Role of AMP deaminase reaction in the response of phosphofructokinase to the adenylate energy charge

Vol. 112, No. 1, 1983 April

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

15, 1983

Pages

96-101

ROLE OF AMP DEAMINASE REACTION IN THE RESPONSE OF PHOSPHOFRUCTOKINASE TO THE ADENYLATE ENERGY CHARGE Yoshino 1) and Keiko

Masataka 1) Department

of Biochemistry,

School

Yokohama

of Medicine,

2)

Murakami

Yokohama

City

232,

University

Japan

and

2) Department versity Received

February

22,

of Laboratory School

Medicine,

of Medicine,

St.

Marianna

Kawasaki

213,

Uni-

Japan

1983

The role of NH; ion and AMP deaminase reaction in the activation Summary of phosphofructokinase with respect to its response to the adenylate energy (a) Phosphofructocharge was investigated using permeabilized yeast cells. kinase and AMP deaminase were activated by the decrease in the adenylate energy charge. The addition of NH: further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5. (b) The increase in NHt ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine. Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NHt in the presence of K?. The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed. Phosphofructokinase of glycolysis

(EC 2.7.1.11)

(l-3).

effect

and the elevated

and the effect (1, level

conditions

(5-lo),

suggested

that

to the

the activation elevated

result

flux

(l-3,

11-14).

of phosphofructokinase

NH: level

0006-291X/83 $1.50 Copvright 0 1983 by Academic Press, Inc. ,411 rights of reproduction in any form reserved.

under

96

the conditions

activator

energy

from

the hypoxic

in the

control

in the mechanism

in the adenylate

a part

in the

as an effective

of ATP under

to play

and of the glycolytic

have shown ascribed

are

synthesis

role

implicated

+ as NH4' which

of Pi as well

and the decreased

to act

has been

The decrease

2, 4).

hydrolysis

fructokinase

a principal

Ammonium ion was shown

of phosphofructokinase, the Pasteur

plays

the

of

charge increased

and ischemic

stimulation

of phcspho-

Our previous

papers

can be essentially of increased

ATP hydro-

Vol

112, No. 1, 1983

lysis

(15-17)

in permeabilized

the modulation

Permeabilized

the

study

equilibrium

ation

increased

the energy

with

the shift

vity

in the

re;.ction

of

kinase

energy

of physiological in

charge

has not

because

reaction, This

level

paper

describes

the

to the vari-

giving

activity

the maximal

of K+: a key role

the activation

of near

can form AMP

the phosphofructokinase value

system

which

of phosphofructokinase

charge

been

experimental

charge,

of ATP (19).

+ of NH4 on

the effect

an excellent energy

NH: activated

the optimal

energy

(EC 2.7.4.3)

hydrolysis

was demonstrated

MATERIALS ~-___.

offer

of the adenylate

charge:

presence

in vivo

by the cells

RESEARCH COMMUNICATIONS

However,

(18).

of NH: in the response

role

in

lated

on control

after

regulatory

yeast

yeast

of adenylate

rapidly

AND BIOPHYSICAL

of phosphofructokinase

examined. for

BIOCHEMICAL

of glycolysis

acti-

of AMP deaminase

under

the

simu-

conditions.

AND -___ METHODS

Materials - All mer~tiGZ~revious vi:;iae) was obtained

chemicals were obtained from commercial sources papers (15-19). Baker's yeast (Saccharomyces locally.

as cere-

Incubation Conditions and Determination -- of Metabolites - Baker's yeast wa:: permeabilized as described previously (18). Reaction mixture of 0.5 or O.:!5'ml contained 4 mM ATP plus-AMP, 8 mM MgC12, 2.4 mM glucose 6-phosphate, 40-60 mM KCl. and toluenized yeast cells 20 mM cacodylate buffer (pH 7.1), (4 or 8 mg/ml) in the absence and presence of NHt, spermine or linolenate. Al.quots of 0.2 ml were deproteinized after 5 min-incubation and then neutralThe supernatant was utilized for the izi:d as described previously (16, 17). de'lermination of ammonia and fructose 1, h-bisphosphate (Fru-1, 6-P2) plus tr;ose phosphates. RE:;LILTS -__ IJhen the phsjsphate,

formation

charge

value

energy

c‘large,

activation tisn K+,

reaction

(Fig.

was started of Fru-1,

1).

and the

of the

by the addition

6-P2 and NH: was plotted

Phosphofructokinase slope

enzyme with

of ATP and glucose

responded

of the energy the

charge

decrease

from

characteristic increased

0.3

to 0.5

(Fig.

1A).

in the energy

of the ATP-regenerating with

the

decrease

Production enzyme:

in the energy 97

charge

the

to variation

response

+ of NH effectively activated phosphofructokinase 4 and the optimal value of the energy charge giving

shifted

against

curve

indicated

in the presence

maximal

activity

was

a typical

the AMP deaminase lB),

an

The addi-

of NH: exhibited

(Fig.

energy in the

charge.

even

6-

but

activity the optimal

of

Vol.

112,

No.

BIOCHEMICAL

1, 1983

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

/‘-; i

05

Energy

Charge

Fig. 1 Response of the activity of phosphofructokinase (A) and AMP deaminase (B) to variation in the adenylate energy charge in the absence and presence of NH1 ion. Reaction mixture of 0.5 ml contained 4 mM ATP plus AMP, 8 mM MgCl,, 2.4 mM glucose 6-phosphate, 20 mM cacodylate buffer (pH 7.1), 60 r&l KCl, and toluenized yeast cells (4 mg/ml) in the absence and presence of NHt. Aliquots of 0.2 ml were deproteinized after 5 min-incubation and then neutralized as described previously (16). The supernatant was utilized for the determination of Fru-I+ 6-P2 plus triose phosphates and ammonia (16). 53 , None; ~3 , 2 mM NH . 0 , 5 mM NH+ added. 4' 4

energy the

charge addition The

on

the

value of

giving

NH:

influence relationship

(data of

the between

maximal not

response

the

enzyme

was

not

of

deaminase

a 'fected

shown).

activation the

or

inhibition

phosphofructokinase

Energy Fig. 2 deaminase (B) presence of 1 was similar to and 40 mll KC1 2 mM linolenate

o

AW

activity

and

reaction the

energy

Charge

Response of the activity of phosphofructokinase (A) and AMP to variation in the adenylate energy charge in the absence and mM spermine or 2 mM linolenate. Reaction mixture of 0.25 ml that of Fig. 1 except that 8 mg/ml permeabilized yeast cells were included. l , 1 mM spermine added; d , 0 , None; added. 98

by

Vol. 112, No. 1, 1983

charge

was investigated

linolenate

enhanced

activity

(Fig.

energy

charge

increased

in the

presence

presence charge

of linolenate value

for

or inhibition produced

(Fig.

through

value

The addition the formation

giving

activation

hand,

was not

2B).

These

of AMP deaminase

of phosphofructokinase

to the physiological

in the

is

the

a little

altered

with

suggest

bv sperrnine

by enhancing

decrease

6-P2,

finding

energy the activation

that

NH: ion

can participate

the sensitivity

energy

and

in the

the optimal

results

or

of phosphofructo-

decreased

On the other

spermine

of Fru-1,

response

but

activity

of

Noteworthy

maximal

of spermine,

(Fig.

RESEARCH COMMUNICATIONS

respectively.

2A).

enzyme

the

in the stimulation response

2).

the AMP deaminase

of the

cells.

or inhibited

the optimal

kinase

AND BIOPHYSICAL

in permeabilized

effectively

the AI% deaminase that

BIOCHEMICAL

charge

of the

in vivo. -~

DISCUSSION Phosphofructokinase, 2),

is

controlled

which

and -__ in viva

characteristic

conditions

charge;

the increased

This

and is

that

mechanism

(see,

charge

can be highly

by the

stimulation

activator

of the

that

for

from

the K+-sites

enzyme,

(4).

depletion

through

the

elevated

of the

enzyme for

99

in AMP level

of total

is extremely

specificity

through

The adenylate

Ka value

The high

of the

21).

but

sites

ATP-inhibition

depletion

to the NH:-activated

its

(11).

in the

(19,

from

in the perme-

AMP produced

belongs

Binding

NH: (16).

resulting

to

resulting

enzyme form

The increase

(11).

by the

of glycolysis

Phosphofructokinase

activator

+ level in NH&

stabilized

charge

of the

the g

to the response

phosphofructokinase

in the

reaction

increase

energy

inhibit-

shows a typical

regard

as in the purified

of AMP deaminase,

and in the

in the

(1,

enzyme is

NH: and K' under

enzyme with

by the reversal

increase

the

regulation

Phosphofructokinase

decrease

explained

kinase

the activity

adenylates

20).

1) as well

Fig.

by the

of adenylate

the

(20):

by AMP, Pi,

of ATP activates

has been

enzyme and further

enhance

is,

hydrolysis

cells

action

activated (l-3,

in glycolytic

mechanism

of the ATP-regenerating

the energy

abilized

a key role

by the multimodulation

ed ty ATP and citrate, vitro __-

plays

type: larger

(16,

K+ can act

energy (19)

and

17). as an

as compared

NH: are possibly

of the activation

can

of total

adenylates

NH: level

the

to

distinct

of phospho-

Vol.

112,

No.

yeast

(22)

activating

and Clostridium

cation.

level,

in the energy

charge

value

presence

charge

giving

These

as demonstrated

most

effectively

-0.9)

under

the condition

where

Spennine

activated

the activity

the increase kinase

activity

ed NH: level lated

with

energy present lation

(16).

the activation giving

further

The decrease

formation

relation charge

effect aminase

might with

charge

can be acti-

24),

charge

(0.6

resulting

ascribed

by spermine with

to the

elevat-

was closely

the shift

role

in

on the phosphofructo-

corre-

of the optimal

These

of the enzyme.

is

where

(19).

closely

data

of NH4 + in the

effect

the adenylate

condition

(4).

NH: ion

be attributed,

may stimu-

enzymes,which

respond

will

kinase-AMP

hydrolysis

elevates

pro-

deaminase

or decreased

the NH: concentration.

was earlier

Combined

the

action

discussed

in

of the decreased

energy

may participate

enhancement

cells.

The Pasteur

to the interaction

of AMP de-

at least

serve

in hypoxic

in part,

in viva. -__

The analysis

presented

as a guide

to a metabolic

regulation

to variation

and ATP-utilizing

with

on phosphofructokinase

phosphofructokinase cells

correlated

The increased

the hypoxic

of the glycolytic

permeabilized

generating

energy

in the

the energy

(18,

and is

of NH: on phosphofructokinase

and increased part

in

of spermine

a substantial

the condition

to the Pasteur

principal

response

supporting

be inhibited

effect

optimal

by spermine.

of ATP under

Activating

Action

of AMP deaminase

in the energy

cannot

decrease

to be indirect

the maximal

of NH: under

reaction

the

phosphofructokinase

of phosphofructokinase

evidence

to the vari-

increased

of AMP deaminase

16).

Activation

of glycolysis

duction

(15,

paper:

presence

NH: was produced.

was demonstrated

charge

that

by the physiological

in NH: level

this

muscle

in the

response

of the enzyme

suggest

vated

in

the

rabbit

as a physiological

phosphofructokinase modified

response

COMMUNICATIONS

enzyme from

and NH: 1s . regarded

activated

results

RESEARCH

the purified

and further

the maximal

of NH:.

BIOPHYSICAL

in

(23),

Ammonium ion

+ of K at physiological ation

AND

by NH: was demonstrated

fructokinase (4),

BIOCHEMICAL

1, 1983

in

the energy

types. 100

charge,

including

in a

here

using

by the ATP-re-

Vol. 112, Nlo. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS- This work was supported in part by Grants-in-Aid for Scientific Research (No. 56570119 and No. 56770190) from the Ministry of Education, Science and Culture of Japan. We are grateful to Professor K. Tsushima from the Department of Biochemistry, Yokohama City University School of Medicine for his interest and encouragement in this work. REFERENCES ---1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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