Comparison of butyrylcholinesterase and acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are compared, the first focus being on the high homologies of the molecular forms, and the homologies in protein sequences. Cholinesterases are the prototype of a new family of related serine hydrolases. Secondly, the distribution and regulation of AChE and BChE is reviewed. In this context the proposed noncholinergic roles for cholinesterases are described. Finally, comparison of the structure and base composition of the genes may give clues to understanding the origin and evolution of AChE and BChE

Full text

Biochem.
J.
(1989)
260,
625-634
(Printed
in
Great
Britain)
REVIEW
ARTICLE
Comparison
of
butyrylcholinesterase
and
acetylcholinesterase
Arnaud
CHATONNET*
and
Oksana
LOCKRIDGEtl
*Departement
de
Physiologie
Animale,
Institut
National
de
la
Recherche
Agronomique,
2
place
Viala,
34060
Montpellier
Cedex,
France,
and
tDepartment
of
Pharmacology,
Medical
School,
The
University
of
Michigan,
Medical
Science
1,
Ann
Arbor,
MI
48109-0626,
U.S.A.
INTRODUCTION
In
vertebrates
two
different
enzymes
hydrolyse
acetyl-
choline.
Acetylcholinesterase
(EC
3.1.1.7;
AChE)
termi-
nates
the
action
of
acetylcholine
at
the
post-synaptic
membrane
in
the
neuromuscular
junction.
The
other
enzyme
hydrolyses
acetylcholine
as
well
as
many
other
esters,
but
has
no
known
physiological
function.
It
is
called
butyrylcholinesterase,
pseudocholinesterase,
non-
specific
cholinesterase,
and
cholinesterase
(EC
3.1.1.8).
In
this
review
it
is
called
butyrylcholinesterase
(BChE),
while
'cholinesterases'
refers
to
both
AChE
and
BChE.
In
vertebrates
both
enzymes
are
inhibited
by
1O-5
M-
eserine,
a
property
which
distinguishes
them
from
non-
specific
esterases.
AChE
and
BChE
can
be
specifically
in-
hibited
by
BW284C51
and
NN'-di-isopropylphosphoro-
diamidic
anhydride
[1].
Butyrylcholinesterase
is
studied
by
pharmacologists
because
it
is
responsible
for
the
hydrolysis
of
succinyl-
choline,
a
drug
used
in
surgery
as
a
short-acting
blocker
of
the
acetylcholine
receptor.
Some
patients
experience
prolonged
apnea
due
to
slow
hydrolysis
of
succinyl-
choline
which
can
be
related
to
a
genetic
variation
of
the
enzyme
[2].
The
role
of
AChE
in
some
tissues,
as
for
example
the
red
cell
membrane,
migrating
neurocrest
cells
[3,4],
and
early
myotendinous
junction
[5],
is
not
clear.
During
embryonic
development
a
pattern
of
organization
or
succession
of
BChE
and
AChE
has
been
reported,
leading
to
the
hypothesis
that
BChE
functions
as
an
embryonic
acetylcholinesterase
[6,7].
Protein
sequencing
[8],
as
well
as
the
recently
reported
cDNA
clones
and
deduced
amino
acid
sequences
for
these
enzymes
[9-13],
allow
a
better
comparison
of
AChE
and
BChE.
A
large
number
of
reviews
have
dealt
with
cholin-
esterases
[14-16]
or
more
specifically
with
AChE
[17-20]
or
BChE
[21-23].
In
this
review
the
two
enzymes
are
compared,
the
first
focus
being
on
the
high
homologies
of
the
molecular
forms,
and
the
homologies
in
protein
sequences.
Cholinesterases
are
the
prototype
of
a
new
family
of
related
serine
hydrolases.
Secondly,
the
dis-
tribution
and
regulation
of
AChE
and
BChE
is
reviewed.
In
this
context
the
proposed
noncholinergic
roles
for
cholinesterases
are
described.
Finally,
comparison
of
the
structure
and
base
composition
of
the
genes
may
give
clues
to
understanding
the
origin
and
evolution
of
AChE
and
BChE.
OCCURRENCE
OF
AChE
AND
BChE
IN
ANIMAL
PHYLA
All
organisms
seem
to
have
some
kind
of
enzyme
capable
of
hydrolysing
acetylcholine
[24],
but
it
is
not
clear
if
they
can
be
called
cholinesterases
by
the
defini-
tions
made
for
vertebrate
AChE
and
BChE,
which
are
inhibited
by
l0-5
M-eserine.
Drosophila
has
only
one
cholinesterase,
an
AChE
[10]
with
substrate
preferences
intermediate
between
AChE
and
BChE
[25].
In
addition,
Drosophila
has
an
unspecific
esterase
with
sequence
similarities
to
the
cholinesterases
[26].
The
presence
of
a
single
cholinesterase
in
insects
cannot
be
generalized
to
all
invertebrates.
Three
genes
coding
for
AChE
seem
to
exit
in
the
nematode
Caenorhabditis
elegans
[27].
In
heart
of
the
squid,
Sepia
officinalis,
the
presence
of
both
BChE
and
AChE
is
suggested
[28].
The
clear
demonstration
of
BChE
as
well
as
AChE
has
been
made
only
in
vertebrates.
The
BChE
of
Torpedo
has
some
of
the
specificities
of
AChE.
It
hydrolyses
acetylcholine
better
than
it
does
butyrylcholine
and
is
responsible
for
the
physiological
hydrolysis
of
acetylcholine
in
the
heart.
This
suggests
that
the
gene
duplication
that
gave
rise
to
the
two
enzymes
occurred
at
the
emergence
of
the
vertebrates
[29].
COMPARISON
OF
MOLECULAR
POLYMORPHISM
Both
BChE
and
AChE
exist
as
polymers
of
catalytic
subunits
(Fig.
1).
The
globular
forms
GI,
G2
and
G4
contain
one,
two,
or
four
subunits.'
These
forms
are
either
readily
extractable
in
low
ionic
strength
buffers
or
tightly
bound
to
membranes
and
require
detergent
for
solubiliz-
ation.
Forms
with
an
elongated
shape
are
called
asym-
metric,
and
these
do
not
interact
with
detergents
but
are
solubilized
in
buffers
with
high
salt
concentration.
The
asymmetric
forms
contain
one
to
three
tetramers
of
subunits
attached
by
disulphide
bonds
to
a
collagen-like
tail.
The
most
complex
form,
A12,
has
12
subunits.
The
forms
can
be
classified
as
either
hydrophilic,
water-
soluble
or
linked
to
a
membrane
or
extracellular
matrix
by
strong
interactions
with
other
molecules.
Hydrophilic,
water-soluble
forms
Both
AChE
and
BChE
exist
as
water-soluble
forms
secreted
into
body
fluids.
Human
plasma
BChE
is
the
soluble
form
which
has
been
most
intensively
studied.
The
G4
tetramer
represents
95
%
of
the
activity
found
in
plasma.
This
tetramer
is
an
association
of
two
dimers
by
strong
hydrophobic
interactions
[30,31].
The
two
sub-
units
in
each
dimer
are
linked
by
one
disulphide
bond
at
Cys-571
[32].
Dimers
and
monomers
found
in
plasma
seem
to
be
degradation
products
of
tetramers
[33].
In
plasma
a
size
isomer
of
BChE
migrating
in
electro-
Abbreviations
used:
AChE,
acetylcholinesterase;
BChE,
butyrylcholinesterase.
t
To
whom
correspondence.
and
reprint
requests
should
be
addressed.
Vol.
260
625

A.
Chatonnet
and
0.
Lockridge
Gl
(
HYDROPHILIC
FORMS
G2
_4
~~~~~~~Al:
?"
Alternative
splicing
and
translation
of
mRNA
t
~~~Gl
AMPHIPHILIC
|
FORMS
l
Di
AChE
and
BChE
7
Vertebrates
>
AChE
and
BChE
,
Vertebrates
secreted
forms
of
vertebrates
WS.
S
AChE
and
BChE
Vertebrates
muscle,
electric
organ
G4
Glycolip
anchor
AChE
and
BChE
Vertebrates
brain
AChE
-
Invertebrates
and
vertebrates;
G2
precursor?
\
AChE
/
Drosophila
head,
Torpedo
electric
G2
organ,
id
mammalian
erythrocytes
Fig.
1.
Schematic
model
of
the
molecular
polymorphism
of
AChE
and
BChE
Open
circles
designate
catalytic
subunits.
Disulphide
bonds
are
indicated
by
S-S.
Hydrophilic
forms
are
GI,
G2
and
G4
forms.
The
asymmetric
A12
forms
have
three
hydrophilic
G4
heads
linked
to
a
collagen
tail
via
disulphide
bonds.
The
G4
amphiphilic
forms
of
brain
are
anchored
into
a
phospholipid
membrane
through
a
20
kDa
anchor.
The
G2
amphiphilic
forms
of
erythrocytes
have
a
glycolipid
anchor.
In
Torpedo
AChE
hydrophilic
forms
and
amphiphilic
G2
forms
are
produced
by
alternative
splicing,
so
that
the
proteins
are
identical
at
535
amino
acids
but
are
nonidentical
at
their
C-termini
[116].
phoreses
faster
than
a
dimer
[31]
was
shown
to
be
an
association
by
a
disulphide
bond
of
a
monomer
of
BChE
with
albumin
(P.
Masson,
personal
communication).
AChE
exists
as
a
water-soluble
G4
form
secreted
by
adrenal
gland
[34],
by
nerve
cell
cultures
[35],
muscle
cell
cultures
[36],
by
peripheral
nerve
cells
in
vivo
or
upon
stimulation
of
nerve
in
hemidiaphragm
preparations
[37].
AChE
is
liberated
by
central
nervous
cells
into
the
cerebrospinal
fluid
[38]
from
which
it
spills
into
the
amniotic
fluid
in
fetuses
with
neural
tube
defect
[39].
The
11
S
form
of
eel
AChE
derived
by
proteolysis
of
asym-
metric
AChE
has
a
hydrophilic
G4
structure,
similar
to
human
plasma
BChE.
AChE
is
found
circulating
in
plasma
in
adult
rabbit
and
rat
and
in
fetal
cow
[24].
Fetal
bovine
serum
AChE
has
the
same
subunit
organization
as
human
BChE
[40].
Immobilized
forms
Asymmetric
forms.
The
structure
of
the
A12
form
of
Torpedo
AChE
is
a
linkage
of
catalytic
subunits
with
the
strands
of
a
triple
helical
collagen
tail
(Fig.
1).
Three
tetramers
of
catalytic
subunits
are
attached
to
a
collagen
tail.
Homologous
catalytic
subunits
are
used
for
asym-
metric
AChE
and
for
hydrophilic
BChE
(Fig.
1).
In
addition,
a
noncatalytic
100
kDa
subunit
was
found
in
Torpedo
californica
[41].
Asymmetric
forms
are
found
in
muscle
of
the
primitive
vertebrate,
the
lamprey
eel
[42],
but
are
not
found
in
invertebrates.
AChE
and
BChE
exist
in
the
A12
form
in
mammals,
and
birds
[15].
In
1-day-old
chick
muscle
three
different
A12
forms
have
been
described:
a
major
hybrid
form
containing
equal
numbers
of
AChE
and
BChE
1989
/II
626

Comparison
of
butyrylcholinesterase
and
acetylcholinesterase
subunits
and
two
minor
homogeneous
forms
with
either
all
BChE
or
all
AChE
subunits.
The
homogeneous
BChE
form
does
not
react
with
monoclonal
antibodies
specific
for
the
collagenous
tail
of
the
hybrid
form,
suggesting
that
the
collagenous
part
of
the
molecule
is
different
in
the
two
forms.
In
chick
muscle
the
AChE
and
BChE
subunits
are linked
individually
to
the
collagen
tail
[43].
In
contrast,
only
half
of
the
subunits
are
covalently
linked
to
the
collagen
tail
in
AChE
from
electric
organ.
A
small
pool
of
AChE
asymmetric
forms
is
found
intracellularly.
The
subunits
become
attached
to
a
coll-
agen
tail
in
the
Golgi
[44]
and
are
secreted.
The
collagen-
tailed
AChE
interacts
with
heparan
sulphate
proteo-
glycans
as
well
as
with
other
components
of
the
basement
membrane
[45].
Linkage
of
the
collagen
tail
with
the
extracellular
matrix
components
is
through
ionic
inter-
actions
[46].
Amphiphilic
globular
forms.
The
membrane-bound
globular
AChE
forms
have
hydrophobic
domains
that
anchor
them
in
the
membrane
phospholipid
bilayers.
Two
different
amphiphilic
forms,
G2
and
G4,
are
attached
to
two
different
hydrophobic
anchors.
G4
AChE
in
mammalian
brain
has
a
hydrophobic
anchor
of
20
kDa
that
is
attached
asymmetrically
to
two
catalytic
subunits
via
disulphide
bonds.
The
20
kDa
anchor
con-
tains
fatty
acids
but
contains
no
inositol
and
no
ethanol-
amine
or
glucosamine
with
free
amino
acid
groups
[47].
Thus
the
20
kDa
anchor
is
different
from
the
glycolipid
anchor
of
erythrocyte
G2
AChE.
The
20
kDa
anchor
has
not
yet
been
characterized
with
respect
to
amino
acid
and
carbohydrate
content.
Amphiphilic
G2
AChE
has
been
found
in
mammalian
erythrocytes
[48],
platelets,
sheep
basal
ganglia
[49],
Drosophila
head
[25,50-52],
and
Torpedo
electric
organ
[19,53].
G2
AChE
has
been
characterized
in
greatest
detail
in
human
erythrocytes,
where
the
C-terminal
amino
acid
of
the
catalytic
subunit,
which
is
glycine,
is
covalently
bound
in
amide
linkage
to
phosphatidylinositol.
The
glycolipid
contains
1
molar
equivalent
each
of
myo-
inositol,
glucosamine
and
ethanolamine
and
2
equivalents
of
fatty
acids
[48].
In
contrast
to
most
glycolipid-
anchored
proteins,
the
G2
AChE
of
human
erythrocytes
is
not
cleaved
by
phosphatidylinositol-specific
phospho-
lipase
C.
The
reason
is
the
presence
of
a
structural
modification
in
phosphatidylinositol.
The
hydrophobic
domain,
which
includes
the
glycolipid,
has
a
size
of
about
3
kDa.
Protease
cleavage
separates
fully
active
hydro-
philic
G2
AChE
from
its
hydrophobic
domain
[54].
Similar
membrane-bound
BChE
forms
may
exist
since
detergent-soluble
G2
BChE
has
been
reported
in
heart
of
Torpedo
marmorata
[29]
and
superior
cervical
ganglion
of
rat
[55].
Detergent-soluble
G4
BChE
has
been
reported
in
mammalian
brain
[55,56].
As
cholinesterases
have been
found
in
all
branches
of
the
animal
kingdom
[24]
and
are
seen
during
development
in
noncholinergic
systems,
one
can
wonder
whether
acetylcholine
and
cholinesterases
first
played
a
role
in
which
the soluble
character
of
cholinesterases
was
necess-
ary.
During
evolution
AChE
may
have
been
recruited
for
a
function
for
which
its
fixation
to
membranes
was
necessary.
COMPARISON
OF
PROTEIN
SUBUNITS
Comparison
of
amino
acid
sequences
To
date
the
only
cholinesterase
sequences
known
are
Torpedo
AChE,
Drosophila
AChE,
85
0O
of
fetal
bovine
AChE,
and
human
BChE
[9-13,57].
Both
enzymes
have
not
been
sequenced
from
the
same
species,
so
it
is
not
possible
to
compare
BChE
and
AChE
from
one
source.
There
is
a
high
degree
of
similarity
between
BChE
and
AChE
despite
the
fact
that
the
enzymes
are
from
species
that
are
far
apart
in
evolution.
Human
BChE
and
Torpedo
AChE
are
5400
identical,
while
human
BChE
and
Drosophila
AChE
are
38
0
identical.
Human
BChE
and
Torpedo
AChE
are
more
similar
to
each
other
than
to
Drosophila
AChE.
However,
bovine
AChE
is
closer
to
Torpedo
AChE
than
to
human
BChE
(about
60
0
and
50
0
similar
respectively)
[57].
The
divergence
between
AChE
and
BChE
probably
occurred
in
the
deuterosto-
mian
lineage.
The
disulphide
bonds
in
human
BChE
[32]
and
Torpedo
AChE
[58]
are
in
exactly
the
same
positions,
and
their
three
disulphide
loops
contain
exactly
the
same
number
of
amino
acids.
For
both
enzymes
the
cysteine
is
found
four
amino
acids
before
the
C-terminus
and
is
used
in
a
disulphide
bond
between
identical
subunits
[32,58].
The
sequence
around
the
active
site
serine
is
conserved
so
that
all
four
have
the
sequence
GESAG.
The
lengths
of
the
catalytic
subunits
are
similar:
574
amino
acids
for
human
hydrophilic
BChE,
575
for
hydrophilic
Torpedo
AChE,
537
for
mature
amphiphilic
Torpedo
AChE,
and
577
for
amphiphilic
Drosophila
AChE.
For
hydrophilic
fetal
bovine
AChE
the
number
of
residues
is
estimated
to
be
577.
The
specificity
of
the
cholinesterases
for
their
substrates
comes
from
the
presence
of
an
'anionic
site'
which
binds
the
choline
residue
during
hydrolysis
of
choline
esters.
A
sequence
GSXF
that
should
be
close
to
the
'anionic
site'
has
been
found
by
photoaffinity
labelling
with
an
inhibitor
of
electric
eel
AChE
[59].
It
corresponds
to
a
sequence
GSFF
in
Torpedo
AChE.
Hasan
et
al.
[60]
argued
that
the
'anionic
site'
could
be
hydrophobic
rather
than
ionic
in
nature
accommodating
the
trimethyl-
ammonium
part
of
the
substrate.
Evidence
for
a
true
anionic
component
of
the
'anionic'
subsite
comes
from
the
discovery
of
a
point
mutation
in
human
BChE
of
patients
carrying
the
atypical
variant
of
the
enzyme.
The
mutation
was
known
to
affect
the
anionic
site
[61],
and
not
the
sequence
around
the
active
serine
[62].
The
discovery
of
a
single
mutation
in
atypical
BChE,
replacing
Asp-70
by
Gly
which
induces
a
change
of
one
charge,
is
in
favour
of
a
true
anionic
site
[63].
Asp-70
is
also
present
in
Torpedo
AChE
and
fetal
bovine
AChE
but
not
in
Drosophila
AChE.
In
Drosophila
AChE
the
tyrosine
which
replaces
the
aspartate
could
play
the
role
of
the
aspartate.
The
importance
of
the
hydrophobic
and
ionic
forces
around
the
'anionic'
site
could
vary
among
species.
Differences
in
hydrolysis
of
charged
substrates
by
AChE
were
found
in
fish
in
response
to
adaptation
to
high
pressure
and
low
temperature
[64].
This
could
correspond
to
a
smaller
hydrophobic
binding
region
around
the
anionic
site.
Comparison
of
glycosylation
Both
AChE
and
BChE
are
glycoproteins.
Torpedo
AChE
has
four
asparagine-linked
carbohydrate
chains
[58]
and
human
BChE
has
nine
[32].
Carbohydrate
moieties
can
differ
for
AChE
from
different
tissues.
This
was
suggested
by
Meflah
et
al.
[65]
who
reported
differ-
ences
in
lectin
binding
for
AChE
from
bovine
lympho-
cytes,
erythrocytes
and
brain
membrane.
Interaction
with
lectins
also
distinguishes
fetal
calf
serum
AChE
Vol.
260
627

A.
Chatonnet
and
0.
Lockridge
400
423
438
519
S
S
Human
BChE
I
H
I
K1
~
AL
I&
A A
402
425
440
521
Torpedo
AChE,
S
s
IH
H
hydrophilic
67
72
93 94
S--S
I
D
Dl
200
254 265
*
S-S
OH
402
425
440
521
S
S
H
H
r~~~~~~~~~
.A
104
130131
276
330345
480503
518
598
Di
OH
XIq
O
H
H
X
69
9798
195
247258
410
441
I
DI
OH
I
HH
65
83
84
188
240
252
408
445
SS
OH
H
H
H
1
2424
2435
S-S
I
I
2573
2697
S
S
Torpedo
AChE,
amphiphilic
Drosophila
AChE,
amphiphilic
Rabbit
esterase
Drosophila
esterase-6
Human
thyroglobulin
I
I
.......A
~~~~~~~~~~~~~~_
Ak
r:
I
nsertion
Regions
which
are
non-homologous
to
cholinesterases
C-terminus
of
hydrophilic
forms
*
C-terminus
of
amphiphilic
forms
.
Position
of
last
introns
Fig.
2.
Schematic
diagram
of
proteins
homologous
to
cholinesterases
Conserved
residues
are
shown,
including
the
active
site
serine
(OH*),
an
aspartic
acid
that
may
be
part
of
the
catalytic
triad
(D-91
in
BChE),
and
two
histidines
(H-423
and
-438
in
BChE)
that
may
be
important
for
catalysis.
Asp-70
of
BChE
is
at
the
anionic
site.
Intron
locations
for
cholinesterases
are
indicated
by
a
triangle.
Disulphide
bonds
(S-S)
for
human
BChE
[32]
and
Torpedo
AChE
[58]
were
determined.
For
other
proteins
the
location
of
disulphide
bonds
is
by
homology.
Amino
acid
sequence
information
is
from
[8]
(human
BChE),
[9]
and
[11]
(Torpedo
AChE),
[10]
(Drosophila
AChE),
[69]
(rabbit
microsomal
liver
esterase),
[26]
(Drosophila
esterase-6)
and
[123]
(bovine
thyroglobulin).
from
bovine
erythrocyte
AChE
[40].
A
decrease
in
sialylation
of
AChE
occurs
during
erythroid
differenti-
ation
in
a
human
leukaemia
cell
line
[66].
A
subset
of
AChE
G2
forms
from
electric
organ
of
Torpedo
is
recognized
by
an
anti-carbohydrate
antibody
[67].
Little
is
known
about
possible
heterogeneity
in
the
glycosyl-
ation
of
BChE.
Comparison
of
the
active
sites
of
the
cholinesterase
and
serine
proteases
Homology
of
the
peptide
containing
the
active
-site
serine
in
trypsin-like
proteases
(GDSGG)
and
cholin-
esterases
(GESAG)
could
indicate
common
ancestry.
However,
the
active
site
serine
is
closer
to
the
N-terminus
in
the
cholinesterases
than
in
serine
proteases
and
the
pattern
of
disulphide
bonds
is
different.
The
cholinester-
ases
have
no
histidine
in
a
location
similar
to
the
catalytic
triad
histidine
of
the
serine
proteases.
This
suggests
that
the
similarity
of
the
active
site
in
the
trypsin-like
proteases
and
cholinesterases
arose
from
convergent
evolution.
The
cholinesterase
family
of
esterases
The
cholinesterases
seem
to
belong
to
a
class
that
is
distinct
from
the
serine
proteases
[32,58,68].
The
new
class
is
not
limited
to
cholinesterases,
as
is
shown
in
Fig.
2.
Similarities
have
been
found
with
other
esterases,
a
rabbit
liver
microsomal
esterase
of
60
kDa
[69]
and
esterase-6
of
Drosophila
[26]
as
well
as
with
thyroglobulin
[8,9].
Disulphide
bonds
on
each
side
of
the
active
serine
are
conserved.
A
third
sulphide
bond
at
the
C-terminus
is
not
present
in
the
two
unspecific
esterases.
No
sequence
homology
is
found
at
the
C-terminus,
particularly
in
the
region
which
can
give
rise
to
different
forms
of
cholinesterases.
Since
the
tertiary
structure
may
be
partly
1989
65
70
91
92
s
--.S
ID
Dl
67
72
93
94
S--S
ID
Di
198
252
263
S
S
OH
I
200
254
265
S.S
OH
2246
2263
S-S
I
- -
I
-
m
628

Comparison
of
butyrylcholinesterase
and
acetylcholinesterase
conserved
it
is
likely
that
amino
acids
involved
in
the
catalytic
triad
are
conserved
in
all
these
enzymes.
Two
histidines
(423
and
438
in
human
BChE)
are
conserved
in
the
cholinesterases
as
well
as
in
rabbit
liver
microsomal
esterase
and
Drosophila
esterase-6.
Both
histidines
could
play
a
role
in
catalysis.
His-441
of
rabbit
esterase
is
phosphorylated
by
di-isopropylphosphofluoridate
at
the
same
time
and
under
the
same
conditions
as
the
active
site
serine,
suggesting
that
this
histidine
is
present
in
the
esteratic
site
[69].
Asp-91
is
found
in
the
same
position
in
all
the
enzymes
and
could
represent
the
third
amino
acid
of
the
catalytic
triad.
COMPARISON
OF
THE
DISTRIBUTION
OF
AChE
AND
BChE
IN
TISSUES
AND
REGULATION
DURING
DEVELOPMENT
The
various
cholinesterase
forms
are
tissue-specific.
Asymmetric
AChE
and
BChE
forms
are
found
only
in
peripheral
nerves
and
muscles
of
vertebrates.
Membrane-
bound
G4
AChE
and
G4
BChE
are
found
in
mammalian
brain
and
membrane-bound
G2
AChE
is
found
in
erythrocytes
[15].
Both
BChE
and
AChE
have
been
described
in
tissues
during
development.
There
is
evidence
that
BChE
is
present
transiently
in
some
embryonic
cells
where
it
can
sometimes
be
replaced
by
AChE.
This
suggests
a
function
for
BChE
as
an
embryonic
acetylcholinesterase.
Em-
bryonic
development
has
been
mostly
studied
in
chicken,
and
in
this
species
the
highest
BChE
activity
is
in
embryonic
tissues,
thus
allowing
better
comparison
of
AChE
and
BChE.
Appearance
and
fate
of
BChE
and
AChE
in
muscle
The
role
of
contractile
activity
in
the
localization
and
regulation
of
the
forms
of
AChE
at
the
neuromuscular
junction
has
been
extensively
reviewed
[44].
In
muscle
fibres
both
enzymes
are
progressively
restricted
to
the
neuromuscular
junction
by
aggregating
factors
[70].
Neurotrophic
factors,
for
example
a
dipeptide
Gly-Gln
in
chicken
and
rat
muscle
cell
cultures,
seem
to
be
involved
in
the
regulation
of
AChE
[71].
In
chicken
skeletal
muscle
the
total
amounts
and
forms
of
AChE
change
in
parallel
with
those
of
BChE
during
development
[72].
There
is
a
period
of
relatively
high
activity
of
BChE
which
lasts
until
just
before
hatching.
The
localization
of
the
two
enzymes
at
this
stage
is
not
clear,
but
the
fact
that
subunits
of
both
participate
in
the
same
asymmetric
forms
implies
an
identical
location
[43].
In
the
rat
a
similar
evolution
of
activities
occurs
in
the
neonate
[73]
and
this
can
be
related
to
the
maturation
of
neuro-muscular
junctions.
Appearance
and
fate
of
BChE
and
AChE
in
nervous
tissue
AChE
associated
with
the
cholinergic
system
is
found
in
the
brain,
but
its
role
is
unclear
in
regions
where
no
acetylcholine
is
present
[24].
BChE
is
present
in
regions
of
the
brain
in
positions
not
related
to
AChE,
namely
in
capillary
endothelial
cells,
in
glial
cells,
and
in
neurones
[74].
Human
BChE
is
clearly
synthesized
in
the
brain,
and
not
derived
from
plasma,
since
cDNA
clones
have
been
found
in
brain
cell
cDNA
libraries
[12,13].
Human
brain
and
liver
BChE
and
water-soluble
G4
BChE
from
plasma
have
identical
amino
acid
sequences
[23].
Although
almost
half
of
the
G4
form
of
brain
BChE
is
Vol.
260
membrane-bound
[56],
no
clones
coding
for
an
alternative
C-terminus
have
been
found.
In
embryonic
chicken
brain
and
eye
the
expression
of
BChE
and
AChE
are
co-regulated
[75,76].
BChE
is
expressed
just
before
and
during
mitosis,
while
AChE
is
expressed
about
11
h
after
mitosis.
The
strong
correlation
between
BChE
and
cell
proliferation
and
between
AChE
and
cellular
differentiation
suggests
cholinesterase
involvement
in
the
regulation
of
these
processes
[75].
Appearance
and
fate
of
BChE
and
AChE
in
blood
The
concentration
of
BChE
in
human
serum
is
corre-
lated
with
growth
hormone,
obesity,
pregnancy
and
parturition
[22].
Average
adult
human
plasma
contains
3300
ng
of
BChE/ml
and
8
ng
of
AChE/ml
as
measured
by
immunosorbent
assay
[77].
Some
9400
of
plasma
BChE
is
in
the
water-soluble
G4
form
and
its
origin
is
thought
to
be
the
liver.
Of
the
AChE
in
plasma
54
0
is
in
the
G4
form
and
44
0
is
GI
and
G2
[78].
While
the
GI
and
G2
AChE
forms
could
possibly
be
released
from
red
blood
cells,
the
G4
form
probably
originates
from
the
neuromuscular
junction,
or
autonomic
ganglia
or
the
central
nervous
system.
Human
fetal
serum
contains
considerably
more
AChE
than
is
present
in
adult
serum
[79].
Fetal
bovine
serum
contains
predominantly
AChE
[40].
The
presence
in
plasma
of
AChE
and
BChE
and
the
presence
of
AChE
on
red
cell
membranes
suggests
a
requirement
for
circulating
cholinesterase
activity
rather
than
a
requirement
for
one
of
the
cholinesterases.
This
would
explain
the
dispensable
character
of
BChE
as
exhibited
in
humans
by
homozygote
'silent'
individuals
with
no
BChE
protein
in
the
plasma.
Appearance
and
fate
of
BChE
and
AChE
in
heart
The
heart
in
higher
vertebrates
is
one
of
the
tissues
containing
a
large
amount
of
BChE.
A
partial
switch
from
BChE
to
AChE
has
clearly
been
shown
in
the
atrioventricular
specialized
tissue
of
the
rat
during
early
post-natal
development
[80,8
1];
the
proportion
of
AChE
increased
from
6
0
to
15
Qo
[82].
The
switch
is
linked
to
the
development
of
adrenergic
innervation
and
can
be
delayed
when
anti-(nerve
growth
factors)
are
used
[80].
Although
the
activity
of
BChE
in
the
total
atria
of
the
adult
rat
is
in
excess
compared
with
AChE
[83],
AChE
is
responsible
for
the
physiological
hydrolysis
of
acetyl-
choline
liberated
by
the
vagus
nerve
[82].
In
contrast,
in
a
more
primitive
vertebrate,
Torpedo,
BChE
is
the
only
cholinesterase
present
in
the
heart
and
is
responsible
for
the
physiological
hydrolysis
of
AChE
[29].
Tissue-specific
regulation
of
AChE
and
homeostatic
regulation
of
BChE
in
adults
Edwards
&
Brimijoin
[84]
measured
AChE
and
BChE
activity
in
tissues
of
three
strains
of
adult
rats
and
showed
that,
whereas
there
was
no
correlation
of
AChE
activity
between
tissues
of
the
rat
there
was
correlation
of
BChE
activity
among
tissues
in
individual
rats.
There
was
greater
variation
between
BChE
activity
in
the
same
tissues
in
different
animals
or
different
strains.
They
also
studied
the
effect
of
hypophysectomy
and
found
that
BChE
was
more
affected
than
AChE
[85].
Their
con-
clusion
was
that
the
regulation
of
AChE
is
tissue-specific.
In
contrast,
BChE
depends
on
a
homeostatic
type
of
regulation
throughout
the
whole
body.The
discrepancies
between
studies
showing
parallel
[86]
or
opposite
regu-
629

A.
Chatonnet
and
0.
Lockridge
lation
of
AChE
and
BChE
could
come
from
the
fact
that
cholinesterases
may
have
more
than
one
function.
ROLES
OF
CHOLINESTERASES
NOT
RELATED
TO
THE
CHOLINERGIC
SYSTEM
Amidase
and
peptidase
activity
In
locations
where
no
acetylcholine
is
released,
the
presence
of
AChE
or
BChE
is
puzzling.
AChE
and
BChE
were
shown
to
possess
other
catalytic
activities
in
addition
to
esteratic
activity.
Both
AChE
and
BChE
have
arylacylamidase
activity
[87,88].
The
arylacyl-
amidase
activity
of
BChE
is
probably
responsible
for
the
C-terminal
deamidation
of
Substance
P.
The
aryl-
acylamidase
activity
of
BChE
is
inhibited
by
the
cholinesterase
inhibitors
eserine
and
tetraisopropylpyro-
phosphoramide,
suggesting
involvement
of
the
active
site
serine
[88].
Though
both
AChE
and
BChE
have
been
reported
to
hydrolyse
Substance
P
[89,90],
the
suggestion
has
been
made
that
a
contamination
by
dipeptidyl-
aminopeptidase
IV
is
responsible
for
Substance
P
hydrolysis
by
BChE
preparations
[91,92].
In
fact,
two
different
types
of
peptidasic
activities
have
been
described
for
human
BChE.
The
first
peptidase
activity
has
the
substrate
specificity
of
dipeptidylaminopeptidase
and
has
an
active
site
that
is
distinct
from
the
esteratic
site
of
BChE
[93].
The
number
of
peptidasic
sites
in
a
highly
purified
preparation
of
BChE
was
very
low
compared
with
the
number
of
esteratic
sites
[94].
The
strong
association
of
peptidase
and
esterase
activities
on
gel
electrophoresis
[92,94]
raises
the
question
of
the
possible
association
in
vivo
of
cholinesterases
with
other
molecules
with
different
activities.
The
second
peptidase
activity
described
for
BChE
is
a
carboxypeptidase
activity
towards
Substance
P
[95].
This
activity
is
inhibited
by
EDTA
and
was
not
found
in
previous
experiments
due
to
the
presence
of
chelating
agents.
This
activity
is
not
inhibited
by
di-isopropyl-
phosphofluoridate
or
other
esterase
inhibitors
like
eserine.
The
possibility
of
another
contaminant
cannot
be
ruled
out,
but
this
activity
is
immunoprecipitated
along
with
the
esteratic
activity.
AChE
hydrolyses
Substance
P,
enkephalins
and
their
precursors
[96,97].
AChE
may
also
hydrolyse
chromo-
granin,
a
protein
secreted
by
adrenal
cells
[98].
AChE
shows
more
than
one
type
of
peptidase
activity,
i.e.
a
trypsin-like
activity
and
a
metalloexopeptidase-like
activity
[99].
The
trypsin-like
activity
is
not
inhibited
by
phosphorylation
of
the
serine
at
the
esterasic
site
but
is
inhibited
by
di-isopropylphosphofluoridate
at
high
concentration.
This
raises
the
same
question
as
for
BChE:
are
the
peptidasic
and
esteratic
sites
different
or
overlapping?
Other
roles
not
related
to
neurotransmission
Other
noncholinergic
roles
have
been
tentatively
attri-
buted
to
AChE
and
BChE.
The
injection
of
purified
AChE
in
the
substantia
nigra
of
the
brain
induced
compartmental
changes
of
long
effect.
BChE
did
not
produce
similar
effects
[100].
Dendritic
release
of
AChE
suggested
a
role
in
choline
re-uptake
[101].
The
presence
of
BChE
in
liver
where
fatty
acid
metabolism
occurs
sugg9sted
a
role
for
BChE
in
this
metabolism
[102].
Cholinesterases
are
found
in
intracellular
locations
such
as
the
nuclear
envelope,
in
membrane
fractions,
the
transverse
tubular
system of
muscle,
and
in
sarcoplasmic
reticulum,
and
could
play
an
intracellular
role
as
only
a
fraction
of
the
subunits
end
up
outside
the
cell
[44].
Whatever
role
is
proposed
for
BChE
it
has
to
be
compatible
with
the
fact
that
homozygotes
for
the
silent
BChE
gene
have
no
BChE
in
plasma
and
liver.
COMPARISON
OF
THE
GENES
AND
THE
TRANSCRIPTIONAL
REGULATION
OF
CHOLINESTERASES
Alleles
and
variants
Genetic
variants
are
known
for
human
BChE.
Two
genetic
loci
determine
BChE
activity
in
plasma.
Locus
E,
is
located
on
chromosome
3 in
the
region
3q21-25
[103].
This
position
has
been
confirmed
by
chromosome
in
situ
hybridization
[104].
The
E1
locus
controls
most
of
the
genetic
variants.
The
best
known
alleles
are
the
usual
gene
E
U,
the
atypical
gene
E1a,
the
fluoride-resistant
gene
E1f,
and
the
silent
gene
Els
[20,21].
Quantitative
variants
E1
and
E1k
have
reduced
activity
[22].
In
addition
to
the
homozygotes
all
combinations
of
heterozygotes
have
been
found.
Both
alleles
are
expressed
in
heterozygotes
and
the
plasma
tetramers
show
all
possible
combinations
of
the
two
kinds
of
subunits
[105].
The
human
BChE
E1
variants
have
reduced
affinity
for
substrates
and
lower
activity
at
standard
substrate
concentration.
The
E1
variants
are
indistinguishable
from
normal
BChE
on
the
basis
of
size
or
electrophoretic
mobility.
The
Ela
variant
enzyme
has
a
low
affinity
for
choline
esters
and
positively
charged
inhibitors
[2,61],
thus
suggesting
that
the
single
amino
acid
mutation
is
at
the
anionic
site.
In
atypical
BChE
a
single
base
substitution
at
nucleotide
209
con-
verts
Asp-70
to
Gly
[63].
In
human
populations
there
seems
to
be
more
than
one
type
of
silent
gene,
so
that
E1s
homozygotes
have
either
no
activity
or
2
0
activity
in
plasma
[21].
Humans
with
no
activity
enjoy
normal
health
unless
they
undergo
surgery
with
injection
of
the
myorelaxant
succinylcholine.
Two
subjects
with
'silent'
BChE
were
found
to
have
a
frame-shift
mutation
at
nucleotide
351
(GGT
-*
GGAG)
which
created
a
stop
codon
at
nucleotide
384
[63].
Their
BChE
protein
should
contain
128
amino
acids,
and
should
not
include
the
active-site
serine.
This
explains
the
complete
absence
of
BChE
activity
in
the
homozygote
'silent'
person.
The
E2
locus
controls
the
appearance
of
a
distinct
band
of
BChE
on
gels
in
approximately
10
%
of
Cauca-
sians
[22].
The
band
appears
to
be
a
slow-moving
tetramer
called
C5.
The
C.
variant
is
an
autosomal
dominant
trait
nonallelic
with
the
E1
locus
[107].
However,
the
C
band
had
the
same
dibucaine
resistance
as
heterozygote
E1uE
a
J107,108],
suggesting
that
C5
was
controlled
by
both
genetic
loci.
To
explain
how
two
genetic
loci
can
affect
a
single
protein
it
was
proposed
that
C5
may
be
a
hybrid
of
cholinesterase
and
asecond
protein
[108].
The
locus
E2
may
be
located
on
chromosome
16
[109].
However,
there
is
no
clear
evidence
that
the
E2
locus
is
a
structural
gene
for
cholinesterase
except
that
probes
of
BChE
cDNA
hybridized
to
chromosome
16
as
well
as
to
chromosome
3
[104].
There
is
only
one
gene
for
AChE
in
Torpedo
[11]
and
one
gene
in
Drosophila
[10].
Genetic
variants
of
AChE
with
two
different
subunit
molecular
masses
of
100
and
105
kDa
but
no
differences
in
activity
have
been
reported
for
chicken.
These
are
allelic
variants
and
only
hetero-
zygotes
show
the
two
forms
[44,1
10J.
Genetic
variants
1989
630

Comparison
of
butyrylcholinesterase
and
acetylcholinesterase
7...
I
ORF
ORF
ORF
mm
-
]ZL1Z
5'
end
of
the
gene
-----
.........
Z!iiIiIiIi
I
_|iii
Human
BChE
j j
:
j
Torpedo
marmorata
AChE
, j
js
j
Torpedo
californica
AChE
IIILIILII
C:j
EEZv////Drosophila
AChE
J0
IIt
I.I
iII
LI
:
Rat
thyroglobulin
1
kb
5'
untranslated
region
D
Coding
region
M
3'
untranslated
region
/\
Alternative
splicing
]
[
Positions
of
introns
*
Hydrophilic
C-terminal
f
Hydrophobic
C-terminal
ORF
Open
reading
frames
Fig.
3.
Comparison
of
gene
structure
of
cholinesterases
and
C-terminus
of
the
thyroglobulin
gene
Intron
locations
but
not
their
lengths
are
shown.
The
coding
region
of
human
BChE
and
Torpedo
AChE
is
interrupted
by
two
introns.
Drosophila
AChE
and
rat
thyroglobulin
have
many
additional
introns
in
the
coding
region.
The
number
of
introns
in
the
5'
untranslated
regions
is
still
uncertain
and
the
Figure
shows
the
minimum
number
based
on
current
data.
Open
reading
frames,
ORF,
containing
potential
translation
initiation
sites
are
present
in
Torpedo
and
Drosophila
AChE.
At
the
3'
end
two
exons
used
in
alternative
splicing
are
indicated.
containing
amino
acid
alterations
have
not
been
reported
for
human
AChE.
Insects
have
genetic
variants
of
AChE
that
are
resistant
to
organophosphate
poisons
[1
1
1]
and
Drosophila
has
variants
with
no
AChE
activity
[112].
Homozygotes
with
no
AChE
cannot
develop
beyond
a
very
early
stage
of
embryogenesis
and
therefore
this
mutation
can
only
be
maintained
in
heterozygote
populations.
Structure
of
the
genes
for
AChE,
BChE
and
thyroglobulin
It
is
now
clear
that
BChE
and
AChE
are
products
of
different
genes.
The
structure
of
the
gene
is
similar
for
Torpedo
AChE
and
human
BChE
(Fig.
3).
A
common
feature
is
a
large
exon
possessing
approx.
800%
of
the
coding
sequence
starting
in
the
5'
untranslated
region
and
ending
far
after
the
active-site
serine.
In
thyro-
globulin,
as
in
Drosophila
AChE,
at
least
five
introns
are
found
in
the
region
where
vertebrate
AChE
and
BChE
have
a
unique
large
exon.
The
processed
character
of
the
coding
region
(homologous
with
a
mature
spliced
mRNA)
could
be
due
to
a
retrotranscription
event
before
duplication
of
the
gene
that
gave
rise
to
AChE
and
BChE.
This
may
have
occurred
at
the
time
of
emergence
of
the
vertebrates.
The
region
upstream
of
the
large
coding
exon
is
complex
in
Torpedo
AChE.
Alternative
splicing
involving
at
least
two
exons
was
deduced
from
isolation
of
multiple
cDNA
and
from
protection
assays
of
mRNA
[11].
T.
marmorata
has
multiple
translation
initiation
sites.
Use
of
some
of
these
initiation
sites
would
not
result
in
a
protein
product
because
of
the
presence
of
in-frame
stop
codons.
Similarly,
Drosophila
AChE
has
multiple
initiation
sites,
some
of
which
are
within
small
open
reading
frames.
Drosophila
AChE
has
an
unusually
long
5'
untranslated
leader
sequence
of
about
1000
bp.
Bingham
et
al.
[113]
have
suggested
that
Drosophila
AChE
is
an
example
of
on/off
regulation
at
the
level
of
splicing.
In
this
interpretation
the
mRNA
containing
small
open
reading
frames
is
nonfunctional,
and
only
fully-spliced
mRNA
is
functional.
Human
BChE
also
has
multiple
ATGs
in
the
5'
leader
sequence.
However,
it
contains
no
in-frame
stop
codons
and
therefore
initiation
at
alternative
ATGs
would
produce
functional
BChE.
The
most
probable
size
of
the
signal
peptide
in
human
BChE
is
28
amino
acids.
The
signal
peptide
in
T.
marmorata
is
24
amino
acids
and
in
T.
californica
is
21
amino
acids
long.
Downstream
of
the
large
coding
exon
there
is
a
small
exon
from
Gly-478
to
Gly-534
in
human
BChE.
The
positions
of
the
two
introns
limiting
this
small
exon
are
identical
in
the
genes
for
Torpedo
AChE,
human
BChE,
Drosophila
AChE
(D.
Fournier,
personal
communi-
cation)
and
rat
thyroglobulin
[114].
The
sequence
of
this
small
exon
is
not
very
well
conserved
but
the
splicing
sites
are
conserved.
Vol.
260
631

A.
Chatonnet
and
0.
Lockridge
The
coding
sequence
terminates
with
a
choice
of
two
possible
exons,
producing
precursors
of
hydrophilic
or
amphiphilic
forms.
When
translated,
the
'hydrophilic'
exon
of
Torpedo
AChE
gives
40
amino
acids.
The
exon
for
G2
amphiphilic
forms
of
Torpedo
AChE
is
translated
in
a
peptide
of
31
amino
acids
[115].
The
terminal
29
amino
acids
are
replaced
by
a
phosphatidylinositol
anchor
[116].
This
'hydrophobic'
exon
is
found
in
a
minor
proportion
of
mRNA
of
Torpedo
AChE
and
has
not
yet
been
found
for
BChE.
The
cDNA
of
BChE
so
far
found
by
McTiernan
et
al.
[13]
and
Prody
et
al.
[12]
has
a
last
exon
similar
to
that
of
the
hydrophilic
form
of
Torpedo
AChE
and
corresponded
to
the
protein
sequence
of
BChE
of
plasma
[8].
Surprisingly,
introns
have
been
found
in
cDNA
clones
of
BChE
and
AChE.
A
cDNA
clone
of
human
BChE
contained
105
bases
at
the
5'
side
of
the
last
exon
[13].
A
clone
of
rabbit
BChE
contained
exon
3
and
portions
of
introns
at
each
extremity
(A.
Chatonnet,
unpublished
work).
An
AChE
cDNA
clone
from
Torpedo
marmorata
had
an
intron
at
the
5'
end
of
the
cDNA
[11].
This
would
explain
why
pools
of
mRNA
with
very
different
sizes
elicited
production
of
cholinesterase
when
injected
into
Xenopus
oocytes
[117].
Slow
maturation of
pre-mRNA
of
AChE
would
explain
the
high-molecular-mass
bands
seen
in
Northern
blots
hybridizing
with
AChE
probes
[9,10,11].
The
presence
of
introns
in
mRNA
may
be
an
indication
of
regulation
at
the
level
of
splicing
[113].
A
cDNA
clone
with
a
sequence
at
the
3'
end
that
corresponded
to
neither
the
exons
coding
for
hydrophilic
or
amphiphilic
AChE
was
found
in
Torpedo.
The
cDNA
terminated
with
a
poly(A)
stretch,
suggesting
that
no
other
exons
followed
it.
This
sequence
could
be
either
recognized
as
an
intron
and
spliced
out
or
used
as
a
continuation
of
the
preceding
exon.
This
sequence
was
present
in
a
small
percentage
of
the
mRNA
[115].
Base
composition
and
codon
usage
in
cholinesterase
mRNA
Human
BChE
mRNA
possesses
40
%
of
C
+
G,
which
is
approximately
the
proportion
of
these
bases
in
the
total
human
genome
(Table
1).
The
dinucleotide
CG
is
very
under-represented
in
human
BChE
mRNA
[12,13,118].
We
found
only
17
CG
in
the
coding
sequence
of
BChE,
whereas
Torpedo
AChE
has
82
CG
and
Drosophila
AChE
has
153
CG.
This
difference
in
base
composition
is
in
marked
contrast
with
the
high
similarity
of
amino
acid
sequences
of
54
%
between
Torpedo
AChE
and
human
BChE.
A
large
number
(40)
of
the
CG
dinucleotides
in
Torpedo
are
at
the
codon-codon
boundary
(C
is
the
third
base
of
a
codon
and
G
is
the
first
base
of
the
next
codon).
In
human
BChE
the
C
is
often
not
present
and
is
replaced
by
a
T.
This
is
found
in
24
out
of
the
40
CG
present
at
the
codon-codon
boundary
in
Torpedo
AChE.
This
reflects
a
striking
difference
in
the
proportion
of
the
four
bases
at
the
last
base
of
codons
of
BChE
when
compared
with
AChE.
When
compared
with
the
human
genes
so
far
sequenced
human
BChE
presents
one
of
the
lowest
C
+
G
content
at
the
third
base
of
the
codons
(34
%,
see
Table
1).
Comparison
with
the
cholinergic
proteins
(nicotinic
and
muscarinic
acetylcholine
receptors,
choline
acetyl-
transferase
and
BChE)
shows
that
in
contrast
with
BChE
the
cholinergic
proteins
of
mammals
and
birds
have
a
high
C
+
G
content.
Table
1.
C
+
G
content
in
total
mRNA
and
at
the
third
base of
codons
in
cholinergic
proteins
Data
were
obtained
from
GENBANK,
EMBL,
and
NBRF.
The
computer
programs
were
from
the
Protein
Identification
Resource,
National
Biomedical
Research
Foundation,
Washington
DC,
U.S.A.
C+G
%
at
third
%
of
Sequences
base
total
Invertebrates
Drosophila
AChE
75.2
58.7
Poikilotherm
vertebrates
Torpedo
AChE
71.9
55.0
Torpedo
nicotinic
ACh
receptor
a
subunit
37.0
39.4
Torpedo
nicotinic
ACh
receptor
3
subunit
84.2
58.9
Torpedo
nicotinic
ACh
receptor
y
subunit
43.7
44.4
Torpedo
nicotinic
ACh
receptor
6
subunit
43.6
41.6
Homeotherm
vertebrates
Human
BChE
34
40.2
Human
nicotinic
ACh
receptor
y
subunit
79
61.1
Human
nicotinic
ACh
receptor
a
subunit
67
51.4
Mouse
nicotinic
ACh
receptor
a
subunit
74.2
56.5
Bovine
nicotinic
ACh
receptor
e
subunit
80.6
59.7
Chicken
nicotinic
ACh
receptor
a
subunit
78
57.6
Chicken
nicotinic
ACh
receptor
y
subunit
80
58.1
Porcine
muscarinic
ACh
receptor
95
66.6
Porcine
choline
acetyltransferase
70.9 56.4
According
to
this
view
AChE
at
the
neuromuscular
junction
should
have
a
high
C+G
content
to
be
con-
sistent
with
the
high
C+G
content
of
muscle
genes
in
higher
vertebrates
[119].
Plasma
BChE
is
produced
by
the
liver
and
as
expected
shows
a
liver-type
C
+
G
content.
It
should
be
noted
that
a
comparison
cannot
be
done
with
Torpedo
genes
where
a
great
variety
of
C
+
G
contents
can
be
found
even
between
the
different
subunits
of
the
receptor
(C
+
G
37-86
%).
Tissue
specificity
of
codon
usage
does
not
seem
to
exist
in
Torpedo
and
might
be
restricted
to
homeotherm
vertebrates.
C
+
G
content
could
be
important
to
regulation
at
the
transcriptional
level.
The
genome
of
warm-blooded
verte-
brates
can
be
separated
into
domains
of
high
and
low
C+G
content
called
isochores
[120].
Studies
of
this
compositional
compartmentalization
of
the
genome
of
vertebrates
showed
that
warm-blooded
vertebrates
have
more
genes
in
isochores
of
higher
C+G
content
[121].
The
C
+
G
content
of
the
genes
reflects
the
proportion
of
these
bases
in
the
domain
in
which
they
lie.
A
low
C
+
G
content
at
the
last
base
of
codons
is
correlated
with
a
low
proportion
of
these
bases
in
untranslated
regions
and
in
introns
[122].
The
position
of
genes
in
isochores
is
conserved
during
evolution.
This
is
verified
for
human
BChE
[12,13]
and
for
rabbit
BChE
(A.
Chatonnet,
un-
published
work)
which
have
conserved
the
low
C
+
G
content.
Other
cholinergic
proteins
are
likely
to
be
found
in
different
isochores.
The
tissue
specificity
of
codon
usage
could
reveal
that
isochores
are
not
used
equally
in
transcription
in
different
tissues.
Thus
AChE
and
BChE,
which
probably
belong
to
two
different
transcriptional
units
of
the
genome
with
opposite
base
content,
could
be
used
successively
or
alternatively
in
different
tissues.
1989
632

Comparison
of
butyrylcholinesterase
and
acetylcholinesterase
CONCLUSIONS
Comparison
of
AChE
and
BChE
shows
extensive
similarities
in
protein
sequences
and
in
molecular
forms.
This
contrasts
with
differences
in
expression
during
tissue
differentiation
and
development.
One
cholinesterase
replaces
another
during
development,
suggesting
a
com-
plementarity
of
roles
for
AChE
and
BChE.
The
existence
of
a
family
of
serine
esterases
structurally
related
to
cholinesterase,
but
unrelated
to
the
serine
proteases,
suggests
that
serine
esterases
have
evolved
to
maximize
their
esterase
function.
We
thank:
Dr.
Bert
N.
La
Du
for
constant
support
and
interest
in
our
work;
Drs.
M.
Arpagaus,
F.
Bacou,
P.
Masson,
J.
Massoulie
and
J.
P.
Toutant
for
helpful
comments;
Drs.
P.
G.
Layer
and
D.
Fournier
for
communication
of
their
most
recent
work.
This
work
was
supported
by
grants
from
U.S.
Army
Medical
Research
and
Development
Command
Contract
No
DAMD17-86-C-6037,
and
A.
I.
P.
'Cholin-
esterases'
INRA
1988.
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