Regulatory properties of phosphofructokinase in the ... - The Storey Lab

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M. SToREY2)and. KENNETH. B. SToREY2). 1)Department of Applied. Biology,. Kyoto ... subunit. When eggs were cold-acclimated, different profiles occurred;eggs ..... at 5℃ and 0℃, 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



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