Purification and characterization of an esterase isozyme from insecticide resistant and susceptible strains of german cockroach, Blattella germanica (L.)

InsectBiochem. 0965-1748(94)00093-X

-

Bid. Vol. 25. No. 4, pp. 519-524. 1995 Copyright c 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-174X/95 $9.50 + 0.00

M&c.

Pergamon

Purification and Characterization of an Esterase Isozyme from Insecticide Resistant and Susceptible Strains of German Cockroach, Blattella germanica (L.) SURESH

K. PRABHAKARAN,*

SHRIPAT

T. KAMBLE*t

Received 30 June 1994; revised and accepted 28 October 1994

The most active forms of esterases (E5, E6 and E7) from the German cockroach, Bhzttellu germanica (L.) were purified from resistant and susceptible strains. About 45-155 fold purification with a ll-16% of total esterase recovery was achieved after different column chromatography and preparative gel electrophoresis. Elution profiles of resistant and susceptible strains were similar, but esterase E6 activity was higher in the resistant strains. Kinetic analyses indicate no differences in K,,, values between the resistant and susceptible strains. However V,,,,, was significantly higher in resistant strains. Inhibition of esterase activity by paraoxon, chlorpyrifos and propoxur did not suggest any structural differences in esterase E6 between strains. From these results we suggest that insecticide resistance in German cockroach is due to the increased production of E6 esterase. The role of E6 may be sequestration of toxic molecules rather than hydrolysis. Blattella germanica

Esterase E6

Purification

Insecticide resistance

INTRODUCTION

MATERIALS

The German cockroach, Blattella germanica (L.) has developed resistance to organophosphate (OP), carbamate, and pyrethroid insecticides (Schal, 1988; Cochran, 1989; Rust and Reierson, 1991; Zhai and Robinson, 1992; Prabhakaran and Kamble, 1993). Enhanced oxidative and hydrolytic enzyme activities have been associated with OP and carbamate resistant German cockroaches (Siegfried and Scott, 1991, 1992; Prabhakaran and Kamble, 1993, 1994). Electrophoretic analysis of German cockroach homogenates revealed ten esterase isozymes differing in composition and activity between insecticide resistant and susceptible strains (Prabhakaran and Kamble, 1993, 1994). All of these isozymes hydrolyzed a-naphthyl acetate at different rates. Isozyme E6 was the most active esterase in insecticide resistant strains having at least a 3-fold increase in specific activity when compared with the susceptible strain. The current study involved purification of this enzyme from insecticide resistant and susceptible strains of German cockroach and characterization of its biochemical properties and toxicological interactions.

Insects Three strains of German cockroach used were: CSMA (susceptible strain reared in the laboratory without insecticide selection pressure) and Baygon-R and Dursban-R (multiresistant strains to OP and carbamate and insecticides [Siegfried et al. 1990; Prabhakaran Kamble, 19931). Cockroaches were reared on Purina’ dog chow (Ralston Purina, St Louis, MO.) and water. The colonies were maintained in Plexiglas containers (30 x 30 x 30 cm) at 27 + 2”C, 60 t_ 10% r.h. and a photoperiod of 12: 12 (L:D) h. Chemicals Insecticides used for inhibition tests were: paraoxon (90%, Sigma Chemical Co, St Louis, MO.), propoxur (99.41%, Environmental Protection Agency, Research Triangle Park, N.C.), and chlorpyrifos (99%, DowElanco, Indianapolis, Ind.). Biochemicals were purchased from Bio-Rad, Hercules, CA and Sigma Chemical Co, St Louis, MO. All the chemicals and buffers were of reagent grade or better. Enzyme

*Department of Entomology and Water Center/Environmental grams, University of Nebraska, Lincoln, NE 68583-0816, tAuthor for correspondence.

ProU.S.A.

AND METHODS

assays

During purification, esterase activity was measured as the change in absorbance at 405 nm using a Spectronic

SURESH

520

K. PRABHAKARAN

601 spectrophotometer equipped with an accessory control module (Milton Roy, Rochester, N.Y.). The reaction mixture (1 ml) consisted of 20 ,ul of 0.01 M p-nitrophenyl acetate (PNPA) in 200 mM phosphate buffer (pH 7.2) and a proper amount of enzyme. Unless otherwise stated, all enzyme assays were conducted at 26°C. Protein concentrations were estimated by the Bradford (1976) method using bovine serum albumin as the standard. Pur@ication

of esterase

E6

About 10 g of adult German cockroaches were frozen at -20°C and homogenized after removing head, wings and legs with a Tissue Tearer@ (Biospec Products, Bartlesville, Okla) in 20 ml of 10 mM Tris-Cl buffer (pH 7.8). The homogenate was centrifuged at 10,OOOg for 20 min at 4°C. The supernatant was applied to a Q-Sepharose Fast flow (Sigma Chemical Co., St Louis, MO.) column (8 x 1.5 cm) equilibrated with the homogenization buffer. The column was eluted with salt gradient (O-l.4 M NaCl in Tris-Cl buffer) at 1 ml/min for 80 min and fractions of 2 ml were collected and tested for activity toward PNPA. Active fractions were pooled and precipitated with ammonium sulfate. The protein was desalted using a Bio-Gel P6 DG column (10 x 1.5 cm, Bio-Rad, Hercules, Calif.). Sodium phosphate buffer (0.02 M, pH 7.2) was used as the elution buffer. Fractions with esterase activity were combined and applied to a hydroxylapatite column (8 x 1.5 cm, Bio-Gel HTP Gel, Bio-Rad, Hercules, Calif.) equilibrated with 20mM phosphate buffer, pH 6.8. Esterase active fraction was eluted with a phosphate buffer gradient (20-200 mM) at 1 ml/min for 50 min and fractions were collected at 2 min intervals. Esterase eluted as a single peak and fractions with activity were pooled together and concentrated in ultrafilter concentrator unit (PGC Scientifics, Gaithersburg, Md). The concentrated sample (with 10% glycerol and bromophenol blue dye) was loaded onto a 9% acrylamide gel (7 cm) with a 4.8% stacking gel (2 cm) in a Model 491 Prep Cell (Bio-Rad, Hercules, Calif.). The gel was run at 15 W constant power with 50 mM Tris 25 mM Boric acid electrode buffer (pH 8.7). The pure enzyme was eluted with 0.02 M phosphate buffer using an Econo System (Bio-Rad, Hercules, Calif.). The active fractions were pooled and analyzed for purity with SDS-PAGE.

and SHRIPAT

T. KAMBLE

manufacturer’s instructions. pf calibration markers (3-9) were used to obtain the calibration curve. The gel was stained with Coomassie blue. The pl values were determined from three replicates. Gel jiltration The molecular weight of cockroach esterases were determined using a Sephacryl S-200 HR column (1.5 x 34 cm) equilibrated with 50 mM phosphate buffer (pH 7.2). The column was calibrated at 4°C using Blue Dextran and standard proteins (M, 12,400-200,000). A sample of 1 ml was applied to the column and eluted at 0.075 ml/min flow rate. Kinetic analysis

Enzyme kinetics of the purified esterase isozyme from resistant and susceptible cockroach strains was determined by recording the activity toward nine concentrations (20-500 PM) of PNPA. Lineweaver-Burk plots were constructed and I’,,,,, and Km values were calculated for each strain. The means of three different preparations were analyzed by Fisher’s least significant difference (LSD, c( = 0.05) test (SAS Institute, 1989). Inhibition

analysis

Inhibition kinetics of the purified enzyme was determined by stopped time inhibition assays. The reaction 100

r

252770 A

(A)

20 -

-60

!.

- 50 - 40 - 30 - 20 - 10 -0

loo E -20

S

4o

5 E

Q400

f

iii ki8-

Gel electrophoresis SDS-PAGE was performed according to Laemmli (1970) using 4.8% acrylamide stacking gel and 12% acrylamide separating gel in a Bio-Rad Protean II mini electrophoresis cell along with protein molecular weight markers (29-205 KDa, Sigma Chemical Co., St Louis, MO.). Proteins were stained with Coomassie blue R250. Isoelectric focusing

Isoelectric focusing was performed using Bio-Lyte 3/10 (Bio-Rad, Hercules, Calif.) ampholytes on a Bio-Rad model 111 mini IEF cell according to the

FRACTION NUMBER PROTEIN . .. . . .

ESTEFiASE ACTIVITY ELUTloN GR+lJ!ENT -

FIGURE 1. Elution profiles for protein and esterase activity using: (A) Q-sepharose chromatography and protein eluted with O-l.4 M NaCl gradient. (B) Hydroxylapatite chromatography and protein &ted with 20-200 mM phosphate buffer.

TABLE

ESTERASE

ISOZYME

OF BLATTELLA

1. Purification

of esterase

from different

Total protein Strain

Step

Baygon-R

Dursban-R

CSMA

“Data

represents

(mg)

GERMANICA

strains

Enzyme activity (p mol/min/mg)

of German

521 cockroach”

Fold purification

Yield W)

Homogenate Q-sepharose Hydroxylapatite Prep Cell E5 E6 E7

1351.2 153.0 10.8

10.9 76.4 308.5

1.0 7.0 28.3

100.0 79.4 22.6

0.4 0.7 0.3

605.6 1666.4 512.2

55.6 152.9 47.0

1.8 8.2 1.2

Homogenate Q-Sepharose Hydroxylapatite Prep Cell E5 E6 E7

1136.2 132.0 11.5

7.6 53.5 212.5

1.0 7.0 27.8

100.0 81.3 28.1

0.4 0.9 0.3

594.2 1111.1 489.3

77.7 145.4 64.0

2.7 12.2 2.0

Homogenate Q-Sepharose Hydroxylapatite Prep Cell ES E6 E7

1034.5 102.4 7.9

4.3 30.2 125.9

1.0 7.1 29.6

100.0 70.5 22.6

0.4 0.4 0.3

527.8 615.4 466.6

124.3 144.9 109.8

4.8 6.3 3.2

a single purification

-

procedure.

mixture consisted of 0.02 M phosphate buffer (pH 7.2), a series of concentrations of insecticides (dissolved in acetone) and proper amounts of the purified enzyme. Aliquots were taken at various intervals and their residual activities were determined using PNPA as described before. The control had only the solvent used to dissolve the insecticide. Slopes and standard errors of inhibition curves were calculated by linear regression and I,, ‘s calculated as described by Main and Iverson (1966). Each inhibition curve was derived from three different enzyme preparations with three determinations at each time point.

utilized were identical for all the strains tested. Data for a single purification procedure are presented in Table 1, and the elution profiles for the ion-exchange (Q-Sepharose) and hydroxylapatite chromatography are presented in Fig. 1. Three most active molecular forms of cockroach esterase (E5, E6 and E7) were collected as pooled fractions after the prep cell electrophoresis (Fig. 2). These fractions were homogenous and active in all the strains used. All preparations resulted in a final

El

Recovery

of esterase

inhibition

The recovery of inhibited esterase was determined according to Cuany et al. (1993). The purified enzyme (2.5 ml) was incubated with 25 ,~l of 0.01 M paraoxon for 1 h on ice. The mixture was passed through a prepacked Sephadex G-50 column to elute unbound insecticide. Protein fractions free from unbound paraoxon were pooled after testing for antiacetylcholinesterase activity. The esterase activity of the recovered protein fraction was determined at 1 and 2 h intervals. The control had only the solvent used to dissolve paraoxon. The experiment was repeated at least three times.

RESULTS

PuriJication

of esterase

E2 E3 E4 ES E6

E7

ES

E9

El0

Et5

The procedure followed for the purification of esterase isozyme was relatively efficient. The elution profiles obtained for the different chromatographic techniques

FIGURE 2. Native PAGE of the most active esterases purified from German cockroach. Lane A, 10,OOOg supernatant; Lane B, esterase E5; Lane C, Esterase E6: and Lane D. Esterase E7.

522

SURESH

K. PRABHAKARAN

and SHRIPAT

T. KAMBLE

TABLE 2. Molecular weight and isoelectric points of the most active forms of esterase in German cockroach strains Esterase

M, (KD)

Pi

ES E6 E7

51& 1.3 55 * 1.5 53 + 1.4

4.9s + 0.04 4.84 f 0.05 4.73 & 0.02

Mean + SEM of three separate

preparations.

recovery of ll-16% of total esterase in all the strains. The purification of the three active esterases ranged between 45-155 fold. Esterase E6 of Baygon-R and Dursban-R had significantly higher activity when compared with CSMA in all the purification experiments conducted. The specific activity of esterase E5 and E7 did not differ between the resistant and susceptible strains. Isoelectric focusing

and molecular

B

C

205 116 91.4

DURS_BAN-R BAYtON-R

55000 f 1500 Da. the M, calculated from the SDSPAGE was 47000 f 3000 Da (Fig. 3). The SDS-PAGE data indicate that the enzyme E6 is monomeric in nature. Enzyme

kinetics

Results of kinetic analyses of esterase E6 from resistant and susceptible strains are presented in Table 3 and Fig. 4. Michaelis constant, K, did not indicate any difference in E6 between the resistant and susceptible strains of German cockroaches (LSD, CI= 0.05). However, the rate of reaction V,,,, was very different for the strains tested. The resistant strains had 2-3 fold higher V,,, when compared with the resistant strain. Similar K,,, values indicate that enzymes from susceptible and resistant strains interact similarly. However, I’,,,,, values suggest that the resistant strains of German cockroaches have higher titres of E6. and recovery

The insecticides paraoxon, chlorpyrifos and propoxur were used in the inhibition kinetics studies. The I,, values (the concentration required to inhibit 50% of esterase activity) obtained for the three German cockroach strains are presented in Table 4. The I,, values of the insecticides tested were similar for both resistant and susceptible strains. This indicates that the esterase E6 from resistant and susceptible strains binds similarly

29

TABLE

3. SDS-PAGE weight markers;

CSMA

FIGURE 4. Lineweaver-Burk plot of kinetics of esterase E6 purified from three strains of German cockroach determined using different concentrations of p-nitrophenyl acetate.

Inhibition

FIGURE Molecular

0.06

l/p-rhOPHEN~*cET*TTATE

weight

The isoelectric points of the most active German cockroach esterases are presented in Table 2. The pl of the purified esterases were somewhat acidic and differed slightly. The molecular weight estimated based on Sephacryl chromatography for esterase E5 and E7 were 57000 &- 1300 and 53000 * 1400 Da, respectively. The molecular weight of the purified esterase E6 was A

0.04

(0.02)

of the purified esterase isozyme. Lane A, Lane B, homogenate; and Lane C, purified esterase E6.

3. Kinetics

of esterase purified from different of German cockroaches

Strain

V,,, & SEM (pmol/min/mg)

Baygon-R Dursban-R CSMA

2667.4 k 89.2a 1798.6 + 105.6b 997.6 + 79.4c

strains

Km + SEM (PM) 118.6 f 20.la 117.4 * 28.2a 117.4 * 34.2a

Mean f SEM of three separate preparations each with three replications. Means within the same column followed by the same letter are not significantly different (LSD, P < 0.05).

ESTERASE TABLE

4. Inhibition different

(expressed as I,,) of esterase strains of German cockroaches

Paraoxon (IO-‘M)

Strain Baygon-R Dursban-R CSMA

ISOZYME purified

Chlorpyrifos (lO-4 M)

3.156 k 0.046a 3.138 + 0.073a 3.162 k 0.024a

2.786 k 0.215a 2.831 k 0.196a 2.812 k 0.167a

OF BLATTELLA from

Propoxur (IO-’ M) 1.766 + 0.124a 1.785 &O.l85a 1.816 k 0.108a

Mean k SEM of three separate preparations each with three replications. Means within the same column followed by the same letter are not significantly different (LSD, P < 0.05).

to the inhibitors. This also validates the similarity in the catalytic properties of purified enzymes from the German cockroach strains. Esterase E6 activity inhibited by 10s4 M paraoxon was not recovered even after 2 h following the removal of unbound insecticide (Table 5). This suggests insignificant dephosphorylation of the inhibited E6. Loss of esterase activity prohibited the study at longer intervals.

DISCUSSION Esterases exhibiting an elevated activity toward model substrates have been associated with insecticide resistance in many insects (Devonshire, 1991). In German cockroaches elevated esterase activity has been shown to be associated with insecticide resistance (Siegfried and Scott, 1991, 1992; Prabhakaran and Kamble, 1993, 1994). As reported earlier, the esterase activity is much higher in resistant German cockroaches when compared with susceptible strain. The purification procedure yielded about 1 mg of most active esterases (E.5, E6 and E7). The estimated molecular weight of these esterase forms ranged between 53-57 kDa. These estimates were slightly lower than the molecular weights reported earlier by Prabhakaran and Kamble, 1994 based on the retardation coefficients of esterases. The pl of these enzymes were acidic and differed slightly. Esterases with similar molecular weights and pl have been reported in mosquitoes, C&x pipiens L. (Fournier et al., 1987) C. quinqwfaciatus Say (Ketterman et al., 1993) and rice brown planthoppers, Nilaparvata lugens Stal (Chen and Sun, 1994). Esterase E6 of the German cockroaches was the predominant form among the purified esterases. It had 2-3 fold higher specific activity in resistant strains when compared with susceptible strains. The similarity in K,,,

TABLE

5. Activity of German cockroach esterase E6 after inhibition with paraoxon and removal of unbound insecticide Time after inhibition ~~. lh

Strain

Baygon-R Dursban-R CSMA

Control

.~_ 2h

Inhibited

Control

(PM of PNPA hydrolyzed/min) 27.35F0.50 0.11+_0.02 29.15+_0.90 18.52 i 0.41 0.12 + 0.01 17.64 f 0.61 10.25 + 0.42 0.08 + 0.02 10.95 rfr 0.15

Inhibited

0.13+0.04 0.10 f 0.02 0.06 + 0.01

GERMANIC-A

523

values and inhibition curves obtained for E6 from the three German cockroach strains suggest that there are no differences in their catalytic ability toward the substrate. Consequently, increase in E6 activity in resistant strains is not due to difference in active sites, but to the quantity of enzyme present. Higher V,,, values in the insecticide resistant strains also demonstrate the quantitative activity differences among strains. In order to demonstrate the involvement of E6 in insecticides resistance by hydrolysis of insecticide or by sequestration, reactivation experiments were conducted. Absence of any significant activity 2 h after removal of unbound oxons strongly suggest that the resistance mechanism is sequestration rather than hydrolysis. Similarly, insecticide resistance by sequestration of insecticide molecules have been reported in peach-potato persicae (Devonshire and Moores, aphids, Myzus 1982) C. pipiens (Cuany et al., 1993) and rice brown planthoppers, N. lugens (Chen and Sun, 1994). Strong inhibition of E6 by paraoxon also supports the role of oxon analogs in civo toxicity of organophosphate insecticides. The current investigation demonstrates that the overproduction of esterase isozyme E6 is likely responsible for the insecticide resistance in German cockroaches. If resistance was due to the qualitative changes in the similarities in kinetic properties, inhibition enzyme, curves and chromatographic behavior would not have been observed. The overproduction of esterase E6 in German cockroaches could have occurred by two mechanisms. The first one may be due to gene amplification which is the presence of multiple coding regions for esterases in the DNA as reported in Myzus persicae Sulzer (Field et al., 1988) and Culex mosquitoes (Mouches et al., 1986). The second one may be by the modification in the esterase E6 gene regulatory area of the DNA. The exact mechanism of overproduction of E6 in German cockroach is not very well understood at this time.

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and SHRIPAT

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Rust M. K. and Reierson D. A. (1991) Chlorpyrifos resistance in German cockroaches from restaurants. J. Econ. Entomol. 84, 7366740. SAS Institute. (1989) SAS User’s Guide: Statistics. SAS Institute. Cary, N.C. Schal C. (1988) Relation among efficacy of insecticides, resistance levels, and sanitation in the control of German cockroach. J. Econ. Entomol. 81, 5366544. Siegfried B. D., Scott J. G., Roush R. T. and Zeichner B. C. (1990) Biochemistry and genetics of chlorpyrifos resistance in the German cockroach, Blattella germanica L. Pestic. Biochem. Physiol. 38, 110-121. Siegfried B. D. and Scott J. G. (1991) Mechanisms responsible for Pestic. Sci. 33, propoxur resistance in the German cockroach. 133-146. Siegfried B. D. and Scott J. G. (1992) Biochemical characterization of hydrolytic and oxidative enzymes associated with chlorpyrifos and propoxur resistance in the German cockroach, Blattellu germanica (L.). J. Econ. entomol. 85, 1092-1098. Zhai J. and Robinson W. H. (1992) Measuring cypermethrin resistance in the German cockroach. J. Econ. Entomol. 85, 348-351.

Acknowledgements-The authors thank Steven R. Skoda and David B. Taylor for their critical review of this manuscript. This research was supported in part by the Center for Biotechnology, UNL. This is published as paper No. 10772, Journal series, Nebraska Agriculture Research Division, and contribution No. 867, Department of Entomology and Water Center/Environmental programs, University of Nebraska, Lincoln.