Kinetic and Molecular Differences in the Amplified and Non-amplified Esterases from Insecticide-resistant and Susceptible Culex quinquefasciatus Mosquitoes
1995; Elsevier BV; Volume: 270; Issue: 52 Linguagem: Inglês
10.1074/jbc.270.52.31124
ISSN1083-351X
AutoresS.H.P.P. Karunaratne, Janet Hemingway, K. G. I. Jayawardena, Vasanthi Dassanayaka, Ashley M. Vaughan,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoTwo non-amplified esterases were purified from the insecticide-susceptible Pel SS strain of Culex quinquefasciatus. These were the two major esterase activity peaks in this strain. The two corresponding amplified carboxylesterases, Estα2 and Estβ2, involved in organophosphate sequestration were purified from two resistant C. quinquefasciatus strains. The Pel SS esterases were significantly less reactive with the organophosphates than those from the resistant strains. One of the Pel SS esterases was electrophoretically identical to amplified Culex Estβ1. However, it differed kinetically, and in its nucleotide and predicted amino acid sequences from the two characterized amplified Estβ1s, it is classified as Estβ13. Restriction fragment analysis suggested Pel SS has only one Estα and one Estβ gene, while the resistant Pel RR has both amplified and non-amplified forms of Estα and Estβ. The EcoRI fragments for both Pel SS esterases were distinct from those of the amplified Estα21, Estβ21, or Estβ11&2. An esterase with the same size EcoRI fragment as Estβ13 was also present in Pel RR. This and restriction enzyme fragment analysis of C. quinquefasciatus field populations suggest that variability of the susceptible alleles may be lower than previously suggested. A non-amplified Estα with a unique EcoRI band was present in Pel RR. The previous esterase purification procedures may not have separated these amplified and non-amplified alleles. Hence, the small differences between the purified esterases from resistant strains may reflect mixtures of identical amplified alleles with different non-amplified alleles, which have significantly different ka values. Two non-amplified esterases were purified from the insecticide-susceptible Pel SS strain of Culex quinquefasciatus. These were the two major esterase activity peaks in this strain. The two corresponding amplified carboxylesterases, Estα2 and Estβ2, involved in organophosphate sequestration were purified from two resistant C. quinquefasciatus strains. The Pel SS esterases were significantly less reactive with the organophosphates than those from the resistant strains. One of the Pel SS esterases was electrophoretically identical to amplified Culex Estβ1. However, it differed kinetically, and in its nucleotide and predicted amino acid sequences from the two characterized amplified Estβ1s, it is classified as Estβ13. Restriction fragment analysis suggested Pel SS has only one Estα and one Estβ gene, while the resistant Pel RR has both amplified and non-amplified forms of Estα and Estβ. The EcoRI fragments for both Pel SS esterases were distinct from those of the amplified Estα21, Estβ21, or Estβ11&2. An esterase with the same size EcoRI fragment as Estβ13 was also present in Pel RR. This and restriction enzyme fragment analysis of C. quinquefasciatus field populations suggest that variability of the susceptible alleles may be lower than previously suggested. A non-amplified Estα with a unique EcoRI band was present in Pel RR. The previous esterase purification procedures may not have separated these amplified and non-amplified alleles. Hence, the small differences between the purified esterases from resistant strains may reflect mixtures of identical amplified alleles with different non-amplified alleles, which have significantly different ka values. INTRODUCTIONThe use of pesticides, both directly and indirectly against the mosquito Culex quinquefasciatus, has resulted in the selection of broad spectrum organophosphate and carbamate resistance. The most commonly selected resistance mechanism is the increased activity of Estα2 and Estβ2 carboxylesterases (EC 3.1.1.1) (A2 and B2 esterases on an earlier classification)(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 49-55Crossref Scopus (43) Google Scholar, 3.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 453-457Crossref Scopus (29) Google Scholar, 4.Raymond M. Pasteur N. Georghiou G.P. Mellon R.B. Wirth M.C. Hawley M. J. Med. Entomol. 1987; 24: 24-27Crossref PubMed Scopus (56) Google Scholar, 5.Raymond M. Beyssat-Arnaouty V. Sivasubramanian N. Mouches C. Georghiou G.P. Pasteur N. Biochem. Genet. 1989; 27: 417-423Crossref PubMed Scopus (77) Google Scholar, 6.Devonshire A.L. Field L.M. Annu. Rev. Entomol. 1991; 36: 1-23Crossref PubMed Google Scholar). Classification of these esterases is based on their preferences for α- or β-naphthyl acetate, their mobility on native polyacrylamide gel electrophoresis (PAGE), ( (1)The abbreviations used are: PAGEpolyacrylamide gel electrophoresiskbkilobase(s)pNPAp-nitrophenyl acetatepNPHp-nitrophenyl hexanoate.) and their nucleotide sequence(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The overproduction of the Estα21 and a series of Estβ esterases is due to gene amplification(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 5.Raymond M. Beyssat-Arnaouty V. Sivasubramanian N. Mouches C. Georghiou G.P. Pasteur N. Biochem. Genet. 1989; 27: 417-423Crossref PubMed Scopus (77) Google Scholar, 7.Mouches C. Pasteur N. Berge J.B. Hyrien O. Raymond M. De Saint Vincent B.R. De Silvestri M. Georghiou G.P. Science. 1986; 233: 778-780Crossref PubMed Scopus (278) Google Scholar, 8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). Identical EcoRI restriction fragment sizes of the amplified Estβ2 from resistant C. quinquefasciatus worldwide has been reported, in contrast to a high level of variability in Estβ from insecticide-susceptible mosquitoes(9.Raymond M. Callaghan A. Fort P. Pasteur N. Nature. 1991; 350: 151-153Crossref PubMed Scopus (241) Google Scholar). A cDNA with 97% identity to Estβ21 has been cloned from an insecticide susceptible strain (Pel SS) of C. quinquefasciatus from Sri Lanka(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar); however, the protein has not been characterized. After native starch or PAGE of homogenates of individual resistant Culex larvae of the amplified Estα2/Estβ2 esterase phenotype, two electromorphs can be visualized. Under the same conditions, no esterase bands are visible in homogenates of susceptible insects. This has lead to the suggestion that the susceptible insects have null alleles for these loci(10.Georghiou G.P. Pasteur N. J. Econ. Entomol. 1978; 71: 201-205Crossref PubMed Scopus (83) Google Scholar).Here, we report the purification and characterization of both an α- and a β-naphthyl acetate-specific esterase from the Pel SS strain of mosquito and compare these at a biochemical and basic molecular level to the elevated esterases Estα21, Estβ12, and Estβ21. The role of the amplified esterases in resistance is sequestration, which is rapid binding and slow turnover of insecticides (11.Ketterman A.J. Jayawardena K.G.I. Hemingway J. Biochem. J. 1992; 287: 355-360Crossref PubMed Scopus (51) Google Scholar, 12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar, 13.Jayawardena K.G.I. Karunaratne S.H.P.P. Ketterman A.J. Hemingway J. Bull. Entomol. Res. 1994; 84: 39-44Crossref Scopus (21) Google Scholar). ( (2)G. J. Small, S. H. P. P. Karunaratne, and J. Hemingway, submitted for publication.) The current study elucidates the relative efficiencies of the purified esterases from a susceptible and two further resistant strains at binding the carbamates and biologically active oxon analogues of the organophosphorus insecticides. Characterization of the amplified and non-amplified esterases will facilitate future site-directed mutagenesis studies on the essential amino acid residues involved in the enzyme-insecticide interaction.EXPERIMENTAL PROCEDURESFour mosquito strains of C. quinquefasciatus were used. Pel was established from a large (>1,000) sample of larvae collected from Peliyagoda (Sri Lanka) in 1984. The population was heterogeneous for insecticide resistance, and both Pel SS and Pel RR were derived from this strain. The insecticide-susceptible Pel SS strain was obtained by selection and pooling of multiple single families for low esterase activity over three generations. The resistant Pel RR strain was selected from the Pel strain by mass larval selection with temephos(2.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 49-55Crossref Scopus (43) Google Scholar, 3.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 453-457Crossref Scopus (29) Google Scholar). The Muheza strain was collected from Tanzania in 1987 and maintained under intermittent chlorpyrifos selection. SPerm was collected from Jeddah (Saudi Arabia) in 1989. It was selected for 20 generations with permethrin and then intermittently with malathion and temephos(15.Hemingway J. Callaghan A. Amin A.M. Med. Vet. Entomol. 1990; 4: 275-282Crossref PubMed Scopus (33) Google Scholar). The three resistant strains both have the elevated esterases Estα2 and Estβ2.Field collections, each of >1000 larvae, were made in six suburbs of Colombo (Sri Lanka) in 1994. The areas included Peliyagoda, where the 1984 collection had been made. The frequency of the amplified esterases in these field populations was analyzed by nonspecific esterase assays of 100-200 larvae(3.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 453-457Crossref Scopus (29) Google Scholar). Batches of 100 larvae of the remaining insects were used for DNA extraction.Esterases were copurified from the resistant and susceptible strains to ensure that any differences in the enzymes seen between this and previous studies were not due to minor variations in our purification and kinetic determination procedures over time.Q-Sepharose Fast Flow, phenyl-Sepharose Fast Flow, PD-10 columns, Nap-5 columns, and Nick spin columns were purchased from Pharmacia (United Kingdom). Hydroxyapatite and the protein assay kit were purchased from Bio-Rad (UK). The p-chloromercuribenzoate was from Pierce (Chester, UK). Biochemicals were purchased from Sigma (UK) except as stated. The diethyl (dimethoxythiophosphorylthio) succinate (malaoxon, 87.5% pure), diethyl-4-nitrophenyl phosphate (paraoxon, 97.4% pure), and 2-isopropoxyphenyl methylcarbamate (propoxur, 97% pure) were purchased from British Greyhound (Birkenhead, Merseyside, UK). The O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate (chlorpyrifosoxon, analytical grade) and O,O-dimethyl-O′-4-nitro-m-tolyl phosphorothioate (fenitrooxon, 98.3% pure) were gifts from Dow Elanco (Midland) and Sumitomo Chemical Co. (Osaka, Japan), respectively.Enzyme activities were assayed with the substrate p-nitrophenyl acetate (pNPA) (1 mM) in 50 mM phosphate buffer (pH 7.4) at 22°C. The protein concentration was estimated by the method of Bradford (16.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar) using bovine serum albumin as the standard protein.Purification of CarboxylesterasesBatches of 4th instar larvae were homogenized and centrifuged at 15,000 × g for 5 min, and the supernatant was taken. Carboxylesterases Estα and Estβ were purified several times from numerous batches of larvae of each strain by sequential column chromatography and preparative electrophoresis as described previously (12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar, 17.Ketterman A.J. Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Pest. Biochem. Physiol. 1993; 47: 142-148Crossref Scopus (32) Google Scholar). Final enzyme preparations were homogeneous as determined by SDS-PAGE. Crude homogenates for the insecticide interaction experiments were prepared in ice-cold 50 mM phosphate buffer (pH 7.4) with 5% (v/v) glycerol and 10 mMDL-dithiothreitol.All specific activities are given in units/mg of protein. A unit corresponds to the hydrolysis of 1 μmol of substrate in 1 min under the assay conditions used. Kinetic constants were determined from at least three experiments for each substrate or inhibitor using enzymes from several different purifications. For the inhibition kinetics, stopped time inhibition assays were performed using pNPA or p-nitrophenyl hexanoate (pNPH) as the substrate. Insecticide stock solutions were prepared in acetonitrile and diluted in phosphate buffer (pH 7.4). The purified enzyme was incubated with a series of concentrations of the test insecticide (acetonitrile concentration of the medium never exceeded 1% (v/v)) for fixed time intervals. Residual activity was determined from the rate of substrate hydrolysis.Inhibitor concentrations were usually in large excess so that linear pseudo-first order kinetics were obtained. The bimolecular rate constants for the formation of acylated enzyme (ka) were derived as described previously(18.Aldridge W.N. Reiner E. Enzyme Inhibitors as Substrates. Frontiers of Biology, North Holland, Amsterdam1972Google Scholar). If inhibitor concentration could not be maintained in large excess, ka values were determined in the presence of substrate(19.Main A.R. Dauterman W.C. Nature. 1963; 198: 551-553Crossref Scopus (45) Google Scholar). To minimize the effect of the reversible enzyme-substrate complex on the rate of acylation, the substrate concentration was maintained at a very low concentration so that the [S]/Km ratio was always less than 0.5(18.Aldridge W.N. Reiner E. Enzyme Inhibitors as Substrates. Frontiers of Biology, North Holland, Amsterdam1972Google Scholar).Electrophoresis of native protein samples was performed in 7.5% acrylamide gels in Tris borate buffer, pH 8.0(20.Davis B.J. Ann. Natl. Acad. Med. Sci. 1964; 121: 404-427Crossref Scopus (15920) Google Scholar). The gels were stained for esterase activity with 0.04% (w/v) α- and β-naphthyl acetate and 0.1% (w/v) Fast Blue B in 100 mM phosphate buffer, pH 7.4. SDS-PAGE was performed with standard proteins using 4-20% acrylamide gradient gels in Tris/glycine/SDS buffer, pH 8.3(21.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205998) Google Scholar).Genomic DNA StudiesDNA was extracted(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar), precipitated with ethanol, resuspended in a small volume of TE, and stored at 4°C until used for Southern blotting. Pel RR Estβ21 and Estα21 esterase cDNA fragments (1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar) were used as probes to determine the haplotype of the esterases in each laboratory strain or field collection of mosquitoes. Genomic DNA (10 μg) was digested to completion with EcoRI, HindIII, or BamHI and separated on 0.8% (w/v) agarose gels. The DNA was transferred to charged nylon membranes (Amersham) and hybridized with 32P-labeled probe (specific activity > 2 × 106 cpm/μg) at 65°C for 16 h in hybridization buffer (5 × Denhardt's solution, 6 × SSC, 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate, 5% (w/v) polyethylene glycol 8000, 100 μg/ml boiled sheared herring sperm DNA). The final membrane washes were at 65°C in 0.1 × SSC and 0.1% (w/v) SDS for 20 min. Membranes were probed first with the Estβ21 cDNA then stripped and probed with Estα21 cDNA.RESULTSOn native PAGE gels, the susceptible Pel SS strain had no visible esterase bands, whereas all the resistant strains had the amplified esterase bands Estα2 and Estβ2 (Fig. 1). The specific activity of Pel SS crude homogenate for the substrate pNPA was 0.02 units/mg, approximately 50-fold less than that routinely observed for resistant crude homogenates. For pNPH, the specific activities were much higher (0.14 units/mg for Pel SS crude homogenate), and this was used as the assaying substrate in subsequent esterase purifications from Pel SS, as the higher specific activity made peak detection simpler. However, the first purification was followed with both pNPA and pNPH, and the major peaks detected were the same with both substrates. Since hydrolysis of pNPH is linear for less than 1 min, pNPA was still the substrate of choice for all purifications from the resistant strains. After purification, only 5-10 μg of each of the Estα and Estβ enzymes were obtained from 10-15 g, wet weight, of Pel SS larvae.SDS-PAGE with purified esterases demonstrated that the molecular masses of the Estα and Estβ enzymes (67 and 62 kDa, respectively) from the susceptible and both resistant strains were similar to those previously reported for Estα21 and Estβ21(11.Ketterman A.J. Jayawardena K.G.I. Hemingway J. Biochem. J. 1992; 287: 355-360Crossref PubMed Scopus (51) Google Scholar, 12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar). The electrophoretic mobility on native PAGE of the Pel SS-purified Estα was reproducibly faster than that of the resistant Estα21 (Fig. 2). In contrast, the Pel SS Estβ had a slower mobility than that of the resistant Estβ21 (Fig. 3) but exactly the same mobility as the elevated C. quinquefasciatus Estβ1(14.Bisset J.A. Rodriguez M.M. Diaz C. Ortiz E. Marquetti M.C. Hemingway J. Bull. Entomol. Res. 1990; 80: 245-250Crossref Scopus (70) Google Scholar).Figure 2:A native PAGE of purified Estα type carboxylesterases from the susceptible Pel SS and five resistant strains of C. quinquefasciatus stained for esterase activity. Crude homogenate of Pel RR, which has elevated Estα21 and Estβ21, is shown for reference.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:A native PAGE of purified Estβ type carboxylesterases from the susceptible Pel SS and five resistant strains of C. quinquefasciatus stained for esterase activity. Crude homogenate of Pel RR, which has elevated Estα21 and Estβ21, is shown for reference.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For the substrates pNPA and pNPH, the Km values of the Pel SS Estβ enzyme were 247.2 ± 23.3 and 12.2 ± 2.21 μM, respectively. Km values of the Pel SS Estα enzyme for pNPA and pNPH were 119.3 ± 5.7 and 40.0 ± 7.2, respectively.Previous studies showed ka to be the most important constant in the interaction between the Culex esterases and the insecticides(12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar). 10-fold differences in ka between the Pel SS and the resistant Muheza and SPerm strains for chlorpyrifos-oxon were seen for Estα (Table 1). Efficiencies in binding chlorpyrifos-oxon and paraoxon of the Pel SS Estβ enzyme were, respectively, 1000- and 100-fold less than those of the resistant strains (Table 2). Thus, the non-amplified enzymes from Pel SS are less able to bind the insecticides than their respective amplified counterparts from various resistant strains. The ratios of the reaction rates with the insecticides for the crude homogenates of Pel RR and Pel SS were similar to those observed for the purified enzymes (Table 3). As with the purified enzymes, the greatest differences were for chlorpyrifos-oxon and paraoxon, which suggests that this method may be valid as a crude means of detecting the level of interaction between these enzymes and insecticides.Table 1: Open table in a new tab Table 2: Open table in a new tab Table 3: Open table in a new tab The genomic EcoRI digests of the Pel SS and Pel RR strains and the field-collected insects sequentially probed for the Estα and Estβ esterases are shown in Fig. 4. The amplified Estα21 and Estβ21 bands in Pel RR are clearly distinguishable. The elevated Estα21 esterase is seen as a 3.5-kb band in the Pel RR strain, along with a fainter (non-amplified) 4-kb band. In Pel SS, a single non-amplified 4.1-kb Estα band is present. In both the Pel RR and Pel SS strains, there is a fainter non-amplified 3.3-kb Estβ band. The six field collections of insects had elevated esterase frequencies ranging from 0.12 to 0.84 on the basis of nonspecific esterase assays. When DNA digests from field-collected insects were probed, the amplified Estα21 and Estβ21 bands had amplification levels broadly in agreement with the different frequencies of elevated esterase individuals in each of the populations. There were three distinct non-amplified bands of each of Estα and Estβ bands in all six field collections analyzed with each of the restriction enzymes (Fig. 4). These bands were identical in all field collections. Blots of DNA digests from individual Pel SS and field-caught insects showed that the amplified bands were easily visible in these samples, but the non-amplified bands were not visible under our experimental conditions. The non-amplified bands from Pel SS were, however, visible in the pooled DNA from 4 to 5 insects. Hence, the non-amplified restriction bands seen in the field material must be present in more than one insect from the pool of 100.Figure 4:Southern blots of EcoRI genomic digests probed with cDNA fragments of the Pel RR Estα21 and Estβ21 esterases. Track 1, Pel RR; track 2, Pel SS probed with Estα21; track 3, Pel RR; track 4, Pel SS; track 5, Kaduwela; and track 6, Peradeniya Culex field samples. 10 μg of DNA were digested per sample. Final washes were at 65°C with 0.1% SDS and 0.1 × SSC (approximately 29 and 84% of the Peradeniya and Kaduwela C. quinquefasciatus populations, respectively, contained elevated esterases).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONBoth Estα and Estβ esterases are expressed in the insecticide-susceptible Pel SS strain of C. quinquefasciatus, although at a lower level than in resistant strains where these esterase genes are amplified(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). The two esterases from Pel SS now need classifying in line with the system adopted for mosquito esterases(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Recently, two Estβ1 esterases were found to be different from each other in their nucleotide sequence and inferred amino acid sequence, demonstrating that multiple alleles of the Estβ locus with identical electrophoretic mobility occur(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). The TEM-R strain of C. quinquefasciatus has an amplified Estβ11EcoRI band of 2.1 kb(9.Raymond M. Callaghan A. Fort P. Pasteur N. Nature. 1991; 350: 151-153Crossref PubMed Scopus (241) Google Scholar), while the MRES Estβ12 has a doublet of bands of 3 and 3.2 kb(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). We have now shown that the Estβ esterase in Pel SS electrophoretically would be classified as Estβ1, but it has an EcoRI band of 3.3 kb, which is distinct from those in both the MRES and TEM-R strains, and has 98% identity at the amino acid level with the amplified Estβ(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar), hence the Pel SS esterase should be classified as Estβ13. The high stringency washing conditions for the Southern blots show that the non-amplified and amplified Estα esterases must be closely related. The Estα esterase in Pel SS has a different electrophoretic mobility to Estα2 and Estα1 (formerly A1), hence it should be classified as Estα3.The Pel SS Estβ13Km for pNPA was significantly higher than those of the resistant Estβ2 previously observed for Pel RR (140.8 ± 5.24 μM), Dar91 (85.41 ± 3.94 μM), and Tanga85 (90.11 ± 6.49 μM)(12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar, 17.Ketterman A.J. Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Pest. Biochem. Physiol. 1993; 47: 142-148Crossref Scopus (32) Google Scholar). In contrast, the Pel SS Estα3 and Estβ13 esterase pNPH Km values are not significantly different from those previously reported for Estα21 and Estβ21(17.Ketterman A.J. Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Pest. Biochem. Physiol. 1993; 47: 142-148Crossref Scopus (32) Google Scholar). The differences (up to 1000-fold) between the inhibition kinetic constants for the enzymes from the susceptible and resistant strains for the oxon analogues of the organophosphorus insecticides are far greater than those observed between resistant strains. The ka values for the purified esterases from the resistant strains used in this study were only slightly different from those determined previously for other resistant strains (see Table 2:, Table 3:)(17.Ketterman A.J. Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Pest. Biochem. Physiol. 1993; 47: 142-148Crossref Scopus (32) Google Scholar). Thus, the high level of variability between the ka values for the susceptible and all the resistant strains cannot be accounted for by minor variations in conditions and experimental techniques between the studies, and we conclude that all the amplified esterases are more efficient at binding the insecticides than their non-amplified Pel SS counterparts. This superiority of insecticide binding suggests that there has been a positive insecticide selection pressure to maintain amplification of favorable resistant alleles.The kinetic differences between the purified amplified Estα2 and Estβ2 enzymes from different strains were previously suggested to be due to either allelic mixtures of the esterases or different single allelic forms of both Estα2 and Estβ2 in each of the resistant strains(12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar, 13.Jayawardena K.G.I. Karunaratne S.H.P.P. Ketterman A.J. Hemingway J. Bull. Entomol. Res. 1994; 84: 39-44Crossref Scopus (21) Google Scholar, 17.Ketterman A.J. Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Pest. Biochem. Physiol. 1993; 47: 142-148Crossref Scopus (32) Google Scholar). However, the Estβ2 EcoRI digest pattern, unlike that for Estβ1, does not appear to vary(9.Raymond M. Callaghan A. Fort P. Pasteur N. Nature. 1991; 350: 151-153Crossref PubMed Scopus (241) Google Scholar), hence the observed kinetic differences may not be reflected at the DNA level. Our current data show that Pel RR has the invariant Estβ21EcoRI band and that it apparently contains an allelic mixture of both Estα and Estβ, with a minor non-amplified band of each of the esterase types being present along with the prominent amplified band. Thus, in this and previous biochemical studies, all the purified Estα and Estβ esterases from the resistant strains may have been mixtures of amplified and non-amplified alleles, as the purification conditions used may not have separated these minor variants from each other. Such allelic mixtures would not have been detected, as both the Pel SS Estβ13 and the Pel RR Estβ21 esterase genes code for proteins of 540 amino acids, whose predicted molecular weights would not allow their separation by SDS-PAGE(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). This may explain the reported differences between purified esterases from different resistant strains, as variability could arise from different proportions of the amplified and non-amplified alleles, given the big differences in ka values between the amplified and non-amplified alleles of both the Estα and Estβ esterases. Alternatively, different non-amplified alleles could be mixed with an identical amplified allele to give a similar result. Our results also suggest that there is limited variability in the non-amplified Estα and Estβ alleles of the Sri Lankan field population, as only single non-amplified Estα and Estβ EcoRI bands were apparent from DNA obtained from mass homogenates of Pel SS, suggesting that this colony contains only a single Estα and a single Estβ esterase, even though the strain originated from numerous pooled single families selected from a large field collection of insects. Several years of laboratory colonization could have resulted in the loss of much of the variability. However, if the latter is true, it is surprising that the non-amplified Estβ allele of Pel RR has an identical EcoRI fragment to the Estβ13 allele of Pel SS, given that the two strains are maintained in separate insectaries. Similarly, restriction digest analysis of recent field collections of C. quinquefasciatus also suggest that the variability of the non-amplified Estα and Estβ alleles in the Sri Lankan field population is limited, with three common restriction fragments, as well as the amplified EcoRI fragment, occurring with each of three different restriction enzymes in six independent field collections from the Colombo area for each esterase. INTRODUCTIONThe use of pesticides, both directly and indirectly against the mosquito Culex quinquefasciatus, has resulted in the selection of broad spectrum organophosphate and carbamate resistance. The most commonly selected resistance mechanism is the increased activity of Estα2 and Estβ2 carboxylesterases (EC 3.1.1.1) (A2 and B2 esterases on an earlier classification)(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 2.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 49-55Crossref Scopus (43) Google Scholar, 3.Peiris H.T.R. Hemingway J. Bull. Entomol. Res. 1990; 80: 453-457Crossref Scopus (29) Google Scholar, 4.Raymond M. Pasteur N. Georghiou G.P. Mellon R.B. Wirth M.C. Hawley M. J. Med. Entomol. 1987; 24: 24-27Crossref PubMed Scopus (56) Google Scholar, 5.Raymond M. Beyssat-Arnaouty V. Sivasubramanian N. Mouches C. Georghiou G.P. Pasteur N. Biochem. Genet. 1989; 27: 417-423Crossref PubMed Scopus (77) Google Scholar, 6.Devonshire A.L. Field L.M. Annu. Rev. Entomol. 1991; 36: 1-23Crossref PubMed Google Scholar). Classification of these esterases is based on their preferences for α- or β-naphthyl acetate, their mobility on native polyacrylamide gel electrophoresis (PAGE), ( (1)The abbreviations used are: PAGEpolyacrylamide gel electrophoresiskbkilobase(s)pNPAp-nitrophenyl acetatepNPHp-nitrophenyl hexanoate.) and their nucleotide sequence(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The overproduction of the Estα21 and a series of Estβ esterases is due to gene amplification(1.Vaughan A. Hemingway J. J. Biol. Chem. 1995; 270: 17044-17049Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 5.Raymond M. Beyssat-Arnaouty V. Sivasubramanian N. Mouches C. Georghiou G.P. Pasteur N. Biochem. Genet. 1989; 27: 417-423Crossref PubMed Scopus (77) Google Scholar, 7.Mouches C. Pasteur N. Berge J.B. Hyrien O. Raymond M. De Saint Vincent B.R. De Silvestri M. Georghiou G.P. Science. 1986; 233: 778-780Crossref PubMed Scopus (278) Google Scholar, 8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar). Identical EcoRI restriction fragment sizes of the amplified Estβ2 from resistant C. quinquefasciatus worldwide has been reported, in contrast to a high level of variability in Estβ from insecticide-susceptible mosquitoes(9.Raymond M. Callaghan A. Fort P. Pasteur N. Nature. 1991; 350: 151-153Crossref PubMed Scopus (241) Google Scholar). A cDNA with 97% identity to Estβ21 has been cloned from an insecticide susceptible strain (Pel SS) of C. quinquefasciatus from Sri Lanka(8.Vaughan A. Rodriguez M. Hemingway J. Biochem. J. 1995; 305: 651-658Crossref PubMed Scopus (54) Google Scholar); however, the protein has not been characterized. After native starch or PAGE of homogenates of individual resistant Culex larvae of the amplified Estα2/Estβ2 esterase phenotype, two electromorphs can be visualized. Under the same conditions, no esterase bands are visible in homogenates of susceptible insects. This has lead to the suggestion that the susceptible insects have null alleles for these loci(10.Georghiou G.P. Pasteur N. J. Econ. Entomol. 1978; 71: 201-205Crossref PubMed Scopus (83) Google Scholar).Here, we report the purification and characterization of both an α- and a β-naphthyl acetate-specific esterase from the Pel SS strain of mosquito and compare these at a biochemical and basic molecular level to the elevated esterases Estα21, Estβ12, and Estβ21. The role of the amplified esterases in resistance is sequestration, which is rapid binding and slow turnover of insecticides (11.Ketterman A.J. Jayawardena K.G.I. Hemingway J. Biochem. J. 1992; 287: 355-360Crossref PubMed Scopus (51) Google Scholar, 12.Karunaratne S.H.P.P. Jayawardena K.G.I. Hemingway J. Ketterman A.J. Biochem. J. 1993; 294: 575-579Crossref PubMed Scopus (74) Google Scholar, 13.Jayawardena K.G.I. Karunaratne S.H.P.P. Ketterman A.J. Hemingway J. Bull. Entomol. Res. 1994; 84: 39-44Crossref Scopus (21) Google Scholar). ( (2)G. J. Small, S. H. P. P. Karunaratne, and J. Hemingway, submitted for publication.) The current study elucidates the relative efficiencies of the purified esterases from a susceptible and two further resistant strains at binding the carbamates and biologically active oxon analogues of the organophosphorus insecticides. Characterization of the amplified and non-amplified esterases will facilitate future site-directed mutagenesis studies on the essential amino acid residues involved in the enzyme-insecticide interaction.
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