M. SToREY2)and. KENNETH. B. SToREY2). 1)Department of Applied. Biology,. Kyoto ... subunit. When eggs were cold-acclimated, different profiles occurred;eggs ..... at 5â and 0â, PFK activity ... No.36). Arrows indicate the elution volumes of standards:rabbit muscle. PFK(a;360 ... 2.78±0.15mM(3.05±0.06),1.02±0.27. mM.
日 蚕 雑68(3),181-194(1999) J.Seric.
Sci.
Jpn.
Regulatory
properties
in the TOSHIHARU
eggs
of the silkworm,
FURUSAWA*1), YuKlo
AYA
SuGIMuRA1),
1)Department
of
Applied
2)Department
of
Biology,
of phosphofructokinase
KONISHI1),
JANET Biology,
M.
Kyoto
Carleton
Institute
University,
Phosphofructokinase(PFK)was silkworm,
column
chromatography.
of
a
and
Partially
whereas purified
PFK
of
Technology, Ontario,
geggs)over
9-days
than
day
initial
a change
geggs
were
kinase
or
oviposition,
values
incubated protein
Diapause
egg
from
form can
to of
day
10
be
PFK
be
and
linked
regulated
showed
the
increase
in
a variety
activity
reversible
factors
protein
when
under
time of
to
of
the
enzyme
appears
diapause
was
prevented
phosphorylation,
and
3.4 mM
at under
to
be
by
the HCl-
in Bombyx
effects the
incubation
incubation
activity
temperature
day
dephosphorylating
effect
PFK
6 and
monitored.
by
the
Thus,
including
was
as
form
PFK.
F6P
HCl 5-fold
protein
unchanged
in So .S over
same
diapause
of
of
S0 .5 for
its
increase
of day
on
a-decrease the
by to
activity
of CAMP-dependent
reduced
of
0.2 U/
rose
action
was
fructose-2,
prevented
of this
the
fructose-6by
6 and
homogenates
which
strongly
virtually
Hence,
the
4 mM
was
was
310
90 kDa.
concentrations
day
mechanism
enzyme,
of incubation
dephosphorylation
by
subunit,
but
was
if the
at
at
activity(about
initiation by
centered
substrate,
PFK
doubled
were profiles
peak
activated
a high
constant
had
promoted
its
was
Sephacryl both
different
a broad
for
by
diapause
of the
on that
activity
in
PFK
ATP-agarose
peaks
PFK
curve 7.0.
and
about
eggs
that
the to
effect
S0 .5 of and
conditions.
phosphorylated
the
high
HCI-treated
at
dephosphorylating
appears
a
conditions
0.9 mM
treatment
and
showed
that
of
primarily
low
determine
conditions
peaks
inhibited
when
state
distinct
cold-acclimated,
and
activity
To
Japan.
at 25℃)of
and
showed
were
at pH
showed
PFK
9.
606-8585,
5B6.
S-300,
two
saturation
whereas
day
phosphatase
PFK
PFK
6 and
by
SToREY2)
eggs(incubated
kDa;SDS-PAGE
broad
mM
phosphorylation
under
phosphorylating
conditions.
state
oviposition
after
KOTANI1),
Kyoto KIS
Sephacryl
activity
phosphate,
after
in the
two
eggs
Sakyo-ku,
non-diapause
eggs
sigmoidal
Diapause
B.
Canada
into
185
showed
1.02±0.27
inorganic
ATP.
one
be
under
and
and When
5℃
a
So.5 of
from
separated
310
at
KENNETH
hydroxylapatite,
exhibited
held showed
AMP
substrate,
treatment
at 0℃
an
6-bisphosphate,
could
subunit.
kDa
eggs
phosphate(F6P)with
higher
48
acclimated
90 kDa,
second
being
EIJI
Dec.241998)
activity
masses
single
occurred;eggs
using
PFK
molecular
composed
day
by
more
SAKANO1),
Ottawa,
purified
Bombyx mori,
S-300,the
DAISUKE
SToREY2)and
(Received
the
Bombyx
on
effects
of
the
eggs native
allosteric
modulators.
Key
words:6-Phosphofructo-1-kinase, reversible
protein
diapause,
egg
development,
allosteric
regulation,
phosphorylation
Introduction Diapausing *To
whom
correspondence
should
be addressed
mori, -181-
have
eggs
the ability
of the silkworm, to endure
adverse
Bombyx environ-
182
The Journal
mental
circumstances,
ture. to
Chief
particularly
among
support
the
diapause
egg
glycogen
and
glycerol
AWA
and
accumulation
against
freezing
initiation
of
bform
phase,
glycogen
to
the
active
a
leading
from
pentose
the
reducing
sis
from
pathway,
activity
of
drogenase
is about
high
as
phofructokinase(PFK)(SuzuKl
the
amounts
of
accumulated
pentose G6P
is
to
1990).Asimilar eggs
the
regulated.
PFK
inhibitory
to
divert
phosphate
most
the
reactions
these must
the
number
the of
synthesis
in
chiocarnpus activity
cited
production
Papilio
high
be may
a
insects
including and
during tion.
CHINO,1982)and
Philosamia
Cynthia
populi(HAYAKAWA is modulated
and by
low
the
treat-
and In
kinetics
of the
and
PFK
enzyme,
states study,
eggs,
and
the
on
Materials
in
present
temperature by
regulatory
developing
the
regulation
and
change
non-diapause
PFK
we char-
influence properties,
reversible
protein
phos-
Methods
the
embryonic laid
identical
enzyme in glycerol Havestrehalose and
CHINO,1982). temperatures.
Tri-
eggs
for
each
to
eggs 12 days,
and
about
acclimated
25℃
to
hours
were the
transferred hatching
80%were eggs,
at
pooled
The
eggs
proceed
were
embryonic at
oviposition. to 25℃
ability
an and
regimes:(1)con-
chilling
after
to
genera-
were
following
or(2)continuous 24
maternal period(i.e.
stage),
the
exposure
for
3 h
experiment.
under
0℃starting these
a
exposure
dark(24D:OL)
life of the within
programby
the
developmental
development, a
race(N4)was
non-diapause
Eggs
convert
Monema
was
eggs
the for
temperature(25℃)in
maintained
key
activity eggs(SUZUKI
However,
clear.
produce
used
cryoprotectants
rnachaon and
in
as
of
cold-hardy
tens(HAYAKAWA production
been
but
dormancy(KAGEYAMA
polyvoltine
to
pentose
that
diapause,
in diapause
from
the
tinuous
has
regulating
been PFK
enzyme
med
is neces-
into
of
and
level,
diapause
Animals:The
sorbitol. PFK
PFK
or
and
flux
PFK
metabodiapause
initiation
trace
break
acclimation
and
activ-
phorylation.
10℃(Fu-
glycolysis
flow
of
PFK
both
in non-diapause
between not
(STOREY,
in
non-
operate
over
carbon
of
sorbitol below
glycolytic
control
pathway
to
of
to
et al.,
To
some
HCI
acterized the
in
a
higher
purified
pentose
occurs
fall
locus
Although
continue,
the
accumulate
al.,1987,1992).
have
fraction
via
temperatures
et
pathways,
G6P
which
when
phos-
10%of
glycerol(YAGINUMA flux
activity
through
Another
carbon
diapause
RUSAWA
oxidized
metabolized
pathway
glycerol
are
of
about
is at
the
1971).
dehy-
of
to-5℃)which
carbohydrate
MIYA,1975,1977)and with
pathway
a combination
during
mechanism(s)responsible
MIYA,1975)
to
pathway.
eventually
phosphate
sary
equivalent
sorbitol
phosphate
that
and
G6P
eggs,
(G6P)
as
At
OHNISHI,
al.,1974;SUZUKI In these
twice
ed
by
changes
on
the
development(KAGEYAMA
activity
and
the
synthesis
investigated
considerably
syntheand
glucose-6-phosphate
influence
been
embryonic
PFK
supplies
glucose(CHINO,1960;KAGEYAMA
have
locus,
temperatures
into
sorbitol eggs,
by
al.,1981).
PFK
warmer
inhibited
into
their
OHNISHI,1971).
sorbitol
et
and
at the
flow
syn-
is induced
temperatures(5
silkworm
and
which
po-der(NADPH)for
carbon
anaerobic
is coupled
sorbitol
at
but
at colder
The
path-
sequential
et
carbon
synthesis
diverts
ity
the
control
active
allow
1982).In
conver-
then
to
being
lism
sorbitol
IVIIYA,1975;KAGEYAMA,1976).
and
the
under
to
the
and
is attributed
its inactive
al.,1975).
glycogen
phosphate
At
larvae,
temperatures(STOREY
of factors
synthesis
by
form
OHNISHI,1971;TAKAHASHI and
both.
from
et
glycerol
glycerol
protection
is stimulated
of
lowering
enzyme
al,1982).
sorbitol
phosphorylase
solidaginis
(10-15℃)to
provide or
Vol.68 No.3
EuYOSta
This
diapause(CHINO,
desiccation
diapause
glycogen
to the
of sorbitol
et
may
conditions(YAMASHITA way
of
of Japan
thesis of
YAMASHITA,1977;FURUS-
or
of
stored
sion
pools
Science In
conversion
SHIKATA,1982;FURUSAWA
Polyol
from
large
initiation and
tempera-
adjustments
is the
into
the
1958;YAGINUMA
low
biochemical
survival
reserves at
of Sericultural
and
of more
observed
in
5℃
incubated than
90
5℃-and
respectively(FURUSAWA
or
When
0℃et
al.,
1989). The
bivoltine
race(Shunrei
x
Shogetsu),
FURUSAWA et al.: reared
on
med
to
fresh
mulberry
produce
diapause
ture(25℃)and during
the
tion.
Eggs
a
Some
laid
within
the
20
eggs for
h after
Eggs
and
the
eggs
the
HCI-treatment.
Enzyme
dation
at
were
20
nm
using
non-diapause
or
a
10th
U
240
were
or
using
30%v/v
7.0,15
natant Sephadex
few
crystals
of
× 40
cm)which
potassium
pellet passed G-25
×g
were
was
5ml eluted
(equilibrated
20
carried
discarded through
solid
and
determine
subunit
PFK.
A
out and
a
small with
the column HB)
40μl
min
an
ice
then
was
of by
enzyme mass
fraction of
applied
and
was
centrifugation
molecular
volume
was
to
a
out
by
the
of
boiled
sample
loading
10%
(w/v)
SDS-polyacrylamide
carried
in
fraction
Purified
of each
equal
gel
mass mass S-300
Standards dicated
above
electro-
method
of
LAEM-
by
at
280
kDa).
by
for
was
in
kDa), kDa),
bovine
same
manner
as
purification
assays
at
340
pyruvate
(99
kDa),
weight
nm
and for
kinase(240
hexokinase
heart
Se-
chromatography.
the
enzyme
molecular
determined
filtration
run
dehydrogenase(90
The
native
PFK
activity
(160
nm
determination:The
gel
for
PFK(360
aldolase malate
of
were
detected
super-
a
ADP
MLI(1970.
muscle or
aliquot
in and
phacryl and
These on
3
5 m1VI
These
by
overnight.
was
temper-
Centricon-30
4℃
and
PFK
at a room Each
a
of HB,
fractions.
22℃). in
to
plus
out
at
used
an
in HB,5ml
Then
1 ml
carried
at
rpm
Seto
washed
KCI
F6P
in
were
was
fractions
through
HB.
5 mM
same
applied
mM
concentrated 5,000
peak
in HB,5ml
in
collected
the
sequentially 500
F6P
of
l 30
× 2 cm)equilibrated
was of
10 mM
fraction
then
cm
ADP
5 ml
molecular
with
min.
and
column
Molecular
homogenate
for
with
with
centrifugation
of HB,2ml
2.5mM
in
l ml
Pooled
above
of 2.5 mM
of
acrylamide
3.5 g)were
The
The
10 ml
pooled
column(1.5
and
eluted
column(1
HB,
mM
a mortar
by as
to
6.8)containing
of each
activity.
G25
were
S-300
col-
used
equilibrated
was
linear
were
2-mercaptoethanol
PFK
desalted
a
was
fractions
buffer(pH
measure
phoresis
of
homogenized
27,000
to
buffer
inhibitor)
using
homogenizer. at
used
eluent
to 200μl
fil-
phosphate(KPi)
of the Peak
mM
100
with
fractions
was
EDTA,10
and
eluted
a Sephacryl
phosphate
The
to homogenization.
of HB
procedures
was
cm
The column
2 ml
100μl
to
min.
× 3.5 cm)equili-
potassium
activity. applied
was
20
eggs(usually
subsequently
centrifuged
4℃.The
from concen-
2-mercaptoethanol
prior
frozen
Turrex
subsequent
PFK
to
1
1.8 cm
or
ature(approximately
plots
fluoride(protease
4 volumes
and
50
then
HB
reduces
purification:
A
immediately of
and
and
for
mM
glycerol.
with
lected,
mM
mM One
fractionations
constants(Ka)
and
phenylmethylsulfonyl
was
that
reciprocal
buffer(HB)contained
Ultra
final
plots.150
F6P
double
pH
pestle,
a
determined
imidazole-HCI,
Samples
0-500
buffer.
of HB,5ml
2 U
1/[activator].
added
of
HB
with
constants
Hanes
Activator
preparation
ground
in
50%)were
indicated.
determined
were
and
affinity
Hill
homogenizing
and
was
in
unit(U)
versus[inhibitor]at
(1/V-V0)versus Enzyme
PFK
7.0),5mM
isomerase
of inhibitor
by
of velocity as
HB.
ATP-agarose
NADH,0.5
Substrate from
concentration
trations
in
gradient
for
hydroxylapatite
brated
were
mM
a or
phadex
ATP,50
dehydrogenase
velocity
plots
onto height
conditions
buffer(pH
mM
1 ml.
oxi-
recording
assay
triosephosphate
determined
enzyme
loaded
rpm
2 cm
buffer
spectrometer
Standard
glycerol-3-phosphate
values(the
after
NADH
Gilford
imidazole-HCI
of
day
(PFK)
Shimadzu
MgSO4,0.12
were
at 25℃
3,000
x
assay
embry-
develop
fructose-6-phosphate(F6P),2mM
volume
HCl
was
183
(1.2cm
in
them
obtain
following
a
or
aldolase,0.5
with
keeping to
gth
by
22-25℃.
KCI,5mM
an
egg.
at
v/vglycerol.
340
mM
treated
to
the
monitored
at
at pooled.
Phosphofructokinase
spectrophotometer UV-1600
were
allowed on
trate
genera-
period(i.e.
of Bombyx
centrifugation
tempera-
maternal
5 min)after
hatched
was
the
oviposition
then
assay:
activity
high
eggs(artificial
were
program-
stage)were
developmental
eggs).
by
a 3 hour
diapause
for
was
photoperiod(16L:8D)
life of
gr.,1.075,46℃
at 25℃ onic
eggs day
developmental
of
(Sp.
long
embryonic
identical
leaves,
Phosphofructokinase
or
by
PFK
rabbit kDa),
kDa),
and
absorbance
cytochrome of
in-
were
c(14.5 was
deter-
184
The Journal
mined
from
mass
for
a
the
To PFK,
plot
40μl
in
buffer
a
and
equal
gel
phoresis
was
each
mass
fraction
a
10%
by
the
method
gel
electro-
The
Reversible
phosphorylation
was
ground
then
one
volume
equal
20
of
mM
D).Buffer
A
tein
and
Buffer
was
plus
ylation
18hours,
was
then
homogenized
×g
a
at 3,000 activity.
PFK
Protein
content
the
estimated
20
min
the
at
Sephadex
for
1 min,
for
Ultra
method
of
In
presenting
the of
that
exists
in
was
(peak
I)form
both
by by
purified
enzyme
of
could
contents eggs et
al.
so
enyzme
was
for
these
low
it
eggs
hexamer
final
method,
a
the
of
yields
kinetic
out
studies
after
results,
B. mori Ⅱ)or
carried
these
used
re-
molecular
development
to
this
be
of stain
band a
from
embryonic
with not
blue
silver
major had
From
Due
detected
with
tetramer(peak
eggs.
enzyme
umn
a
SDS-
molecu-
brilliant
the
PFK
during
Each
contaminants
subunit
native
in
on
was
noted
cases,
was ADP
subunit
band
minor
48 kDa.
the
non-diapause
PFK
single
PFK
de-
PFK 5 mM
the
several
both
about
The
followed
LOWRY
(Fig.2b).
weight
and
were
buffers
collected.
Coomassie
preparation
exten-
analyzed
with
whereas
PFK
then
After
plus
and
A
stained
appears
G-25
non-diapause
and
gels
was 5℃.
eggs
the
by
and
of
were
its purity
weight(Fig.2).
on
(Fig.2a),
10mM
fractions
PFK
desalted
Methods,
F6P
concentrated,
to check
185kDa, the
G25
sequence and
I and
and
column.
the
the
peaks(Fig.1).
pooled,
of 5 mM
1 ml was
applied
of Peaks
Sephadex
Materials
5 ml
and
the
supernatant
of
rpm
the
100
lmM
an
Protein
by
lar
at 4-5℃
determination:
HCI-treated
EDTA
plus
with
fraction
were
U
from
purification,
ATP-agarose
the
then
310kDa
Ⅱ
and
× 10-2
eluted
main
further
with
in
was and
weights
through an
8.11
enzyme
about
Peak
washing
HB
enzyme
and
D
in
pooled
was
column
mM
fractions
KPi)were
in two
For
140
Active
4ml
molecular were
to
sive
mM
column
PFK
fractions
PAGE
plus
with
for
column
assay
pro-
homogenate
and
centrifugation
were
of
scribed
dephosphor-
incubated
The
discarded,
through
of
ATP enzyme Buffer
27,000
all
consist-
D
200 these
S-300
estimated
U/ml)at
peak.
The
Sephacryl
applied
mM
both and
Buffer
was
at
passed
inhibit
promoted
contained
homogenizer.
centrifuged
of
in
eluted
NaF,5mM
mixture
and
Turrex
to
B
contained
Each
buffers,
phosphatases
Cpromoted
and
MgCl2.
pellet
and
centrifugation
with
7.0),15
MgCl2,10mM
Buffer
of and
mixed
three
100mM
and
gram
10%(v/v)glycerol(Buffer
Buffer
NaF,10mM
cAMP.
of
designed
EGTA.
phosphorylation mM
110
filtration
II
LAEM-
of HB
was
one
protein
D
5 mM
One 5 ml
imidazole-HCI(pH and
kinases
PFK: with
of homogenate
2-mercaptoethanol
of
of
in a mortar
volume
containing
and
a
× 10_2
single
respectively.
eggs
ed
a
activity
total
to
MLI(1970).
an
activity(3.38 with
(between
(w/v)
of
Vol.68 No.3
(54.4%recovery).
loading
SDS-polyacrylamide out
the
boiled
sample
to
of
on was
of
applied
carried
of Japan
KPi
volume
and
Science
maximum
molecular
chromatography
then
acrylamide
log
molecular
of
column
min
versus
subunit
aliquot
ATP-agarose 3
Ka
standards.
determine
a
for
of
protein
of Sericultural
of
studies
with
the
partially
pure
purified
hydroxylapatite
col-
chromatography. To
(1951).
assess
the
cold-acclimated
characteristics
non-diapause
of
eggs,
PFK
of
samples
of
Results eggs Enzyme
purification
nation
of
(kept
was
25℃).
natant(1.3
ity
purified
eluted
to
from
old)of After
ml;total
applied
weight
determi-
tion,
the
highspeed
activity,14.9
110-200mM
develop-
non-diapause
desalting,
a hydroxylapatite between
embryonic
× 10_2
column;PFK KPi
1).In activity
U)was
on a
310
enzyme
at 25℃
then
After
the was kDa
24
hours
gel
PFK
non-diapause
and
stored
in
filtration
present 90
distribution
after
fractionation
column, by
for
continuously
days.
analyzed
super-
had
30
ylapatite
type
activand
kept
and
for
eggs(6-day at
molecular
PFK.
PFK mental
and
were
these
on
kDa. in
at
5℃
or
on
a
hydrox-
samples
Sephacryl
eggs in two
at
5℃
broad This
embryonic
oviposi0℃
was
5-300(Fig. and
peaks
contrasts
0℃,
PFK
centered with
developmental
the
FURUSAWA et al.: Phosphofructokinase
Fig.1. of
Elution
B.
mori.
circles),
or
circles)or
profiles The
were
applying
to
fraction
the
5and
0℃,
at
U/ml(25℃,
kDa),
aldolase(c;160
for
the
The
the
Sephacryl
containing
the
Tube indicate
at
25℃
respectively.
for
first
24
enzyme S-300
Relative
kDa),
U/ml(5℃,
Tube
standards:rabbit
hexokinase(d;99
kDa)and
in
×,1.15
each
as
per
1.77
then
1.77
either
on
hydroxylapatite
is
U/ml
in
eggs
U/ml(0℃
kDa)
of
at
25
,
4 .1 × 10_2
, Tube
, pyruvate
dehydrogenase(e;90
relative kept
activity
× 10_2
5℃(open
plotted
the
maximum
PFK(a;360
eggs
at
preparation
the
diapause
development(solid
chilled
× 10-2
to
No.54)and
malate
from
chromatography
cent
muscle
activity
enzyme
×, and
185
embryonic
and by
egg.
PFK for
oviposition purified
Activity
is expressed
for
oviposition
after
partially
column.
of
after
hours was
activity
volumes
chromatography
6-days
activity;4.1
× 10-2
elution
column
25℃
highest
No.47),1.15 the
S-300
kept
squares). to
Arrows
Sephacryl
were
kept
0℃(open
before
of
eggs
of Bombyx
No.36).
kinase(b;240
kDa)
, and
cytochrome
c(f;14kDa).
eggs
at
activity The
The
also
differed
185kDa
diapause eggs
25℃.
at
gent
in
that
low
eggs
induces
su
the
was
whereas 5℃.
eggs
distribution
between
PFK
eggs,
kept
relative
The kept
temperature
the
the the
90
PFK at
three
majority kDa
0℃.
in the
in
more
Thus, of
native
PFK
groups.
enzyme
became
exposure
changes
of
non-
of
HCI-treated
Shogetsu)were column
chromatography,
purified
enzymes
diver-
substrate
it appears
of
units.
PFK
The
was
diapause
was
for
used
values(Hill
at
F6P
higher eggs
all
coefficients,
of
nH,
are
affinity. Extracts
of
(1.61±0.08),and 6-day
old
eggs
of
non-diapause
pH
6.5,7.0
0.97±0.27 and
8.0,
and
8.0.
non-diapause HCI-treated (Fig.3).
So .s
in brackets)were
2.78±0.15mM(3.05±0.06),1.02±0.27 Substrate
enzyme
7.0
the
values
partially of
pH
from
that pH
the
analysis
6.5,
PFK
x
hydroxylapatite
then
for
pH
of
than at
eggs(Shunrei
by and
affinities
affinity
eggs
diapause
fractionated
in the
non-diapause state
(N4)and
mM mM(2.20±0.48)at
respectively,
for
PFK
from
186
The Journal
of Sericultural
Science
of Japan
strongly
Vol .68 No.3
activated
μMand
0.33μM,
from
even
which
eggs
at
fully
two
Ka
values
for
Pi
no
effect
the
supernatant;B,
pooled
ylapatite
peak
weight
used
were
hydrox-
from
column.
Standard
myosin(200
kDa),
rose
above
and
8,0.
ATP
phosphorylase
serum
albumin(66
carbonic
pH
proteins
kDa),
hibitor(21.5
8.0,
mM
kDa),
ovalbumin(45 kDa),
mM(1.50),4.85 pH
and
trypsin
treated
diapause
saturation
in-
diapause
eggs but
diapause
those
eggs
in The
F6P
for
for
rather
at
PFK
from
the
the
at
for
PFK
at
least
from all
eggs
pH
activator, PFK
effects
6.5,7.2
diapause HCI-treated
non-
three
pH
HCI-treated
and
inhibitors.
of
the
potent
are eggs
shown and
to
6.5,7.2
occur
for
Fig.4.
PFK eggs
from was
The
values
pH7.0
at
over
ATP
3.2,2.2
of and
10 HCI-
5.0 mM
for
pH
6.5,7.2 Km
and
0.1-0.22U/g
of diapause
5
5.0 mM also
at
for
eggs,
pH
6.5
whereas
in and
8.0,
at
shows
0.08,0.42
and non4.29
respectively. ATP
were
and0.5mM
and
were
were
values
diapause
oviposition(Fig.6).
pH8.0
all mM,
HCI-treated
0.1 mM
values
andpH7.0
which
Figure
were
16.3
6.5, and
For
at
Km
ATP
pH
were
and
for
at
whereas
values
9.4,7.8,
Iso values
0.2mM
Phosphorylation
eggs
at
ATP
ATP
eggs,
between
eggs
affinity.
eggs at
8.0, respectively,
respectively.
and
diapause
In non-
non-diapause
were
and
3.8 mM 8.0,
(F2,6P2),on
in
pH
particularly
began
higher
substrate
mM
allosteric
diapause(HCI-treatment)and eggs
either
ATP,
non-diapause
3-fold
diapause
fructose-2,6-bisphosphate from
levels
eggs(N4)was
values
eggs
from
were
ATP Km
hyperbolic.
activators The
HCI-treated when
at
by
I50
diapause
atpH6.5 Allosteric
by
from
ATP
inhibition The
7.2and HCI-
substrate
PFK
sigmoidal
7.69
mM(1.20)at
respectively,
for were
with
3.29
eggs(Fig.3b).
curves
values,
compared
8.0,
0℃.
inhibition
substrate
non-diapause
inhibited
where
ATP.
ATP
kDa),
and
mM(1.36)and
6.5,7.2
enzyme and
occurred
2 mM from
respectively. eggs(Fig.3a),
5℃
bovine
kDa).
non-diapause
the
at
asses-
β-galactosidase
b(97.4
anhydrase(31
about
less
treated (116kDa),
PFK
was
for eggs
inhibition
PFK
somewhat
molecular
standards;1-4:fractions
agarose-affinity
crude
after
eggs
Pi
when
differ-
are:A,
chromatography;C,
eggs,
For
obtained
sigmoidal
For
(Fig.5).
9.98
old), but
eggs.
was
non-diapause
to
not
of
eggs(6-day
subject
and
egg
values
of diapause
was
and
eggs
diapause
Ka
non-diapause
was
0.13μM
(data
on
with
curve
old
old
diapause
to be
effect
PFK
curve
PFK
Lanes
other
of PFK
respectively
no
on
6-day
30-day
ATP
(N4),
a hyperbolic
but
from
activator
of non-diapause
from
sed,
purification.
F2,
2.2μM
the
diapause
activated
PFK
has
PFK
of
as
from
allosteric
had
also
activation,
steps
by
high
determined
egg
Pi
mM
ent
of 0.1 PFK
enzyme
HCI-treated
AMP
PFK.
an
were the
shown).
at
values
activation as
the
is also
non-diapause
PFK
no
concentrations
10.3μMfor
of
Ka
sources.
and
analysis
with
showed
activated
AMP
SDS-PAGE
F2,6P2
respectively.However,
diapause
6P2,
Fig.2.
by
In 0.1 mM
atpH8.0.
dephosphorylation. eggs,
PFK eggs
activity up
In embryonic prevented
from
to
fluctuated 9-days
after
developmental diapause
initia-
FURUSAWA et al.: Phosphofructokinase
Fig.3.
Effect
of
polyvoltine,
pH
hydroxylapatite G-25.
6.5,7.0
or
A
as
8.0
tion about
the
by
again
HCI
g
activity
was
eggs
at least
less
and
then day
before
in PFK
reaction
at
diapause
eggs,6-days
Material
and
was
mixed
25℃.
PFK was
for
n=3
from
950μl
by
of
20
mM
KCI,0.12mM
aliquots
of
purified
desalting
by
through
imidazole-HCI
N and
separate
4 g
partially
Methods)followed
with
MgSO4,50mM
means
187
old(b),
dehydrogenase,
show
natural
40
to
as
buffer
DH,0.5Ualdolase,0.
different
concentrations
preparations;SEM
at
change
bars
are
of
contained
eggs(N4),
PFK
and
l day
level
protein,
was
after
up
these
seen, due not
of 0.1 U/g
to
to
activity
oviposition,
about
of 0.85
U/g
showing a specific result
just were
patterns that
of
the
rise of
a
general
could
content
of
the
or
This
a less
latter
could
from
developing
B.
extracts
three
inhibit phatases, ylation,
activity
to
active
to
the
result
which
egg
enzyme existing
of
from
6
buffer both
or
kept
could
types.
and
Buffer
B
old were Buffer
protein
Buffer
kinases promoted C
active
state.
reversible
pro-
to to
be
regulate
determine
modified
crude
9-day
at 25℃
the
order
phosphorylation,
eggs
promoted
HCI-
diapause
of a more
In
PFK
the
increased
is known
sources.
mori
in
versus
conversion
be
other
reversible
PFK
either
the
phosphorylation
PFK
of
due to
from
tein
of
developmental
be
synthesis
treated
induction of
eggs
whether
0.4 U/g
activities
parallel
the
In the
increase
embryonic
enzyme
PFK
protein
larvae. The
just
5℃,
overall
treated
eggs
level shown).
When
was
0.98U/g
at
low
5
increasing
not
a maximum
were
activity
a
eggs
day
in
increased,
then
continuously
gradually
U/mg
through
of
rise
was
non-diapause
days(data
0.1 U/g
6, and
0
6 and
maximum
kept
activity
activity
day
a
increased
enzyme
Data
day
In
hatching.
activity
on
maintained
than
expressed
in
extract
ATP,50mM
PFK
developmental
was
this
to
were
embryonic
enzyme
2mM
figure.
from
2-fold
day
for
of
Thereafter,
hatching.
(N4)which
PFK-catalyzed
described
treatment, eggs
about
on
before
on
the
oviposition.
rising
the
HCI-treated
egg.
symbols.
0.25U/g
after
of
or
aliquot
containing
in
the
by
rate
isomerase,2Uglycerol-3-phosphate
shown
within
initial
eggs(a),
50μl
5Utriosephosphate F6P
the
chromatography(as
Sephadex (pH
on
non-diapause
of Bombyx
diapause
and
incubated A
was
and
by
enzyme HCI-
with
one
designed protein
enzyme enzyme
to phos-
phosphordephos一
188
The Journal
Fig.4. from
Effect
of
diapause
3.Standard assays
of
Fig.5.
egg
of
oviposition conditions
as at
on
the
described
in
except
for
Results
0.8
mM
rate
Fig.3.
mean
pH
values
egg
partially
purified
described
concentration
PFK
non-diapause
extract(100μl), of
of
final
non-diapause
reaction. or
of
the
was
eggs(b)as of
PFK.
F6P
in was
Results
are
Figure
3 mM
for
means
of
preparations.
PFK
values
PFK
non-diapause
that with
eggs(b), Aliquots
different
except
the
reaction.
or
separate
of
of Japan Vol.68 No.3
PFK-catalyzed
assays
two
diapause
the
used for
on
initial
are
of
Science
untreated(a),
were
performed
ATP
rate and
25℃
and
HCI-treated
25℃
concentration.
preparations.
initial
at
PFK
determinations
eggs(a),
the
HCI-treated
conditions
diapause
Effect
diapause
on
both
assay
duplicate
ATP
F2,6P2
eggs,
of Sericultural
the
duplicate
was
were
imidazole
prepared
eggs(c),
buffer(pH
determinations
all
assayed
from
4 g
sampled
6
under 6.5,7.0
performed
samples
standard
days
or
8.0)and on
two
of after assay
varying separate
FURUSAWA et al.: Phosphofructokinase
Fig.6.
Changes
in
mental
eggs)(b),
and
and
data
are
scale).
PFK
activity
expressed
Results
are
in
non-diapause both
as
means
diapause
eggs(a),
eggs(c).
of
U/g
PFK
eggs(filled
duplicate
of Bombyx
HCl-treated
was circle,
diapause
measured left
determinations
egg.
under
scale)and on
eggs(embryonic
standard as
performed
189
assay
U/mg
two
develop-
conditions
protein(open
separate
at circle,
25℃ right
preparations.
phorylation. Effects of incubation on enzyme substrate affinity for F6P were then assessed
state of PFK as it was at the time of sampling. Table 1 shows that PFK in both 6-and 9-day old
(Table 1), F6P affinity being a key parameter that is typically modified by the phosphorylation state
diapause eggs had similar F6P affinity constants with values of about 4-4.5 mM for enzyme prepar-
of the kinases
ed in buffer A. Incubation of extracts from diapause eggs under conditions promoting
enzyme. By inhibiting and protein phosphatases,
tion in Buffer A preserves
both protein homogeniza-
the phosphorylation
enzyme phosphorylation
(Buffer B) had no effect
Table 1. Effect of Phosphorylation and/or dephosphorylation bufferes on enzyme affinities F6P and Hill coefficient (nH) of PFK of diapause and HCl-treated eggs
The
diapause
for
embryonic
mixed
an
at
(±SEM)of
in
the
volume
4-5℃
HCI-treated
diapause
18
Buffer
hours,
b or
are
A, and
B S0.5
or and
C
eggs,
9-day
old,which
with as
shown
nH
were
performed
significantly
HCI-treated
6-and
homogenized
determinations
a and
eggs
were of
for
triple
symbols each
and
development, equal
incubated
with
eggs
on
different respectively,
HB
and
in
Materials
estimated two
separate
from
the by
prevented then
and from
the
from
Student's
diapause
volume
of
Methods. Hill
plot.
t-test.
A P
