Multiple forms of acetylcholinesterase in Allolobophora caliginosa: Purification and partial characterization

Comp. Biochem. Physiol. Vol. 61C, pp. 147 151

0306-4492/78/0901-0197502.00/0

© Pergamon Press Ltd 1978. Printed in Great Britain

MULTIPLE FORMS OF ACETYLCHOLINESTERASE IN ALLOLOBOPHORA CALIGINOSA: PURIFICATION A N D PARTIAL CHARACTERIZATION GIOVANNI B. PRINCIPATO,* M. VITTORIA AMBROSINI, ANNARITA MENGHINI, ELWO GIOVANNINI and MASSIMO DELL'AGATA Institute of General Biology, Faculty of Medicine and Surgery, University of Perugia, Perugia, Italy and II Chair of General Biology and Zoology, Faculty of Medicine and Surgery, University of Chieti, Chieti, Italy

(Received 28 November 1977) Abstract--1. The authors studied certain characteristics of the acetylcholinesterase present in Allolobophora caliginosa; a good purification of the enzyme was achieved by homogenization, ultracentrifugation and then by Sephadex G-200 and DEAE-cellulose chromatographies. 2. Three enzymatic forms, probably monomeric, dimeric and tetrameric were isolated. The monomeric one is quantitatively prevailing and shows a higher specific activity; it seems to be composed of two subunits with the same molecular weight. 3. The various active fractions are inhibited by eserine and hydrolyse acetylthiocholine more rapidly than butyrylthiocholine; besides, this latter does not cause substrate inhibition. 4. The enzyme is classifiable as acetylcholine hydrolase (E.C. 3.1.1.7).

INTRODUCTION Acetylcholinesterase (ACHE) is an enzyme widely present in the animal kingdom and it plays, as is wellknown, an important role in the activity of the nervous cells. However, an examination of the literature relative to this subject shows that extensive biochemical studies about the AChE in Invertebrata concern so far mainly Insecta (Hellenbrand & Krupka, 1970; Habibulla & Newburgh, 1973; Huang & Dauterman, 1973) and Mollusca (Emson & Kerkut, 1971 ; Bevelaqua et al., 1975). On the other hand, this enzyme was also detected in N e m a t o d a (Nolan & Sehnitzerling, 1975), Platyhelminthes (Lentz, 1968), Coelenterata (Lentz & Barrnett, 1961) and Porifera (Lentz, 1966); it was even observed in Protozoa (Seaman & Houlihun, 1951). Therefore, the likely presence of A C h E in the various zoological groups, also including the most primitive, suggests the possibility of carrying out a comparative study concerning molecular and catalytic properties of it, in order to detect adaptations and modifications in an enzyme system during the animal evolution. Therefore, we thought that it would be of interest to undertake purification and characterization of the A C h E in several phyla of Metazoa; the present research, concerning the Annelidae Oligochaeta, was carried out in Allolobophora caliginosa. MATERIALS AND METHODS

Purification of the enzyme Specimens of Allolobophora caliginosa of various sizes, collected in agrarian soil during May, were quickly dissected in order to remove the mould contained in the gut and cut into 1-2 cm pieces. The material obtained in this

* Present address: Istituto di Biologia generale dell'Universit& Via del Giochetto, 1-06100 Perugia, Italia.

way (550 g), after adding 550 ml of 0.05 M NaCl, was kept at -20°C for a few days; it was then thawed and, after further adding 1100 ml of the same solution, homogenized for 15 min in a mincing homogenizer refrigerated by an ice-water mixture. The very foamy homogenate was then subjected to a preliminary centrifugation at 700g for 10min in order to eliminate the foam and to sediment the most heavy debris; the supernatant was further centrifuged at 100,000g for 1 hr. Such operations were carried out at 5°C using a refrigerated ultracentrifuge. The supernatant finally recovered (1650 ml) was dialysed in cellulose tubing for 24 hr at 5°C against two changes of deionized water (40 vol each) and lyophilized. The material thus obtained was subjected to two successive chromatographies on Sephadex G-200-120 column, carried out in a cold room (5°C). In particular, 1-g fractions of lyophilized material were each dissolved in 50 ml of 0.15 M NaCI-0.02~ NaN3, after having verified that no enzyme activity reduction occurred with NaN a in such a concentration. Each solution, after centrifuging (10,000 g for l0 rain) in order to sediment denatured materials, was stratified on a 40 x 2.5-cm column equilibrated and then eluted (0.5 ml/min) by 0.15M NaCI-0.02~ NaN3; 7-ml fractions were collected and the most active of them were combined, dialysed as above described and iyophilized. The subsequent chromatography was performed using 0.1-g fractions of the previously lyophilized material; each of them was again dissolved in 5 ml of NaCI-NaN3 solution and applied on a 50 x 2.5-cm column, equilibrated and eluted according to the above-stated procedure. Fractions of 5 ml were collected; also in this case, the most active fractions, corresponding to each peak of enzymatic activity, were combined, dialysed and iyophilized. These active materials were then chromatographed on DEAE-cellulose (Sigma, 0.84m-equiv/g, medium mesh), using a l0 x 2.5-cm column; the elution was carried out by 50 mM phosphate buffer at pH 7.2, containing a NaCl (0-1 M) linear gradient in a 200 ml volume, at a flow of 3 ml/min and at a 10°C temperature. The same buffer was used to dissolve the lyophilized materials (1/10w/v) and wash the column (50 ml) just after they had been applied. Fractions of 8 ml were collected and, as above, the most active of them were selected and recovered.

147

148

GIOVANNI B. PRINCIPATOet al.

When DEAE-cellulose chromatography led to separation of several peaks of enzyme activity, the fractions corresponding to each of them were again eluted with the same procedure on a 6 x 2.5-cm column; 5-ml fractions were thus collected and among these, those containing enzyme in a higher concentration were selected. The lyophilized materials obtained at the various steps of the procedure described so far were stored at -20°C until subsequent use.

pH 8.8. The enzyme was localized by its catalytic activity, spraying the strips with 75 mM ATC and successively with 10 mM dithiobisnitrobenzoate (DTNB); the protein bands were stained with amido-black. The protein concentrations were determined according to the technique of Lowry et al. (1951), using bovine serum albumin as a standard.

Analytical methods

The homogenization initially performed led to solubilization of nearly half of the enzyme present in wet tissue; about 42Vo of the total activity detectable in the homogenate was recovered following dialysis and lyophilization of the supernatant resulting from the centrifugation at 100,000g; furthermore, a starting material was thus obtained already showing a considerable specific activity (Table 1, step 1). Sephadex G-200 chromatographies led to enzymatic activity and absorbance profiles represented in Fig. 1, obtained with the second elution (Table 1, step 3); the separation of two enzymatic activity peaks (P,N), respectively eluted at 105 and 150 ml was thus achieved. The subsequent DEAE-cellulose chromatography (Table 1, step 4) achieved a partial dissociation of the former (P), provided with a lower specific activity, into two enzymatic fractions (FI, F2) with activity peaks respectively eluted at 88 and 104ml (Fig. 2). The most active material of each form was again (and separately) chromatographed on DEAE-cellulose (Table 1, step 5), leading to a better separation and purification; the peaks were eluted at 60 (F1) and 80 (F2) ml (Fig. 3). The molecular weight estimation in each purified form, carried out by chromatography on Sephadex G-200 column, gave the following values: FI ~ 700,000 (the elution occurred in the void volume, after blue dextran and together with thyreoglobulin); F2 ~ 350,000; N ~ 180,000. The N form is further dissociable by 6 M guanidine hydrochloride; in fact, the subsequent elution on Sephadex G-200 shows a sole protein component with a molecular weight of about 80,000. The results of electrophoresis show that all the active materials migrate toward the anode. In particular, the P fraction eluted on Sephadex G-200 separ-

The molecular weight of purified materials provided with enzymatic activity was evaluated according to Andrews (1965). technique by chromatography on Sephadex G-200-120 column (50 x 2.5cm) equilibrated and eluted by 0.15 M NaCI (3 ml/min); 5-ml fractions were collected. The proteins used as a standard were monomeric serum albumin (Sigma), lactic dehydrogenase (Boehringer), human 7-globufin II fraction (Sigma), catalase (Sigma) and thyreoglobulin (Sigma); blue dextran (Sigma) was used to evaluate the void volume. The proteins were detected by light-extinction measurements at appropriate wavelengths or by suitable enzyme assays; blue dextran was estimated by extinction at 625 rim. AChE was detected on the basis of its catalytic activity. The dissociation into subunits of the N native form was carried out in a 6 M guanidine hydrochloride solution (Rosenberry et al., 1974); an evaluation of molecular weight in the subunits was obtained, also in this case, by chromatography on Sephadex G-200-120 column equilibrated and eluted with 6 M guanidine hydrochloride (1.5 ml/hr, 1-ml fractions). Sigma protein preparations (rabbit phosphorylase A, monomeric BSA, ovalbumin and cytochrome c) were used as a standard. Kinetic measurements were performed according to the method of Ellman et al. (1961), by evaluating spectrophotometrically at 405 nm the hydrolysis of acetylthiocholine (ATC) and butyrylthiocholine (BTC) in a 50-mM phosphate buffer at pH 7.2. One enzymatic unit was defined as the amount of enzyme which catalyses the hydrolysis of 1 #mole of substrate/min at 25°C. Inactivation tests of the enzyme were also carried out using 10 5 M eserine, which, as is well-known, inactivates the cholinesterases. The values of Michaefis constant (K,,) relevant to the purified enzymatic forms were measured in the 0.1-25 mM range of substrate concentration. The electrophoresis of the materials provided with enzymatic activity was carried out at various stages of purification procedure; it was performed at 160 V for 1 hr on cellulose acetate strips in a 50-mM Tris--barbital buffer at

RESULTS

Table 1. Summary of purification procedure. The enzyme units were evaluated by using acetylthiocholine (ATC) as a substrate

Purification step

Total protein (mg)

Total activity (mU)

1. Supernatant after 100,000 g centrifugation 1 0 , 0 0 0 804,588 2. Sephadex G-200 chromatography (I) 2223 657,156 3. Sephadex G-200 P 380 69,195 chromatography (II) N 570 528,523 4. DEAE-cellulose F1 39 9000 chromatography (I) Fz 36 15,000 N 118 239,400 5. DEAE-cellulose F1 14 6046 chromatography (II) F2 10.3 6011

Specific activity (mU/mg) 80.5 296 182 927 231 417 2029 432 584

Recovery (Yo)

Purification factor

100

1

81.7 74.3 32.7

3.68 2.26 11.52 2.87 5.18 25.20 5.37 7.25

Multiple forms of acetylcholinesterase

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All the three enzymatic forms are completely inhibited by 10-s M eserine. The Km values, obtained by the statistical method of Wilkinson (1961), are shown in Table 2. Using ATC as a substrate, the N and F1 forms show an tO0

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150

GIOVANNIB. PRINCIPATOet al.

Table 2. K,. values (M) relative to the three enzymatic forms N, F~, Fz and to acetylthiocholine (ATC) or butyrylthiocholine (BTC) as a substrate Substrate ATC BTC

N

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8.94 x 10-5 8.67 x 10-4

identical K,, value, while it is more than doubled in F 2. Otherwise, using BTC, the K , values differ widely in the three enzymatic forms; F2 shows the highest value. Figure 4 shows the behaviour of the various active fractions in increasing concentrations of substrate. When using ATC, the form with a higher K,, (F2) does not show, unlike the others, substrate inhibition in the considered concentration range, whereas, in the presence of BTC, no enzymatic form shows substrate inhibition. DISCUSSION

The results obtained demonstrate the existence in A. caliginosa of three enzymatic forms to be classified as acetylcholine hydrolase (E.C. 3.1.1.7). In fact, they are completely inhibited by 10 -5 M eserine and hydrolyse more rapidly ATC than BTC.

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The three active fractions show molecular weights almost in progression (N ~ 180,000; F2 ~ 350,000; F 1 ~ 700,000); therefore, they are likely multiple forms of ACHE, respectively monomeric, dimeric and tetrameric. In particular, the molecular weight that we attached to N form is a mean value among those reported by other authors about AChE in certain species of Insecta (Krysan & Chadwick, 1966; Krysan & Kruckeberg, 1970; Huang & Dauterman 1973). On the other hand, the existence of AChE multiple forms was widely reported as regards the nervous system (Wenthold et aL, 1974; Rosenberry, 1976) and erythrocytes (Wright & Plummer, 1973) in Mammals and also concerning the nervous system of Insecta (Huang & Dauterman, 1973). Moreover, the smallest native enzyme unit (N) observed by us is formed of subunits with a lower molecular weight; these are probably identical or very similar. The existence of a quaternary structure in the native AChE was also reported as regards both Vertebrata (Rosenberry, 1976) and Invertebrata (Emson & Kerkut, 1971; Habibulla & Newburgh, 1973). On the basis of our results, it seems unlikely that in A. caliginosa there are isoenzymes of ACHE, because each of the three enzymatic forms isolated by us migrates as a single band in cellulose acetate strips electrophoresis. The K,. values which we observed by using ATC as a substrate, show the same size (10 -5 M) reported by other authors as regards the AChE in Insecta (Heilenbrand & Krupka, 1970; Kawal Dhari et al., 1976) and Nematoda (Nolan & Schnitzerling, 1975). Moreover, K,, measurements in the various enzymatic forms, carried out using two different substrates, lead us to believe it probable that polymerization modifies the enzyme affinity for both ATC and BTC. Substrate inhibition of the N and F~ forms is caused by ATC but not by BTC; a similar difference was also reported by Guilbault et al. (1970) as regards AChE extracted from the bee and using acetylcholine and butyrylcholine as substrates. The possible physiological role of what we observed still remains to be determined and requires further and more extensive research. Studies about the kinetic characterization of the smaller native AChE (N) isolated by us in A. caliainosa are now in progress, in order to carry out a comparison with the analogous enzymatic forms already described in other species.

0s Fig. 4. Substrate inhibition curves relative to N, F1 and F2 enzymatic fractions with acetylthiocholine (O) and butyrylthiocholine (O).

The authors carried out the purification of acetylcholinesterase (ACHE) present in Allolobophora caliginosa and studied certain characteristics of it. The enzyme was obtained in a soluble form by homogenizing specimens of A. caliginosa and centrifuging the homogenate at 100,000g; the material obtained by lyophilization of the supernatant was then subjected to repeated chromatographies, performed on Sephadex G-200 and DEAE-cellulose. A good purification degree of the enzyme was thus obtained and three active fractions (F~, F 2, N) were isolated. They show a higher substrate affinity for acetylthiocholine (ATC) than butyrylthiocholine (BTC) and are inhibited by eserine; therefore, they are classifiable

Multiple forms of acetylcholinesterase as acetylcholine hydrolase (E.C. 3.1.1.7). Moreover, they show molecular weights in progression and are probably monomeric (N ~ 180,000), dimeric (F2 ~ 350,000) and tetrameric (F~ ~ 700,000) forms. The monomeric one is quantitatively prevailing over the others and shows the highest specific activity; it probably consists of two identical subunits. Each of the three enzymatic forms electrophoretically migrates as a single band and therefore the existence of A C h E isoenzymes in A. caliginosa is unlikely. The monomeric (N) and tetrameric (F1) forms show substrate inhibition with ATC, whereas none of the three forms shows substrate inhibition with BTC.

REFERENCES

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KAWAL DHARI, NAM PI~KASH ~ MEHROTRA K. N. (1976)

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Properties of acetylcholinesterase from rat brain. J. Neurochem. 22, 945-949. WILKINSON G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. ~0, 324-332. WRIGHT D. L. & PLUMMER D. T. (1973) Multiple forms of acetylcholinesterase from human erythrocyte. Biochem. J. 133, 521-527.