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Purification and Characterization of Carboxylesterases of a Rice Green LeafhopperNephotettix cincticepsUhler
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PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO. 0022
54, 181–189 (1996)
Purification and Characterization of Carboxylesterases of a Rice Green Leafhopper Nephotettix cincticeps Uhler SHIH-WEN CHIANG
AND
CHIH-NING SUN
Department of Entomology, National Chung-Hsing University, Taichung, Taiwan 40227, Republic of China Received September 6, 1995; accepted February 5, 1996 More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper, Nephotettix cincticeps Uhler, could be resolved by isoelectric focusing electrophoresis. A combination of ammonium sulfate fractionation, gel filtration, and chromatofocusing chromatography was used to isolate and purify these isozymes. Four fractions, i.e., E1, E2, E3, and E4, with pI’s ranging from 5.1 to 4.85, were obtained. The most abundant E3 had a molecular mass of 58.6 kDa and appeared electrophoretically homogeneous on SDS–PAGE. [1,3-3H]Diisopropyl fluorophosphate-labeling experiment revealed that the proteins of 58.6 kDa, a minor component of E2 and the major component of E4, were the carboxylesterase isozymes sought. A protein of the same molecular weight which existed in a very minute amount in E1 and was barely detectable on SDS–PAGE by Coomassie blue staining was actually the carboxylesterase isozyme of pI 5.1. All four fractions exhibited significant activity toward several model substrates with a-naphthyl butyrate being the most preferred. Their activity toward malathion, permethrin, and cypermethrin was ca. 106-fold lower than their activity toward the model substrates. The pyrethroids were hydrolyzed more readily than malathion by these hydrolases, and cis-permethrin was more preferred than the trans-isomer. E4 was the only fraction that cross-reacted with the antiserum against carboxylesterases of a rice brown planthopper, Nilaparvata lugens. Among the four isozyme fractions, E3, the most abundant, showed surprisingly low activity toward all four insecticides and was actually the least active fraction toward cis-permethrin and cypermethrin. A field strain of N. cincticeps had 26- to 37-fold higher carboxylesterase activity toward the model substrates than a susceptible strain. Yet, little, if any, difference in the hydrolysis of malathion, permethrin, and cypermethrin was observed between these two strains. The field strain produced at least eight times more carboxylesterases than the susceptible strain. © 1996 Academic Press, Inc.
INTRODUCTION
Nephotettix spp. are widely distributed green leafhoppers of rice. Of the three known species in Taiwan Nephotettix cincticeps Uhler is the predominant one and an important vector of viruses that cause rice dwarf, transitory yellowing, tungro, and yellow dwarf diseases (1). A variety of chemicals including organophosphorus (OP),1 carbamate, pyrethroid insecticides, and their mixtures have long been used for the control of these leafhoppers (2). Several reports on the occurrence of OP, carbamate, and pyrethroid resistance in this insect have appeared since mid-1970s in Taiwan (3, 4). In Japan, malathion resistance of this leafhopper was first observed in early 1960s (5) and considerable 1 Abbreviations use: DFP, diisopropyl fluorophosphate; IEF, isoelecrtric focusing; a-NA, a-naphthyl acetate; b-NA, b-naphthyl acetate; b-NB, b-naphthyl butyrate; pNPA, p-nitrophenyl acetate; OP, organophosphorus; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
efforts have been made since then to investigate the resistance mechanisms (6). The OP resistance of this leafhopper has been found to associate with a high carboxylesterase activity by many researchers. Ozaki and his co-workers (7, 8) used thin-layer agar gel electrophoresis to resolve carboxylesterase isozymes of N. cincticeps and found that E2, E3, and E4 bands were more active in resistant than in susceptible strains. Subsequently, Miyata (9) reported that E1, E2, and E3 were able to degrade [14C]malathion with E2 being the most active form. Motoyama et al. (10) resolved five esterase peaks using chromatofocusing from an OP-resistant strain of this leafhopper and four peaks were found active toward malathion, paraoxon, and fenvalerate. They proposed a dual role for these esterases in resistance mechanisms: a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides. The present study attempted to isolate and
181 0048-3575/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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purify the major isozymes of carboxylesterases in N. cincticeps, to determine their activity toward some general substrates and insecticides, and to compare the isozyme makeup of this detoxifying enzyme in susceptible and resistant strains of this leafhopper. MATERIALS AND METHODS
Insects The susceptible strain of the green rice leafhopper was obtained from the Agricultural Research Station, ICI Japan Ltd., in 1993. The resistant strain was collected from the rice fields in central Taiwan. Chemicals The chemicals used in the enzyme assays were obtained from Sigma (St. Louis, MO) and E. Merck (Darmstadt, Germany) and those used for protein purification were products of Pharmacia (Uppsula, Sweden). Protein standards for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) were products of Bio-Rad (Richmond, CA), and reagents for protein quantitation were purchased from Pierce (Rockford, IL). [1,3-3H]Diisopropyl fluorophosphate (DFP) with a specific activity of 10 Ci/mmol and the autoradiography enhancer Enlightening were obtained from DuPont NEN (Boston, MA). Malathion (98%) and cypermethrin (97%) were products of Riedel-de Haen AG (Seelze, Germany), and trans-permethrin (99%) and cispermethrin (99%) were provided by the U.S. Environmental Protection Agency (Triangle Park, NC). Purification of Carboxylesterases Three grams of adult hoppers of the field (F) strain was ground with Polytron PT10-35 homogenizer for 2 min in 5 vol of 0.05 M Tris– HCl buffer (pH 7.2). The cheesecloth-filtered homogenate was centrifuged first at 10,000g for 15 min and then at 100,000g for 1 hr at 4°C. The supernatant was subject to ammonium sulfate fractionation and the fraction precipitated between 30 and 60% ammonium sulfate had 93% of the total carboxylesterase activity using
a-naphthyl acetate (a-NA) as substrate. This fraction was subsequently introduced onto a Sepharose 6B column (2.2 × 45 cm) and the column was eluted with 0.05 M Tris–HCl buffer (pH 7.2). Fractions with activity toward a-NA were pooled and concentrated and the buffer was changed to 0.025 M histidine–HCl buffer (pH 6.2) using a Centricon-30. For chromatofocusing, this solution (ca. 3 ml) was put onto a preequilibrated PBE 94 column (1 × 20 cm) which was first eluted with 5 ml of the histidine–HCl buffer and subsequently with polybuffer 74–HCl buffer (pH 4.0) at 6 ml/hr. A total of 120 fractions (one fraction every 15 min) were collected. Fractions with activity toward a-NA were individually analyzed (concentrated if necessary) on IEF and SDS–PAGE for purity. Fractions which were not electrophoretically homogeneous were further resolved using Sephadex G-75 and Sephacryl S-200 chromatography. Both the Sephadex G-75 column (1.6 × 45 cm) and the Sephacryl S-200 column (1 × 45 cm) were eluted with 0.05 M Tris–HCl (pH 7.2) at 6 ml/hr and fractions (every 15 min) were tested for activity toward a-NA. Active fractions were concentrated and analyzed on SDS–PAGE for purity. Enzyme Assay For the hydrolysis of a-NA, a-naphthyl butyrate (a-NB) and b-naphthyl acetate (b-NA), the method of van Asperen (11) was adpoted. A Beckman DU-70 spectrophotometer was used to measure the products of the reaction, i.e., 600 nm for a-NA and a-NB, and 555 nm for b-NA. a-Naphthol and b-naphthol were used to construct the standard curves. For the hydrolysis of p-nitrophenyl acetate (p-NPA) the method of Ljungquist and Augustinsson (12) was followed. A molar extinction coefficient of 16.36 mM−1 cm−1 was used to convert the absorbance changes at 400 nm into the amounts of product. The partially purified fractions or the supernatant of 10,000g centrifugation of the homogenate of the hoppers was used as the enzyme source. Buffer solution of equivalent volume was added to the controls to replace the enzyme solution. Three replicates were performed for all treatments.
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In vitro degradation of several insecticides by the homogenates (prepared as described above) and the four carboxylesterase fractions was determined by adding 10 ml of enzyme source and 10 ml of insecticide solution (1 mg/ml of ethylene glycol monoethyl ether) in 0.1 M Tris–HCl buffer (pH 8.0) to make a final volume of 0.5 ml. Incubation at 37°C was carried out overnight for malathion and for 5hr for permethrin and cypermethrin. At the end of the incubation, 0.5 ml n-hexane was added with vigorous shaking. The unreacted insecticides in the hexane layer were quantitated using a Varian 3400 gas chromatograph with a 15% OV-17 and 1.95% OV-210 Chrom W HP glass column (2 mm × 2 m) at 240°C, and a flame photometric detector (300°C) for the OPs and a Ni-63 electron capture detector (320°C) for the pyrethroids. For the controls, buffer solution was added to replace the enzyme source. All treatments were duplicated or triplicated. Proteins were quantitated by bicinconinic acid assay according to the manufacturer’s (Pierce) instruction. Electrophoresis and Immunoblotting SDS–PAGE was performed according to Laemmli (13) using a 3.75% stacking gel and a 12.5% running gel with a Bio-Rad Mini-Protean II electrophoresis cell at 100 V. IEF electrophoresis was performed between pH 4.5 and 5.4 using a Bio-Rad 111 mini-IEF cell according to the manufacturer’s instruction. The carboxylesterase activity was stained using a-NA as substrate. Proteins on SDS–PAGE were transferred to NC paper with a Hoefer TE77 Semiphor Transfer Unit (San Francisco, CA) and immunoblotting was performed according to Blake et al. (14). Primary antiserum to carboxylesterase El of a brown planthopper, Nilaparvata lugens, raised in rabbit (15) (2000-fold dilution) and an alkaline phosphatase-conjugated goat antirabbit IgG (5000-fold dilution) were used. [1,3-3H]Diisopropyl Fluorophosphate Labeling of Carboxylesterases Adult leafhoppers were homogenized in 0.02M sodium phosphate buffer (pH 7.8) and
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the homogenate was centrifuged at 10,000g twice. The supernatant or the carboxylesterase fraction obtained above was incubated with 3 mCi [3H]DFP dissolved in propylene glycol at room temperature for 1hr and dialyzed overnight at 4°C in the above buffer to remove the unbound DFP. The supernatant was subsequently analyzed by SDS–PAGE or IEF. The gels were fixed for 30 min in acetic acid and methanol, submerged in Enlightening solution for 30 min. The SDS–PAGE gel was vacuum dried and the IEF gel was air-dried before processing for fluorography. RESULTS
Purification of Carboxylesterase Isozymes of N. cincticeps In the crude homogenate of this leafhopper, more than four carboxylesterase isozymes with pI’s between 4.5 and 5.4 could be observed using a-NA as substrate on IEF (Fig. 1). The supernatant after 1hr of 100,000g centrifugation of the homogenate retained more than 83% of the total carboxylesterase activity. The proteins precipitated between 30 and 60% ammonium sulfate contained 84% of the activity in this supernatant (Table 1). While Sepharose 6B chromatography removed only a very limited amount of large, unwanted proteins (Fig. 2A), subsequent chromatofocusing removed a significant amount of proteins with higher pI’s (Fig. 2B). IEF analysis of each tube with enzyme activity from chromatofocusing showed that four major fractions
FIG. 1. IEF of carboxylesterases in the homogenates of N. cincticeps. Lanes 1–7, susceptible strain (1/10 of an adult per lane); and lanes 8–15, field strain (1/80 of an adult per lane).
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CHIANG AND SUN TABLE 1 Purification of Carboxylesterase Isozymes (E1, E2, E3, and E4) of a Field (F) Strain of N. cincticeps
Step Crude homogenate 100,000g supernatant 30–60% (NH4)2SO4 precipitate Sepharose 6B Chromatofocusing E1 E2 E3 E4 a b
Total activitya (mmol/min)
Specific activity (mmol/min/mg protein)
129.1 59.5
208.4 173.0
1.6 2.9
100 83
1.0 1.8
21.7 17.3
145.6 121.0
6.7 7.0
70 58
4.2 4.4
17.2 13.2 49.8 0.9
(13.2) (16.5) 38.3 (4.5)
Total protein (mg)
(1.3)b (0.8) 1.3 (0.2)
Yield (%)
Purification (fold)
8.3 6.3 24 0.43
(8.3) (10.3) 23.9 (2.9)
a-NA was used as substrate. Since E1, E2, and E4 fractions had contaminating proteins, some of their data are thus put in parentheses.
could be resolved (Fig. 3). Tubes showing only one band with enzyme activity on IEF were pooled and concentrated, and the four fractions were designated as E1, E2, E3, and E4 (Fig. 4A). Their pI’s were 5.1, 5.0, 4.9, and 4.85, respectively. SDS–PAGE analysis revealed that while E1 fraction appeared to have only one protein of 83.7 kDa, the other three fractions all consisted of three proteins of 83.7, 58.6, and 42 kDa (Fig. 4B). Subsequent Sephadex G-75 chromatography removed the two contaminating proteins from E3 (Fig. 5A), and the remaining protein of E3 had a molecular weight of 58.6 kDa (Fig. 5B) with carboxylesterase activity representing
24-fold purification (Table 1). However, Sephadex G-75 followed by Sephacryl S-200 chromatography failed to resolve the three proteins in E2 and E4. Fluorographic analysis clearly showed that in all four fractions, only one protein of 58.6 kDa was labeled by [1,3-3H]DFP (Fig. 4C). Therefore, the 83.7-kDa protein in E1 fraction was only a contaminant, and the E1 isozyme (58.6 kDa) present in very minute proportion was barely detectable (Fig. 4B). The 83.7 and 42-kDa proteins in both fractions E2 and E4 were also contaminating proteins. Therefore, all four carboxylesterase isozymes of this leafhopper had the same molecular mass of 58.6 kDa but varied pI’s (5.1 to 4.85). Although
FIG. 2. (A) Sepharose 6B chromatography of carboxylesterases after ammonium sulfate fractionation of the homogenate of a field strain of N. cincticeps, and (B) subsequent chromatofocusing.
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FIG. 3. IEF of carboxylesterases in the fractions collected from chromatofocusing. The gel was stained for activity toward a-NA.
three of the four fractions were not purified carboxylesterase isozymes, they were still used for further studies. Certain trends with regard to their biochemical and toxicological properties still could be seen. Biochemical and Toxicological Properties of Carboxylesterase Isozymes of N. cincticeps All four isozymes were able to hydrolyze the model substrates tested, i.e., a-NA, a-NB, b-NA, and p-NPA, with a-NB being the most preferred one (Table 2). The presence of contaminating proteins in all fractions except E3 makes comparison of specific activity among the four isozymes difficult.
The hydrolysis of malathion, permethrin, and cypermethrin by these four carboxylesterase fractions was at least 106-fold less than the hydrolysis of model substrates (Table 2). Yet, for the same isozymes, malathion was hydrolyzed 3- to 10-fold less efficiently than the two pyrethroids. All four isozymes of this leafhopper hydrolyzed cis-permethrin 2- to 5-fold faster than trans-permethrin. Despite of the presence of contaminating proteins, some tendency among the four fractions can still be seen rather clearly. The general ability of E3, the electrophoretically pure and most abundant form, to degrade these three insecticides appeared to the the lowest. E4 exhibited especially high activity toward the two pyrethroids. In an immunoblotting experiment, a protein (or proteins) of ca. 58.6 kDa in this leafhopper cross-reacted weakly with an antiserum raised against the dominant carboxylesterase isozyme El of N. lugens, a rice brown planthopper (Fig. 6A). When the four fractions were tested, only the protein of 58.6 kDa in E4 showed apparent crossreactivity (Fig. 6B). Carboxylesterases of Susceptible and Field Strains of N. cincticeps Although both the susceptible (S) and field (F) strains demonstrated extensive carboxylesterase activity toward several model substrates with the latter having 26- to 37-fold higher activity, their ability to degrade malathion, permethrin, and cypermethrin was again very limited and differed only slightly (Table 3). IEF analysis of carboxylesterases in the homogenates of this leafhopper revealed that while more than
TABLE 2 Hydrolysis of Some Model Substrates and Insecticides by the Four Carboxylesterase Fractions of N. cincticeps Specific activity
mmol/min/mg protein Isozyme E1 E2 E3 E4 a b c
a-NA
a b,c
13.2 ± 0.9 16.5 ± 0.4 38.3 ± 0.6 4.6 ± 0.1
pmol/hr/mg protein
a-NB
b-NA
p-NPA
Malathion
trans-Permethrin
cis-Permethrin
Cypermethrin
16.9 ± 2.1 19.5 ± 0.6 52.2 ± 1.7 9.6 ± 0.2
13.5 ± 1.2 16.5 ± 2.7 40.9 ± 0.7 6.3 ± 0.2
12.5 ± 1.3 15.4 ± 0.7 27.8 ± 1.2 5.2 ± 0.7
37.5 ± 0.2 20.8 ± 0.3 25.6 ± 1.0 23.7 ± 0.1
68.3 ± 0.1 20.5 ± 1.4 27.0 ± 0.2 140 ± 1
163 ± 1 102 ± 1 83 ± 1 235 ± 1
98.3 ± 0.3 52.6 ± 0.4 23.8 ± 0.2 213 ± 1
a-NA, a-naphthyl acetate; a-NB, a-naphthyl butyrate; b-NA, b-naphthyl acetate; and p-NPA, p-nitrophenyl acetate. Mean ± SE of two or three replicates. Since E1, E2, and E4 have contaminating proteins, the specific activities in this table are underestimated.
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FIG. 4. (A) IEF of the four carboxyelsterase fractions obtained in chromatofocusing and the homogenate (H) of N. cincticeps. The gel was stained for activity toward a-NA; (B) SDS–PAGE of the same fractions and the gel was stained for proteins by Coomassie blue; and (C) SDS–PAGE of the [3H]DFP-labeled carboxylesterase fractions.
four isozymes could be detected in F strain, only the most abundant E3 was clearly observed in S strain (Fig. 1). Fluorographic analysis of [3H]DFP-labeled carboxylesterases following SDS–PAGE shows that while three weak bands appeared in S strain, only one intensely labeled band corresponding to a molecular mass of 58.6 kDa appeared in F strain (Fig. 7A) The latter was at least 8-fold higher in quantity than the equivalent protein in S strain. Similar analysis following IEF shows that all four major carboxylesterase isozymes were present in higher amounts in F strain than in S strain, and E3 and E4 were the most abundant forms (Fig. 7B). DISCUSSION
Among the four carboxylesterase isozymes of F strain of N. cincticeps obtained in this study, only E1 and E3 could be purified to electrophoretic homogeneity (Figs. 4B and 5B). Al-
though the molecular mass of the three proteins in E2 and E4 fractions differed sufficiently for their separation by gel filtration, Sephadex G-75 and Sephacryl S-200 chromatography was unable to resolve them. Glycoproteins have been reported to interact sometimes with gel filtration media that are dextran-based, such as Sephadex gels, and thus exhibit irregular chromatographic behavior (16). This might account for the failure of our attempt to purify E2 and E4 fractions. [3H]DFP-labeling experiments showed that only the 58.6-kDa protein(s) in these four carboxylesterase fractions (Fig. 4C) and the homogenate of F strain of N. cincticeps (Fig. 7A) were bound to DFP. Therefore, the four carboxylesterase isozymes of this leafhopper had to be proteins of the same molecular mass 58.6 kDa, whose activity toward a-NA was observed on IEF gel with pI’s of 5.1, 5.0, 4.9, and 4.85 (Fig. 4A). This protein was of
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FIG. 5. (A) Sephadex G-75 chromatography of E3 fraction obtained in chromatofocusing; and (B) subsequent SDS–PAGE of fractions 25–34 with Coomassie blue staining.
such a minute amount in E1 fraction that it was barely observed on SDS–PAGE and the dominant 83.7-kDa protein was not the isozyme sought (Fig. 4B). Similarly, the 83.7- and 42kDa proteins in E2 and E4 fractions were not the isozymes either (Fig. 4B). The present study found that all four carboxylesterase isozymes of N. cincticeps in general were able to hydrolyze the two pyrethroids, permethrin and cypermethrin, much more efficiently than malathion, the OP insecticide readily hydrolyzed by this enzyme in many insects (17). This observation does not quite agree with data reported by Motoyama et al. for the same leafhopper (10). In their work, one of the five forms of carboxylesterases was found to degrade malathion twice as fast as the pyrethroid fenvalerate, and three other forms exhibited approximately equal activity toward these two insecticides. In both mammals and insects carboxylesterases have been reported to prefer permethrin, a pyrethroid of primary alcohol ester, to cypermethrin, a pyrethroid of secondary alcohol ester (18, 19), and trans-permethrin to cis-permethrin (19–23). In this study, cispermethrin was degraded at a rate 2- to 5-fold higher than its trans-isomer and all four isozymes hydrolyzed cypermethrin to a significant extent. Similarly, in another study, larvae of the green lacewing, Chrysopa carnea Stephens, an important predator to many agricultural pests, showing a considerable tolerance to pyrethroids, has unusually high activity for hydro-
lyzing the cis-isomers of permethrin and cypermethrin, 2- to 3-fold faster than the corresponding trans-isomers (24). The same phenomenon was also observed in a rice brown planthopper N. lugens (15). The presence of contaminating proteins in three of the carboxylesterase fractions obtained in the present study results in an underestimation of their specific activities toward various substrates, especially of E1 and E2, and makes comparisons difficult. Nevertheless, some conclusions still could be drawn. The most abundant E3 isozyme (as shown in Figs. 1 and 7B) possessed the lowest activity toward practically all three insecticides tested (Table 2). The E4 fraction which had an overall lower activity toward the model substrates showed unusual preference toward the two pyrethroids, permethrin and cypermethrin. Motoyama et al. (10) found that in N. cincticeps peak V of chromatofocusing of this hydrolase, compared with the other four peaks, had relatively high activity toward fenvalerate compared with malathion and paraoxon. This peak V might correspond to the carboxylesterase isozyme in E4 fraction of this study. Significant amount of this isozyme was shown in the DFP-labeling experiment to be present in the F strain (Fig. 7B), collected from the rice paddy in central Taiwan where a number of pyrethroids have been applied for the control of various leafhoppers and planthoppers. Among the four isozyme, E4 was the only one showing cross-reactivity toward the antise-
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FIG. 6. Western blot analysis of immuno-cross-reactivity between E1 of Nilaparvata lugens (BPH) and carboxylesterases of N. cincticeps (RGLH). (A) SDS–PAGE of homogenates of BPH and RGLH, M, molecular marker; and (B) SDS–PAGE of homogenate of BPH, lanes 1 to 5 loaded with 1/8, 1/16, 1/32, 1/64, and 1/128 of a planthopper, and 50 µg each of E1, E2, E3, and E4 of RGLH.
rum against carboxylesterase E1, the isozyme overproduced in resistant rice brown planthopper, N. lugens (Fig. 6B). The significance of this immunorelationship is unclear. TABLE 3 Hydrolysis of Some Model Substrates and Insecticides by the Homogenates of a Susceptible (S) and a Field (F) Strains of N. cincticeps Specific activity Substrate
a-NAa a-NB b-NA p-NPA
Malathion trans-Permethrin cis-Permethrin Cypermethrin
S
F
F/S
mmol/min/mg protein 0.10 ± 0.01b 2.77 ± 0.42 0.16 ± 0.03 4.12 ± 0.27 0.09 ± 0.01 2.45 ± 0.65 0.07 ± 0.01 2.59 ± 0.11
27.7 25.8 27.2 37.0
pmol/hr/mg protein ND 0.8 ± 0.3 0.6 ± 0.07 0.6 ± 0.2 1.6 ± 0.01 1.4 ± 0.05 0.7 ± 0.02 1.0 ± 0.02
— 1.0 0.9 1.4
a a-NA, a-naphthyl acetate; a-NB, a-naphthyl butyrate; b-NA, b-naphthyl acetate; and p-NPA, p-nitrophenyl acetate. b Mean ± SE of two or three replicates.
FIG. 7. Fluorographic analysis of [1,3-3H]DFP-labeled carboxylesterases. (A) SDS–PAGE; and (B) IEF of homogenates of a susceptible (S) and a field (F) strains of N. cincticeps.
The correlation between high carboxylesterase activity and malathion resistance in N. cincticeps has been observed by several researcher (6–9). However, the action of this enzyme remained ambiguous until Motoyama et al. (10) proposed that it played a dual role as the overproduced carboxylesterase isozyme E4 in Myzus persicae (25): a catalyst for hydrolysis of malathion and fenvalerate and a binding protein for the oxygen analogs of other OP insecticides. The field strain of this leafhopper in the present study exhibited up to ca. 40-fold higher carboxylesterase activity toward the model substrates than the susceptible strain (Table 3, Fig. 1). However, practically no difference in the hydrolysis of malathion and the two pyrethroids was detected between these two strains (Table 3). In addition, 58.6-kDa proteins (Fig. 7A) with varying pIs (Fig. 7B), which presumably are the carboxylesterase isozymes seen in Fig. 1, were overproduced at least eight times more in the F strain than in S strain. All these data seem to suggest that the carboxylesterase isozymes in resistant N. cincticeps might serve more as binding proteins than as catalysts. The two ad-
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ditional weak bands in S strain (Fig. 7A) could be other serine hydrolases. ACKNOWLEDGMENTS We thank Japan ICI Ltd. and Dr. C. C. Chen of Taichung Agricultural Improvement Station for supplying the susceptible and field strains, respectively, of the green rice leafhopper. This work was supported by Grants NSC83-0409B005-002 and NSC83-0618-B005-001 of the National Science Council, Taiwan, Republic of China.
REFERENCES 1. C. C. Chen, The occurrence and control of homopterous insects transmitting virus diseases of rice in Taiwan, in “Proceedings of Insect Ecology and Control,” pp. 113–122, Academia Sinica, Taipei, 1978. 2. Anonymous, “Plant Protection Manual,” pp. 38–56, Taiwan Provincial Department of Agriculture and Forestry, Taichung, 1992. 3. T. Y. Ku and S. C. Wang, The efficacy of pesticides for the control of major insect pests on rice, in “Proceedings of Insect Ecology and Control,” pp. 143– 162, Academia Sinica, Taipei, 1978. 4. H. L. Kao, M. Y. Liu, and C. N. Sun, Nephotettix cincticeps (Homoptera: Cicadellidae) resistance to several insecticides in Taiwan, J. Econ. Entomol. 75, 495 (1982). 5. K. Ozaki, The resistance to organophosphorus insecticides of the green rice leafhopper, Nephotettix cincticeps Uhler and the smaller brown planthopper, Laodelphax striatellus Fallen, Rev. Plant Protect. Res. 2, 1 (1969). 6. H. Hama, Mechanism of insecticide resistance in green rice leafhopper and small brown planthopper, Rev. Plant Protect. Res. 13, 54 (1980). 7. K. Ozaki, Y. Kurosu, and H. Koiki, The relation between malathion resistance and esterase activity in the green rice leafhopper, Nephotettix cincticeps Uhler, SABCO J. 2, 98 (1966). 8. K. Ozaki and T. Kassai, Relationship between patterns of esterase zymogram and resistance to insecticides in green rice leafhopper, Nephotettix cincticeps Uhler, Botyu-Kagaku 42, 181 (1970). 9. T. Miyata, Detection and monitoring methods for resistance in arthropods based on biochemical characteristics, in “Pest Resistance to Pesticides” (G. P.Georghiou and T.Saito, Eds.), pp. 99–116, Plenum, New York, 1983. 10. N. Motoyama, L. R. Kao, P. T. Lin, and W. C. Dauterman, Dual role of esterases in insecticide resistance in the green rice leafhopper, Pestic. Biochem. Physiol. 21, 39 (1984).
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11. K. van Asperen, A study of housefly esterases by means of a sensitive colorimetric method, J. Insect Physiol. 8, 401 (1962). 12. A. Ljungquist and K. Augustinsson, Purification and properties of two carboxylesterases from rat-liver microsomes, Eur. J. Biochem. 23, 303 (1971). 13. U. K. Laemmli, Cleavage of structural protein during assembly of the head of bacteriophage T4, Nature 227, 680 (1970). 14. M. S. Blake, G. J. Johnson, G. J. Russell-Johnes, and E. C. Gotschlich, A rapid, sensitive method for detection of alkaline phosphatase-conjugated antibody on Western blots, Anal. Biochem. 236, 175 (1984). 15. W. L. Chen and C. N. Sun, Purification and characterization of carboxylesterases of a rice brown planthopper, Nilaparvata lugens Stal, Insect Biochem. Mol. Biol. 24, 247 (1994). 16. R. K. Scopes, “Protein Purification: Principles and Practice,” 2nd ed., Springer-Verlag, New York, (1987). 17. F. Matsumura, “Toxicology of Insecticides,” Plenum, New York, (1985). 18. C. O. Abernathy, K. Ueda, J. L. Engel, L. C. Gaughan, and J. E. Casida, Substrate-specificity and toxicological significance of mouse liver microsomes, Pestic. Biochem. Physiol. 3, 300 (1973). 19. J. S. Holden, Absorption and metabolism of permethrin and cypermethrin in the cockroach and the cottonleafworm larvae, Pestic. Sci. 10, 295 (1979). 20. I. Ishaaya and J. Casida, Properties and toxicological significance of esterase hydrolyzing permethrin and cypermethrin in Trichoplusia ni larval gut and integument, Pestic. Biochem. Physiol. 14, 178 (1980). 21. C. O. Abernathy and J. E. Casida, Pyrethroids insecticides: Esterase cleavage in relation to selective toxicity, Science 179, 1235 (1973). 22. L. T. Jao and J. E. Casida, Insect pyrethroidhydrolyzing esterases, Pestic. Biochem. Physiol. 4, 465 (1974). 23. T. Shono, T Unai, and J. E. Casida, Metabolism of permethrin isomers in American cockroach adults, and cabbage looper larvae, Pestic. Biochem. Physiol. 9, 96 (1978). 24. I. Ishaaya and J. Casida, Pyrethroid esterases may contribute to natural pyrethroid tolerance of larvae of the common green lacewing, Environ. Entomol. 10, 684 (1981). 25. A. L. Devonshire and G. D. Moores, A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach–potato aphids (Myzus persicae), Pestic. Biochem. Physiol. 18, 235 (1982).
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY ARTICLE NO. 0022
54, 181–189 (1996)
Purification and Characterization of Carboxylesterases of a Rice Green Leafhopper Nephotettix cincticeps Uhler SHIH-WEN CHIANG
AND
CHIH-NING SUN
Department of Entomology, National Chung-Hsing University, Taichung, Taiwan 40227, Republic of China Received September 6, 1995; accepted February 5, 1996 More than four carboxylesterase isozymes in the homogenate of a rice green leafhopper, Nephotettix cincticeps Uhler, could be resolved by isoelectric focusing electrophoresis. A combination of ammonium sulfate fractionation, gel filtration, and chromatofocusing chromatography was used to isolate and purify these isozymes. Four fractions, i.e., E1, E2, E3, and E4, with pI’s ranging from 5.1 to 4.85, were obtained. The most abundant E3 had a molecular mass of 58.6 kDa and appeared electrophoretically homogeneous on SDS–PAGE. [1,3-3H]Diisopropyl fluorophosphate-labeling experiment revealed that the proteins of 58.6 kDa, a minor component of E2 and the major component of E4, were the carboxylesterase isozymes sought. A protein of the same molecular weight which existed in a very minute amount in E1 and was barely detectable on SDS–PAGE by Coomassie blue staining was actually the carboxylesterase isozyme of pI 5.1. All four fractions exhibited significant activity toward several model substrates with a-naphthyl butyrate being the most preferred. Their activity toward malathion, permethrin, and cypermethrin was ca. 106-fold lower than their activity toward the model substrates. The pyrethroids were hydrolyzed more readily than malathion by these hydrolases, and cis-permethrin was more preferred than the trans-isomer. E4 was the only fraction that cross-reacted with the antiserum against carboxylesterases of a rice brown planthopper, Nilaparvata lugens. Among the four isozyme fractions, E3, the most abundant, showed surprisingly low activity toward all four insecticides and was actually the least active fraction toward cis-permethrin and cypermethrin. A field strain of N. cincticeps had 26- to 37-fold higher carboxylesterase activity toward the model substrates than a susceptible strain. Yet, little, if any, difference in the hydrolysis of malathion, permethrin, and cypermethrin was observed between these two strains. The field strain produced at least eight times more carboxylesterases than the susceptible strain. © 1996 Academic Press, Inc.
INTRODUCTION
Nephotettix spp. are widely distributed green leafhoppers of rice. Of the three known species in Taiwan Nephotettix cincticeps Uhler is the predominant one and an important vector of viruses that cause rice dwarf, transitory yellowing, tungro, and yellow dwarf diseases (1). A variety of chemicals including organophosphorus (OP),1 carbamate, pyrethroid insecticides, and their mixtures have long been used for the control of these leafhoppers (2). Several reports on the occurrence of OP, carbamate, and pyrethroid resistance in this insect have appeared since mid-1970s in Taiwan (3, 4). In Japan, malathion resistance of this leafhopper was first observed in early 1960s (5) and considerable 1 Abbreviations use: DFP, diisopropyl fluorophosphate; IEF, isoelecrtric focusing; a-NA, a-naphthyl acetate; b-NA, b-naphthyl acetate; b-NB, b-naphthyl butyrate; pNPA, p-nitrophenyl acetate; OP, organophosphorus; SDS– PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
efforts have been made since then to investigate the resistance mechanisms (6). The OP resistance of this leafhopper has been found to associate with a high carboxylesterase activity by many researchers. Ozaki and his co-workers (7, 8) used thin-layer agar gel electrophoresis to resolve carboxylesterase isozymes of N. cincticeps and found that E2, E3, and E4 bands were more active in resistant than in susceptible strains. Subsequently, Miyata (9) reported that E1, E2, and E3 were able to degrade [14C]malathion with E2 being the most active form. Motoyama et al. (10) resolved five esterase peaks using chromatofocusing from an OP-resistant strain of this leafhopper and four peaks were found active toward malathion, paraoxon, and fenvalerate. They proposed a dual role for these esterases in resistance mechanisms: a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides. The present study attempted to isolate and
181 0048-3575/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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purify the major isozymes of carboxylesterases in N. cincticeps, to determine their activity toward some general substrates and insecticides, and to compare the isozyme makeup of this detoxifying enzyme in susceptible and resistant strains of this leafhopper. MATERIALS AND METHODS
Insects The susceptible strain of the green rice leafhopper was obtained from the Agricultural Research Station, ICI Japan Ltd., in 1993. The resistant strain was collected from the rice fields in central Taiwan. Chemicals The chemicals used in the enzyme assays were obtained from Sigma (St. Louis, MO) and E. Merck (Darmstadt, Germany) and those used for protein purification were products of Pharmacia (Uppsula, Sweden). Protein standards for sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) were products of Bio-Rad (Richmond, CA), and reagents for protein quantitation were purchased from Pierce (Rockford, IL). [1,3-3H]Diisopropyl fluorophosphate (DFP) with a specific activity of 10 Ci/mmol and the autoradiography enhancer Enlightening were obtained from DuPont NEN (Boston, MA). Malathion (98%) and cypermethrin (97%) were products of Riedel-de Haen AG (Seelze, Germany), and trans-permethrin (99%) and cispermethrin (99%) were provided by the U.S. Environmental Protection Agency (Triangle Park, NC). Purification of Carboxylesterases Three grams of adult hoppers of the field (F) strain was ground with Polytron PT10-35 homogenizer for 2 min in 5 vol of 0.05 M Tris– HCl buffer (pH 7.2). The cheesecloth-filtered homogenate was centrifuged first at 10,000g for 15 min and then at 100,000g for 1 hr at 4°C. The supernatant was subject to ammonium sulfate fractionation and the fraction precipitated between 30 and 60% ammonium sulfate had 93% of the total carboxylesterase activity using
a-naphthyl acetate (a-NA) as substrate. This fraction was subsequently introduced onto a Sepharose 6B column (2.2 × 45 cm) and the column was eluted with 0.05 M Tris–HCl buffer (pH 7.2). Fractions with activity toward a-NA were pooled and concentrated and the buffer was changed to 0.025 M histidine–HCl buffer (pH 6.2) using a Centricon-30. For chromatofocusing, this solution (ca. 3 ml) was put onto a preequilibrated PBE 94 column (1 × 20 cm) which was first eluted with 5 ml of the histidine–HCl buffer and subsequently with polybuffer 74–HCl buffer (pH 4.0) at 6 ml/hr. A total of 120 fractions (one fraction every 15 min) were collected. Fractions with activity toward a-NA were individually analyzed (concentrated if necessary) on IEF and SDS–PAGE for purity. Fractions which were not electrophoretically homogeneous were further resolved using Sephadex G-75 and Sephacryl S-200 chromatography. Both the Sephadex G-75 column (1.6 × 45 cm) and the Sephacryl S-200 column (1 × 45 cm) were eluted with 0.05 M Tris–HCl (pH 7.2) at 6 ml/hr and fractions (every 15 min) were tested for activity toward a-NA. Active fractions were concentrated and analyzed on SDS–PAGE for purity. Enzyme Assay For the hydrolysis of a-NA, a-naphthyl butyrate (a-NB) and b-naphthyl acetate (b-NA), the method of van Asperen (11) was adpoted. A Beckman DU-70 spectrophotometer was used to measure the products of the reaction, i.e., 600 nm for a-NA and a-NB, and 555 nm for b-NA. a-Naphthol and b-naphthol were used to construct the standard curves. For the hydrolysis of p-nitrophenyl acetate (p-NPA) the method of Ljungquist and Augustinsson (12) was followed. A molar extinction coefficient of 16.36 mM−1 cm−1 was used to convert the absorbance changes at 400 nm into the amounts of product. The partially purified fractions or the supernatant of 10,000g centrifugation of the homogenate of the hoppers was used as the enzyme source. Buffer solution of equivalent volume was added to the controls to replace the enzyme solution. Three replicates were performed for all treatments.
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In vitro degradation of several insecticides by the homogenates (prepared as described above) and the four carboxylesterase fractions was determined by adding 10 ml of enzyme source and 10 ml of insecticide solution (1 mg/ml of ethylene glycol monoethyl ether) in 0.1 M Tris–HCl buffer (pH 8.0) to make a final volume of 0.5 ml. Incubation at 37°C was carried out overnight for malathion and for 5hr for permethrin and cypermethrin. At the end of the incubation, 0.5 ml n-hexane was added with vigorous shaking. The unreacted insecticides in the hexane layer were quantitated using a Varian 3400 gas chromatograph with a 15% OV-17 and 1.95% OV-210 Chrom W HP glass column (2 mm × 2 m) at 240°C, and a flame photometric detector (300°C) for the OPs and a Ni-63 electron capture detector (320°C) for the pyrethroids. For the controls, buffer solution was added to replace the enzyme source. All treatments were duplicated or triplicated. Proteins were quantitated by bicinconinic acid assay according to the manufacturer’s (Pierce) instruction. Electrophoresis and Immunoblotting SDS–PAGE was performed according to Laemmli (13) using a 3.75% stacking gel and a 12.5% running gel with a Bio-Rad Mini-Protean II electrophoresis cell at 100 V. IEF electrophoresis was performed between pH 4.5 and 5.4 using a Bio-Rad 111 mini-IEF cell according to the manufacturer’s instruction. The carboxylesterase activity was stained using a-NA as substrate. Proteins on SDS–PAGE were transferred to NC paper with a Hoefer TE77 Semiphor Transfer Unit (San Francisco, CA) and immunoblotting was performed according to Blake et al. (14). Primary antiserum to carboxylesterase El of a brown planthopper, Nilaparvata lugens, raised in rabbit (15) (2000-fold dilution) and an alkaline phosphatase-conjugated goat antirabbit IgG (5000-fold dilution) were used. [1,3-3H]Diisopropyl Fluorophosphate Labeling of Carboxylesterases Adult leafhoppers were homogenized in 0.02M sodium phosphate buffer (pH 7.8) and
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the homogenate was centrifuged at 10,000g twice. The supernatant or the carboxylesterase fraction obtained above was incubated with 3 mCi [3H]DFP dissolved in propylene glycol at room temperature for 1hr and dialyzed overnight at 4°C in the above buffer to remove the unbound DFP. The supernatant was subsequently analyzed by SDS–PAGE or IEF. The gels were fixed for 30 min in acetic acid and methanol, submerged in Enlightening solution for 30 min. The SDS–PAGE gel was vacuum dried and the IEF gel was air-dried before processing for fluorography. RESULTS
Purification of Carboxylesterase Isozymes of N. cincticeps In the crude homogenate of this leafhopper, more than four carboxylesterase isozymes with pI’s between 4.5 and 5.4 could be observed using a-NA as substrate on IEF (Fig. 1). The supernatant after 1hr of 100,000g centrifugation of the homogenate retained more than 83% of the total carboxylesterase activity. The proteins precipitated between 30 and 60% ammonium sulfate contained 84% of the activity in this supernatant (Table 1). While Sepharose 6B chromatography removed only a very limited amount of large, unwanted proteins (Fig. 2A), subsequent chromatofocusing removed a significant amount of proteins with higher pI’s (Fig. 2B). IEF analysis of each tube with enzyme activity from chromatofocusing showed that four major fractions
FIG. 1. IEF of carboxylesterases in the homogenates of N. cincticeps. Lanes 1–7, susceptible strain (1/10 of an adult per lane); and lanes 8–15, field strain (1/80 of an adult per lane).
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CHIANG AND SUN TABLE 1 Purification of Carboxylesterase Isozymes (E1, E2, E3, and E4) of a Field (F) Strain of N. cincticeps
Step Crude homogenate 100,000g supernatant 30–60% (NH4)2SO4 precipitate Sepharose 6B Chromatofocusing E1 E2 E3 E4 a b
Total activitya (mmol/min)
Specific activity (mmol/min/mg protein)
129.1 59.5
208.4 173.0
1.6 2.9
100 83
1.0 1.8
21.7 17.3
145.6 121.0
6.7 7.0
70 58
4.2 4.4
17.2 13.2 49.8 0.9
(13.2) (16.5) 38.3 (4.5)
Total protein (mg)
(1.3)b (0.8) 1.3 (0.2)
Yield (%)
Purification (fold)
8.3 6.3 24 0.43
(8.3) (10.3) 23.9 (2.9)
a-NA was used as substrate. Since E1, E2, and E4 fractions had contaminating proteins, some of their data are thus put in parentheses.
could be resolved (Fig. 3). Tubes showing only one band with enzyme activity on IEF were pooled and concentrated, and the four fractions were designated as E1, E2, E3, and E4 (Fig. 4A). Their pI’s were 5.1, 5.0, 4.9, and 4.85, respectively. SDS–PAGE analysis revealed that while E1 fraction appeared to have only one protein of 83.7 kDa, the other three fractions all consisted of three proteins of 83.7, 58.6, and 42 kDa (Fig. 4B). Subsequent Sephadex G-75 chromatography removed the two contaminating proteins from E3 (Fig. 5A), and the remaining protein of E3 had a molecular weight of 58.6 kDa (Fig. 5B) with carboxylesterase activity representing
24-fold purification (Table 1). However, Sephadex G-75 followed by Sephacryl S-200 chromatography failed to resolve the three proteins in E2 and E4. Fluorographic analysis clearly showed that in all four fractions, only one protein of 58.6 kDa was labeled by [1,3-3H]DFP (Fig. 4C). Therefore, the 83.7-kDa protein in E1 fraction was only a contaminant, and the E1 isozyme (58.6 kDa) present in very minute proportion was barely detectable (Fig. 4B). The 83.7 and 42-kDa proteins in both fractions E2 and E4 were also contaminating proteins. Therefore, all four carboxylesterase isozymes of this leafhopper had the same molecular mass of 58.6 kDa but varied pI’s (5.1 to 4.85). Although
FIG. 2. (A) Sepharose 6B chromatography of carboxylesterases after ammonium sulfate fractionation of the homogenate of a field strain of N. cincticeps, and (B) subsequent chromatofocusing.
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FIG. 3. IEF of carboxylesterases in the fractions collected from chromatofocusing. The gel was stained for activity toward a-NA.
three of the four fractions were not purified carboxylesterase isozymes, they were still used for further studies. Certain trends with regard to their biochemical and toxicological properties still could be seen. Biochemical and Toxicological Properties of Carboxylesterase Isozymes of N. cincticeps All four isozymes were able to hydrolyze the model substrates tested, i.e., a-NA, a-NB, b-NA, and p-NPA, with a-NB being the most preferred one (Table 2). The presence of contaminating proteins in all fractions except E3 makes comparison of specific activity among the four isozymes difficult.
The hydrolysis of malathion, permethrin, and cypermethrin by these four carboxylesterase fractions was at least 106-fold less than the hydrolysis of model substrates (Table 2). Yet, for the same isozymes, malathion was hydrolyzed 3- to 10-fold less efficiently than the two pyrethroids. All four isozymes of this leafhopper hydrolyzed cis-permethrin 2- to 5-fold faster than trans-permethrin. Despite of the presence of contaminating proteins, some tendency among the four fractions can still be seen rather clearly. The general ability of E3, the electrophoretically pure and most abundant form, to degrade these three insecticides appeared to the the lowest. E4 exhibited especially high activity toward the two pyrethroids. In an immunoblotting experiment, a protein (or proteins) of ca. 58.6 kDa in this leafhopper cross-reacted weakly with an antiserum raised against the dominant carboxylesterase isozyme El of N. lugens, a rice brown planthopper (Fig. 6A). When the four fractions were tested, only the protein of 58.6 kDa in E4 showed apparent crossreactivity (Fig. 6B). Carboxylesterases of Susceptible and Field Strains of N. cincticeps Although both the susceptible (S) and field (F) strains demonstrated extensive carboxylesterase activity toward several model substrates with the latter having 26- to 37-fold higher activity, their ability to degrade malathion, permethrin, and cypermethrin was again very limited and differed only slightly (Table 3). IEF analysis of carboxylesterases in the homogenates of this leafhopper revealed that while more than
TABLE 2 Hydrolysis of Some Model Substrates and Insecticides by the Four Carboxylesterase Fractions of N. cincticeps Specific activity
mmol/min/mg protein Isozyme E1 E2 E3 E4 a b c
a-NA
a b,c
13.2 ± 0.9 16.5 ± 0.4 38.3 ± 0.6 4.6 ± 0.1
pmol/hr/mg protein
a-NB
b-NA
p-NPA
Malathion
trans-Permethrin
cis-Permethrin
Cypermethrin
16.9 ± 2.1 19.5 ± 0.6 52.2 ± 1.7 9.6 ± 0.2
13.5 ± 1.2 16.5 ± 2.7 40.9 ± 0.7 6.3 ± 0.2
12.5 ± 1.3 15.4 ± 0.7 27.8 ± 1.2 5.2 ± 0.7
37.5 ± 0.2 20.8 ± 0.3 25.6 ± 1.0 23.7 ± 0.1
68.3 ± 0.1 20.5 ± 1.4 27.0 ± 0.2 140 ± 1
163 ± 1 102 ± 1 83 ± 1 235 ± 1
98.3 ± 0.3 52.6 ± 0.4 23.8 ± 0.2 213 ± 1
a-NA, a-naphthyl acetate; a-NB, a-naphthyl butyrate; b-NA, b-naphthyl acetate; and p-NPA, p-nitrophenyl acetate. Mean ± SE of two or three replicates. Since E1, E2, and E4 have contaminating proteins, the specific activities in this table are underestimated.
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FIG. 4. (A) IEF of the four carboxyelsterase fractions obtained in chromatofocusing and the homogenate (H) of N. cincticeps. The gel was stained for activity toward a-NA; (B) SDS–PAGE of the same fractions and the gel was stained for proteins by Coomassie blue; and (C) SDS–PAGE of the [3H]DFP-labeled carboxylesterase fractions.
four isozymes could be detected in F strain, only the most abundant E3 was clearly observed in S strain (Fig. 1). Fluorographic analysis of [3H]DFP-labeled carboxylesterases following SDS–PAGE shows that while three weak bands appeared in S strain, only one intensely labeled band corresponding to a molecular mass of 58.6 kDa appeared in F strain (Fig. 7A) The latter was at least 8-fold higher in quantity than the equivalent protein in S strain. Similar analysis following IEF shows that all four major carboxylesterase isozymes were present in higher amounts in F strain than in S strain, and E3 and E4 were the most abundant forms (Fig. 7B). DISCUSSION
Among the four carboxylesterase isozymes of F strain of N. cincticeps obtained in this study, only E1 and E3 could be purified to electrophoretic homogeneity (Figs. 4B and 5B). Al-
though the molecular mass of the three proteins in E2 and E4 fractions differed sufficiently for their separation by gel filtration, Sephadex G-75 and Sephacryl S-200 chromatography was unable to resolve them. Glycoproteins have been reported to interact sometimes with gel filtration media that are dextran-based, such as Sephadex gels, and thus exhibit irregular chromatographic behavior (16). This might account for the failure of our attempt to purify E2 and E4 fractions. [3H]DFP-labeling experiments showed that only the 58.6-kDa protein(s) in these four carboxylesterase fractions (Fig. 4C) and the homogenate of F strain of N. cincticeps (Fig. 7A) were bound to DFP. Therefore, the four carboxylesterase isozymes of this leafhopper had to be proteins of the same molecular mass 58.6 kDa, whose activity toward a-NA was observed on IEF gel with pI’s of 5.1, 5.0, 4.9, and 4.85 (Fig. 4A). This protein was of
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FIG. 5. (A) Sephadex G-75 chromatography of E3 fraction obtained in chromatofocusing; and (B) subsequent SDS–PAGE of fractions 25–34 with Coomassie blue staining.
such a minute amount in E1 fraction that it was barely observed on SDS–PAGE and the dominant 83.7-kDa protein was not the isozyme sought (Fig. 4B). Similarly, the 83.7- and 42kDa proteins in E2 and E4 fractions were not the isozymes either (Fig. 4B). The present study found that all four carboxylesterase isozymes of N. cincticeps in general were able to hydrolyze the two pyrethroids, permethrin and cypermethrin, much more efficiently than malathion, the OP insecticide readily hydrolyzed by this enzyme in many insects (17). This observation does not quite agree with data reported by Motoyama et al. for the same leafhopper (10). In their work, one of the five forms of carboxylesterases was found to degrade malathion twice as fast as the pyrethroid fenvalerate, and three other forms exhibited approximately equal activity toward these two insecticides. In both mammals and insects carboxylesterases have been reported to prefer permethrin, a pyrethroid of primary alcohol ester, to cypermethrin, a pyrethroid of secondary alcohol ester (18, 19), and trans-permethrin to cis-permethrin (19–23). In this study, cispermethrin was degraded at a rate 2- to 5-fold higher than its trans-isomer and all four isozymes hydrolyzed cypermethrin to a significant extent. Similarly, in another study, larvae of the green lacewing, Chrysopa carnea Stephens, an important predator to many agricultural pests, showing a considerable tolerance to pyrethroids, has unusually high activity for hydro-
lyzing the cis-isomers of permethrin and cypermethrin, 2- to 3-fold faster than the corresponding trans-isomers (24). The same phenomenon was also observed in a rice brown planthopper N. lugens (15). The presence of contaminating proteins in three of the carboxylesterase fractions obtained in the present study results in an underestimation of their specific activities toward various substrates, especially of E1 and E2, and makes comparisons difficult. Nevertheless, some conclusions still could be drawn. The most abundant E3 isozyme (as shown in Figs. 1 and 7B) possessed the lowest activity toward practically all three insecticides tested (Table 2). The E4 fraction which had an overall lower activity toward the model substrates showed unusual preference toward the two pyrethroids, permethrin and cypermethrin. Motoyama et al. (10) found that in N. cincticeps peak V of chromatofocusing of this hydrolase, compared with the other four peaks, had relatively high activity toward fenvalerate compared with malathion and paraoxon. This peak V might correspond to the carboxylesterase isozyme in E4 fraction of this study. Significant amount of this isozyme was shown in the DFP-labeling experiment to be present in the F strain (Fig. 7B), collected from the rice paddy in central Taiwan where a number of pyrethroids have been applied for the control of various leafhoppers and planthoppers. Among the four isozyme, E4 was the only one showing cross-reactivity toward the antise-
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FIG. 6. Western blot analysis of immuno-cross-reactivity between E1 of Nilaparvata lugens (BPH) and carboxylesterases of N. cincticeps (RGLH). (A) SDS–PAGE of homogenates of BPH and RGLH, M, molecular marker; and (B) SDS–PAGE of homogenate of BPH, lanes 1 to 5 loaded with 1/8, 1/16, 1/32, 1/64, and 1/128 of a planthopper, and 50 µg each of E1, E2, E3, and E4 of RGLH.
rum against carboxylesterase E1, the isozyme overproduced in resistant rice brown planthopper, N. lugens (Fig. 6B). The significance of this immunorelationship is unclear. TABLE 3 Hydrolysis of Some Model Substrates and Insecticides by the Homogenates of a Susceptible (S) and a Field (F) Strains of N. cincticeps Specific activity Substrate
a-NAa a-NB b-NA p-NPA
Malathion trans-Permethrin cis-Permethrin Cypermethrin
S
F
F/S
mmol/min/mg protein 0.10 ± 0.01b 2.77 ± 0.42 0.16 ± 0.03 4.12 ± 0.27 0.09 ± 0.01 2.45 ± 0.65 0.07 ± 0.01 2.59 ± 0.11
27.7 25.8 27.2 37.0
pmol/hr/mg protein ND 0.8 ± 0.3 0.6 ± 0.07 0.6 ± 0.2 1.6 ± 0.01 1.4 ± 0.05 0.7 ± 0.02 1.0 ± 0.02
— 1.0 0.9 1.4
a a-NA, a-naphthyl acetate; a-NB, a-naphthyl butyrate; b-NA, b-naphthyl acetate; and p-NPA, p-nitrophenyl acetate. b Mean ± SE of two or three replicates.
FIG. 7. Fluorographic analysis of [1,3-3H]DFP-labeled carboxylesterases. (A) SDS–PAGE; and (B) IEF of homogenates of a susceptible (S) and a field (F) strains of N. cincticeps.
The correlation between high carboxylesterase activity and malathion resistance in N. cincticeps has been observed by several researcher (6–9). However, the action of this enzyme remained ambiguous until Motoyama et al. (10) proposed that it played a dual role as the overproduced carboxylesterase isozyme E4 in Myzus persicae (25): a catalyst for hydrolysis of malathion and fenvalerate and a binding protein for the oxygen analogs of other OP insecticides. The field strain of this leafhopper in the present study exhibited up to ca. 40-fold higher carboxylesterase activity toward the model substrates than the susceptible strain (Table 3, Fig. 1). However, practically no difference in the hydrolysis of malathion and the two pyrethroids was detected between these two strains (Table 3). In addition, 58.6-kDa proteins (Fig. 7A) with varying pIs (Fig. 7B), which presumably are the carboxylesterase isozymes seen in Fig. 1, were overproduced at least eight times more in the F strain than in S strain. All these data seem to suggest that the carboxylesterase isozymes in resistant N. cincticeps might serve more as binding proteins than as catalysts. The two ad-
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CARBOXYLESTERASES OF RICE LEAFHOPPER
ditional weak bands in S strain (Fig. 7A) could be other serine hydrolases. ACKNOWLEDGMENTS We thank Japan ICI Ltd. and Dr. C. C. Chen of Taichung Agricultural Improvement Station for supplying the susceptible and field strains, respectively, of the green rice leafhopper. This work was supported by Grants NSC83-0409B005-002 and NSC83-0618-B005-001 of the National Science Council, Taiwan, Republic of China.
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