396 L. Prapanthadara et al. Insect Biochemistry and Molecular Biology 30 2000 395–403
show remarkable similarities in their primary sequences within their respective classes. Despite only minor
sequence differences, the functional properties of iso- forms within a class are also distinct. The heterogeneity
of substrate specificity between isoforms in any given class has significant physiologic and pathophysiologic
importance in detoxication of endogenous and exogen- ous compounds for review see Beckett and Hayes,
1993.
Heavy use of chemicals for pest control has increased the rate of insect resistance to insecticides. It has lead
to a public health problem in many countries, especially for tropical insect borne diseases. The glutathione
mediated reaction catalyzed by glutathione S-transferase is one of the important mechanisms that allow insects to
survive in a contaminated environment. Some examples of insecticides that have been recognized as substrates
for glutathione conjugation are DDT 1,1,1-trichloro- 2,2-bis[p-chlorophenyl]ethane, diazinon, fenitrothion
and parathion Lamoureux and Rusness, 1989. Several insecticide-resistant strains of housefly have been
reported to have elevated GST activity in crude extracts Motoyama and Dauterman, 1975; Clark and Dauter-
man, 1982; Clark et al., 1986. The DDT-resistant An. gambiae
has also been shown to have GST as a resist- ance mechanism Hemingway et al., 1985.
Many preliminary studies of insect GSTs reveal mul- tiple forms exist. Those include housefly Clark et al.,
1984; Fournier et al., 1992, grass grub Clark et al., 1985 and Drosophila Cochrane et al., 1987; Toung et
al., 1990. There are at least three GST isoenzymes present in mosquitoes, three in Aedes aegypti Grant and
Matsumura, 1989; Grant et al., 1991 and seven in An. gambiae
Prapanthadara et al., 1993. Different forms of GST exhibited varying specificities for the insecticides
studied. In a DDT-resistant strain compared with a sus- ceptible strain of the African mosquito An. gambiae,
there was an increased synthesis of different isoenzymes of GSTs that possessed a greater DDT dehydrochlorin-
ase activity Prapanthadara et al. 1993, 1995. Observed differences in the GSTs from the two strains demon-
strated that expression of the enzymes is influenced by environmental factors such that qualitatively distinct
forms can be selected at the genetic level and differen- tially expressed.
The GSTs from An. gambiae were fractionated into seven isoenzymes using sequential column chromato-
graphy Prapanthadara et al., 1993. These seven enzymes were divided into two groups according to elu-
tion properties shown on a S-hexylglutathione affinity column. A comparison study in DDT-resistant and sus-
ceptible strains of An. gambiae demonstrated that there was an eight-fold increase in DDT-dehydrochlorinase
activity in the resistant insects as a result of increased activity in every isoenzyme. Kinetic characterization of
the isolated GST isoenzymes was restricted for An. gam- biae
due to unsuccessful purification. Using An. dirus B as a model anopheline, less diversity was shown and
GST-4a was purified to homogeneity Prapanthadara et al., 1996. In this report we have continued to isolate
GST isoenzymes from An. dirus B and have partially characterized them with various substrates and insecti-
cides. One isoenzyme from the peak four GSTs, GST- 4c, has been purified to homogeneity. This isoenzyme
possesses the highest specific activity for DDT in this species.
2. Material and methods
2.1. Chemicals Trizma base, dithiothreitol DTT, 1-chloro-2,4-
dinitrobenzene CDNB, ethacrynic acid, cumene hydro- peroxide, bromosulfophthalein, 4-nitropyridine-N-oxide,
1,2-epoxy-3-p-nitrophenoxypropane, nicotinamide
adenine dinucleotide
phosphate reduced
form NADPH, glutathione reductase, glutathione GSH, S-
hexylglutathione, and S-hexylglutathione agarose were from Sigma Chemical Co. St Louis, MO, USA. 1,2-
dichloro-4-nitrobenzene DCNB,
trans -4-phenyl-3-
buten-2-one, and
p -nitrophenethyl
bromide were
obtained from Aldrich Chemical Co. Milwaukee, WI, USA. Q-Sepharose and phenyl Sepharose were pur-
chased from Pharmacia LKB Uppsala, Sweden. Hydroxylapatite and protein assay reagent were pur-
chased from Bio-Rad Richmond, CA. High purity stan- dard p,p-DDT 98.5, p,p-DDE 1,1,1-trichloro-2,2-[p-
chlorophenyl]ethylene 99, dicofol and other insecti- cides
were purchased
from British
Greyhound Birkenhead, Merseyside, UK.
2.2. Mosquitoes An established laboratory colony of mosquito Anoph-
eles dirus species B at the Department of Parasitology,
Faculty of Medicine, Chiangmai University was used. Species B had been confirmed by both morphological
and chromosomal properties. The starting material for the purification protocol was fourth instar. These were
snap-frozen in liquid nitrogen and stored at 270
° C
until used. 2.3. Purification of An. dirus glutathione S-
transferases Unless otherwise stated, all extractions and procedures
in the purification scheme were performed at 4 °
C and all buffers contained 10 mM DTT. The larvae 30 g
were homogenized in 150 ml of 25 mM Tris–HCl, pH 7.4 buffer A using a glass homogenizer with a motor-
driven Teflon pestle. The homogenate was centrifuged
397 L. Prapanthadara et al. Insect Biochemistry and Molecular Biology 30 2000 395–403
at 10,000 g for 20 min and the resultant supernatant was filtered through Whatman No. 1 filter paper using Swin-
nex Disc Filter Holders Millipore to remove floating lipids. The supernatant was applied to a Q-Sepharose
column 60 ml, 4.4
× 4 cm, equilibrated with buffer A.
The column was washed with five bed volumes of this buffer and a linear gradient of 150 ml of buffer A con-
taining 0–0.30 M NaCl was applied. GST activity detected in the wash-through fraction was designated
peak 1 and GST activity eluted from this column was designated peak 2.
Peak 2 was applied directly to a S-hexylglutathione agarose column 25 ml, 2.2
× 6.6 cm equilibrated with
buffer A. The column was washed with buffer A con- taining 0.2 M NaCl until no protein appeared in the elu-
ate. Bound proteins were eluted with 100 ml of 5 mM S- hexylglutathione in washing buffer. Two peaks of GST
activity were recovered from this column, designated peak 3 unbound fraction and peak 4 bound fraction.
Peak 3 and peak 4 from the S-hexylglutathione col- umn were concentrated and further purified separately,
using an hydroxylapatite column at room temperature 26
° C. Peak 3 was applied to a 20 ml 2.2
× 5.3 cm
column whereas a 10 ml column was used for peak 4. Starting buffer was 10 mM phosphate buffer pH 6.5
buffer B containing 0.2 M NaCl. After washing with buffer B until no protein was detected in the eluate, the
bound proteins were eluted, first with four column vol- umes of buffer B containing no NaCl, second with 10
column volumes of a linear gradient of 10–200 mM phosphate buffer pH 6.5. In total, five GST activity
peaks were resolved, with GST 4a, GST 4b and 4c being from peak 4 and GST 5 as well as GST 6 originating
from peak 3.
GST 4a was further purified as previously described Prapanthadara et al., 1996. GST 5 and GST 6 from the
hydroxylapatite column were diluted with 2 volumes of 0.3 M phosphate buffer, pH 6.5, containing 2 M NaCl.
The dilution buffer was used previously to equilibrate a phenyl Sepharose column 10 ml; 1.6
× 4 cm. Diluted
enzyme was applied to the phenyl Sepharose column at room temperature and the column was washed with three
bed volumes of equilibration buffer. Bound proteins were step eluted with 30 ml 0.3 M sodium phosphate
buffer pH 6.5, then a 60 ml gradient of 0.3–0.01 M sodium phosphate buffer pH 6.5, followed by 40 ml 25
mM Tris–HCl buffer pH 7.4 containing 30 ethylene glycol.
The GST activity of peak 5 was fractionated from other contaminating proteins and eluted out at the last
step. Step gradient elution of GST-6 was the same as for GST-5 but the GST activity was eluted in the first step.
SDS–PAGE to detect homogeneity of the isolated iso- enzymes was performed with standard proteins M
r
14.2–66 kD using a 15 resolving gel and a 4.5 stacking gel, Laemmli, 1970. Coomassie Blue R250
was used to stain for protein. 2.4. Determination of enzyme activity
The methods for determination of glutathione S-trans- ferase activity with CDNB as well as DCNB were modi-
fied as described below Habig et al., 1974. Activity with 1 mM CDNB and 10 mM GSH was measured at
340 nm in 0.1 M phosphate buffer, pH 6.5, at 22 °
C. This was the standard assay for GST activity during the
purification procedure. A unit of GST activity is defined as
µ mole CDNB-GSH conjugated product formation per
minute. The activity with DCNB was measured at 340 nm in the presence of 10 mM GSH and 1 mM DCNB.
With all other substrates, the enzyme activity was meas- ured as previously described Habig et al., 1974. Gluta-
thione peroxidase activity with cumene hydroperoxide as substrate was determined Wendel, 1981. Stock sol-
utions of GSH were prepared in buffer. The concen- tration of ethanol in the assays was kept constant at 5
vv. These ethanol concentrations did not affect the GST activity.
Protein was assayed by the method of Bradford Bradford, 1976 using the Bio-Rad protein reagent with
bovine serum albumin as the standard protein. 2.5. Determination of DDT-dehydrochlorinase activity
DDT-dehydrochlorinase or DDTase activity is the GST catalyzed DDT dehydrochlorination reaction to
yield the product DDE. This GST catalyzed dehydroch- lorination requires the presence of reduced glutathione.
The method to determine DDTase activity has been described previously Prapanthadara et al., 1996.
2.6. Inhibition study An inhibition study with various GST substrates was
performed to examine interaction with the enzymes using the standard assay conditions 10 mM GSH and 1
mM CDNB in the absence and presence of inhibitors. Inhibitor concentrations were fixed to be the same as
when the compounds are used as substrates. If it was necessary to solubilize the compounds, the final concen-
tration of ethanol used was kept constant at 5 in the assay. Determination of IC
50
was performed for GST- 4c with Cibacron Blue 3GA, bromosulfophthalein and
ethacrynic acid by varying the inhibitor concentrations. The IC
50
were calculated by producing a competitive binding curve using GraphPad PRISM Version 2.01
software. 2.7. Kinetic studies
The initial rate of the enzymatic reaction was meas- ured at 0.5–10 mM GSH with CDNB concentration
398 L. Prapanthadara et al. Insect Biochemistry and Molecular Biology 30 2000 395–403
varied from 0.025–2 mM. The K
m
and V were determ- ined by non-linear regression analysis Leatherbarrow,
1992.
3. Results
