- Email: [email protected]
Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe)
Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe) ¨ Forget*, Sandrine Livet, Francois Joelle Leboulenger ¸ Laboratoire d’Ecotoxicologie (LEMA), UPRES-EA3222, Faculte´ des Sciences et Techniques, Universite´ du Havre, 25 rue Philippe Lebon, BP 540, 76058 Le Havre cedex, France Received 2 December 2001; received in revised form 5 March 2002; accepted 8 March 2002
Abstract Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 8C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Acetylcholinesterase; Biochemical characterization; Isoelectric focusing electrophoresis; Biomarker; Invertebrates; Copepod; Eurytemora affinis; Pollutants
1. Introduction Intensive use of organophosphate and carbamate pesticides has caused various ecotoxicological problems. The primary toxicity of these substances is due to an irreversible inhibition of acetylcholinesterase (AChE), a key enzyme of the nervous system. Phosphorylation or carbamoylation of the serine residue at the active site of AChE blocks metabolization of the neurotransmitter acetylcholine. The postsynaptic membrane remains depolarized, and therefore, synaptic transmission is altered. Anticholinesterase agents such as organophosphate and carbamate insecticides are widely *Corresponding author. Tel.: q33-2-3274-4379; fax: q332-3274-4314. E-mail address: [email protected] (J. Forget).
used to control various agricultural pests (insects, acari and nematodes), but are potentially toxic for other animals. Individuals poisoned by these compounds experience partial cholinesterase inhibition, which can be lethal (Alfthan et al., 1989). However, the enzyme is also present in body regions where inhibition does not affect viability (Massoulie´ et al., 1993). For many years, cholinesterases (ChEs) have been considered as interesting biomarkers for the monitoring of environmental contamination by compounds such as organophosphate and carbamate insecticides, heavy metals and pulp waste (Livingstone, 1993; Payne et al., 1996). In the marine environment, bivalve mollusks such as the blue common mussel or the oyster have been the favorite target species for biomonitoring. However,
1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 0 5 0 - 9
86
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
mollusk AChE have proved less sensitive to inhibitors than those of crustacean or vertebrate species ´ 1990). Moreover, acetyl(Galgani and Bocquene, choline has been actually identified as a neurotransmitter in crustacean central nervous system (Baker et al., 1972; Braun and Mulloney, 1994), and AChE has been the only form of ChE found in the ectoparasitic copepod sealouse (Lepeophtheirus salmonis) (Walday and Fonnum, 1989a,b). Cholinesterases have been extensively studied because, they are highly polymorphic enzymes in most species, they play an important role in the transmission of nervous influx and they are the specific targets for most nerve agents and insecticides. The number of genes encoding ChEs varies according to species: one in insects, two in vertebrates and three in nematodes (Massoulie´ et al., 1993). In vertebrates, use of acetylcholine and butyrylcholine has allowed differentiation of the two proteins as AChE and butyrylcholinesterase (BuChE). Results from molecular biology indicate that AChE and BuChE have approximately 50% amino-acid identity within species (Massoulie´ et al., 1993; Silver, 1974). For each protein, various molecular forms originate either from alternative gene splicing or degradation. These forms may be either soluble or linked to the basal lamina via a collagen tail or to the membrane via a glycolipid or a hydrophobic peptide. They have been classified as asymmetrical, globular asymmetrical or collagen-tailed forms, existing in all vertebrate classes but apparently absent in invertebrates. Globular forms are encountered as monomers, dimers and tetramers. The two enzymes AChE and BuChE are distinguished functionally, primarily on the basis of substrate specificity (Radic et al., 1991). AChE hydrolyzes acetylcholine much faster than other choline esters (like propionylcholine) and is virtually inactive on butyrylcholine, whereas BuChE not only hydrolyzes butyrylcholine well but also acetylcholine at an appreciable rate. The two enzymes may also be distinguished by their susceptibility to diagnostic inhibitors such as isoOMPA for AChE (Austin and Berry, 1953). AChE also exhibits excess substrate inhibition, which may be mediated by a peripheral substrate binding site (Radic et al., 1991). Among marine crustaceans, ChEs have been characterized in Decapoda (Maia verrucosa, Palinurus vulgatus) (Talesa et al., 1990), and Stomatopoda (Squilla mantis)
(Principato et al., 1988), and Copepoda (Tigriopus ´ 1999). brevicornis) (Forget and Bocquene, To investigate the apparent sensitivity of the copepod, Eurytemora affinis ChE was purified by affinity chromatography and its specific kinetic behavior towards substrates and inhibitors was determined. 2. Materials and methods 2.1. Materials Specimens of the copepod Eurytemora affinis (E. affinis) were collected by plankton netting along the Seine estuary (France), near the Tancarville bridge and conveyed to the laboratory in plastic containers for immediate freezing at y80 8C. Acetylthiocholine (AcSChI), butyrylthiocholine (BuSChI), propionylthiocholine (PrSChI), eserine, iso-OMPA, nonylphenol, 17b-oestradiol, DTNB w5,59-dithio-bis(2-nitrobenzoic acid)x, decamethonium and EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline) were obtained from Sigma Aldrich, France. Carbofuran and atrazine were obtained from Cluzeau Info Labo, France. All chemicals used in this study were of analytical grade. 2.2. Extraction of cholinesterases from copepods Extraction was performed using 20 mM phosphate buffer, pH 7, plus 0.1% Triton X-100. E. affinis were homogenized in toto 1:5 (WyV) for 1 min on ice using an ultraturrax (type Diax 900). Extracts were then centrifuged at 10 000 g for 30 min, and this operation was repeated on the pellet after resuspension. The extract was then diluted 1:3 in the extraction buffer. 2.3. Purification procedure All purification steps were conducted at 4 8C. Affinity chromatography was performed on procainamide, a ligand specific for the choline-binding site. The diluted extract was directly applied to the affinity column. Procainamide-containing gel was prepared using ECH-Sepharose 4B (Pharmacia) as the coupling gel and EEDQ for ligand immobilization. The loading buffer was 20 mM phosphate buffer, pH 7, plus 0.1% Triton X-100, and the elution buffer was 20 mM phosphate buffer, pH
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
9.5, plus 0.1% Triton X-100 containing 20 mM decamethonium. Fractions exhibiting ChE activity were pooled and stored at y80 8C until use (Bocquene´ et al., 1997). 2.4. Non-denaturing electrophoresis Electrophoresis experiments were performed under non-denaturing conditions in a 2–23% acrylamide gradient gel containing 0.02% Triton X100. Samples were prepared as follows: to the protein solution was added an equal volume of glycerol (50%) and 2% bromophenol blue (wyv), and 20 ml of this mixture were then applied to each well of the gel. After equilibration of the gel, migration was carried out at 8 8C for approximately 16 h at a constant voltage (150 V). Cholinesterase bands were stained according to the procedure of Karnovsky and Roots (1964) based on hydrolysis of a thiocholine ester and formation of an insoluble copper complex. Apparent molecular weights were estimated with respect to the migration of high-molecular-weight standard proteins for non-denaturing electrophoresis gels (Pharmacia). 2.5. Isoelectric focusing electrophoresis Isoelectric point was estimated by isoelectric focusing under non-denaturing conditions using PhastSystem apparatus, Ampholine PAGplate IEF gel (Pharmacia) (pH range 3–9.5) and pI calibration kit as marker proteins from Pharmacia. After focusing, gels were stained with Coomassie blue for protein detection, or according to the Karnovsky and Roots method for esterase activity. 2.6. Activity, substrate and inhibitor specificities Cholinesterase activity was routinely monitored according to Ellman et al. (1961) using AcSChI at 1 mM as substrate in 20 mM phosphate buffer, pH 7. One unit corresponded to the hydrolysis of 1 nmole of substrate per min. Bradford’s method (Bradford, 1976) was used for quantitative determination of proteins with bovine serum albumin as standard. These methods were adapted for measurement with a microplate reader. Cholinesterase activities are expressed as units miny1, and specific activities are given as units mgy1 protein (one unit corresponding to the metabolization of 1 nmole of acetylcholine miny1).
87
The hydrolysis of three different substrates— AcSChI, BuSChI and PrSChI—was followed for each molecular form at a concentration of substrate ranging from 5 mM to 10 mM at 25 8C. The Michaelis constant Km was determined by analysis of the Lineweaver–Burk transformation. The sensitivity of ChE to inhibition by pollutants wisoOMPA (tetraisopropyl pyrophosphoramide) a specific inhibitor of human BuSCh, the two carbamates (eserine and carbofuran), atrazine, nonylphenol and 17b oestradiolx was investigated. Stock solutions of inhibitors were prepared in ethanol. The generally accepted inhibition mechanism of AChE by organophosphate and carbamate compounds has been described by Aldridge and Reiner (1969). The formula describing the inhibition mechanism is as follows:
where Esenzyme, CXscarbamate or organophosphate, Xsleaving group, Kdsky1yk1, k2sthe carbamoylation or phosphorylation rate constant, k3sthe decarbamoylation or dephosphorylation rate constant, and kisthe bimolecular rate constant (sk2yKd). For ki determination, enzymes were incubated for various times with different insecticide concentrations wIx, in conditions such as wIx always being at least ten-fold superior to wEx, in 20 mM phosphate buffer, pH 7, at 25 8C. The time-course variation of free enzyme wEx was estimated by the Ellman method w1961x. This variation follows a pseudo-first-order function, Ln wEx y wE0xsyki y wIx t, where t represents the time of incubation and I the inhibitor. All graphs obtained for each inhibitor were linear, whatever the variable (wIx or t), suggesting that reactivation (k3) remained negligible during the time of the experiment (20 min). 3. Results 3.1. Extraction, purification and separation All ChE activity from the copepod homogenates was extracted by 20 mM phosphate buffer, pH 7, 0.1% Triton X-100. The low ionic strength buffer did not release extra activity. When applied on a procainamide affinity column, most ChE activity
88
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
Fig. 1. Electropheretic analysis of cholinesterase from the copepod Eurytemora affinis. Non-denaturing electrophoresis was performed in gradient polyacrylamide gels in the presence of 0.1% Triton X100. Lane M, high-molecular-weight markers. Lane D, migration of the dimeric form of Drosophila melanogaster AChE. Lanes C, migration of the cholinesterase from copepod Eurytemora affinis.
remained bound to the column, with recovery ranging from 50–70% of original activity. 3.2. Characterization of the single form Electrophoresis of the purified extract performed in non-denaturing conditions on gradient gel in the presence of Triton X-100 showed one band migrating at 140 KDa (Fig. 1). When the detergent migrated in the gel, ChE migrated until it reached the gradient zone corresponding to its apparent molecular weight (Sine et al., 1992). This single band reached the gradient zone between the lactate dehydrogenase and the dimeric form of Drosophila melanogaster AChE (Fournier et al., 1987). The purified enzyme was also subjected to analytical isoelectric focusing in a pH gradient of 3 to 9.5. Results indicated that ChE was homogeneous with an apparent pI of 6.2 (Fig. 2). This single band reached the pI between the two markers bovine carbonic anhydrase B (pI: 5.85) and human carbonic anhydrase B (pI: 6.55). 3.3. Substrate specificity, inhibition and kinetic analysis of AChE activity The specificity of the enzyme toward AcSChI, BuSChI and PrSChI was determined with 1 mM of substrate (Table 1). E. affinis ChE was most active against AcSChI (100%), and almost equally much less active against BuSChI (10%) and PrSChI (9%).
This form displayed an apparent Michaelian behavior in the 5 mM to 5 mM range (Fig. 3) when AcSChI was used as substrate, with a Km of 32 mM (Table 1). The ChE of E. affinis appeared to be an AChE, i.e. it hydrolyzed AcSChI at a higher rate than other substrates and was inhibited by eserine but not by Iso-OMPA (Table 2). Inhibition experiments with several compounds tested showed a high sensitivity of the copepod ChE to pesticides, 17b oestradiol and Nonylphenol, with ki values in the order of 105 for carbofuran to 103 for atrazine, 17b oestradiol and Nonylphenol (Table 2). 4. Discussion Our results indicate the presence of a single ChE in the copepod E. affinis as previously shown in another copepod Tigriopus brevicornis (Forget ´ 1999) and in other crustaceans, and Bocquene, such as Squilla mantis (Principato et al., 1988), Maia verrucosa and Palinurus vulgaris (Talesa et al., 1995). However, the question still arises as to whether this enzyme is actually ChE or a nonspecific esterase, which also metabolizes acetylcholine. Cholinesterases have been described and classified by many authors into two groups, AChE or BuChE (pseudo-cholinesterase), depending on substrate hydrolysis and sensitivity to inhibitors. The single form in the copepod E. affinis is most
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
active against AcSChI and shows far less activity against PrSChI and BuSChI, as does the enzyme in the ectoparasitic copepod sealouse (Walday and Fonnum, 1989b), the intertidal copepod T. brevi´ 1999) and the deccornis (Forget and Bocquene, apod Maia verrucosa (Talesa et al., 1995). Moreover, this enzyme, with a Km in the range of 32 mM, is also inhibited by eserine and by an excess of AcSChI, as most AChEs are, and is insensitive to iso-OMPA, which has been identified as a specific BuChE inhibitor (Toutant, 1989; Sine et al., 1992). Although it is tempting to classify the single ChE found in E. affinis as true AChE, this classification has only been clearly established in vertebrate species in which the two enzymes are very closely related. Conversion of AChE into a BuChE-like enzyme has been performed by substitution of a single amino acid in the acyl pocket of the enzyme (Harel et al., 1992; Vellom et al., 1993). The classification of invertebrate ChEs is more ambiguous. In insects, only one ChE
89
Table 1 Kinetic characteristics of AChE from the copepod Eurytemora affinis. N.M. not measurable
AcSChI BuSChI PrSChI
Relative rate of hydrolysis
Km (mM)
Kss (mM)
100% 10% 9%
32 N.M. N.M.
14.8 N.M. N.M.
metabolizes both acetylcholine and butyrylcholine to various degrees (Toutant, 1989; Gnagey et al., 1987; Zhu and Clark, 1995). The isoelectric point (6.2) of purified E. affinis enzyme matched with what was observed for the ChE found in the bacteria Pseudomonas fluorescens (Rochu et al., 1998). Concerning phylogenetic relationships, ChEs have been found in all invertebrate phyla except for some species of Cnidaria (Scemes et al., 1982). Particularly, ChEs have been identified in members of the Crustacea wTigriopus brevicor-
Fig. 2. Isoelectrofocusing (IEF) gel. Lane M, markers from kit for IEF range 3.5–9.3. Lane C, acetylcholinesterase from E. affinis.
90
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
´ 1999), Squilla mantis nis (Forget and Bocquene, (Principato et al., 1988), Lepeophtheirus salmonis (Walday and Fonnum, 1989b), Maia verrucosa and Palinurus vulgaris (Talesa et al., 1992)x; Mollusca wMurex brandaris (Talesa et al., 1990), Crassostrea gigas (Bocquene´ et al., 1997)x; Sipunculida wSipunculus nudus (Talesa et al., 1993)x; Oligochaeta wAllobophora caliginosa (Principato et al., 1989)x; and Hirudinea wthe leech Hirudo medicinalis (Principato et al., 1981, 1983; Talesa et al., 1995)x. From a kinetic point of view, the single form of ChE from E. affinis exhibited Km for AcSChI in the range of 32 mM. Similar results have been reported using the same substrate for AChE from the crustacean T. brevicornis (20 mM), Squilla mantis (20 mM), the crab Maia verrucosa (44 mM), the lobster Palinurus vulgaris (33 mM), the leech (25 and 70 mM), the oyster (18 and 77.6 mM), the murex (78 mM) and class A and B from most nematodes (in the range of 10 mM). Comparative analysis of Km values of the AChE from the copepod E. affinis shows characteristics similar to those of other known ChE from both invertebrates and vertebrates. For instance, the ki values (Table 2) for carbofuran and eserine fit with inhibition constants for Tigriopus brevicornis (For´ 1999), Electrophorus electricus get and Bocquene, and Drosophila melanogaster AChE, respectively,
Table 2 Bimolecular reaction constants (ki, in My1 miny1) of Eurytemora affinis AChE to different inhibitors and comparison with those of another copepod Eurytemora affinis AChE Eserine Iso-OMPA Atrazine Carbofuran Nonylphenol 17 b oestradiol
1.7 8.3 3.6 2.0 7.4 6.2
106 101 103 105 103 103
Copepod AChEa 2.5 106 6.1 101 2.8 105
´ AChE from Tigriopus brevicornis (Forget and Bocquene, 1999). a
as reported by Villatte et al. (1998). Values of ki in E. affinis were compared with relevant data for invertebrates and especially for the AChE of the fly Drosophila melanogaster, which is considered to be among the most sensitive ChEs to organophosphate (Op) and carbamate (C) compounds and thus used as a biosensor (Villatte et al., 1998). Inhibition constants (ki) observed with atrazine, nonylphenol and 17b oestradiol are relevant for using Eurytemora AChE as a good indicator of environmental contamination. As AChE is considered to be an allosteric enzyme, numerous conformational changes can occur that could be
Fig. 3. Substrate affinities of purified AChE from Eurytemora affinis.
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
influenced by the binding of environmental contaminants other than Ops and C pesticides. In summary, our study shows that the copepod E. affinis contains a single form of AChE with an average molecular weight of 140 kDa, a pI of 6.2 and a high sensitivity to several inhibitors investigated. As the measurement of AChE inhibition in copepods can be used as a biomarker of the effects of neurotoxic contaminants wincluding organophosphate and carbamate insecticides but also complex mixtures of pollutants (Forget et al., 1998)x, the existence of only one form of cholinesterase and the wide distribution of E. affinis in estuaries favor the use of this copepod as a potential bioindicator of inhibitory effects in the aquatic environment. Acknowledgments This work is a part of the ‘Seine Aval’ interdisciplinary research program sponsored by the ´ Region Haute-Normandie and the Agence de l’Eau Seine Normandie. References Aldridge, W.N., Reiner, E., 1969. Acetylcholinesterase. Two types of inhibition by an organophosphorous compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147–162. ¨ Alfthan, K., Kenttamaa, H., Zukale, T., 1989. Characterization and semiquantitative estimation of organophosphorous compound based on inhibition of cholinesterases. Anal. Chim. Acta 217, 43–51. Austin, L., Berry, W.K., 1953. Two selective inhibitors of cholinesterase. Biochem. J. 54, 695–700. Baker, D.L., Molinoff, P.B., Kravitz, E.A., 1972. Octopamine in the lobster nervous system. Nature New Biol. 236, 61–63. ´ G., Roig, A., Fournier, D., 1997. Cholinesterases Bocquene, from the common oyster (Crassostrea gigas). Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors. FEBS Lett. 407, 261–266. Bradford, M., 1976. A rapid and sensitive assay of protein utilizing the principle of dye binding. Anal. Biochem. 772, 248–264. Braun, G., Mulloney, B., 1994. Acetylcholinesterase activity in neurons of crayfish abdominal ganglia. J. Comp. Neurol. 350, 272–280. Ellman, G.L., Courtney, K.O., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. ´ G., 1998. Joint action of Forget, J., Pavillon, J.F., Bocquene, pollutant combinations (pesticides and metals) on survival (LC50) values and acetylcholinesterase activity of Tigriopus
91
brevicornis (Copepoda, Harpacticoida). Environ. Toxicol. Chem. 40, 239–244. ´ G., 1999. Partial purification and enzyForget, J., Bocquene, matic characterization of acetylcholinesterase from the intertidal marine copepod Tigriopus brevicornis. Comp. Biochem. Physiol. B123, 345–350. ´ J.B., 1987. Fournier, D., Cuany, A., Bride, J.M., Berge, Molecular polymorphism of head acetylcholinesterase from adult houseflies (Musca domestica L). J. Neurochem. 49, 1455–1461. ´ G., 1990. In vitro inhibition of acetylGalgani, F., Bocquene, cholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxico. 45, 243–249. Gnagey, A.L., Forte, M., Rosenberry, T.J., 1987. Isolation and characterization of AchE from Drosophila. Biol. Chem. 262, 13290–13298. Harel, M., Sussman, J.L., Krejci, E., Bon, S., Chanal, P., ´ J., Silman, I., 1992. Conversion of acetylcholinMassoulie, esterase to butyrylcholinesterase, modeling and mutagenesis. Proc. Natl. Acad. Sci. USA 89, 10827–10831. Karnovsky, M.J., Roots, L.A., 1964. Direct coloring thiocholine method from cholinesterases. J. Histochem. Cytochem. 12, 219–222. Livingstone, D.R., 1993. Biotechnology and pollution monitoring: use of molecular biomarkers in the aquatic environment. J. Chem. Tech. Biotechnol. 57, 195–211. ´ J., Pezzementi, L., Bon, S., Kejci, E., Valette, F.M., Massoulie, 1993. Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91. Payne, J.F., Mathieu, A., Melvin, W., Fancey, L., 1996. Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Mar. Pollut. Bull. 32, 225–231. Principato, G.B., Ambrosini, M.V., Liotti, F.S., Giovannini, E., 1981. Propionylcholinesterase in Hirudo medicinalis: purification, partial characterization and comparative study with a mammalian acetylcholinesterase. Comp. Biochem. Physiol. C70, 209–213. Principato, G.B., Contenti, S., Talesa, V., Mangabiene, G., Pascolini, R., Rosi, G., 1989. Propionylcholinesterase from Allobophora caliginosa. Comp. Biochem. Physiol. C94, 23–27. Principato, G.B., Rosi, G., Biagioni, M., Giovannini, E., 1983. Kinetic studies on the acetylcholinesterase from Hirudo medicinalis. Comp. Biochem. Physiol. C75, 185–192. Principato, G.B., Talesa, V., Giovannini, E., Pascolini, R., Rosi, G., 1988. Characterization of the soluble cholinesterase from Squilla mantis. Comp. Biochem. Physiol. C90, 413–416. Radic, Z., Gibney, G., Taylor, P., 1991. Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives. Molec. Pharmacol. 39, 98–104. Rochu, D., Rothlisberger, C., Taupin, C., Renault, F., Gagnon, J., Masson, P., 1998. Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity. Biochim. Biophys. Acta 1385, 126–138. Scemes, E., Mendes, E.G., Chaimovich, H., 1982. Absence of cholinesterase activity in body wall homogenates from the ˆ Comp. Biochsea anemone Bunodosoma caissarum Correa. em. Physiol. C73, 415–418.
92
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
Silver, A., 1974. The Biology of Cholinesterases. Elsevier, Amsterdam. Sine, J.P., Toutant, J.P., Weigel, P., Colas, B., 1992. Amphiphilic forms of butyrylcholinesterase in mucosal cell of rat intestine. Biochemistry 31, 10893–10900. Talesa, V., Contenti, S., Mangiabene, C., Pascolini, R., Rosi, G., Principato, B., 1990. Propionylcholinesterase from Murex brandaris: comparison with other invertebrate cholinesterase. Comp. Biochem. Physiol. C96, 39–43. Talesa, V., Contenti, S., Principato, G.B., Pascolini, R., Giovannini, E., Rosi, G., 1992. Cholinesterases from Maia verrucosa and Palinurus vulgaris: a comparative study. Comp. Biochem. Physiol. C101, 499–503. Talesa, V., Grauso, M., Giovannini, E., Rosi, G., Toutant, J.P., 1995. Solubilization, molecular forms and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis). Biochem. J. 306, 687–692. Talesa, V., Principato, G.B., Giovannini, E., Di Giovannini, M.V., Rosi, G., 1993. Dimeric forms of cholinesterase in Sipunculus nudus. Eur. J. Biochem. 215, 267–275.
Toutant, J.P., 1989. Insect acetylcholinesterase: catalytic properties, tissue distribution and molecular forms. Prog. Neurobiol. 32, 423–446. Vellom, D.C., Radic, Z., Li, Y., Pickering, N.A., Camp, S., Taylor, P., 1993. Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry. 32, 12–17. Villatte, F., Marcel, V., Estrada-Mondac, S., Fournier, D., 1998. Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens. Electron. 13, 157–162. Walday, P., Fonnum, F., 1989a. A comparative pharmacological characterization of cholinesterases in salmon (Salmo salar) brain and sealice (Lepeophtheirus salmonis). Comp. Biochem. Physiol. C92, 197–199. Walday, P., Fonnum, F., 1989b. Cholinergic activity in different stages of sealice (Lepeophtheirus salmonis). Comp. Biochem. Physiol. C93, 143–147. Zhu, K.Y., Clark, J.M., 1995. Comparison of kinetic properties of acetylcholinesterase purified from azinphosmethyl-susceptible and resistant strains of Colorado potato beetle. Pest. Biochem. Physiol. 51, 57–67.
Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe) ¨ Forget*, Sandrine Livet, Francois Joelle Leboulenger ¸ Laboratoire d’Ecotoxicologie (LEMA), UPRES-EA3222, Faculte´ des Sciences et Techniques, Universite´ du Havre, 25 rue Philippe Lebon, BP 540, 76058 Le Havre cedex, France Received 2 December 2001; received in revised form 5 March 2002; accepted 8 March 2002
Abstract Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 8C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Acetylcholinesterase; Biochemical characterization; Isoelectric focusing electrophoresis; Biomarker; Invertebrates; Copepod; Eurytemora affinis; Pollutants
1. Introduction Intensive use of organophosphate and carbamate pesticides has caused various ecotoxicological problems. The primary toxicity of these substances is due to an irreversible inhibition of acetylcholinesterase (AChE), a key enzyme of the nervous system. Phosphorylation or carbamoylation of the serine residue at the active site of AChE blocks metabolization of the neurotransmitter acetylcholine. The postsynaptic membrane remains depolarized, and therefore, synaptic transmission is altered. Anticholinesterase agents such as organophosphate and carbamate insecticides are widely *Corresponding author. Tel.: q33-2-3274-4379; fax: q332-3274-4314. E-mail address: [email protected] (J. Forget).
used to control various agricultural pests (insects, acari and nematodes), but are potentially toxic for other animals. Individuals poisoned by these compounds experience partial cholinesterase inhibition, which can be lethal (Alfthan et al., 1989). However, the enzyme is also present in body regions where inhibition does not affect viability (Massoulie´ et al., 1993). For many years, cholinesterases (ChEs) have been considered as interesting biomarkers for the monitoring of environmental contamination by compounds such as organophosphate and carbamate insecticides, heavy metals and pulp waste (Livingstone, 1993; Payne et al., 1996). In the marine environment, bivalve mollusks such as the blue common mussel or the oyster have been the favorite target species for biomonitoring. However,
1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 0 5 0 - 9
86
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
mollusk AChE have proved less sensitive to inhibitors than those of crustacean or vertebrate species ´ 1990). Moreover, acetyl(Galgani and Bocquene, choline has been actually identified as a neurotransmitter in crustacean central nervous system (Baker et al., 1972; Braun and Mulloney, 1994), and AChE has been the only form of ChE found in the ectoparasitic copepod sealouse (Lepeophtheirus salmonis) (Walday and Fonnum, 1989a,b). Cholinesterases have been extensively studied because, they are highly polymorphic enzymes in most species, they play an important role in the transmission of nervous influx and they are the specific targets for most nerve agents and insecticides. The number of genes encoding ChEs varies according to species: one in insects, two in vertebrates and three in nematodes (Massoulie´ et al., 1993). In vertebrates, use of acetylcholine and butyrylcholine has allowed differentiation of the two proteins as AChE and butyrylcholinesterase (BuChE). Results from molecular biology indicate that AChE and BuChE have approximately 50% amino-acid identity within species (Massoulie´ et al., 1993; Silver, 1974). For each protein, various molecular forms originate either from alternative gene splicing or degradation. These forms may be either soluble or linked to the basal lamina via a collagen tail or to the membrane via a glycolipid or a hydrophobic peptide. They have been classified as asymmetrical, globular asymmetrical or collagen-tailed forms, existing in all vertebrate classes but apparently absent in invertebrates. Globular forms are encountered as monomers, dimers and tetramers. The two enzymes AChE and BuChE are distinguished functionally, primarily on the basis of substrate specificity (Radic et al., 1991). AChE hydrolyzes acetylcholine much faster than other choline esters (like propionylcholine) and is virtually inactive on butyrylcholine, whereas BuChE not only hydrolyzes butyrylcholine well but also acetylcholine at an appreciable rate. The two enzymes may also be distinguished by their susceptibility to diagnostic inhibitors such as isoOMPA for AChE (Austin and Berry, 1953). AChE also exhibits excess substrate inhibition, which may be mediated by a peripheral substrate binding site (Radic et al., 1991). Among marine crustaceans, ChEs have been characterized in Decapoda (Maia verrucosa, Palinurus vulgatus) (Talesa et al., 1990), and Stomatopoda (Squilla mantis)
(Principato et al., 1988), and Copepoda (Tigriopus ´ 1999). brevicornis) (Forget and Bocquene, To investigate the apparent sensitivity of the copepod, Eurytemora affinis ChE was purified by affinity chromatography and its specific kinetic behavior towards substrates and inhibitors was determined. 2. Materials and methods 2.1. Materials Specimens of the copepod Eurytemora affinis (E. affinis) were collected by plankton netting along the Seine estuary (France), near the Tancarville bridge and conveyed to the laboratory in plastic containers for immediate freezing at y80 8C. Acetylthiocholine (AcSChI), butyrylthiocholine (BuSChI), propionylthiocholine (PrSChI), eserine, iso-OMPA, nonylphenol, 17b-oestradiol, DTNB w5,59-dithio-bis(2-nitrobenzoic acid)x, decamethonium and EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2dihydroquinoline) were obtained from Sigma Aldrich, France. Carbofuran and atrazine were obtained from Cluzeau Info Labo, France. All chemicals used in this study were of analytical grade. 2.2. Extraction of cholinesterases from copepods Extraction was performed using 20 mM phosphate buffer, pH 7, plus 0.1% Triton X-100. E. affinis were homogenized in toto 1:5 (WyV) for 1 min on ice using an ultraturrax (type Diax 900). Extracts were then centrifuged at 10 000 g for 30 min, and this operation was repeated on the pellet after resuspension. The extract was then diluted 1:3 in the extraction buffer. 2.3. Purification procedure All purification steps were conducted at 4 8C. Affinity chromatography was performed on procainamide, a ligand specific for the choline-binding site. The diluted extract was directly applied to the affinity column. Procainamide-containing gel was prepared using ECH-Sepharose 4B (Pharmacia) as the coupling gel and EEDQ for ligand immobilization. The loading buffer was 20 mM phosphate buffer, pH 7, plus 0.1% Triton X-100, and the elution buffer was 20 mM phosphate buffer, pH
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
9.5, plus 0.1% Triton X-100 containing 20 mM decamethonium. Fractions exhibiting ChE activity were pooled and stored at y80 8C until use (Bocquene´ et al., 1997). 2.4. Non-denaturing electrophoresis Electrophoresis experiments were performed under non-denaturing conditions in a 2–23% acrylamide gradient gel containing 0.02% Triton X100. Samples were prepared as follows: to the protein solution was added an equal volume of glycerol (50%) and 2% bromophenol blue (wyv), and 20 ml of this mixture were then applied to each well of the gel. After equilibration of the gel, migration was carried out at 8 8C for approximately 16 h at a constant voltage (150 V). Cholinesterase bands were stained according to the procedure of Karnovsky and Roots (1964) based on hydrolysis of a thiocholine ester and formation of an insoluble copper complex. Apparent molecular weights were estimated with respect to the migration of high-molecular-weight standard proteins for non-denaturing electrophoresis gels (Pharmacia). 2.5. Isoelectric focusing electrophoresis Isoelectric point was estimated by isoelectric focusing under non-denaturing conditions using PhastSystem apparatus, Ampholine PAGplate IEF gel (Pharmacia) (pH range 3–9.5) and pI calibration kit as marker proteins from Pharmacia. After focusing, gels were stained with Coomassie blue for protein detection, or according to the Karnovsky and Roots method for esterase activity. 2.6. Activity, substrate and inhibitor specificities Cholinesterase activity was routinely monitored according to Ellman et al. (1961) using AcSChI at 1 mM as substrate in 20 mM phosphate buffer, pH 7. One unit corresponded to the hydrolysis of 1 nmole of substrate per min. Bradford’s method (Bradford, 1976) was used for quantitative determination of proteins with bovine serum albumin as standard. These methods were adapted for measurement with a microplate reader. Cholinesterase activities are expressed as units miny1, and specific activities are given as units mgy1 protein (one unit corresponding to the metabolization of 1 nmole of acetylcholine miny1).
87
The hydrolysis of three different substrates— AcSChI, BuSChI and PrSChI—was followed for each molecular form at a concentration of substrate ranging from 5 mM to 10 mM at 25 8C. The Michaelis constant Km was determined by analysis of the Lineweaver–Burk transformation. The sensitivity of ChE to inhibition by pollutants wisoOMPA (tetraisopropyl pyrophosphoramide) a specific inhibitor of human BuSCh, the two carbamates (eserine and carbofuran), atrazine, nonylphenol and 17b oestradiolx was investigated. Stock solutions of inhibitors were prepared in ethanol. The generally accepted inhibition mechanism of AChE by organophosphate and carbamate compounds has been described by Aldridge and Reiner (1969). The formula describing the inhibition mechanism is as follows:
where Esenzyme, CXscarbamate or organophosphate, Xsleaving group, Kdsky1yk1, k2sthe carbamoylation or phosphorylation rate constant, k3sthe decarbamoylation or dephosphorylation rate constant, and kisthe bimolecular rate constant (sk2yKd). For ki determination, enzymes were incubated for various times with different insecticide concentrations wIx, in conditions such as wIx always being at least ten-fold superior to wEx, in 20 mM phosphate buffer, pH 7, at 25 8C. The time-course variation of free enzyme wEx was estimated by the Ellman method w1961x. This variation follows a pseudo-first-order function, Ln wEx y wE0xsyki y wIx t, where t represents the time of incubation and I the inhibitor. All graphs obtained for each inhibitor were linear, whatever the variable (wIx or t), suggesting that reactivation (k3) remained negligible during the time of the experiment (20 min). 3. Results 3.1. Extraction, purification and separation All ChE activity from the copepod homogenates was extracted by 20 mM phosphate buffer, pH 7, 0.1% Triton X-100. The low ionic strength buffer did not release extra activity. When applied on a procainamide affinity column, most ChE activity
88
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
Fig. 1. Electropheretic analysis of cholinesterase from the copepod Eurytemora affinis. Non-denaturing electrophoresis was performed in gradient polyacrylamide gels in the presence of 0.1% Triton X100. Lane M, high-molecular-weight markers. Lane D, migration of the dimeric form of Drosophila melanogaster AChE. Lanes C, migration of the cholinesterase from copepod Eurytemora affinis.
remained bound to the column, with recovery ranging from 50–70% of original activity. 3.2. Characterization of the single form Electrophoresis of the purified extract performed in non-denaturing conditions on gradient gel in the presence of Triton X-100 showed one band migrating at 140 KDa (Fig. 1). When the detergent migrated in the gel, ChE migrated until it reached the gradient zone corresponding to its apparent molecular weight (Sine et al., 1992). This single band reached the gradient zone between the lactate dehydrogenase and the dimeric form of Drosophila melanogaster AChE (Fournier et al., 1987). The purified enzyme was also subjected to analytical isoelectric focusing in a pH gradient of 3 to 9.5. Results indicated that ChE was homogeneous with an apparent pI of 6.2 (Fig. 2). This single band reached the pI between the two markers bovine carbonic anhydrase B (pI: 5.85) and human carbonic anhydrase B (pI: 6.55). 3.3. Substrate specificity, inhibition and kinetic analysis of AChE activity The specificity of the enzyme toward AcSChI, BuSChI and PrSChI was determined with 1 mM of substrate (Table 1). E. affinis ChE was most active against AcSChI (100%), and almost equally much less active against BuSChI (10%) and PrSChI (9%).
This form displayed an apparent Michaelian behavior in the 5 mM to 5 mM range (Fig. 3) when AcSChI was used as substrate, with a Km of 32 mM (Table 1). The ChE of E. affinis appeared to be an AChE, i.e. it hydrolyzed AcSChI at a higher rate than other substrates and was inhibited by eserine but not by Iso-OMPA (Table 2). Inhibition experiments with several compounds tested showed a high sensitivity of the copepod ChE to pesticides, 17b oestradiol and Nonylphenol, with ki values in the order of 105 for carbofuran to 103 for atrazine, 17b oestradiol and Nonylphenol (Table 2). 4. Discussion Our results indicate the presence of a single ChE in the copepod E. affinis as previously shown in another copepod Tigriopus brevicornis (Forget ´ 1999) and in other crustaceans, and Bocquene, such as Squilla mantis (Principato et al., 1988), Maia verrucosa and Palinurus vulgaris (Talesa et al., 1995). However, the question still arises as to whether this enzyme is actually ChE or a nonspecific esterase, which also metabolizes acetylcholine. Cholinesterases have been described and classified by many authors into two groups, AChE or BuChE (pseudo-cholinesterase), depending on substrate hydrolysis and sensitivity to inhibitors. The single form in the copepod E. affinis is most
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
active against AcSChI and shows far less activity against PrSChI and BuSChI, as does the enzyme in the ectoparasitic copepod sealouse (Walday and Fonnum, 1989b), the intertidal copepod T. brevi´ 1999) and the deccornis (Forget and Bocquene, apod Maia verrucosa (Talesa et al., 1995). Moreover, this enzyme, with a Km in the range of 32 mM, is also inhibited by eserine and by an excess of AcSChI, as most AChEs are, and is insensitive to iso-OMPA, which has been identified as a specific BuChE inhibitor (Toutant, 1989; Sine et al., 1992). Although it is tempting to classify the single ChE found in E. affinis as true AChE, this classification has only been clearly established in vertebrate species in which the two enzymes are very closely related. Conversion of AChE into a BuChE-like enzyme has been performed by substitution of a single amino acid in the acyl pocket of the enzyme (Harel et al., 1992; Vellom et al., 1993). The classification of invertebrate ChEs is more ambiguous. In insects, only one ChE
89
Table 1 Kinetic characteristics of AChE from the copepod Eurytemora affinis. N.M. not measurable
AcSChI BuSChI PrSChI
Relative rate of hydrolysis
Km (mM)
Kss (mM)
100% 10% 9%
32 N.M. N.M.
14.8 N.M. N.M.
metabolizes both acetylcholine and butyrylcholine to various degrees (Toutant, 1989; Gnagey et al., 1987; Zhu and Clark, 1995). The isoelectric point (6.2) of purified E. affinis enzyme matched with what was observed for the ChE found in the bacteria Pseudomonas fluorescens (Rochu et al., 1998). Concerning phylogenetic relationships, ChEs have been found in all invertebrate phyla except for some species of Cnidaria (Scemes et al., 1982). Particularly, ChEs have been identified in members of the Crustacea wTigriopus brevicor-
Fig. 2. Isoelectrofocusing (IEF) gel. Lane M, markers from kit for IEF range 3.5–9.3. Lane C, acetylcholinesterase from E. affinis.
90
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
´ 1999), Squilla mantis nis (Forget and Bocquene, (Principato et al., 1988), Lepeophtheirus salmonis (Walday and Fonnum, 1989b), Maia verrucosa and Palinurus vulgaris (Talesa et al., 1992)x; Mollusca wMurex brandaris (Talesa et al., 1990), Crassostrea gigas (Bocquene´ et al., 1997)x; Sipunculida wSipunculus nudus (Talesa et al., 1993)x; Oligochaeta wAllobophora caliginosa (Principato et al., 1989)x; and Hirudinea wthe leech Hirudo medicinalis (Principato et al., 1981, 1983; Talesa et al., 1995)x. From a kinetic point of view, the single form of ChE from E. affinis exhibited Km for AcSChI in the range of 32 mM. Similar results have been reported using the same substrate for AChE from the crustacean T. brevicornis (20 mM), Squilla mantis (20 mM), the crab Maia verrucosa (44 mM), the lobster Palinurus vulgaris (33 mM), the leech (25 and 70 mM), the oyster (18 and 77.6 mM), the murex (78 mM) and class A and B from most nematodes (in the range of 10 mM). Comparative analysis of Km values of the AChE from the copepod E. affinis shows characteristics similar to those of other known ChE from both invertebrates and vertebrates. For instance, the ki values (Table 2) for carbofuran and eserine fit with inhibition constants for Tigriopus brevicornis (For´ 1999), Electrophorus electricus get and Bocquene, and Drosophila melanogaster AChE, respectively,
Table 2 Bimolecular reaction constants (ki, in My1 miny1) of Eurytemora affinis AChE to different inhibitors and comparison with those of another copepod Eurytemora affinis AChE Eserine Iso-OMPA Atrazine Carbofuran Nonylphenol 17 b oestradiol
1.7 8.3 3.6 2.0 7.4 6.2
106 101 103 105 103 103
Copepod AChEa 2.5 106 6.1 101 2.8 105
´ AChE from Tigriopus brevicornis (Forget and Bocquene, 1999). a
as reported by Villatte et al. (1998). Values of ki in E. affinis were compared with relevant data for invertebrates and especially for the AChE of the fly Drosophila melanogaster, which is considered to be among the most sensitive ChEs to organophosphate (Op) and carbamate (C) compounds and thus used as a biosensor (Villatte et al., 1998). Inhibition constants (ki) observed with atrazine, nonylphenol and 17b oestradiol are relevant for using Eurytemora AChE as a good indicator of environmental contamination. As AChE is considered to be an allosteric enzyme, numerous conformational changes can occur that could be
Fig. 3. Substrate affinities of purified AChE from Eurytemora affinis.
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
influenced by the binding of environmental contaminants other than Ops and C pesticides. In summary, our study shows that the copepod E. affinis contains a single form of AChE with an average molecular weight of 140 kDa, a pI of 6.2 and a high sensitivity to several inhibitors investigated. As the measurement of AChE inhibition in copepods can be used as a biomarker of the effects of neurotoxic contaminants wincluding organophosphate and carbamate insecticides but also complex mixtures of pollutants (Forget et al., 1998)x, the existence of only one form of cholinesterase and the wide distribution of E. affinis in estuaries favor the use of this copepod as a potential bioindicator of inhibitory effects in the aquatic environment. Acknowledgments This work is a part of the ‘Seine Aval’ interdisciplinary research program sponsored by the ´ Region Haute-Normandie and the Agence de l’Eau Seine Normandie. References Aldridge, W.N., Reiner, E., 1969. Acetylcholinesterase. Two types of inhibition by an organophosphorous compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147–162. ¨ Alfthan, K., Kenttamaa, H., Zukale, T., 1989. Characterization and semiquantitative estimation of organophosphorous compound based on inhibition of cholinesterases. Anal. Chim. Acta 217, 43–51. Austin, L., Berry, W.K., 1953. Two selective inhibitors of cholinesterase. Biochem. J. 54, 695–700. Baker, D.L., Molinoff, P.B., Kravitz, E.A., 1972. Octopamine in the lobster nervous system. Nature New Biol. 236, 61–63. ´ G., Roig, A., Fournier, D., 1997. Cholinesterases Bocquene, from the common oyster (Crassostrea gigas). Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors. FEBS Lett. 407, 261–266. Bradford, M., 1976. A rapid and sensitive assay of protein utilizing the principle of dye binding. Anal. Biochem. 772, 248–264. Braun, G., Mulloney, B., 1994. Acetylcholinesterase activity in neurons of crayfish abdominal ganglia. J. Comp. Neurol. 350, 272–280. Ellman, G.L., Courtney, K.O., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. ´ G., 1998. Joint action of Forget, J., Pavillon, J.F., Bocquene, pollutant combinations (pesticides and metals) on survival (LC50) values and acetylcholinesterase activity of Tigriopus
91
brevicornis (Copepoda, Harpacticoida). Environ. Toxicol. Chem. 40, 239–244. ´ G., 1999. Partial purification and enzyForget, J., Bocquene, matic characterization of acetylcholinesterase from the intertidal marine copepod Tigriopus brevicornis. Comp. Biochem. Physiol. B123, 345–350. ´ J.B., 1987. Fournier, D., Cuany, A., Bride, J.M., Berge, Molecular polymorphism of head acetylcholinesterase from adult houseflies (Musca domestica L). J. Neurochem. 49, 1455–1461. ´ G., 1990. In vitro inhibition of acetylGalgani, F., Bocquene, cholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxico. 45, 243–249. Gnagey, A.L., Forte, M., Rosenberry, T.J., 1987. Isolation and characterization of AchE from Drosophila. Biol. Chem. 262, 13290–13298. Harel, M., Sussman, J.L., Krejci, E., Bon, S., Chanal, P., ´ J., Silman, I., 1992. Conversion of acetylcholinMassoulie, esterase to butyrylcholinesterase, modeling and mutagenesis. Proc. Natl. Acad. Sci. USA 89, 10827–10831. Karnovsky, M.J., Roots, L.A., 1964. Direct coloring thiocholine method from cholinesterases. J. Histochem. Cytochem. 12, 219–222. Livingstone, D.R., 1993. Biotechnology and pollution monitoring: use of molecular biomarkers in the aquatic environment. J. Chem. Tech. Biotechnol. 57, 195–211. ´ J., Pezzementi, L., Bon, S., Kejci, E., Valette, F.M., Massoulie, 1993. Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91. Payne, J.F., Mathieu, A., Melvin, W., Fancey, L., 1996. Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Mar. Pollut. Bull. 32, 225–231. Principato, G.B., Ambrosini, M.V., Liotti, F.S., Giovannini, E., 1981. Propionylcholinesterase in Hirudo medicinalis: purification, partial characterization and comparative study with a mammalian acetylcholinesterase. Comp. Biochem. Physiol. C70, 209–213. Principato, G.B., Contenti, S., Talesa, V., Mangabiene, G., Pascolini, R., Rosi, G., 1989. Propionylcholinesterase from Allobophora caliginosa. Comp. Biochem. Physiol. C94, 23–27. Principato, G.B., Rosi, G., Biagioni, M., Giovannini, E., 1983. Kinetic studies on the acetylcholinesterase from Hirudo medicinalis. Comp. Biochem. Physiol. C75, 185–192. Principato, G.B., Talesa, V., Giovannini, E., Pascolini, R., Rosi, G., 1988. Characterization of the soluble cholinesterase from Squilla mantis. Comp. Biochem. Physiol. C90, 413–416. Radic, Z., Gibney, G., Taylor, P., 1991. Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives. Molec. Pharmacol. 39, 98–104. Rochu, D., Rothlisberger, C., Taupin, C., Renault, F., Gagnon, J., Masson, P., 1998. Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity. Biochim. Biophys. Acta 1385, 126–138. Scemes, E., Mendes, E.G., Chaimovich, H., 1982. Absence of cholinesterase activity in body wall homogenates from the ˆ Comp. Biochsea anemone Bunodosoma caissarum Correa. em. Physiol. C73, 415–418.
92
J. Forget et al. / Comparative Biochemistry and Physiology Part C 132 (2002) 85–92
Silver, A., 1974. The Biology of Cholinesterases. Elsevier, Amsterdam. Sine, J.P., Toutant, J.P., Weigel, P., Colas, B., 1992. Amphiphilic forms of butyrylcholinesterase in mucosal cell of rat intestine. Biochemistry 31, 10893–10900. Talesa, V., Contenti, S., Mangiabene, C., Pascolini, R., Rosi, G., Principato, B., 1990. Propionylcholinesterase from Murex brandaris: comparison with other invertebrate cholinesterase. Comp. Biochem. Physiol. C96, 39–43. Talesa, V., Contenti, S., Principato, G.B., Pascolini, R., Giovannini, E., Rosi, G., 1992. Cholinesterases from Maia verrucosa and Palinurus vulgaris: a comparative study. Comp. Biochem. Physiol. C101, 499–503. Talesa, V., Grauso, M., Giovannini, E., Rosi, G., Toutant, J.P., 1995. Solubilization, molecular forms and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis). Biochem. J. 306, 687–692. Talesa, V., Principato, G.B., Giovannini, E., Di Giovannini, M.V., Rosi, G., 1993. Dimeric forms of cholinesterase in Sipunculus nudus. Eur. J. Biochem. 215, 267–275.
Toutant, J.P., 1989. Insect acetylcholinesterase: catalytic properties, tissue distribution and molecular forms. Prog. Neurobiol. 32, 423–446. Vellom, D.C., Radic, Z., Li, Y., Pickering, N.A., Camp, S., Taylor, P., 1993. Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry. 32, 12–17. Villatte, F., Marcel, V., Estrada-Mondac, S., Fournier, D., 1998. Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides. Biosens. Electron. 13, 157–162. Walday, P., Fonnum, F., 1989a. A comparative pharmacological characterization of cholinesterases in salmon (Salmo salar) brain and sealice (Lepeophtheirus salmonis). Comp. Biochem. Physiol. C92, 197–199. Walday, P., Fonnum, F., 1989b. Cholinergic activity in different stages of sealice (Lepeophtheirus salmonis). Comp. Biochem. Physiol. C93, 143–147. Zhu, K.Y., Clark, J.M., 1995. Comparison of kinetic properties of acetylcholinesterase purified from azinphosmethyl-susceptible and resistant strains of Colorado potato beetle. Pest. Biochem. Physiol. 51, 57–67.