Lamprey brain contains globular and asymmetric forms of acetylcholinesterase

Neurochem. Int. Vol. 12, No. 2, pp. 131-135, 1988 Printed in Great Britain

0197-0186/88 $3.00+0.00 Pergamon Press pie

LAMPREY BRAIN CONTAINS GLOBULAR AND ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE LI~ P E z ~ , * $ HUGH C. NICKSON*§and RONALD J. B ~ t a r c t *Department of Biology, Birmingham-Southern College, Birmingham, AL 35254, U.S.A. and ~'The Neuropsychiatric Research Program, School of Medicine, The University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. (Received 13 April 1987; accepted 7 August 1987) Almam:t--To obtain more information about the evolution of acetylcholinesterase in the vertebrates, we studied the choline~rase activity from the brain of the lamprey Petromyzon marinus. We found that the enzyme is true acetylcholinesterase and that 98% of it is present in the (34 globular form. Only I% of the enzyme was found distributed among the asymmetric forms A4, As and An; an additional 1% of the activity could not be extracted from the brain. The identity of the asymmetric forms was configmed by cotlagena~ digestion. These data demonstrate that asymmetric acetylchofinesterase is present in the CNS of organisms representing all classes of vertebrates. However, our results are incomistent with an evolutionary trend that has been observed for vertebrate brain acetyleholinesterase.

Vertebrate acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; ACHE) exists in various globular and asymmetric molecular forms. On the basis of experiments with AChE from the electric organ of the eel Electrophorus electricus, Bon et al. (1979) have developed a heuristic model which describes the polymorphism of the enzyme. The globular forms, Gi, (32 and (34 consist of 1, 2 or 4 catalytic subunits; the asymmetric forms, A,, A8 and A~2 consist of 1, 2 or 3 globular tetramers attached to an elongated collagenous tail. Various combinations of globular and asymmetric AChE have been found in skeletal muscle from organisms representing all classes of vertebrates (Massouli~ and Bon, 1982; Brimijoin, 1983; Massouli6 et al., 1984; Pezzementi et al., 1987;

Toutant and Massouli6, 1987). Globular and asymmetric forms of AChE have also been found in the central nervous system (CNS) of members of all vertebrate classes that have been examined thus far: Chondrichthyes (Witzemann and Boustead, 1981; Massouli~ and Bon, 1982), Osteichthyes (Guillon and Massouli~, 1976; Rodriguez-Borrajo et aL, 1982), Amphibia (Nicolet and Rieger, 1982; RodriguezBorrajo et o2., 1982), Reptilia (Rodriguez, Borrajo et al., 1982; Massouli~ and Bon, 1982), Aves (Rieger et al., 1980; Barat et aL, 1980), and Mammafia (Rieger et al., 1980; Grassiet al., 1982). Moreover, it has been suggested that there is an inverse relationship between the position of an organism on the phylogenetic scale and the proportion of asymmetric AChE present in the brain, with lower vertebrates containhag relatively more asymmetric enzyme (Bon et al., 1980; Rodriguez-Borrajo et al., 1982; Massouli~ and Bon, 1982; Brimijoin, 1983; Ramirez et al., 1984). However, data on AChE from the CNS of a representative of the most primitive class of vertebrates, the Agnatha, have not been reported. Therefore, to obtain more information about the evolution of AChE in the CNS of vertebrates, we investigated the polymorphism of AChE in the brain of the lamprey Petromyzon mar/nus, a member of the Agnatha.

~Address correspondence to: Dr Leo Pezzementi, Department of Biology, Birmingham-Southern College, 800 8th Ave, West, Birmingham, AL 35254, U.S.A. Tel.: 205-226-4869. §Present address: School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Abbreviations used: ACHE, acctylcholinesterase; BW284c51, 1,5-bis(4-ailyldimethylammoniumphenyl)pentane-3-one dibromide; CNS, central nervous system; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycolbis(beta-aminoethyl ether) N,N,N'N'-tetraacetic acid; HIS buffer, hish ionic strength NaHPO4 buffer with protease inhibitors; iso-OMPA, tetramonoisopropyl ~ A L PROCEDURES pyrophusphortetramide; LIS buffer, low ionic strength NaHPO4 buffer with prote.ase inhibitors; pseudc~ChE, Materials Spawning lampreys were obtained from Dr James Seelye l~eudocholinesterase; S1, S2, $3, supernatants from sequenfiai extractions; H4, pellet from sequential extraction. of the Hammond Bay Biological Station, U.S. Fish and 131

132

LEo PEZZEMENTIet al.

Wildlife Service, Rogers City, Mich. and kept in artificial pond water at 4°C. Clostridium histolyticum collagenase CLSPA was obtained from Worthington. [3H]Acetylcholine (50 mCi/mmol) was purchased from New England Nuclear. All other reagents were of analytical grade and obtained from Fisher or Sigma. Extraction o f AChE and velocity sedimentation

Adult spawning lampreys were killed and their brains were removed and homogenized at 4°C in a ground glass homogenizer. For pharmacological and kinetic experiments, brains were homogenized in 19 volumes of a high ionic strength extraction buffer (10mMNaHPO4, pH7.4, 1% Triton X-100, 1 M NaCI, 0.02% azide) and centrifuged at 30,000g for 30 rain at 4°C. For sequential extraction of globular and asymmetric forms of ACHE, the method of Younkin et al. (1982) was used. Both LIS buffer (10 mM NaHPO4, pH 7.4, 1% Triton X-100) and HIS buffer (10mMNaHPO 4, pHT.4, 1% Triton X-100, I M NaC1) contained a battery of protease inhibitors (Melanson et al., 1985). Velocity sedimentation was performed in 5-20% linear sucrose gradients as described previously (Pezzementi et al., 1987). Apparent sedimentation coefficients were calculated relative to the sedimentation ofcatalase (11.3 S) as described by Rosenberry and Scoggin (1984). AChE assay

AChE was assayed spectrophotometrically by the method of Ellman et al. (1961) and radiometrically by the method of Johnson and Russell (1975) as modified by Younkin et al. (1982).

Table 1. Sequentialextraction of globular and asymmetrictbrms of AChE AChE activity Fraction BuffeP pmol/min-mg b % extracted S1 LIS 401 + 135 95 ± 32 $2 LIS 15 _+9 3~ 5 $3 HIS 5+ 1 I :~: i H4 HIS 3+ 1 I ± t aSequentialextractionwas performed in low ionicstrengthextraction buffer (LIS) and high ionic strength extraction buffer (HIS) as described in ExperimentalProcedures. bUnitsof acetylcholinesteraseactivityare pmol of [3H]acetylcholine hydrolyzed per rain and mg (wet weight) of tissue. Means and SD of 4 separate experimentsare given. We found that 98% of the A C h E is extracted into LIS buffer (Sl and S2; Table 1), while only 1% is extracted into HIS buffer ($3); an additional I % of the activity could not be extracted (H4). To estimate the sedimentation coefficients and identify the forms of the A C h E in the LIS and HIS fractions, we analyzed samples of SI and $3 on sucrose gradients. The activity in SI sedimented predominantly at 9.8 S (Fig. 1A, cf. Table 2), which is consistent with the behavior of G4. In a few cases,

lO

Collagenase treatment

Collagenase digestion of asymmetric forms of AChE was done according to the method of Younkin et al. (1982), except the digestion contained 1 mg/ml of Clostridium histolyticum collagenase and were incubated for 2 h at 25°C. Parallel control incubations lacked collagenase.

RESULTS

To determine if the cholinesterase activity of lamprey brain is due to ACHE, we performed a series of pharmacologic and kinetic experiments. Approximately 95% of the acetylthioeholine-hydrolyzing activity was inhibited by 1 # M BW2Mc51, an inhibitor of ACHE, while 1 ~ M iso-OMPA and ethopropazine, inhibitors of pseudoeholinesterase (pseudo-ChE), had no effect on the enzyme. The cholinesterase activity hydrolyzed acetylthiocholine with a K m of 36 # M and exhibited inhibition by excess substrate. These data indicate that the vast majority of the enzyme is ACHE, not pseudo-ChE. To characterize the molecular forms of A C h E present in lamprey brain, we performed sequential extraction, homogenizing tissue first in LIS buffer, which preferentially extracts globular ACHE, and then in HIS buffer, which extracts asymmetric forms (Younkin et al., 1982; Fernandez et al., 1984).

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A

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40

Fig. 1. Sedimentation profiles of globular and asymmetric forms of AChE extracted from lamprey skeletal imuscle. AChE in S1 (A) and $3 (13) were separated on 5-20% sucrose ~ e n t s and assayed for [3H]acetylcholine hydrolysis as described in Experimental Procedures. Arrows indicate the position of catalase (11.3 S). Top of gradient is at left.

AChE from lamprey brain Table 2. SummmTof mdimeutat/oacoeffa~mtsof molecularforms of AChE from the tmdn of the lamlm~ Na~ra~ o c c ~ form Form M u n ± SD (Number)

G,

A,

(4)

(6)

Prodttcts of colktgenase digestionb Form A~ M(um :1:SD

A,

9.8+0.8 9.4+0.3 13.2:[:I.3

10.44-0.3 (3)

(4)

A.2 17.0+ I.I (5)

A~

A~2

15.4-1-0.5 (3)

20.5 ±0.9 (3)

(Nmber) qn the text,the sedimentationcoefficientsof the molecularform are referred to by the averat~ in this table. bTbeseformsare assumedto be the resultsof collagenasedigestion of A4, As and AI2. small, diffuse peaks of activity, probably corresponding to Gl and/or (32, were observed. AChE in S3 sedimented at 9.4, 13.2 and 17S (Fig. 1B), which probably represent the A4, As and A12 forms of the enzyme. To confirm the identity of the asymmetric forms, we treated enzyme in $3 with collagenase. Collagenase digestion converted the 9.4, 12.6 and 17 S AChE to more rapidly sedimenting forms with sedimentation coe~cients of 10.4, 15.4 and 20.5S (Fig. 2A). This shift in sedimentation coefficient is consistent with the removal of a collagen=like tail from the asymmetric enzyme. The sedimentation coetficients of the asymmetric AChE were not altered in parallel control incubations lacking collagenase (Fig. 2B). These sedimentation data suggest that 98% of the AChE corresponds to globular enzyme (SI and $2), while only 1% represents asymmetric forms ($3). A summary of the molecular forms of AChE from lamprey brain is presented in Table 2. DISCUSSION

133

Borrajo et aI., 1982; Grassi et a/., 1982; Ramirez et a/., 1982), the finding that this form of the enzyme is conserved throughout the evolution of the vertebrates argues that it plays a significant physiological function. Previous studies with Gnathostomata have led to the suggestion that the proportion of asymmetric AChE present in the brain decreases throughout vertebrate evolution. In the lower vertebrate classes (Chondrichthyes, Osteichthyes, Amphibia, and Reptilia), asymmetric AChE accounts for 5-15% of the brain ACHE; whereas, in higher vertebrate classes (Ayes, Mammalia), this percentage decreases to < 1% (Rodriguez-Borrajo et al., 1982; Ramirez et a/., 1984; Massouli~ and Bon, 1982). Our finding that asymmetric AChE comprises only 1% of the brain enzyme in a member of the most primitive vertebrate class contradicts this proposed evolutionary trend. It is therefore important to consider whether this low value for asymmetric AChE in lamprey brain i s accurate. Although the sequential extraction technique that we used preferentially extracts and separates globular and asymmetric forms into LIS

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A

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On the basis of data from sequential extraction, collagenase digestion, and velocity sedimentation on sucrose gradients, we conclude that globular and asymmetric forms of AChE are present in the brain of the lamprey Petromyzon marinus. Thus, asym. metric AChE has now been found in the CNS as well as the skeletal muscle of representatives of all classes of vertebrates. In contrast, asymmetric forms of AChE have not been found in organisms from the deuterostome invertebrate classes that have been investigated thus far--Urochordata and Echinoidea (Ozaki, 1974, 1975; Mcedel and Whittaker, 1979; Meedei, 1980; Akasaka eta/., 1986), even when their possible presence has been considered (Mcedel, 1980). This distribution of AChE molecular forms suggests that asymmetric AChE appeared early in vertebrate evolution. Although the role of asymmetric AChE in the CNS is unknown (Rodriguez-

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20 ~ Froction Number

40

Fig. 2. Effect of collagenase on sedimentation profile of asymmetricforms of ACHE. Molecular forms of AChE were digested with mllalma~, ,eparated on mcrme ~adients, and assayed for [~I]acetylcholine hydrolyms as described in Experimental Procedures. (A) collasem~ d/gestion; (B) control. Arrows indicate the posit/on of catalase (II.3S). Top of 8radient is at left,

134

LEO PEZZEMENTIel al.

and HIS buffer fractions, a certain amount of crosscontamination can occur. The HIS fraction could contain small amounts of globular enzyme (Grassiet al., 1982); and, in fact, small shoulders or peaks that correspond to G~/G2 can be seen in the $3 gradients. Small amounts of G4 would be obscured by the prominent A4 peak. However, the presence of globular contaminants in $3 would have led us to overestimate the amount of asymmetric AChE present. Conversely, in some tissues, up to 50% of the asymmetric enzyme can be extracted into LIS buffer (Bon, 1982). Because of the size of the G4 peak, we cannot determine whether any asymmetric enzyme was extracted into S 1. However, even if half of the collagentailed enzyme were solubilized by LIS buffer, the proportion of asymmetric AChE would increase only to 2%. Another extraction characteristic of asymmetric AChE may also have confounded the data. On the basis of sensitivity to the presence of Ca 2+chelators in the extraction buffer, two classes of asymmetric AChE have been identified. Class I AChE requires high ionic strength for extraction, while solubilization of class II AChE also requires high concentrations of a chelating agent (RodriguezBorrajo et al., 1982). It is possible that we extracted only class I enzyme from lamprey brain because of the low concentrations of EDTA and EGTA in our extraction buffers and, therefore, underestimated the amount of asymmetric ACHE. However, in the lower vertebrates, the class I enzyme is the predominant collagen-tailed form (Ramirez et al., 1984). Also, only 1% of the lamprey brain AChE activity remained unextracted after sequential extraction. Finally, it is possible that the asymmetric forms of lamprey brain AChE were degraded by proteolysis during the experiments. Such degradation was probably minimal since a battery of protease inhibitors was included in the extraction buffers, and incubation of the asymmetric AChE at 25"C for 2 h did not alter the sedimentation pattern of the enzyme appreciably. Thus, we conclude that our results are an accurate estimate of the amount of asymmetric AChE present in lamprey brain. But, we are also cognizant of the fact that there is considerable interspecific variation in the polymorphism of AChE (Massouli6 and Bon, 1982), and that other agnathans may possess different amounts of asymmetric esterase. In any case, it seems likely that the physiological role of asymmetric AChE in the CNS will have to be elucidated before any evolutionary trends can be rationalized. Acknowledgements--We thank George Kemp, George Brown, Mark Edge and Maureen L. Pezzementi for advice,

equipment, supplies and laboratory space. This research was supported by a Cottrell College Science Grant from Research Corporation to L.E

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