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Characterization of Acetylcholinesterase Purified from the Lesser Grain Borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)
Comp. Biochem. Physiol. Vol. 119C, No. 2, pp. 205–210, 1998 Copyright 1998 Elsevier Science Inc. All rights reserved.
ISSN 0742-8413/98/$19.00 PII S0742-8413(97)00208-9
Characterization of Acetylcholinesterase Purified from the Lesser Grain Borer, Rhyzopertha dominica (Coleoptera: Bostrichidae) R. N. C. Guedes,1 K. Y. Zhu,2 S. Kambhampati,2 and B. A. Dover 2 1
Departamento de Biologia Animal, Universidade Federal de Vic¸osa, Vic¸osa, MG 36571.000, Brasil; and 2 Department of Entomology, Kansas State University, Manhattan, Kansas 66506, USA ABSTRACT. 1. Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the lesser grain borer (Rhyzopertha dominica) was significantly inhibited by higher concentrations of the substrates acetylthiocholine (ATC), acetyl(β-methyl) thiocholine (Aβ MTC) and propionylthiocholine (PTC). 2. The efficiency of AChE for hydrolyzing different substrates was ATC . Aβ MTC . PTC . S-butyrylthiocholine. The enzyme activity was completely inhibited by 1025 M eserine or BW284C51, but was only partially inhibited by ethopropazine at the same concentration. These results confirmed that the purified enzyme was an typical insect AChE. 3. Non-denaturing and SDS polyacrylamide gel electrophoresis (PAGE) showed only one major molecular form in the purified AChE with a molecular weight of about 107,000 prior to reduction and about 56,000 after reduction, suggesting the homodimer of AChE linked with disulfide bonds. comp biochem physiol 119C;2:205–210, 1998. 1998 Elsevier Science Inc. KEY WORDS. Acetylcholinesterase, affinity chromatography, enzyme kinetics, enzyme purification, lesser grain borer, molecular weight, Rhyzopertha dominica, substrate specificity
INTRODUCTION Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme involved in the nerve impulse transmission across cholinergic synapses, where it hydrolyzes the excitatory neurotransmitter acetylcholine into choline and acetic acid (4,20). The enzyme also is the target site of inhibition by organophosphate and carbarnate insecticides (17,22). Increased incidence of insecticide resistance due to the reduced sensitivity of AChE to inhibition by organophosphate and carbamate insecticides as a resistance mechanism further adds to the toxicological significance of AChE and has stimulated further research of this important enzyme in insects (28,29). Most of the studies on insect AChE have been carried out with relatively crude preparations which contain other esterases with possible overlapping substrate specificities (9,22). The use of purified AChE has obvious advantages over crude homogenates in kinetic studies of substrate and inhibitor interactions, especially when other esterases are capable of hydrolyzing compounds under investigation (14). Although many different methods have been used for the isolation of insect AChE (1,2,6,9,10,11,14,15,21,23,25– 28), affinity chromatography has been demonstrated to be Address reprint requests to: Dr. Kun Yan Zhu, Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, U.S.A. Tel. (785) 532-4721; FAX (785) 532-6232; E-mail: [email protected] Received 31 March 1997; accepted 30 October 1997.
the most effective technique for purification of AChE from insects. It usually provides a high yield with an adequate purity of AChE which is particularly desirable in many toxicological studies. In this paper, we present the following: (i) purification of AChE from the lesser grain borer (Rhyzopertha dominica), an important pest of stored cereals throughout the world, by affinity chromatography; (ii) enzyme kinetics, and substrate and inhibitor specificities of the purified enzyme; and (iii) determination of molecular weights of the reduced and non-reduced forms of the purified AChE. Knowledge obtained from this study should facilitate further investigation of this important enzyme in lesser grain borer and other insect species. MATERIALS AND METHODS Insects Lesser grain borers were reared on whole wheat under constant conditions in the Laboratory of Stored Product Insects, Department of Entomology, Kansas State University, Manhattan (KS) as described previously (7,8). Adults were used exclusively in this study. Chemicals Acetic anhydride, acetyl-(β-methyl) thiocholine iodide (Aβ MTC), acetylthiocholine iodide (ATC), 1,5-bis (4-
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allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), aprotinin, bicinchoninic acid (BCA) solution, bromophenol blue, S-butyrylthiocholine iodide (BTC), cyanogen bromide activated Sepharose 4B, 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB), dithiothreitol (DTT), eserine hemisulfate, ethopropazine hydrochloride, leupeptin, pepstatin, phenylmethanesulfonyl fluoride (PMSF), propionylthiocholine iodide (PTC), silver stain SDS-PAGE molecular weight standard mixtures, sodium dodecyl sulfate (SDS) and Triton X-100 were purchased from the Sigma Chemical Company (St. Louis, MO). Coomassie brilliant blue G250, 1-cyclohexyl-3 (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonale, 3dimethylaminobenzoic acid, ethanolamine, iodoethane, 1,6-hexamethylenediamine, sodium azide and tetraethylammonium iodide (NEt4I) were purchased from Aldrich Chemical Company (Milwaukee, WS). Acrylamide, ammonium persulfate, N,N′-methylene-bis-acrylamide, bovine serum albumin, ethylenediamine-tetraacetic acid (EDTANa2), glycine and protein silver stain kit were purchased from Bio-Rad Laboratories (Hercules, CA). Purification of AChE AChE was purified from lesser grain borer by affinity chromatography using 3-(carboxyphenyl) ethyldimethyl ammonium as affinity ligand (16) as described by Zhu and Brindley (26) with some modifications. Briefly, samples were prepared by homogenizing 4 g of adult insects (whole body) in 60 ml of ice-cold 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% (v/v) Triton X-100, 0.2 M NaCl, and five protease inhibitors (0.1 µM aprotinin, 1 mM EDTANa2, 1 µM leupeptin, 1 µM pepstatin and 10 µM PMSF). The soluble fraction was obtained as previously described (26,28) and applied to an affinity column (1.0 3 7.5 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 and 0.2 M NaCl. After the column was washed with 180 ml of the same buffer, bound AChE was eluted with 0.2 M NEt4I in the same buffer without NaCl. Constant flow rate was maintained at 30 ml/hr with a peristaltic pump, and fractions of 1 ml were collected after the eluting solution was applied to the column. The affinity purified enzyme was dialyzed against 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 to remove NEt4I. Enzyme Activity Assay AChE activity was determined according to the method of Ellman et al. (5) at 405 nm using a Vmax kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA). Inhibition of AChE by three distinct inhibitors, eserine, BW284C51 and ethopropazine were carried out based on the procedures described by Zhu and Clark (28). Kinetic activity of purified AChE was studied by using four sub-
strates (ATC, Aβ MTC, PTC and BTC), each with 12 concentrations ranged from 8 mM to 3.9 µM, at 25°C and pH 7.5. The enzyme activity was determined based on the reaction for 2 min, whereas the initial velocity was determined based on the reaction for 30 sec for ATC, Aβ MTC and PTC, and 2 min for BTC. Michaelis constant (Km) and maximal velocity (Vmax) values were determined by Hanes transformations (18). Protein concentration was determined by the BCA method (19) using bovine serum albumin as standard. Electrophoretic Analysis Non-denaturing polyacrylamide gel electrophoresis (PAGE) (13) was carried out with a Penguin P8DS dualgel electrophoresis system (Owl Scientific, Inc., Woburn, MA) coupled with a cold water circulating system. The gels (4 and 9% acrylamide in stacking and separating gels respectively) were run at a constant voltage of 150V for 2 hr. The AChE bands were visualized by incubating the gels in AChE staining mixture (12) overnight at room temperature for its activity. Staining for total protein was conducted with a silver staining technique using a commercial kit (Bio-Rad) according to the manufacturer’s instructions. Denaturing SDS-PAGE was used to estimate the molecular weights of the reduced and non-reduced AChE as described by Zhu and Clark (28). The procedure was similar to the non-denaturing PAGE except that a final concentration of 0.1% (w/v) SDS was incorporated into both gels and the electrode buffers, and all marker proteins and AChE samples were treated by incubating them in 2% (w/v) SDS solution containing 25 mM DTT in a boiling water bath for 2 min. The molecular weight of the non-reduced or reduced AChE was determined by using a calibration cure of the following molecular weight markers: bovine erythrocyte carbonic anhydrase, 29,000; porcine heart fumarase, 48,500; bovine serum albumin, 66,000; rabbit muscle phosphorylase b, 97,000; and E. coli β-galactosidase, 116,000. RESULTS AND DISCUSSION Purification of AChE AChE was successfully purified from lesser grain borer by affinity chromatography following extraction with Triton X-100. The purification factor was 771 and the yield was 54%, which were typical of nine additional batches of purification during the course of the study (Table 1). The purified AChE had a specific activity of about 4.7 µmol/min/ mg protein, but showed low stability after purification. Addition of glycerol to a final concentration of 20% (v/v) increased the enzyme stability, and the enzyme could be maintained for about three days at 4°C without significant loss of the AChE activity. The degree of purification of AChE from lesser grain borer was comparable to those obtained from western tar-
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TABLE 1. Summary of purification of acetylcholinesterase from R. dominica by affinity chromatography following extraction
by Triton X-100
Procedure Triton X-100 extraction Affinity chromatography
Volume (ml)
Protein (mg/ml)
Total protein (mg)
60
7.83
469.80
3
0.11
0.33
nished plant bug (26,27) and horn fly (23), but smaller than that from Colorado potato beetle (28,29). Using ATC as substrate, the specific activities of purified AChE were 1350 µmol/min/mg protein for Drosophila heads (6), 385 for horn fly (23), 50 for western tarnished plant bug (27), 128 for Colorado potato beetle (28) and 5 for lesser grain borer (this study). The variation in the specific activity of the purified AChE among different insect species may be partially due to the degree of the purity of AChE, but also can be due to insect species variations. Our results are consistent with the notion that insects in Hemiptera, Coleoptera and Lepidoptera exhibit lower levels of AChE activity than in Diptera (22). This is particularly true in Colorado potato beetle (28), in which AChE was apparently purified to homogenous but its specific activity was only less than 10% of the activity determined in AChE purified from Drosophila heads (6) when the same substrate (i.e., ATC) was used.
Specific activity (nmol/min/mg)
Total activity (nmol/min)
Yield (%)
Purification factor (-fold)
6.09
2861.08
100
1
4698.07
1550.36
54
771
indicated that AChE hydrolyzed ATC, Aβ MTC and PTC, 31-, 22-, and 15-fold, respectively, more efficiently than it did BTC. The relative efficiency of purified AChE hydrolyzing BTC vs ATC, as determined by the Vmax (BTC): Vmax (ATC) ratio, was 0.032 for lesser grain borer (this study) and Colorado potato beetle AChE (28). Although smaller ratios were obtained for western tarnished plant bug (0.098) (26) and Drosophila (0.6) (6) AChEs, these ratios are still larger than those found in vertebrates such as electric eel (0.005) and human erythrocytes (0.01) (6). It seems that the substrate specificity of insect AChEs is lower than that of vertebrates as judged by these two substrates. AChE was completely inhibited by 1025 M eserine (a
Substrate Specificity, and Kinetic and Inhibitory Properties of Purified AChE Four substrates, ATC, Aβ MTC, PTC and BTC were used to study the effect of substrate concentration on AChE activity (Fig. 1). Although optimal substrate concentrations were rather broad for ATC, Aβ MTC and PTC, which ranged between 0.06 and 1.00 mM, AChE activity was significantly inhibited by higher concentrations of ATC, Aβ MTC and PTC. Higher concentrations of BTC also inhibited AChE activity, but the inhibition was in a much smaller magnitude. This substrate inhibition phenomenon could be due to the binding of excess substrate to the peripheral regulatory site of the enzyme (3), and is an important distinguishing feature of the AChEs (22). Kinetic studies determined the affinity of AChE to its different substrates. The overall differences in affinity were relatively small among these four substrates. The Km 6 SE values were 24.2 6 2.4 µM for ATC, 13.4 6 3.5 for Aβ MTC, 20.4 6 7.9 for PTC, and 8.4 6 1.7 for BTC. The hydrolyzing efficiencies of AChE for the four substrates, as indicated by their Vmax values were 18.7 6 1.6 µmol/min/ mg protein for ATC, 13.4 6 0.6 for Aβ MTC, 9.3 6 0.6 for PTC, and 0.6 6 0.03 for BTC (Fig. 2). These results
FIG. 1. Substrate specificity and effect of substrate concentration on the hydrolysis of acetylthiocholine (ATC), acetyl(b-methyl) thiocholine (AbMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) by AChE purified from R. dominica. Each point represents the mean of four determinations (n 5 4). Vertical bars indicate standard errors of the mean.
208
FIG. 2. Hanes plots of [s] vs [s]/v for AChE hydrolyzing
acetylthiocholine (ATC), acetyl-(b-methyl) thiocholine (AbMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC). Each point represents the mean of four determinations. All correlation coefficients (r) . 0.98 (P , 0.01).
R. N. C. Guedes et al.
partially contributed to the relatively low specific activity of AChE in lesser grain borer. Molecular weight of purified subunits of AChE was determined in the presence of the reducing agent DTT. Only a major band was verified with an estimated molecular weight of 56,000 (data not shown). The SDS-PAGE analysis with reducing agent indicated the molecular weight of the AChE subunit of lesser grain borer was 56,000, virtually identical to those purified from Drosophila (55,000) (6) and horn fly (54,000) (24). However, the estimated molecular weight was 107,000 for non-reduced AChE, suggesting that each molecule of AChE consisted of two similar subunits connected with disulfide bonds, as found in other insect AChEs (22). In summary, our present study firmly established that the AChE purified from lesser grain borer was a typical insect AChE possessing many common characteristics as found in other insect species (22,28). The major features include: 1) the substrate inhibition at higher concentrations of ATC, AβMTC and PTC; 2) high sensitivity to inhibition by eserine and BW284C54 but less sensitive to ethopropazine; 3) much higher efficiency in hydrolyzing ATC than BTC; 4) comparable molecular size to other AChEs found in other insects; and 5) relatively low activity as compared with AChEs found in vertebrates. All these findings should help further biochemical and molecular studies on AChE from this important pest as were achieved in a few other insect species (24,26,28–31).
general inhibitor for AChE and butyrylcholinesterase, BuChE) and BW284C51 (a relatively specific inhibitor for AChE), but only 60% of the AChE activity was inhibited by ethopropazine (a relatively specific inhibitor of BuChE) at this concentration (Fig. 3). The I50 values expressed as mean 6 SE (n 5 3) were 0.09 (60.03) 3 1028 M for eserine, 8.0 (60.4) 3 1028 M for BW284C51, and 105.0 (649.7) 3 1028 M for ethopropazine. This inhibitor specificity further supported our conclusion that the cholinesterase activity in lesser grain borer was due to AChE, and agreed with other three previously suggested criteria (22): a) the enzyme activity was inhibited by ATC at high concentrations; b) the enzyme hydrolyzed ATC more efficiently than BTC; and c) the enzyme hydrolyzed Aβ MTC at a fairly rapid rate. Molecular Properties of Purified AChE Results from non-denaturing PAGE in the absence of Triton X-100 showed only one major band of AChE when the enzyme was stained for activity. This band corresponded to a major band stained for protein by silver stain (Fig. 4), which suggested only a single major molecular form of AChE in our purified AChE samples and other small bands in the protein-staining gel were probably due to contaminating proteins. These contaminating proteins probably
FIG. 3. Inhibition of R. dominica AChE by eserine,
BW284C51, or ethopropazine. Each point represents the mean of three determinations. Vertical bars indicate standard error of the means.
Acetylcholinesterase Purified from R. dominica
209
8.
9.
10.
11. 12. 13. 14. FIG. 4. Electrophoretic analysis of purified AChE from R.
dominica with non-denaturing PAGE. The gel was stained either for the enzyme activity or protein.
15. 16.
The authors thank the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior-CAPES, UFV Department of Animal Biology, and KSU Department of Entomology for the financial support provided. This manuscript is contribution no. 97-362-J from the Kansas Agricultural Experiment Station.
17. 18. 19.
References 1. Bonning, B.C.; Malcolm, C.A.; Hemingway, J. Purification and characterization of acetylcholinesterase from Culex pipiens pipiens Say. Pestic. Sci. 24:278–280;1988. 2. Brestkin, A.P.; Maizel, E.B.; Moralev, S.N.; Novozhilov, K.V.; Sazonova, I.N. Cholinesterases of aphids-1: Isolation, partial purification and some properties of cholinesterases from spring grain aphid Schizaphis gramina (Rond.). Insect Biochem. 15: 309–314;1985. 3. Cohen, S.G.; Chishti, S.B.; Bell, D.A.; Howard, S.I.; Salih, E.; Cohen, J.B. General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate. Biochem. Biophys. Acta 1076: 112–122;1991. 4. Eldefrawi, A.T. Acetylcholinesterases and anticholinesterases. In: Kerkut, G.A.; Gilbert, L.I. (eds). Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 12. New York: Pergamon Press; 1985:115–130. 5. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Featherstone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88–95;1961. 6. Gnagey, A.L.; Forte, M.; Rosenberry, T.L. Isolation and characterization of acetylcholinesterase from Drosophila. J. Biol. Chem. 262:13290–13313;1987. 7. Guedes, R.N.C.; Dover, B.A.; Kambhampati, S. Resistance to chlorpyrifos-methyl, pirimiphos-methyl, and malathion in
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Brazilian and U.S. populations of Rhyzopertha dominica (Coleoptera: Bostrichidae). J. Econ. Entomol. 89:27–32;1996. Guedes, R.N.C.; Kambhampati, S.; Dover, B.A.; Zhu, K.Y. Biochemical mechanisms of organophosphate resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae) from the United States and Brazil. Bull. Entomol. Res. 87:581–586; 1997. Guilbault, G.G.; Kuan, S.S.; Sadar, M.H. Purification and properties of cholinesterases from honeybees—Apis melifera Linnaeus—and boll weevils—Anthonomus grandis Boheman. J. Agric. Food Chem. 18:692–697;1970. Hama, H.; Iwata, T.; Miyata, T.; Saito, T. Some properties of acetylcholinesterases partially purified from susceptible and resistant green rice leafhoppers, Nephotettix cincticeps Uhler (Hemiptera: Deltocephalidae). Appl. Ent. Zool. 15:249–261; 1980. Huang, C.T.; Dauterman, W.C. Purification of fly head cholinesterase. Insect Biochem. 3:325–334;1973. Karnovsky, M.I.; Roots, L. A ‘direct coloring’ thiocholine method for cholinesterases. J. Histochem. Cytochem. 12:219– 221;1964. Laemmi, U.K. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–687; 1970. Lee, A.-H.; Metcalf, R.L.; Kearns, C.W. Purification and some properties of house cricket (Acheta domesticus) acetylcholinesterase. Insect Biochem. 4:267–280;1974. Lord, K.A. The partial purification and properties of a cholinesterase from Blatella germanica L. Biochem. J. 78:483–490; 1961. Massoulie´, J., Bon, S. Affinity chromatography of acetylcholinesterase: The importance of hydrophobic interactions. Eur. J. Biochem. 68:531–539;1976. Matsumura, F. Toxicology of Insecticides, 2nd Edition. New York: Plenum Press; 1985. Schulz, A.R. Enzyme Kinetics: From Diastase to Multi-enzyme Systems. New York: Cambridge University Press; 1994. Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Analyt. Biochem. 150:76–85;1985. Soreq, H.; Zakut, H. Human cholinesterases and anticholinesterases. San Diego: Academic Press; 1993. Steele, R.W.; Smallman, B.N. Acetylcholinesterase of the house-fly head: Affinity purification and subunit composition. Biochem. Biophys. Acta 445:147–157;1976. Toutant, J.-P. Insect acetylcholinesterase: Catalytic properties, tissue distribution and molecular forms. Prog. Neurobiol. 32:423–446;1989. Xu, G.; Bull, D.L. Acetylcholinesterase from the horn fly (Diptera: Muscidae): Distribution and purification. J. Econ. Entomol. 87:20–26;1994. Xu, G.; Bull, D.L. Acetylcholinesterase from the horn fly (Diptera: Muscidae) II: Biochemical and molecular properties. Arch. Insect Biochem. Physiol. 27:109–121;1994. Wood, E.; Zerba, E.; Picollo, M.; de Licastro, S. Partial purification and characterization of Triatoma infestans head acetylcholinesterase. Insect Biochem. 9:595–601;1979. Zhu, K.Y.; Brindley, W.A. Enzymological and inhibitory properties of acetylcholinesterase purified from Lygus hesperus Knight (Hemiptera: Miridae). Insect Biochem. Molec. Biol. 22:245–251;1992. Zhu, K.Y.; Brindley, W.A.; Hsiao, T.H. Isolation and partial purification of acetylcholinesterase from Lygus hesperus (Hemiptera: Miridae). J. Econ. Entomol. 84:790–794;1991.
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28. Zhu, K.Y.; Clark, J.M. Purification and characterization of acetylcholinesterase from the Colorado potato beetle, Leptinotarsa decemlineata (Say). Insect Biochem. Mol. Biol. 24:453– 461;1994. 29. Zhu, K.Y.; Clark, J.M. Comparisons of kinetic properties of acetylcholinesterase purified from azinphosmethyl-susceptible and resistant strains of Colorado potato beetle. Pestic Biochem. Physiol. 51:57–67;1995.
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30. Zhu, K.Y.; Lee, S.H.; Clark, J.M. A point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in Colorado potato beetle. Pestic Biochem. Physiol. 55:100–108;1996. 31. Zhu, K.Y.; Clark, J.M. Validation of a point mutation of acetylcholinesterase in Colorado potato beetle by polymerase chain reaction coupled to enzyme inhibition. Pestic Biochem. Physiol. 57:28–35;1997.
ISSN 0742-8413/98/$19.00 PII S0742-8413(97)00208-9
Characterization of Acetylcholinesterase Purified from the Lesser Grain Borer, Rhyzopertha dominica (Coleoptera: Bostrichidae) R. N. C. Guedes,1 K. Y. Zhu,2 S. Kambhampati,2 and B. A. Dover 2 1
Departamento de Biologia Animal, Universidade Federal de Vic¸osa, Vic¸osa, MG 36571.000, Brasil; and 2 Department of Entomology, Kansas State University, Manhattan, Kansas 66506, USA ABSTRACT. 1. Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the lesser grain borer (Rhyzopertha dominica) was significantly inhibited by higher concentrations of the substrates acetylthiocholine (ATC), acetyl(β-methyl) thiocholine (Aβ MTC) and propionylthiocholine (PTC). 2. The efficiency of AChE for hydrolyzing different substrates was ATC . Aβ MTC . PTC . S-butyrylthiocholine. The enzyme activity was completely inhibited by 1025 M eserine or BW284C51, but was only partially inhibited by ethopropazine at the same concentration. These results confirmed that the purified enzyme was an typical insect AChE. 3. Non-denaturing and SDS polyacrylamide gel electrophoresis (PAGE) showed only one major molecular form in the purified AChE with a molecular weight of about 107,000 prior to reduction and about 56,000 after reduction, suggesting the homodimer of AChE linked with disulfide bonds. comp biochem physiol 119C;2:205–210, 1998. 1998 Elsevier Science Inc. KEY WORDS. Acetylcholinesterase, affinity chromatography, enzyme kinetics, enzyme purification, lesser grain borer, molecular weight, Rhyzopertha dominica, substrate specificity
INTRODUCTION Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme involved in the nerve impulse transmission across cholinergic synapses, where it hydrolyzes the excitatory neurotransmitter acetylcholine into choline and acetic acid (4,20). The enzyme also is the target site of inhibition by organophosphate and carbarnate insecticides (17,22). Increased incidence of insecticide resistance due to the reduced sensitivity of AChE to inhibition by organophosphate and carbamate insecticides as a resistance mechanism further adds to the toxicological significance of AChE and has stimulated further research of this important enzyme in insects (28,29). Most of the studies on insect AChE have been carried out with relatively crude preparations which contain other esterases with possible overlapping substrate specificities (9,22). The use of purified AChE has obvious advantages over crude homogenates in kinetic studies of substrate and inhibitor interactions, especially when other esterases are capable of hydrolyzing compounds under investigation (14). Although many different methods have been used for the isolation of insect AChE (1,2,6,9,10,11,14,15,21,23,25– 28), affinity chromatography has been demonstrated to be Address reprint requests to: Dr. Kun Yan Zhu, Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, U.S.A. Tel. (785) 532-4721; FAX (785) 532-6232; E-mail: [email protected] Received 31 March 1997; accepted 30 October 1997.
the most effective technique for purification of AChE from insects. It usually provides a high yield with an adequate purity of AChE which is particularly desirable in many toxicological studies. In this paper, we present the following: (i) purification of AChE from the lesser grain borer (Rhyzopertha dominica), an important pest of stored cereals throughout the world, by affinity chromatography; (ii) enzyme kinetics, and substrate and inhibitor specificities of the purified enzyme; and (iii) determination of molecular weights of the reduced and non-reduced forms of the purified AChE. Knowledge obtained from this study should facilitate further investigation of this important enzyme in lesser grain borer and other insect species. MATERIALS AND METHODS Insects Lesser grain borers were reared on whole wheat under constant conditions in the Laboratory of Stored Product Insects, Department of Entomology, Kansas State University, Manhattan (KS) as described previously (7,8). Adults were used exclusively in this study. Chemicals Acetic anhydride, acetyl-(β-methyl) thiocholine iodide (Aβ MTC), acetylthiocholine iodide (ATC), 1,5-bis (4-
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allyldimethylammonium phenyl)-pentan-3-one dibromide (BW284C51), aprotinin, bicinchoninic acid (BCA) solution, bromophenol blue, S-butyrylthiocholine iodide (BTC), cyanogen bromide activated Sepharose 4B, 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB), dithiothreitol (DTT), eserine hemisulfate, ethopropazine hydrochloride, leupeptin, pepstatin, phenylmethanesulfonyl fluoride (PMSF), propionylthiocholine iodide (PTC), silver stain SDS-PAGE molecular weight standard mixtures, sodium dodecyl sulfate (SDS) and Triton X-100 were purchased from the Sigma Chemical Company (St. Louis, MO). Coomassie brilliant blue G250, 1-cyclohexyl-3 (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonale, 3dimethylaminobenzoic acid, ethanolamine, iodoethane, 1,6-hexamethylenediamine, sodium azide and tetraethylammonium iodide (NEt4I) were purchased from Aldrich Chemical Company (Milwaukee, WS). Acrylamide, ammonium persulfate, N,N′-methylene-bis-acrylamide, bovine serum albumin, ethylenediamine-tetraacetic acid (EDTANa2), glycine and protein silver stain kit were purchased from Bio-Rad Laboratories (Hercules, CA). Purification of AChE AChE was purified from lesser grain borer by affinity chromatography using 3-(carboxyphenyl) ethyldimethyl ammonium as affinity ligand (16) as described by Zhu and Brindley (26) with some modifications. Briefly, samples were prepared by homogenizing 4 g of adult insects (whole body) in 60 ml of ice-cold 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% (v/v) Triton X-100, 0.2 M NaCl, and five protease inhibitors (0.1 µM aprotinin, 1 mM EDTANa2, 1 µM leupeptin, 1 µM pepstatin and 10 µM PMSF). The soluble fraction was obtained as previously described (26,28) and applied to an affinity column (1.0 3 7.5 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 and 0.2 M NaCl. After the column was washed with 180 ml of the same buffer, bound AChE was eluted with 0.2 M NEt4I in the same buffer without NaCl. Constant flow rate was maintained at 30 ml/hr with a peristaltic pump, and fractions of 1 ml were collected after the eluting solution was applied to the column. The affinity purified enzyme was dialyzed against 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1% Triton X-100 to remove NEt4I. Enzyme Activity Assay AChE activity was determined according to the method of Ellman et al. (5) at 405 nm using a Vmax kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA). Inhibition of AChE by three distinct inhibitors, eserine, BW284C51 and ethopropazine were carried out based on the procedures described by Zhu and Clark (28). Kinetic activity of purified AChE was studied by using four sub-
strates (ATC, Aβ MTC, PTC and BTC), each with 12 concentrations ranged from 8 mM to 3.9 µM, at 25°C and pH 7.5. The enzyme activity was determined based on the reaction for 2 min, whereas the initial velocity was determined based on the reaction for 30 sec for ATC, Aβ MTC and PTC, and 2 min for BTC. Michaelis constant (Km) and maximal velocity (Vmax) values were determined by Hanes transformations (18). Protein concentration was determined by the BCA method (19) using bovine serum albumin as standard. Electrophoretic Analysis Non-denaturing polyacrylamide gel electrophoresis (PAGE) (13) was carried out with a Penguin P8DS dualgel electrophoresis system (Owl Scientific, Inc., Woburn, MA) coupled with a cold water circulating system. The gels (4 and 9% acrylamide in stacking and separating gels respectively) were run at a constant voltage of 150V for 2 hr. The AChE bands were visualized by incubating the gels in AChE staining mixture (12) overnight at room temperature for its activity. Staining for total protein was conducted with a silver staining technique using a commercial kit (Bio-Rad) according to the manufacturer’s instructions. Denaturing SDS-PAGE was used to estimate the molecular weights of the reduced and non-reduced AChE as described by Zhu and Clark (28). The procedure was similar to the non-denaturing PAGE except that a final concentration of 0.1% (w/v) SDS was incorporated into both gels and the electrode buffers, and all marker proteins and AChE samples were treated by incubating them in 2% (w/v) SDS solution containing 25 mM DTT in a boiling water bath for 2 min. The molecular weight of the non-reduced or reduced AChE was determined by using a calibration cure of the following molecular weight markers: bovine erythrocyte carbonic anhydrase, 29,000; porcine heart fumarase, 48,500; bovine serum albumin, 66,000; rabbit muscle phosphorylase b, 97,000; and E. coli β-galactosidase, 116,000. RESULTS AND DISCUSSION Purification of AChE AChE was successfully purified from lesser grain borer by affinity chromatography following extraction with Triton X-100. The purification factor was 771 and the yield was 54%, which were typical of nine additional batches of purification during the course of the study (Table 1). The purified AChE had a specific activity of about 4.7 µmol/min/ mg protein, but showed low stability after purification. Addition of glycerol to a final concentration of 20% (v/v) increased the enzyme stability, and the enzyme could be maintained for about three days at 4°C without significant loss of the AChE activity. The degree of purification of AChE from lesser grain borer was comparable to those obtained from western tar-
Acetylcholinesterase Purified from R. dominica
207
TABLE 1. Summary of purification of acetylcholinesterase from R. dominica by affinity chromatography following extraction
by Triton X-100
Procedure Triton X-100 extraction Affinity chromatography
Volume (ml)
Protein (mg/ml)
Total protein (mg)
60
7.83
469.80
3
0.11
0.33
nished plant bug (26,27) and horn fly (23), but smaller than that from Colorado potato beetle (28,29). Using ATC as substrate, the specific activities of purified AChE were 1350 µmol/min/mg protein for Drosophila heads (6), 385 for horn fly (23), 50 for western tarnished plant bug (27), 128 for Colorado potato beetle (28) and 5 for lesser grain borer (this study). The variation in the specific activity of the purified AChE among different insect species may be partially due to the degree of the purity of AChE, but also can be due to insect species variations. Our results are consistent with the notion that insects in Hemiptera, Coleoptera and Lepidoptera exhibit lower levels of AChE activity than in Diptera (22). This is particularly true in Colorado potato beetle (28), in which AChE was apparently purified to homogenous but its specific activity was only less than 10% of the activity determined in AChE purified from Drosophila heads (6) when the same substrate (i.e., ATC) was used.
Specific activity (nmol/min/mg)
Total activity (nmol/min)
Yield (%)
Purification factor (-fold)
6.09
2861.08
100
1
4698.07
1550.36
54
771
indicated that AChE hydrolyzed ATC, Aβ MTC and PTC, 31-, 22-, and 15-fold, respectively, more efficiently than it did BTC. The relative efficiency of purified AChE hydrolyzing BTC vs ATC, as determined by the Vmax (BTC): Vmax (ATC) ratio, was 0.032 for lesser grain borer (this study) and Colorado potato beetle AChE (28). Although smaller ratios were obtained for western tarnished plant bug (0.098) (26) and Drosophila (0.6) (6) AChEs, these ratios are still larger than those found in vertebrates such as electric eel (0.005) and human erythrocytes (0.01) (6). It seems that the substrate specificity of insect AChEs is lower than that of vertebrates as judged by these two substrates. AChE was completely inhibited by 1025 M eserine (a
Substrate Specificity, and Kinetic and Inhibitory Properties of Purified AChE Four substrates, ATC, Aβ MTC, PTC and BTC were used to study the effect of substrate concentration on AChE activity (Fig. 1). Although optimal substrate concentrations were rather broad for ATC, Aβ MTC and PTC, which ranged between 0.06 and 1.00 mM, AChE activity was significantly inhibited by higher concentrations of ATC, Aβ MTC and PTC. Higher concentrations of BTC also inhibited AChE activity, but the inhibition was in a much smaller magnitude. This substrate inhibition phenomenon could be due to the binding of excess substrate to the peripheral regulatory site of the enzyme (3), and is an important distinguishing feature of the AChEs (22). Kinetic studies determined the affinity of AChE to its different substrates. The overall differences in affinity were relatively small among these four substrates. The Km 6 SE values were 24.2 6 2.4 µM for ATC, 13.4 6 3.5 for Aβ MTC, 20.4 6 7.9 for PTC, and 8.4 6 1.7 for BTC. The hydrolyzing efficiencies of AChE for the four substrates, as indicated by their Vmax values were 18.7 6 1.6 µmol/min/ mg protein for ATC, 13.4 6 0.6 for Aβ MTC, 9.3 6 0.6 for PTC, and 0.6 6 0.03 for BTC (Fig. 2). These results
FIG. 1. Substrate specificity and effect of substrate concentration on the hydrolysis of acetylthiocholine (ATC), acetyl(b-methyl) thiocholine (AbMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) by AChE purified from R. dominica. Each point represents the mean of four determinations (n 5 4). Vertical bars indicate standard errors of the mean.
208
FIG. 2. Hanes plots of [s] vs [s]/v for AChE hydrolyzing
acetylthiocholine (ATC), acetyl-(b-methyl) thiocholine (AbMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC). Each point represents the mean of four determinations. All correlation coefficients (r) . 0.98 (P , 0.01).
R. N. C. Guedes et al.
partially contributed to the relatively low specific activity of AChE in lesser grain borer. Molecular weight of purified subunits of AChE was determined in the presence of the reducing agent DTT. Only a major band was verified with an estimated molecular weight of 56,000 (data not shown). The SDS-PAGE analysis with reducing agent indicated the molecular weight of the AChE subunit of lesser grain borer was 56,000, virtually identical to those purified from Drosophila (55,000) (6) and horn fly (54,000) (24). However, the estimated molecular weight was 107,000 for non-reduced AChE, suggesting that each molecule of AChE consisted of two similar subunits connected with disulfide bonds, as found in other insect AChEs (22). In summary, our present study firmly established that the AChE purified from lesser grain borer was a typical insect AChE possessing many common characteristics as found in other insect species (22,28). The major features include: 1) the substrate inhibition at higher concentrations of ATC, AβMTC and PTC; 2) high sensitivity to inhibition by eserine and BW284C54 but less sensitive to ethopropazine; 3) much higher efficiency in hydrolyzing ATC than BTC; 4) comparable molecular size to other AChEs found in other insects; and 5) relatively low activity as compared with AChEs found in vertebrates. All these findings should help further biochemical and molecular studies on AChE from this important pest as were achieved in a few other insect species (24,26,28–31).
general inhibitor for AChE and butyrylcholinesterase, BuChE) and BW284C51 (a relatively specific inhibitor for AChE), but only 60% of the AChE activity was inhibited by ethopropazine (a relatively specific inhibitor of BuChE) at this concentration (Fig. 3). The I50 values expressed as mean 6 SE (n 5 3) were 0.09 (60.03) 3 1028 M for eserine, 8.0 (60.4) 3 1028 M for BW284C51, and 105.0 (649.7) 3 1028 M for ethopropazine. This inhibitor specificity further supported our conclusion that the cholinesterase activity in lesser grain borer was due to AChE, and agreed with other three previously suggested criteria (22): a) the enzyme activity was inhibited by ATC at high concentrations; b) the enzyme hydrolyzed ATC more efficiently than BTC; and c) the enzyme hydrolyzed Aβ MTC at a fairly rapid rate. Molecular Properties of Purified AChE Results from non-denaturing PAGE in the absence of Triton X-100 showed only one major band of AChE when the enzyme was stained for activity. This band corresponded to a major band stained for protein by silver stain (Fig. 4), which suggested only a single major molecular form of AChE in our purified AChE samples and other small bands in the protein-staining gel were probably due to contaminating proteins. These contaminating proteins probably
FIG. 3. Inhibition of R. dominica AChE by eserine,
BW284C51, or ethopropazine. Each point represents the mean of three determinations. Vertical bars indicate standard error of the means.
Acetylcholinesterase Purified from R. dominica
209
8.
9.
10.
11. 12. 13. 14. FIG. 4. Electrophoretic analysis of purified AChE from R.
dominica with non-denaturing PAGE. The gel was stained either for the enzyme activity or protein.
15. 16.
The authors thank the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior-CAPES, UFV Department of Animal Biology, and KSU Department of Entomology for the financial support provided. This manuscript is contribution no. 97-362-J from the Kansas Agricultural Experiment Station.
17. 18. 19.
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