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• W2107115483 abstract "Carboxylesterases are enzymes that catalyze the hydrolysis of a wide range of ester-containing endogenous and xenobiotic compounds. Although the use of pyrethroids is increasing, the specific enzymes involved in the hydrolysis of these insecticides have yet to be identified. A pyrethroid-hydrolyzing enzyme was partially purified from mouse liver microsomes using a fluorescent reporter similar in structure to cypermethrin (Shan, G., and Hammock, B. D. (2001) Anal. Biochem. 299, 54-62 and Wheelock, C. E., Wheelock, A. M., Zhang, R., Stok, J. E., Morisseau, C., Le Valley, S. E., Green, C. E., and Hammock, B. D. (2003) Anal. Biochem. 315, 208-222) and subsequently identified as a carboxylesterase (NCBI accession number BAC36707). The expressed sequence tag was then cloned, expressed in baculovirus, and purified to homogeneity. Kinetic constants for a large number of both type I and type II pyrethroid or pyrethroid-like substrates were determined. This esterase possesses similar kinetic constants for cypermethrin and its fluorescent-surrogate (kcat = 0.12 ± 0.03 versus 0.11 ± 0.01 s-1). Compared with their cis- counterparts, trans-permethrin and cypermethrin were hydrolyzed 22- and 4-fold faster, respectively. Of the four fenvalerate isomers the (2R)(αR)-isomer was hydrolyzed at least 1 order of magnitude faster than any other isomer. However, it is unlikely that this enzyme accounts for the total pyrethroid hydrolysis in the microsomes because both isoelectrofocusing and native PAGE indicate the presence of a second region of cypermethrin-metabolizing enzymes. A second carboxylesterase gene (NCBI accession number NM_133960), isolated during a cDNA mouse liver library screening, was also found to hydrolyze pyrethroids. Both these enzymes could be used as preliminary tools in establishing the relative toxicity of new pyrethroids. Carboxylesterases are enzymes that catalyze the hydrolysis of a wide range of ester-containing endogenous and xenobiotic compounds. Although the use of pyrethroids is increasing, the specific enzymes involved in the hydrolysis of these insecticides have yet to be identified. A pyrethroid-hydrolyzing enzyme was partially purified from mouse liver microsomes using a fluorescent reporter similar in structure to cypermethrin (Shan, G., and Hammock, B. D. (2001) Anal. Biochem. 299, 54-62 and Wheelock, C. E., Wheelock, A. M., Zhang, R., Stok, J. E., Morisseau, C., Le Valley, S. E., Green, C. E., and Hammock, B. D. (2003) Anal. Biochem. 315, 208-222) and subsequently identified as a carboxylesterase (NCBI accession number BAC36707). The expressed sequence tag was then cloned, expressed in baculovirus, and purified to homogeneity. Kinetic constants for a large number of both type I and type II pyrethroid or pyrethroid-like substrates were determined. This esterase possesses similar kinetic constants for cypermethrin and its fluorescent-surrogate (kcat = 0.12 ± 0.03 versus 0.11 ± 0.01 s-1). Compared with their cis- counterparts, trans-permethrin and cypermethrin were hydrolyzed 22- and 4-fold faster, respectively. Of the four fenvalerate isomers the (2R)(αR)-isomer was hydrolyzed at least 1 order of magnitude faster than any other isomer. However, it is unlikely that this enzyme accounts for the total pyrethroid hydrolysis in the microsomes because both isoelectrofocusing and native PAGE indicate the presence of a second region of cypermethrin-metabolizing enzymes. A second carboxylesterase gene (NCBI accession number NM_133960), isolated during a cDNA mouse liver library screening, was also found to hydrolyze pyrethroids. Both these enzymes could be used as preliminary tools in establishing the relative toxicity of new pyrethroids. Because of the removal of organophosphates from the market, pyrethroids are now the major class of insecticides used in the United States with more than 1200 metric tons used in American agriculture in 1997 (USGS Pesticide National Synthesis Project, available at ca.water.usgs.gov/pnsp). Both agricultural and residential usage is continuing to grow (4Landrigan P.J. Claudio L. Markowitz S.B. Berkowitz G.S. Brenner B.L. Romero H. Wetmer J.G. Matte T.D. Gore A.C. Godbold J.H. Wolff M.S. Environ. Health Perspect. 1999; 107: 431-437Crossref PubMed Scopus (269) Google Scholar, 5Whyatt R.M. Camann D.E. Kinney P.L. Reyes A. Ramirez J. Dietrich J. Diaz D. Holmes D. Perera F.P. Environ. Health Perspect. 2002; 110: 507-514Crossref PubMed Scopus (192) Google Scholar), leading to increased human exposure to these compounds. Pyrethroids are ester-containing insecticides that generally have low acute oral mammalian toxicity due to their rapid metabolism (6Casida J.E. Gammon D.W. Glickman A.H. Lawrence L.J. Annu. Rev. Pharmacol. Toxicol. 1983; 23: 413-438Crossref PubMed Scopus (390) Google Scholar, 7Soderlund D.M. Xenobiotica. 1992; 22: 1185-1194Crossref PubMed Scopus (20) Google Scholar). The major routes of pyrethroid metabolism in humans include ester hydrolysis by carboxylesterases (8Abernathy C.O. Ueda K. Engel J.L. Gaughan L.C. Casida J.E. Pestic. Biochem. Physiol. 1973; 3: 300-311Crossref Scopus (63) Google Scholar) and oxidation by cytochrome P450s (8Abernathy C.O. Ueda K. Engel J.L. Gaughan L.C. Casida J.E. Pestic. Biochem. Physiol. 1973; 3: 300-311Crossref Scopus (63) Google Scholar, 9El-Tawil O.S. Abdel-Rahman M.S. Pharm. Res. (N. Y.). 2001; 44: 33-39Crossref Scopus (32) Google Scholar, 10Hodgson E. J. Biochem. Mol. Toxicol. 2003; 17: 201-206Crossref PubMed Scopus (53) Google Scholar) and alcohol dehydrogenases (11Choi J. Rose R.L. Hodgson E. Pestic. Biochem. Physiol. 2002; 73: 117-128Crossref Scopus (74) Google Scholar). Carboxylesterases are also responsible for the hydrolysis of a large number of other endogenous and xenobiotic ester-containing compounds such as long chain, acyl-CoA esters, and many drugs (12Satoh T. Hosokawa M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 257-288Crossref PubMed Scopus (635) Google Scholar). Their importance in the hydrolysis of pyrethroids in mammals was first demonstrated via the use of carboxylesterase inhibitors (8Abernathy C.O. Ueda K. Engel J.L. Gaughan L.C. Casida J.E. Pestic. Biochem. Physiol. 1973; 3: 300-311Crossref Scopus (63) Google Scholar). These observations were further confirmed when Suzuki and Miyamoto (13Suzuki T. Miyamoto J. Pestic. Biochem. Physiol. 1978; 8: 186-198Crossref Scopus (41) Google Scholar) isolated a pyrethroid-hydrolyzing carboxylesterase from rat liver. Although pyrethroids have relatively low mammalian toxicity, a natural variation in esterase activity or the presence of other esterase substrates and/or inhibitors may compromise the ability of an organism to detoxify these pesticides (12Satoh T. Hosokawa M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 257-288Crossref PubMed Scopus (635) Google Scholar). Furthermore, both the levels of cytochrome P450s and carboxylesterases have been shown to be significantly lower in prenatal/newborn rats than in adults (14Miyamoto J. Kaneko H. Tsuji R. Okuno Y. Toxicol. Lett. 1995; 82/83: 933-940Crossref Scopus (71) Google Scholar, 15Cantalamessa F. Arch. Toxicol. 1993; 67: 510-513Crossref PubMed Scopus (160) Google Scholar). These data suggest an increased susceptibility of fetuses and young infants to pyrethroids. Although it has been established that carboxylesterases are involved in the detoxification of pyrethroids (16Miyamoto J. Environ. Health Perspect. 1976; 14: 15-28Crossref PubMed Scopus (184) Google Scholar), most of the work on pyrethroid toxicity in mammals has been based solely on investigations in whole animals (16Miyamoto J. Environ. Health Perspect. 1976; 14: 15-28Crossref PubMed Scopus (184) Google Scholar) or liver microsomes (8Abernathy C.O. Ueda K. Engel J.L. Gaughan L.C. Casida J.E. Pestic. Biochem. Physiol. 1973; 3: 300-311Crossref Scopus (63) Google Scholar, 17Soderlund D.M. Abdel Y.A.I. Helmuth D.W. Pestic. Biochem. Physiol. 1982; 17: 162-169Crossref Scopus (24) Google Scholar). Therefore, the specific carboxylesterases that hydrolyze pyrethroids have not yet been characterized (18Sogorb M.A. Vilanova E. Toxicol. Lett. 2002; 128: 215-228Crossref PubMed Scopus (452) Google Scholar). Several carboxylesterase isozymes with various substrate specificities have been isolated from a variety of organisms (12Satoh T. Hosokawa M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 257-288Crossref PubMed Scopus (635) Google Scholar). However, none have displayed any specificity for pyrethroids. Therefore, we decided to identify a pyrethroid-hydrolyzing esterase(s) and to clone, express, and purify it for further characterization. This esterase was isolated from a common laboratory mouse strain (Swiss-Webster) due to the use of mice as animal models for studying the effects of both endogenous and xenobiotic compounds (19Miyata M. Yakugaku Zasshi. 2003; 123: 569-576Crossref PubMed Scopus (1) Google Scholar). By isolating specific carboxylesterases we may begin to understand their relative importance in the hydrolysis of a range of different esters. Chemicals—All substrates used in this study are numbered according to the system outlined in Fig. 1. The compounds (R/S)-α-cyano(6-methoxy-2-naphthyl)-methyl acetate (A1), (R/S)-α-cyano(6-methoxy-2-naphthyl)methyl-(R/S)-trans/cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (A2), (R/S)-α-cyano(6-methoxy-2-naphthyl)-methyl-(R)-(-)-2-(4-chlorophenyl)-3-methyl butanoate ((αR/S)(2R)-A3), (R/S)-α-cyano(6-methoxy-2-naphthyl)-methyl-(S)-(+)-2-(4-chlorophenyl)-3-methyl butanoate ((αR/S)(2S)-A3), and fenvalerate isomers ((αR)(2R)-B3), ((αR)(2S)-B3), ((αS)(2R)-B3), ((αR)(2R/S)-B3), and ((αS)(2R/S)-B3) were all previously synthesized in this laboratory (1Shan G. Hammock B.D. Anal. Biochem. 2001; 299: 54-62Crossref PubMed Scopus (45) Google Scholar, 2Wheelock C.E. Wheelock A.M. Zhang R. Stok J.E. Morisseau C. Le Valley S.E. Green C.E. Hammock B.D. Anal. Biochem. 2003; 315: 208-222Crossref PubMed Scopus (48) Google Scholar, 20Shan G. Stoutamire D.W. Wengatz I. Gee S.J. Hammock B.D. J. Agric. Food Chem. 1999; 47: 2145-2155Crossref PubMed Scopus (74) Google Scholar). All fenvalerate isomers were ∼96% enantiomeric excess. Malathion, cypermethrin (B2), esfenvalerate ((αS)(2S)-B3), (trans/cis)-permethrin (C2), and (cis)(αS)(1R,3R)deltamethrin ((cis)(αS)(1R,3R)-B4) were obtained from Chem Services Inc. (West Chester, PA). Both cis- and trans-permethrin (C2) were gifts from ICI Agrochemicals. Both cis- and trans-cypermethrin (B2), α-cypermethrin ((αR)(1S,3S)- and (αS)(1R,3R)-B2) and ζ-cypermethrin ((αS)(1R/S,3R/S)-B2) (S/R ratio, 89.7/10.3) were gifts from FMC Corp. (Princeton, NJ). All isomers unless otherwise stated were ∼99% of the isomer mixture indicated. All other chemicals used in this study were either purchased from Sigma-Aldrich or Fisher. Synthesis of compounds A4-8 is outlined in the supplemental material. Isolation of a Cypermethrin-hydrolyzing Carboxylesterase—The following procedures were performed at 4 °C unless otherwise stated. Livers from male Swiss-Webster mice (Charles River Laboratories Inc., Wilmington, MA) were perfused with 1.15% KCl (∼10 °C) and homogenized (2 × 30 s, 10,000 rpm, Polytron homogenizer, Brinkmann) in Buffer A (20 mm sodium phosphate buffer, pH 7.4, 5 mm EDTA, and 1 mm 1-phenyl-2-thiourea). The supernatant was collected by centrifugation of the homogenized sample (10,000 g, 20 min). The pellet was resuspended in buffer A (ratio 1:10), and the supernatant was collected by centrifugation (10,000 × g, 20 min). After centrifugation of the combined supernatants (100,000 × g, 1 h), the microsomes were washed in fresh buffer A, re-pelleted (100,000 × g, 1 h), resuspended in 10 mm sodium phosphate, pH 7.0, 20% glycerol, 0.5 m EDTA, and 1 mm 1-phenyl-2-thiourea, and stored at -80 °C. Microsomes were solubilized using the previously described protocol (21Huang T.L. Shiotsuki T. Uematsu T. Borhan B. Li Q.X. Hammock B.D. Pharm. Res. (N. Y.). 1996; 13: 1495-1500Crossref PubMed Scopus (35) Google Scholar). Briefly, octyl pyranoglucoside (final concentration, 1% w/v) was added to the microsomes, which were then placed on a rotating wheel for 1 h. The resulting protein mixture was concentrated, mixed 1:1 with Tris-glycine native sample buffer (Invitrogen), loaded onto a 7% polyacrylamide gel tube, and run overnight on a preparative electrophoresis unit (Mini Prep Cell, Bio-Rad). The pyrethroid activity was screened with A2. Active fractions were pooled and concentrated by filtration (30-kDa cutoff; Millipore, Billerica, MA). Identification of a “cypermethrin-hydrolyzing carboxylesterase” was achieved via separation of the semi-purified protein on two-dimensional PAGE. A pH 4-7 immobilized pH gradient strip (13 cm, Amersham Biosciences) was rehydrated overnight in a solution (250 μl) containing semi-purified protein (10-15 μg), 5% glycerol, and 2% immobilized pH gradient (4Landrigan P.J. Claudio L. Markowitz S.B. Berkowitz G.S. Brenner B.L. Romero H. Wetmer J.G. Matte T.D. Gore A.C. Godbold J.H. Wolff M.S. Environ. Health Perspect. 1999; 107: 431-437Crossref PubMed Scopus (269) Google Scholar, 5Whyatt R.M. Camann D.E. Kinney P.L. Reyes A. Ramirez J. Dietrich J. Diaz D. Holmes D. Perera F.P. Environ. Health Perspect. 2002; 110: 507-514Crossref PubMed Scopus (192) Google Scholar, 6Casida J.E. Gammon D.W. Glickman A.H. Lawrence L.J. Annu. Rev. Pharmacol. Toxicol. 1983; 23: 413-438Crossref PubMed Scopus (390) Google Scholar, 7Soderlund D.M. Xenobiotica. 1992; 22: 1185-1194Crossref PubMed Scopus (20) Google Scholar) buffer and then focused on a 2117 Multiphore II IEF 1The abbreviations used are: IEF, isoelectrofocusing; pNPA, p-nitrophenyl acetate; GCMS, gas chromatography-mass spectroscopy. unit (LKB, Bromma, Sweden). A second SDS-PAGE dimension was run by first equilibrating the strip in SDS equilibration buffer (50 mm Tris-HCl, pH 8.8, 6 m urea, 30% v/v glycerol, 2% w/v SDS, and bromphenol blue) with 1% (w/v) dithiothreitol for 15 min. This solution was then replaced with SDS equilibration buffer that included 0.25% (w/v) iodoacetamide, and the strip was equilibrated for 15 min. This strip was then loaded onto a 10% SDS-PAGE gel and sealed with 1% IsoGelagarose. The gel was run at a constant current of 30 mA in a vertical slab gel unit (Hoeffer) at 10 °C. After the isoelectrofocusing step, a second immobilized pH gradient strip was cut into ∼1-2-mm slices and screened for A2 activity. The strip was then aligned with the SDS-PAGE gel, and the appropriate spots were sequenced. In-gel trypsin digestion followed by peptide mass-mapping and mass spectrometry protein sequencing were performed by the Molecular Structure Facility (University of California at Davis). cDNA Library Screening—The mRNA from Swiss-Webster mouse liver (∼4 g, pooled from 4 mice) was isolated via a two-step procedure in which total RNA was initially extracted (RNeasy and Oligotex Kits, Qiagen, Valencia, CA). First strand cDNA was synthesized using Superscript reverse transcriptase II (Invitrogen) and the BD SMART mRNA amplification Kit (BD Biosciences) according to the manufacturer's procedure. The cDNA generated was used as the template in the subsequent PCR reaction. PCR primers were designed to one of the sequenced peptides, NLFGSEDLK (5′-AATTTGTTYGGTTCAGAGGATCTGAAG-3′) and, in addition, to either the conserved catalytic serine region IFGESAGGT (5′-TGTGCCACCTGCTGACTCTCCAAAAAT-3′) or the C-terminal region of the putative mouse carboxylesterase (NCBI accession number BAC36707) (5′-CTACAACTCTTTGTGCCTCTCCTGAGA-3′). A DNA fragment (399 bp or 1.4 kilobases) was amplified with Taq DNA polymerase (Invitrogen) using the above primers and employing the following conditions: 95 °C for 3 min, 30 cycles of 95 °C for 45 s, 45 °C for 5 min, and 72 °C for 2 min, and completed with 72 °C for 10 min. The DNA fragment was ligated into a pCR2.1-TOPO treated vector (TA cloning kit, Invitrogen), and the sequence was verified (DNA Sequencing Facility, University of California at Davis). Approximately 1% of a mouse (C57Bl/6J) liver cDNA library (1 × 107 independent clones) (Invitrogen) was screened using the horseradish peroxidase-labeled PCR fragment generated above (ECL, Amersham Biosciences). Potentially positive clones were transformed into Escherichia coli DH5α and verified by DNA sequencing. Cloning, Expression, and Purification of the Pyrethroid-hydrolyzing Carboxylesterase—An Expressed Sequence Tag (Clone ID 1450336, Invitrogen) was purchased that contained the gene (NCBI accession number BC055062) corresponding to the protein identified by mass spectrometry. The gene was excised from the original vector (pME18S-FL3) using StuI/XbaI and ligated into a similarly cut vector pBacPAK8 (BD Biosciences). The recombinant baculovirus containing the esterase gene was generated (22OReilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman and Co., New York1994Google Scholar) and expressed in High five Trichoplusia ni cells (2 liters, 1 × 106/ml) with a multiplicity of infection of ∼0.1. At 72 h post-infection the cells were pelleted (2,000 × g, 20 min), resuspended in 100 mm Tris-HCl, pH 8.0 (containing 1 mm dithiothreitol, 1 mm EDTA, and 1 mm 1-phenyl-2-thiourea), and homogenized (2 × 30 s; Polytron homogenizer, Brinkmann). The supernatant that was generated after centrifugation (10,000 × g, 20 min) underwent further centrifugation (100,000 × g, 1 h) to pellet the microsomal fraction. The latter supernatant was diluted 5-fold with buffer B (20 mm Tris HCl, pH 8.0, 1 mm dithiothreitol), loaded onto a DEAE ion exchange column (3 × 15 cm; Amersham Biosciences), and washed in the same buffer. After a second wash with 75 mm NaCl in Buffer B, the esterase was eluted with 125 mm NaCl in Buffer B and detected by measuring p-nitrophenyl acetate (pNPA) hydrolysis. After concentration, the targeted protein was further purified using a preparative isoelectrofocusing unit (Bio-Rad; pH 3-8). The esterase-containing fractions were then combined, mixed with an equal volume of Buffer B containing 4 m KCl, and loaded onto a phenyl-Sepharose column (1.6 × 9 cm; Amersham Biosciences) that had been equilibrated in advance with buffer B containing 2 m KCl. The esterase was eluted at ∼50 mm KCl by running a linear salt gradient (2 to 0 m KCl) and was then stored at -80 °C. The enzyme could be purified in a similar fashion from the microsomal fraction generated after the second centrifugation step (100,000 × g). In this case, solubilization (1% (w/v) octyl glucopyranoside) was necessary before application on the DEAE column. Characterization of the Carboxylesterases—Protein concentration was determined with the Pierce BCA assay (Pierce) using bovine serum albumin as the standard. SDS-PAGE and native PAGE were performed using 12% Tris-glycine gels (Invitrogen), whereas IEF PAGE was performed using IEF 3-7 gels (Invitrogen). The pI was estimated by incubating 2.5-mm IEF gel strips at room temperature for 30 min in water (500 μl) and measuring the pH with a pH meter. De-glycosylated protein was obtained by employing N-glycosidase F (New England Biolabs, Beverly, MA) using the procedure provided by the manufacturer. Briefly, the esterase (100 μg) was denatured with the addition of 0.5% SDS and 1% β-mercaptoethanol (final concentration) for 10 min at 100 °C. After cooling on ice, 50 mm sodium phosphate, pH 7.5, 1% Nonidet P-40 (final concentration), and N-glycosidase F (5000 units) were added, and the consequent solution (final volume 100 μl) was incubated for 1 h at 37 °C. Enzyme Activity Assays—Esterase activity was detected using a number of different substrates: pNPA, malathion, individual pyrethroid isomers or racemic mixtures, and fluorescent-pyrethroid surrogates (A1-8). Because all isomers or isomer mixtures were at least 98% of the stereochemistry indicated, quantities were sufficient to satisfy Michaelis-Menten conditions. Briefly, pNPA hydrolysis was monitored for 2 min at 405 nm according to the methods of Ljungquist and Augustinsson (23Ljungquist A. Augustinsson K.B. Eur. J. Biochem. 1971; 23: 303-313Crossref PubMed Scopus (90) Google Scholar), as modified in Wheelock et al. (24Wheelock C.E. Severson T.F. Hammock B.D. Chem. Res. Toxicol. 2001; 14: 1563-1572Crossref PubMed Scopus (58) Google Scholar). The malathion assay was conducted according to the method previously described (25Talcott R.E. Toxicol. Appl. Pharmacol. 1979; 47: 145-150Crossref PubMed Scopus (69) Google Scholar, 26Huang T.L. Villalobos S.A. Hammock B.D. J. Pharm. Pharmacol. 1993; 45: 458-465Crossref PubMed Scopus (19) Google Scholar). GCMS assays (2Wheelock C.E. Wheelock A.M. Zhang R. Stok J.E. Morisseau C. Le Valley S.E. Green C.E. Hammock B.D. Anal. Biochem. 2003; 315: 208-222Crossref PubMed Scopus (48) Google Scholar) were modified by adding 1 μl of substrate (B2-4, 7, C2, D2, and E7; 25 mm in ethanol) to 0.5 ml of the enzyme (1-30 μg/ml). The enzyme mixture was incubated for 20 s to 10 min depending on the substrate. Ethyl acetate (250 μl) and brine (250 μl) were added to each sample, after which the mixture was vortexed. An internal standard (3-(4-methoxy)-phenoxybenzaldehyde; 80 μm final concentration) was added to 100 μl of the ethyl acetate solution, and this solution was analyzed by GCMS. The method detection limits for 3-phenoxybenzaldehyde and 4-fluoro-3-phenoxybenzaldehyde were calculated by heat-killing the carboxylesterase (3 μg) and running a mock assay without the addition of substrate. The assay solutions were spiked with either 3-phenoxybenzaldehyde or 4-fluoro-3-phenoxybenzaldehyde (0.5 μm final concentration; 5× lowest detectable standard), brine (250 μl) and ethyl acetate (250 μl) were added, and the mixture was vortexed. This procedure was repeated seven times, and the ethyl acetate extract of each replicate was analyzed by GCMS. Fluorescent assays were conducted by measuring the production of 6-methoxynaphthaldehyde for 9 min after the addition of the substrates (A1-8) using an excitation wavelength of 330 nm (bandwidth, 35 nm) and an emission wavelength of 465 nm (bandwidth, 35 nm) (2Wheelock C.E. Wheelock A.M. Zhang R. Stok J.E. Morisseau C. Le Valley S.E. Green C.E. Hammock B.D. Anal. Biochem. 2003; 315: 208-222Crossref PubMed Scopus (48) Google Scholar). Protein concentration varied from 1 ng to 50 μg for these fluorescent assays. All assays were performed in buffer B at 30 °C. No more than 10% of the substrate was hydrolyzed during the assay, and solvent content never exceeded 1% of the total assay volume. Isolation of a Pyrethroid-hydrolyzing Carboxylesterase from Mouse Liver—To isolate a “pyrethroid-hydrolyzing esterase” microsomes were prepared from murine livers. Cypermethrin and A2 were employed to characterize the behavior of esterases on both native and IEF PAGE. At least 90% of the A2 activity was associated with esterases located in the lower section of both native and IEF gels (Fig. 2). Further analysis revealed a second region of pyrethroid-hydrolyzing esterases accounting for 50-60% of the total cypermethrin activity (Fig. 2). Because the esterases that migrate faster on native PAGE (Fig. 2A) had higher apparent hydrolase activity specific for cypermethrin and A2 when compared with pNPA (5-10% of the total pNPA activity), we concentrated on purifying a pyrethroid hydrolase from this area using A2 as a reporter. Effective separation of the pyrethroid-hydrolyzing carboxylesterase(s) from the majority of the other esterases was achieved using a preparative electrophoresis unit (Table I). To separate the remaining proteins, IEF PAGE was employed together with SDS-PAGE to run a two-dimensional gel. Three protein spots on this two-dimensional gel were found to have A2 hydrolysis activity.Table IPartial purification of a pyrethroid-hydrolyzing carboxylesterase from mouse liver microsomesTotal proteinTotal activityaEnzyme activity was measured using A2 as described under “Materials and Methods.”Specific activityPurification foldActivitymgnmol/minnmol/min/mg%Microsomes22NDbNot determined.NDSolubilized microsomescMicrosomes were solubilized according to the methods of Huang et al. (21).132.30.181.0100Preparative electrophoresis0.0540.79158134a Enzyme activity was measured using A2 as described under “Materials and Methods.”b Not determined.c Microsomes were solubilized according to the methods of Huang et al. (21Huang T.L. Shiotsuki T. Uematsu T. Borhan B. Li Q.X. Hammock B.D. Pharm. Res. (N. Y.). 1996; 13: 1495-1500Crossref PubMed Scopus (35) Google Scholar). Open table in a new tab Identification of a Pyrethroid-hydrolyzing Carboxylesterase—Mass spectral sequence analysis of the protein spot with the highest pyrethroid activity produced two peptide sequences (PYTEEEE; LQFWTK) that were identical to sequences in a putative carboxylesterase (NCBI accession number BAC36707). A third peptide (QNDNLFGSEDLK) was also found to correspond to the same putative carboxylesterase with a one-amino acid difference from the reported sequence (reported sequence, QNDNLMGSEDLK). The remaining two protein spots that possessed pyrethroid activity were also analyzed. Although they contained some of the same peptides that corresponded with the first carboxylesterase, they were both contaminated with other proteins, and therefore, no conclusions were drawn (peptides observed were GFFSTGDQHAK and QQNLVHFGG; residues that are different in NCBI accession number BAC36707 are underlined). Screening cDNA Library—Using the information gathered from protein sequencing we attempted to isolate the corresponding gene (NCBI accession number BC055062) from a mouse liver cDNA library using a probe generated by PCR. Although the gene of interest was not isolated from these attempts, two other putative carboxylesterase genes were found (NCBI accession numbers NM_133960 and NM_144930). Expression and Purification—The pyrethroid-hydrolyzing carboxylesterase (NCBI accession number BAC36707) was expressed by infecting T. ni High five cells with the recombinant baculovirus. Esterase activity was subsequently found in both the microsomal pellet and the supernatant of centrifugation (100,000 × g), and thus, the esterase was purified separately from both sources. The most effective step in the purification of this esterase was performed by the preparative IEF unit, which increased the specific activity of the enzyme 10-fold (Table II). The two additional carboxylesterase genes were both expressed and purified in a similar fashion to the first (data not shown).Table IIPurification of a recombinant pyrethroid-hydrolyzing carboxylesterase BAC36707 expressed in baculovirusStepTotal activityaEnzyme activity was measured using A2 as described under “Materials and Methods.”Total proteinActivitySpecific activityPurification foldnmol/minmg%nmol/min/mgCrude1900 ± 2303600 ± 1301000.54 ± 0.061.0010,000 × g1200 ± 572100 ± 45630.56 ± 0.031.04Supernatant 100,000 × g980 ± 801700 ± 31510.58 ± 0.051.08DEAE200 ± 1131 ± 3116.5 ± 0.912Preparative IEF (3-8)280 ± 123.5 ± 0.11478 ± 10146Phenyl-Sepharose159 ± 11.4 ± 0.18110 ± 6209a Enzyme activity was measured using A2 as described under “Materials and Methods.” Open table in a new tab Characterization of the Pyrethroid-hydrolyzing Carboxylesterase—The N-terminal sequence (DSASPIRNTH) confirmed that the expressed protein gave an exact match to the mature protein with the removal of the signal peptide (27 amino acids). IEF PAGE analysis revealed 3 separate bands that had an average pI of 5.8 ± 0.2, similar to that determined from the mouse liver microsomes. Initial experiments using mass spectrometry indicated that the esterase was heavily glycosylated, and hence, it was difficult to determine the mass of the protein. SDS-PAGE indicated that the purified enzyme had two equally intense bands. Only the lower of the two bands remained after removal of the glycosylation, suggesting that approximately half of the protein was not glycosylated. Kinetic Analysis of the Pyrethroid-hydrolyzing Carboxylesterase—To determine the specificity of the esterase BAC36707 toward pyrethroids, the kinetic constants of a number of general esterase substrates were compared with both pyrethroid mimics (A2, A3, and A5) and cypermethrin (Table III). For this particular enzyme A2 seems to be an excellent mimic of cypermethrin because there is little difference between these two substrates (Km and kcat). In addition, the esterase was found to have similar Km values to other pyrethroids, such as (αR/S)(2R)-A3 and A5 but with much lower kcat values. Comparison to acetate substrates such as A1 and pNPA suggests that the enzyme predominantly recognizes the acid component of the pyrethroid rather than the alcohol. Although pNPA has an extremely high kcat value (94 ± 2 s-1), the Km value for this substrate was ∼200-600-fold higher than that of the pyrethroids.Table IIIKinetic constants determined for the purified recombinant pyrethroid-hydrolyzing carboxylesterase (NCBI accession number BAC36707)SubstrateKmkcatkcat/KmμMs−1μM−1 s−1p-Nitrophenyl acetate286 ± 1694 ± 20.33 ± 0.01Cypermethrin (B2)0.50 ± 0.150.12 ± 0.030.24 ± 0.06A1NMaNM, not measurable. Km and kcat values could not be calculated due to substrate insolubility.NM1.2 ± 0.1A22.2 ± 0.20.11 ± 0.010.040 ± 0.004(αR/S) (2R)-A30.89 ± 0.080.067 ± 0.0120.077 ± 0.021A50.96 ± 0.130.099 ± 0.0050.11 ± 0.01a NM, not measurable. Km and kcat values could not be calculated due to substrate insolubility. Open table in a new tab Specific activity was determined for both type I and II pyrethroids and their mimics (Table IV). In general, the highest specific activity in all the purified enzymes and solubilized microsomes was associated with esters with simple acids such as acetate and butyrate (pNPA, A1" @default.
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• W2107115483 date "2004-07-01" @default.
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• W2107115483 title "Identification, Expression, and Purification of a Pyrethroidhydrolyzing Carboxylesterase from Mouse Liver Microsomes" @default.
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• W2107115483 doi "https://doi.org/10.1074/jbc.m403673200" @default.
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