Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelecypoda: Filibranchia) from the northern Adriatic sea

Chemico-Biological Interactions 134 (2001) 151– 166

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Soluble and membrane-bound acetylcholinesterases in Mytilus gallopro6incialis (Pelecypoda: Filibranchia) from the northern Adriatic sea Vincenzo Talesa, Rita Romani, Cinzia Antognelli, Elvio Giovannini, Gabriella Rosi * Department of Experimental Medicine, Di6ision of Cellular and Molecular Biology, Uni6ersity of Perugia, Via del Giochetto, 06122 Perugia, Italy Received 28 July 2000; received in revised form 9 December 2000; accepted 11 December 2000

Abstract Three forms of acetylcholinesterase (AChE) were detected in samples of the bivalve mollusc Mytilus gallopro6incialis collected in sites of the Adriatic sea. Apart from the origin of the mussels, two spontaneously soluble (SS) AChE occur in the hemolymph and represent about 80% of total activity, perhaps hydrolyzing metabolism-borne choline esters. These hydrophilic enzymes (forms A and B) copurified by affinity chromatography (procainamideSepharose gel) and were separated by sucrose gradient centrifugation. They are, respectively, a globular tetramer (11.0–12.0 S) and a dimer (6.0– 7.0 S) of catalytic subunits. The third form, also purified from tissue extracts by the same affinity matrix, proved to be an amphiphilic globular dimer (7.0 S) with a phosphatidylinositol tail giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. Such an AChE is likely functional in cholinergic synapses. All three AChE forms show a good substrate specificity and are inactive on butyrylthiocholine. Studies with inhibitors showed low inhibition by eserine and paraoxon, especially on SS forms, high sensitivity to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51) and no inhibition with propoxur and diisopropylfluorophosphate (DFP). The ChE forms in M. gallopro6incialis are possibly encoded by different genes. Some kinetic features of these enzymes suggest a genetic 

The first and second authors contributed equally to this study. * Corresponding author. Tel.: + 39-75-5857484/7480; fax: + 39-75-5857491. E-mail address: [email protected] (G. Rosi).

0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 1 ) 0 0 1 5 2 - 1

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polymorphism. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mytilus; Acetylcholinesterase; Carbamates; Organophosphates; Molecular polymorphism

1. Introduction Cholinesterases are a ubiquitous class of serine hydrolases that hydrolyze choline esters with various efficiency. In Vertebrata, two forms of cholinesterase occur, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3 1.1.8), encoded by two distinct genes; they differ in substrate specificity and affinity for selective inhibitors [1,2]. AChEs are involved in the cholinergic neurotransmission, providing for rapid elimination of acetylcholine in the cholinergic synapse. They are found in nervous tissues and muscles and display a complex molecular polymorphism. In particular, asymmetric forms (A) are high-Mr oligomers including one (A4), two (A8) or three (A12) catalytic tetramers attached, via an elongated collagenous tail, to the extracellular matrix (basal lamina). Globular forms, monomeric (G1), dimeric (G2) or tetrameric (G4), may be amphiphilic or hydrophilic; the former ones interact with non-denaturing detergents and are linked to membrane phospholipid bilayers through a hydrophobic domain [3,4]. AChEs in Vertebrata show two main types of catalytic subunits encoded by distinct mRNAs, resulting from alternative splicing of two exons, H and T, in 3% terminal region of a single primitive transcript. BChEs in Vertebrata are mainly localized in the serum as soluble tetrameric proteins and are also found in several tissues [5,6]. Their function is not as yet well-understood [2]. As regards Invertebrata, asymmetric forms of AChE have been detected in Cephalochordata [7], while only globular forms occur in all the other phyla. In particular, a dimeric AChE anchored to the cell membrane by phosphatidylinositol (PtdIns) is mostly present in the species so far studied [2,8 –12]. Such a G2 AChE seems to play a key role in cholinergic transmission. Moreover, in Invertebrata the encoding of AChE subunits can be done by one or several genes (four in Caenorhabditis)6 , thus giving a complex polymorphism of the enzyme [13]. Our previous reports on cholinesterases from Invertebrata [9,14,15] often pointed out a less-defined substrate specificity when compared with the AChEs from vertebrate nervous system and a wide variability in the kinetic behavior. The research has now been addressed to Bivalvia, a class of Mollusca showing a well-developed organ system and a worldwide distribution, mostly in marine habitats. Moreover, although the existence of cholinergic neurotransmission in both the motor and sensory systems of these organisms is well-known [16,17], an extensive characterization of their AChE is so far lacking. The present study was focused on detecting and characterizing distinct AChE forms in Mytilus gallopro6incialis that might be recovered from the hemolymph and from the other tissues by sequential extraction. The M. gallopro6incialis species is regularly cultured for commercial production in shallow waters along the Italian coasts of the Adriatic sea.

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2. Materials and methods

2.1. Materials Live specimens of M. gallopro6incialis each weighing about 20 g and belonging to the same growth level, were collected during April in three different sites along the coast of the northern Adriatic sea near the Italian towns of Porto Garibaldi (station 604), Ravenna (station 608) and Cesenatico (station 614). Such sites are placed in a north – south succession and spaced about 30 km from one another. Moreover, the mussels collected at each marine station were from one particular culture at that site. The animals thus gathered were quickly transferred to the laboratory in dry ice and stored at − 80°C until subsequent use. Acetyl- (ATC), propionyl- (PTC), butyryl-thiocholine (BTC) used as substrates for ChE activity measurements, eserine sulfate, procainamide, 1,5-bis (4-allyldimethylammoniumphenyl)pentan-3-one-dibromide (BW284c51), diisopropylfluorophosphate (DFP), diethyl-p-nitrophenylphosphate (paraoxon) and propoxur (pestanal), used as ChE inhibitors, bacitracin and aprotinin (protease inhibitors), Escherichia coli alkaline phosphatase as a marker protein, Sephadex G-50, were purchased from Sigma-Aldrich S.r.l. (Milano, Italy). 5,5%-dithiobis(-2-nitrobenzoic acid) (DTNB) was from Merck (Darmstadt, RFG). Procainamide-containing affinity resin for ChE purification was prepared according to Ralston et al. [18] using 1-cyclohexyl-3-(2-morpholynil-4-ethyl) carbodiimide metho-p-toluene sulfonate (Sigma-Aldrich) for ligand coupling to epoxy-activated Sepharose. Electrophoresis purity reagents were from Bio-Rad (Melville, NY, USA). BenchMark Protein Ladder, a protein Mr standard for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), was supplied by Life Technologies Italia S.r.l. (San Giuliano Milanese, Italy). Bacterial Ptdlns-specific phospholipase C (B. cereus) and b-galactosidase (E. coli ) were bought from Boehringer (Mannheim, FRG). All other reagents were analytical grade products from various sources and all solutions were made using twice distilled water.

2.2. Composition of buffers used for extractions, purification procedure and sedimentation analysis Low salt (LS) buffer contained 20 mM Tris –HCl, pH 7.4, 1 mM ethylene diamine tetraacetic acid (EDTA), 5 mM MgCl2, 0.1 mg/ml bacitracin and 8× 10 − 3 TIU/ml aprotinin to minimize proteolysis. Low-salt-Triton (LST) and high-salt (HS) buffers contained LS buffer supplemented as above plus 1% Triton X-100 or 1.0 M NaCl, respectively. High-salt-Triton (HST) and high-salt-Brij (HSB) buffer contained HS buffer plus 1% Triton X-100 or 0.5% Brij 96 (10-oleyl ether), respectively.

2.3. Extraction of ChE Extraction of ChE was performed at 5°C. Three groups of 20 mussels, each

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collected in the above said stations (604, 608 and 614), were thawed and their tissues pulled out from the shell in pieces. This operation released about 1 ml of hemolymph per animal; this reddish fluid, showing a valuable ChE activity (spontaneously soluble, SS ChE), was pooled within each group of animals and, after adding bacitracin (0.1 mg/ml) and aprotinin (8× 10 − 3 TIU/ml), stored at − 80°C. Pooled body tissues of each group of mussels (about 80 g) were minced into small pieces and, after adding 5 vol. of LS buffer, homogenized in an Ultraturrax blender for 4 min. The resulting homogenate was centrifuged at 100 000× g for 1 h. The supernatant (low-salt soluble, LSS extract), showing only traces of ChE activity, was discarded. The recovered pellet underwent three consecutive cycles of homogenization (1/5 mass/vol.; LST, HS and HST buffer) and centrifugation giving detergent-soluble (DS), high-salt soluble (HSS) and high-salt-detergent soluble (HSD) extracts, respectively. DS extract resulted to contain the bulk of ChE activity in the body tissues and was used, together with hemolymph, as starting material for enzyme purification. The HSS and HDS fractions, showing a poor ChE activity, were discarded.

2.4. Purification of ChE Operating at 5°C, the pooled hemolymph recovered from each group of mussels was filtered (1 ml/min) on a Sephadex G-50 column (2.5× 80 cm) equilibrated with LST buffer. In this way, a partial removal of medium –low Mr compounds as well as the sample equilibration were achieved, while ChE was quickly collected in the void volume. The active material from hemolymph and DS extract from the same animals underwent affinity chromatography on a procainamide-Sepharose gel, carried out at 5°C. The column (1.5× 2 cm, equilibrated with LST buffer) was washed with the same buffer containing 50 mM NaCl up to A280 = 0. Bound ChE was eluted (0.25 ml/min) by LST buffer plus 50 mM NaCl and 50 mM procainamide; 2 ml fractions were collected. Procainamide was removed from the pooled active fractions by dialysis against LST buffer. Electrophoresis with enzymatic staining showed two ChE forms and a single one in the purified materials from hemolymph or body tissues of Mytilus, respectively. Such results were consistently obtained performing the purification procedure with animals from stations 604, 608 and 614. Small amounts of distinct ChE forms present in the hemolymph were separately recovered for analytical purposes by centrifugation on a sucrose density gradient (5 – 20%) in HST buffer, as detailed below. They were indicated as A and B based on the decreasing value of the sedimentation coefficient. All the active materials obtained by the above procedure were stored at − 80°C for subsequent use. This purification procedure was repeated to obtain the amount of enzyme for the various analytical procedures.

2.5. Assay methods Protein concentrations were measured according to Bradford [19], using bovine serum albumin as a standard.

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ChE activity in the various crude or purified preparations from hemolymph or body tissues of Mytilus specimens collected in the different marine stations was evaluated at 20°C according to a modification of the spectrophotometric method described earlier by Ellman et al. [20], using thiocholine esters (ATC, PTC and BTC) as substrates (1 mM final concentration). Details of such a procedure were given in previous papers [9,21]. One enzyme unit (IU) was defined as the amount of enzyme, which catalyzes the hydrolysis of 1 mmol of substrate per min at saturating substrate concentration. Inhibition studies of purified ChE forms were carried out in the presence of ATC as substrate and using well-known inhibitors of ChEs, previously employed to study these enzymes: eserine [1,10], BW284c51 [1,22], DFP [10,23], paraoxon [1,24] and propoxur [25]. Inhibitor concentrations in the 10 − 2 – 10 − 9 M range were used in the assay and residual ChE activities were determined. In particular, the reaction was started after a 1 min extract-inhibitor incubation by adding the substrate solution.

2.6. Non-denaturing and SDS-electrophoresis Non-denaturing PAGE of hemolymph and DS extract, as well as of purified materials after affinity chromatography (SS and DS ChE, respectively), was performed running 20 ml samples in a vertical 8× 7.3× 0.1 cm slab-gel apparatus (20 mA current, 0.025 M Tris/0.192 M glycine running buffer, pH 8.3, 5°C). In particular, hemolymph and purified SS ChE were run on a gradient gel (4–7% acrylamide, 0.2% bis-acrylamide), while with DS extract and purified DS ChE a homogeneous gel (7% acrylamide, 0.2% bis-acrylamide) was used, also adding 0.5% Triton X-100. Staining for ChE activity was achieved according to Karnovsky and Roots [26] using 3 mM ATC as substrate. SDS-PAGE in reducing conditions (5 mM dithiothreitol) for the various purified ChE forms with subsequent silver staining was carried out as detailed in a previous paper [9] to evaluate the purification degree. In particular, the BenchMark Protein Ladder was used as a standard for evaluation of Mr values of the enzyme subunits in the 10 000 –220 000 range

2.7. Density gradient centrifugation Samples (250 mm) of crude starting material (hemolymph or DS extract) and of purified fractions from either hemolymph (SS ChE) or DS extract (DS ChE) after affinity chromatography were layered on to 5–20% sucrose density gradients (10 ml in polyallomer tubes) in HS, HST or HSB buffer. In the last case, samples were incubated first with 0.5% Brij 96 for 30 min. E. coli alkaline phosphatase (6.1 S) and b-galactosidase (16.0 S) were included as internal standards for calculation of ChE sedimentation coefficients (S). Centrifugations were performed at 5°C in a Beckman L60 ultracentrifuge equipped with a SW 41 Ti rotor at 36 000 rpm (222 000× g). The gradients were emptied from the bottom and 40 fractions of 250 ml each

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collected. ChE activity was measured on the basis of the A412 change through 30 min in the usual assay mixture containing 1 mM ATC and 0.05 ml of each fraction.

2.8. Treatment of DS ChE with phospholipase C and sedimentation analysis The possible presence in DS ChE of a Ptdlns anchor which binds the enzyme to the cell membrane was studied by digestion with Ptdlns-specific phospholipase C, carried out as detailed in some of our previous reports [9–12], and also by subsequent sedimentation analysis. This procedure was performed, as described above (Section 2.7) by using a detergent-devoid buffer (HS) and also running a control of undigested sample.

3. Results

3.1. Extraction and purification of ChE Based on several extraction experiments carried out separately using specimens of M. gallopro6incialis from distinct marine sites (stations 604, 608 and 614), the bulk of ChE activity (about 80%) of these animals lies in the hemolymph as SS ChE. A sequential extraction from the other tissues gave the remaining 20% activity essentially as DS ChE. Such a distribution of the ChE activity can also be deduced from the total activity values reported in Table 2 for enzyme purification. As regards hemolymph, the ChE specific activity levels do not show significant differences in animals from the various marine stations. On the contrary, ChE specific activities of DS extracts significantly differ from one site to another with the highest and the lowest values in animals from stations 608 and 604, respectively (Table 1).

Table 1 ChE activity in the hemolymph (SS ChE) and in DS extract of total body tissues (DS ChE) from specimens of M. gallopro6incialis collected at different sites of the Adriatic sea (stations 604, 608 and 614)a ChE form

Marine station

ChE activity (mIU/mg)

SS

604 608 614 604 608 614

175 9 28 220 9 44 250 9 38 140 9 27 800 9 176 390 979

DS

Comparison 604–608–614

NS

PB0.01

a ChE activity was measured using ATC as substrate. Single values are given as mean 9 S.D. of four experiments. Statistical comparisons were made by variance analysis. NS, not significant.

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The brief purification of ChE from both hemolymph and DS extract, carried out by affinity chromatography on a procainamide-containing matrix, gave for either starting materials a 12 – 35% yield and a purification of 74–111-fold (Table 2). As regards hemolymph, affinity matrix consistently bound two enzyme forms subsequently separated by density gradient centrifugation. The fairly low recoveries of enzyme with the affinity column are not far different from those previously obtained by us purifying in the same way ChEs from other Invertebrata as Mollusca [9] and Annelida [27]. On the other hand, the ChE forms thus recovered are representative of the entire set of such enzymes, since purified fractions contain the same ChE forms present in the crude starting materials, as shown by electrophoresis as well as sedimentation analysis.

3.2. Non-denaturing and SDS-electrophoresis Electrophoresis in non-denaturing conditions and staining for ChE activity, carried out in duplicate with purified fractions (Fig. 1), displayed two enzyme forms in the hemolymph and a single one in the DS extract from mussels collected in the various marine sites. Parallel experiments using crude materials gave quite similar results as for the number of ChE activity bands. Such results fully agree with those of sedimentation analysis (see below). SDS-slab gel electrophoresis (Fig. 2), in duplicate as well, of purified ChEs from either hemolymph or DS extract, gave, under reducing conditions, single protein bands showing for these presumed monomers the common Mr value of 68 000. A good purification degree was achieved, since only some faint residual bands of proteins are detectable.

3.3. Density gradient centrifugation Four experiments were carried out for each of the crude or purified enzyme preparations from specimens of M. gallopro6incialis collected in the various marine sites. Fig. 2 reports the sedimentation profiles of purified SS and DS ChE, as result from the mean values of activity found in each fraction. The sedimentation pattern of SS ChE in animals from the various stations shows two distinct activity peaks (forms A and B) at about 11.0 – 12.0 and 6.0 –7.0 S position, respectively, either in a detergent-free gradient or in the presence of Triton X-100 or Brij 96. Relative amounts of forms A and B, as estimated from the areas of the activity peaks in the sedimentation profiles, turned out to be about 60 and 40%. Sedimentation analysis of DS ChE from animals of the various origins in a detergent-free gradient yielded a diffuse aggregate, fully resolved by addition of Triton X-100 or Brij 96, with single activity peaks at 7.0 – 7.9 and 5.0 –5.5 S, respectively. Density gradient centrifugation of crude starting materials (hemolymph and DS extract) gave quite similar sedimentation patterns of ChE activity (results not shown).

a

614

608

604

614

660 1.25 474 0.99 640 1.03 152 0.25 30.7 0.14 108 0.26

Total protein (mg)

110 16.5 103 21.6 152 18.2 19 3.6 23 8 40 8.8

Total activity (IU)

The enzyme activity was evaluated using ATC as substrate.

Hemolymph Aff. chrom. Hemolymph Aff. chrom. Hemolymph Aff. chrom. DS extract Aff. chrom. DS extract Aff. chrom. DS extract Aff. chrom.

604 608

Purification step

Marine station

0.17 13.20 0.22 21.82 0.24 17.67 0.13 14.40 0.75 57.14 0.37 33.85

Specific activity (IU/mg)

100 15 100 21 100 12 100 19 100 35 100 22

Recovery (%)

1 78 1 99 1 74 1 111 1 76 1 91

Purification factor

Table 2 Purification procedure of the ChE from hemolymph (SS ChE) or DS extract of total body tissues (DS ChE) from specimens of M. gallopro6incialis collected in different sites of the Adriatic sea (stations 604, 608 and 614)a

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Fig. 1. Non-denaturing PAGE carried out prior to (I) or after (II) purification of SS ChE (A) and DS ChE (B) from specimens of M. gallopro6incialis collected in distinct sites of Adriatic sea (stations 604, 608 and 614). Samples of crude starting material (hemolymph, A; I or DS extract, B, I) and purified SS or DS ChE after affinity chromatography (A, II or B, II, respectively) were run as described in the text. Staining for ChE activity was performed according to Karnovsky and Roots [26].

3.4. Treatment of DS ChE with phospholipase C and sedimentation analysis Sedimentation analysis (four experiments) in a sucrose density gradient with HS buffer of purified DS ChE of mussels from the various marine stations gave, after

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phospholipase C treatment, single activity peaks at 8.0 S position. Undigested controls showed, as expected, a diffuse aggregation of the enzyme (Fig. 3, squares DS).

3.5. Pharmacological properties of SS and DS ChE Table 3 gives specific activity values of the various ChE forms in M. gallopro6incialis specimens from different marine sites, after purification and, as to SS enzyme, after further separation of A and B forms that copurify in the affinity gel. Activity was determined using different substrates. ATC seems to be the best one for all ChE forms, that also show a good, although lower and varied, activity level with PTC. In contrast, BTC is not hydrolyzed at all by the majority of the ChEs studied and only SS enzymes of the animals from station 604 show traces of activity. The ratios ATC activity/PTC activity for SS ChE forms A and B of each station are always very close, but different than the ratio of fraction DS. Moreover, these ratios, either concerning SS ChEs A and B or DS ChE, are different from one station to another. As for experiments with ChE inhibitors, ATC hydrolyzing ability of ChE SS forms A and B of animals from the various marine sites was completely inhibited by 10 − 4 M BW284c51, while all these enzymes still showed a valuable activity in the presence of 10 − 2 M eserine. DS ChEs were totally inhibited by 10 − 4 –10 − 5 M

Fig. 2. SDS-PAGE analysis of purified SS and DS ChE from specimens of M. gallopro6incialis collected in distinct sites of Adriatic sea (stations 604, 608 and 614), carried out using 5 mM dithiothreitol as reducing agent. Protein bands were revealed by silver staining. The mobility of four standard proteins included in the BenchMark Protein Ladder run in parallel are indicated with their Mr values.

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Fig. 3. Sedimentation pattern in sucrose density gradient of purified SS ChE and DS ChE from M. gallopro6incialis collected in distinct sites of Adriatic sea (stations 604, 608 and 614). Gradients (5 – 20%) were made in HS (), HST ( ) or HSB ( ) buffer, as well as in HS buffer for sedimentation of DS ChE prior to or after digestion with Ptdlns-specific phospholipase C (). ChE activity was measured using ATC as substrate. The arrowheads indicate the positions of E. coli b-galactosidase (16 S) and alkaline phosphatase (6.1 S) used as internal standards.

BW284c51 and 10 − 2 M eserine. Paraoxon was ineffective on SS ChEs, while gave a total inhibition of DS ChEs at about 10 − 3 M concentration. Propoxur and DFP, at the used concentrations, proved to be in any case ineffective on both SS and DS ChEs. Table 4 reports the values of IC50 regarding all the ChE forms studied with the above-mentioned inhibitors.

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Table 3 Specific activity of ChEs purified from hemolymph (SS ChEs A and B) or DS extract of total body tissues (DS ChE) from specimens of M. gallopro6incialis, collected in different sites of the Adriatic sea (stations 604, 608 and 614)a Marine station

604

608

614

ChE from

SS A B DS SS A B DS SS A B DS

ChE activity (mIU/mg)

ATC/PTC activity ratio

ATC

%

PTC

%

BTC

%

11095 80 94 11509 63 11595 13098 38009170 330917 280913 20509120

100 100 100 100 100 100 100 100 100

90 95 62 93 598 934 38 93 46 92 3230 9224 175 910 140 9 11 1107 941

82 77 52 33 35 85 53 50 54

39 1 290.5 ND ND ND ND ND ND ND

3 2

1.22 1.29 1.92 3.03 2.83 1.18 1.89 2.00 1.85

a

ChE activity was measured using ATC, PTC and BTC as substrates. Activity values are given as mean9 S.D. of four experiments. ND, not detectable.

4. Discussion The present report points out the existence of a polymorphism of ChE in M. gallopro6incialis with three forms mainly differing as to amount, tissue localization and molecular size. About 80% of ChE activity (SS form) lies in the hemolymph; such a condition shows marked analogies with those reported for the annelid Hirudo medicinalis [10], as well as the gastropod molluscs Murex brandaris [9] or Table 4 IC50 values for inhibitors (eserine, propoxur, BW284c51, paraoxon, DFP) of ChEs purified from hemolymph (SS ChEs A and B) or DS extract of total body tissues (DS ChE) in specimens of M. gallopro6incialis collected in different sites of Adriatic sea (stations 604, 608 and 614)a Marine station

604

608

614

ChE form

SS A B DS SS A B DS SS A B DS

IC50 (M) Eserine

Propoxur

BW284c51

Paraoxon

DFP

ND ND 10−4 10−3 10−3 10−4 ND ND 10−4

ND ND ND ND ND ND ND ND ND

10−7 10−7 10−7 10−7 10−7 10−7 0.5×10−8 0.5×10−8 10−8

ND ND 0.5×10−4 ND ND 0.5×10−4 ND ND 0.5×10−4

ND ND ND ND ND ND ND ND ND

a ChE activity was evaluated using ATC as substrate. Inhibitor concentration was in the 10−2–10−9 M range. Each value is the mean of two determinations. ND, not determinable.

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Helix pomatia [28]. The results of electrophoresis and density gradient centrifugation experiments show the presence of two molecular forms of ChE in the hemolymph: based on the sedimentation coefficients, the prevailing form A (about 60%) is presumably a tetramer (11.0 –12.0 S) of catalytic subunits, while form B seems to be a dimer (6.0 – 7.0 S). Such sedimentation features are close to those of several dimeric and tetrameric ChEs from Vertebrata [2,22] or Invertebrata [8,9,11,29]. Moreover, according to sedimentation analysis with or without detergents, these enzymes prove to be hydrophilic proteins devoid of detergent interaction or self-aggregation. Both the forms A and B of SS ChE consist of subunits showing an Mr value (68 000), close to those of several ChEs from Invertebrata [9,11,28]. The presence of tetrameric besides dimeric ChEs has been also observed in Mollusca of the Gastropoda [9] and Cephalopoda [29] classes. Among Invertebrata of a more ancient origin tetrameric forms of ChE have been so far detected only in Nematoda [23,30]. Based on analogies with vertebrate enzymes, ChE forms na A and B of M. gallopro6incialis might be classified as a Gna 4 tetramer and a G2 dimer, respectively [31]. The DS form present in mussels collected in the various marine sites is an amphiphilic protein, as evidenced by solubilization behavior and results of sedimentation analysis. In fact, it shows a diffuse self-aggregation, which can be avoided by addition of Triton X-100 or Brij 96 to the gradient. These features are probably due to phosphatidylinositol in the DS ChE molecule, since digestion by specific phospholipase C gave a hydrophilic enzyme devoid of self-aggregation, as evidenced by sedimentation analysis in a detergent-free gradient. Phosphatidylinositol likely anchors in vivo DS ChE to the cell membrane, as occurs in a number of other amphiphilic G2 ChE forms [2,3]. Furthermore, the apparent sedimentation coefficient of DS ChE before (7.0 S) or after (8.0 S) phosphatidylinositol removal is roughly similar to those of the above-mentioned G2 forms of ChE from Vertebrata and Invertebrata. According to the classification proposed by Bon et al. [31], DS ChE can be identified as a type I Ga2 dimer consisting of subunits with a Mr value of 68 000. In particular, DS ChE recovered from the whole body tissues of M. gallopro6incialis likely corresponds to that found by Mora et al. [24] in the gills of the same mussel. The results so far discussed in the present study suggest that in M. gallopro6incialis most of ChE, consisting of the SS forms in the hemolymph, is not involved in the cholinergic neurotransmission. It could hydrolyze metabolism-borne choline esters of usual or non-usual composition, thus playing important protective roles for the nervous system. In any case, DS ChE, Ptdlns linked to the cell membrane, is likely present and functional in the cholinergic synapses. Both SS and DS ChE, respectively, present in hemolymph and body tissues of M. gallopro6incialis no matter at which marine site they were collected, are AChEs hydrolyzing ATC at the highest rate. Moreover, all these enzymes, although showing a valuable efficiency with PTC as substrate, lack catalytic activity with BTC. A similar behavior has been described by us in a recent report as to an AChE from the optic lobe of the cephalopod mollusc Loligo opalescens [32]. Molecular cloning of the relative cDNA suggested that the marked specificity for ATC as a

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substrate and the inability to hydrolyze BTC is likely due to the presence of a polar serine residue, instead of a non-polar one, in the acyl pocket of the active site. According to inhibitor studies, all SS ChEs from M. gallopro6incialis specimens of the various origins clearly show a poor sensitivity for eserine, which, at difference, gives a moderate inhibition (IC50, 10 − 4) of DS ChEs from the same animals. Such an objective difference in sensitivity for a competitive inhibitor binding to both the anionic and esteratic moiety of the active site [1] suggests possible differences in details of active site conformation and, perhaps, of the catalytic mechanism in SS and DS ChEs. This idea is further on supported by parallel differences in the inhibition of SS and DS ChEs by paraoxon, an organophosphate which binds to the esteratic site [1]. The marked sensitivity of both SS and DS ChE from various origins towards BW284cS1 is a typical feature of AChEs [1,22]. IC50 values for such an inhibitor with the enzymes studied are comparable to those recently observed for AChEs in the annelids Dendrobaena [11] and Spirographis [27] or in the mollusc Loligo [32]. A peculiarity of both SS and DS ChE of mussels from the various marine sites is the lack of sensitivity for propoxur, a carbamate inhibitor of ChEs commonly used as pesticide, and for DFP, an organophosphorus compound. The lack of inhibition by DFP and the moderate inhibition by paraoxon of DS ChE well-agree with the observations of Mora et al. [24] regarding a ChE from the gills of M. gallopro6incialis and confirms that such an enzyme corresponds to the DS ChE studied in the present report. In addition, the same behavior in the presence of DFP has been also observed with the above-mentioned AChE from optic lobe of L. opalescens [32]. The differences in some pharmacological properties (inhibition by eserine and paraoxon) between DS and SS forms of ChE from M. gallopro6incialis of any origin support the possibility that these enzymes are encoded by two distinct genes. One could give the subunit of DS form, Ptdlns linked to the cell membrane and likely functional in cholinergic synapses. Another gene could originate a catalytic polypeptide giving, after post-translational modifications, either a tetrameric (A) or a dimeric (B) SS form showing close similarities in substrate specificity and inhibition pattern. The hypothesis of two separated genes for ChEs in M. gallopro6incialis is also suggested by the kinetic behavior of the enzymes. In fact, in the mussels from each marine site SS forms show quite similar values of the ATC activity/PTC activity ratios, differing from those of DS ChE. On the other hand, the encoding of ChE by several genes is not a novelty in Invertebrata [13,33]. The origin from different marine sites does not seem to affect significantly molecular polymorphism and behavior with several inhibitors, as shown by the various ChE forms in M. gallopro6incialis. However, the above-mentioned sets of ATC activity/PTC activity ratio values (SS forms A–B on the one hand and DS form on the other) are different from one station to another. Such a behavior suggests the existence of structural diversities, coming from mutations, in the ChE active site of mussels from different marine areas. This could be due to a polymorphism in the genes encoding the catalytic subunits.

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Acknowledgements This research was supported by grants of the Italian ‘Ministero dell’Universita´ e della Ricerca Scientifica e Tecnologica’ and of the ‘Fondazione Cassa Risparmio Perugia’. The authors are grateful to Salwa Jazmati for the language revision of the manuscript and to Andrea Piazzoli and Francesco Fabi for technical assistance.

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