Insect Biochemistry and Molecular Biology 30 2000 395–403 www.elsevier.comlocateibmb
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
c
a
Research Institute for Health Sciences, Chiangmai University, Chiangmai 50200, Thailand
b
Department of Parasitology, Faculty of Medicine, Chiangmai University, Chiangmai 50200, Thailand
c
Institute 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 the Anopheles GST isoenzymes preferred 1-chloro-2,4-dinitrobenzene CDNB as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V
max
of GST-4c was 24.38 µ
moleminmg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K
m
s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V
max
for 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 GS
2
on electrophilic cen-
Corresponding author. Tel.: +
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66-53-221966. E-mail address:
inhso001chiangmai.ac.th L. Prapanthadara.
0965-174800 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 1 3 - 8
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.
Extensive knowledge about GSTs is mostly from stud- ies in mammals. GST isoenzymes present in human
tissue belong to at least six major gene families, Alpha, Mu, Pi, Theta, Sigma and Zeta Mannervik et al., 1985;
Mannervik and Danielson, 1988; Meyer et al. 1991, 1996; Pemble and Taylor, 1992. These gene families
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
