Proc.
Nati.
Acad.
Sci.
USA
Vol.
76,
No.
12,
pp.
6656-6660,
December
1979
Neurobiology
Purification
and
primary
structure
of
the
neuropeptide
egg-laying
hormone
of
Aplysia
californica
(bag
cells/amino
acid
sequence/neurosecretory
peptide/molluskan
neuropeptide/microsequence
analysis)
A.
Y.
CHIU,
M.
W.
HUNKAPILLER,
E.
HELLER,
D.
K.
STUART*,
L. E.
HOOD,
AND
F.
STRUMWASSERt
Division
of
Biology,
California
Institute
of
Technology,
Pasadena,
California
91125
Communicated
by
Theodore
H.
Bullock,
August
15,1979
ABSTRACT
Egg-laying
hormone
(ELH),
a
neuropeptide
synthesized
by
the
bag
cell
neurons,
induces
egg
laying
and
its
correlated
behavior
in
Aplysia
californica.
In
present
study,
ELH
has
been
purified
to
homogeneity
and
its
primary
structure
has
been
determined.
We
find
this
molecule
to
have
36
amino
acid
residues
with
a
M,
of
4385
and
a
calculated
isoelectric
point
of
9.7.
Direct
microsequence
analysis
revealed
a
single
amino
acid
sequence
that
is
in
agreement
with
the
amino
acid
composition
determined
after
acid
hydrolysis
of
ELH:
HRIle-
Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-
Thr-Glu-Gln-Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-
Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH.
Enzyme
data
indicate
that
the
COOR-terminal
lysine
may
be
modified
but
its
exact
nature
remains
to
be
determined.
There
is
no
similarity
between
the
amino
acid
sequence
of
ELH
and
that
of
presently
known
ver-
tebrate
neuropeptides.
The
two-step
urification
procedure,
starting
with
a
homogenate
of
bag
ce~l
clusters,
consisted
of
cation
exchange
chromatography
on
SP
C25
(Sephadex)
followed
by
gel
filtration
on
Bio-Gel
P4.
Our
purification
results
in
a
10-fold
enrichment
of
ELH
from
bag
cell
homogenates
and
a
36%
recovery
of
purified
radiolabeled
marker
ELH.
Analysis
of
purified
ELH
radiolabeled
with
[35S]methionine
or
[3HVleu-
cine
on
isoelectric
focusing
gels
and
on
8
M
urea/sodium
do-
decyl
sulfate
gels
showed
only
a
single
peak
containing
90%
of
the
radiolabel.
Radiolabeled
ELH
migrated
with
a
pI
of
9.0-9.2
and
an
apparent
M,
of
3500-5700.
ELH
retained
egg-laying
bioactivity
when
eluted
from
this
segment
of
the
gel.
We
find
that
2.5
nmol
of
pure
ELH
consistently
induces
egg
laying
at
200C.
In
molluscs,
a
clear
example
of
neurohormone-induced
be-
havior
is
the
egg
laying
in
Aplysia
californica
evoked
by
a
neuropeptide
egg-laying
hormone
(ELH)
(1,
2).
ELH
is
syn-
thesized
by
two
symmetrical
populations
of
about
400
neuro-
secretory
bag
cells
each,
clustered
around
the
base
of
the
pleurovisceral
connectives
at
the
rostral
edge
of
the
abdominal
ganglion
(3).
When
a
homogenate
of
bag
cells,
containing
ELH,
is
injected
into
the
hemocoel
of
a
sexually
mature
animal,
a
pattern
of
egg-laying
behavior
is
triggered
which
results
in
egg
laying
within
65
min
at
14'C
(1,
4).
Egg-laying
behavior
typ-
ically
consists
of
locomotion
to
a
stationary
position
on
a
vertical
surface,
puckering
of
mouth
musculature,
inhibition
of
feeding,
and
increased
frequency
of
head
weaving
movement,
culmi-
nating
in
the
extrusion
and
winding
of
the
egg
string
into
a
compact
knot
attached
to
the
vertical
substrate
(5,
6).
Bag
cell
neurons
are
normally
electrically
silent
but
can
be
triggered
to
produce
a
synchronous
afterdischarge
of
action
potentials
lasting
20-60
min
when
electrically
stimulated
(7).
Recent
findings
of
gap
junctions
in
bag
cell
clusters
support
the
premise
that
this
synchrony
of
activity
is
due
to
electrical
coupling
between
bag
cell
neurons
(8).
In
vitro
experiments
have
demonstrated
the
release
of
radiolabeled
and
bioactive
ELH
only
when
bag
cells
afterdischarge
(9).
In
vivo,
such
an
afterdischarge,
whether
induced
or
spontaneous,
is
always
followed
by
egg
laying
(10,
11).
Previous
studies
have
characterized
ELH
as
a
basic
peptide
of
Mr
approximately
6000
with
an
isoelectric
point
of
9.0-9.3
(12,13).
We
have
purified
ELH
by
a
simple
two-step
procedure
to
yield
a
homogeneous
product
with
biological
activity.
We
find
the
molecule
to
be
a
polypeptide
of
36
amino
acids
with
a
calculated
Mr
of
4385
and
migrating
with
a
pI
of
9.0-9.2.
We
now
report
its
amino
acid
sequence.
MATERIALS
AND
METHODS
Egg-Laying
Bioassay.
Material,
at
various
stages
of
purifi-
cation,
was
checked
for
its
ability
to
induce
egg
laying
by
in-
jection
through
the
foot
into
the
hemocoel
of
a
sexually
mature
A.
californica
maintained
at
20'C
as
described
(9).
A
test
was
considered
to
be
positive
if
eggs
were
sighted
within
90
min
of
injection.
Injection
volumes
ranged
from
1
to
3
ml
and
usually
consisted
of
an
aliquot
of
test
material
in
low-ionic
strength
buffer
mixed
with
an
equal
volume
of
filtered
seawater.
Bag
Cell
Clusters,
Radiolabeling,
and
Homogenization.
A.
californica
(>270
g)
were
dissected
within
2
days
after
collection
from
the
Palos
Verdes
Peninsula
during
October
and
November
of
1977
and
1978.
There
is
some
correlation
between
body
weight
and
reproductive
tract
weight,
and
the
latter,
in
particular,
is
an
indicator
of
sexual
maturity
(1).
Our
starting
material
of
bag
cell
clusters
was
obtained
from
sexually
mature
animals.
Bag
cell
clusters
were
cut
free
from
the
rest
of
the
abdominal
ganglia
in
ice-cold
filtered
seawater
and
stored
at
-200C.
For
radiolabeling
ELH,
Millipore-filtered
(0.22
,gm)
natural
seawater
with
a
final
concentration
of
10
mM
glucose
was
buffered
to
pH
8.0
at
250C
by
20
mM
Na
Hepes
and
supple-
mented
with
150
1Ag
each
of
K
penicillin
G
and
streptomycin
sulfate
per
ml.
L-[4,53H]Leucine
[250,uCi
(1
Ci
=
3.7
X
1010
becquerels);
Amersham;
specific
activity,
100
Ci/mmoll
or
[35Slmethionine
(350
,Ci;
New
England
Nuclear;
specific
ac-
tivity,
450
Ci/mmol)
was
taken
to
dryness
under
forced
air
in
a
boiling
water
bath
and
redissolved
in
the
above
medium.
Freshly
dissected
abdominal
ganglia
were
incubated
in
radio-
labeled
medium
for
16
hr
at
room
temperature
(200C)
and
then
subjected
to
a
series
of
rinses
in
filtered
seawater
lasting
2
hr.
The
bag
cell
clusters
were
dissected
out
and
kept
frozen
at
-20'C
until
used.
To
obtain
radiolabeled
ELH,
6
pairs
of
la-
beled
clusters
were
ground
with
34
pairs
of
unlabeled
tissue.
In
a
typical
preparation,
40
pairs
of
frozen
bag
cell
clusters
Abbreviations:
ELH,
egg-laying
hormone;
>PhNCS,
phenylthiohy-
dantoin;
IEF,
isoelectric
focusing;
NaDodSO4,
sodium
dodecyl
sul-
fate.
*
Present
address:
Department
of
Molecular
Biology,
University
of
California,
Berkeley,
CA
94720.
t
To
whom
correspondence
should
be
addressed
at
Division
of
Biology,
California
Institute
of
Technology,
Pasadena,
CA
91125.
6656
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"ad-
vertisement"
in
accordance
with
18
U.
S.
C.
§1734
solely
to
indicate
this
fact.
Proc.
Natl.
Acad.
Sci.
USA
76
(1979)
6657
were
homogenized
by
hand
in
ice-cold
10
mM
Na
phosphate
pH
6.0
(at
250C;
standard
buffer)
with'phenylmethylsulfonyl
fluoride.
The
homogenate
was
centrifuged
in
a
Sorvall
RC
2B
at
12,000
X
g
for
20
min
and
the
supernatant
was
dialyzed
(Spectrapor
3
membrane,
cutoff
at
Mr
3500)
against
two
changes
of
200
vol
of
standard
buffer
for
2
hr
at
4VC.
The
re-
tained
material
was
then
ready
for
purification.
Total
protein
in
samples
was
measured
by
absorbance
at
215
nm
with
a
Beckman
DB-G
spectrophotometer.
The'
sensitivity
was
0.052
A215
units/10
,g
of
bovine
serum
albumin
(used
to
generate
a
standard
curve)
per
ml.
Amino
Acid
Composition.
For
analysis
of
amino
acid
con-
tent,
ELH
was
hydrolyzed
in
two
ways:
in
6
M
HC1
under
re-
duced
pressure
at
1100C
for
24
hr,
and
in
3
M
mercaptoe-
thanesulfonic
acid
under
reduced
pressure
at
1100C
for
24
hr.
Peptide
fragments
were
subjected
to
the
HCl
hydrolysis
only.
All
amino
analyses
were
performed
on
a
Durrum
D500
amino
acid
analyzer.
Radiolabeled
ELH
was
cleaved
by
24-hr
incubation
in
6-fold
(wt/wt)
excess
cyanogen
bromide
in
70%
formic
acid
under
nitrogen
at
200C.
The
reaction
was
stopped
by
dilution
with
distilled
water,
and
the
sample
was
frozen
and
lyophilized.
In
one
experiment,
the
reaction
products,
a
mixture
of
whole
hormone
and
its
cleavage
products,
were
subjected
directly
to
sequence
determination
in
tandem.
In
a
second
experiment,
the
reaction
products
were
redissolved
in
distilled
water
and
separated
on
Bio-Gel
P-6
in
10
mM
Na
phosphate,
pH
7.5/50
mM
NaCl.
The
radioactive
peak
migrating
with
a
much
higher
Kay
than
ELH
was
again
chromatographed
on
Bio-Gel
P-2
and
then
on
Bio-Gel
P-4,
both
equilibrated
in
1
M
acetic
acid.
After
these
two
additional
steps
of
gel
filtration,
the
major
peak
of
radiolabel
was
subjected
to
microsequence
analysis
and
to
6
M
HCI
hydrolysis
and
was
found
to
be
fragment
14-36
of
ELH.
Sequence
Determination.
Automated
Edman
degradation
was
performed
on
the
intact
hormone
and
on
the
two
peptides
generated
by
cyanogen
bromide
cleavage
by
using
a
sequenator
program
similar
to
that
described
(14).
The
program
included
a
30-min
coupling
step
using
0.3
M
Quadrol
buffer,
a
single
5-min
cleavage
step,
and
automated
conversion
of
the
anili-
nothiazolinones
to
phenylthiohydantoin
(>PhNCS)
amino
acids
by
treatment
with
25%
aqueous
trifluoroacetic
acid
for
45
min.
The
cup
and
conversion
flask
were
maintained
at
520C.
The
spinning
cup
sequenator
used
for
the
Edman
degrada-
tion
was
designed
and
built
at
Caltech
and
will
be
described
in
detail
elsewhere.
It
incorporates
many
of
the
design
features
described
by
Wittmann-Liebold
(15)
and
Hunkapiller
and
Hood
(14),
with
further
refinements
in
the
delivery
head
as-
sembly
and
reagent/solvent
reservoir
system,
and
is
equipped
with
a
straight-edge
cup
from
Beckman.
Polybrene
(Aldrich,
6
mg)
and
glycylglycine
(100
nmol)
dissolved
in
0.5
ml
of
distilled
water
were
loaded
into
the
se-
quenator
cup
and
subjected
to
five
complete
degradation
cycles.
The
peptide
to
be
sequenced
was
then
loaded
and
the
se-
quencing
program
commenced
with
the
coupling
stage.
>PhNCS
amino
acids
were
identified
by
high-performance
liquid
chromatography
(HPLC)
on
a
DuPont
Zorbax
CN
col-
umn.
Details
on
identification
of
>PhNCS
amino
acids
and
standard
chromatograms
are
described
elsewhere
(16).
Isoelectric
Focusing
Gels
(IEF).
IEF
gels
were
prepared
and
run
as
reported
(9).
On
extrusion,
gels
were
immediately
sliced
into
1.4-mm
segments.
To
locate
peaks
of
radioactivity,
each
slice
was
added
to
5
ml
of
Wilson's
cocktail
(17)
for
assay
in
a
Beckman
LS
scintillation
counter.
Bioactivity
was
determined
by
incubation
of
gel
segments
in
two
changes
of
standard
buffer
and
injecting
the
combined
perfusates
(total
volume,
2
ml)
into
receptive
test
animals.
To
obtain
the
pH
gradient,
an
equivalent
gel
was
run
concurrently
and
sliced
into
2.8-mm
segments,
each
of
which
was
extracted
in
1
ml
of
100
mM
KC1
for
several
hours;
pH
was
then
measured
directly
with
a
Beckman
Futura
39505
electrode
and
a
Beck-
man
Model
76
pH
meter.
When
8
M
urea
1EF
gels
were
used,
normal
IEF
gels
were
made
to
8
M
urea
and
samples
were
brought
to
a
final
con-
centration
of
3
M
urea.
Electrode
buffers
and
other
conditions
were
identical
to
those
of
normal
IEF
gels.
Gradients
of
pH
were
similar
in
normal
and
urea
IEF
gels,
and
in
both
cases,
cytochrome
c
'was
used
in
every
gel
as
a
reference
marker.
Sodium
Dodecyl
Sulfate
(NaDodSO4)
Gel
Electrophoresis.
NaDodSO4/8
M
urea
slab
gels
were
made
according'
to
the
method
of
Swank
and
Munkres
(18)
with
a
final
concentration
of
12.5%
acrylamide
and
a
bisacrylamide/acrylamide'ratio
of
1:10.
Standard
mixtures
of
myoglobin
(17,200),
cytochrome
c
(12,300),'
a-bungarotoxin
(7800),
bovine
pancreatic
trypsin
inhibitor
(6160),
insulin
(5700),
and
glucagon
(3500)
were
ap-
plied
to
wells
on
either
side
of
the
sample
and
run
simulta-
neously.
Gels
were
run
at
150C,
to
minimize
diffusion,
at
a
constant
voltage
of
65
V
for
12-18
hr.
The
length
of
the
gel
containing
the
sample
was
then
cut
out
and
sliced,
crosswise,
into
1.4-mm
segments
for
assay
of
radioactivity
as
described
for
IEF
gels.
The
flanking
slabs
were
stained
and
destained
to
reveal
standards.
RESULTS
Hormone
Purification.
All
operations
were
carried
out
at
40C.
Cation
exchange
chromatography
(SP
C25
Sephadex).
A
dialyzed
homogenate
of
bag
cell
clusters
was
diluted
with
standard
buffer
to
a
final
volume
of
30
ml
and
applied
to
a
6.0
X
1.5
cm
column
of
SP
C25,
equilibrated
to
the
standard
buffer.
After
a
rinse
with
50
ml
of
standard
buffer,
a
linear
gradient
made
with
50
ml
of
standard
buffer
and
50
ml
of
20
mM
Na
phosphate,
pH
6.5/250
mM
NaCl
was
applied.
Two
major
peaks
of
protein
'were
eluted
at
approximately
65
and
144
mM
NaCl
(Fig.
1).
Biological
activity
was
observed
only
in
the
first
peak.
Excluded
material
and
rinse
fractions
also
did
not
show
any
biological
activity.
A
flow
rate
of
30
ml/hr
was
generated
by
gravity,
and
a
Gilson
microfractionator
collected'80
fractions
(1.1
ml).
00.36
300
60~~~~~~~~~~~~~~
I
0240
0
~~~~~~~~~~~~~~0
EE
E.30
E
-0.12
ioo3Q
0
20
40
60
80
Fraction
FIG.
1.
Sephadex
SP
C25
chromatography
of
supernatant
of
bag
cell
extract
after
dialysis
in
standard
buffer.
Purified
[3H]leucine-
labeled
ELH
was
added
as
a
marker
to
the
supernatant.
The
column
(6.0
X
1.5
cm)
was
equilibrated
with
standard
buffer,
and
a
gradient
of
50
ml
each
of
standard
buffer
and
20
mM
Na
phosphate,
pH
6.5/250
mM
NaCl
was
applied.
The
fraction
volume
was
1.1
ml
and
flow
rate
was
30
ml/hr.
A
background
of
20
cpm
was
subtracted.
Bioactivity
coincided
with
the
labeled
marker
ELH
peak.
Salt
concentration
was
measured
by
conductivity
on
a
conductivity
meter
(type
CDM
2d,
Radiometer,
Copenhagen).
Neurobiology:
Chiu
et
al.