THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276:102-lll(1996) Presence of (anAcetylcholinesterase in the Cnidarian Actinia equina (Anthozoa:Actiniaria) and of a Thiocholine Ester-Hydrolyzing Esterase in the Sponge Spongia ofjCicinaZis (Demospongiae:Keratosa) VINCENZO TALESA, RITA ROMANI, GABRIELLA ROSI, AND ELVIO GIOVANNINI Department of Experimental Medicine, Division of Cell and Molecular BLology, University of Perugia, 06100 Perugia, Italy ABSTRACT Cholinesterase (ChE) was studied in Cnidaria (Actinia equina) and in Porifera (Spongia oficinalis). In A. equina a single enzyme form was detected, likely membrane-bound through weak ionic or hydrophobic interactions. According to gel-filtration chromatography and sedimentation analysis, it seems a GI globular monomer (78 kDa, 6.1 S) including some hydrophobic domain. This enzyme shows a good active site specificity with differently sized substrates. The behaviour with specific ChE inhibitors and substrate inhibition is typical of the acetylcholinesterases and makes it quite distinct from non-specific esterases also present in A. equina. In S. officinalis, ChE-like activity is due to a smaller hydrophilic protein (50 kDa, 4.8 S). This enzyme shows a very low substrate affinity for thiocholine esters, a poor sensitivity for positively charged ChE inhibitors and for eserine, as well as absence of substrate inhibition with acetylthiocholine. These results, together with those of electrophoretic analysis, suggest that in S. officinalis a particular esterase form has also fitted for hydrolyzing choline esters with a low catalytic efficiency. 01996 Wiley-Liss, Inc. Cholinesterases (ChEs") are a class of serine hydrolases ubiquitous in the animal kingdom. They likely originated from the addition of a negative charge into the active site of an ancestral esterase, thus gaining a better specificity for choline esters as substrates. As is well known, according to substrate specificity and sensitivity t o specific inhibitors, a ChE can be classified as acetylcholinesterase (AChE, EC 18.104.22.168) or less specific butyrylcholinesterase (BChE, EC 22.214.171.124) (Silver, '74). In Vertebrata, AChEs are mainly involved in the cholinergic neurotransmission, while BChEs are present as soluble proteins in the serum and also occur in various tissues (Silver, '74; Toutant et al., '85; Massoulie et al., '93).AChEs and BChEs from Vertebrata are encoded b,y two distinct genes. They both are polymorphic enzymes with asymmetric and globular forms. Asymmetric AChEs, typical of neuromuscular junctions, consist of high molecular weight oligomers (A4, b,Alz) anchored through a collagenous structure t o the basal lamina within the synaptic cleft. Globular forms (GI, G2, G4) include either amphiphilic or hydro0 1996 WILEY-LISS, INC. philic catalytic subunits, based on the capacity of interacting with non-ionic detergent. Detergentsoluble enzymes are membrane-linked through a hydrophobic domain (Massoulie and Bon, '82; Silman and Futerman, '87; Massoulik et al., '93). In Invertebrata, a number of studies concerning ChEs from Insecta (Gnagey e t al., '87; Toutant, %9),Nematoda (Arpagaus et al., '92, '941, Sipunculida (Talesa et al., '93), Mollusca (Talesa et al., '94, '95a,b), Annelida (Talesa et al., '95c) only showed globular forms. Sometimes a dimeric (G,) ChE, glycolipid-anchored to the cell mem- *Abbreviations used: AChE = acetylcholinesterase; ATC = acetylthiocholine; BChE = butyrylcholinesterase; Brij 96 = 10-old ether; BTC = butyrylthiocholine; BW284c51 = 1:5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide; ChE = cholinesterase; DTNB = 5,5'-dithiobis-(-2 nitrobenzoic acid); HS = high-salt; HSB = high-salt-Brij; HSDS = high-salt-detergent-soluble; HSS = high-saltsoluble; HST = high-salt-Triton; LS = low-salt; LSDS = low-salt-detergent-soluble; LSS = low-salt-soluble;LST = low-salt Triton; p-NPhB = p-nitrophenylbutyrate; PTC = propionylthiocholine; PtdIns = phosphatidylinositol. Received November 8, 1995; revision accepted May 10, 1996. Address reprint requests to Dr. Vincenzo Talesa, Dipartimento di Medicina Sperimentale, Sezione di Biologia Cellulare e Molecolare, Universita di Perugia, Via del Giochetto, 06100 Perugia, Italy. CHOLINESTERASE IN ACTINIA AND SPONGIA brane, coexists with another form contained in the hemolymph (Talesa et al., '94, '95a). ChEs from Invertebrata often display a less defined substrate specificity than the corresponding enzymes from Vertebrata and a wide variability in the kinetic behaviour (Talesa et al., '90, '94). Our previous research (Talesa et al., '92) studied soluble forms of AChE in Cnidaria (VeZeZZa uelella, Actinia equina), a phylum in which tissue differentiation gave the inception of the nervous system in the animal kingdom as a primitive nerve net. However, the presence of what appeared to be ChE has been observed in Porifera as well (Lentz, '661, even if such a phylum did not evolve a nervous system or sense organs and shows only the simplest of contractile elements (Hickman, '67; Hickman et al., '88). Goals of the present work were further studies of ChE in Cnidaria (Actinia equina) as well as a more reliable identification and characterization of possible ChE forms in Porifera (Spongia oficinalis). In addition, bearing in mind the remote phylogenic origin of these organisms, we tried t o detect in them some evidence for a possible origin of ChEs from esterases (likely carboxylesterases) also ubiquitous in the animal kingdom, based on changes in molecular and kinetic features, MATERIALS AND METHODS Materials Acetyl- (ATC), propionyl- (PTC), butyrylthiocholine (BTC) iodide as well as p-nitrophenylbutyrate (p-NPhB) used as substrates for ChE or esterase activity measurements, respectively, eserine sulfate, edrophonium chloride, procainamide, and 1:5-bis(4-allyldimethylammoniumphenyl)-pentan-3-onedibromide (BW284c51)used as ChE inhibitors, diethyl-p-phenylphosphateand bis-p-nitrophenylphosphateused as esterase inhibitors, bacitracin and aprotinin (protease inhibitors), Escherichia coli alkaline phosphatase, marker proteins, and blue dextran for M, evaluation, fast blue BB salt, and a-naphthylacetate for esterase staining after electrophoresis, were purchased from Sigma Chemical Co. (St. Louis, MO). Ultrogel AcA 44 for gel-filtration chromatography was bought from LKB, Bromma, Sweden. 5,5'-dithiobis(-2-nitrobenzoicacid) (DTNB) was from Merck (Darmstadt, FRG). Electrophoresis purity reagents were from Bio-Rad (Melville, NY).Bacterial phosphatidylinositol (Ptd1ns)-specific phospholipase C (Bacillus cereus) and P-galactosidase (E. coli) were bought from Boehringer (Mann- 103 heim, FRG). All other reagents used were analytical grade products from various sources and all solutions were made in twice-distilledwater. Specimens of Actinia equina (3-5 cm in diameter, 8-10 cm in length) and Spongia officinalis (15-20 cm in diameter) were collected during May on the rocky bottom in shallow waters along the coast of the Thyrrenian sea near Livorno (Italy). Both materials were quickly frozen at -20°C and transferred t o the laboratory in dry ice. Composition of buffers used for extractions, sedimentation analysis, and gel-filtration chromatography Low-salt (LS) buffer contained 20 mM Tris-HC1, pH 7.4, 1mM EDTA, 5 mM MgC12,0.1 mg/ml bacitracin, and 0.008 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 NaCI, respectively. High-salt-Triton (HST) and high-saltBrij (HSB) buffer contained HS buffer plus 1% Triton X-100 or 0.5% 10-oleil ether (Brij 961, respectively. Assay methods ChE activity measurements were carried out at 20°C according t o a modification of the method early described by Ellman et al. (,61), using ATC as substrate. In particular, as a general rule, the assay mixture was composed of 0.89 ml of 0.1 M Na-phosphate buffer, pH 7.2, containing 0.5 mM DTNB, 0.1 ml of 1.0 mM substrate (final concentration), and 0.01 ml of enzyme solution (HSDS or other extract). Esterase activity is commonly evaluated measuring the hydrolysis of p-nitrophenylesters as substrates (Van Lith e t al., '89). In the present study, p-NPhB at 5 mM final concentration was used, since it is not hydrolyzed by ChE from A. equina, as observed in a previous report (Talesa et al., '92). Esterase activity measurements in HSDS o r other extracts were made with the same assay mixture used for ChE except the absence of DTNB. The product of thiocholine reaction with DTNB (ChE) or p nitrophenol production (esterase) were spectrophotometrically determined at 412 nm (E = 13,600 M-' ern-') or 400 nm (E = 13,200 M-' cm-I), respectively, by continuous recording. The rate of absorbance change was linear for at least 2 min for both activities and the slope was used to calculate the initial rate. One enzyme unit (IU) was defined as the amount of enzyme which catalyzes the hydrolysis of 1 pmol of substrate/min. V. TALESA ET AL. 104 Extraction of ChE and esterase for calculation of the sedimentation coefficients ~~. . (S).The gradients were emptied from the bottom using a peristaltic pump and 40 fractions of 250 p1 each were collected. ChE or esterase activity in each fraction was measured on the basis of the bI2 or boo change through 30 min in the usual assay mixture containing 1 mM ATC or 5 mM p NPhB and 0.05 ml of each fraction. Alkaline phosphatase and P-galactosidase activities were determined according to Principato et al. ('84a) or Massouli6 and Rieger ('69), respectively. The extraction of enzymes was carried out on the whole from 50 g of A. equina specimens and from 100 g of S. officintrlis, operating at 5°C. The typical procedure, whi ch was repeated several times, started from 5 g of A. equina or 10 g of S. officinalis. Either material, after washing with bidistilled water and adding 20 ml of HST buffer, was carefully minced by scissors in small pieces. In a preliminary expeniment the resulting materials were centrifuged a t 100,OOOg for 30 min (Beckman L 60 ultracentrifuge, SW 41 Ti rotor, Gel-filtration chromatography and M r 24,000 rpm; Beckman, Palo Alto, CA); the superevaluation of ChEs natants were found almost devoid of ChE as well The M, value of ChE forms from A. equina or as esterase activity and discarded. Therefore, as S. officinalis were estimated from the Svedberg a general rule, either minced material in HST equation M, = 67cq NS RJ(l-p/pm), where N is the buffer was homogenized with an Ultra-Turrax T Avogadro number, while for q (viscosity of water), 25 homogenizer and centrifuged a s above at p and pm(densities of water and enzyme protein) 100,OOOg for 1h. The supernatants thus obtained the values reported by Osterman ('86) were used. from either source (20-22 ml) and noted as high- The Stokes radii (R,) were determined according salt-detergent-soluble (HSDS) extracts, showed to Laurent and Killander ('64). A sample (5 ml) of marked ATC- and p-NPhB-splitting activities that HSDS extract was applied to an Ultrogel AcA 44 were also assayed in the presence of 10-4M es- column (2.5 x 114 cm) equilibrated and eluted (0.5 erine (ChE inhibitor). Tentative experiments ml/min) with HST buffer. Fractions of ,5 ml were with S. officinalis extracts were also carried out collected. Horse heart cytochrome c (17 A), chicken adding to the assay mixture 10" M diethyl-p-nitro- egg 9lbumin (30 A), E. coli alkaline phosphatase phenylphosphate or bis-p-nitrophenylphosphate, (33 A), and yeast alcohol dehydrogenase (46 A) well-known esterase inhibitors (Van Lith et al., were used as standards. The void volume was es'89). It is also noticeable that similar amounts of timated using blue dextran. ChE activity was solubilized enzyme activity were obtained as well evaluated as described under Density gradient using in the extraction procedure LS, HS, or LST centrifugation. The sedimentation coefficients ( S ) buffer (LSS, HSS, LSDS extract, respectively). In to be used in the M, calculation were obtained by addition, preliminary electrophoretic analyses (see sedimentation analysis as reported above. Results: PAGE) displayed in the extracts from each of the sources the Eiame patterns of ChE and Polyacrylamide gel electrophoresis (PAGE) esterase forms. However, HSDS extracts were choElectrophoretic patterns of ChE as well as of sen for analytical studies since they showed esterase activity from either source were studied slightly higher specific activities (IU/ml). They by PAGE (7% acrylamide, 0.2% bis-acrylamide) were stored at -80°C until subsequent use. carried out at non-denaturing conditions. In particular, after a dialysis against LS buffer, 20 pl of Density gradient centrifugation HSDS extracuane (4 mIU ChE and 13 mIU esterase activity, A. equina; 3 mIU ChE and 25 mIU Sedimentation pattern of ChE and esterase acesterase activity, S. officinalis) were run on a 16 tivity in HSDS extract from either A. equina and x 16 x 0.15 cm slab-gel (20 mA current, 0.025 M S. officinalis was studied by centrifugation of Tris-0.192 M glycine buffer, pH 8.3, 5°C). A presamples (200 pl) layered. onto 5 2 0 % sucrose denliminary electrophoretic analysis of LSS, HSS, and sity gradients (10 ml in polyallomer tubes) in HS, LSDS extracts was also carried out in the same HST, or HSB buffer. In the last case, samples were way. Staining for ChE activity was obtained acpreincubated with 0.5% Brij 96 for 30 min before cording to the method of Juul('68) as modified by applying. Centrifugations were performed at 5°C 3 mM ATC as substrate. In Stenersen (' 8 0) using in a Beckman L60 ultracentrifuge equipped with particular, after the incubation in a copper/ATC a SW 41 Ti rotor at 36,000 rpm (222,OOOg); E. solution, the gel was washed 2-3 hours in twicecoli alkaline phosphataEle (6.1 S) and P-galactosidase (16 S) were included as internal standards distilled water prior to the final dithiooxamide CHOLINESTERASE IN ACTZNIA AND SPONGZA treatment. Staining for esterase activity was performed according to Hirose et al. ('90) with fastblue BB salt using 0.05% a-naphthyl acetate as substrate. Treatment of ChE with phospholipase C and non-denaturing PAGE Since ChE from A. equina interacts with detergents (see Results: Density gradient centrifugation), the possible presence in this enzyme of a PtdIns anchor was studied by PtdIns-specific phospholipase C digestion and subsequent PAGE. Samples of HSDS extract (200 p1,40 mIU of ChE activity) were incubated with 5 IU of phospholipase C (5 pl of pure commercial preparation) for 90 minutes at 25°C under continuous stirring. Non-denaturing PAGE was carried out as previously detailed (Talesa et al., '93) running aliquots (20 pl? 4 mIU) of the above described mixture, as well as an undigested control, in a vertical slab gel (8 x 7.3 x 0.1 cm) apparatus (BioRad). Gel and running buffer contained 0.5% Triton X-100; gel staining for ChE activity was performed following the method of Juul ('68) as modified by Stenersen ('80). The actual activity of phospholipase C preparation was checked by a parallel experiment with a DS extract from the annelid Hirudo medicinalis, containing a PtdInstailed ChE (Talesa et al., '95c). These trials were carried out by digestion of H. medicinalis extract as above (200 pl, 200 mIU of ChE activity) and subsequent running of 20 pl aliquots (20 mIU) of phospholipase C-treated or undigested enzyme. 105 Lineweaver-Burk double reciprocal plots of experimental data, using ATC, PTC, and BTC as substrates in the 0.1-1 mM concentration range (five concentrations for each substrate). Inhibition studies of ChE activity in HSDS extracts from both A. equina and S. officinalis were carried out in the presence of ATC as substrate and using well-known inhibitors of ChEs, previously employed t o study invertebrate enzymes: procainamide (Principato et al., '89; Talesa et al., '90, '92), edrophonium (Talesa et al., ,931, eserine (Silver, '74; Arpagaus et al., '92; Talesa et al., '95a,b) and BW284c51 (Silver, '74). Inhibitor concentrations in the 10-3-10-9M range were used in the assay and residual ChE activities were determined (two distinct determinations for each inhibitor concentration). In particular, the reaction was started after an extract-inhibitor incubation of 1minute by adding the substrate solution. Substrate inhibition tests of ChE from either origin were also performed using ATC in the 0.1-50 mM concentration range. RESULTS Extraction of ChE and esterase No significant amount of ChE or esterase activity was recovered from either A. equina or S. officinalis in a free form by dilacerating the tissues without homogenization. The enzyme extraction, carried out by homogenization in LS, HS, LST, or HST buffer, respectively, gave comparable recoveries of both enzyme activities from either source; however, the maximal yield as total Kinetic and inhibition studies amount and specific activity (IU/ml) was obtained Kinetic parameters K, and V,, were deter- using HST buffer (HSDS extracts) (Table 1). The presence of lo4 M eserine in the assay submined for thiocholine ester-splitting enzymes present as a single form in either studied species stantially unaffected esterase activity, while ChE (see Results: PAGE) by computer analysis of activity was suppressed or significantly reduced TABLE 1. Extraction of cholinesterase (ChE) and esterase from Actinia equina (5 g) and Spongia officinalis (10 g) carried out by homogenization i n high-salt-detergent (HSDS extract), i n low-salt-detergent (LSDS extract), high-salt (HSS extracts), or low-salt (LSS extract) buffer, as detailed in the text' Extract A. equina HSDS LSDS HSS S. officinalis LSS LSDS LSDS HSS LSS ChE (total activity [IUD Esterase (total activity [IUI) 4.0(ND) 13.3 (13.7) 9.5 11.0 9.6 25 (23.9) 22 22 19 3.5 3.3 3.3 2.5 (1.75) 2.3 2.3 2.1 'ChE and esterase activities were evaluated using acetylthiocholine (ATC) and p-nitrophenylbutyrate (p-NPhB) as substrate, respectively. The values in parentheses were obtained in the presence of lo4 M eserine. One enzyme unit (IU) was defined as the amount of enzyme catalyzing the hydrolysis of 1pmol of substrateimin. ND: not detectable. V. TALESA ET AL. 106 in the extracts from A. equina and S. officinalis, respectively. Assays carried out (S. oficinalis) with esterase inhibitors displayed total suppression of both ChE and esterase activity (results not shown). Density gradient centrifigation Sedimentation analysis of ChE and esterase activity in the HSDS extracts from A. equina or S. officinalis were performed with groups of four experiments. The apparent sedimentation coefficients (peaks of enzyme activity) are given as means SD, while the sedimentation profiles result from the mean values of activity in each fraction. Sucrose gradient centrifugation of HSDS extract from A. equina, carried out in the absence of detergent (HS buffer), showed a single peak of ChE activity at 10.1 2 0.4 S, shifted t o apparent 6.7 f 0.2 S and 6.1 r 0.2 S positions when Brij 96 or Triton X-100 was, respectively, added to the gradient (Fig. 111).The same allalysis for esterase, with a detergent-free gradient, gave a diffuse sedimentation pattern at high S values, likely due to partial aggregation of enzyme, besides an activity peak at 6.7 2 0.1 S. Addition of Triton X-100 or Brij 96 gave double peak-shaped sedimentation profiles (9.4 2 0.3 S , 4.8 f 0.1 S, and 8.7 2 0.2 S , 4.8 2 0.2 S, respectively), thus suggesting the presence of at least two esterase forms (Fig. 1B). The same sedimentation analysis of HSDS extract from S. officinalis gave, in a detergent-devoid gradient, a single peak of ChE activity at 4.8 2 0.2 S, which remained unchanged after adding Triton X-100 (4.8 & 0.3 s) or Brij 96 (4.8 & 0.4 S) to the gradient (Fig. 10. The sedimentation profile of esterase also showed a single, although less sharply shaped, activity peak at 4.8 f 0.4 S position in a detergent-free gradient. It was unchanged in the presence of Triton X-100 (4.8 2 0.3 S) or Brij 96 (4.8 2 0.2 S)(Fig. ID). 0.0 n ol 0.6 4 c c 3 0.4 3) L 0.2 b I 0.0 n D .6d - c > I V I V I 30 40 .tr 0 4 aJ 0 (I? 0.3 W Lo Q L W c u 0.2 0) .tr Lo w 0.i 0.0 0 10 20 30 40 0 10 20 Frectlon number Fig. 1. Sedimentation pattern in sucrose density gradient of cholinesterase (ChE) and esterase forms in high-saltdetergent-soluble (HSDS) extracts from Actinia equina (A,B) and Spongia officinalis (C,D) prepared as detailed in the text. Gradients (5-20% were made in HS buffer with 1%Triton X- 100 (01, 0.5% Brij 96 (O), or no detergent (0). ChE and esterase activities were evaluated using ATC orp-NPhB as substrate, respectively. The arrows indicate the position of E. coli P-galactosidase (16 S) and alkaline phosphatase (6.1 S ) used as internal standards. CHOLINESTERASE IN ACTINIA AND SPONGIA The fairly widened shape of such a n activity peak suggests a n adjoining sedimentation of several molecular forms of enzyme. ChE 0- 107 E ChE E Gel-filtrationchromatography and M revaluation of ChEs Ultrogel AcA 44 chromatography of HSDS extract from A. equina or S. officinalis gave for both a single ChE activity peak at Stokes radius (R,) values of 31 and 25 A, respectively. A repetition of such experiments displayed quite similar results. The M, values, calculated setting in the Svedberg equation R, and S from gel-filtration and sedimentation analysis (6.1 S and 4.8 S), were 78,000 (A. equina) and 50,000 (S. officinalis). Polyaciylamide gel electrophoresis (PAGE) According to the results of PAG-electrophoresis, both ChEs from A. equina and S. officinalis migrated towards the anode as single activity bands. On the contrary, electrophoretic patterns of esterases showed several bands of activity (two for 1 2 3 4 A. equina, more numerous for S. officinalis). In addition, as to A. equina, a correspondence occurs Fig. 2. Non-denaturing PAGE of cholinesterase (ChE) and between the ChE activity band and that of the faster migrating esterase form (Fig. 2, lanes 1, esterase (E) forms in high-salt-detergent-soluble (HSDS) extract from Actinia equina (lanes 1,2: 4 mIU ChE and 13 2), while the ChE activity band of S. officinalis mIU E activity, respectively) and Spongia oficinalis (lanes corresponds in position to the predominant one of 3,4: 3 mIU ChE and 25 mIU E activity, respectively) preesterase activity (Fig. 2, lanes 3,4). Such experi- pared as detailed in the text. Samples of 20 pl were run. ments were repeated several times with quite Staining for ChE and E activity was performed according t o the method of Juul ('68) as modified by Stenersen ('80) and similar results. A preliminary electrophoretic analysis of LSS, Hirose et al. ('90) respectively. 0: origin of migration. HSS, and LSDS extracts from A. equina and S. officinalis gave substantially the same patterns A. equina and S. oficinalis. Such kinetic constants of ChE and esterase activity observed with HSDS were determined using different substrates. Based on the sets of V, and Vm,/Km values, ATC is the extract (results not shown). best substrate for both enzymes, followed by PTC, lkeatment of ChE with phospholipase C while BTC is by far the less suitable one: the and non-denaturing PAGE Vmax(~~~)/Vmax(ATC) ratio is 0.18 and 0.42 for the ChE Digestion with PtdIns-specific phospholipase C from A. equina and S. officinalis, respectively. In of HSDS extract gave no change of electrophoretic addition, while V, values for both enzymes lie pattern of ChE from A. equina in non-denaturing roughly into the same size order, ChE from S. conditions and in the presence of Triton X-100. The officinalis shows far higher K, values (nearly two ChE activity band migrated towards the anode in magnitude orders). In consequence, the V,JK, the same way as undigested control (Fig. 3, lanes values are even 100-fold lower (ATC) in compari1,2). The experiment was repeated with identical son with those of the ChE from A. equina. results. The positive control with a ChE-containAs regards the studies with some ChE inhibiing DS extract from Hirudo medicinalis verified tors (Fig. 4), ATC-hydrolyzing activity of ChE from the activity of phospholipase C, giving the expected A. equina showed, as a rule, a higher sensitivity conversion of the amphiphilic slow-migrating en- than the corresponding enzyme from S. oficinalis. zyme to a faster hydrophilic form (Fig. 3, lanes 3,4). Residual activity of the latter exceeded 50% even with 10 M procainamide, BW284c51, or eserine, Kinetic and inhibition studies while Is0 was about 5 x lo4 M with edrophonium. Table 2 gives V, and K, values for thiocholine On the contrary, ChE from A. equina showed only ester-splitting enzymes present as single forms in traces of residual activity with M BW284c51 V. TALESA ET AL. 108 T C T C 1 2 3 4 Fig. 3. Non-denaturing PAGE of cholinesterase (ChE) in high-salt-detergent-soluble(HSDS) extract from Actinia equina (lanes 1,2:4 mIU of ChE activity) prior to and after digestion with PtdIns-specific phospholipase C , carried out as described in the text. Posritive control of phospholipase C activity with a ChE-conta ning DS extract from Hirudo medicinalis (lanes 3,4: 20 riIU of ChE activity). Samples of 20 pl were run. Staining for ChE activity was performed according t o the method of Juiil('68) as modified by Stenersen ('80). T,phospholipase C-treated sample; C, control undigested sample. or eserine, while lo3 M procainamide or edrophonium left a detectable activity. Ih0values for this enzyme were roughly lo4 M (procainamide and edrophonium), M (eserine), and 5 x lo-' M (BW284c51). The results of substrate inhibition tests, carried out in the presence of ATC, evidenced that such an inhibition only concerns ChE from A. equina and starts beyond 5 mlVI substrate (Fig. 5). DISCUSSION The joined results of sedimentation and electrophoretic analysis suggest the presence both in A. equina and S. offiiciizaZis of a single thiocholine ester-hydrolyzing enzyme and, in addition, a set of esterases, more numerous in the latter organism. Such enzymes can be solubilized at comparable yields by homogenization in a low or high salt buffer, with or without a detergent. ChE from A. equina does not overlap its activity with esterase, being inactive on p-NPhB (Talesa et al., '92) and strongly inhibited by eserine. Sedimentation pattern in a detergent-free gradient shows t h a t such a n enzyme is a n amphiphilic protein with some hydrophobic domain, giving aggregated oligomers (10.1 S position), fully resolved by adding detergent to the gradient (peak shifting to 6.1-6.7 S). However, such a hydrophobicity is not due to a PtdIns, giving the in vivo membrane-anchoring of several amphiphilic ChEs (Silman and Futerman, '87; Ferguson and Williams, '88; Massoulib et al., '93). In fact, the enzyme shows no change of electrophoretic pattern in the presence of Triton X-100 after treatment with a specific phospholipase C. The M, value (78,000) emerging from the results of sedimentation analysis and gel-filtration chromatography approaches that (83,000) which we found previously by SDS-PAGE of the same ChE from A. equina (Talesa et al., '92). Therefore we believe that the studied enzyme is a monomeric globular form (GJ, while the M, value (330,000) and tetrameric structure suggested in Talesa et al. ('92) for the presumed native ChE likely concerned an aggregate. The esterases ofA. equina are as well amphiphilic forms with hydrophobic detergent-binding domains giving aggregation of the enzymes, as shown by centrifugation in a detergent-devoid gradient. According to solubilization behaviour, both ChE and esterase forms from A. equina could be weakly attached t o the cell membrane through either ionic interactions or the hydrophobic domains evidenced by sedimentation analysis. In S. officinalis an enzyme assay with different substrates and inhibitors cannot state whether ChE-like and esterase activities are mutually exclusive or overlap, since the former is only in part inhibited by eserine and suppressed by usual esterase inhibitors. The thiocholine ester-splitting enzyme, based on results of density gradient centrifugation, is likely a hydrophilic protein, devoid of detergent interaction or self-aggregation.Its sedimentation coefficient (4.8 S ) approaches those of globular monomeric (GJ ChEs from Vertebrata (Massoulie et al., '93) or Invertebrata (Arpagaus et al., '92), while the M, value (50,000) is rather lower than those usually observed for monomeric 109 CHOLINESTERASE IN ACTZNZA AND SPONGZA TABLE 2. Kinetic constants of cholinesterase (ChE) activities in high-salt-detergent-soluble (HSDS) extracts from Actinia equina and Spongia officinalis, prepared as described in the text (IU/ml) 0.216 k0.060 0.120 f 0.020 0.040 2 0.010 0.142 2 0.060 0.134 f: 0.028 0.060 2 0.014 ATC PTC BTC ATC PTC BTC A. equina S. officinalis K, (mM) V,,, Substrate Vmax/K, 0.098 k 0.014 0.145 0.035 0.300 f: 0.086 8 * 1.09 14 k 4.20 19 4.13 2.20 0.83 0.13 1.8 x 9.6 x 3.2 x (min-3 lo-' lo3 lo3 'The enzyme activity was determined using actyl- (ATC), propionyl- (PTC), and butyryl-thiocholine (BTC)as substrates.,,V and K, values are given as M f SD of four experiments. One enzyme unit (IU)was defined as the amount of enzyme catalyzing the hydrolysis of 1 pmol of substratelmin. ChEs or catalytic subunits of these enzymes. The set of esterases from s.officinalis shown by PAGelectrophoresis holds hydrophilic forms as well, with a sedimentation pattern unaffected by the presence or absence of detergent in the sucrose gradient. Probably, both ChE-like enzyme and esterases from S. oficinalis occur in vivo as soluble cytosolic proteins; otherwise, they could be once ... A 80 - 60 - 40 - 20 - n be W n 4 - .C > again membrane-linked by weak electrostatic interactions. As from kinetic studies, thiocholine ester-hydrolyzing enzymes from both A. equina and S . officinalis could be classified as AChEs according to the higher values of V,, and Vm,Km (Fersht, '85; Principato et al., '88) with ATC as substrate. Moreover, both the enzymes show a good specific- 4 0 43 W 100 .c u r_ 80 43 3 U c 60 u) a Q! 40 20 0 -10 -8 -6 -4 Fig. 4. Inhibition of cholinesterase (ChE) forms in highsalt-detergent-soluble (HSDS)extracts from Actinia equina ( 0 )and Spongia officinalis (0) assayed as detailed in the -2 -10 -8 -6 -4 -2 text. ChE activity was evaluated using ATC as substrate. A, procainamide; B, edrophonium; C, BW284c51; D, eserine. V. TALESA ET AL. 110 AChEs, high sensitivity to BW284c51, and eserine as well as substrate inhibition with ATC (Silver, '74). Moreover, the degree of inhibition by procainamide and edrophonium is comparable to that of some invertebrate ChEs (Talesa et al., .d '95a). The AChE from A. equina also hydrolyzes detectably a charge-devoid substrate (a-naphthyl 0.04 3 acetate), thus giving an apparent electrophoretic u band of esterase activity. u 0.02 Therefore, keeping in mind the course of anic u 0 .oo mal phylogeny, it is likely that Porifera, devoid of -1 .5 -0.5 0.5 1 .5 a nervous system and sense organs, lack enzymes classifiable as ChEs, even if some of their esterases already gained the capacity of hydrolyzing, with a low catalytic efficiency, choline esters. On the contrary, in Cnidaria, where the onset of Fig. 5. Substrate inhibition curves of cholinesterase (ChE) forms in high-salt-detergent-soluble (HSDS) extracts from tissue differentiation has given a primitive nerActinia equina (@) and Spon<giaoffcinaZis (0). ChE activity vous system, there is a ChE showing kinetic was evaluated using ATC as mbstrate. and molecular features typical of such a class of enzymes. 0.10 n c .C ity level of the active site with differently sized ATc) values approach those substrates: Vmm(BTC)/Vma ofAChEs from Vertebrata and Jnsecta (Silver, '74; Gnagey et al., '87). However, apart from such analogies, the ChElike enzyme from 5'. officinalis shows a far lower catalytic efficiency (V,,JK, values), due t o a poor substrate affinity for tl~iocholineesters: indeed, Km values exceed by far those of A. equina enzyme and even those ol' well-known BChEs from Vertebrata (Andersen and Mikalsen, '78; Principato et al., '84b). Such findings suggest a reduced role of electrostatic interactions in the enzyme-substrate complex formation. Considering also the very low sensitivity for competitive positively charged inhibitors of ChEs (procainamide, edrophonium, BW284~51)and the absence of substrate inhibition with ATC, it is likely that the active site of S. ofj'icinalis enzyme is devoid of the ChE-typical anionic moiety. Furthermore, the low inhibition by eserine di$sagreeswith a distinctive feature of ChEs, while it is usual for aryl- and carboxyl-esterases (Silvw, '74). On the other hand, the existence of an active site conformation and catalytic mechanism dif'ferent from those of ChEs and more suited to estsrases is in keeping with the results of electrophoretic analysis of the enzymes from s.officinalis, where the ATC-hydrolyzing activity seems to be axnmitted to a predominant esterase form. On the contrary, the thiocoline ester-splitting enzyme from A. equina seems a true AChE, based on substrate affinity, close to that of vertebrate ACKNOWLEDGMENTS We are grateful t o Mr. Marco and Maurizio Rosi, Livorno, Italy, for collecting the specimens of Actinia equina and Spongia officinalis used in this study. We thank Andrea Piazzoli and F'rancesco Fabi for technical assistance. This work was supported by a grant from the Italian CNR (94.02479.CT04). LITERATURE CITED Andersen, R.A., and A. Mikalsen (1978) Substrate specificity, effect of inhibitars and electrophoretic mobility of brain and serum cholinesterase from frog, chicken and rat. Gen. Pharmacol., 9:177-181. Arpagaus, M., P. Richier, J.-B. Berge, and J.-P. Toutant (1992j Acetylcholinesterases of the nematode Steinernemu carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association. Eur. J. Biochem., 207:llOl-1108. Arpagaus, M., Y. Fedon, X. Cousin, A. Chatonnet, J.-B. Berg6, D. Fournier, and J.-P. Toutant (1994) cDNA sequence, gene structure and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. J. Biol. Chem., 269A 101-1108. Ellman, G.L., D.K. Courtney, V. Andres, and R.M. Featherstone (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7:88-95. Perguson, M.A.J., and A.F. Williams (1988) Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Ann. Rev. Biochem., 57:285-320. Fersht, A. (1985) Enzyme Structure and Mechanism. W.H. Freeman, New York, pp. 98-106. Gnagey, A.L., M. Forte, and T.L. Rosenberry (1987) Isolation and characterization of acetylcholinesterase from Drosophila. J. Biol. Chem., 262:13290-13298. Hickman, C.P. (1967) Biology of the Invertebrates. C.V. Mosby, St. Louis, MO, pp. 86-104. Hickman, C.P., L.S. Roberts, and F.M. Hickman (1988) Inte- CHOLINESTERASE IN ACTINIA AND SPONGIA grated Principles of Zoology. C.V. Mosby, St. Louis, MO, pp. 166-179. Hirose, T., A. Morita, H. Nikaido, J. Hayakawa, and 0. Nikaido (1990) Selective purification of sex-influenced esterase from rat serum by immunoaffinity chromatography. Eur. J . Biochem., 189:431-436. Juul, P. (1968) Human plasma cholinesterase isoenzymes. Clin. Chim. Acta, 19~205-213. Laurent, T.C., and J. Killander (1964) A theory of gel filtration and its experimental verification. J. Chromatogr., 14~317-330. Lentz, T.L. (1966) Histochemical localization of neurohumors in a sponge. J . Exp. Zool., 162:171-180. Massoulie, J., and S. Bon (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates. Annu. Rev. Neurosci., 557-106. Massoulie, J., and F.Rieger (1969) L'acetylcholinestbrase des organes Blectriques de Poissons (torpille et gymnote); complexes membranaires. Eur. J. Biochem., 11:441-455. Massoulib, J., L. Pezzementi, S. Bon, E. Krejci, and F.M. Vallette (1993) Molecular and cell biology of cholinesterases. Prog. Neurobiol., 41:3 1-91. Osterman, L.A. (1986) Methods of Protein and Nucleic Acid Research. Springer Verlag, Berlin, Heidelberg, pp. 130-139. Principato, G.B., V. Bocchini, G. Rosi, M.C. Aka, and E. Giovannini (1984a) Purification and characterization of four different alkaline phosphatases from Spirographis spallanzanii. Comp. Biochem. Physiol., 78B:485-491. Principato, G.B., G. Rosi, V. Bocchini, and E. Giovannini (1984b) Active site kinetics of horse serum cholinesterase. Biochem. Int., 8:135-141. Principato, G.B., V. Talesa, E. Giovannini, R. Pascolini, and G. Rosi (1988) Characterization of the soluble cholinesterase from Squilla mantis. Comp. Biochem. Physiol., 9OC~413-416. Principato, G.B., S. Contenti, V. Talesa, C. Mangiabene, R. Pascolini, and G. Rosi (1989) Propionylcholinesterase from Allolobophoru caliginosa. Comp. Biochem. Physiol., 94C:23-27. Silman, I., and A.H. Futerman (1987) Modes of attachment of acetylcholinesterase t o the surface membrane. Eur. J. Biochem., 17O:ll-22. Silver, A. (1974) The biology of cholinesterases. In: Frontiers of Biology, vol. 36. A. Neuberger, E.L. Tatum, eds. NorthHolland, Amsterdam. Stenersen, J. (1980) Esterases of earthworms. Part 11: Characterization of the cholinesterases in the earthworm Eisenia 111 foetidu (Savigny) by ion exchange chromatography and electrophoresis. Comp. Biochem. Physiol., 66C:45-51. Talesa, V., S. Contenti, C. Mangiabene, R. Pascolini, G. Rosi, a n d G.B. Principato (1990) Propionylcholinesterase from Murex branduris: Comparison with other invertebrate cholinesterases. Comp. Biochem. Physiol., 98C:39-43. Talesa, V., G.B. Principato, C. Mangiabene, E. Giovannini, S.J. Norton, and G. Rosi (1992) Cholinesterase in the cnidarians Velella velella (Hydrozoa: Syphonophora) and Actinia equinu (Anthozoa: Actiniaria): A comparative study. J. Exp. Zool., 263:367-373. Talesa, V., G.B. Principato, E. Giovannini, M.V. Di Giovanni, and G. Rosi (1993) Dimeric forms of cholinesterase in Sipunculus nudus. Eur. J. Biochem., 215:267-275. Talesa, V., G.B. Principato, E. Giovannini, S.J. Norton, and G. Rosi (1994) Presence of a soluble tetrameric and membranebound dimeric forms of cholinesterase in Murex brundaris (Mollusca, Gastropoda). J. Exp. Zool., 270:233-244. Talesa, V., M. Grauso, G.B. Principato, E. Giovannini, and G. Rosi (1995a) Cholinesterase in Helix pomatia (Gastropoda, Stylommatophora): Presence of a soluble (hemolymph) and membrane-bound form. Comp. Biochem. Physiol., 110B:649-656. Talesa, V., M. Grauso, E. Giovannini, G. Rosi, and J.-P. Toutant (1995b) Acetylcholinesterase in tentacles of Octopus vulgaris (Cephalopoda). Histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms. Neurochem. Int., 27~201-211. Talesa, V., M. Grauso, E. Giovannini, G. Rosi, and J.-P. Toutant (1995~)Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis). Biochem. J., 306~687-692. Toutant, J.-P. (1989) Insect acetylcholinesterase: Catalytic properties, tissue distribution and molecular forms. Prog. Neurobiol., 32:423-446. Toutant, J.-P., J. Massoulie, and S. Bon (1985) Polymorphism of pseudocholinesterase in Torpedo marmoratu tissues: Comparative study of the catalytic and molecular properties of this enzyme with acetylcholinesterase. J . Neurochem., 44580-592. Van Lith, H.A., M. Den Bieman, and B.F.M. Van Zutphen (1989) Purification and molecular properties of rabbit liver esterase ES-1A. Em. J. Biochem., 184:545-551.