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Isoenzymes of glutathione S-transferase from the mosquito
Anopheles dirus
species B: the purification, partial characterization
and interaction with various insecticides
L. Prapanthadara
a,*, N. Promtet
a, S. Koottathep
a, P. Somboon
b, A.J. Ketterman
caResearch Institute for Health Sciences, Chiangmai University, Chiangmai 50200, Thailand bDepartment of Parasitology, Faculty of Medicine, Chiangmai University, Chiangmai 50200, Thailand
cInstitute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Thailand
Received 8 June 1999; received in revised form 30 December 1999; accepted 10 January 2000
Abstract
Previously we have purified and characterized a major glutathione S-transferase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a model mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koottathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996)Insect Biochem. Mol. Biol.26:3, 277–285]. In this report we have purified an isoenzyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purified and characterized for comparison. All of theAnophelesGST isoenzymes preferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the Vmax of GST-4c was 24.38
µmole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different Kms for CDNB and glutathione.
Despite being only partially pure GST-4b had nearly a four-fold greater Vmaxfor CDNB than GST-4c. In contrast, GST-4c possessed
the greatest DDT-dehydrochlorinase specific activity among the purified insect GST isoenzymes and no activity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of inhibiting CDNB conjugating activity to different extents for the different isoenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inhibitors. The inhibition studies demonstrate different degrees of interaction of the An. dirus isoenzymes with various insecticides. The GSTs were inhibited more readily by organochlorines and pyrethroids than by the phosphorothioates and carbamate. In a comparison between An. dirus and previous data from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of three GST classes in anopheline insects. 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Glutathione S-transferase; GST; Mosquito;Anopheles dirus
1. Introduction
Glutathione S-transferases (GSTs; EC 2.5.1.18) belong to a multigene family of dimeric multifunctional proteins that play a central role in detoxication of xeno-biotic compounds including drugs, herbicides and insec-ticides (Mannervik, 1985; Hayes and Wolf, 1988; Pickett and Lu, 1989; Tsuchida and Sato, 1992; Daniel, 1993; Hayes and Pulford, 1995). The enzymes catalyze the attack of ionized glutathione (GS2) on electrophilic
cen-* Corresponding author. Tel.:+66-53-221849; fax:+66-53-221966.
E-mail address:[email protected] (L. Prapanthadara).
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ters of lipophilic compounds. In mammals, the resulting conjugates can then be either excreted in bile or further metabolized to mercapturic acid for urinary excretion (Habig et al., 1974). The GSTs are involved in metab-olism of organophosphorus and organochlorine insecti-cides (Clark et al., 1984; Clark and Drake, 1984; Clark and Shamaan, 1984; Hayes and Wolf, 1988; Lamoureux and Rusness, 1989).
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 inAedes 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 forAn.
gam-biaedue to unsuccessful purification. UsingAn. dirusB 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-nitrophenoxy)propane, 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-dardp,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
at 10,000gfor 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 (Mr
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 asµmole 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% (v/v). 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 IC50 was performed for
GST-4c with Cibacron Blue 3GA, bromosulfophthalein and ethacrynic acid by varying the inhibitor concentrations. The IC50 were calculated by producing a competitive
binding curve using GraphPad PRISM Version 2.01 software.
2.7. Kinetic studies
varied from 0.025–2 mM. The Km and V were
determ-ined by non-linear regression analysis (Leatherbarrow, 1992).
3. Results
3.1. Purification of GST isoenzymes from An. dirus
The sequential column chromatography described pre-viously (Prapanthadara et al. 1993, 1996) was modified and employed to isolate 5 GST isoenzymes from An. dirusB (Scheme 1). GST 4a, GST-4b and GST-4c were the isoenzymes retained on the affinity column whereas GST-5 and GST-6 were isolated from the unbound frac-tion. The GST 4a had previously been purified to hom-ogeneity and partially characterized (Prapanthadara et al., 1996). In this paper GST-4c was purified whereas GST-4b, GST-5 and GST-6 were partially purified.
Scheme 1. Schematic diagram for isolation of GSTs fromAn. dirusB larvae.
Table 1 and Fig. 1 show purification factors and purity of theAn. dirusGST isoenzymes. GST-4b and GST-4c were purified to 266.6- and 67.1-fold, respectively, after the hydroxylapatite column. GST-4c was purified to homogeneity after hydroxylapatite chromatography. Further purification of GST-4b on the phenyl Sepharose column was not successful because the enzyme did not bind to the column even though very high concentrations of phosphate buffers (0.3 M sodium phosphate and 2 M sodium chloride) were used. This characteristic suggests few hydrophobic residues are exposed on the enzyme. SDS–PAGE of GST-4b shows four major bands of which one of them has the same molecular size as GST-4a. GST-4c contains a single band with the same relative mobility as GST-4a.
respect-Table 1
Purification table of GSTs isoenzymes from 4thinstar ofAn. dirusBa
Fractions Protein (mg) GST Specific % Yield Fold
activity(µmol/min) activity(µmol/min/mg)
Crude supernatant 958.72 308.99 0.322 100 1
Q-Sepharose column
Peak I 353.80 12.88 0.036 4.2 –
Peak II 97.55 168.67 1.73 54.6 5.37
S-hexylglutathione column
Peak III 95.55 18.83 0.20 6.10 –
Peak IV 1.24 57.78 46.74 18.7 145.2
Hydroxylapatite column
GST-4a ND 60.41 ND 19.6 ND
GST-4b 0.033 4.08 85.86 1.3 266.6
GST 4c 0.167 3.62 21.62 1.2 67.1
GST-5 9.80 4.90 0.50 1.55 1.55
GST-6 11.66 7.20 0.62 2.3 1.92
Phenyl sepharose column
GST-4a 0.302 55.13 149.08 17.8 462.6
GST-5 0.089 2.10 23.51 0.7 73.0
GST-6 1.74 3.41 1.96 1.1 6.1
aThe purification was performed as explained in Materials and Methods using 30 g of fourth instar larvae as the starting material.
Fig. 1. SDS–PAGE on 15% polyacrylamide minislab gel. Lane 1 is standard molecular weight marker, lane 2=10µg GST-4a, lane 3=10µg GST-4b and lane 4=10µg GST-4c.
ively. The final purifications were 73-fold for GST-5 and 6.1-fold for GST-6. This result demonstrates less diver-sity of the isoenzymes in An. dirus compared to An. gambiae reported earlier (Prapanthadara et al., 1993). Interaction of An. dirusGST-5 and GST-6 with phenyl Sepharose column was also different from the similar isoenzymes from An. gambiae. GST-5 was eluted only in an extremely non-polar condition (30% ethylene gly-col in Tris–HCl buffer) whereas GST-5a and GST-5b from An. gambiaewere eluted by using a reverse gradi-ent of 300–10 mM sodium phosphate buffer. In contrast, GST-6 from An. dirus possesses a higher polarity than GST-6a and GST-6b from An. gambiae such that GST 6 was eluted from the column when 0.3 M sodium phos-phate buffer without sodium chloride was applied.
3.2. Kinetic parameters
The steady state kinetic studies were performed on GST-4b and GST-4c with CDNB and GSH as substrates. The kinetic parameters are presented in Table 2 with GST-4a from the previous study for comparison. The kcatvalue was not calculated for GST-4b because of the
presence of impurities. However, the present data sug-gests that 4b is most similar to 4a. For GST-4a and GST-4c, the Kms for CDNB and GSH are
sig-nificantly different. The lower kcatand the kcat/Km
indi-cate lower substrate specificity for GSH conjugation with CDNB for GST-4c compared to GST-4a.
3.3. DDTase activity
The DDTase activity for each isolated isoenzyme was determined and the data is presented in Table 3. Every
Table 2
Summary of the kinetic constants forAn. dirus B GST-4a, GST-4b and GST-4c with GSH and CDNB as substratesa
Constant GST-4* GST-4b GST 4c
Vm 179.75±5.64 92.89±8.56 24.38±1.84 (µmole/min/mg)
Km(GSH)(mM) 0.87±0.10 0.97±0.51 0.30±0.07
Km(CDNB)(mM) 0.21±0.02 0.23±0.09 0.10±0.01
kcat(s-1) 149.85 ND 18.69
kcat/Km(GSH) 172.24 ND 62.30
(mM-1s-1)
kcat/Km(CDNB) 713.57 ND 186.9
(mM-1s-1)
Table 3
DDT-dehydrochlorinase (nmole DDE formation) and GST (unit=µmol/min) CDNB activities of GST isoenzymes from fourth instar larvae ofAn. dirusBa
Isoenzymes DDTase GST DDTase/GST
nmol/mg nmol/g Units/g (nmol/unit) protein larvae larvae
GST 4a 15.80 0.27 2.52 0.11±0.02 GST 4b 90.93 0.18 0.17 1.06±0.74 GST 4c 1,308.74 9.08 0.15 60.53±14.55 GST 5 ,0.71 ,0.01 0.20 ,0.03 GST 6 272.83 41.76 0.30 139.20±48.37
aIsoenzymes are named as indicated in Scheme 1 and Table 1. The
numbers were means from at least four experiments and calculated based on the purification yield indicated in the purification table (Table 1).
isoenzyme except GST-5 possessed DDT-dehydrochlor-inase activity. These activities are not correlated with CDNB conjugating activities. For example, the majority of GST CDNB-conjugating activity is found in GST-4a, whereas the majority of DDT-dehydrochlorinase activity is found in GST-6. The GST-6 also possesses the highest relative DDT-dehydrochlorinase/GST activity (139.20±48.37 nmole DDE formation per unit GST). However, GST-4c has the greatest DDTase specific activity. In a comparison between the two purified isoen-zymes, GST-4c has 83-fold greater DDT-dehydrochlori-nase specific activity than GST-4a.
3.4. Substrate specificity
Nine GST general substrates were tested for utiliz-ation by the isolated isoenzymes. Those were CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), cumene hydroperoxide, 4-nitropyridine-N-oxide, p-nitrophenethyl bromide, 1,2-epoxy-(3-p -nitrophenoxy)propane, bromosulfophthalein, trans -4-phenyl-3-buten-2-one and ethacrynic acid. GST-4a and GST-6 showed activity with DCNB (0.60±0.02 and 0.88±0.04 µmole/min/mg, respectively) whereas the other isoenzymes did not use this substrate. Of the remaining substrates tested only CDNB was used by these anopheline GST isoenzymes.
3.5. Inhibition of CDNB conjugating activity by various GST general substrates
It has been documented that many GST substrates are inhibitors to standard CDNB conjugating activity of many GST isoenzymes (Mannervik and Danielson, 1988). In this paper, a simple inhibition study was per-formed to determine the interaction between the enzymes and those general substrates. The percent
inhi-bitions are presented in Table 4. All the isoenzymes were inhibited to different extents. 1,2-epoxy-3-(p -nitrophenoxy)propane andtrans-4-phenyl-3-buten-2-one have the least effect on the CDNB conjugating activity. The first compound has been identified as a specific sub-strate for the vertebrate class Theta GST (Meyer et al., 1991). The lack of activity and the inhibitory effect indi-cate that insect GSTs although they may possess some sequence similarity to the Theta class (Wilce et al., 1995) they are not very closely related. Mammalian Theta GSTs also do not have activity with CDNB. This result suggests that a distinct classification of GST isoenzymes in insects should be applied.
3.6. IC50 study
IC50s were determined for three putative GST
inhibi-tors to study the homogenous property of GST-4c. The typical IC50plots (Fig. 2) are symmetrical for all
inhibi-tors studied with a maximum slope of20.58 at the point of inflexion. This result indicates the inhibition charac-teristic of a homodimeric enzyme (Tahir and Mannervik, 1986; Mannervik and Danielson, 1988). The IC50values
were 0.002±0.001, 0.001±0.001 and 1.58±0.50 µM for Cibacron Blue 3GA, bromosulfophthalein and ethac-rynic, respectively. These numbers are 10–102-fold
dif-ferent from the values determined for GST-4a.
3.7. Inhibition of CDNB conjugating activity by various insecticides
An indirect measurement by a simple inhibition study was performed to demonstrate interaction of various insecticides with GST. Table 5 presents the percent inhi-bition of An. dirusGST isoenzymes by a fixed concen-tration of each insecticide. The high percent inhibition demonstrates a strong interaction between the enzymes and the insecticides. Most of the insecticides gave a strong interaction except gamma-HCH, diazinon and bendiocarb. A large difference between peak 3 and peak 4 isoenzymes is that peak 3 GSTs show fewer interac-tions with all insecticides tested. It is obvious that GST-6 is the most different from all other isoenzymes in that its CDNB conjugating activity was not inhibited by any insecticide. This is in contrast to the result that GST-6 possesses the majority of DDTase activity in this mos-quito species. In terms of kinetic properties, non-inhibi-tory effects of DDT to CDNB conjugating activity may be due to a very low affinity (high Km) of the enzyme
for DDT when compared to CDNB.
4. Discussion
Table 4
Inhibition ofAn. dirusB GSTs CDNB conjugating activity with various GST general substratesa
Substrates % inhibition
mM GST4a GST4b GST4c GST5 GST6
4-NitropyridineN-oxide 0.19 7.35 5.74 13.20 25.18 2.17
p-Nitrophenethyl bromide 0.09 22.84 18.20 42.11 11.08 0
1,2-Epoxy-3-(p-nitrophenoxy)propane 4.76 0 0 12.44 8.99 0
Bromosulfophthalein 0.01 95.1 92.00 84.80 93.24 87.02
trans-4-phenyl-3-buten-2-one 0.05 0 0 0 10.17 16.34
Ethacrynic acid 0.19 99.74 100 100 100 95.02
1,2-Dichloro-4-nitrobenzene 0.95 11.48 6.61 32.92 14.31 0
aThe interaction was measured as a percentage of inhibition by the substrates on CDNB conjugating activity. Each number is the mean of at
least four separate experiments.
Fig. 2. IC50plots of GST-4C inhibition of CDNB conjugating activity
by various inhibitors.
difficulties in isolating individual isoforms from the large variety present. In this study we have isolated vari-ous GST isoenzymes fromAn. dirusB, a Thai mosquito which is used as a model for anopheline malaria vectors. Previously we were able to purify and characterize the first GST isoenzyme, GST-4a (Prapanthadara et al., 1996). In this report we have purified an additional iso-enzyme, GST-4c, and several other isoenzymes were partially purified.
GST-4c possesses the highest DDTase specific activity among all known insect GST isoenzymes. It has been fractionated from the previously reported GST-4b (Prapanthadara et al., 1996). GST-4c is distinctly differ-ent from GST-4b as shown by DDTase activity (Table 3). SDS–PAGE and the IC50plots demonstrate the purity
of GST-4c. The relative molecular weight of the subunit is comparable to GST-4a, which is about 23 kD. The purification yield for this isoenzyme expressed as CDNB conjugating activity was 1.2% and it contributes 17.7% of the total DDTase activity. Although some kinetic properties such as Kmfor CDNB of GST-4c is
compara-ble to GST-4a, the catalytic efficiency for glutathione
Table 5
Inhibition of CDNB conjugating activity by various insecticides with GST isoenzymes fromAn. dirusBa
% inhibition
Insecticides GST 4a GST 4b GST 4c GST 5 GST 6
Organochlorines
DDT 95.6 70.9 62.1 6.1 0
DDE 89.8 66.8 49.0 38.7 0
Gamma-HCH 0 0 27.3 0 0
Pyrethroids
Permethrin 100 60.8 62.4 7.7 0
Lambda 100 76.4 69.1 0 0
cyhalothrin
Deltamethrin 100 83.3 77.7 20.0 0 Phosphorothioates
Diazinon 24.7 0 14.9 0 0
Fenitrothion 51.8 47.9 44.9 37.3 0 Pirimiphos- 34.4 25.0 20.4 13.5 0 methyl
Chlorpyriphos-80.2 46.0 41.7 9.3 0 ethyl
Temephos 100 76.9 68.2 46.7 0 Carbamate
Bendiocarb 12.9 0 19.7 2.4 0
a Final concentrations of all insecticides were 0.1 mM except for
malathion and diazinon, which was 1.0 mM. The GSH and CDNB concentrations were 10.0 and 1.0 mM, respectively. The data were the mean of at least four separate experiments, each of which was perfor-med in duplicate.
conjugation with CDNB is three-fold greater for 4a than for 4c. CDNB conjugating activity of GST-4a is 7.4-fold greater than GST-4c whereas DDTase activity of GST-4c is 83-fold greater than GST-4a. The IC50studies with three putative GST inhibitors also
The involvement of GSTs in resistance to insecticides other than DDT has been reported in houseflies (Clark et al., 1986). Their role as a secondary resistance mech-anism in detoxication of the oxon analog of fenitrothion was reported inAn. subpictus(Hemingway et al., 1991). In a previous study of recombinant class I GSTs from both An. dirus and An. gambiae the isoenzymes were observed to strongly interact with various insecticides ranging from organophosphorus compounds and pyr-ethroids to organochlorine insecticides (Prapanthadara et al., 1998). To determine whether the wild-type enzymes from An. dirus were able to interact with insecticides, the ability of the insecticide to inhibit the CDNB conju-gating activity was determined. Every peak 4 GST and also GST-5 interacted with most insecticides tested although to different extents, with GST-4a being the most interactive. In contrast, GST-6 with the highest DDTase activity did not interact with any insecticide tested. Although the Km was not determined for DDT
for any isoenzyme, for GST-6 the Km is must be very
high relative to the Km for CDNB.
Biochemical characterization of the five isolated iso-enzymes from An. dirus compared with the previously isolated seven isoenzymes from An. gambiae
(Prapanthadara et al., 1996) reveal differences. The elu-tion profiles ofAn. dirusGST-5 and GST-6 are different from the profiles ofAn. gambiaeenzymes. TheAn. dirus
5 is more hydrophobic than either 5a or GST-5b from An. gambiae. The elution of An. dirus GST-5 from the phenyl Sepharose column required 30% ethyl-ene glycol. However GST-6 possesses less hydro-phobicity than GST-5 and was eluted from the phenyl Sepharose column with the same conditions as GST-6b from An. gambiae.
Although the total DDTase activity fromAn. gambiae
(Prapanthadara et al., 1993) is comparable to the activity in An. dirus in this study, the distribution of DDTase activity among the isoenzymes from the twoAnopheles
are different. The majority of DDTase activity in An. dirus is in GST-6 (81%) whereas no activity was detected in GST-5 (,0.71 nmole/mg). In An. gambiae
80% of total DDTase activity was in GST-5a. Peak 4 GSTs in An. gambiae contained no detectable DDTase activity whereas all three isoenzymes from An. dirus
peak 4 showed DDTase activity and GST-4c possessed 18% of the total activity. This result suggests that although the two anopheline species possess a similar pattern of GST isoenzymes as shown by elution from sequential column chromatography, each of the isoen-zymes may be very different at the functional level.
The biochemical studies of the wild-type GSTs from theAnophelesmosquitoes suggested two classes of GST occurred. In class I, GST-4a, GST-4b and GST-4c, are the enzymes retained on a S-hexylglutathione affinity column. They possess a relatively high CDNB conjugat-ing activity but low DDTase activity. Class II are those
proteins that are unbound on the affinity column and which are fractionated into peak 5 and peak 6. Two classes of GSTs have also been reported for housefly andDrosophila(Fournier et al., 1992). The class I from both Anopheles species and the flies have proven to be the same class based on the nucleotide sequence hom-ology (Ranson et al., 1997; Prapanthadara et al., 1998). However An. dirusGST-4a has an N-terminal sequence distinct from the known class I sequences. Yet this N-terminus is identical to Aedes aegyptiGST-2 (Grant et al., 1991). It is therefore not yet confirmed how many subclasses or distinct classes of GST isoenzymes exist in the observationally defined class I.
In some studies it has been suggested that insect GSTs belong to Theta class. These studies include the GSTs from housefly,Drosophilaand Australian sheep blowfly (Pemble and Taylor, 1992; Wilce et al., 1995). However the amino acid sequence identity of these insect GSTs to the vertebrate Theta class is about 40% or less. In the nomenclature for human glutathione S-transferases it was proposed that the overall amino acid sequence ident-ities between any two members within a class should be .50% (Mannervik et al., 1992). In addition, the ver-tebrate Theta class GSTs do not use CDNB as an electro-philic substrate but prefer 1,2-epoxy-3-(p -nitrophenoxy)propane and p-nitrophenethyl bromide (Meyer et al., 1991). The anopheline mosquito GSTs prefer only CDNB (this study and Prapanthadara et al., 1996). Therefore we suggest that insect GSTs should be classed differently from the mammalian Theta class. A recent phylogenetic study with available nucleic acid sequences showed two insect GST groups as distinctly different from theta (Snyder and Maddison, 1997). The available biochemical data suggests there may be a mini-mum of three GST classes in Anopheles mosquitoes. Recently the insect Theta class was renamed Delta class (Board et al., 1997).
References
Beckett, G.J., Hayes, J.D., 1993. Glutathione S-transferases: biomed-ical applications. Adv. Clin. Chem. 30, 281–380.
Board, P.G., Baker, R.T., Chelvanayagam, G., Jermiin, L.S., 1997. Zeta, a novel class of glutathione transferase in a range of species from plants to humans. Biochem. J. 328, 929–935.
Bradford, M.M., 1976. A rapid and sensitive method for the quantit-ation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248–254.
Clark, A.G., Dauterman, W.C., 1982. The characterization by affinity chromatography of glutathioneS-transferases from different strains of house fly. Pestic. Biochem. Physiol. 17, 307–314.
Clark, A.G., Dick, G.L., Martindale, S.M., Smith, J.N., 1985. Gluta-thione S-transferases from the New Zealand grass grub,Costelytra zealandica. Their isolation and characterization and the effect on their activity of endogenous factors. Insect Biochem. 15, 35–44. Clark, A.G., Drake, B., 1984. Purification and properties of glutathione
Clark, A.G., Shamaan, N.A., 1984. Evidence that DDT-dehydrochlori-nase from the house fly is a glutathione S-transferase. Pestic. Biochem. Physiol. 22, 249–261.
Clark, A.G., Shamaan, N.A., Dauterman, W.C., Hayaoka, T., 1984. Characterization of multiple glutathione transferases from the house fly Musca domestica (L). Pestic. Biochem. Physiol. 22, 51–59.
Clark, A.G., Shamaan, N.A., Sinclair, M.D., Dauterman, W.C., 1986. Insecticide metabolism by multiple glutathione S-transferases in two strains of the house flyMusca domestica(L). Pestic. Biochem. Physiol. 25, 169–175.
Cochrane, B.J., Morrissey, J.J., LeBlanc, G.A., 1987. The genetics of xenobiotic metabolism inDrosophila—IV. Purification and charac-terization of the major glutathione S-transferase. Insect Biochem. 17, 731–738.
Daniel, V., 1993. Glutathione S-transferases: gene structure and regu-lation of expression. CRC Crit. Rev. Biochem. Molec. Biol. 28, 173–207.
Fournier, D., Bride, J.-M., Poirie´, M., Berge´, J.-B., Plapp, F.W., 1992. Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecti-cides. J. Biol. Chem. 267, 1840–1845.
Grant, D.F., Dietze, E.C., Hammock, B.D., 1991. Glutathione S-trans-ferase isozymes inAedes aegypti: purification, characterization, and isozyme-specific regulation. Insect Biochem. 21, 421–433. Grant, D.F., Matsumura, F., 1989. Glutathione S-transferase 1 and 2
in susceptible and insecticide resistant Aedes aegypti. Pestic. Biochem. Physiol. 33, 132–143.
Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transfer-ases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139.
Hayes, J.D., Pulford, D.J., 1995. The glutathione S-transferase super-gene family: regulation of GST and the contribution of the isoen-zymes to cancer chemoprotection and drug resistance. CRC Crit. Rev. Biochem. Molec. Biol. 30, 445–600.
Hayes, J.D., Wolf, C.R., 1988. Role of glutathione transferase in drug resistance. In: Sies, H., Ketterer, B. (Eds.), Glutathione Conju-gation. Mechanisms and Biological Significance. Academic Press, London, pp. 315–355.
Hemingway, J., Malcolm, C.A., Kissoon, K.E., Boddington, R.G., Curtis, C.F., Hill, N., 1985. The biochemistry of insecticide resist-ance inAnopheles sacharovi: comparative studies with a range of insecticide susceptible and resistantAnophelesandCulexspecies. Pestic. Biochem. Physiol. 24, 68–76.
Hemingway, J., Miyamoto, J., Herath, P.R.J., 1991. A possible novel link between organophosphorus and DDT insecticide resistance genes inAnophelessupporting evidence from fenitrothion metab-olism studies. Pesticestic. Biochem. Physiol. 39, 49–56.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lamoureux, G.L., Rusness, D.G., 1989. The role of glutathione and
glutathione-S-transferases in pesticide metabolism, selectivity, and mode of action in plants and insects. In: Dolphin, D., Avroamovic, O., Poulson, R. (Eds.), Glutathione. Chemical, Biochemical, and Medical Aspects. John Wiley and Sons, New York, pp. 153–196. Leatherbarrow, R. J. Grafit. (Version 3.0). 1992. Staines, U.K.,
Eri-thacus Software. (GENERIC) Ref Type: Computer Program Mannervik, B., 1985. The isoenzymes of glutathione transferase. Adv.
Enzymol. Relat. Areas Mol. Biol. 57, 357–417.
Mannervik, B., Awasthi, Y.C., Board, P.G., Hayes, J.D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M.,
Pear-son, W.R., Pickett, C.B., Sato, K., Widersten, M., Wolf, C.R., 1992. Nomenclature for human glutathione transferases. Biochem. J. 282, 305–306.
Mannervik, B., A˚ lin, P., Guthenberg, C., Jensson, H., Tahir, M.K., Warholm, M., Jo¨rnvall, H., 1985. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic proper-ties. Proc. Natl. Acad. Sci. USA 82, 7202–7206.
Mannervik, B., Danielson, U.H., 1988. Glutathione transferases— structure and catalytic activity. CRC Crit. Rev. Biochem. 23, 283–337.
Meyer, D.J., Coles, B., Pemble, S.E., Gilmore, K.S., Fraser, G.M., Ketterer, B., 1991. Theta, a new class of glutathione transferases purified from rat and man. Biochem. J. 274, 409–414.
Meyer, D.J., Muimo, R., Thomas, M., Coates, D., Isaac, R.E., 1996. Purification and characterization of prostaglandin-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli. Biochem. J. 313, 223–227.
Motoyama, N., Dauterman, W.C., 1975. Interstrain comparison of glut-athione-dependent reactions in susceptible and resistant houseflies. Pestic. Biochem. Physiol. 5, 489–495.
Pemble, S.E., Taylor, J.B., 1992. An evolutionary perspective on gluta-thione transferases inferred from class-Theta glutagluta-thione transfer-ase cDNA sequences. Biochem. J. 287, 957–963.
Pickett, C.B., Lu, A.Y.H., 1989. Glutathione S-transferases: gene struc-ture, regulation, and biological function. A. Rev. Biochem. 58, 743–764.
Prapanthadara, L., Hemingway, J., Ketterman, A.J., 1993. Partial puri-fication and characterization of glutathione S-transferases involved in DDT resistance from the mosquitoAnopheles gambiae. Pestic. Biochem. Physiol. 47, 119–133.
Prapanthadara, L., Ketterman, A.J., Hemingway, J., 1995. DDT-resist-ance in Anopheles gambiaeGiles from Zanzibar Tanzania based on increased DDT-dehydrochlorinase activity of glutathione S-transferases. Bull. Entomol. Res. 85, 267–274.
Prapanthadara, L., Koottathep, S., Promtet, N., Hemingway, J., Ketter-man, A., 1996. Purification and characterization of a major gluta-thione S-transferase from the mosquito Anopheles dirus (species B). Insect Biochem. Molec. Biol. 26, 277–285.
Prapanthadara, L., Ranson, H., Somboon, P., Hemingway, J., 1998. Cloning, expression and characterization of an insect class I gluta-thione S-transferase from Anopheles dirus species B. Insect Biochem. Mol. Biol. 28, 321–329.
Ranson, H., Prapanthadara, L., Hemingway, J., 1997. Cloning and characterization of two glutathione S-transferases from DDT-resist-ant strain ofAnopheles gambiae. Biochem. J. 324, 97–102. Snyder, M.J., Maddison, D.R., 1997. Molecular phylogeny of
gluta-thione-S-transferases. DNA and Cell Biology 16, 1373–1384. Tahir, M.K., Mannervik, B., 1986. Simple inhibition studies for
dis-tinction between homodimeric and heterodimeric isoenzymes of glutathione transferase. J. Biol. Chem. 261, 1048–1051.
Toung, Y.-P.S., Hsieh, T.-S., Tu, C.-P.D., 1990. Drosophila gluta-thione S-transferase 1-1 shares a region of sequence homology with the maize glutathione S-transferase III. Proc. Natl. Acad. Sci. USA 87, 31–35.
Tsuchida, S., Sato, K., 1992. Glutathione transferases and cancer. CRC Crit. Rev. Biochem. Molec. Biol. 27, 337–384.
Wendel, A., 1981. Glutathione peroxidase. Meth. Enzymol. 77, 325– 333.
