journal of biological chemistry 262, no. 25, pp. q by … · 2017-10-16 · acetylcholinesterase...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, No. 27, Issue of September 25, pp. 1329&13298,1987 Printed in U.S.A.

Isolation and Characterization of Acetylcholinesterase from Drosophila”

(Received for publication, July 17, 1986)

Ann L. GnageySB, Michael ForteST, and Terrone L. RosenberryII** From the Departments of 11 Pharmacology and $Biology, Case Western Reserve University, Cleveland, Ohio 44106

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none ( ~ 4 % ) corresponded to collagen-tailed forms. The membrane- bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an M, of 165,000 similar to that observed for dimeric (G,) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfide reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified Gz enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other Gz acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more effi- ciently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.

* This investigation was supported by a postdoctoral fellowship (to A. L. G.) from the American Heart Association, Northeast Ohio Affiliate, by Grant NS-16577 from the National Institutes of Health and a grant from the Muscular Dystrophy Association U. S. A. (to T. L. R.), and by Grants BNS-8302600 and PCM-8309110 from the National Science Foundation (to M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Current address: Dept. of Human Physiology, Flinders Medical School, Flinders University, Bedford Park, South Australia 5042.

7 Current address: Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201.

** To whom reprint requests should be addressed.

Acetylcholinesterase (AChE,’ EC 3.1.1.7) is associated with cholinergic synapses where it hydrolyzes the neurotransmitter acetylcholine. AChE from several species is known to exist in a number of molecular forms which have been divided into two classes, asymmetric and globular (for reviews, see Mas- souli6 and Bon, 1982; Rosenberry, 1985). The globular forms correspond to monomers (GI), dimers (G2), and tetramers (G,) of similar catalytic subunits. The asymmetric forms are char- acterized by the presence of a collagen-like tail associated with one, two, or three catalytic subunit tetramers. Globular forms can be further subdivided into two categories: soluble forms and detergent-binding forms which contain a hydrophobic domain that presumably anchors the enzyme in phos- pholipid membranes (Lazar and Vigny, 1980; Massouli6, 1980).

In Drosophila and other insects, most of the AChE activity is found in the central nervous system rather than in the periphery (Jan and Jan, 1976). Since Drosophila is an excellent model for a genetic study of the cholinergic system, there has been considerable interest in Drosophila AChE (Dewhurst and Seecoff, 1975; Hall and Kankel, 1976; Dudai, 1977; Green- span et al., 1980; Morton and Singh, 1980; Zingde et al., 1983; Melanson et al., 1985). On a genetic level, both conditional and nonconditional mutations exist which appear to affect AChE activity. These mutations map to a single locus, the Ace locus, and have a dramatic impact not only on the development and elaboration of the nervous system but also on the behavioral responses of mutant adults. Biochemical reports agree that both soluble and membrane-bound AChE forms are present in Drosophila, but information to date has largely involved AChE in crude extracts. We report here the first purification of Drosophila AChE to homogeneity. Since the complete open reading frame of cDNAs corresponding to the Ace locus has recently been sequenced and shown to correspond to AChE (Hall and Spierer, 1986), we find a structural comparison of Drosophila AChE to other AChEs to be of particular interest. We also document the presence of glucosamine and ethanolamine in Drosophila AChE, components which together with phosphatidylinositol or a close derivative are found in covalently attached C-terminal glycolipid anchors in GZ AChEs and certain other membrane proteins (Haas et al., 1986; see Low et al., 1986). In addition, we show the substrate specificity of Drosophila AChE to be intermediate to those of mammalian AChEs and ChEs.

butyrylcholinesterase; DS AChE, detergent-solubilized AChE LSS The abbreviations used are: AChE, acetylcholinesterase; ChE,

AChE, AChE solubilized at low ionic strength in the absence of detergent; HS AChE, AChE solubilized at high ionic strength; BW 284~51, 1:5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one di- bromide; DFP, diisopropyl fluorophosphate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EGTA, [ethyl- enebis(oxyethylenenitrilo)]tetraacetic acid.

13290

Acetylcholinesterase from Drosophila 13291

EXPERIMENTAL PROCEDURES

Materials-Drosophila melanogaster were maintained in the De- partment of Developmental Genetics and Anatomy at Case Western Reserve University. AChE was purified from human erythrocytes (Rosenberry and Scoggin, 1984) and eel electric organ (Rosenberry et al., 1974) as described previously. Bovine catalase and serum albumin were obtained from Sigma and polypeptide standards for SDS-PAGE were from Bethesda Research Laboratories. [3H]DFP (6.5 Ci/mmol) was from Amersham Corp., and ["CIHCHO (40 Ci/mol) was from ICN.

Protein Determination-Absolute protein determinations were obtained from corrected amino acid analyses (Rosenberry and Scoggin, 1984) and were observed to be 91.3 f 1.5% (n = 4) of the spectrophotometric protein estimates obtained by following the procedures of Markwell et al. (1978) based on the methods of Lowry et al. (1951) with bovine serum albumin as a protein standard. Spectrophotometric protein estimates by the Markwell et al. (1978) procedure that included this calibration factor were used routinely.

Extraction of AChE from Drosophila-Flies were frozen quickly on dry ice, stored at -70 'C. and decapitated by shaking vigorously in a large flask. Heads were separated from other material by sieving (Schmidt-Nielsen et al., 1977). Heads were suspended in buffer a t low ionic strength (10 mM sodium phosphate, 1 mM EDTA, 1 mM iodoacetamide, 0.1 pg/ml pepstatin, pH 6.9) by adding 8-10 ml of buffer/g of heads, and the suspensions (20-600 ml) were homogenized with a Brinkmann Polytron (see Younkin et al., 1982) in six 15-5 bursts at 4 "C. Homogenates were centrifuged at 100,OOO X g for 45 min, and the supernatant was passed through eight layers of cheesecloth (LSS AChE extract). The pellet was washed where indicated by adding fresh buffer equivalent to the original volume followed by rehomogen- ization and recentrifugation. The pellet (1 volume) was resuspended in 3 volumes of buffer plus 1% Triton X-100, homogenized, stirred for 1 h at 4"C, and centrifuged at 100,000 X g for 45 min. The supernatant corresponded to the DS AChE extract. The pellet was subjected to the following sequential rehomogenizations and recen- trifugations where indicated: buffer plus 1% Triton X-100; buffer plus 1.0 M NaCl followed by buffer plus 1.0 M NaCI, 1% Triton X- 100 (HS AChE extracts).

Purification of AChE-DS AChE extract from 70-100 g of heads (700-900 ml) was mixed with 10 ml of affinity resin (acridinium linked to Sepharose CL-4B; Rosenberry and Scoggin, 1984) in a 500- ml centrifuge bottle and swirled for 1 h at 4°C. The resin with adsorbed AChE was sedimented at 3000 X g for 10 min, washed by resedimentation in 5 mM sodium phosphate, 1% Triton X-100 (pH 7), and packed in a column (1.5 X 20 cm). The column was washed with a series of solutions, all of which were buffered with 5 mM sodium phosphate, pH 7: 1% Triton X-100 (300 ml); 0.1 M NaCl in 1% Triton x-100 (500 ml); 0.1 M NaCl (200 ml); 0.5 M NaCl (300- 500 ml); buffer alone (100-250 ml). AChE was eluted with 5 mM sodium phosphate, 50 mM NaCI, 0.1% Triton X-100, 5 mM decamethonium bromide (Sigma), pH 7, at a flow rate of 40-50 pllmin. The affinity resin was regenerated for subsequent reuse by an overnight wash with 1% SDS at 25 "C and stored in 5 mM sodium phosphate, pH 7.

Sucrose Gradient Centrifugation and M, Estimation-Isokinetic sucrose gradients (5-25% or 5-15%) were prepared, centrifuged in a Beckman SW 41-Ti rotor, and analyzed as described in Younkin et al. (1982). All gradients contained 1 M NaCl and 10 mM sodium phosphate (pH 7), and Triton X-100 (1%) was included where indicated. Apparent sedimentation coefficients (S) were calculated as the migration relative to catalase (11.4 S). Stokes radii (Rs) were obtained by chromatography on Sepharose CL-4B in 1 M NaCl, 20 mM sodium phosphate (pH 7) with Triton X-100 (1%) included where indicated. Rs values were determined from plots of (-log uersus Rs (Bon et al., 1976) with catalase (Rs = 5.2 nm) as an internal marker and a calibration curve obtained previously (Rosenberry and Scoggin, 1984). Values of M, were estimated from the Svedberg equation M, = (sm,J(Rs)(L)/(l - u ) (see Rosenberry and Scoggin, 1984) assuming S Z O , ~ = S and the partial specific volume u = 0.715 ml/g (Bon et al., 1976, 1979).

SDS-PAGE-PAGE on gradient slab gels (separating gel 5 1 3 % ) was conducted according to Dreyfuss et al. (1984) and silver staining according to Morrissey (1981). Samples exposed to disulfide reduction were incubated in 1% SDS sample buffer containing 40 mM &thio- threitol a t 50 "C for 20 min. Nonreduced samples were treated simi- larly in the absence of dithiothreitol. Polypeptide molecular masses in kilodaltons were estimated by comparison of their migrations

relative to those of protein standards of known mass (Rosenberry and Scoggin, 1984).

Enzyme Assays-AChE activity was monitored by a spectrophotometric assay based on the procedure of Ellman et al. (1961) unless otherwise noted. Substrates were added to a standard assay mixture that contained sodium phosphate (0.10 M in total phosphate), 0.33 mM 5,5'-dithiobis-(2-nitrobenzoic acid), 1% Triton X-100 in 3.0 ml at 25 "C. Routine assays also included 0.5 mM acetylthiocholine and were adjusted to pH 7.0. Radioisotopic assay of AChE activity with [3H]acetylcholine was conducted as outlined by Younkin et al. (1982). Catalase was monitored by its Amnm.

At AChE substrate concentrations low enough to avoid substrate inhibition (tl-100 mM for acetylcholine), AChE activity corresponds to Equation 1.

In Equation 1, u is the observed hydrolysis rate, V is the maximum extrapolated hydrolysis rate as the concentration of substrate [SI approaches infinity, and rC,,.,,) is the apparent Michaelis constant. AChE catalysis is known to be inhibited by protonation of an active site group(s) as indicated in Equation 1, with KH the inhibition constant associated with Km(app)/V and K'R, that with 1/V (Rosen- berry, 1975). Values of pKH and ~ K ' H are about 6.3 with cationic substrates for eel (Rosenberry, 1975) and bovine erythrocyte AChE (Krupka, 1966), and similar values (6.4 f 0.3) were estimated in this study for Drosophila AChE. Estimates of Km(spp) for acetylthiocholine with human erythrocyte and Drosophila AChE average about 100 pM (Ott et al., 1975; Niday et al., 1977; see Table VI below). Insertion of these values into Equation 1 indicates that, in the routine acetylthiocholine assay noted above, u corresponds to 70% of V. The literature standard for AChE activity units is pmol of acetylcholine hydrolyzedper min with 2.7 mM acetylcholine in 0.1 M NaCl and 0.02 M MgCl, at pH 7.4 and 25°C (Rosenberry, 1975). Under these standard assay conditions, u corresponds to 90% of V. One unit of activity is defined here as 1 pmol of acetylthiocholine hydrolyzed per min under standard assay conditions ( u = 90% of V), and the routine assay rates are adjusted to these conditions by assuming 3.67 AA1lznm/

min/unit of activity (see Rosenberry and Scoggin, 1984). Butyrylthiocholine was assayed in the spectrophotometric assay

mixture described above at pH 8. Benzoylcholine was assayed spec- trophotometrically in 67 mM sodium phosphate at pH 8 with Atzanm = 6700 M" cm" (Kalow and Lindsay, 1955; Lockridge and La Du, 1978). Benzoylcholine assay concentrations ranged from 25 p~ (1-cm pathlength cuvettes) to 1000 PM (0.1-cm pathlength cuvettes). All assays were conducted at 25 "C. Km(app) values were determined from unweighted least squares analysis of [ S ] / u uersw [SI at pH 8.0 with 4-9 data points.

Titrations with the fluorogenic reagent 1-methyl-7-dimeth- ylcarbamoyloxyquinolinium iodide were carried out in 0.4 ml of 20 mM sodium phosphate (pH 7) as outlined by Rosenberry and Bern- hard (1971).

Radiomethylation and Amine Analysis-Purified Drosophila AChE in 0.1% Triton X-100 and 5 mM decamethonium bromide was reduc- tively methylated with 10 mM ["CIHCHO and 50 mM NaCNBH, in 5 mM sodium phosphate (pH 7) and repurified by a second cycle of affinity chromatography as outlined in Haas and Rosenberry (1985). Samples were subjected to SDS-PAGE, and gels were sliced and scintillation counted according to Barnett and Rosenberry (1979). Alternatively silver-stained bands were cut from the gels and hydrolyzed for radiomethylated amine analysis in a four-buffer amino acid analyzer system (Haas and Rosenberry, 1985).

RESULTS

Extraction and Purification of AChE-A sequential extraction procedure has been applied to mammalian tissue to resolve globular AChEs which are solubilized at low ionic strength from asymmetric AChEs which remain particulate at low ionic strength and are solubilized at high ionic strength (Younkin et al., 1982). An additional extraction step was included here to separate LSS AChE solubilized at low ionic strength without added detergent from DS AChE that re- quired 1% Triton X-100 for solubilization at low ionic strength (Lazar and Vigny, 1980). We found that LSS AChE

13292 Acetylcholinesterase from Drosophila

accounts for 40% of the total activity in the fly heads (Table I), a value in agreement with other estimates of 40-45% AChE solubilization under these conditions (Zingde et al., 1983; Melanson et al., 1985). Most of the remaining AChE corresponds to DS AChE. Little additional activity is solubilized at high ionic strength (HS AChE extracts in Table I), an indication that Drosophila heads contain little or no amounts of asymmetric AChE forms.

The extraction procedure was scaled up to permit AChE purification as indicated in Table 11. LSS AChE was partially removed prior to extraction with Triton X-100, and additional washing of the substantial pellet remaining after the initial Triton X-100 extraction was omitted. Adsorption of Drosoph- ila AChE in the Triton X-100 extract to the affinity resin during batch exposure was quite efficient. About 93% of the AChE was adsorbed to new resin, and >85% was adsorbed with regenerated resin. Subsequent wash of the resin as outlined under “Experimental Procedures” resulted in nonspecific elution of about 15% of the AChE, and 70-80% of the total applied AChE was recovered on specific elution with decamethonium bromide. The specific activity of the purified peak AChE fractions averaged 1350 -+ 200 units/mg for six preparations, about 4000 times that of the initial homogenate. Thus, AChE accounts for about 0.02% of the total protein in the initial homogenate. A second cycle of affinity chromatography gave no further increase in specific activity. The observed amino acid composition of the purified AChE is in

TABLE I Summary of sequential extraction of AChE from Drosophila

Fly heads (2-5 g) were extracted sequentially as outlined under “Experimental Procedures” with 10 mM sodium phosphate buffer, pH 7 (LSS AChE), buffer plus 1% Triton X-100 (DS AChE), and buffer plus 1.0 M NaCl and 1% Triton X-100 (HS AChE). Nonsolubilized activity remained in the pellet. Protease inhibitors included throughout were 10 mM EGTA, 5 mM iodoacetamide, 0.1 pg/ml pepstatin, 5 mM N-ethylmaleimide, 2 mM benzamidine, 1 mM EDTA. The enzyme activity with 0.5 mM acetylthiocholine in each extract fraction is expressed as a percentage of the total activity recovered. The data represent averages of three preparations except where noted.

AChE extract fraction Activity

%I

LSS DS

39.7 f 7.8

HS 56.7 f 7.4

2.5 f 0.3” Pellet 1.7 f 1.1

Average of two preparations.

TABLE I1 Summary of affinity chromatography purification of Drosophila

AChE Procedures are described under “Experimental Procedures,” and

the data represent one typical preparation from 70 g of fly heads assayed with 0.5 mM acetylthiocholine.

Fraction Total Specific activity activity

units unitslmg

Initial homogenate 3160 0.30 LSS AChE extract 220 0.08 Pellet resuspended in Triton X-100 2670 0.35 DS AChE extract 1610 0.65 DS AChE extract activity not re- 190 0.10

Peak fractions from affinity chro- 715 1305”

protein

tained by affinity resin

matography The specific activity for six preparations averaged 1350 & 200

units per mg of protein.

reasonable agreement with that calculated from the cDNA sequences (Table 111).

Hydrodynamic Properties of AChE-Sucrose gradient centrifugation provides information both about the size of indi- vidual AChE forms and about their interactions with nonionic detergents. A shift in apparent S value of an AChE form on adding detergent to the gradient indicates binding of detergent molecules to a hydrophobic domain in the enzyme and suggests that this AChE form may be membrane-bound in situ (Massoulit!, 1980). Both the LSS and the DS AChE extracts contain predominant AChE forms of about 7.5 S in the absence of detergent (also see Melanson et al., 1985). These forms are shifted to apparent 6.3-6.7 S positions when 1% Triton X-100 is added to the gradient (Fig. 1, A and E?). Purified AChE exhibits similar sedimentation coefficients and, in addition, partially aggregates in the absence of detergent (Fig. IC). Chromatography of the purified AChE on Sepharose CL-4B in 1 M NaCl, 20 mM sodium phosphate (pH 7) gave two peaks of enzyme activity, a broad early peak with a Stokes radius (Rs) of about 14 nm and a narrower peak of 5.6 & 0.1 nm that presumably corresponded to aggregate and 7.4 S species, respectively (data not shown). Addition of 1% Triton X-100 to the column solvent resulted in a single peak that eluted sharply at 6.6 f 0.1 nm, the shift again indicating a detergent interaction. Combining the 7.4 S and 5.6-nm Rs values with a partial specific volume previously used with AChE forms from several species (Bon et ab, 1979) results in an M, estimate of 165,000 for Drosophila AChE from the Svedberg equation. This M, is similar to that previously determined for G2 AChE from human erythrocytes (Rosen- berry and Scoggin, 1984; Dutta-Choudhury and Rosenberry, 1984) and indicates that Drosophila AChE also is a Gz form. An M, estimate of 160,000 for the predominant AChE purified from frozen heads of a strain of the housefly Musca domestica was derived by different techniques (Steele and Smallman,

TABLE 111 Amino acid composition of Drosophila AChE

Observed mole percentages are the means of seven determinations (each 15-20 pg of protein) from four purified preparations. Amino acids were detected with ninhydrin and corrected for decomposition and other changes during hydrolysis as outlined in Rosenberry and Scoggin (1984). Mole percentages were also calculated from Drosoph- ila AChE cDNA sequences (Hall and Spierer, 1986) with the approx- imation that the mature protein extended from Glya to SersZ1 (see “Discussion”). The observed mole percentage sum totals 96.0 and assumes the calculated values for cysteine and tryptophan, amino acids that are destroyed during hydrolysis. The corresponding mean residue weight was 110.34. Standard errors for the values listed did not exceed 0.25 mol % except for methionine (0.31 mol %).

Amino acid Observed Calculated mol %

Aspartic acid 11.2 11.3 Threonine 5.3 5.5 Serine 7.0 6.3 Glutamic acid 9.9 9.6 Proline 5.3 5.8 Glycine 9.0 9.2 Alanine 8.9 8.7 Valine 5.3 5.8 Methionine 3.7 3.1 Isoleucine 4.7 4.8 Leucine 7.0 6.7 Tyrosine 3.7 4.3 Phenylalanine 4.0 4.4 Histidine 2.3 2.4 Lysine 4.1 3.6 Arginine 4.6 4.6 Cysteine 1.4 Tryptophan 2.6


t I 0 20 40

FRACTION

i I 0 10 30

FRACTION

i I 0 10

FRACTION 30

FIG. 1. Sucrose gradient centrifugation of Drosophila AChE in crude extracts and following purification. Samples were applied to 5-25% ( A ) or 5-15% ( E , C) sucrose gradients that either contained Triton X-100 (+) or were free of detergent (0) and centrifuged as outlined under “Experimental Procedures.” A, LSS AChE extract (Table I; onput: 200 pl, 0.03 unit). Apparent S values of peak activity: 0, 7.6; +, 6.7 k 0.1 ( n = 3). E , DS AChE extract (Table I); +, 12-fold dilution into 10 mM sodium phosphate (onput: 350 pl, 0.07 unit); 0 (onput: 200 rl, 0.4 unit). Apparent S values of peak activity: 0, 7.4; +, 6.3 f 0.1 ( n = 5). C, purified AChE (Table 11) was diluted 100-fold into 10 mM sodium phosphate (pH 7) with (+) or without (0) 1% Triton X-100 (onputs: 240 pl, 0.4 unit). Apparent S values of peak activity: 0, 7.5 k 0.2 (n = 3); +, 6.6 f 0.2 ( n = 3). Enzyme activity in each fraction was determined by radioisotopic assay and is expressed as a percentage of the total activity recovery. Total activity recoveries ranged from 80 to 30%. The distance from the top of the gradient to the peak of the catalase marker (V) was normalized for the two gradients in each panel.

1976a) and suggests a close similarity between the two fly head AChEs.

The 6.3-6.7 S peaks observed for Drosophila AChE in crude LSS and DS AChE extracts appeared to have slight shoulders of trailing AChE activity. A second cycle of sedimentation applied to a DS AChE extract fraction from this region resulted in the emergence of two peaks of about 6.2 and 4.2 S (Fig. W). The latter S value was similar to that reported for GI AChEs produced by disulfide reduction of GS AChE forms from other species (Bon and Massoulie, 1980; Lee et al., 1982; Rosenberry and Scoggin, 1984). Reduction of the purified Drosophila enzyme converted the 6.6 S peak to a 4.7 S peak in the presence of Triton X-100 (Fig. 2B) or a 5.3 S peak in the absence of detergent, an indication that GI AChE was produced by reduction of the GS form. Thus, the small amount of more slowly sedimenting component in crude Drosophila extracts appears to be GI AChE that could arise by the action of either an endogenous protease (Vigny et al., 1979) or residual endogenous reducing agents (see Chang and Bock, 1977).

SDS-PAGE-Drosophila AChE purified by the procedure in Table I1 appeared free of significant protein contaminants by SDS-PAGE (Fig. 3, lanes 1 and 3 ) . The predominant band in the absence of disulfide reduction appeared to be about

A. DS SHOULDER

15

0 20 FRACTION

20

10

0

D

0 10 20 30 FRACTION

FIG. 2. Conversion of Drosophila AChE to a slower sedimenting form by disulfide reduction. Samples were applied to 5- 25% sucrose gradients that contained Triton X-100 and analyzed as in Fig. 1. A : 0, DS AChE extract (Table I; 0.2 ml, 0.4 unit); +, Fraction 32 (M) was diluted 5-fold into 10 mM sodium phosphate (pH 7) and 350 pl (0.02 unit) was applied. B: +, purified AChE was reduced with 10 mM dithiothreitol for 20 min at 25 “C and alkylated with 100 mM iodoacetamide, and 0.2 ml (0.15 unit) was applied; 0, a sample identical to + except that no dithiothreitol nor iodoacetamide was added.


200-

97-

68-

43-

26 -

18-

1 2 3 4 FIG. 3. PAGE analysis in SDS of purified AChE from Dro-

sophila. Samples were reduced with dithiothreitol except where indicated, arrayed on slab gels, and silver stained (Morrisey, 1981). Polypeptide standards: myosin, phosphorylase b, bovine serum albumin, ovalbumin, or-chymotrypsinogen, and P-lactoglobulin with molecular masses of 200, 97, 68, 43, 26, and 18 kDa, respectively, as indicated. Lanes 1 and 3, Drosophila AChE purified by affinity chromatography as indicated in Table I1 (1.4 pg of protein/lane). Lanes 2 and 4, AChE purified from human erythrocytes (Rosenberry and Scoggin, 1984; 0.4 pg/lane). Samples in lanes 1 and 2 were run in the absence of reducing agent.

twice the size of the predominant band following reduction, a relationship observed with purified G, AChEs from other species (Grossmann and Lieflander, 1975; Ott et al., 1975; Viratelle and Bernhard, 1980; Lee et al., 1982; Rosenberry and Scoggin, 1984; Stieger and Brodbeck, 1985) and consistent with an intersubunit disulfide linkage as suggested by the sedimentation profiles in Fig. 2B (see Vigny et al., 1979). An unusual feature of the predominant purified Drosophila AChE SDS-PAGE bands was their correspondence to apparent sizes of 100 kDa prior to reduction and 55 kDa after reduction, values that are 20-30% smaller than those reported for the purified G, AChEs from other species. This size difference was apparent from a comparison of the Drosophila AChE with purified human erythrocyte AChE (Fig. 3, lunes 2 and 4 ) . However, the Drosophila enzyme samples also showed faint bands that corresponded to those of the human AChE, 125- 130 kDa prior to reduction and 70 kDa after reduction. Since the native molecular weights of these two AChEs coincided, proteolysis of the Drosophila AChE subunits was suspected. If proteolysis generated 55-kDa polypeptides from intact 70- kDa subunits in the ratio indicated by the reduced sample in lane 3, and if the 55-kDa polypeptides retained sulfhydryl VOUP(S) involved in the intersubunit disulfide linkage, then prior to reduction one would expect an intense band corresponding to a dimer of 55-kDa polypeptides, a band of intermediate intensity corresponding to a dimer of one 55-kDa and one 70-kDa polypeptide, and a faint band representing a dimer of 70-kDa subunits. This is precisely the pattern obtained in lane 1 . An additional band a t 16 kDa was apparent in both the nonreduced and the reduced Drosophila AChE samples that could correspond to another AChE proteolytic fragment.

To investigate the possibility of proteolysis of Drosophila AChE, both purified AChE and crude membrane-bound AChE that corresponded to the DS AChE fraction were labeled at the active site with t3H]DFP and compared by

SDS-PAGE. Nondialyzable label ranged from 1500 to 2000 3H counts/min/original unit of AChE activity in both the crude and the purified AChE extracts. The constant ratio indicated that AChE was the predominant [3H]DFP-labeled protein in the crude Drosophila membrane preparation, a conclusion also reached by Zingde et al. (1983) by somewhat different criteria. By labeling the membrane-bound enzyme in the presence of a large battery of protease inhibitors (including some like benzamidine that were not included previously because they interfere with affinity chromatography) and extracting directly into SDS sample buffer, it was hoped that proteolysis would be minimized. However, the results in Fig. 4 show little effect of these protease inhibitors on the patterns of either purified or membrane-bound AChE. Labeled 100-kDa bands were apparent when no dithiothreitol was added to the SDS sample buffer, and 55-kDa bands predominated after reduction. Some conversion of the 100- kDa to the 55-kDa band occurred in the crude membrane extracts even when no dithiothreitol was added, but this observation is consistent with the endogenous proteolysis or reduction inferred from the partial formation of GI AChE in Fig. 2A. A minor band a t about 63 kDa is somewhat more intense in the crude membrane fractions prepared in the presence of protease inhibitors, and a minor band at about 70 kDa can be detected in the purified AChE following reduction. These labeled bands confirm that AChE polypeptides larger than those in the 55-kDa band exist. Thus, the data are consistent with proteolytic cleavage of an intact 70-kDa sub-

200 -

97-

68-

43-

2 6-

1 2 3 4 5 6 FIG. 4. Fluorographic analysis of [3H]DFP-labeled mem-

brane-bound polypeptides and purified AChE following SDS- PAGE. Purified AChE (16 units; lanes 1 and 2) was labeled with ['HH]DFP (6 p ~ ) for 1 h at 25 "C after which the labeled enzyme was dialyzed for 48 h at 4 "C and centrifuged at 10,000 X g. Sedimented pellets from LSS AChE extraction (Table I) were resuspended by homogenization in 10 mM sodium phosphate (pH 7). No protease inhibitors were present in one set (lanes 3 and 4 ) while in a second set, protease inhibitors (1 mM EDTA, 5 mM EGTA, 4 mM iodoacetamide, 2.5 mM N-ethylmaleimide, 0.1 pg/ml pepstatin, 2 mM benzamidine, and 1 mM aprotinin) were present throughout the homoge- nizations (lanes 5 and 6). ['HIDFP (6 p ~ ) was added directly to the suspensions containing 2 units of AChE activity for labeling and dialysis as outlined above, and 25 pl (7000 cpm) was taken for electrophoresis. Samples in lanes 1, 3, and 5 were nonreduced while those in lanes 2,4, and 6 were reduced with dithiothreitol in the SDS sample buffer. Calibration standards correspond to those in Fig. 3.


unit similar in size to that of human erythrocyte AChE. The protease mixture used in Fig. 4 did not prevent this putative cleavage. It is noteworthy that a report on the subunit struc- ture of purified housefly head AChE shows several parallels to our observations (Steele and Smallman, 1976b). SDS- PAGE following reduction of [3H]DFP-labeled housefly AChE revealed a predominant labeled band of 59 kDa, a minor labeled band of 82 kDa, and an unlabeled band of 20- 23 kDa.

Radiomethylation of AChE and Demonstration of Proteolytic Fragments-Radiomethylation is a sensitive and convenient method for the identification of free amine groups in proteins (Haas and Rosenberry, 1985; Haas et al., 1986). N-terminal amino acids (Haas and Rosenberry, 1985) as well as ethanolamine and glucosamine in a novel glycolipid at the C terminus of human erythrocyte AChE (Haas et al., 1986) have been quantitated. Radiomethylation of Drosophila AChE also reveals these putative glycolipid components. As shown in Table IV, analysis of Drosophila AChE revealed 0.6 f 0.1 residues of glucosamine and 2.1 f 0.4 residues of ethanolamine per 70- kDa polypeptide, values similar to those reported earlier for the glycolipid anchors of three other proteins, AChE, and decay-accelerating factor of human erythrocytes (Haas et al., 1986; Medof et al., 1986), and Thy-1 of rat brain (Fatemi et al., 1987). About 15 of the total of 21 lysine residues predicted from the cDNA sequences (see Table 111) were radiomethylated on their t-amino groups under the nondenaturing conditions employed (Table IV). Only one N“-radiomethylated amino acid ( “ X ) could be detected in significant amounts. This assignment as a Ne-amino acid was based both on a rate of radiomethylation that was much greater than those of all other amines in the protein (data not shown; see Haas and Rosenberry, 1985). X eluted from the analyzer near the position of radiomethylated lysine, but its elution position could not be correlated with any of the usual amino acids in Table 111.

Since glycolipid anchors containing glucosamine and ethanolamine have only been observed at the C terminus of membrane proteins (Haas et al., 1986; see Low et al., 1986), these amine groups should provide convenient markers for

TABLE IV Radiomethylation of Drosophila AChE and identification of amines

by amino acid analysis Radiomethylated samples prepared and repurified by affinity chro-

matography as outlined under “Experimental Procedures” were analyzed as described previously (Haas and Rosenberry, 1985; Haas et al., 1986). The radiomethylated residues/70-kDa polypeptide were calculated from the radiolabel specific activity and the content (Table 111) of the observed amino acids valine, phenylalanine, and arginine (Haas and Rosenberry, 1985). Confirmation of labeled “X” was obtained by radiomethylation of the purified enzyme preparation at 0.2 mM [“CIHCHO (data not shown), conditions under which Nu- amino acids are preferentially radiomethylated (Haas and Rosen- berry, 1985). This was the only Na-amino acid observed with a stoichiometry greater than 0.2 residue/polypeptide. Bands corresponding to those in Fig. 3 were cut from SDS-PAGE gels after staining with silver and analyzed as outlined under “Experimental Procedures.” Percentages were calculated from the distribution of total radioactivity among the bands (70 kDa, 9%; 55 kDa, 78%; and 16 kDa, 13%) and the distribution of label in each band. Values are averages from two radiomethylated preparations.

Amine Residues/ Percent of residues in band

po’YIJeptide 70 kDa 55 kDa 16 kDa

Glucosamine 0.6 +- 0.1 9 72 19 Ethanolamine 2.1 & 0.4 10 58 32 Lysine 15.1 f 3.8 9 82 9 N“-Amino acids

“X” 0.9 & 0.1 13 5 82

assessing the relationship of the 70-, 55-, and 16-kDa polypeptide bands. However, analysis of the radiomethylated polypeptides separated by SDS-PAGE gave complex results (Ta- ble IV). Glucosamine and ethanolamine in a roughly fixed ratio were associated with all three bands, although the 16- kDA band was somewhat enriched in ethanolamine. In contrast, X was associated primarily with the 70- and 16-kDa bands. Although the moles of each peptide in the SDS-PAGE bands are difficult to quantitate, the observation that about 10% of each labeled amine is present in the 70-kDa band suggests that about 90% of the subunits are fragmented to the 55- and 16-kDa bands. The presence of the glycolipid anchor components in all three bands clearly indicated that 55- and 16-kDa species are fragments of the 70-kDa polypeptide. However, the presence of these components in both bands suggests that the bands contain polypeptide mixtures.

Preliminary radiomethylation of the purified Drosophila LSS AChE also indicated ethanolamine and glucosamine.

Catalytic Properties of Drosophila AChE-The specific activity observed for purified Drosophila AChE is considerably less than that reported for purified AChEs from other species, as indicated in column 2 of Table V. To assess whether this lower value arose from incomplete purification or from an intrinsic lower activity of the active sites in the Drosophila enzyme, active site normalities were determined by titration with the fluorogenic reagent 1-methyl-7-dimethylcar- bamoyloxyquinolinium iodide (Rosenberry and Bernhard, 1971). These titrations permitted calculation of the turnover numbers of Drosophila AChE and of human erythrocyte AChE and their comparison to previously reported turnover numbers for eel AChE and human serum ChE as shown in column 3 of Table V. The Drosophila AChE turnover number is 3-6-fold lower than those of the other two AChEs in Table V. To demonstrate that this lower turnover number accounts entirely for the lower specific activity, the ratio of the turnover number to the specific activity was calculated. This ratio corresponds to the equivalent weight of each enzyme active site as shown in column 4 of Table V, and the equivalent weights for all four enzymes are the same within experimental

TABLE V Active site turnover numbers with acetylthiocholine and active site

equivalent weights for purified AChEs and ChE Specific activities are adjusted to standard assay conditions as

outlined under “Experimental Procedures.” Turnover numbers are defined here as units of activity under standard assay conditions per pmol of active sites, where the pmol of active sites was determined by titration with the fluorogenic reagent 1-methyl-7-dimeth- ylcarbamoyloxyquinolinium iodide as outlined under “Experimental Procedures.” Equivalent weights are calculated as the ratio of the turnover number to the specific activity.

Species Specific Turnover Equivalent activity number weight

unitslmg protein nin” X 1 O P mol sites g protein/

Eel AChE (11 S) 10,800“ 823” 76,000” Human erythro- 5,850 f 360’ 400 f 16 68,000 f 6,000

Drosophila AChE 1,350 +_ 200 107 f 3 79,000 f 12,000 Human serum 930‘ 70d 75,000‘

cyte AChE

ChE Data from Rosenberry (1975).

*Data from Rosenberry and Scoggin (1984). e Calculated from relative specific activity data in Das and Liddell

(1970) and the maximal specific activity with benzoylcholine reported by Lockridge and La Du (1982).

Calculated from the data in Footnotes c and e.

benzoylcholine (Lockridge and La Du, 1978, 1982). e Calculated from specific activity and turnover number values with


error. Furthermore, the equivalent weights are consistent with the subunit masses of about 70 kDa observed SDS-PAGE, compelling evidence that the enzymes are purified to a state free of contaminants.

The observations in Table V that the specific activity of Drosophila AChE toward acetylthiocholine was considerably lower than that of other AChEs but only about 50% higher than that of human serum ChE raised the question of whether the Drosophila enzyme was in fact an AChE rather than a ChE. To address this point, the substrate specificities of the Drosophila enzyme were compared to those of other AChEs and ChE in Table VI. AChEs typically have a very low efficiency of butyrylthiocholine hydrolysis relative to acetylthiocholine hydrolysis, and this is illustrated by V,,/V,, ratios of about 0.005-0.01 for the human erythrocyte and eel AChEs. However, this ratio for Drosophila AChE is about 0.6, only 3- fold less than that of human serum ChE and barely below the ratio of one traditionally used to designate an esterase as an AChE rather than a ChE. The efficiency of benzoylcholine hydrolysis roughly paralleIs that of butyrylthiochoIine for all four enzymes in Table VI, as ratios of Vb,/Vbu differ only by about a factor of 10.

Relative V values in Table VI do not vary between crude and purified Drosophila AChE preparations. Thus, there appears to be only one significant cholinesterase enzyme in Drosophila heads, and this enzyme has a substrate specificity intermediate to those of mammalian AChEs and ChEs.

DISCUSSION

The purification outlined in Table I1 results in a 2000-fold purification of AChE in the DS AChE extract to apparent homogeneity in one affinity chromatography step. Incomplete removal of LSS AChE prior to detergent extraction may have resulted in some LSS AChE in addition to DS AChE in the purified product. However, these two AChEs have very similar sedimentation properties as indicated in Fig. 1, and the extent of any structural divergence between them is unclear. In contrast to our observations with DS AChE extracts, preliminary experiments with [3H]DFP labeling of LSS AChE extracts indicated considerable nonspecific label incorporation.

TABLE VI Relative maximum velocities of hydrolysis of acetylthiocholine W m ) ,

butyrylthiocholine (Vbu) and benzoylcholine ( V b z ) for AChEs and ChE Purified enzymes were assayed except where indicated. AChE

assays utilized 0.5 mM acetylthiocholine, 1.0 mM butyrylthiocholine, or 50-500 p~ benzoylcholine at pH 8 as outlined under “Experimental Procedures.” Maximum velocities were calculated from the observed assay velocities according to Equation 1 with the following measured values of Km(app, in WM. Acetylthiocholine: Drosophila AChE, 79 f 3; human erythrocyte AChE, 168 f 4; eel AChE, 50 (Nolte et al., 1980). Butyrylthiocholine: Drosophila AChE, 104 f 7; human erythrocyte AChE, 210 -e 33; eel AChE, 820 f 30. Benzoylcholine: Drosophila AChE, 48 8; human erythrocyte AChE, 296 f 35; eel AChE, 480 +- 80.

Enzyme Vhl v, vbd v b u

Drosophila AChE Initial homogenate 0.56 & 0.02 LSS AChE extract 0.63 f 0.05 DS AChE extract 0.57 f 0.03 0.052 & 0.001 Purified stock 0.56 * 0.02 0.062 +- 0.008

Human erythrocyte AChE 0.011 f 0.001 0.61 f 0.07 Eel AChE 0.0055 f 0.0003 0.153 f 0.021 Human serum ChE 1.4,b 2.2‘ 0.153’ Benzoylcholine velocities were too low for accurate measurement. Reported maximum velocity ratios for human serum ChE (Das

Reported maximum velocity ratios for human serum ChE (Heil- and Liddell, 1970).

This may account for a previous report of a difference in SDS-PAGE profiles between 13H]DFP-labeled AChE in LSS and DS AChE extracts (Zingde et al., 1983). We have now purified LSS AChE by the same affinity chromatography procedure used here for DS AChE. No differences in silver staining profiles on SDS-PAGE are apparent for the two enzymes, and both contain the glycolipid anchor components glucosamine and ethanolamine (data not shown).

The sedimentation properties of purified 0, DS AChE forms from Drosophila are similar to those of purified GP DS AChEs from Torpedo marrnorata electric organ and human erythrocytes. Purified G, AChEs from all three species sedi- ment at apparent values of 6.3-6.6 S in the presence of Triton X-100 and aggregate in the absence of detergent (Fig. 1; Ott et al., 1975; Rosenberry and Scoggin, 1984; Stieger and Brod- beck, 1985). However, aggregation of the Drosophila G, AChE in the absence of detergent is only partial (Fig. lC), and the nonaggregated G, is shifted to 7.4 S. In this respect the Drosophila G, enzyme resembles purified G, DS AChE from human caudate nucleus, which only partially aggregates in the absence of detergent and shows a shift in the nonaggregated G, peak from 10.0 to 10.6 S (Gennari and Brodbeck, 1985). The magnitudes of these shifts in S value are consistent with the binding of these AChEs to detergent micelles (Ro- senberry and Scoggin, 1984). The S values of Drosophila enzyme in both the LSS and DS AChE crude extracts are also shifted in the absence of detergent, but only slight aggregation is apparent (Fig. 1, A and B ) . Detergent-binding AChE forms in crude LSS AChE extracts are also observed from torpedo electric organ (Bon and Massoulie, 1980; Lee et a t , 1982), mammalian brain (Rieger and Vigny, 1976; Grassi et al., 1982; Gennari and Brodbeck, 1985) and a cultured neu- roblastoma x sympathetic ganglion hybrid cell line (Lazar and Vigny, 1980). Although slight immunochemical differences between AChE in LSS and DS AChE extracts have been reported (see Rakonczay and Brimijoin, 1985), the extent to which LSS AChE which binds detergent differs from DS AChE and from LSS AChEs which do not bind detergent (Lazar and Vigny, 1980; Ralston et al., 1985; Gennari and Brodbeck, 1985) in membrane binding and cell localization in situ remains unclear.

The hydrodynamic data indicate that the Gz AChEs from both Drosophila and human erythrocytes have native M, values of 160,000-165,000 and are converted to active monomers by disulfide reduction. We ascribe the difference between these M, estimates for the dimers and the 125-130 kDa estimates from the largest nonreduced SDS-PAGE bands of these enzymes to intrinsic inaccuracies in the techniques rather than to additional polypeptides separated by SDS- PAGE. Radiomethylation of Drosophila AChE reveals glucosamine and ethanolamine in amounts comparable to those reported in mammalian glycolipid-anchored proteins (Haas et al., 1986; Medof et al., 1986; Fatemi et al., 1987). Ethanolamine and glucosamine susceptible to radiomethylation occur only in the C-terminal glycolipid anchor of the human erythrocyte G2 AChE (Haas et al., 1986), and the presence of these components in the Drosophila GP AChE strongly suggests a similar C-terminal glycolipid anchor. Since other evidence of a glycolipid anchor has been obtained in G2 torpedo AChE (Futerman et al., 1985), it appears likely that all membrane- bound Gz AChEs have similar C-terminal glycolipid anchors.

While the catalytic subunits of the asymmetric forms of Torpedo californica were the first AChEs to be cloned and sequenced (Schumacher et al., 1986), Drosophila AChE is the first G, AChE for which a complete sequence has been estab-

bronn,-1959). lished by cDNA cloning (Hall and Spierer, 1986). The two


sequences show extensive homologies, but the C-terminal sequences of the coding regions diverge. In particular, the Drosophila AChE C-terminal sequence contains mostly hydrophobic amino acids. cDNAs of other glycolipid-anchored proteins also indicate C-terminal hydrophobic sequences, and such sequences of 17-23 residues in trypanosome variant surface glycoproteins (Cross, 1984) and 31 residues in Thy-1 (Tse et al., 1985) are removed in a post-translational processing step which presumably is associated in general with at- tachment of the glycolipid anchor. The C-terminal sequences of AChEs also are of interest because, in the torpedo asymmetric AChE, a cysteine four residues from the C terminus is the only site of intersubunit disulfide linkage (MacPhee- Quigley et al., 1986). While a corresponding cysteine residue does not occur in the Drosophila AChE cDNA sequence, the putative glycolipid post-translational processing should place CYS"~ close (probably within 2-10 residues) to the C terminus of the mature protein. One can then ask whether Cys6I5 of Drosophila AChE is involved in intersubunit disulfide linkages. On initial inspection, the data in Table IV appear to argue against this site of linkage. The 16-kDa band with the amines expected in the C-terminal glycolipid anchor is observed on SDS-PAGE analysis in the absence of disulfide reduction. Since this band contains lysine residues and no lysine residue is found C-terminal to in the putative mature AChE, these observations could be interpreted as indicating that Cys6'' is not a site of intersubunit disulfide linkage. However, we have recently sequenced residues 2-5 of a peptide in the 16-kDa band and found that they correspond to Ile40-Leu43 (Hall and Spierer, 1986).2 Residue 39 is an excellent candidate for the N terminus of the mature protein based on the torpedo AChE sequence (Schumacher et al., 1986), and removal of a leader sequence corresponding to the first 38 residues is reasonable. Thus, the sequenced peptide in the 16-kDa band corresponds to an N-terminal fragment released by a single proteolytic cleavage of the mature 70-kDa subunit into 16- and 55-kDa fragments. The ethanolamine and glucosamine in the 16-kDa band would appear to indicate a second fragment from the C terminus. This second fragment could be a very short peptide with a cleavage site C-terminal to Cys6I5, since the glycolipid anchor could cause this fragment to migrate in the 16-kDa region on SDS-PAGE. It is unclear why no N"-amino acid corresponding to this fragment is observed. This interpretation permits Cys6I5 to be the site of intersubunit disulfide linkage, consistent with the pattern of disulfide linkages proposed for the torpedo asymmetric AChE (MacPhee-Quigley et al., 1986).

The Drosophila AChE fragmentation observed in this study occurs prior to denaturation in SDS. Radiomethylation of the enzyme in 1% SDS resulted only in a 10% increase in Ne- amino lysine radiomethylation and revealed no new amine residues (data not shown). However, the purified enzyme may not be completely free of contaminant proteases, as suggested by the relative instability of the purified Drosophila AChE preparations. These preparations progressively lose their ca- pacity for aggregation on Sepharose CL-4B in the absence of detergent over a period of months, and preparations stored for several months no longer show nonionic detergent interactions and exhibit a shift in the predominant SDS-PAGE band from 55 to 47 kDa (data not shown). Fragmentation occurring in the native enzyme i s not accompanied by fragment release, because the smaller 16- and 55-kDa polypeptides cannot be removed by gel exclusion chromatography and because the observed amino acid composition of the purified

R. Haas, T. L. Marshall, and T. L. Rosenberry, manuscript in preparation.

enzyme agrees with that calculated for the mature protein (Table 111). Subunit fragmentation has not been observed in purified human erythrocyte or torpedo Gz AChEs, but the 75- kDa catalytic subunits derived from asymmetric AChE in eel electric organ do undergo an apparent single cleavage to produce 50-kDa fragments that contain the active site serine and 23-27-kDa fragments that do not (Rosenberry et al., 1974).

Table VI indicates that purified Drosophila AChE has a substrate specificity for butyrylthiocholine and acetylthiocholine intermediate to those of mammalian AChEs and ChEs. The ratio of 0.56 for the maximum velocities of these two substrates is consistent with previous reports on impure fly head extracts. Cherbas et al. (1977) noted that at optimal substrate concentrations butyrylthiocholine was hydrolyzed about half as rapidly as acetylthiocholine by crude Drosophila extracts, and Krupka and Hellenbrand (1974) determined the maximum velocity ratio to be 0.50 for partially purified housefly head AChE. Substrate specificities are difficult to assign in crude extracts because of the possibility that several enzymes are contributing to the observed reaction rates. How- ever, the maximum velocity ratios in Table VI are the same with crude Drosophila extracts and the purified Drosophila AChE and thus indicate that there is only one detectable cholinesterase activity in Drosophila heads. Aside from the substrate specificity, the Drosophila AChE appears similar to mammalian AChEs. The Drosophila enzyme in crude extracts showed substrate inhibition with 10 mM acetylthiocholine, pronounced inhibition by 0.01-0.1 PM BW 284~51, and little inhibition by 0.1 mM tetramonoisopropylpyrophosphor- tetramide (Melanson et al., 1985). These characteristics are typical of mammalian AChEs but not ChEs.

The relationship between mammalian AChEs and ChEs is unclear. In addition to their distinct substrate and inhibitor specificities, an antiserum to human serum ChE showed no immunochemical cross-reactivity with human erythrocyte AChE (Eckerson and La Du, 19781, in contrast to the quan- titative cross-reactivity shown by an antiserum to human erythrocyte AChE with human brain and muscle A C ~ E S . ~ ChEs also differ from AChEs in appearing to have little or no role in acetylcholine hydrolysis at neuromuscular junctions (Ferry and Marshall, 1973), and the functional significance of ChEs is unknown. However, AChEs and ChEs have similar catalytic subunit masses (Lockridge, et al., 1979), exhibit parallel series of asymmetric and globular forms (see Massou- lib and Bon, 1982), and show considerable amino acid sequence homology (Schumacher et al., 1986; Hall and Spierer, 1986; Lockridge et al., 1987). Residues in the sequence that contains the active site serine of human serum ChE, for example, are indentical at 16 of 20 positions with those in the corresponding sequence of torpedo AChE and at 16 of 21 positions with those of the Drosophila AChE sequence.

AChEs and ChEs, therefore, are distinct yet related proteins. On a gene level, this observation is characteristic of proteins which have evolved from a common ancestral form by mechanisms such as gene duplication followed by functional and structural divergence. The functional rationale for the divergence of AChE and ChE in mammalian systems remains to be elucidated. Our observation that Drosophila extracts contain only a single cholinesterase with a substrate specificity intermediate to those of mammalian AChE and ChE suggests that the presumed gene duplication leading to the two cholinesterase forms occurred subsequent to the point at which the vertebrate and invertebrate lines diverge.

T. L. Rosenberry, C. Rosenstein, and M. Sheldon, manuscript in preparation.

13298 Acetylcholinesterase from Drosophila Acknowledgments-We thank Todd L. Marshall for excellent tech-

nical assistance and Dr. Robert Haas for helpful discussions.

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