Metabolite-induced activation of hepatic phosphofructokinase

Vol. 118, No. 2, 1984 Jctnuary 30, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 567-572

BIOCHEMICAL

~TABOLITE-INDUCEDACTIVATION OF HEPATICPHOSPHOFRUCTOKINASE Christopher Biological

Laboratory,

G. PROUD

University of Kent, Canterbury, Great Britain.

CT2 7NJ,

Received November 14, 1983 SIMfARY: Hepatic phosphofructokinase, isolated in a mediumcontaining 100 I&! This metabolite-induced activation was can be activated by ATP. m-4> 2m+, investigated in view of the suggestion that it is related to phosphorylation The results obtained do not support this interpretof phosphofructokinase. ation. Inhibitors of protein phosphatases (NaF) and kinases (the I@++chelator, ethylene diamine tetraacetic acid) did not affect the recovery of In contrast, media of high ionic strength reduced the phosphofructokinase. phosphofructokinase activity and rendered the enzyme sensitive to ATP-induced act ivat ion. Activation was also induced by other known effecters of phosph ofructokinase (nucleoside triphosphates, fructose bisphosphates) and was not dependent on Mg++-ions. It is suggested that activation represents ligandinduced reversal of the inactivation of phosphofructokinase which occurs at high ionic strength. sensitivity of phosphofructokinase The differential from fed or starved animals to inactivation and reactivation is discussed.

INI’RODUCTION: Hepatic phosphofructokinase 1.11;

PFK) occupies a key position

metabolism and its activity substrates,

products,

bisphosphate) (l-3). dependent protein

for the control

and the recently

of liver

discovered activator,

kinase, although the significance regulation

fructose

2,6-

by cyclic

AMP-

of this modification

of

remains unclear (4-6).

supernatants prepared in the presence of

phosphate and ammoniumsulphate.

The degree of activation

nutritional

The activation

state of the animal.

of PFK by an endogenouscyclic

AMP-independent protein

phosphorylation

kinase.

The

mechanismhas however remained unpro.ren,

do not support the contention

mediated by protein phosphorylation

varied with the

was ascribed to phosphory-

and this report describes experiments designed to clarify The results

(including

(7-9) have described an ATP- and time-dependent

of PFK in rat liver

case for this putative

carbohydrate

PFK is also subject to phosphorylation

Brand and sling

lation

1-kinase, EC 2.7.

is modulated by several metabolites

the enzyme for its physiological

activation

(6-phosphofructo

this question.

that ATP-induced activation

is

and show that other metabolites can 0006-291X/84 $1.50 567

Copyright 0 1984 by Academic Press. Iw. ,411 rights of reproduction in unv,form rrserrvd.

Vol. 118, No. 2, 1984

induce a similar bility

BIOCHEMICAL

activation

of PFK.

to metabolite-induced

from livers

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It is clear

activation

however that

of PFK differs

of fed or starved animals.

The likely

the suscepti-

in supernatants

derived

cause of these differences

is discussed. M4TERIAL.S AND ME’IHOIXS: Coupling enzymes and NADH were from Boehringer &ewes, and other biochemicals were from Sigma (Poole, England) and of the grade available. Bats (CD-strain) were kept on a 12h dark/light cycle (1700, 1900) and allowed free access to food and water. Where appropr’.ate, food deprivation was begun at 0900. All animals were sacrificed by cervical dislocation between 0830 and 0900. Livers were rapidly removed, chilled on ice and homogenised in a Teflon/ glass homogeniser in Mediwn A (20 mM potassim phosphate, 100 IIM (NH,J2SO~ , 0.5 n&l MgCla, 5 n&l 8-mercaptoethanol, pH 7.6 (7)) unless otherwise indicated. The homogenate was centrifu ed at 75 Ooo x 9 for 40 min at 4’ and an aliquot was gelfiltered on a Sepha 8ex G-25 column in 20 II$! potassim phosphate, 0.5 mM MgClp, 5 I&! f+metcaptoethanol, pH 7.6. The main protein-containing fraction was used in the subsequent experiments. Phosphofructokinase activity was determined under optimal conditions as described by (10). Where appropriate, preincubation of samples prior to Protein was determined assay was at 20°C for 10 min unless otherwise stated. as in (11). RESULTS: When rat livers a substantially

higher

were homogenised and fractionated

PFK activity

from fed as compared to 24h-starved trate

with 5 mMATP resulted

was detected animals.

in gelfiltered

in a substantial

increase

fed or fasted animals,

in agreement with (8) . (Table I).

concentration

by NaCl or NaCl plus NaF (protein

(a) a substantially

Medium A and (b) little

Only when medium containing

relatively

EDNA.

high ionic

were performed

using

fell,

extent by ATP-preincubation

inhibitor)

or in

The results

568

showed

with ATP (Table I). ATP-activation or to its

high KC1 concentrations

but the enzyme could be activated (Table 1).

ammonium

than in samples prepared using

of preincubation

media containing

by ATP ATP

(Mt,)zS01, was used was substantial

strength,

for

effective

To test whether this was due to (NH4)2S04 itself

the measured PFK actively greater

basal PFK activity

or no effect

Activation

by Tris-HCl,

phosphatase

by the chelating-agent

higher

now being similar

experiments

media in which phosphate was replaced

which MgC12 was replaced

observed.

activity

and the half-maximally Further

was ~2 mM (Fig.1).

homogenisation sulphate

the final

of the gelfil-

in the PFK-activity

conditions,

5 min preincubation

supernatants

Preincubation

measured under optimal

was maximal after

in Medium A (8)

An ident ical

to a

time-course

BIOCHEMICAL

Vol. 118, No. 2, 1984

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

TABLE1 PFK Activity

in Gelfiltered

Supematants

MedilPn

from Fed or Starved Rats

PFK mu

Activity -1 mg protein

24h-fasted -ATP

Fed

+ATF’

Ra

+A#

‘o

-AT!’

+ATp % m

m

5.03UI.5 (3

24.622.2 (7)

4.9

11.3+1.0 (7)

23.821.5 (7)

2.1

10.74.4 0

11.9+1.8 0

1.1

19.7+2.9 0

19.0+4.4 0

0.96

C: 2W Tris-HCl, O.OSM 9.6a.7 733) KCl, 0.05 M KF, O.Sndil Wl2, 5M f+mercaptoethanol, pH7.6

10.65.7 (3)

1.1

16.721.2 (3)

18.55.8 (3)

1.1

D: 2W

6.43.2 (3)

24.622.1 (3)

3.9

N.D

N.D

N.D

10.121.3 (5)

14.221.8 (5)

1.4

12.722.3 (3)

14.9+3.2 (5

1.2

2.64.3 G-1

22.224.9 (3)

8.6

5.022.7 (3)

23.523.3 (3)

4.7

A:

(8)

B: ZO& Tris-HCl, O.lM KCl, O.SnM Mg’.&, WI 8-mercaptoethanol, pH7.6

Tris-HCl, 0.5M 0.1 M M,),so,, 5n&I B-mercaptoethanol, pH 7.6

W-32,

E: as B, but 0.2 M KC1 F: as B, but 0.4

M

KC1

Livers were hoamgenised in the medium indicated, and PFK-activity was determined after preincubation of gelfiltered supernatants without or with of ATP (5 JIM). Results are expressed + standard deviation and the ntier experiments in each case is indicate3 in parentheses.

and concentration-dependence

was shown for ATP-mediated activation

of PFK in

samples prepared in Medium A or in high KC1 (not illustrated). Table 2 illustrates filtration

that activation

by ATP Persisted after

further

gel-

on Sephadex G-25 and did not require Mg2+-ions (although they

5

0 Time

10 0

2.5 Concentration

bin)

Activation

5 Crnt.4)

of PFK by ATP (0)) fructose 1,6-bisphosphate (0) or (A). Gelfiltered supernatants of liver (from 24h-starved rat, homogenised in medium A) were preincubated at 200 with (A) 5 IIM ATP, 2 n&I fructose 1,6-bisphosphate or 2 I+! fructose 2,6-bisphosphate for the times shown or (B) with a range of ATP or fructose 1,6-bisphosphate concentrations for 10 min, prior to assay of PFK under optimal conditions. Fructose 2,6-bisphosphate was effective at umolar concentrations (see Results).

F&se.2,6-bisphosPhate

569

Vol. 118, No. 2, 1984

BIOCHEMICAL

Activiation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLEII of PFKby Adenine Nucleotides PFK-Activity

mU mg protein Preincubation Condition

Expt.1 (Fed)

H20

11.0

ATP

(5 ti

ADp mfJ Al-P A-P

(5ti (5 m (5 t&f) (5ti

+ EDTA(4 +Wl2(5ti

enhanced the activation).

Slight

ADP, but AMP was ineffective. activated

as ATP, UTP rather

as effective that

as ATP.

for ATP (Fig.1) .

concentrations

(half

molar concentrations

but variable

of fructose

24.9

activation

was obtained

triphosphates

Fructose 6-phosphate or fructose

tested

effective

1,6-bisphosphate

also

as effective activated was

time course to

was effective

concentration,

with

2,6-bisphosphate

occurred over a similar

Fructose 2,6-bisphosphate maximally

19.6

22.8

Gl’P and CTP being approximately

1,6-bisphosphate

Activation

19.0

A.

less so (Table III).

only weakly, but fructose

8.4 24.2 21.8 IO.5 9.8

6.2 21.5 23.9

Other nucleotide

PFK in preincubations,

5.0 21.1 18.4 6.0 5.1

7.9

9.7 20.8 25.2

Livers were homogenised in Mediun

(Sta4md)

23.4 20.5

21.3 11.9

IIN)

(StaLeed)

6.1

24.2

ATP (5 nM), then gelfiltered

(Staked)

-1

at micmlar

%8 I.MJ whilst

were required

milli-

(Fig.1).

TABLEIII Metabolite-induced Activation of PFK Metabolite

Concentration Relative Efficiency of Activation (Activation by Metabolite/Activation a-1 by ATP) x 100 (z Standard deviation)

ATP m CTP UP Fructose 6-phosphate Fructose 1,6-bisphosphate Fructose 2,6-bisphosphate ATP plus Fructose 1,6-bisphosphate ATP plus Fructose 2,6-bisphosphate

100 85 + 9

100 + 16 65 ; 16 4,13

(171 (51 (5)

loo _+3 91 ,+ 12 111 + 6

(91 WI

109 + 7

(4)

(31

Livers were homogenised in MediumA. The nunber of experiments in each case is shownin parentheses. 570

BIOCHEMICAL

Vol. 118, No. 2, 1984

Activation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b ATP and the fructose bisphosphates was not additive

ation of PPKby GTP or fructose of the activated

2,6-bisphosphate persisted after Similar activation

sample (not illustrated).

nucleoaide triphosphates

and activgelfiltraticll by ATP,

and the fructose bisphosphates was observed in ten

experiments in which livers

were homogenised in Medim A or a mediumcontain-

ing 0.4 M KC1 (Mediwn F of Table I). DISWSSION: The present results

concerning the ATP-induced activation

of PFK

confirm and extend those of Brand and .S&ing (7,8) who ascribed the large differences

in PFK-activity

ing nutritional the ability

status,

of supernatants from livers

and the susceptibility

of PFK to activation

of Medium A to maintain PFK in a phosphorylation It was suggested that

that in vivo.

PFK-kinase and phosphate inhibited results

of animals of differ-

(NH4)2SO~inhibited

NaF (phosphatase inhibitor)

state resetiling

the (putative)

the corresponding phosphatase.

do not support this interpretation

by ATF’, to

Our

since use of media containing

and EDTA (chelating

agent for Mg2+, required

cosubstrate for protein kinases) did not reproduce the results obtained with MediumA.

Instead, the effect

inactivation

of PFK occurring at high ionic strengths since media containing

high KC1 concentrations susceptibility

generate a similar

to activation

suggest activation process ;

of Medium A appears to be related to an

by ATP.

whilst protein

(reviewed in 12,13),

(ii)

a protein phosphorylation

equally effectively kinases are

phosphate donor and also exhibit

and enhanced

Several other lines of evidence

of PFK by ATP does not reflect

(i) PPK is activated

triphosphates,

loss of PFK-activity

by several nucleoside

generally

specific

for ATP as

a much lower Km than that apparent here

nucleoside triphosphate-induced

activation

did

not require Mg2+-ions, whilst NTP-fg is the normal substrate in phosphotransfer

reactions

(12) and (iii)

similar

activation,

to that induced by nucleoside triphosphates, bisphosphates, established effecters that the metabolite-induced structural

activation

of PFK.

which was not additiw?

was mediated by the fructose It therefore

appears likely

of PFK is due to a ligand-induced

change in the enzyme rather than to its phosphorylation. 571

It is

BIOCHEMICAL

Vol. 118, No. 2, 1984

well-known

that PFK undergoes complex

by metabolites

(5,14)

accompanied

by association

of the catalytic preparation

subunit

to high-salt

either

is

of 32P-labelled

PFK (9) may reflect

AMP-dependent protein

in susceptibility

the presence

kinase in the PFK

PFK against in liver

to the enzyme itself

In preliminary (16)) of fructose

inactivation. falls

: 0.3 nmol g-l

regulation

of PFK from fed and starved animals and thus to metabolite

a modification

(umolar,

metabolite-induced ical

of cyclic

showed ATP-activation

The incorporation

inmnmoprecipitable

concentrations.

concentrations

starved

of PFK (8).

induced inactivation

may reflect

metabolite

themselves

which are influenced

used (15).

The difference

protect

oligomerisations,

and Brand et al.

phosphate from ATP into

nmtabolite

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(15)).

activation

The elucidation

further

were found to

concentration

of this

(fed : 8 nmol g

of the structural

of PFK and its possible

of the enzyme requires

near-physiological

2,6-bisphosphate

in fasting

activation

or changes in cellular

experiments,

The intracellular

precipitously

mediated

role

-1

; 18h-

basis of

in the physiolog-

work, both with the purified

enzyme and in uivo. REFERENCES

1.

2. 3. 4. 5. 6. 87: 9. 10. 11. 12. :43: 15. 16.

:

Uyeda, K. (1978) Adv. Ensymol. Relat. Areas Mol. Biol. 48, 193-244. van Schaftingen, E., Hue, L. and Hers, H.-G. (1980) Biaem. J. 192, 897-901. Pilkis, S.J., El-Maghrabi, M.R., Pilkis, J., Claus, T.H. and Cumning, D.A. (1980). J. Biol. Chem. 256, 3171-3174. Kag&to, T. and Uyeda, K. PO> Arch. Biochem. Biophys. 203, 792-799. Pilkis, S.J., El-Maghrabi, M.R., Pilkis, J. and Claus, T.ml982) [email protected]. 215, 379-389. Soling, H.D. and Bran0.A. (1981) Curr. Topics Cell. Regul. z, 107-138. Brand, I.A. and Sijling, H.D. (1975) FEBS Letters 57, 163-168. Brand, I.A., MUller, M.K., Unger, C. and sling, m. (1976) FEBS Letters, 68, 271-274. Soling, Hn. and Brand, I.A. (1975) in 'Metabolic Interconversions of (Springer-Verlag, Berlin) pp. 203-212. Enzymes, Arad". Ed. S. Shaltiel Brand, I.A. and Siiling, H.D. (1974) J. Biol. Chem. 249, 7824-7831. Lowry, O.H., Rosebrough, N.J., Fax-r, A.L. and RandaT R.J. (1951) J. Biol. C&em. 193, 257-265. Flockhart, D.A. and Corbin, J.D. (1982) CRC Critical Reviews in Biochemistry 12, 133-186. Beavo, J.A. and Krebs, E.G. (1979) Ann. Rev. Biochem. 48, 923-959. Hesterberg, L.K. and Lee, J.C. (1982) Biochemistry 21,216-222. Brand, I.A., Mieskes, G. and Sbling, H.D. (1983) FE= Letters l& 65-69. Pilkis, S.J., El-Maghrabi,M.R., McGrane, M., Pilkis, J. and Claus, T.H. (1982) Federation Proc. 41, 2623-2628. 572