fructose-2,6-bisphosphatase cycle

Vol.

131,

No. 2, 1985

September

16,

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

899-904

Pages

QUASI-STATIONARY CONCENTRATIONS OF FRUCTOSE-2,6-BISPHOSPHATE IN THE PHOSPHOFRUCTOKINASE-2/FRUCTOSE-2,6-BISPHOSPHATASE CYCLE Matthias

Kretschmer, Institute

Received

August

Wolfgang

Schellenberger

and Eberhard

Hofmann

of Biochemistry, Karl-Marx-University, Leipzig, German Democratic Republic

5, 1985

of phosphofructokinase-2 and fructose-2,6SUMMARY: The cooperation bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructoseThe two enzyme activities were 2,6-bisphosphate to quasi-stationary levels. The quasi-stationary levels of found to exert strong temperature dependence. are independent on temperature. 0 1985 fructose-2,6-bisphosphate, however, Academic

Press,

Inc.

The tissue dynamic

levels

of fructose-2,6-bisphosphate

characteristics

bisphosphatase

of the

cycle

same enzyme protein

and can be active

description

cycle

of the

individual

requires

measurements

have been derived.

These

cooperation

of the cycle

CAMP dependent activities the cycle Owing

protein

the effect

(2,3).

knowledge

and of their laws

taken

enzymes. kinases

PFK-2 and FBPase-2

detailed

rate

were

(1,Z).

simultaneously

enzyme activities

From kinetic

depend

on the

phosphofructokinase-2/fructose-2,6-

(PFK-2/FBPase-2)

of this

(F-2,6-P2)

for

Because

exerts

it

(4)

the

known

reciprocal

of phosphorylation

kinetic

behavior

cooperation.

for is

on the

A quantitative

of the

the PFK-2

as a basis

reside

and for

FBPase-2

investigation that

control

of the

phosphorylation over

on the quasi-stationary

by

the two enzyme dynamics

of

was investigated. to a strong

dependence

appeared

interesting

to study

stationary

levels

of F-2,6-P2

of the two activities whether

in the PFK-2/FBPase-2 MATERIALS

PFK-2/FBPase-2 Wistar rats after

the temperature

on temperature, influences

it

the quasi-

cycle.

AND METHODS

was prepared from liver starvation for 72 hours

899

according to (9,lO) using and 48 hours of refeeding.

male The

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

131,

No. 2, 1985

BIOCHEMICAL

AND

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RESEARCH

COMMUNICATIONS

purified enzyme was phosphorylated as described in (5). The pure C subunit of CAMP dependent protein kinase type II from beef heart was a gift from Prof. F. Hofmann, Homburg, FRG. All experiments were carried out in 100 mM imidazole/HCl, pH 6.6, 20 mM KH PO /K HP0 , 10 mM MgCl , 10 mM mercaptoethanol and 100 mM KC1 ?Buffe? A)!The PFK-2 an 4 FBPase-2 activities were assayed according to (10,ll) and (7), respectively. For the investigation of the heat stability 0.25 mU of native PFK-2 (corresponding to 0.11 mU FBPase-2 at 30 "C) were incubated for 45 minutes in 100 pl of Buffer A at various temperatures. Then temperature was adjusted to 30 'C and the remaining activities were determined with 3 mM ATP and 2 mM fructose-6phosphate (F-6-P) for PFK-2 and 20 pM F-2,6-P for FBPase-2. The same substrate concentrations were used for the de z ermination of PFK-2 and FBPase2 activities at different temperatures. Aliquots of the reaction mixtures were removed after 5, 10 and 15 minutes and treated as described in (11). The dynamics of the PFK-2/FBPase-2 cycle was investigated by following the time evolution of the F-2.6-P concentration to quasi-stationary values. For this the native and phosphory 1ated enzyme was incubated in 100 pl with the indicated concentrations of ATP and F-6-P or ATP and F-2,6-P2, respectively. In all experiments a protein concentration was applied referring to 5 mu/ml PFK-2 (30 "C). The F-2,6-P2 concentrations obtained from these experiments were compared with the solution of the respective balance equations by means of nonlinear regression analysis. The expression for the PFK-2 activity in the differential equations was taken from (4). The kinetics of FBPase-2 was reinvestigated under the present experimental conditions (data not shown). It can be described by the following equation : [F-2,6-P2] 1 V (1) 'FBPase-2 = FBPase-2. 1+[F-6-P],K . Ks+[F-2,6-P2] i with

K = 0.51 pM and Ki = 25 p. S

RESULTS AND DISCUSSION Fig.

1 presents

temperature

the results

of the experiments

dependence of PFK-2 and FBPase-2.

found stable

up to a temperature

minutes

1C and D). Above this

(Fig

of PFK-2 and FBPase-2 occurred. two activities

were observed.

temperature

No differences Fig.

The two activities

"C. The Arrhenius

plots

are linear

an incubation fast

nearly

between

of the

the temperature

influence

fivefold

of 45

inactivation

in the heat stability

A strong

were

period

irreversible

1A and B demonstrate

increase

and

The two enzyme activities

of 40 "C within

dependence of the two enzyme activities. was found.

about heat stability

of temperature

between 25 "C and 35

10 'C and 40 'C (insets

of Fig.

and B). The time course

of the metabolites

PFK-2/FBPase-2

is governed

W-2,6-P21/dt

= vpFK-2 - VFBPase-2

d[ATP]/dt

= - vPFKm2

in a closed

by two coupled

differential

reaction

chamber containing

equations

:

1A

Vol.

131,

No. 2, 1985

Fig.

BIOCHEMICAL

1 : Effect

AND

of temperature

BIOPHYSICAL

on the

RESEARCH

native

COMMUNICATIONS

enzyme

A: PFK-2 ( 3mM ATP, 2mM F-6-P, Buffer A ) B : FBPase-2 ( 20 pM F-2,6-P2 , Buffer A ) C and D : Relative activity remaining after 45 minutes of incubation in Buffer A at various temperatures

denote

where “PFK-2 and "FBPase-2 respectively.

Integration

concentrations reactants closed

effective into

quasi-stationarity

weakly

Km value

account

of the

that

competitively

to ATP (2,4).

Experiments

stationary system

with

are

concentrations was started

Quasi-stationarity the investigated

kinase

with

for

of F-2,6-P2 range.

continuous

can be attained

for

is

not

The PFK-2/FBPase-2 901

must

on the

initial

the

evolution

time

identical

altered could

of Fquasi-

independently and ATP,

the

be taken

PFK-2

F-2,6-P2

system

of F-2,6-P2

ADP inhibits

attained

of the

sufficiently it

2A and B. Nearly

significantly

of ATP in the because

exceeds

enzyme demonstrating

and ATP or with

of the other

level

ATP depends

because

were

dependent

decrease

ATP. For calculation

Km value

of F-2,6-P2

time

the concentrations

as the ATP level

shown in Fig.

F-6-P

yields

The quasi-stationary

nucleotides

the native

concentrations

of the

enzymes.

the effective

of the adenine

and (3)

of F-2,6-P2

on ATP as long

concentration

2,6-P2

Despite

of the two cycle only

(2)

of PFK-2 and FBPase-2,

and ATP from which

2

may be calculated.

cooperation depends

of equations

of F-2,6-P

system

the activities

whether

the

respectively.

by temperature

satisfactorily

in be

Vol.

131,

No.

2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

100 -I

@

50

Fig.

50

100

Time

Imln)

A :

= 2360 pM 27 “C

;;I;-;;~

= =

50 P’ 100 pM 50 PM

=

100

=

B : [F-2,6-P2100 [F-2,6-P210

32

37

“C

by equations (1)

were

applied.

used as parameters shown

in Table

dependence the

(2)

in

1 are

28 f 2 71 f 3 30+2 68+4

0 I

and (3)

are

temperature

Quasi-stationary 2/FBPase-2 native

are

(Fig.

enzyme

Table

(Fig.

1 : V,,,

3).

values

'PFK-2 bU/ml ) VFBPasem2 (mu/ml) VPFK-2'VFBPase-2

given

the

1).

The ratio

hence,

the

(4)

and in

of PFK-2 and FBPase-2

The computed

with

in

shape

were

maximum activities

of the

temperature

of PFK-2/FBPase-2

quasi-stationary

is

nearly

concentrations

independent.

F-2,6-P2 two orders

parameters

procedure.

in good agreement

same at 27, 32 and 37 "C and,

of F-2,6-P2

when the

pM pM

the experiments

maximum activities

the fitting

of PFK-2/FBPase-2

[F-2,6-P21stat

;

@

Only

to quasi-stationary ‘C

ATP did not drop below 2100 t&i during

equation

Cm1n)

2 : Time evolution of F-2,6-P2 concentrations values with the native enzyme

[ATPI0

described

100

Time

concentrations of magnitude

They also

obtained

obtained lower

do not

than

depend

with those

32 "C 4.30 f 0.59 1.63 + 0.38 2.6

902

approached

with

on the temperature.

from data shown in Fig.

27 "C 1.70 f 0.53 0.64 f 0.26 2.7

phosphorylated

2 (native

enzyme)

37 OC 7.07 f 1.04 2.86 * 0.49 2.5

PFKthe

Vol.

131,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

L 50

100 Time

Fig.

150

imInI

3 : Time evolution of F-2,6-P concentrations values with the phosphory 1 ated enzyme

[ATPI

= 2360 pM

[F-2,6-P210 [F-2,6-P210

= =

27 "C

32 "C

37 'C

it

is

parameters

given

0.2 f 0.1 pM 0.5 f 0.2 pl

sufficient

description

the

ratio

mainly is

between

(4)

the

independent increments characteristic

values

by the FBPase-2

defined(Table with

although

the native

is

and (3)

with

possible

The maximum

the

enzyme a by switching

of the curves

and phosphorylated

concentrations

temperature.

Table

(2)

For the phosphorylated cycle

activity.

the individual

times

equations

The shape

only.

the experiments

in Fig.

3 is

of the PFK-2

activity

2).

quasi-stationary

with

(1).

of the F-6-P/F-2,6-P2

The experiments that

to apply

and in equation

the Vm,,

determined

not well

possible

still

in

[F-2,6-P21stat

50 pM 100 PM

ATP did not drop below 2200 pM during

Interestingly,

to quasi-stationary

of F-2,6-P2

are

enzyme activities

However,

in which

PFK-2/FBPase-2

temperature

quasi-stationary

temperature

exhibit influences

states

27 "C 0.34 f 0.12 1.88 + 0.31 0.18

32 'C 0.65 f 0.46 2.82 f 0.54 0.23

903

strong the

are approached.

2 : Vm,, values obtained from data shown in Fig. (phosphorylated enzyme)

'PFK-2 W/ml) VFDpases2 (mu/ml> VPFK-2'VFBPase-2

show

3

37 "C 1.20 + 0.64 4.62 f 0.64 0.26

The

Vol.

131,

rate

No. 2, 1985

laws

for

the

respectively, 2/FBPase-2

out

low

enzyme are

metabolite

reported

obtained

with

hepatic

levels

reported

native

for

in

(4)

RESEARCH

COMMUNICATIONS

and in equation

a quantitative

which

are

of these cycle

for

F-2,6-P2

(l),

description

for

glucagon

fed rats

of the PFK-

are

rather This

metabolites

on the

cycle

quasi-stationary

of further

high

by the

concentrations

hepatocytes

ando(-glycerophosphate of the

adjusted

the cellular

treated

(12,13).

to be effecters

subject

with

PFK-2/FBPase-2

known

is

concentrations

in good agreement

citrate

of phosphoenolpyruvate,

2/FBPase-2

given

sufficient

quasi-stationary

values

action

BIOPHYSICAL

cycle.

phosphorylated

system,

AND

PFK-2 and FBPase-2

turned

The very

of this

BIOCHEMICAL

(12.13). compared

The with

may be due to the absence in the enzymes states

in vitro (2,4,5,7).

of the PFK-

investigation.

REFERENCES 1. Van Schaftingen, E., and Hers, H.-G. (1984) in : Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol. 17 (eds. Greengard, P. et al.,) pp 343-349, Raven Press, New York 2. Pilkis, S.J., Regen, D.M., Stewart, B.H., Chrisman, T., Pilkis, J., Kountz, P., Pate, T., MC Grane, M., El-Maghrabi, M.R., and Claus, T.H. (1984) in: Enzyme Regulation by reversible Phosphorylation, (ed. Cohen, P.) pp 95-122, Elsevier Science Publishers B.V. 3. Pilkis, S.J., Regen, D.M., Stewart, B.H., Pilkis, J., M.R. Pate, T.M., and El-Maghrabi, (1984) J. Biol. Chem. 259, 949-958 4. Kretschmer, M., and Hofmann, E. (1984) Biochem. Biophys. Res. Commun. 124, 793-796 5. Van Schaftingen, E., Davies, D.R., and Hers, H.-G. (1981) Biochem. Biophys. Res. Commun. 103, 362-368 6. El-Maghrabi, M.R., Claus, T.H., Pilkis, J., and Pilkis, S.J. (1982) Proc. Natl. Acad. Sci. USA 79, 315-319 7. Van Schaftingen, E., Davies, D.R., and Hers, H.-G. (1982) Eur. J. Biochem. 124, 143-149 8. El-Maghrabi, M.R., Claus, T.H., Pilkis, J., Fox, E., and Pilkis, S.J. (1982) J. Biol. Chem. 257, 7603-7607 9. Sakakibara, R., Kitajima, S., and Uyeda, K. (1984) J. Biol. Chem. 259, 41-46 10. Van Schaftingen, E., and Hers, H.-G. (1981) Biochem. Biophys. Res. Commun. 101, 1078-1084 11. Van Schaftingen, E., Lederer, B., Bartrons, R., and Hers, H.-G. (1982) Eur. J. Biochem. 129, 191-195 12. Hue, L., Blackmore, P.F., Shikama, H., Robinson-Steiner, A., and Exton, J.H. (1982) J. Biol. Chem. 257, 4308-4313 13. Bartrons, R., Hue, L., Van Schaftingen, E., and Hers, H.-G. (1983) Biochem. J. 214, 829-837

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the

The