substrate specificity of glutathione s-transferases from the fall armyworm

Substrate specificity of glutathione S-transferasesfrom the fall armywormq

S.J. Yu*

Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA

Received 7 June 2002; accepted 21 August 2002

Abstract

Ten cytosolic glutathione S-transferase (GST) isozymes isolated from midguts and fat bodies of control

and allelochemical-induced fall armyworm (Spodoptera frugiperda) larvae were tested for their activity

toward 13 model substrates belonging to halogenated compounds, nitro compounds, a; b-unsaturatedcarbonyl compounds, and organic hydroperoxides. Based on the pattern of activity toward these sub-

strates, these GST isozymes exhibited different but overlapping substrate specificities. With a few excep-

tions, 1-chloro-2,4-dinitrobenzene (CDNB) was the best substrate for these isozymes. The isozymes were

active toward numerous toxic a; b-unsaturated carbonyl allelochemicals including trans-2-octenal, trans-2-

nonenal, 2,4-hexadienal, trans,trans-2,4-heptadienal, trans,trans-2,4-nonadienal, and trans,trans-2,4-deca-

dienal, suggesting that GSTs play an important role in the feeding strategies of lepidopterous insects. These

GSTs also possessed glutathione peroxidase activity toward cumene hydroperoxide and conjugating ac-

tivity toward 4-hydroxy nonenal, a lipid peroxidation product, and therefore they are antioxidant enzymes.

Microsomal glutathione S-transferase from fat bodies of fall armyworm larvae metabolized a variety of

model substrates such as CDNB, 1,2-dichloro-4-nitrobenzene (DCNB), p-nitrophenyl acetate, and cumene

hydroperoxide, but had no activity toward a; b-unsaturated carbonyl compounds. With the exception of

ethacrynic acid, glutathione S-transferase activities toward these substrates were all inducible by allelo-

chemicals such as xanthotoxin and indole 3-acetonitrile in midguts and fat bodies of fall armyworm larvae.

Induction ranged from 1.3- to 20.2-fold for midgut GSTs and 1.4- to 48.8-fold for fat body GSTs, de-

pending on the inducer and substrate used. In all instances, DCNB-conjugating activity was most inducible

based on percentage of control.

� 2002 Elsevier Science (USA). All rights reserved.

1. Introduction

Glutathione S-transferases (GSTs)1 are a

group of multifunctional detoxification enzymes

qFlorida Agricultural Experiment Station Series No.

R-08831.* Fax: 352-392-0190.

E-mail address: [email protected].

1 Abbreviations used: GSTs, glutathione S-transfer-

ases; GSH, glutathione; CDNB, 1-chloro-2,4-dinitro-

benzene; DCNB, 1,2-dichloro-4-nitrobenzene; TPBO,

trans-4-phenyl-3-buten-2-one; PAGE, polyacrylamide

gel electrophoresis.

Pesticide Biochemistry and Physiology 74 (2002) 41–51

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0048-3575/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

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mail to: [email protected]

that catalyze the conjugation of reduced gluta-

thione (GSH) with various xenobiotics and en-

dogenous compounds possessing an electrophilic

center [1]. GSTs are important in phase I metab-

olism of organophosphorus and organochlorine

compounds and play a significant role in resis-

tance to these insecticides in insects [2–5]. They

are also important in phase II metabolism of re-

active metabolites formed by microsomal oxida-

tion [6]. Recently, GSTs were found to be

involved in pyrethroid tolerance through seques-

tration [7] and pyrethroid resistance through an-

tioxidant defense [8] in insects.

Insect glutathione S-transferases have been

shown to be active toward numerous electro-

philic xenobiotics including halogenated com-

pounds (e.g., 1-chloro-2,4-dinitrobenzene), nitro

compounds (e.g., p-nitrophenyl acetate), a; b-unsaturated carbonyl compounds (e.g., trans-4-

phenyl-3-buten-2-one), isothiocyanates (e.g., allyl

isothiocyanate), organothiocyanates (e.g., benzyl

thiocyanate), oxides (e.g., styrene oxide), or-

ganophosphates (e.g., diazinon), and organic

hydroperoxides (cumene hydroperoxide) [3].

However, very little is known about substrate

specificity of individual GST isozymes in insects.

This knowledge is very important for under-

standing the molecular mechanisms of detoxifi-

cation in insects.

Therefore, the purpose of this research was to

study the substrate specificity of various GST

isozymes isolated from midguts and fat bodies of

fall armyworm larvae. Attempts also were made

to learn if substrate specificity of glutathione S-

transferases changes during larval development

and after induction.

2. Materials and methods

2.1. Insects

Larvae of the fall armyworm. Spodoptera fru-

giperda (J.E. Smith), were reared on an artificial

diet [9] and maintained in environmental cham-

bers at 25 �C with a 16:8 light:dark photoperiod.

2.2. Chemicals

The chemicals (analytical grade) used in this

study and their sources were glutathione–agarose

(thiol linked), reduced glutathione (GSH), gluta-

thione reductase, 1-chloro-2,4-dinitrobenzene (CD

NB), ethacrynic acid, xanthotoxin, indole 3-ace-

tonitrile, cumene hydroperoxide, and sodium py-

ruvate (Sigma Chemical, St. Louis, MO); trans-4-

phenyl-3-buten-2-one (TPBO), 2,4-hexadienal,

trans,trans-2,4-heptadienal, trans,trans-nonadie-

nal, trans,trans-decadienal, trans-2-octenal, trans-

2-nonenal (Aldrich, Milwaukee, WI); 1,2-di-

chloro-4-nitrobenzene (DCNB); and p-nitrophe-

nyl acetate (Eastern Kodak, Rochester, NY). All

other chemicals were the highest purity available

commercially.

2.3. Treatment of insects

In induction experiments, groups of 25 larvae

(newly molted sixth instars) were individually fed

an artificial diet containing allelochemicals for 2

days prior to enzyme preparation. Control larvae

were fed the artificial diet only.

2.4. Enzyme preparation

Groups of midguts and fat bodies were dis-

sected from 2-day-old sixth instars. In the case of

midguts, gut contents were removed. All tissues

were then washed in 1.15% KCl and homogenized

in 25ml of ice-cold 0.1M sodium phosphate

buffer, pH 7.5, in a motor-driven tissue grinder for

30 s. The crude homogenate was filtered through

cheese cloth and filtered homogenate was centri-

fuged at 10,000gmax for 15min in a Beckman L5-

50E ultracentrifuge. The pellet (cell debris, nuclei,

and mitochondria) was discarded and the super-

natant was recentrifuged at 105,000gmax for 1 h to

obtain the soluble fraction (supernatant). In some

experiments, fat body microsomes (pellet) were

washed twice with 0.1M sodium phosphate buf-

fer, pH 7.5, by recentrifigation at 105,000gmax for

1 h. The washed microsomes were finally sus-

pended in 0.1M sodium phosphate buffer, pH 7.5.

2.5. Purification of cytosolic glutathione S-trans-

ferases

Glutathione S-transferases were purified from

the soluble fraction according to a method de-

scribed previously [10,11]. Briefly, the soluble

fraction was first fractionated with ammonium

sulfate to obtain a protein fraction that corre-

sponded to 45–75% saturation. Precipitated pro-

teins were suspended in 22mM sodium phosphate

buffer, pH 7.0, and dialyzed against 250 vol of the

same buffer for 2 h to eliminate the minute amount

of ammonium sulfate in the preparation. The dia-

lyzed sample was then applied to a thiol-linked

42 S.J. Yu / Pesticide Biochemistry and Physiology 74 (2002) 41–51

glutathione–agarose column (1� 10 cm) previ-

ously equilibrated with 22mM sodium phosphate

buffer, pH 7.0. The column was eluted with the

same buffer until no further protein was detected

by monitoring the absorbance at 280 nm with an

ISCO Model UA-5 absorbance/fluorescence de-

tector. The bound enzyme was then released by

eluting with 0.05M Tris–HCl buffer, pH 9.6, con-

taining 5mM GSH. Fractions containing GST

activity toward CDNB were combined and con-

centrated by ultrafiltration on an Amicon Diaflo

PM-10 membrane. GST isozymes were separated

using nondenaturing gel electrophoresis as de-

scribed below. All samples were stored at �70 �Cfor further analysis. Protein concentrations were

determined by the method of Bradford [12] using

bovine serum albumin as standard.

2.6. Enzyme assays

Glutathione S-transferase activities toward

CDNB and DCNB were measured as reported

previously [13]. GST activity toward p-nitrophenyl

acetate was determined by the method of Keen and

Jakoby [14]. GST activities toward ethacrynic acid

and trans-4-phenyl-3-buten-2-one were measured

as described by Habig et al. [15]. GST activities

toward trans,trans-2,4-alkadienals and trans-2-al-

kenals were measured by the method of Brophy et

al. [16]. GST activity toward 4-hydroxy nonenal

was assayed as described by Alin et al. [17].

GST peroxidase activity was determined using

a method slightly modified from that of Ahmad

and Pardini [18] using cumene hydroperoxide as

substrate. Briefly, the 2.9-ml reaction mixture

which contained 50mM sodium phosphate buffer,

pH 7, 0.1mM EDTA, 1mM GSH, 0.2mM

NADPH, and 10U glutathione reductase was first

incubated for 3min at 25 �C, followed by the ad-

dition of 20 ll cumene hydyroperoxide solution

(prepared in methyl Cellosolve) to yield 1.2mM

final concentration. The reaction was started by

the addition of 0.1ml of the enzyme. The rate of

NADPH oxidation was recorded at 340 nm

against the same reaction mixture in the absence

of enzyme in a Beckman Model 5260 UV/Vis

spectrophotometer. Lactate dehydrogenase activ-

ity was determined as described by Berstein and

Everse [19].

2.7. Electrophoresis

Nondenaturing polyacrylamide gel electro-

phoresis (PAGE) was conducted according to the

method of Davis [20]. The 10-cm separating gel

contained 7.5% acrylamide and the 1-cm stacking

gel contained 3% acrylamide. Electrophoresis was

carried out at 2mA/gel at 4 �C. Two gel tubes

were stained briefly with a solution containing

3.5% perchloric acid and 0.04% Coomassie bril-

liant blue G [21] and used as references to locate

and remove the protein bands from the non-

staining gels. GST isozymes were eluted from the

appropriate gel sections containing protein bands

using a Bio-Rad Model 422 electro-eluter ac-

cording to manufacturer�s instructions (Bio-Rad).

Occasionally, the gels were stained for proteins

with Coomassie brilliant blue R according to

Fairbank et al. [22].

2.8. Kinetic studies

The Michaelis constant ðKmÞ and maximum

velocity (Vmax) for purified GST isozymes were

determined by Lineweaver–Burk plots using

CDNB (0.1–0.8mM) as substrate.

2.9. Statistical analysis

Whenever appropriate, data were analyzed by

Student�s t test.

3. Results

In previous reports [10,23], fall armyworm

larval midgut was found to possess six cytosolic

GST isozymes, namely, MG GST-1, MG GST-2,

MG GST-3, MG GST-4, MG GST-5, and MG

GST-6, whereas the fat body contained three

isozymes, namely, FB GST-1, FB GST-2, and FB

GST-3. In the present study, substrate specificity

of these isozymes was determined using 13 model

substrates belonging to halogenated compounds,

nitro compounds, a; b-unsaturated carbonyl

compounds, and organic hydroperoxides. Data in

Table 1 show that the pattern of activity toward

these substrates was different among isozymes.

MG GST-3 was not active toward p-nitrophenyl

acetate, whereas MG GST-4 was not active to-

ward TPBO. Furthermore, DCNB-conjugating

activity was not detected in any of these isozymes.

In most instances, CDNB was the best substrate

among those tested. The purifications were 4- to

294-fold, depending on the isozyme and substrate

used.

The substrate specificity of GST isozymes from

fat bodies is shown in Table 2. Due to the poor

S.J. Yu / Pesticide Biochemistry and Physiology 74 (2002) 41–51 43

Table 1

Substrate specificity of cytosolic glutathione S-transferases from midguts of fall armyworm larvae

Substrate Specific activity (nmol/min/mg protein)a

Cytosol MG MG MG MG MG MG

GST-1b GST-2b GST-3 GST-4 GST-5 GST-6

CDNB 228.5� 9.76 2189.8� 5.03 1662.9� 177.1 1372.9� 8.12 1525.3� 37.3 5338.5� 130.6 2249.0� 261.2

DCNB 14.95� 0.21 0 0 0 0 0 0

p-Nitrophenyl acetate 192.2� 9.87 866.2� 66.3 948.0� 17.4 0 853.2� 122.2 995.4� 71.3 775.7� 51.8

Ethacrynic acid 66.1� 3.18 852.2� 172.1 1121.2� 91.8 2857.9� 368.7 1642.8� 143.2 1625.0� 250.7 1465.9� 216.5

TPBO 3.72� 0.30 109.9� 18.3 79.4� 17.3 88.74� 17.8 0 154.6� 9.54 489.6� 43.3

trans-2-Octenal 5.14� 0.18 125.1� 2.30 113.6� 6.00 318.3� 29.3 355.1� 23.7 287.8� 15.2 438.3� 10.3

trans-2-Nonenal 2.85� 0.26 145.0� 4.19 172.8� 3.54 750.6� 15.3 504.5� 16.3 245.1� 12.3 837.0� 93.2

4-Hydroxy nonenal 14.45� 1.10 787.9� 20.3 654.6� 36.5 1240.6� 150.1 856.0� 53.2 721.1� 66.8 1246.7� 73.4

2,4-Hexadienal 5.78� 0.51 203.4� 8.15 196.2� 3.85 326.8� 17.2 188.9� 15.3 197.4� 3.46 370.7� 39.3

trans,trans-2,4-Heptadienal 4.64� 0.31 115.5� 16.5 160.9� 20.7 271.8� 19.4 436.1� 11.8 5886.9� 51.7 341.2� 41.3

trans,trans-2,4-Nonenal 7.09� 26.0 160.6� 13.1 156.3� 8.25 2225.2� 6.50 195.3� 26.0 208.3� 20.9 504.2� 21.4

trans,trans-2,4-Decadienal 8.42� 1.00 149.7� 18.8 177.2� 8.87 257.5� 19.8 336.7� 35.2 305.8� 3.95 428.5� 61.3

Cumene hydroperoxide 33.6� 1.07 318.1� 9.14 1272.6� 17.1 1513.1� 94.8 1272.8� 94.7 1085.2� 56.8 1709.9� 182.5

aMeans�SE of three experiments, each with duplicate determinations.bData from Yu [28] except for 4-hydroxy nonenal.

44

S.J

.Y

u/P

esticide

Bio

chem

istryand

Physio

logy

74

(2002)

41–51

resolution between FB GST-2 and FB GST-3,

combined activity was measured for these two

isozymes as FB GST2/3. As found in the MG

GSTs, the pattern of activity toward these sub-

strates was different between isozymes. No activ-

ity was detected against DCNB, p-nitrophenyl

acetate, and ethacrynic acid for FB GST-1. In the

case of FB GST-2/3, no activity was detected to-

ward DCNB. The results also showed that CDNB

was the best substrate for these isozymes, followed

by cumene hydroperoxide and 4-hydroxy none-

nal. The purifications ranged from 7- to 230-fold,

depending on the isozyme and substrate used.

We previously showed that induction of glu-

tathione S-transferases by xanthotoxin in fall ar-

myworm larvae resulted in production of two new

cytosolic isozymes (FB GST-A and FB GST-B) in

fat bodies [23]. Table 3 shows that these two in-

duced GST isozymes from fat bodies exhibited

different catalytic pattern toward these substrates.

However, for both isozymes, ethacrynic acid was

the best substrate, followed by CDNB and cum-

ene hydroperoxide, and no activity was detected

toward DCNB or trans-4-phenyl-3-buten-2-one.

Table 4 shows the activities of microsomal

glutathione S-transferase prepared from fat

Table 2

Substrate specificity of cytosolic glutathione S-transferases from fat bodies of fall armyworm larvae


Cytosol FB GST-1 FB GST-2/3

CDNB 393.4� 7.85 25992.0� 199.0 3074.9� 170.7

DCNB 6.39� 0.58 0 0

p-Nitrophenyl acetate 10.56� 0.41 0 689.1� 71.3

Ethacrynic acid 0 0 749.9� 27.2

TPBO 1.14� 0.22 57.60� 9.62 100.8� 7.77

trans-2-Octenal 0 204.5� 22.7 485.1� 27.8

trans-2-Nonenal 0 468.7� 26.1 505.8� 37.7

4-Hydroxy nonenal 5.56� 0.70 745.4� 63.8 1279.6� 49.1

2,4-Hexadienal 6.03� 0.18 336.2� 29.3 261.6� 36.7

trans,trans-2,4-Heptadienal 7.37� 0.63 239.2� 24.8 257.0� 15.8

trans,trans-2,4-Nonadienal 7.78� 0.20 273.4� 27.4 210.3� 12.0

trans,trans-2,4-decadienal 6.81� 1.07 286.2� 16.9 194.2� 12.9

Cumene hydroperoxide 49.94� 1.37 1588.6� 27.1 1762.3� 31.0

aMeans � SE of three experiments, each with duplicate determinations.

Table 3

Substrate specificity of xanthotoxin-induced glutathione S-transferase isozymes from fat bodies of fall armyworm lar-

vaea

Substrate Specific activity (nmol/min/mg protein)b

FB GST-A FB GST-B

CDNB 1915.9� 167.9 1264.9� 261.2

DCNB 0 0

p-Nitrophenyl acetate 365.7� 40.8 121.9� 20.4

Ethacrynic acid 4107.1� 895.5 1392.9� 35.8

TPBO 0 0

trans-2-Octenal 251.6� 8.14 255.7� 28.4

trans-2-Nonenal 306.9� 28.0 227.9� 6.57

4-Hydroxy nonenal 974.0� 65.1 519.5� 65.1

2,4-Hexadienal 234.9� 78.5 161.9� 21.0

trans,trans-2,4-Heptadienal 294.7� 11.8 138.5� 8.86

trans,trans-2,4-Nonadienal 156.2� 11.2 0

trans,trans-2,4-Decadienal 195.4� 45.2 147.3� 3.01

Cumene hydroperoxide 1062.2� 96.4 732.1� 43.2

aNewly molted sixth instars were fed an artificial diet containing xanthotoxin (0.01%) for 2 days prior to enzyme

purification.bMeans � SE of three experiments, each with duplicate determinations.


bodies of fall armyworm larvae. In this experi-

ment, the second wash of microsomes removed all

detectable traces of cytosolic contamination as

indicated by an absence of lactate dehydrogenase

activity in the preparation (data not shown). It

can be seen that, in addition to CDNB, the mi-

crosomal GST was active toward DCNB, p-

nitrophenyl acetate, and cumene hydroperoxide,

but had no activity against a; b-unsaturated car-

bonyl compounds. Attempts were made to learn

whether microsomal GST activity could be acti-

vated by a sulfhydryl reagent in fall armyworm

larvae. Washed microsomes from fat bodies were

incubated with N-ethylmaleimide at 1mM for

10min at 25 �C prior to addition of GSH and

CDNB for the GST assay. The results showed

that N-ethylmaleimide (1mM) significantly in-

hibited GST activity (30% reduction) compared

with the control.

Using the midgut soluble fraction as the en-

zyme source, we found that the pattern of GST

activities toward various substrates was slightly

different among 4th, 5th, and 6th instar (Table 5).

In all instances, CDNB was the best substrate for

each instar, however. It is interesting to note that

there is a positive correlation between the enzyme

activity (toward CDNB and p-nitrophenyl ace-

tate) and the instar. On the other hand, a negative

correlation was observed between the enzyme ac-

tivity (toward ethacrynic acid and trans-2-none-

nal) and the instar.

The results obtained from the induction of

GSTs by allelochemicals in midguts and fat bodies

of fall armyworm larvae are summarized in Tables

6 and 7. From Table 6, it is seen that xanthotoxin

induced GST activities toward all substrates with

the exception of ethacrynic acid, trans-4-phenyl-3-

buten-2-one, and trans-2-nonenal. In the case of

indole 3-acetonitrile, it induced GST activities

toward all substrates except ethacrynic acid. In all

instances, DCNB-conjugating activity was most

inducible based on percentage of control. Induc-

tion ranged from 1.3- to 20.2-fold depending on

Table 4

Microsomal glutathione S-transferase activities in fat

bodies of fall armyworm larvae

Substrate Specific activity

(nmol/min/mg

protein)a

CDNB 157.3� 1.10

DCNB 8.62� 0.57

p-Nitrophenyl acetate 16.67� 0.98

Ethacrynic acid 0

TPBO 0

trans-2-Octenal 0

trans-2-Nonenal 0

4-Hydroxy nonenal 0

2,4-Hexadienal 0

trans,trans-2,4-Heptadienal 0

trans,trans-2,4-Nonadienal 0

trans,trans-Decadienal 0

Cumene hydroperoxide 18.52� 0.21

aWashed microsomes from fat bodies were used as

enzyme source. Means � SE of three experiments, each

with duplicate determinations.

Table 5

Cytosolic glutathione S-transferase activities in midguts of different instars of fall armyworm


4th Instar 5th Instar 6th Instar

CDNB 142:9� 16:7 (1)b 179:8� 8:58 (1) 228:5� 9:76 (1)

DCNB 9:97� 0:40 (6) 9:04� 0:82 (5) 14:95� 0:21 (5)

p-Nitrophenyl acetate 78:05� 5:46 (3) 84:20� 5:62 (2) 192:2� 9:87 (2)

Ethacrynic acid 108:5� 8:68 (2) 75:62� 3:30 (3) 66:1� 3:18 (3)

TPBO 5:15� 0:26 (9) 2:98� 0:33 (11) 3:72� 0:30 (12)

trans-2-Octenal 2:18� 0:15 (13) 1:87� 0:37 (13) 5:14� 0:18 (10)

trans-2-Nonenal 7:80� 0:50 (7) 4:11� 0:17 (10) 2:85� 0:26 (13)

4-Hydroxy nonenal 5:10� 0:91 (10) 4:78� 0:24 (7) 14:45� 1:10 (6)

2,4-Hexadienal 2:89� 0:12 (12) 2:31� 0:38 (12) 5:78� 0:51 (9)

trans,trans-2,4-Heptadienal 4:37� 0:37 (11) 4:33� 0:22 (9) 4:64� 0:31 (11)

trans,trans-2,4-Nonadienal 6:73� 0:25 (8) 5:29� 0:16 (6) 7:09� 0:68 (8)

trans,trans-2,4-Decadienal 11:28� 0:54 (5) 4:76� 0:11 (8) 8:42� 1:00 (7)

Cumene hydroperoxide 32:06� 1:28 (4) 52:86� 4:22 (4) 33:60� 1:07 (4)

aMidgut cytosol was used as enzyme source. Means � SE of three experiments, each with duplicate determinations.bNumbers in parentheses denote the ranking (from high to low) for each activity of the respective preparation.


the inducer and substrate used. Similar results

were also obtained when fat bodies were used as

the enzyme source (Table 7). Both xanthotoxin

and indole 3-acetonitrile induced GST activities

toward all substrates with the exception of eth-

acrynic acid. In all instances, DCNB-conjugating

activity was most inducible based on percentage

of control. Induction ranged from 1.4- to

Table 6

Effect of allelochemicals on substrate specificity of cytosolic glutathione S-transferases from midguts of fall armyworm

larvaea

Substrate Substrate specificity (nmol/min/mg protein)b

Inducer

Control Xanthotoxin Indole 3-acetonitrile

CDNB 236.0� 22.5 639.6� 45.9� 657.3� 14.1�

DCNB 15.16� 1.00 307.2� 24.9� 150.6� 7.56�

p-Nitrophenyl acetate 202.0� 13.6 345.9� 19.8� 338.0� 16.6�

Ethacrynic acid 52.5� 2.56 53.4� 2.24 51.16� 4.45

TPBO 5.55� 0.13 6.24� 0.28 7.79� 0.78�

trans-2-Octenal 7.68� 0.01 13.06� 1.43� 11.51� 0.77�

trans-2-Nonenal 4.40� 0.63 4.15� 0.88 13.95� 2.40�

4-Hydroxy nonenal 13.35� 0.70 35.48� 4.57� 35.04� 0.20�

2,4-Hexadienal 4.95� 0.13 6.09� 0.35� 7.97� 0.07�

trans,trans-2,4-Heptadienal 4.62� 0.22 6.77� 0.56� 8.60� 0.64�

trans,trans-2,4-Nonadienal 6.41� 0.53 8.31� 0.76� 12.63� 0.35�

trans,trans-2,4-Decadienal 8.38� 0.57 11.96� 1.06 12.77� 0.34�

Cumene hydroperoxide 33.75� 1.93 72.13� 2.68� 55.46� 0.56�

aNewly molted sixth instars were fed artificial diets containing xanthotoxin (0.01%) or indole 3-acetonitrile (0.2%) for

2 days prior to enzyme assays.bMeans � SE of three experiments, each with duplicate determinations.*Value significantly different from the control (p < 0:05).

Table 7

Effect of allelochemicals on substrate specificity of cytosolic glutathione S-transferases from fat bodies of fall armyworm

larvaea

Substrate Specific activity (nmol/min/mg protein)b

Inducer

Control Xanthotoxin Indole 3-acetonitrile

CDNB 393.4� 7.85 1460.5� 73.8� 2495.4� 162.7�

DCNB 6.39� 0.58 311.9� 6.93� 182.8� 18.5�

p-Nitrophenyl acetate 10.56� 0.41 138.4� 2.18� 248.9� 10.0�

Ethacrynic acid 14.92� 2.10 16.44� 0.65 11.84� 1.32�

TPBO 1.14� 0.22 2.57� 0.14� 2.45� 0.11�

trans-2-Octenal 4.41� 0.16 12.88� 1.70� 19.43� 1.83�

trans-2-Nonenal 5.44� 0.94 13.75� 0.76� 18.41� 0.51�

4-Hydroxy nonenal 5.56� 0.70 36.16� 0.39� 63.16� 0.71�

2,4-Hexadienal 6.03� 0.18 9.62� 0.96� 11.58� 0.31�

trans,trans-2,4-Heptadienal 7.37� 0.63 11.46� 0.54� 20.05� 0.97�

trans,trans-2,4-Nonadienal 7.78� 0.20 18.23� 0.86� 23.74� 1.13�

trans,trans-2,4-Decadienal 6.81� 1.07 21.48� 0.31� 18.82� 1.10�

Cumene hydroperoxide 49.94� 1.37 134.8� 7.95� 120.0� 1.59�

aNewly molted sixth instars were fed artificial diets containing xanthotoxin (0.01%) and indole 3-acetonitrile (0.2%)

for 2 days prior to enzyme assays.bMeans � SE of three experiments, each with duplicate determinations.* Value significantly different from the control (p < 0:05).


48.8-fold depending on the inducer and substrate

used. The results also showed that fat bodies were

more inducible than midguts with respect to

GSTs.

Table 8 showed that all the GST isozymes were

different based on their Km, Vmax, and Kcat values.

4. Discussion

The results of this study clearly demonstrated

that the 10 GST isozymes isolated from fall ar-

myworm larvae were different catalytically based

on the order of substrate specificity toward 12

substrates as summarized in Table 9. DCNB-

conjugating activity was not included here be-

cause activity toward this substrate was not

detected. These isozymes exhibited different but

overlapping substrate specificities. With a few

exceptions, CDNB was the best substrate among

those employed. Thus, CDNB can be used as a

general substrate for measuring GST activity in

insects. These isozymes were active toward the

a; b-unsaturated carbonyl compounds. The results

are in agreement with Wadleigh and Yu [24] who

found that several a; b-unsaturated carbonyl al-

lelochemicals including trans-cinnamaldehyde,

benzaldehyde, trans, trans-2,4-decadienal, and

trans-2-hexenal were metabolized by cytosolic

GSTs from fall armyworm larvae. These a; b-un-saturated carbonyl allelochemicals are commonly

presented in corn, wheat, and oats [25–27], all of

which are preferred host plants for the fall ar-

myworm. Our results showed that, in addition to

these toxic allelochemicals, numerous other a; b-

unsaturated carbonyl allelochemicals including

trans-2-octenal, trans-2-nonenal, 2,4-hexadienal,

trans, trans-heptadienal, trans, trans-2,4-nonadie-

nal, and trans, trans-2,4-decadienal were also me-

tabolized by the enzymes. The present findings

further support the notion that GSTs play an

important role in the feeding strategies of lepi-

dopterous insects.

In the present study, we were unable to detect

GST activity toward DCNB for all of the purified

isozymes. However, DCNB-conjugating activity

was observed in the affinity-purified preparations

[28], indicating that GSH–agarose was capable of

retaining the enzyme during chromatography. It is

highly possible that low yields of enzyme coupled

with low specific activity resulted in undetectable

quantities of the enzyme.

Interestingly, all of the purified cytosolic GST

isozymes possessed glutathione peroxidase activ-

ity toward cumene hydroperoxide and conjugat-

ing activity toward 4-hydroxy nonenal, the latter

being a cytotoxic product of microsomal lipid

peroxidation. The results indicated that all these

GST isozymes are antioxidant enzymes. The lipid

peroxidation products formed by the free-radical-

mediated attack on membrane lipids can lead to

membrane destruction and DNA damage [29,30].

Therefore, the detoxification of lipid peroxidation

products is an essential process in animals.

Cumene hydroperoxide peroxidase activity was

detected in the cabbage looper, southern army-

worm, and black swallowtail and has been

characterized as GSTs with peroxidase activity

[31]. Moreover, a GST isolated from Drosophila

exhibited high activity toward 4-hydroxy nonenal

Table 8

Kinetics of glutathione S-transferase isozymes from fall armyworm larvae

Isozyme Km (mM)c Vmax (lmol/min/mg protein)c Kcatd (min�1) KcatKm

e (mM�1 min�1)

MG GST-1a 0.91 2.35 128 141

MG GST-2a 2.26 3.00 164 73

MG GST-3 1.11 3.33 190 171

MG GST-4 0.65 6.67 381 586

MG GST-5 3.33 10.00 571 171

MG GST-6 1.00 1.42 83 83

FB GST-1 0.95 32.26 648 682

FB GST-2/3 2.86 14.27 407 142

FB GST-Ab 0.62 10.00 280 452

FB GST-Bb 0.51 11.11 311 610

aData from Yu [28].bData from Yu [23].c CDNB as substrate.d Turnover number.e Substrate specificity constant.


further illustrating the antioxidant capacity of

these enzymes [32].

Previously, we reported no quantitative differ-

ence in GST isozyme composition (i.e., same

number of isozymes) during larval development of

fall armyworm [11]. The present study indicated

that changes in levels of these GST isozymes may

have occurred during development because a

slight difference in substrate specificity was ob-

served among the larval instars examined.

In previous work [23], we showed that induc-

tion of GSTs by xanthotoxin and indole 3-aceto-

nitrile in fall armyworm larvae resulted in the

production of two non-constitutive isozymes in

fat bodies. This is reflected in the present study

showing different activity patterns in both groups

of induced GSTs as compared with the control

(Table 7). Although xanthotoxin did not induce

any new isozyme in midguts of fall armyworm

larvae [23], slight changes in levels of the existing

isozymes did occur based on activity patterns of

these GSTs (Table 6). It is interesting to note that

GST antioxidant activities (toward 4-hydroxy

nonenal and cumene hydroperoxide) were also

inducible by allelochemicals in midguts and fat

bodies of fall armyworm larvae. Therefore, in-

duction of GSTs by allelochemicals will also help

insects detoxify endogenous lipid peroxidation

products. However, we were perplexed to learn

that GST activity toward ethacrynic acid was not

induced by the allelochemicals in midguts and fat

bodies of fall armyworm larvae.

Our results showed that microsomal GST from

fat bodies exhibited a narrower substrate speci-

ficity as compared with fat body cytosolic GSTs,

showing no activity toward a; b-unsaturated car-

bonyl compounds. Similar results were also ob-

tained with midgut microsomal GST of this insect

[28]. However, because microsomal cytochrome

P450 monooxygenases catalyze the transforma-

tion of xenobiotics to reactive metabolites (e.g.,

epoxides), microsomal GST which is immedi-

ately available in the microsomal membrane may

be more important than the cytosolic GSTs in

the detoxification of such reactive metabolites.

Moreover, because both microsomal GSTs from

larval midguts [28] and fat bodies possessed

cumene hydroperoxide peroxidase activity, they

are presumably important in the detoxification of

microsomal membrane hydroperoxides generated

during lipid peroxidation.

Microsomal GST from rat liver was found to

be activated by numerous sulfhydryl reagents in-

cluding N-ethylmaleimide, indoacetamide [33] and

Table 9

Substrate specificity of glutathione S-transferase isozymes from fall armyworm larvae

Substrate Order of specific activity

CDNB MG GST-5>FB GST-2/3>FB GST-1>MG GST-6>MG GST-1>FB GST-

A> MG GST-2>MG GST-4>MG GST-3>FB GST-B

p-Nitrophenyl acetate MG GST-5>MG GST-2>MG GST-1>MG GST-4> MG GST-6>FB GST-

2/3>FB GST-A>FB GST-B>MG GST-3 ¼ FB GST-1

Ethacrynic acid FB GST-A>MG GST-3>MG GST-4>MG GST-5>MG GST-6> FB GST-

B>MG GST-2>MG GST-1>FB GST-2/3 >FB GST-1

TPBO MG GST-6>MG GST-5>MG GST-1>FB GST-2/3>MG GST-3>MG GST-

2 >FB GST-1>FB GST-A ¼ FB GST-B ¼ MG GST-4

trans-2-Octenal FB GST-2/3>MG GST-6>MG GST-4>MG GST-3>MG GST-5>FB GST-

B>FB GST-A>FB GST-1>MG GST-1>MG GST-2

trans-2-Nonenal MG GST-6>MG GST-3>FB GST-2/3>MG GST-4>FB GST-1>FB GST-

A>MG GST-5>FB GST-B>MG GST-2> MG GST-1

4-Hydroxy nonenal FB GST-2/3>MG GST-6>MG GST-3>FB GST-A>MG GST-4>MG GST-

1>FB GST-1>MG GST-5>MG GST-2>FB GST-B

2,4-Hexadienal MG GST-5>FB GST-1>MG GST-6>FB GST-2/3> FB GST-A>MG GST-

1>MG GST-4>MG GST-2>MG GST-3> FB GST-B

trans,trans-2,4-Heptadienal MG GST-5>MG GST-4>MG GST-6>FB GST-A>MG GST-3>FB GST-

2/3>FB GST-1>MG GST-2>FB GST-B>MG GST-1

trans,trans-2,4-Nonenal MG GST-6>FB GST-1>MG GST-3>FB GST-2/3>MG GST-5>MG GST-

4>MG GST-1>MG GST-2>FB GST-A>FB GST-B

trans,trans-2,4-Decadienal MG GST-6>MG GST-4>MG GST-5>FB GST-1>MG GST- 3>FB GST-

A>FB GST-2/3>MG GST-2>MG GST-1>FB GST-B

Cumene hydroperoxide FB GST-2/3>MG GST-6>FB GST-1>MG GST-3 >MG GST-4>MG GST-

2>MG GST-5>FB GST-A>FB GST-B>MG GST-1


herbicides (chloranil, captan, and acrolein) [34].

As found in midgut microsomal GST from fall

armyworm larvae [28], in the present study fat

body microsomal GST was not stimulated by

N-ethylmaleimide, indicating that this GST is dif-

ferent from livermicrosomalGST. In the case of rat

liver microsomal GST, the activation involves the

binding of onemolecule ofN-ethylmaleimide to the

single cysteine residue present in each polypeptide

chain of the enzyme [35].MicrosomalGST from fat

bodies and midguts of fall armyworm larvae may

not contain the cysteine residue for such interac-

tion. Additional work is needed to verify this point.

Since N-ethylmaleimide is a known GSH depleter

[36], it may explain the inhibitory effect of this

compound on microsomal GST activity observed

in the fall armyworm.

Acknowledgments

The author thanks Drs. S.M. Valles (USDA-

ARS) and F. Slansky (University of Florida) for

critical reviews of the manuscript. The technical

assistance of Sam Nguyen is also appreciated.

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substrate specificity of glutathione s-transferases from the fall armyworm

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