzyme expression in marsupials may include both endogenous and exogenous
in¯uences, and remain to be elucidated. Importantly, sex-speci®c expression of
xenobiotic metabolising enzymes may be of particular signi®cance in a species, such
as the brown antechinus, in which all males die at the end of the annual breeding
season and GST expression during this period in the life history of the brown
antechinus is worthy of further investigation.
In summary, ®ve GST isoenzymes representing the alpha, mu and pi classes
were puri®ed from the hepatic cytosol of an insectivorous marsupial, the brown
antechinus. The predominance of an alpha GST, similarly to that observed in an
herbivorous marsupial, the brushtail possum, suggests an important functional role
for these GSTs which is yet to be elucidated.

Hepatic glutathione transferases
585
Acknow ledgem ents
R. M. B. is the recipient of a John Storey Junior Memorial Scholarship (1991)
and an Australian Postgraduate Research Award (1992±4).
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