Purification and characterization of acetylcholinesterase from oriental fruit fly [Bactrocera dorsalis (Hendel)] (Diptera: Tephritidae)

Article abstract of DOI:10.1021/jf0494377

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the head of the insecticide susceptible oriental fruit fly, Bactrocera dorsalis (Hendel), by affinity chromatography of Triton X-100 extract. The degree of purification was about 8183-fold with recoveries of 52%. The molecular mass of purified AChE was 116 kDa for its native protein (nonreduced form) and 61 kDa for its subunits (reduced form) as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting that the homodimer of AChE linked with disulfide bonds. Nondenaturing PAGE of the purified AChE revealed only one molecular form. The maximum velocities (V_max) for hydrolyzing acetylthiocholine (ATC), propionylthiocholine, and S-butyrylthiocholine were 833.3, 222.2, and 57.5 μmol/min/mg, and the Michaelis constants (K _m) were 87.9, 26.9, and 195.3 μM, respectively. More than 97% of AChE activity was inhibited by 10 μM eserine or BW284C51, but only 53% of the activity was inhibited by ethopropazine at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nevertheless, the purified AChE exhibited some distinctive characteristics including (i) a lack of the substrate inhibition phenomenon when using ATC as the hydrolyzing substrate and (ii) a higher V_m value for ATC than AChE from other insect species. These biochemical properties may show that AChE purified from the oriental fruit fly may have structural differences from those of other insect species.

Full text of DOI:10.1021/jf0494377

5340 J. Agric. Food Chem. 2004, 52, 5340−5346
Purification and Characterization of Acetylcholinesterase from
Oriental Fruit Fly [Bactrocera dorsalis (Hendel)]
(Diptera: Tephritidae)
‡
YI-MIN HSIAO,*,†,‡ JING-YING LAI,† HSIU-YING LIAO,‡ AND HAI-TUNG FENG
Center for Research and Development, Chungtai Institute of Health Sciences and Technology,
11 Pu-tzu Lane, Pei-tun District 40605, Takun, Taichung, Taiwan, Republic of China, and Pesticide
Chemistry Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute,
11 Kuang-Ming Road, Taichung Hsien 41301, Wufeng, Taiwan, Republic of China
An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the head of the insecticide susceptible
oriental fruit fly, Bactrocera dorsalis (Hendel), by affinity chromatography of Triton X-100 extract.
The degree of purification was about 8183-fold with recoveries of 52%. The molecular mass of purified
AChE was 116 kDa for its native protein (nonreduced form) and 61 kDa for its subunits (reduced
form) as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
suggesting that the homodimer of AChE linked with disulfide bonds. Nondenaturing PAGE of the
purified AChE revealed only one molecular form. The maximum velocities (Vmax) for hydrolyzing
acetylthiocholine (ATC), propionylthiocholine, and S-butyrylthiocholine were 833.3, 222.2, and 57.5
µmol/min/mg, and the Michaelis constants (Km) were 87.9, 26.9, and 195.3 µM, respectively. More
than 97% of AChE activity was inhibited by 10 µM eserine or BW284C51, but only 53% of the activity
was inhibited by ethopropazine at the same concentration. On the basis of the substrate and inhibitor
specificities, the purified enzyme appeared to be a true AChE. Nevertheless, the purified AChE
exhibited some distinctive characteristics including (i) a lack of the substrate inhibition phenomenon
when using ATC as the hydrolyzing substrate and (ii) a higher Vm value for ATC than AChE from
other insect species. These biochemical properties may show that AChE purified from the oriental
fruit fly may have structural differences from those of other insect species.
KEYWORDS: Acetylcholinesterase; kinetics; purification
INTRODUCTION
body (9-15). Crude AChE extracts, containing contaminating
non-AChE factors, such as carboxylesterases, can affect the
measurement of AChE and produce misleading results.
AChE has been purified from only a few insect species,
including Musca domestica (14), Lygus hesperus Knight (16),
Leptinotarsa decemlineata (17), HelicoVerpa armigera (18),
Diabrotica Virgifera Virgifera (6), Galleria mellonella L. (19),
Schizaphis graminum (20), and the cotton aphid, Aphis gossypii
Glover (21).
In Taiwan, the oriental fruit fly, Bactrocera dorsalis (Hendel)
(Diptera: Tephritidae), is the most serious fruit pest, damaging
up to 180 000 ha of fruit trees annually. Organophosphorus and
carbamate insecticides are commonly used to control this pest.
Field populations have developed resistance progressively to
the organophosphorus and carbamate insecticides as revealed
from recent resistance monitoring (22). Altered AChE is
considered a common cause of resistance in the fruit fly toward
organophosphorus insecticides (23, 24). However, the biochemi-
cal characteristics of AChE in the oriental fruit fly have not
been done.
Acetylcholinesterase (AChE, EC 3.1.1.7) plays an important
role in neurotransmission at cholinergic synapses by rapidly
hydrolyzing the excitatory neurotransmitter acetylcholine into
choline and acetic acid (1-4). Because it is the key enzyme in
an insect’s central nervous system, several inhibitors of this
enzyme have been developed (5, 6). Two classes of compounds,
organophosphorus and carbamate insecticides, are commonly
used to quasi-irreversibly inhibit AChE. However, more than
30 years of use of these insecticides has resulted in several
resistant insect species whose altered AChEs are less sensitive
to insecticide with these compounds (7, 8).
Because it is biologically significant to insect neurophysiology
and pest resistance, AChE has attracted a great deal of attention.
Much research has explored how AChE alteration may relate
to insect resistance. However, most of these studies have used
unpurified AChE, the homogenates of body parts, or the whole
* To whom correspondence should be addressed. Tel: 886-4-2239-1647
ext. 6915. Fax: 886-4-2239-5474. E-mail: ymhsiao@chtai.ctc.edu.tw.
† Chungtai Institute of Health Sciences and Technology.
We present here a simple, effective, two-step method for the
purification of oriental fruit fly AChE and have characterized
‡ Taiwan Agricultural Chemicals and Toxic Substances Research Institute.
10.1021/jf0494377 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/30/2004

Acetylcholinesterase from Oriental Fruit Fly
J. Agric. Food Chem., Vol. 52, No. 17, 2004 5341
of 1.0 mL were collected after the elution solution was applied to the
column. The purity of AChE from oriental fruit fly was confirmed by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). The fractions containing purified AChE were pooled, dialyzed
against PTS buffer to remove the Net4I, and concentrated using a
concentrator (Amicon, model 8050) at 4 °C.
the biochemical and molecular properties of the purified enzyme.
The knowledge obtained from this study should facilitate further
investigations on this important enzyme in the oriental fruit fly
and be helpful in the development of strategies for the resistance
management of this pest.
Determination of Protein Concentration. The protein content was
determined by the Bradford method using BSA as the standard (28).
The spectrophotometric assays were carried out with a spectrophotom-
eter at a wavelength of 595 nm.
MATERIALS AND METHODS
Insects. The oriental fruit fly used in this study came from an
insecticide susceptible laboratory strain. Three to five days old adults
were either used immediately or frozen under -80 °C for further
purification of AChE.
Electrophoretic Analysis of Purified AChE. Electrophoresis was
carried out with Hoefer Mighty Small II SE 250 and SE 260 Mini-
Vertical Gel Electrophpresis Units (Piscataway, NJ). The SDS-PAGE
(29) was used to estimate the molecular masses of the nonreduced and
reduced AChE. The AChE samples were treated by incubation in 2%
(w/v) SDS solution with or without 1.25% (v/v) â-mercaptoethanol in
a dry bath at 100 °C for 5 min, and they were loaded onto an 10%
SDS polyacrylamide gel. The gel was run for 1.5 h at 100 V at room
temperature. AChE in the gels was localized by silver staining (30).
The procedure of nondenaturing PAGE was similar to the SDS-
PAGE except that no SDS was used and it was performed in a 4 °C
cold room during electrophoresis. For the analysis of the interaction
between AChE and Triton X-100, both the gels and the electrode buffer
were incorporated with or without 0.1% (v/v) Triton X-100 (16). The
purified AChE was loaded (in duplicate) onto each lane of a 3-12%
gradient polyacrylamide gel. After the gel was run for 2 h at 8 mA, it
was cut into two halves. One half of the gel was used to stain the AChE
activity with ATC as a substrate for 1 h at 37 °C by the method of
Karnovsky and Roots (31) based on hydrolysis of a thiocholine ester
and formation of an insoluble copper complex, whereas the other half
was used to stain total proteins using the silver stain method (30).
Effect of pH and Temperature. The optimum pH for AChE activity
was determined at 37 °C by varying the pH of the reaction from 5.0 to
8.8. The optimum temperature for AChE activity was determined by
assaying the enzyme at various temperatures ranging from 15 to 45
°C.
Chemicals. Acetylthiocholine iodide (ATC), 1,5-bis(4-allyldimethyl-
ammonium phenyl)pentan-3-one dibromide (BW284C51), S-butyryl-
thiocholine iodide (BTC), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB),
eserine hemisulfate, ethopropazine hydrochloride, â-mercaptoethanol,
propionylthiocholine iodide (PTC), and Triton X-100 were purchased
from the Sigma Chemical Company (St. Louis, MO). The ECH
Sepharose 4B molecular markers (High Molecular Weight-SDS Cali-
bration Kit for Electrophoresis and High Molecular Weight Calibration
Kit for Native Electrophoresis) were purchased from Amersham
Pharmacia Biotech (Piscataway, NJ). Tetraethylammonium iodide
(Net4I), procainamide, and N-ethoxycarbonyl-2-ethoxy-1, 2-dihydro-
quinoline (EEDQ) were purchased from ACROS Organics (New Jersey,
United States). The bovine serum albumin (BSA) protein assay standard
II was purchased from Bio-Rad Laboratories (Hercules, CA).
Optimization of Conditions for AChE Extraction. To determine
the optimal pH for the AChE extraction, 0.1 g of frozen oriental fruit
fly heads was homogenized in 5 mL of ice-cold 0.1 M phosphate buffer
containing 0.5% (v/v) Triton X-100 at each of seven pH conditions
ranging from 5.8 to 8.0. The homogenates were centrifuged for 15 min
at 13000g at 4 °C, and the supernatants were used as the enzyme sources
for the AChE assay according to the spectrophotometric method of
Ellman et al. (25) using ATC as the substrate. One unit of AChE activity
was defined as the amount of enzyme needed to hydrolyze 1 µmol of
ATC per min at 37 °C under the assay condition.
To determine the optimal concentration of Triton X-100 to extract
AChE from the oriental fruit fly, six batches of 0.1 g of frozen oriental
fruit fly heads were homogenized in 5 mL of ice-cold 0.1 M phosphate
buffer (pH 7.4). Each batch contained a different concentration of Triton
X-100, ranging from 0 to 1.0% (v/v). The homogenates were
centrifuged, and the supernatants were used for the AChE assay as
described above.
Purification of AChE. After extraction by Triton X-100, AChE was
purified from the heads of adult fruit fly by affinity chromatography
using procainamide, a ligand specific for the choline binding site. The
procainamide affinity column was prepared according to manufacturer’s
instructions and stored at 4 °C using ECH Sepharose 4B as the coupling
gel and EEDQ for ligand immobilization (20, 26, 27). Briefly,
procainamide and EEDQ solutions were prepared by dissolving 0.68 g
of procainamide in 5 mL of water and 0.062 g of EEDQ in 5 mL of
ethanol. The two solutions were mixed and then added to 10 mL of
ECH Sepharose 4B. The procainamide-ECH Sepharose 4B coupling
reaction was carried out by gently mixing the solution with a rotator at
4 °C overnight. The procainamide-coupled Sepharose 4B was poured
into an 1 cm × 7.5 cm glass column and washed successively with
50% ethanol, 20 mM Tris-HCl buffer (pH 7.0), and 1 M NaCl solution.
The column then was equilibrated with 0.1 M phosphate buffer (pH
7.4) containing 0.5% (v/v) Triton X-100.
To extract AChE from the fly head, 8 g of frozen heads was
homogenized in 80 mL of ice-cold 0.1 M phosphate buffer (pH 7.4)
containing 0.5% (v/v) Triton X-100 and centrifuged at 13000g for 15
min at 4 °C. The supernatant was first filtered through two layers of
cheesecloth and then through a Whatman no. 1 filter to remove the
lipids. The filtrate, containing AChE, was applied to the procainamide-
based Sepharose 4B affinity column equilibrated with the same
extraction buffer. The column was washed extensively with 50 mM
phosphate buffer, 0.05% (v/v) Triton X-100 (pH 7.4), and 50 mM NaCl
(PTS) buffer until the absorbance at 280 nm fell below 0.01. The AChE
was then eluted with 30 mM Net4I in PTS buffer. The constant flow
rate was maintained at 30 mL/h with a peristaltic pump, and fractions
Thermal Stability. Thermal inactivation of the AChE was deter-
mined by incubating the purified enzyme in a water bath for 10 min at
various temperatures and then cooling it to room temperature for 5
min. This procedure was repeated at seven different temperatures
ranging from 25 to 60 °C each in triplicate.
Substrate Specificity and Kinetics of Purified AChE. The substrate
specificity of purified AChE was studied with three thiocholine esters
(ATC, PTC, and BTC) according to the method of Ellman et al. (25)
as modified by Zhu and Clark (17). We studied the AChE activities at
12 different concentrations (3-30000 µM) of each substrate. Controls
in the absence of AChE were included in the assays to correct
nonenzymatic hydrolysis of each substrate at high concentrations. The
maximum velocities (Vmax) and the Michaelis constants (Km) for each
substrate were determined according to a Lineweaver-Burk plot (32).
The turnover number (kcat) was calculated according to the molecular
mass of purified AChE and Vmax. The substrate specificity constant
(kcat/Km) was determined based on kcat and Km (33).
Inhibition of Purified AChE. The inhibitor specificity of purified
AChE was studied according to the method of Zhu and Brindley (34).
Purified AChE was preincubated with nine different concentrations of
each of three inhibitors (eserine, BW284C51, and ethopropazine) at
25 °C for 5 min. The remaining activity of AChE was determined.
The median inhibition concentration (IC50) for each inhibitor was
determined based on log-concentration vs probit (% inhibition) regres-
sion analysis.
RESULTS
Optimization of Conditions for AChE Extraction. The
optimal pH value for oriental fruit fly AChE extraction was
indicated by an increased enzyme activity from pH 5.8 to 6.8
and reached its peak at pH 7.4 (Figure 1A). No significant
difference in the enzyme activity was noted between the ranges
of pH 6.8 and 7.8. At pH 8.0, however, the enzyme activity

5342 J. Agric. Food Chem., Vol. 52, No. 17, 2004
Hsiao et al.
Figure 2. SDS−PAGE analysis of purified AChE. The purified AChE
samples were mixed with the sample buffer with or without reducing agent
and heated at 100 °C for 5 min in a dry bath. The treated samples were
loaded onto a 10% SDS−polyacrylamide gel. Electrophoresis was run for
1.5 h at 100 V at room temperature. The protein bands were visualized
by silver staining. Lane 1, molecular weight markers (High Molecular
Marker-SDS Calibration Kit for Electrophoresis, Amersham Pharmacia
Biotech); lane 2, AChE treated with the reducing agent â-mercaptoethanol;
and lane 3, AChE not treated with reducing agent.
either treated or not treated with the reducing agent â-mercap-
toethanol after the gel was stained by silver nitrate (Figure 2).
The molecular mass of the purified AChE was 116 kDa for
nonreduced (native) and 61 kDa for reduced AChE (subunit).
These results indicated that each molecule of AChE consisted
of two very similar or identical subunits connected with a
disulfide bond.
Figure 1. Optimal pH condition and Triton X-100 concentration for AChE
extraction. (A) Effects of pH on AChE extraction. The percentage of AChE
activity was calculated based on the highest AChE activity at pH 7.4.
The results are the means of three replications (n ) 3). (B) Effects of
Triton X-100 on AChE extraction. The percentage of AChE activity was
calculated based on the highest activity at 0.5% Triton X-100. The results
are the means of three replications (n ) 3).
Affinity chromatographically purified AChE was also ana-
lyzed by nondenaturing PAGE in the absence or presence of
Triton X-100 in both the gels and the electrode buffer system.
There was only one band in both electrophoretic conditions
(Figure 3). The band migrated much more slowly in the
presence of Triton X-100, which indicated that the purified
AChE and Triton X-100 may interact.
Characteristics of AChE. The AChE was shown to have a
pH optimum of about pH 7.4 (Figure 4A) and a temperature
optimum of about 37 °C (Figure 4B). Irreversible denaturing
of purified AChE mainly occurred above 45 °C (Figure 5),
which made it seem as though AChE decreased sharply after
exposition at 40 °C, after already being partially inactivated at
45 °C.
Substrate and Inhibitor Specificities and Kinetic Analysis
of Purified AChE. Three substrates, including ATC, PTC, and
BTC, were used to study the effect of substrate concentration
on the activity of purified AChE from the oriental fruit fly
(Figure 6). The optimal substrate concentrations were rather
broad, and the substrate inhibition phenomenon at higher
concentrations was not seen. Kinetic studies determined the
affinity of AChE to its different substrates. The Km ( SD
values were 87.9 ( 9.5 µM for ATC, 26.9 ( 1.4 for PTC, and
195.3 ( 22.0 for BTC (Table 2). The hydrolyzing efficiencies
of AChE for the three substrates, as indicated by their Vmax
values, were 833.3 ( 19.3 µmol/min/mg protein for ATC, 222.2
( 11.0 for PTC, and 57.5 ( 3.6 for BTC. These results
indicated that the affinities of purified AChE to ATC and BTC
were 3.3- and 7.3-fold, respectively, lower than that to PTC, as
indicated by their Km values. In addition, the hydrolyzing
efficiencies of purified AChE for ATC and PTC were 14.5-
and 3.9-fold higher than that for BTC, as indicated by their
Vmax values. The turnover numbers (kcat) of AChE from oriental
Table 1. Summary of the Purification Steps for Oriental Fruit Fly
AChE by Triton X-100 Extraction and Procainamide-Based Affinity
Chromatography
total activity total protein specific activity
yield
(%)
purification
factor (fold)
steps
(µmol/min)
(mg)
(µmol/min/mg)
Triton X-100
extraction
affinity column
35.095
413.369
0.085
100
51.966
1
18.237
0.026
690.931
8183
decreased. The condition for AChE extraction was further
optimized by the addition of 0.5% (v/v) Triton X-100 (Figure
1B). The enzyme activity increased with increases in the Triton
X-100 concentration and reached a maximum at 0.5% (v/v).
The AChE activity was slightly inhibited at 1.0%. In summary,
the optimal extraction condition for AChE purification would
be 0.1 M phosphate buffer, pH 7.4, containing 0.5% (v/v) Triton
X-100.
Purification of AChE from the Oriental Fruit Fly. The
AChE from the oriental fruit fly was successfully purified by
procainamide-based affinity chromatography following Triton
X-100 extraction. Typically, the overall purification factor and
yield were 8183-fold and 52%, respectively (Table 1). This yield
was considerably higher than those found in previous purifica-
tions of other AChEs (6, 19-21, 35-38). Approximately 26
µg of purified AChE with a specific activity of about 690 µmol/
min/mg protein was obtained from 8 g of the oriental fruit fly
heads. These results were reproducible over several purification
attempts during the course of the study. The degree of
purification was further confirmed by SDS-PAGE using the
silver staining method (see below).
Molecular Properties of Purified AChE. The SDS-PAGE
analysis showed one major protein band for AChE samples

Acetylcholinesterase from Oriental Fruit Fly
J. Agric. Food Chem., Vol. 52, No. 17, 2004 5343
Figure 4. pH (A) and temperature (B) activity profiles of oriental fruit fly
AChE. The activities are expressed as a percentage of the maximal activity,
and the results are the means of three replications (n ) 3). The purified
enzyme was used.
Figure 3. Nondenaturing PAGE analysis of the purified AChE in the
absence (A) or presence (B) of 0.1% Triton X-100. The purified AChE
was loaded to each lane of a 3−12% gradient polyacrylamide gel. The
gel was run for 2 h at 8 mA in a 4 °C cold room. After electrophoresis,
the gel was cut into two halves. One half was stained for AChE activity
(lane 3) using ATC as a substrate. The color was developed for 1 h at
37 °C. The other half was stained for total protein with silver (lanes 1
and 2). Lane 1 was a molecular weight marker (High Molecular Marker
Calibration Kit for Native Electrophoresis, Amersham Pharmacia Biotech).
fruit fly for ATC, PTC, and BTC were 97000, 26000, and 6700,
respectively. The substrate specificity constants (kcat/Km) of
AChE from oriental fruit fly for ATC, PTC, and BTC were
1103, 968, and 34, respectively. The substrate specificity
constant for ATC was 32-fold higher than that for BTC,
indicating that AChE had a much higher specificity to ATC
than to BTC.
The purified AChE was highly sensitive to inhibition by
eserine, a general inhibitor for both AChE and butyrylcholin-
esterase (BChE, EC 3.1.1.8) and BW284C51, a relatively
specific inhibitor for AChE, but was much less sensitive to
inhibition by ethopropazine, a relatively specific inhibitor for
BChE (Figure 7). At a concentration of 10 µM, both eserine
and BW284C51 inhibited 99 and 97% of the enzyme activity,
respectively. However, only 53% of the enzyme activity was
inhibited by ethopropazine at the same concentration. The
median inhibition concentrations (IC50, mean ( SD) were
Figure 5. Thermal stability of oriental fruit fly AChE. The activities are
expressed as a percentage of the maximal activity, and the results are
the means of three replications (n ) 3). The purified enzyme was used.
DISCUSSION
The two-step fly head AChE purification procedure that we
developed, Triton X-100 extraction and procainamide-based
affinity chromatography, afforded a 8183-fold purification factor
with a yield of 52%. The purified enzyme was shown to be
(2.80 ( 0.57) × 10-8 M for eserine, (8.59 ( 1.67) × 10-8
M
for BW284C51, and (1.17 ( 0.65) × 10-5 M for ethopro-
pazine.

5344 J. Agric. Food Chem., Vol. 52, No. 17, 2004
Hsiao et al.
Figure 7. Effect of inhibitor concentrations on AChE activity. The inhibitor
with a different concentration was mixed with diluted AChE. The remaining
AChE activity was determined after the mixture was incubated for 5 min
at 25 °C. Results are the means of five determinations (n ) 5). Eserine
(O), BW284C51 (9), and ethopropazine (2).
Figure 6. Effect of substrate concentrations on AChE activity. The effect
of substrate concentration was investigated using the model substrates
ATC (O), PTC (9), and BTC (2). The results are the means of five
determinations (n ) 5).
tarnished plant bug (36), 128 for Colorado potato beetle (17),
5 for lesser grain borer (5), 1456 for cotton aphid (21), and 691
for oriental fruit fly (this study). The variation in the specific
activity of the purified AChE among different insect species
may be partially due to the degree of the purity of AChE but
might also be due to variation in the insect species. Our results
are also consistent with the notion that insects in Hemiptera,
Coleoptera, and Lepidoptera exhibit lower levels of AChE
activity than in Diptera (41).
Table 2. Kinetic Parameters of AChE Purified from Oriental Fruit Fly
in Hydrolyzing Three Model Substratesa
Vmax
Vmax/Km
kcat
kcat/Km
(µM-1 min-1
)
substrates
Km (µM)
(µmol/min/mg) (ratio) (min-1
)
ATC
PTC
BTC
87.92 ± 9.50 833.33 ± 19.34 9.48
26.87 ± 1.42 222.22 ± 11.02 8.27
97000
26000
6700
1103
968
34
195.29 ± 21.96 57.47 ± 3.56
0.29
The SDS-PAGE analysis revealed a rather distinctive 116
kDa protein for the AChE sample untreated with the reducing
agent and a single 61 kDa band for the treated sample. The
result indicates that AChE purified from oriental fruit fly was
dimeric, as has been found in the AChEs of other insects (41).
The molecular mass of the AChE subunit of oriental fruit fly
was virtually the same as those purified from Drosophila (56
kDa) (39), horn fly (54 kDa) (42), Colorado potato beetle (65
kDa) (17), lesser grain borer (56 kDa) (5), waxmoth (60 kDa)
(19), greenbug (72 kDa) (20), and cotton aphid (63.5 kDa) (21).
As 60-65 kDa is almost universally reported as the subunit
size from invertebrate and vertebrate sources (19), this value is
consistent with previously reported results.
Insect AChE exists mostly in different native forms. For
example, M. domestica and L. hesperus Knight AChE have been
reported to have three forms (14, 16), and in the Colorado potato
beetle, L.. decemlineata (Say), the purified native AChE had
two molecular forms (17). In contrast, results with the purified
AChE from the oriental fruit fly obtained from nondenaturing
PAGE, whether in the presence or absence of Triton X-100,
showed only one major band, suggesting a single molecular
form. This band corresponded to the silver-stained protein band,
which suggests that we had only one major molecular form of
AChE in our purified AChE samples. Because there was a
striking difference in migration during nondenaturing electro-
phoresis with or without Triton X-100, the major molecular form
a The results are presented as the means ± SD (n ) 5).
electrophoretically homogeneous with a single band resolved
on SDS-PAGE using silver staining.
The degree of purification is not readily comparable between
species as it obviously reflects the initial amount of extraneous
tissue. However, in this study, extracts were made from only
the heads of the insects. Differences caused by insect materials
and assay conditions usually preclude direct comparisons of
studies by different investigators. The increase of specific
activity from 0.085 units/mg protein in the initial extract from
heads to 691 units/mg protein in the purified AChE preparation
indicated that AChE in the initial extract accounted for only
0.0123% of the total proteins. This amount is approximately
half of the 0.02% value reported in the initial homogenate of
Drosophila heads (39). The purification factor was larger than
AChE purified from heads of G. mellonella (283-fold) (19) but
smaller than that from heads of L. decemlineata (39000-fold)
(17, 35) and D. Virgifera Virgifera (20000-fold) (6). However,
the activity yield of 52% was comparable to the published yields
using the same method for other species, e.g., 54% for
Rhyzopertha dominica (5) and 51% for L. decemlineata (17).
Using ATC as a substrate, the specific activity of purified
AChE has been reported to be 1350 units/mg protein for
Drosophila heads (39), 385 for horn fly (40), 50 for Western
Table 3. Kinetic Parameters Using ATC as Substrate and IC50 Values of Different Inhibitors of AChE Purified from Different Insect Species
IC50 (M) for different inhibitor
BW284C51
Km
(µM)
Vmax
(µmol/min/mg)
species
eserine
ethopropazine
Colorado potato beetle
lesser grain borer
Western corn rootworm
greenbug
14.9
24.2
19.7
57.6
177.7
18.7
184.8
78.0
2.3 × 10-8
0.9 × 10-9
2.6 × 10-8
4.8 × 10-7
5.4 × 10-9
8.0 × 10-8
1.9 × 10-8
1.5 × 10-7
8.7 × 10-6
1.1 × 10-6
1.1 × 10-5
1.0 × 10-4

Acetylcholinesterase from Oriental Fruit Fly
J. Agric. Food Chem., Vol. 52, No. 17, 2004 5345
ACKNOWLEDGMENT
of AChE purified here could be an amphiphilic form (43). In
other words, the AChE purified from oriental fruit fly was
affected by the nature of the detergent, and the slower migration
may have been due to binding between the detergent and the
hydrophobic domain of the amphiphilic AChE molecule, which
then retarded the migration of the protein (43).
We thank Ms. Ju-Chun Hsu for her generous supply of the fruit
fly used in this study.
LITERATURE CITED
Substrate inhibition of enzyme activity at high concentrations
is a typical characteristic for AChE but not for BChE and,
therefore, has been accepted widely as one of the criteria used
to distinguish between AChE and BChE (41). However, our
study has not indicated any significant substrate inhibition with
AChE purified from oriental fruit fly. Similar results have been
reported previously in studies of AChE from Western flower
thrip (44) and altered AChE from organophosphorus and/or
carbamate insecticide resistant tobacco budworm (45) and
Colorado potato beetle (35), none of which exhibited excess
substrate inhibition. Several researchers have hypothesized that
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peripheral anionic sites, leading to an inactive enzyme-
substrate-substrate complex (46-48). This hypothesis suggests
that peripheral anionic sites of oriental fruit fly AChE might
be structurally different from those on AChE of other insect
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Received for review April 5, 2004. Revised manuscript received June
22, 2004. Accepted June 25, 2004. This study was funded by the Taiwan
Agricultural Chemicals and Toxic Substances Research Institute,
Republic of China.
JF0494377