Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover)

Archives of Insect Biochemistry and Physiology 51:37–45 (2002)

© 2002 Wiley-Liss, Inc.DOI: 10.1002/arch.10048 Published online in Wiley InterScience (www.interscience.wiley.com)

Purification and Characterization ofAcetylcholinesterase From Cotton Aphid(Aphis gossypii Glover)

Fei Li and Zhaojun Han*

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphisgossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamideaffinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturingpolyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in threeforms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer,pH7.2, 0.02 mM 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25°C. Investigation also revealed that crude extract andpurified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI,and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive toeserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophosand pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract fromthe resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observa-tions. Arch. Insect Biochem. Physiol. 51:37–45, 2002. © 2002 Wiley-Liss, Inc.

KEYWORDS: Aphis gossypii Glover; acetylcholinesterase; purification; procainamide affinity column; kinetic char-acterization; inhibition

Key Laboratory of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China

Contract grant sponsor: National Natural Science Foundation of China (NNSF); Contract grant number: 39970488; Contract grant sponsor: Projects ofDevelopment Plan of the State Key Fundamental Research (973 Projects); Contract grant number: J2000016207.

*Correspondence to: Zhaojun Han, College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu province, 210095, P.R. China.E-mail: [email protected]

Received 18 August 2001; Accepted 21 May 2002

INTRODUCTION

Acetylcholinesterase (AChE, EC3.1.1.7) plays animportant role in neurotransmission by hydrolyz-ing the neurotransmitter acetylcholine and is thetarget site of organophosphorus (OP) and carbam-ate insecticides. Both AChE and BuChE are foundin vertebrates whereas only AChE is detected ininsects. Numerous experiments revealed that re-duced AChE sensitivity causes resistance to OP andcarbamate insecticides (Fournier et al., 1994; Li et

al., 2001). With the significance of AChE in neu-rotransmission and pest resistance, much attentionhas been paid to studies of AChE from both mam-mals and insects. Generally, most of these studiesused unpurified AChE, mainly from homogenatesof body parts or whole body (Fournier et al., 1987;Graham et al., 1994; Sharon et al., 1999). Com-pared with purified AChE, crude extracts containconsiderable amounts of contaminants. These non-AChE factors, such as carboxylesterases, can affectthe measurement of AChE characteristics. They may

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Archives of Insect Biochemistry and Physiology

degrade or bind insecticides, which dramaticallyinfluences the accuracy of the following AChE as-says. It follows that experiments with crude extractsmay produce misleading results. However, AChEshave been purified from only a few insect species,including Musca domestica (Fournier et al., 1987),Lygus hesperus Knight (Zhu et al., 1992), Leptinotarsadecemlineata (Zhu and Clark, 1994), Helicoverpaarmigera (Gao et al., 1998a), Diabrotica virgifera-virgifera (Gao et al., 1998b) Galleria mellonella L.(Sharon et al., 1999), Blattella germanica (Prab-hakaran and Kamble, 1996) and the greenbug,Schizaphis graminum (Siegfried et al., 1997). Here,we report the purification of AChE from the cottonaphid, Aphis gossypii Glover, by using procainamideaffinity column chromatography, and its character-ization by additional pharmacological experiments.

MATERIALS AND METHODS

Cotton Aphid and Rearing Conditions

The susceptible strain, 81-17lB, is kindly of-fered by the Rothamsted experimental station,Untied Kingdom, and originally collected fromuntreated weed hosts in 1980. A resistance straincotton aphid was originally collected from thecampus of Nanjing Agricultural University, China.After selection, this strain is 120-fold resistant tomethamidophos and 35-fold resistant to pirimi-carb (Li and Han, 2001b). All aphids were rearedroutinely on cotton plants in the laboratory us-ing the caging method (Li and Han, 2001a). Apter-ous adults were collected and stored under –20°Cprior to use.

Chemicals

Acetylcholine iodide (ATChI), 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) and eserine werepurchased from Fluka Chemical Company (Mil-waukee, WI). Acrylamide (acr), N, N¢-methylene-bis-Acrrlamide (bis), procainamide, SDS molecularweight markers (range 29,000–205,000 Da) andbovine serum albumin (BSA) were purchased fromSigma Chemical Company (St. Louis, MO). ECH

sepharose 4B, Sephadex G-25, and Sephadex G-200 were from Pharmacia (Piscataway, NJ). 2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ) was from Aldrich Chemical Company(Milwaukee, WI). Sodium Dodecyl Sulfate (SDS)and Tris-(hydroxymethyl)-aminomethane (Tris)were from Amersco Chemical Company. Insecti-cides used for AChE inhibition studies were of tech-nical grade, with purity over 90%. Other chemicalswere from Chinese chemical companies, all of ana-lytical grade.

Purification of Cotton Aphid AChE

Step 1: Preparation of crude extract. About 0.25 gapterous aphid adults was collected and homog-enized in 3 ml phosphate buffer (pH 8.0, 0.01M,containing 1 mM EDTA, 1% Triton X-100, 1MNaC1). After filtering via fiberglass, homogenateswere centrifuged at 21,000g for 10 min at 4°C. Thesupernatant was used as crude extract.

Step 2: Chromatography on sephadex G-25. SephadexG-25 column with a gel bed height about 45 cmand diameter of 0.8 cm was equilibrated in 0.02M-phosphate buffer, pH7.0, containing 1 mM EDTA(buffer A). The supernatant about 2.4 ml from step1 was loaded onto the prepared sephadex G-25 col-umn and the gel was rinsed with about 80 mlbuffer A. The fractions (2 ml each) were collectedat a constant flow rate (40 ml·h–1) with an auto-matic fraction collector at 4°C. Fractions contain-ing the AChE activity were collected as the enzymeresource for step 3.

Step 3: Chromatography on sephadex G-200. The col-lected fractions from step 2 were loaded onto asephadex G-200 column (length 55 cm and diam-eter 0.8 cm) previously equilibrated in buffer A.The gel was rinsed with buffer A at 4°C at the flowrate of 4 ml ml·h–1. Approximately 5 fractions (2ml per fraction) containing the highest enzyme ac-tivity were collected for the next step.

Step 4: Procainamide affinity chromatography. A pro-cainamide affinity column was made according toinstructions from the manufacturer and stored at4°C (De La Hoz et al., 1986; Ralston et al., 1983).Partial purified enzyme eluted from the sephadex

Purification of AChE From A. gossypii 39

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G-200 column was loaded on the procainamideaffinity column (length 15 cm and diameter 0.55cm) equilibrated in buffer A. The affinity columnwas eluted with buffer A at a constant flow rate(24 ml·h–1) until almost no protein could bemonitored in the effluent. Then, AChE was selec-tively eluted with 0.02M-phosphate buffer, pH 7.0,containing 1 mM EDTA and 100 mM procainamide(buffer B) at a constant flow rate (24 ml·h–1). Thefractions containing high enzyme activity were col-lected and pooled together.

Step 5: Removal of procainamide and condensa-tion. Additional NaC1 was added to the fractionscontaining purified AChE until the concentrationreaches 1M. The sample was dialyzed 3 timesagainst 0.02M-phosphate buffer overnight at 4°C.Purified AChE was condensed with vacuum icecondenser (ThermoSavant, ModulyoD).

Protein content was estimated by the Bradfordmethod using bovine serum albumin as the stan-dard (Bradford, 1976).

Non-Denaturing and Denaturing Polyacrylamide GelElectrophoresis

For both non-denaturing (PAGE) and denatur-ing polyacrylamide gel electrophoresis (SDS-PAGE), 4% stacking gel was overlaid on a 7.5%resolving gel. Samples were run with constant volt-age of 70 V at the stacking gel and 150 V at theresolving gel. PAGE were performed at 4°C whereasSDS-PAGE were at room temperature. Molecularweight markers for SDS-PAGE were a high molecu-lar weight standard mixture including carbonic an-hydrase, 29,000 Da; egg albumin, 45,000; bovineserum albumin, 66,000; phosphorylase B, 97,400;13-galactosidase, 116,000, and Myosin, 205,000.All gels were silver stained using the method rec-ommended by Bio-Rad (Richmond, CA).

Enzyme Dynamics of Crude Extract and Purified AChE

Km values were determined by nonlinear re-gression of enzyme activity measurements over 2min with at least 15 concentrations of ATChI rang-ing from 10,000 to 1.0625 mM according to the

method described by Graham et al. (1996). Theeffect of DTNB concentration, pH, temperature,and ion concentration on enzyme activity was alsomeasured. The DTNB concentrations ranged from1 to 0.005 mM, the buffer ion concentrations from500 to 5 mM, the pH from 8.0 to 5.8, and thetemperature from 75–5°C. To determine the irre-versibility of heat denaturing, AChE was denaturedby incubating for 20 min at different temperatures,and then allowed to recover for 5 min at roomtemperature.

AChE Activity and Sensitivity to Different Inhibitors

AChE activity was determined as described byHan et al. (1998) according to the method ofEllman et al. (1961), with some modification thatallowed for the use of a kinetic assay with a Mo-lecular Devices BioRad microplate reader. Fifty mi-croliters of each enzyme preparation (crude orpurified) was placed in a well of a microplate, andthen 50 ml phosphate buffer (pH 7.2, 0.02M) and100 ml DTNB (45 mM) were added and mixed. Re-action was initiated and monitored at 405 nm for20 min after the addition of 100 ml, 1.5 mM ATChI.Each treatment was replicated 3 times. For a sensi-tivity test, inhibitors were prepared together withthe ATChI and added instead of the correspond-ing substrate solution. Inhibitors and their concen-tration ranges were as follows: eserine 0.75–0.025mml·L–1, methamidophos 400–0.1 mM, and pirimi-carb 50–0.05 mM. Each compound was used atleast with 7 concentrations.

RESULTS

Purification of Cotton Aphid AChE

AChE from both susceptible and resistancestrains of cotton aphid were purified by pro-cainamide affinity chromatography. The AChEfrom the susceptible strain was purified to about35,100-fold with a yield of 30.3%, while that fromthe resistance strain was purified to 33,680-foldwith a yield of 29.8% (see Table 1). The purifiedAChE was resolved as a single band by denaturing

40 Li and Han


polyacrylamide electrophoresis. The relative mo-lecular mass of purified AChE monomer in thepresence of SDS was calculated to be 63,500Daltons. Furthermore, the purified AChE was re-solved into three bands by PAGE, which meansAChE of cotton aphid has 3 native forms (Fig. l).

AChE Activity Under Different Reaction Conditions

Enzyme activity of crude extract and purifiedAChE from both strains was measured under dif-ferent conditions. No apparent difference wasfound between susceptible and resistant strains. So,the data of susceptible strain were shown here todemonstrate the difference between AChE crudeextract and purified enzyme.

DTNB and buffer ion concentration had a simi-lar influence on the activity of crude extract and pu-rified AChE. Extremely high or low concentrationsdecreased the enzyme activity, especially for the pu-rified AChE (Figs. 2, 3). The optimal pH for the pu-rified AChE was 7.2. Higher or lower pH yieldeddecreased activity. Crude extract showed similar char-acteristics as purified AChE at low pH, whereas theenzyme activity of crude extracts remained high upto pH 7.6, and then decreased at pH 7.8 and 8.0(Fig. 4). Irreversible denaturation of both crude ex-tract and purified AChE mainly occurs above 45°C,which was indicated by the dramatically decreasedAChE activity when the enzyme was incubated at orabove 45°C (Fig. 5). In conclusion, the optimal con-dition for measuring the activity of purified AChE

Fig. 1. Denaturing polyacrylamide gel electrophoresis(SDS-PAGE) and non-denaturing polyacrylamide gel elec-trophoresis (PAGE) of purified AChE from A. gossypiiGlover. All gels were silver stained. Lane A: SDS-PAGE

TABLE 1. Summary of AChE Purification From Susceptible and Resistant Strains of Cotton Aphid

Total Total Specific PurificationVolume protein activity Yields activity factor

Strains Purification procedure (ml) (mg) (I.U.) (%) (I.U. · mg–1) (fold)

Susceptible strain (171B) Crude extract 3 5621.3 8184.0 100 1.456 1Procainamide affinity column 6 0.0485 2479.75 30.3 51119.62 35,100

Resistant strain Crude extract 3 4889.4 4334.55 100 1.128 1Procainamide affinity column 5.5 0.034 1291.7 29.8 37991.04 33,680

marker; Lane B: purified AChE on SDS-PAGE gel, show-ing a single band; Lane C: purified AChE on PAGE gel,showing three bands.


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from A. gossypii would be 0.02 mM DTNB, 20 mMphosphate buffer, pH 7.2, at 25–30°C.

Kinetic Characteristics and Inhibitor Sensitivity ofCrude and Purified Enzyme

AChE from A. gossypii was insensitive to sub-strate inhibition. With crude extract, the highestactivity appeared at the ATChI concentration of1,500 mmol·L–1, and the activity did not diminishat higher substrate concentrations. The activity ofpurified AChE was the highest at 3,000 mmol·L–1,again with no decrease at higher substrate concen-trations (Fig. 6). Inhibitory experiments indicated

that one or more factors in the crude extract ap-parently enhances the insensitivity of AChE to in-hibitors such as eserine, methamidophos, andpirimicarb. And the purification procedures removethese influences, which result in decreasing AChEinsensitivity to inhibitors. For the resistant strain,the I50 value of methamidophos decreased from202.04 mmol·L–1 (crude extract) to 34.22 mmol·L–1

(Purified AChE), and I50 value of pirimicarb de-creased from 28.26 mmol·L–1 (crude extract) to2.41 mmol·L–1 (Purified AChE). For the susceptiblestrain, the I50 Value of methamidophos decreasedfrom 0.6491 mmol·L–1 (crude extract) to 0.5471mmol·L–1 (Purified AChE), and I50 value of pirimi-

Fig. 2. AChE activities of crude extract and purifiedAChE in the presence of different DTNB concentrations.Each point represents the mean of four replicate assays,

Fig. 3. AChE activities of crude extract and purified AChEunder different buffer ion concentrations. Each point rep-resents the mean of four replicate assays, and the error

and the error bars represent 1 SEM. Asterisks indicatesignificant differences between crude and purified AChE(P < 0.05).

bars represent 1 SEM. Asterisks indicate significant differ-ences between crude and purified AChE (P < 0.05).

42 Li and Han


carb decreased from 1.203 mmol·L–1 (crude extract)to 0.8613 mmol·L–1 (Purified AChE). Furthermore,the decreasing extent of the AChE insensitivity tomethamidophos and pirimicarb after purificationwas different between the resistant and susceptiblestrains. For the resistant strain, the methamidophosand pirimicarb I50 values ratios of crude extract topurified AChE were 6.43 and 11.73 times, sepa-rately, whereas the ratios of crude extract to puri-fied AChE were only 1.19 and 1.40 times for thesusceptible strain, which indicates that the un-known factors in the crude extract from the resis-tant strain have more influence on the AChEactivity than that from the susceptible strain. (Fig.7, Table 2).

DISCUSSION

Because of its biological significance both in in-sect neurophysiology and pest resistance, AChE at-tracts a great deal of attention. Numerous studieshave been performed on insect AChE to explorethe relationship between AChE alteration and in-sect resistance. In this article, cotton aphid AChEwas purified to electrophoretic homogeneity, witha single band resolved on SDS-PAGE. Due to theinstability of cotton aphid AChE, the purificationprocedure of a good yield should be simple andshort in the operation time. The purification pro-cedure detailed here took approximately 6 h andgave about 30% final yield. So, this method, with

Fig. 4. AChE activities of crude extract and purified AChEunder different phosphate buffer pH. Each point repre-sents the mean of four replicate assays, and the error bars

Fig. 5. The influence of reaction temperature on AChEactivities of crude extract and purified AchE. Each pointrepresents the mean of four replicate assays, and the error

represent 1 SEM. Asterisks indicate significant differencesbetween crude and purified AChE (P < 0.05).



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some modification, potentially could be usable forAChE purification from other insects.

Insect AChE exists mostly in different nativeforms. Electrophoresis on polyacrylamide gradi-ent gel revealed three different native forms of pu-rified M. domestica ACHE. Two of them werehydrophilic with molecular weights of 75,000 and150,000, respectively, and the third form de-pended on the nature and concentration of deter-gents (Fournier et al., 1987). L. hesperus AChE wasalso reported with three forms, a, b, and c. Forma evaluated as a native or reduced protein formhas molecular weights of 199,000 or 94,000 Dr.As hydrophilic, form b is 150,000 and 79,000 Dr.And as amphiphilic, form c is 82,000 and 86,000Dr (Zhu et al., 1992). In the Colorado potato

beetle, L. decemlineata (Say), the purified nativeAChE consisted of two molecular forms; the ma-jor form was hydrophilic and dimer while the mi-nor was an amphilic dimer (Zhu and Clark, 1994).The waxmoth, G. mellonella, AChE is reported tobe a tetramer composed of similar or identicalsubunits (Sharon et al., 1999). In this paper, pu-rified cotton aphid AChE shows three bands onnon-denaturing PAGE. The exact molecular weightfor each was not determined. There could be twopossible explanations. One is that native cottonaphid AChE exists in polymers, which consistedof similar or identical subunits of 63,500 Dr thatresolved as a single band in denaturing SDS-PAGE.The other is that the three bands in the PAGE gelmay correspond to the amphiphilic dimer, the hy-

Fig. 6. AChE activities of crude extract and purified AChEunder different substrate ATChI concentration. Each pointrepresents the mean of four replicate assays, and the error

Fig. 7. The influence of the inhibitor eserine on AChEactivities of crude extract and purified AChE. Each pointrepresents the mean of four replicate assays, and the error



44 Li and Han


drophilic dimer, and the hydrophilic monomer asin some other insects.

Traditionally, AChE activity was determinedwith endpoint measurement, in which the enzymewas incubated with substrate, and excessive DTNBwas added to colorize only after the reaction wasstopped. In this protocol, the influence of DTNBon AChE activity was avoided. However, the kineticmethod has been broadly adopted to measureAChE activity. In this method, DTNB and AChE areincubated together (Graham et al., 1988; Zhao etal., 1997). The high concentration DTNB, whichwas always used in the endpoint measurementmethod, could result in the inhibition of AChE ac-tivity. So the reaction system of kinetic measure-ment method should be modified carefully toobtain proper results. An optimum condition forthe measurement of purified cotton aphid AChEwas proposed as follows: 0.02M phosphate buffer,pH 7.2, 0.2 mM DTNB, and 1.5 mM ATChI at 25°C.

Dissimilar behavior of crude extracts and thepurified AChE indicated that there might be someunknown factors in the crude extracts, which in-fluence the measurement of AChE activity. AChEsensitivity to eserine and conventional pesticides,methamidophos and pirimicarb, was also seriouslyaffected. Moreover, the changing range in sensitiv-ity varied with the susceptible and resistant strains.The purified AChE from the resistant strain was6.43- and 11.73-fold more sensitive to methamid-ophos and pirimicarb, respectively, but that fromsensitive strain was only 1.19- and 1.40-fold moresensitive to methamidophos and pirimicarb. Thedissimilarity of biochemical characteristics and themagnification of AChE insensitivity to inhibitorsand pesticides have a close relation to the non-

AChE protein, especially factors related to pesti-cide resistance, such as esterase, MFO, and glu-tathione S-transferase. Another cause for thedissimilarity may refer to the composition of cot-ton aphid AChE. Molecular studies on the Acegene of the cotton aphid in our laboratory haverevealed two Ace genes in the cotton aphid (un-published data).

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Susceptible strain (171B) 0.6491 ± 0.033 0.5471 ± 0.059 1.19 1.203 ± 0.0273 0.8613 ± 0.0916 1.40Ba Ba Ba Bb

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Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover)