Purification and characterization of a phosphorotriester hydrolase from methyl parathion-resistant Heliothis virescens

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

36, 1-13 (1990)

Purification and Characterization of a Phosphorotriester Hydrolase from Methyl Parathion-Resistant Heliothis virescens T. KoNNo,'~.

KASAI, R. L. ROSE, E. HODGSON, AND W.C.

DAUTERMAN

Toxicology Program, North Carolina State University, Raleigh, North Carolina 27695 Received May 31, 1989; accepted July 26, 1989 The enzyme that hydrolyzes methyl paraoxon in a methyl parathion-resistant strain of Heliothis virescens was characterized using methyl paraoxon as a substrate. It was localized mainly in the soluble fraction, total activity being highest in the integument/muscle, followed by the fat body and the intestine, although specific activity was highest in the Malpighian tubules, followed by the head. The optimal pH was 8 to 9 and 1 x lo-’ M Co*+ and Mn2+ ions significantly activated the enzyme, while 1 x lop3 M Ca” and EDTA had no effect. Hg + and phosphate ions inhibited it. The Isa’s of some esterase inhibitors such as DEF, TOCP, DFP, K-2, OTFP, propoxur, ethyl paraoxon, n-propyl paraoxon, etc. were all greater than 1 x 10m4 M. Purification of the enzyme from the 100,000g supematant of a whole body homogenate by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration resulted in a specific activity of 0.488 pm01 methyl paraoxon hydrolyzed/min/mg protein with 267-fold puriiication. Methyl paraoxon hydrolysis was attributed to a major PAGE band as determined with I-naphthyl acetate. The molecular weight was estimated as 120 kDa by Sephadex G-200 chromatography. The difference in the relative toxicological half-life value, (to.& for methyl paraoxon and ethyl paraoxon corresponded to the difference in resistance factors for these compounds in vivo. The results obtained indicate that the methyl paraoxon hydrolase of H. virescens is a true phosphorotriester hydrolase responsible in part for methyl parathion resistance in this strain of insect. 8 1990 Academic press. IK.

INTRODUCTION

The

tobacco

budworm,

ase activity responsible for oxidative desulfuration of methyl parathion to methyl paraoxon and to an increase in phosphorotriester hydrolase activity responsible for the hydrolysis of methyl paraoxon (7). Investigations on phosphorotriester hydrolases have been conducted mainly on mammalian tissues (8-15) whereas only a limited number of studies have been conducted on insects (16). Phosphorotriester hydrolases have been demonstrated in the house fly (17-19), lepidopterous insects (20), and in the Reduviid bug (21) and have a requirement for Mn2+ and Co2+, whereas the mammalian enzyme requires Ca2’ (16). However, little information is available in resistant insects. In resistant aphids, the hydrolase which could hydrolyze organophosphates was identified as a carboxylesterase (22, 23) and a modified esterase was reported in the organophosphate-resistant house fly (24) and other studies on resistance have failed to demonstrate the presence of the enzymes (25). The question arises as to what extent phos-

Heliothis

virescens (F.), is an important

pest of cotton and has developed resistance to organophosphorus compounds (1-5) and synthetic pyrethroids (6). The strain collected in North Carolina in 1986 (NC-86) has a 60fold resistance to methyl parathion, but is susceptible to carbamates and pyrethroids (7). Biochemical studies showed that resistance was due mainly to a decrease in the cytochrome P450-dependent monooxygen’ Present address: Nihon Nohyaku Co. Ltd., Biological Research Center, 4-31 Honda-cho, KawachiNagano, Osaka 586, Japan. 2 Abbreviations used: DEF, S,S,S,-tributylphosphorotrithioate; TOCP, triorthocresyl phosphate; TCPB, 2,3,6-trichloro-l-propynyloxy benzene; K-2,2phenoxy-4H-1,3,2-benzodioxaphosphorin-2-oxide; DFP, diisopropyl fluorophosphate; OTFP, 3octylthio- 1, 1, I-trifluoro-2-propanone; TLC, thin-layer chromatography; PTU, phenyl thiourea; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PNA, p-nitrophenyl acetate; PNB, pnitrophenyl butyrate; AChE, acetylcholinesterase. 1

OO48-3575190 $3.00 Copyright 8 1!390 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

KONNO

phorotriester hydrolases, also known as “A” esterases, occur in insects (26). The term “A” esterases was introduced by Aldridge (8) and refers to enzymes which hydrolyze p-nitrophenyl acetate faster than pnitrophenyl butyrate, hydrolyze aromatic esters, are inhibited by PCMB but are not inhibited by paraoxon, but hydrolyze it. The present study focuses on the characterization and the purification of a phosphorotriester hydrolase from a resistant strain of insects.

ET AL.

supplied by Dr. R. M. Roe of North Carolina State University. Ring-labeled [U-‘4C]methyl paraoxon with a specific activity of 7.68 mCi/mmol was synthesized as described previously (7). n-Propyl paraoxon was prepared as described by Dauterman and O’Brien (29). DE-52 and Bio-Gel A were purchased from Whatman Inc. (Clifton, NJ) and BioRad Laboratories (Richmond, CA), respectively. Polybuffer 96 and 74 and Sephadex G-200 were purchased from Pharmacia Fine Chemicals (Piscataway, NJ). All other MATERIALS AND METHODS chemicals used were of the highest purity available commercially. The purity of the Experimental Animals radiolabeled compound was greater than Larvae of a methyl parathion-resistant 99% as determined by thin-layer chromastrain of H. virescens were obtained from a tography (TLC). laboratory culture (NC-86) established from insects collected in North Carolina in Distribution Studies 1986. Methyl parathion-susceptible larvae Two-day-old fifth instar larvae of the rewere obtained from a culture which has sistant strain were homogenized in 0.1 M been maintained at North Carolina State Tris-HCl buffer, pH 7.6 (200 mg larvae/ml), University since 1977 (27). Both of the containing 0.5% bovine serum albumin strains were reared on an artificial diet (28) (BSA) and 1 x lop4 M phenyl thiourea at 25°C with a 16:8 (L:D) photoperiod with- (PTU) with a Polytron homogenizer (Brinkout any exposure to insecticides. mann Instruments, Westbury, NY) for 30 sec. The homogenate was centrifuged at Chemicals 1OOOgfor 15 min and the precipitate was utilized for the enzyme assay as 1OOOg (ppt) Methyl parathion and (S,S,Stributylphosphorotrithioate (DEF)’ were after it was diluted to the initial volume. obtained from Chem Service, Inc. (West The supematant was centrifuged at 10,OOOg Chester, PA). Methyl paraoxon and ethyl for 10 min and the 10,OOOgprecipitate was paraoxon were kindly supplied by Ameri- diluted as stated above and utilized as (ppt). The lO,OOOgsupematant was can Cyanamid Co. (Princeton, NJ). EPN 10,OOOg and propoxur were obtained from EPA filtered through glass wool and recentrifor 1 hr and the 100,OOBg (RTP, NC) and pirimicarb was kindly sup- fuged at 100,OOOg plied by Nihon Nohyaku Co. Ltd. (Osaka, supernatant and the microsomal fraction Japan). Triorthocresyl phosphate (TOCP) were separated, diluted, and utilized as was purchased from Pfaltz and Bauer Inc. 100,OOOg(sup) and 100,000g (ppt), respectively. Total activities were computed from (Stamford, CN) and 2,3,6-trichloro1-propynyloxy benzene (TCPB) was a gift the total protein content of the fractions from Dr. T. M. Brown of Clemson Univer- and their specific activities. Hydrolase activity was determined using sity , South Carolina. 2-Phenoxymethyl paraoxon as the substrate and based 4H- 1,3,2-benzodioxaphosphorin-2-oxide (K-2) was a gift from Dr. M. Eto of Kyushu on the amount of p-nitrophenol formed. University, Japan, and diisopropyl fluoro- Unless otherwise stated, the incubation mixture consisted of 0.5 ml of soluble fi-acphosphate (DFP) and 3-octylthio1,1 ,l-trifluoro-2-propanone (OTFP) were tion and 10 nmol of [t4C]methyl paraoxon

METHYL

PARAOXON

HYDROLASE

(170,000 dpm) in 2 g,l of acetone in a total of 1 ml. Duplicate samples were incubated in a water bath, with shaking at 30°C for 1 hr, and the reaction was stopped by the addition of 0.5 ml ethanol. The radioactive material was extracted into ethyl acetate and the quantitative determination of metabolites was conducted using the TLC method described previously (7). When larval tissues were used, 20 larvae were dissected to provide the following tissues: head capsule, intestine with their contents removed, fat body, Malpighian tubules, silk glands, ovaries, hemolymph, and cuticle, including epidermis and body wall muscles. Except for the hemolymph, tissues were homogenized with an all-glass homogenizer in 10 ml buffer. Approximately 0.7 ml hemolymph was collected and diluted with buffer to 2 ml. Twenty to 100 ~1 of the crude solution was used for enzyme assay. Hydrolase activity was determined as described above and an incubation at 0°C for 1 set was also conducted in order to estimate the amount of pnitrophenol formed by the phosphorylation of nonspecific esterases and other proteins (7, 25). The tissue distribution was computed from the total protein content of the tissue homogenates and their specific activities with a correction for the p-nitrophenol released by phosphorylation of nonspecific esterases. Effects of Metal Ions and pH

The 100,OOOgsupematant was used as the enzyme source after it had been dialyzed overnight against 0.1 M Tris buffer (pH 7.6) containing 1 x 10m4M PTU using standard cellulose dialysis tubing (Spectrum Medical Industries Inc., Terminal Annex, LA) with MW cut-off of 12,000 to 14,000. Each metal ion and chelating agent was added to the incubation mixture at a concentration of 1 x 10K3 M. Hydrolase activity was determined as described above. When the effects of pH were studied, 0.1 ml of the supematant was used and diluted to 1 ml of the incubation mixture

OF

3

H. virescens

with the following buffers: citrate (O.lM) for pH 3 to 7, Tris (0.1 M) for pH 7 to 9, glycine (0.1 M) for pH 9 to 11. The original buffer of the supematant did not affect the pH of the buffer added. The reaction was stopped after a 2-hr incubation instead of 1 hr by the addition of 0.5 ml ethanol and the hydrolase activity was determined as described above. In all of the above experiments, boiled tissues were used as controls and the amount of protein was determined by the method of Bradford et al. (30) using BSA as the standard. Purification of Phosphorotriester

Hydrolase

Five hundred fifth instar larvae of the NC-86 strain were dissected and the contents of the intestine were removed with an aspirator. After washing in ice-cold 0.1 M Tris buffer, pH 8.0, containing 1 x 10e4 M PTU to remove hemolymph, they were homogenized and centrifuged as described previously. The 100,000g supematant was used as the enzyme source. The first step in the purification was fractional precipitation with ammonium sulfate. The precipitates containing methyl paraoxon hydrolase activity were collected and dissolved with 0.01 M MOPS buffer, pH 7.8, containing 1 x 10s3 M Co2+ and 1 x low4 M PTU (designated MOPS buffer). Before application to a DE-52 ion-exchange column, the solution was dialyzed overnight. The DE-52 column was equilibrated with MOPS buffer and packed to make a 2 x 30-cm column. After applying the enzyme preparation, 5 bed vol of the starting buffer were passed through the column and the enzyme was eluted with the buffer containing 0.1 M NaCl and 0.15 M NaCl, respectively. Three-milliliter fractions were collected and the tubes containing hydrolase activity were combined and concentrated by ultrafiltration using a Diaflo (PM30) membrane (Amicon Dix, Danvers, MD) and applied to a 1.5 X 13-cm DEAE Bio-Gel A column equilibrated with 0.025 M ethanolamine, pH 9.4. Polybuffer % was diluted 10

4

KONNO

times and used as an eluant after adjusting the pH to 6.5 in order to produce a pH gradient from 9 to 6.5. The column was then washed with MOPS buffer containing 0.1 M NaCl and the bound protein was eluted with a linear gradient consisting of 100ml of MOPS buffer containing 0.1 M NaCl in the mixing chamber and 100 ml of the buffer containing 0.4 M NaCl in the feed chamber. Fractions with hydrolase activity were collected, concentrated, and applied to a 2 x 90-cm Sephadex G-200 gel filtration column equilibrated with MOPS buffer containing 0.1 M NaCl. During the purification, the elution of the protein was monitored at 280 nm with an Aminco DW-2C uv-vis recording spectrophotometer. The amount of protein in the combined fraction was determined by the method of Bohlen et al. (31) using BSA as a standard. Hydrolase activity was also measured spectrophotometrically. The reaction mixture contained 10 ~1 enzyme solution, 1 mg methyl paraoxon in 10 ~1 of acetone, and MOPS buffer in a total volume of 1 ml. The reaction was recorded at 405 nm at 30°C and converted to nanomoles of p-nitrophenol from a standard curve. Nonenzymatic degradation of methyl paraoxon was corrected in the reference cell. During purification, hydrolase activity was only measured spectrophotometrically. Molecular

Weight

The molecular weight of methyl paraoxon hydrolase was determined using Sephadex G-200 gel filtration (22). The standard proteins used were P-amylase (MW, 200,000), alcohol dehydrogenase (MW, lSO,OOO),BSA (MW, 66,000), carbonic anhydrase (MW, 29,000), and cytochrome c (MW, 12,400). The elution volume of standard protein was divided by the void volume and plotted against the logarithmic value of their molecular weight.

ET AL.

ried out by the method of Omstein (32) and Davis (33). Two identical gels of the purified preparation were developed simultaneously. One gel was stained with Fast Blue RR salt in phosphate buffer, 0.1 M, pH 7.6, for the detection of esterase bands with ol-naphthyl acetate. The other gel was utilized for the detection of the methyl paraoxon hydrolase activity. The enzymatic activity was determined calorimetrically as described above after the gel was sliced into small pieces and homogenized in MOPS buffer. Kinetic

Parameters

and ISo’s of Inhibitors

Michaelis constants were obtained from Lineweaver-Burk plots of l/v vs l/[sl. The incubation mixture consisted of 10 pl of purified preparation and 10 ~1 of acetone solution containing various concentrations of methyl paraoxon or another substrate in a total of 1 ml. At least five substrate concentrations with a concentration range of 0.1 to 9 mM were utilized in the determination of each Km and I’,.,,, value. For the inhibition studies, each compound evaluated was dissolved in acetone at various concentrations and 10 ~1 of the solution was added to the incubation mixture and coincubated with 3.8 mM methyl paraoxon as a substrate. Is0 values were calculated based on the probit analysis of the inhibition as described previously (7). Toxicity Tests

Toxicity tests were conducted as previously described (7). Briefly, groups of lday-old fifth instar larvae were collected from the culture and 1 ~1 of acetone containing the test insecticide was applied topically to the dorsal surface of the abdomen. Twenty larvae were treated at each dose, mortality counts were performed 24 hr after treatment, and probit analysis was performed. When synergists were used, they Electrophoresis were topically applied 4 hr prior to the application of the insecticides at the dosage of Polyacrylamide gel electrophoresis (PAGE) of the purified hydrolase was car- 100 pg per larva.

METHYL

PARAOXON

HYDROLASE

oxon hydrolase activity. Total activity was computed from the specific activities given in column (A-B). In larval tissue, approximately 50% of the total activity was located in the integument, followed by the fat body and intestine with 19 and 17% of the total activity, respectively. The Malpighian tubules and the head capsule contained low total activity, although their specific activity was high while the hemolymph, silk glands, and ovaries had no activity. The effects of metal ions were studied using the 100,OOOg supematant of the whole body homogenate. Cobaltous and manganous ions increased hydrolase activity by 76 and 21%, respectively, while cesium, cadmium, copper, silver, and mercurial ions inhibited it (Table 3). Other ions such as calcium or magnesium had no measurable effect. Chelating agents such as EDTA and citrate also had no effect on hydrolase activity. It should, however, be pointed out that phosphate ions inhibited the reaction significantly. The effect on pH of hydrolase activity is shown in Fig. 1. When the pH was below 5, no activity was observed, whereas hydrolase activity increased progressively and reached an optimum at 8 to 9. Above pH 9.5 the activity decreased but was relatively stable even at pH 11. The starting material for the purification of the phosphorotriester hydrolase was the 100,000gsupematant of the whole body homogenate. The pH of the buffer was adjusted to 8.0 (Tris) or 7.8 (MOPS) and 1 x lop3 M Co2+ was added in order to activate the enzyme. The protein was precipi-

Statistical Analysis Each experiment was replicated three times, except for the purification and a part of the characterization of the hydrolase, and all of the data were analyzed by Student’s t test. RESULTS

Phosphorotriester hydrolase activity was found in all of the subcellular fractions tested, but the 100,OOOgsupematnant had the highest specific activity and contained 72% of the total activity in Heliothis larvae (Table l), indicating that the hydrolase was associated with the soluble fraction. Of the tissues tested, Malpighian tubules had the highest specific activity, followed by the head, integument/muscle, fat body, and intestine, while the hemolymph had the lowest activity and the silk glands and ovaries had no detectible activity (Table 2, column A). Since these activities include the phosphorylation of nonspecific esterases as well as the hydrolysis of methyl paraoxon, the reaction rate was also studied at 0°C for 1 set in order to estimate the amount of pnitrophenol formed by the phosphorylation of non-specific esterases (Table 2, column B). The head had the highest activity, followed by Malpighian tubules and hemolymph and other tissues had lower activity. Values in the column (A-B) represent the corrected activity of methyl paraoxon hydrolase. The results were similar as stated above except for hemolymph. It was clear that hemolymph had no methyl paraTABLE The Subcellular

Distribution

Subcellular fraction lc@(-k PPt ww? PPt l@ww? PPt 100,000g sup

of Methyl

Paraoxon virescens

+ 0.008 rt 0.007 ‘- 0.001 -+ 0.016

LIValues represent the mean 2 standard deviation.

1

Hydrolase Larvae”

Specific activity (nmoUhr/mg protein) 0.058 0.025 0.047 0.164

5

OF H. virescens

in Whole

Body

Homogenates

of H.

Total activity (nmotikrva) 1.760 0.277 0.618 6.828

+ 2 2 f

0.258 0.087 0.008 0.683

% 18.6 2.9 6.5 72.0

--_

6

KONNO ET AL. TABLE 2 of Methyl Paraoxon Hydrolase in Fifth Instar Larvae of H. virescens”

Specific Activity and Tissue Distribution

Specitic activity Tissue

Ab

Cuticle and muscle

0.403 0.370 0.079 1.264 0.791 0.014 0.0

Fat body Intestine Malpighian tubules Head Hemolymph Silk gland and ovary

2 ” + 2 k f

Total activity’

BE 0.037 0.018 0.003 0.080 0.134 0.007

0.005 0.0 0.004 0.097 0.135 0.016 0.0

(A-B)d

* 0.002 2 + k +

0.399 0.370 0.074 1.168 0.657 0.0 0.0

o.OtNI2 0.019 0.036 0.009

o Values represent the mean + standard deviations. b The reaction mixture was incubated at 3O’c for 1 hr (nmol/br/mg c The reaction mixture was incubated at 0°C for 1 set (nmoyhr/mg

k + 2 i -c

(nmobbr/larva) 0.032 0.018 0.002 0.010 0.097

7.262 2.676 2.499 1.238 0.719 0.0 0.0

k + -e 2 +

0.637 0.130 0.068 0.010 0.106

% 50.5 18.6 17.4 8.6 5.0 0.0 0.0

protein). protein).

d run01 of methyl paraoxon hyd./hr/mg protein. e Total activity

was calculated

based on the specitic activity

tated with ammonium sulfate and the hydrolase activity was found in the 25 to 50% saturated fractions as shown in Fig. 2. The fractions from 35 to 45% saturation were combined, dialyzed, and applied to a DE-52 column. As shown in Fig. 3A, the main hydrolase peak was eluted with buffer containing 0.15 M NaCl, and this fraction was concentrated and applied to Bio-Gel A column. The hydrolase did not elute between pH 9.4 to 6.5 (Fig. 3B). When the elution was continued using Polybuffer 74 from pH 7.4 to 4.0 in the column equilibrated with 0.025 M imidazole buffer, pH 7.4, the phosTABLE

(A-B).

phorotriester hydrolase was denatured (data not presented). Therefore, the eluant was stopped at pH 6.5 and the bound protein was eluted with MOPS buffer utilizing a linear gradient from 0.1 to 0.4 M NaCl. The fractions containing the hydrolase were combined, concentrated, and applied to a Sephadex G-200 column equilibrated with MOPS buffer containing 0.1 M NaCl. Most of the protein impurities eluted from the column just after the void volume and the hydrolase was eluted later (Fig. 3C). The above procedures are summarized in Table 4. The most effective purification 3

Effect of Metal Ions and Chelating Agents on Methyl Paraoxon Hydrolase Activity in H. viresce& Ion Control co2+ Mn2+ Ba2+ Ca2+ Mg2+ Fe’+

Pb2+ Li+ Fe’+

Zn2+

Specific activity (nmol/hr/mg protein) 0.778 1.373 0.940 0.835 0.831 0.818 0.792 0.790 0.781 0.776 0.774

+ f + + + f T f k + +

0.025 0.044* 0.040* 0.021 0.024 0.018 0.050 0.086 0.026 0.050 0.097

Specific activity (nmol/hr/mg protein)

%b

IOll

100 176 121 107 107 105 102 102 100 100 99

Al’+ Mi2+ Ce’+ CU2+ Cd’+ &+ Hii?+

0.767 0.730 0.724 0.617 0.452 0.156 0.100

+ 0.029 2 0.021 2 0.004* k 0.048* rt 0.032* + 0.008’ -+ 0.003*

EDTA

0.813

+ 0.012

citrate phosphate

0.761 f 0.038 0.660 + 0.002*

%b 99 94 93 79 58 20 13 104

98 85

n Ions were added in the incubation mixture at a concentration of 1 X 10m3 M. Values represent the mean k standard deviation. b Percentage

activity

* P < 0.05 vs control.

compared

to the control.

METHYL

3

1

5

6

7

PARAOXON

HYDROLASE

OF

H. virescens

7

8

PH

FIG. 1. Effect of pH on methyl paraoxon hydrolase activity in H. virescens. Mean f standard deviation. 0, citrate buffer; 0, Tris buffer; & glycine buffer.

step was ammonium sulfate fractionation, which resulted in a 1Cfold purification with an increase in specific activity from 1.8 to 24.7 nmol/min/mg protein. Ultimately, after a Sephadex G-200 gel filtration, an activity of 480 nmol/min/mg protein was reached with 267-fold purification. The major loss occurred with the DE-52 cellulose column and the final yield after Sephadex G-200 chromatography was only 1%. The purified preparation was applied to PAGE (Fig. 4-P) and three esterase bands (I, II, and III) were detected using a-naphthyl acetate as the substrate. However, only esterase III, the major band, hydrolyzed methyl paraoxon. This enzyme was found in the 100,OOOgsupernatant of the whole body homogenate of the NC-86 strain (Fig. 431, the arrow), but it was 3 Z 2 5f L s %

40 30 20

z

10

‘:2E

500 L

FIG. 2. Distribution of methyl paraoxon hydrolase activity in various ammonium sulfate fractions.

: i ,. 2 9 8 6

4 ? D 0

FIG. _ -_ 3. Purification of a methyl paraoxon hydrolase from H. virescens by (A) DEAE-52, (B), Bio-Gel A, and(C), Sephadex G-200 column chromatography. 0, methyl paraoxon hydrolase activity; , . . , A,; -, pH; a, MOPS byffer with 0.1 M NaCl added; b, MOPS buffer with 0.15 M NaCl added; c, a linear gradient (0. I-o.4

M).

hardly detectable in the susceptible strain (Fig. 4S2). The molecular weight of methyl paraoxon hydrolase as determined by Sephadex G-200 chromatography was approximately 120,000 (Fig. 5). Effects of inhibitors on methyl paraoxon hydrolase activity were studied using this preparation (Table 5). Of the 12compounds tested, DEF, TOCP, TCPB, K-2, DFP, OTFP, and EPN showed some inhibitory effect, but their Isa’s were greater than 1 x 10V4 M. Other compounds, such as carbamates and methyl paraoxon homologs, had no effect below 1 x 10V3 M. Based on in vitro experiments, DEF and TCPB were chosen and utilized in in vivo toxicity tests (Table 6). When they were applied to the susceptible reference strain, no

KONNO ET AL.

8

TABLE 4 Purification of Methyl Paraoxon Hydrolase from Whole Body of the Fifth Instar Larvae of H. virescens

Fraction lww?(suP) Ammonium sulfate DE-52 Bio-Gel A (chromatofocusing) Sephadex G-200

Vol (ml)

Total activity (~moYmin)

Total protein (mg)

Specific activity nmole/min/mg protein

Yield (%I

130 13 17

16.46 9.87 0.81

8920 400 7.4

1.8 24.7 109.5

100 60 5

1 14 61

331.3 480.0

3 1

184 267

1.2 1.0

0.53 0.24

synergistic effect with either methyl parathion or methyl paraoxon was observed. On the other hand, a 1.5 and 2.5fold synergistic effect with methyl parathion and methyl paraoxon, respectively, was observed in the NC-86 strain. Phosphorotriester hydrolase activity toward a series of paraoxon homologs and other substrates was studied (Table 7). When ethyl paraoxon was used as a substrate, the Km value was similar to that of methyl paraoxon, but the V,, value was 30 times less than that of methyl paraoxon and the relative enzymatic half-life (t&was 33 times higher. When n-propyl paraoxon was used as a substrate, no enzymatic activity was observed. The V,,, values for pnitrophenyl acetate (PNA) and pnitrophenyl butyrate (PNB) were similar, but the Km value was 30 times less for PNB than for PNA. Consequently, the relative

FIG. 4. Polyacrylamide gel electrophoresis of a methyl paraoxon hydrolase from H. virescens. P, the pur$ed preparation from the NC-86 strain; SI, the ltW,OOttg supernatant from the NC-i% strain; S2, the loO,tXWg supernatant from the control strain, the arrow, and I, II, and III in text. The gel was stained with a-naphthyl acetate and Fast Blue RR’.

1.6 0.50

Purification

enzymatic half-life for PNB was 30 times less than for PNA and 71 times less than for methyl paraoxon. Toxicities of methyl paraoxon and ethyl paraoxon to fifth instar larvae from the two strains are shown in Table 8. The resistance factor for methyl paraoxon was 36, while it was only 1.8 for ethyl paraoxon, indicating that the NC-86 strain is not resistant to ethyl paraoxon. DISCUSSION

Phosphorotriester hydrolases, including A-esterase, arylesterase, paraoxonase, DFPase, etc., are important enzymes for the detoxication of toxic phosphorotriester compounds. They have been reported to be present in high levels in mammals, low lev-

FIG. 5. Molecular weight determination of a methyl paraoxon hydrolase from H. virescens on a Sephadex G-200 column. I, p- amylase; 2, alcohol dehydrogenase; 3, bovine serum albumin; 4, carbonic anhydrase; 5, cytochrome c; k, methyl paraoxon hydrolase; V,, elution volume; V,, void volume.

The Effect

of Esterase

METHYL

PARAOXON

Znhibitors

on Partially

HYDROLASE

OF

TABLE

5

Purified

Methyl

Paraoxon

Chemical

z, (lo-4 M)

Chemical

DEFb TOW= TCPBd K-2 Propoxur

4.40 + 0.19 5.07 + 0.20 3.74 + 0.23 7.70 2 1.24 >lO >lO

DFPf OTFP8 EPN Methyl parathion Ethyl paraoxon n-Propyl paraoxon

F’irimicarb

9

H. virescens

Hydrolase

from

H. virescens’

z,e w-4

M)

> 1.O (24%)” > 1.o (44%) 4.00 + 0.57 >lO >lO >lO

a Values represent the mean 2 standard deviation. b S,S,S-Tributyl phosphorotrithioate. ’ Triorthocresylphosphate. d 2,3,6-Trichloro-l-propynyloxy benzene. ’ 2-Phenoxy-4H-l,3,2benzodioxaphosphorin29xide. f Disopropyl fluorophosphonate. g 3-Octylthio- 1,l , l-tritluoro-2-propanone . h Percentage of inhibition compared to the control.

els in birds (34), and in a few insect species (35). The hydrolase found in the methyl parathion-resistant tobacco budworm is responsible in part for the resistance in this strain of insect (7) and this enzyme may be the first true phosphorotriester hydrolase responsible for resistance in insects. Some of the data to support that the enzyme is a true phosphorotriester hydrolase is (I) the specific activity of the partially purified hydrolase to methyl paraoxon was 0.480 ~mol/min/mg protein (Table 4) which is higher than that of the house fly esterase

which was 0.44 nmol/hr/mg protein to ethyl paraoxon in the Hirokawa strain (24) and almost equal to that of the partially purified arylesterase, from rabbit serum to ethyl paraoxon, 0.642 pmoVmin/mg protein (15); (II) all of the compounds tested which are known inhibitors of B-esterases or catboxylesterases such as DEF, TOCP, K-2, propoxur (36), DFP, ethyl paraoxon, npropyl paraoxon (23), and even a potent inhibitor of JH-esterase, OTFP (37), failed to inhibit the hydrolase completely at a concentration of 1 x 10e4 A4 (Table 5); (III) a

TABLE Eflects

of DEF

and TCPB

on the Toxicity

Chemicalb Methyl parathion + DEF” + TCPBd Methyl paraoxon + DEF + TCPB

6

of Methyl Parathion of H. virescens

and Methyl

NC-86 618 381 433 182 70.9 76.4

2 f k + + f

8 13* 81* 10 6.2* 19.8*

Paraoxon

to Fifth

Znstar

Larvae

Control (1.0) (1.6) (1.4) (1.0) (2.6) (2.4)

10.8 11.8 12.7 5.0 5.4 4.7

k k 5 f k +

0.4 0.8 2.0 0.9 1.2 0.8

(1.0) -(0.9) (0.9) (1.0) (0.9) (1.1)

LIValues represent the mean k standard deviation. b DEF and TCPB were topically applied at a dosage of 100 p&larva, 4 hr prior to the application insecticide. c S,S,S-Tributyl phosphorotrithioate. d 2,3,6-Trichloro-1-propynyloxy benzene. ’ Fiis in parentheses indicate synergistic ratios; LD, for insecticide alone/LD,, for the mixture. * P < 0.05 vs insecticide alone.

of the

10

KONNO

TABLE 7 Activity of a Partially Purified Preparation of Methyl Paraoxon Hydrolase toward a Series of Methyl Paraoxon Homologs and Other Substrates

v K,,,” (n&)

Chemical Methyl paraoxon Ethyl paraoxon n-Propyl paraoxon p-Nitrophenyl acetate p-Nitrophenyl butyrate

a

(~rno~~nhng protein)

0.678 0.738

(to&”

0.300 0.010 ND” 2.36 2.29

?F 0.073

1.57 51.3 0.645 0.022

’ Figures represent mean values of two replicates. b Relative enzymatic half-life = 0.695 (K,,,IV,,). c Not detectable.

number of investigators have observed with mammalian arylesterases (15,38) that phosphate ions inhibit the activity of this enzyme (Table 3), whereas this effect has not been reported with B-esterase or carboxylesterases; and (IV) cobaltous and manganese ions activate this enzyme (Table 3), as reported previously with arylesterases which hydrolyze ethyl paraoxon or methyl paraoxon in a different insect species (1719). Finally, this enzyme can be considered as an “A” esterase (39) since Hg2+ ions inhibit the enzyme (Table 3), and ethyl paraoxon serves as a substrate (Table 7). Despite these findings, a few questions remain as to the properties of this enzyme. Aldridge stated that A-esterases hydrolyzed acetate esters faster than butyrate esters (8) and this concept has been confirmed in a variety of studies dealing with mammalian enzyme sources (40). In the present investigation, the reverse was observed (Table 7), suggesting that this enTABLE 8 Toxicities of Methyl Paraoxon and Ethyl Paraoxon to Fifth Instar Larvae of H. virescens LDso &W’ Chemical Methyl paraoxon Ethyl paraoxon

NC-86 182 k lO* 18* 3

Control

Resistance factor

5.0 * 0.9 9.8 f 1.2

a Values represent the mean k standard deviation. * P < 0.05 vs control.

36.4

1.8

ET AL.

zyme has B-esterase properties. Taking into account the statement of Walker and Mackness (41) that the classification of esterases is in a confused state because esterases tend to have broad and overlapping substrate specificities, the hydrolase found in the tobacco budworm may be different from those reported hitherto. Therefore, we referred to this enzyme as methyl paraoxon hydrolase in order to distinguish it from other arylesterases. The methyl paraoxon hydrolase was mainly distributed in the integument/ muscle of the larvae of the tobacco budworm (Table 2), and this localization was coincident with that of an arylesterase in adult Triutoma infestans (42). This finding was interesting in terms of the development of resistance to contact toxicants because the cuticle is considered the first barrier against intoxication by such compounds. On the other hand, the specific activity of the methyl paraoxon hydrolase was the highest in the Malpighian tubules followed by the head (Table 2). Since the head contains the highest amount of acetylcholinesterase (AChE), which is the target for organophosphate inhibition, the localization of the methyl paraoxon hydrolase in the head of the resistant larvae may serve to protect the AChE, which is as sensitive as that of the susceptible larvae (7), from inhibition by methyl paraoxon. Malpighian tubules are excretory organs in insects and the specific activity of carboxylesterase in this organ is also higher in the case of gypsy moth larvae (43). Considering the K, values for methyl paraoxon and other substrates such as PNB (Table 7), the methyl paraoxon hydrolase may have an important role in the detoxication of undesirable compounds formed in the body other than the hydrolysis of methyl paraoxon, as mentioned with carboxylesterase in the previous report (43). This may be supported by the study on the sensitivity of methyl paraoxon hydrolase to cations, as well. Shishido and Fukami (12) reported that the diazoxon hydrolase present in rat liver was

METHYL

PARAOXON

HYDROLASE

inhibited almost completely by metal ions such as Ce3+, Cu”, Cd*‘, and Hg*+ and was inhibited from 20 to 70% by Ba*‘, Ni*‘, Ag+, Co*+, Pb*+, Mn*‘, and Zn*’ at a concentration of 1 x 10Y4 M. Among these metal ions, Hg*+ and Ag+ ions strongly inhibited the methyl paraoxon hydrolase of the tobacco budworm. Ce3+, Cu*+, and Cd*+ inhibited the enzyme slightly and other ions had no effect at a concentration 10times greater (Table 3), indicating that the insect enzyme had a lower sensitivity to metal ions than the mammalian enzyme. Studies on the hydrolysis of methyl paraoxon homologs by methyl paraoxon hydrolase demonstrated that the enzyme was able to recognize the length of the alkoxy group (R) in the structure of (RO), P(O)-0-@-NO*, that is, it hydrolyzed methoxy > ethoxy and no reaction with npropoxy groups (Table 7). Such recognition has been reported for carboxylesterases (39) and glutathione S-transferase (44)) where the recognition, hydrolysis, or transfer occurs at the alkoxy group. However, with methyl paraoxon hydrolase, the recognition occurs with the alkoxy group but the hydrolysis is at the p-nitrophenyl group. Since the enzyme had a lower V,, value for ethyl paraoxon than for methyl paraoxon, one would expect the NC-86 strain of the tobacco budworm not to be as resistant to ethyl paraoxon. The results obtained in the toxicity tests clearly show a relationship between the in vitro relative enzymatic half-life values and in vivo resistance factors (Table 8). That is, the (to.&. was 33 times less for methyl paraoxon than for ethyl paraoxon which resulted in 20-fold higher resistance to the former than to the latter. In vitro, TCPB inhibited the methyl paraoxon hydrolase as much as DEF (Table 5) and this inhibition was confirmed by the synergistic effect on the toxicity of methyl parathion and methyl paraoxon in vivo (Table 6). TCPB was originally synthesized as a synergist for carbamates in the house fly

OF H. virescens

11

(45,46) and is considered to inhibit the monooxygenases responsible for the detoxication of carbamates (47). Previously it was reported that monooxygenase activity responsible for the desulfuration of methyl parathion to methyl paraoxon was less in the NC-86 strain than in the control strain (7). Therefore, the synergistic effect of TCPB may not be attributable entirely to the inhibition of monooxygenases but also to the inhibition of methyl paraoxon hydrolase. Thus, it may be useful to develop inhibitors of methyl paraoxon hydrolase in order to solve the resistance problem, although such compounds were not found in the present screening. In summary, the methyl paraoxon hydrolase responsible for resistance to methyl parathion in the NC-86 strain (7) appears to be a true phosphorotriester hydrolase, and this may be the first clear demonstration that a relationship exists between in vivo resistance factors and in vitro kinetic parameters of phosphorotriester hydrolase activity in insects. ACKNOWLEDGMENTS Paper No. 12176 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named nor criticism of similar ones not mentioned. Work supported in part by PHS Grant ES-00044 from the National Institute of Environmental Health Services, U.S. Public Health Sciences. The authors are indebted to Dr. M. Lewandowski for advice in the purification. T. Konno, Nihon Nohyaku Co. Ltd., and Y. Kasai, Kao Corp., are grateful to their respective companies for financial support during this study. REFERENCES 1. D. L. Bull and C. J. Whitten, Factors influencing organophosphorus insecticide resistance in tobacco budworms, J. Agric. Food Chem. 20,561 (1972). 2. F. M. Szeicz, F. W. Plapp, Jr., and S. B. Vinson, Tobacco budworm: Penetration of several insecticides into the larva, J. Econ. Entomol. 66, 9 (1973). 3. C. J. Whitten and D. L. Bull, Metabolism and absorption of methyl parathion by tobacco bud-

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