Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains.

Abstract

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.

Full text

Page 1

THE JOURNAL
0 1984 by The American Society of Biological Chemists, Inc.
OF BIOLOGICAL
CHEMISTRY
Vol. 259, No. 21, Issue of November 10, pp. 13186-13194 1984
Printed in (i.S.A.
Purification and Properties of the Membrane-bound Form of
Acetylcholinesterase from Chicken Brain
EVIDENCE FOR TWO DISTINCT POLYPEPTIDE CHAINS*
(Received for publication, January 23,1984)
Richard L. Rotund08
From the Carmgie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210
The major molecular form
(AChE) from chicken brain is a membrane-bound gly-
coprotein with an apparent sedimentation coefficient
of 11.4 S. Analysis of the purified protein by
tion, velocity sedimentation, and sodium dodecyl sul-
fate-gel electrophoresis shows that the solubilized ‘en-
zyme is a globular tetramer with an apparent
420,000. This membrane-bound form of AChE is hy-
drophobic and readily aggregates in the
detergent. These aggregates
pendent, relatively stable in the presence of high salt
concentrations, yet readily dissociate upon addition of
detergent to the 11.4 S form, indicating that the inter-
actions are hydrophobic.
Polyclonal and monoclonal antibodies raised against
chicken brain AChE purified by ion exchange chro-
matography, affinity chromatography, and prepara-
tive gel electrophoresis precipitate AChE enzyme ac-
tivity. However, these antibodies do not cross-react
with the enzyme from chicken muscle which prefer-
entially hydrolyses butyrylcholine. Immunoprecipita-
tion of isotopically labeled enzyme molecules from tis-
sue cultured brain cells and analysis by sodium dodecyl
sulfate-gel electrophoresis shows that AChE consists
of two polypeptide chains with apparent
(a) and 100,000 (8) in a 1:l ratio. Immunoblotting of
brain AChE with either the polyclonal or monoclonal
antibodies indicates that the a and / 3 chains share an-
tigenic determinants. Furthermore, both polypeptide
chains can be labeled with [3H]diisopropyl fluorophos-
phate, indicating that they each contain
This is the first indication that globular forms of AChE
may consist of multiple polypeptide chains.
of acetylcholinesterase
gel filtra-
M, =
absence of
are concentration-de-
M,= 105,000
a catalytic site.
Acetylcholinesterase (EC 3.1.1.7) is an important compo-
nent of cholinergic synapses in the peripheral and central
nervous systems, where it is in part responsible for terminat-
ing the actions of the neurotransmitter acetylcholine. Using
histochemical methods, this enzyme has been localized to the
region of nerve/muscle and nerve/nerve contact in numerous
electrophysiologically defined cholinergic systems. Because of
* This work was supported by Grant BNS 8015778 from the Na-
tional Science Foundation and a Grant from the Muscular Dystrophy
Association of America. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “aduertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
$Recipient of a Sloan Foundation fellowship in Neuroscience.
Present address, Department of Anatomy and Cell Biology (R-124),
University of Miami School of Medicine, 1600 N. W. 10th Avenue,
Miami, FL 33101.
its physiological importance, and the ability to localize this
enzyme using a variety of biochemical and histochemical
techniques, it is an important marker for studying the process
of synaptogenesis and subsequent nerveltarget cell interac-
tions.
The acetylcholinesterase of nerves and muscle constitutes
not one but a family of structurally related molecular forms,
the most common of which are monomers, dimers, tetramers,
and a complex asymmetric form consisting of three tetramers
covalently linked to a three-stranded collagen-like tail (see
Refs. 1-3 for reviews). In some species, this asymmetric form
of AChE’ appears to be concentrated at the regions of nerve/
muscle contact (4-6). Most of our knowledge concerning the
structure of AChE forms derives from studies on the enzyme
isolated from the electric organs of eel and ray (7-11); how-
ever, sufficient evidence exists to indicate that AChE from
rat, mouse, and chicken exhibits homologous series of oligo-
meric forms (12-14).
In addition to distinctions based upon subunit structure,
the AChE of nerve and muscle can also be classified into
membrane-bound, soluble or secretory forms, and by their
intracellular uerszm cell surface localization (15-18). In tissue-
cultured muscle cells, for example, about one-third of the total
cell-associated AChE is located on the external plasma mem-
brane (15, 17, 18). These cells also contain a rapidly turning
over intracellular pool of AChE molecules, the majority des-
tined for secretion (16, 19). However, a small portion of the
intracellular enzyme is destined for incorporation into the
external plasma membrane (15). This membrane-bound form
of AChE is solubilized only in the presence of detergents and
behaves in many ways like an integral membrane protein.
Thus, it is clear that a single cell type can synthesize not only
multiple oligomeric forms of AChE, but also molecules with
multiple subcellular destinations. The molecular basis for this
diversity is at present unknown.
In this paper, the purification and properties of a hydro-
phobic, membrane-bound form of AChE from chicken brain
are described. This particular AChE form is unique in that it
aggregates in the absence of detergents to give a new set of
molecular forms similar to those produced by collagenase
digestion of the 19.5 S asymmetric AChE from muscle (12,
14). Additional studies indicate that the AChE from brain
consists of two distinct polypeptide chains, each containing a
catalytic site, which share many of their antigenic determi-
nants. These results are discussed in terms of the heteroge-
neity of nerve and muscle acetylcholinesterase.
I The abbreviations used are: AChE, acetylcholinesterase; ACh,
acetylcholine, BuCh, butyrylcholine; le, immunoglobulin G;
BW284c51, 1,5-bis-(4-allyldimethylammon~umphenyl)pen~n-3-one
dibromide; NaDodS04, sodium dodecyl sulfate.
13186

Page 2

Chicken Brain AChE
13187
EXPERIMENTAL PROCEDURES
Materiaki-Benzyl chloroformate, 6-aminocaproic acid, N,N-di-
methyl-m-phenylenediamine HC1, dicyclohexyicarbodiimide, and
ethyl iodide were purchased from Aldrich. Hydrogen bromide (30%
in acetic acid) was from J. T. Baker Chemical Co. The marker enzyme
p-galactosidase and alkaline phosphatase were from Worthington.
Decamethonium, tetraisopropyl pyrophosphoramide, bacitracin,
benzamidine, and N-ethylmaleimide were from Sigma. Adult chicken
brains were purchased from Pel-Freeze Biologicals; DEAE-cellulose
was from Whatman; [3H]DFP and [%]methionine were from Amer-
sham Corp.; ["CIACh, ["CIBuCh, lSI-protein A, and endoglycosidase
F were purchased from New England Nuclear.
Synthesis and Coupling of Affinity Ligand to Sepharose-The syn-
thesis of (N-(6-aminocaproyl)-m-aminophenyl)dimethylethyl-am-
monium bromide hydrobromide was essentially as described by Ro-
senberry et al. (20) for (N-(6-aminocaproyl)-p-aminophenyl)-
trimethylammonium bromide hydrobromide with the following mod-
ifications. First, the meta rather than thepara isomer was synthesized
using N,N-dimethyl-m-phenylenediamine HCl as the starting com-
pound. During the subsequent alkylation step, ethyl iodide was sub-
stituted for methyl iodide, and the gummy precipitate was treated
with hydrogen bromide in glacial acetic acid without prior crystalli-
zation. The product was separated and washed using anhydrous ethyl
ether as described (20), and the final product was crystallized from
methanol and ethyl acetate. The affinity ligand has a X ,
and t = 11,100 in 0.1 M NaHC03, pH 10, with 0.5 M NaCl.
The ligand was coupled to Sepharose CL-4B as described by March
et al. (21) using 220 mg of ligand for 50 ml of settled beads. Approx-
imately 5.5 pmol of ligand were coupled per ml of beads. After
extensive washing, the beads were stored in 10 m M Tris, pH 7, 1 m M
EDTA, and 0.1% NaN3. Under these conditions, they remain stable
for at least 1 year.
Purification of Brain AChE-Adult chicken brains (300 g, stored
at -70 'C) were thawed and homogenized in 4 volumes of 10 m M
Tris, pH 7.0, 1 mM EDTA, and protease inhibitors consisting of 2
m M benzamidine, 5 m M N-ethylmaleimide, and 1 mg/ml bacitracin
(22), using a Tekmar homogenizer, followed by centrifugation for 30
min at 27,000 X g. The supernatant was discarded and the pellets
homogenized in 1,200 ml of Buffer A (10 m M Tris, pH 7.0, 1 m M
EDTA, 0.5% Triton X-100) containing protease inhibitors as de-
scribed above. After centrifuging for 45 min at 27,000 X g, the
supernatant was set aside and the pellets rehomogenized in 1,200 ml
of Buffer A with protease inhibitors, and centrifuged. The Triton
supernatants were pooled and pumped into a 1.2-liter DEAE column,
equilibrated with Buffer A, at 200-300 ml/h. This step, although
providing but modest enrichment, is necessary to remove several
proteins which interfere with affinity chromatography. The large
DEAE column is absolutely necessary as the presence of Triton X-
100 and the protease inhibitors severely reduce the capacity of the
resin. The column was washed with Buffer A + 25 m M NaCl, and the
enzyme was eluted with Buffer A + 150 m M NaCl. The peak AChE-
containing fractions (usually about 400 ml) were pooled and dialyzed
against 3,600 ml of Buffer A containing sufficient NaCl to give 50
m M final at equilibrium, assuming the peak fractions to contain 150
mM NaCl. Following dialysis, the enzyme was chromatographed
through a 25-ml affinity column, pre-equilibrated with Buffer A + 50
m M NaCl, at the rate of 0.5-1 column volumes/h. The affinity column
was washed with 2 column volumes of Buffer A + 50 m M NaC1, 2
column volumes of Buffer A alone, and the AChE eluted with 10 m M
decamethonium in Buffer A. Under these conditions, more than 95%
of the enzyme is bound to the column and subsequently eluted. The
peak fractions were pooled, concentrated using a 500-pl DEAE col-
umn, and stored at -70 "C.
Assay of AChE Activity-Acetylcholinesterase activity was assayed
using the radiometric method of Johnson and Russell (23). Fractions
were monitored during AChE purification by assaying 2-pl aliquots
of enzyme on ice in the presence of 10 m M decamethonium bromide
which inhibits enzyme activity by 95-98%. This is necessary due to
the very high turnover number of this enzyme. Even under these
conditions, and with short incubation times (2-3 min), enzyme activ-
ity often exceeded the linear range of the assay. Thus, in the final
stages of purification, the AChE activity in peak fractions is quali-
tative rather than quantitative and serves only to locate the desired
fractions.
To quantitate AChE activity, the enzyme was diluted up to several
thousandfold and assayed in Buffer A at 25 "C using 1.2 m M ["C]
of 238 nm
ACh as substrate. Butyrylcholinesterase was measured using the same
assay procedure with ["CIBuCh as substrate. Protein was measured
by the method of Lowry et al. (24) using bovine serum albumin as
standard.
Velocity Sedimentation-Acetylcholinesterase molecular forms and
aggregation states were determined by velocity sedimentation as
previously described (12) using @-galactosidase (16 S) and bacterial
alkaline phosphatase (6.1 S) as markers. Gradients were 5-20%
sucrose in 50 m M Tris, pH 7, 1 M NaC1, and 1 m M EDTA with or
without 1% Triton X-100. The samples were centrifuged in an SW
50.1 rotor at 5 "C. P-dditional details of centrifugation conditions,
sample size, and presence or absence of 60% sucrose cushions are
described in the figure legends. Unless noted, fractions from deter-
gent-free gradients were collected in polypropylene microfuge tubes
containing sufficient 25% Triton X-100 solution to give 0.5% final in
order to prevent absorption and/or denaturation of AChE and to give
a true estimate of AChE activity (see "Results").
Immunological Studies-Polyclonal antibodies were raised in rab-
bits by immunization with the 105,000-dalton AChE protein. Affinity-
purified AChE was precipitated with acetone, dissolved in NaDodSO,
sample buffer with 10% 0-mercaptoethanol, and heated to 100 "C
before running on preparative 515% polyacrylamide gradient gels.
After briefly staining the gel with Coomassie Blue, the 105,000-dalton
band was cut out, minced, and the protein electroeluted from the gel
and concentrated before use as antigen. Each injection consisted of
100-200 pg of protein in complete or incomplete Freund's adjuvant.
Rabbits were bled prior to immunization to obtain control serum.
Purified IgG was prepared from preimmune or immune serum by 50%
ammonium sulfate fractionation followed by DEAE-cellulose chro-
matography. The purified IgG fraction was dialyzed against and
stored in 10 m M phosphate-buffered saline at -20 "C. See figure
legends for details of the immunoprecipitation procedure.
Monoclonal antibodies were generated following the procedure of
Kennett et al. (25). Mice were immunized with affinity-purified
chicken AChE, 50 pglmouse in complete Freund's adjuvant, and
boosted twice with 50 pg/mouse in phosphate-bufferedsaline by direct
injection into the tail vein. Spleen cells were fused with SP3 myeloma
cells which is an IgG-secreting cell line. The hybridoma culture well
supernatants were initially screened for anti-chicken AChE antibod-
ies by the procedure of Manson et al. (261, using purified chicken
AChE coupled to cyanogen bromide-activated Whatman 540 filter
paper discs and '*SI-rabbit anti-mouse Fab as the probe. Positive
hybridomas were then screened using a second procedure to select for
antibodies with high affinity for AChE. Briefly, 25-pl aliquots of
rabbit-anti-mouse immunobeads @io-Rad Laboratories) were incu-
bated with 500-p1 culture well supernatants followed by extensive
washing with 10 m M phosphate buffer, pH 7.2, 150 m M NaCl. The
beads were then incubated with 500 pl of a dilute chicken AChE
solution prepared by homogenizing brain tissue in 120 volumes of
Buffer A containing 0.5% bovine serum albumin, 150 m M NaCl and
centrifuging 30 min at 27,000 X g before use. The beads were then
washed extensively with the above buffer and resuspended in 30 p1 of
buffer. AChE activity was assayed as described above using 10-pl
aliquots of bead suspension. Of the six anti-AChE-secreting hybri-
doma clones isolated in this manner, one was found to be an IgG
which bound very tightly to chicken AChE, bound to protein A from
Staphylococcus aureus, and did not interfere with acetylcholine hy-
drolysis. This hybridoma was subcloned in soft agar, and the resulting
hybridoma lines were selected for fast growth rate and high levels of
anti-AChE secretion. One hybridoma selected in this manner, here-
after referred to as 1A2 anti-AChE, was propagated as an ascites
tumor in Balb/c mice. The 1A2 IgG was purified from the ascites
fluid as described above. Control antibody consists of the SP3 IgG
originally secreted by this cell line.
Immunoprecipitation-Chicken embryo brain tissue was labeled
with [=S]methionine, 0.5 mCi in 3 ml of
minimal essential medium containing 10% horse serum and 2%
embryo extract. The labeled cells were washed with Hanks' balanced
salt solution and extracted into Buffer A containing 150 m M NaCI,
0.5% bovine serum albumin, and protease inhibitors. The extracts
were centrifuged 20 min at 27,000 X g, and the supernatants were
incubated at 5 "C on a rocker for 2 h with either 125 pg of preimmune
IgG or rabbit anti-chicken AChE IgG. To each sample was added 20
pl of a 1:l suspension of protein A Sepharose beads in 10 m M Tris,
pH 7, 150 m M NaCl, and NaN3, and incubation was continued
overnight. The beads were washed twice with 4 ml of
resuspended in 1 ml of Buffer A in a microfuge tube, and centrifuged
methionine-free Eagle's
Buffer A,

Page 3

13188
Chicken Brain AChE
TABLE I
Summary o f AChE Purification
is based upon 250 g of starting material, and each homogenization step was
2-4 aliquota of sample, diluted when necessary, incubated
in 1.2 m M [“CIACh at 25 “C in total volume of 22 PI. All samples were assayed in the absence of inhibitor; there
is no effect of dilution on AChE activity under these conditions. The pooled
column could not be assayed in this series due to the presence of inhibitor.
T O ~ ~ ~ A C ~ E 2~2~ Specific
This particular purification scheme
in 1 liter of buffer. Enzyme activity was measured using
peak fractions from the affinity
Fraction Volume
Purification
activity factor
rmoljminjmg
protein
AChE
recovery
ml
mmoljmin
mg
%
Whole homogenate
23,938
First
Pooled Triton supernatants
DEAE peak
Affinity column peak“
Pool I AChE from second DEAE
Pool I1 AChE from second DEAE
Total AChE
1,250
930
1,680
330
12
2193.0
0.78 6.3
5.35 0.224
supernatant (without )
100
10

0.53
3.81
2.61 1.06
5,115
6,938
2,459
0.104
0.558 2.5X
4.7x
72
49
0.78 1.34
0.335
0.611

9790X 25

(pools I and 11)
31.3
Specific activity not determined due to presence of 10 mM decamethonium.
through a 100 pl of 60% sucrose cushion in Buffer A. The beads were
washed again with
two 1-ml aliquota each of Buffer A, Buffer B
(Buffer A + 1 M NaCl), and Buffer C (Buffer A + 0.1% NaDodS04
and 0.3 M NaC1). The beads were boiled
buffer containing 50 m M dithiothreitol prior to analysis by polyacryl-
amide gel electrophoresis. Additional details are given in the figure
legends.
Immumblotting-Purified AChE was precipitated with acetone,
solubilized with NaDoDS04 sample buffer containing
threitol, and electrophoresed on 7.5% acrylamide gels using
method of Laemmli (27). The proteins were then transferred
nitrocellulose paper following the procedure of Towbin et al. (28).
Strips of nitrocellulose with bound AChE were incubated with 5-ml
aliquot8 of either preimmune rabbit IgG (25 pg/ml), rabbit anti-AChE
IgG (25 pg/ml), SP3 IgG (10 pg/ml), or 1A2 anti-AChE IgG (10 fig/
ml). The bound antibodies were localized using ‘%I-labeled protein
followed by autoradiography.
in 50 pl of NaDodSO, sample
150 mM dithio-
the
to
A
RESULTS
Purification of Brain Acetylcholinesterase-Approximately
95% of acetylcholinesterase in adult chicken brain occurs as
a membrane-bound glycoprotein with a sedimentation coef-
ficient of 11.4 S; the remainder consists of a 7.4 S form which
is soluble in the absence of detergents (data not shown).
Acetylcholinesterase appears to be heterogeneous with respect
to charge and is eluted from DEAE over a wide range of ionic
strength. For preparative scale purification, the enzyme is
stepped off the column using 150 m M NaC1, with ensuing
losses in recovery, since above this salt concentration other
proteins are eluted which interact with the affinity column
and either interfere with AChE retention or contaminate the
eluted peak. In our experience, preliminary fractionation of
brain AChE by ion exchange chromatography is necessary to
remove those interfering proteins. Other investigators have
not found this step necessary (29); however, this may reflect
the fact that they were purifying the enzyme from isolated
caudate nuclei, which contain predominantly cells bodies,
whereas our starting material is a much cruder whole brain
preparation.
Following DEAE chromatography, the pooled AChE-con-
taining fractions are dialyzed overnight against Buffer A
containing sufficient NaCl to yield 50 m M final concentration
at equilibrium. This step is important
NaCl, AChE does not bind well to the affinity column, and
below this concentration, several proteins bind nonspecifi-
cally to the column and displace AChE (data not shown).
Under these conditions, more than 95% of the enzyme is both
bound to the column and eluted, and the increase in specific
since above 50 m M
activity at this stage is greater than 2,000-fold (Table I). The
purified AChE migrates as a prominent broad band upon
NaDodSO,-gel electrophoresis with an apparent mean M, -
105,000 (Fig. 1). Several faint bands are still apparent after
affinity chromatography, most of which appear to be break-
down products of AChE (see later sections). These minor
contaminants can be removed by preparative gel electropho-
resis (data not shown).
Evidence for the Purification of Acetylcholinesterase-The
turnover number of chicken AChE has been determined by
titration with a stable irreversible inhibitor (30). Based on
the enzyme activity in crude brain homogenates, a turnover
number of 1 x lo7 ACh molecules/active site/h (30), and a
molecular mass of 105,000 daltons for each AChE subunit
(Fig. l), we calculate a concentration of about 10 pg AChE/g
of brain, wet weight (or about 0.01% of total protein). Our
actual values based on yield are about 9.8 pg of enzyme/g of
brain (Table I). Furthermore, the purification factor (9,780,
Table I) is in good agreement. with the predicted value of
10,000.
When purified AChE is labeled with [3H]DFP and analyzed
by NaDodS04-gel electrophoresis (Fig. l), a major broad band
of about 105,000 daltons is apparent, as well as several small
molecular weight bands which correspond to a subset of the
ones visualized by Coomassie stain. These minor bands most
likely consist of breakdown products of AChE because they
co-migrate with the 105,000-dalton subunit during gel chro-
matography on Bio-Gel P-200 in the presence of 1%
NADodSO, and the peak of AChE activity during velocity
sedimentation on 5-20% sucrose gradients.* However, the best
evidence that the 105,000-dalton band is the acetylcholines-
terase subunit derives from immunological studies (see later
sections).
The Purified Protein Is True Acetylcholinesterase-One
distinction between acetylcholinesterase and butyrylcholines-
terase is based upon the substrate specificities of the two
enzymes and their sensitivity to inhibitors which preferen-
tially react with one or the other active sites (31). For purified
chicken AChE, the rate of ACh hydrolysis is 120-150-fold
greater than the rate of BuCh hydrolysis, although the K,,, for
BuCh is about 3-fold lower than that for ACh (100 versus 300
p ~ ) . ’ Incubation of brain AChE with increasing concentration
of the specific AChE inhibitor BW284c51 indicates that ACh
hydrolysis is reduced with an Iso of about 5 X
M (Fig.
R. L. Rotundo, unpublished observations.

Page 4

Chicken B
cI1
116 -
94 3
68
- ? 1
57
6
43
-
31
FIG. 1. Gel electrophoresis of purified AChE. Enzyme from
the pooled affinity column fractions was reduced in the presence of
1% NaDodSO, and electrophoresed on a 10% polyacrylamide gel. Left
l a n e , purified AChE stained with Coomassie Blue. Approximately 30
pg of protein was loaded on the gel. Right lane, purified AChE was
labeled with [3H]DFP,
electrophoresed, and fluorographed. The num-
bers indicate the positions of molecular mass markers (in kilodaltons).
2A). On the other hand, tetraisopropyl pyrophosphoramide,
specific inhibitor of butyrylcholinesterase, has no effect on
ACh hydrolysis at concentrations up to
are obtained using butyrylcholine as substrate (Fig. 2B); its
hydrolysis is inhibited by BW284c51 rather than tetraisopro-
pyl pyrophosphoramide. The shift in the inhibition curve
depicted for BuCh in the presence of BW284c51 probably
reflects the higher affinity of acetylcholinesterase for BuCh,
in spite of its lower rate of hydrolysis, and hence lower
efficiency of the inhibitor.
Immunological Studies: Evidence for Two AChE Polypeptide
a
M. Similar results
'rain AChE
13189
Chins-Antibodies to chicken AChE, polyclonal and mono-
clonal, are highly specific in that they precipitate AChE, but
not butyrylcholinesterase enzyme activity (data not shown).
Both types of antibodies are species specific and do not cross-
react with AChE from several other laboratory animals, with
the exception of quail (Table 11). However, the multiple
molecular forms of chicken AChE are indistinguishable using
either our polyclonal or monoclonal antibodies. Due to space
limitations, a more detailed description of these antibodies
will be presented elsewhere.
Immunoprecipitation of [35S]methionine-labeled AChE
from organ cultured brain tissue and analysis by NaDodS04-
gel electrophoresis on 7.5% polyacrylamide gels indicate that
brain AChE consists of two distinct polypeptide chains (Fig.
3). Approximately 0.01% of the total label incorporated into
protein is immunoprecipitated under conditions
itate all of the AChE enzyme activity. The a and j3 chains
have apparent M, = -105,000 and -100,000, respectively.
Both polypeptides are present in equal amounts based upon
ratio of label incorporated. Immunoblotting of acetylcholin-
esterase purified from chicken brain with either polyclonal
antibodies (Fig. 4A) or monoclonal antibody 1A2 (Fig. 4C)
shows that the a and /3 chains share common antigenic
determinants. By either metabolic labeling or immunoblot-
ting, the a and j3 chains are always present in a ratio of 1:l.
The lower molecular weight bands detected by immunoblot-
ting correspond to many of those detected by Coomassie
staining of purified brain AChE (Fig. 1) and therefore are
most likely proteolytic fragments of AChE. It should be em-
phasized here that based upon the labeling studies presented
in Figs. 3-5, as well as recent studies from this laboratory on
the assembly of AChE forms (see "Discussion"), it is highly
unlikely that the j3 chain is a degradation product of the a
chain, but rather constitutes a separate polypeptide.
Both a and j3 AChE Polypeptide Chins Are Active Catalytic
Site-containing Glycoproteins-Endoglycosidase F is an en-
zyme that can remove both high mannose and complex N-
linked oligosaccharides from denatured glycoproteins (32).
Incubation of purified AChE, previously labeled with [3H]
DFP, with endoglycosidase F results in a marked decrease in
the apparent size of the protein determined by NaDodS04-
gel electrophoresis (Fig. 5). After removal of asparagine-linked
oligosaccharides, the a and j 3 AChE polypeptides are more
readily discernible, presumably due to a decrease in hetero-
which precip-
0 -10
-8
-6
-4
0 -10
-8
(log M )
-6
-4
Inhibitor Concentration
FIG.
amide. Aliquots of purified AChE (5 pl, 1 ng of protein) were mixed with 5 pl of buffer either with or without the
appropriate concentration of inhibitor. The reaction was started by the addition of 10 pl of ["CIACh or ["CIBuCh
to give 1.2 mM final. In this assay, the absolute rate of ACh hydrolysis was 130-fold greater than the rate of BuCh
hydrolysis. A, inhibition of ACh hydrolysis by BW284c51 and tetraisopropyl pyrophosphoramide. B, inhibition of
BuCh hydrolysis under identical conditions. All values are expressed in terms of their respective controls incubated
in the absence of inhibitor.
2. Inhibition of purified acetylcholinesterase by BW284cSl and tetraisopropyl pyrophosphor-

Page 5

13190
Chicken Brain AChE
TABLE
I1
Species specificity of polyclonul antibodies to chicken brain AChE
Brain tissue from each species was homogenized in 10 volumes of
Buffer A containing 0.15 M NaCI, 0.5% bovine serum albumin, and
protease inhibitors and centrifuged for 30 min at 27,000 X g. Electric
eel AChE was purchased from Worthington and diluted in the same
buffer. All samples were further diluted with the extraction buffer to
give approximately equal enzyme activities per unit volume. Aliquots
of each enzyme sample were incubated with varying concentrations
of anti-chicken AChE IgG and precipitated as described in the legend
to Fig. 3. The antibody concentration is
concentration exhibiting binding detectable by immunoprecipitation,
or the highest antibody concentration tested in the case of negative
results. Identical species specificity is obtained using the monoclonal
1A2 anti-AChE IgG except that binding is detectable at lower anti-
body concentrations. Even at the highest antibody concentrations,
we do not detect binding to butyrylcholinesterase assayed using [“C]
BuCh as substrate.
Antibody
bindine
expressed as the lowest
Species
Antibody
concentration
~~
Chicken
Quail
Rat
Mouse
Rabbit
Frog (Xenopus)
Electric eel
d m 1
0.1-0.3
0.3-1.0
100
100
100
100
100
+
+
-
-
-
-
-
PI -
-1 16
-94
-68
- 57
- 43
-31
FIG. 3. Immunoprecipitation of [35S]methionine-labeled
brain AChE. Brain tissue was obtained from 12-day-old chick em-
bryos, rinsed in balanced salt solution, and minced in 3 ml of methi-
onine-free Eagle’s minimal medium containing 10% horse serum and
0.5 mCi of [‘“SS]methionine. The tissue was incubated for 4 h at 37 “C,
then washed in Hanks’ balanced salt solution, extracted in 4 ml of
Buffer A containing 150 mM NaCI, 0.5% bovine serum albumin, and
protease inhibitors. After centrifugation, aliquots of the supernatant
were incubated with either preimmune IgG or anti-AChE IgG, fol-
lowed by protein A Sepharose 4B. The beads were washed, and the
bound material was eluted with NaDodSO. sample buffer containing
50 mM dithiothreitol. Approximately 0.01% of the total labeled pro-
tein is AChE. Aliquots were then analyzed by NaDodS04-gel electro-
phoresis in 7.5% polyacrylamide gels according to Laemmli (27) and
fluorographed. Lune P, preimmune I&. Lune I, precipitation with
anti-AChE IgG. About 2,000 cpm were loaded onto the gel. The
apparent M, of the two subunits are (Y = 105,000 and @ =
100,000.
1 1 1
9,
6 1
5
4
3
ABCD
”w’
6.
d
w
4. I ,
:
3. b’
1.
-a
- B
FIG. 4. Immunoblotting of purified brain AChE. Identical
aliquots of purified brain AChE (approximately 10 pg of protein/
lane) were analyzed by NaDodS0.-gel electrophoresis and transferred
to nitrocellulose paper, as described under “Experimental
dures.” Individual lanes were cut out and probed with either rabbit
anti-AChE IgG (A), rabbit preimmune IgG (B), monoclonal 1A2 anti-
AChE IgG (C), or nonspecific monoclonal
myeloma cell line iU). Antibody concentrations were 25 pg/ml (lnnes
A and B) or 10 pg/ml (lanes C and D) in a total volume of 5 ml. The
bound antibodies were localized using ’2sII-labeled protein A followed
by autoradiography.
Proce-
IgG derived from P3
116-
94-
. a
.l3
FIG. 5. Treatment of [3H]DFP-labeled acetylcholinesterase
with endoglycosidase F. Purified AChE was labeled with [‘HJDFP
(1 pCi/lO pg of AChE), precipitated and
remove unreacted DFP, and resuspended in 10 mM sodium phosphate
buffer, pH 6.1, 50 mM EDTA, 0.1% NaDodS04, 0.1% !j-mercaptoeth-
anol and heated. All procedures were as described by Elder and
Alexander (31). Each incubation mixture consisted of 5 pg of AChE
(about 10,000 cpm of [3H]DFP/pg of protein), 100 mM sodium phos-
phate buffer, pH 6.1, 1% Nonidet P-40,
without 0.2 units of endoglycosidase F, in a total volume of 30 pl.
Incubation was for 1 h at 37 “C. The reaction was stopped by the
addition of 20 pl ( 4 ~ ) NaDodSO. sample buffer containing dithio-
threitol and heated. Aliquots were analyzed on a 7.5% polyacrylamide
gel according to Laemmli (27) and fluorographed.
washed with acetone to
50 mM EDTA, either with or
geneity associated with the carbohydrate portions of the mol-
ecules. The decrease in apparent molecular mass corresponds
to about 10-12,000 daltons of asparagine-linked oligosaccha-
ride, which in turn could correspond to 3-4 such groups/
polypeptide chain. In addition, this experiment shows that
both the a and p polypeptide chains contain active catalytic
sites since both can be labeled with the active site directed
ligand [“HIDFP.
The 11.4 S Brain AChE Is a Tetramer-Analysis of the
purified membrane-bound form of AChE by gel filtration on