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
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.
dirus B 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 from An. dirus B larvae.
Table 1 and Fig. 1 show purification factors and purity of the An. dirus GST 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.
Peak 5 and peak 6 from the hydroxylapatite column were further purified using a phenyl Sepharose column.
There was only one isoenzyme fractionated from each of peak 5 and peak 6, named GST-5 and GST-6, respect-
399 L. Prapanthadara et al. Insect Biochemistry and Molecular Biology 30 2000 395–403
Table 1 Purification table of GSTs isoenzymes from 4
th
instar of An. dirus B
a
Fractions Protein mg
GST Specific
Yield Fold
activity µ
molmin activity µ
molminmg 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
a
The 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 =
10 µ
g GST-4a, lane 3 =
10 µ
g GST-4b and lane 4
= 10
µ g 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. dirus GST-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. gambiae were 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 k
cat
value was not calculated for GST-4b because of the presence of impurities. However, the present data sug-
gests that GST-4b is most similar to GST-4a. For GST- 4a and GST-4c, the K
m
s for CDNB and GSH are sig- nificantly different. The lower k
cat
and the k
cat
K
m
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 for An. dirus B GST-4a, GST-4b
and GST-4c with GSH and CDNB as substrates
a
Constant GST-4
GST-4b GST 4c
Vm 179.75
± 5.64
92.89 ±
8.56 24.38
± 1.84
µ moleminmg
K
mGSH
mM 0.87
± 0.10
0.97 ±
0.51 0.30
± 0.07
K
mCDNB
mM 0.21 ±
0.02 0.23
± 0.09
0.10 ±
0.01 k
cat
s
-1
149.85 ND
18.69 k
cat
K
mGSH
172.24 ND
62.30 mM
-1
s
-1
k
cat
K
mCDNB
713.57 ND
186.9 mM
-1
s
-1 a
Data from Prapanthadara et al., 1996. ND =
not determined.
400 L. Prapanthadara et al. Insect Biochemistry and Molecular Biology 30 2000 395–403
Table 3 DDT-dehydrochlorinase
nmole DDE
formation and
GST unit
=µ molmin CDNB activities of GST isoenzymes from fourth
instar larvae of An. dirus B
a
Isoenzymes DDTase
GST DDTaseGST
nmolmg nmolg
Unitsg nmolunit
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
a
Isoenzymes 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-dehydrochlorinaseGST 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-
nitrophenoxypropane, 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 µ
moleminmg, 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-
nitrophenoxypropane and trans-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. IC
50
study IC
50
s were determined for three putative GST inhibi- tors to study the homogenous property of GST-4c. The
typical IC
50
plots Fig. 2 are symmetrical for all inhibi- tors studied with a maximum slope of 20.58 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 IC
50
values were 0.002
± 0.001, 0.001
± 0.001 and 1.58
± 0.50
µ M for
Cibacron Blue 3GA, bromosulfophthalein and ethac- rynic, respectively. These numbers are 10–10
2
-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. dirus GST 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 K
m
of the enzyme for DDT when compared to CDNB.
4. Discussion
