Two interconvertible forms of yeast phosphofructokinase with different sensitivity to endproduct inhibition

Vol. 15, No. 3, 1964

BlOCHEMlCAi

TWO INTERCONVERTIBLE WITH E.

DIFFERENT 2

Viiiuela,

FORMS

of

L.

February

of

metabolites

and

and

has

kinetics

variety

of

several

been

recently

as

kinetics

cyclic

3',

recently,

5'-AMP Mansour

direct

activation

3'.

PFK, 5'-APIP

1 This Health

de

Spain

with

Fellow

here

PFK the

(see

by

has

a yeast second

in

inhibitory

and

Mansour

a liver what

(1962) by

fluke.

seems

More

to

be

of an

effect

two

forms

of

cyclic

enzyme.

was Research the

supported Grant Comisaria 243

by United ~~-08041. de

a

3',5'-AMP.

cyclic

and

been

stimulated

identification

interconversion,

a

Passonneau

gives

system

observed

compliof

with

from

has

very

ATP-Mg

Mansour

preparations

heart

PFK

fructose-6-P

activating

(1963),

of

in

a number

suggestion

that

1.963).

an

by The

laboratory,

et

al.,

phosphofructokinase

the

inhibition

a conversion

investigation Service

this

to

of

their on

2 Research

Sols

Centro

tissues

excess

respect

for

We report yeast

by in

with

to

sites

endproduct

evidence

led

Inhibition

(Viiiuela

reported

A.

Madrid,

reported.

binding

1963).

of animal

have

characterized

conditions

inhibition

observed

preparation, order

and

Mar-an&,

C.S.I.C.,

from

regulatory

Lowry,

recently

Salas

Instituto

1

INIIIBITION

21, 1964

preparations

cated

PHOSPHOFRUCTOKINASE

2 Margarita

Salas,

Biol6gicas,

Activation (PFK)

RESEARCH COMMUNICATIONS

TO ENDPRODUCT

Enzymology,

Investigaciones

Received

OF YEAST

SENSITIVITY

Maria

Department

AND BIOPHYSICAL

Protecci6n

States

Public Escolar.

of

Vol. 15, No. 3, 1964

BIOCHEMICAC

AND BIOPHYSICAL

RESEARCH COMMJNICATIONS

EXPERIMENTAL Incubation of

fluoride

tivity

leads of

the

extracts

PFK

additive

that

may

with

change appear

a high by

tered

EDTA,

when

ATP

This

effect

hanced

and

the

reappears.

These two

to

could fluoride

is

to

has 0.1

effect

the

PFK

to

suggested that

there

are

two

with

the

3',5'-AMP

of

cyclic

detected

at

seems 3’ ,5'-AMP

concentrations

mM.

244

of

on

When

fluoride

is

ATP PFK

activities

activity

by it

activate the during

can

activities

these

that to

enI),

enzymic

hypothesis

in

extract,

yeast

other

fil-

decrease

by

One

be

gel

(Table

in

tested

markedly

inhibition

the

This

mixture,

with

interconversion. of

suggest-

can with

was

that

could

when

preincubation

incubation

both

during

involved.

dialyzed

3',5'-AMP

by

at

and

Some

the

I

that

place

ATP-Mg

cyclic

Inhibition

Cyclic

been

of

and

consistent

a phosphatase. No direct

with

results

a kinase.

in the

Table

changes

enzyme

extract.

to

in

"activation"

take

place

added

of

the

not

dialyzed

took

were

forms,

catalyze

be

or

the

This

Crude

ATP,

was

of

does

"activated"

sensitivity

able

it

addition

dialyzed,

in

activation

preincubation

extract

exist

itself

ATP

Mg+' of

by

a crude

enzyme

ATP.

out

high

the

sensi-

activity,

metabolite

in

of

I),

PFK

presence

marked

some with

and

to

of

the

as

rule

enzyme

G-25)

sensitivity

to

the

an

the

(Table

for

seemed

level

in

fluoride

of

as

(Sephadex

without

This

concentration

prevented

ATP

together

the

activity

extract of

by

and

ATP. in

some

change

PFK

inhibition

asaayed

of

the

PFK

to

yeast

disappearance

with

preincubation

affect ed

the

when

concentrations

a crude

to

incubated

were

the

of

could the

activity the

be kinase.

of assay

yeast up

BIOCHEMICAC

Vol. 15, No. 3, 1964

AND BIOPHYSICAL TABLE

"Activation"

of

RESEARCH COMMUNICATIONS

I

yeast

PFK

in

extracts

A crude extract was obtained by grinding commercial pressed baker's yeast (Danubio) with alumina (Alcoa A-301), diluting with 10 volumes of 50 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, 1 mM ethanethiol, and elimination of the insoluble residue by centrifugation at 20,000 x g for 15 minutes. Dialysis, when indicated, was carried out against several changes of 50 mM potassium phosphate, pH 7.5, 0,l I& EDTA, 5 ITIM MgC12. 0.1 ml of extract (containing 0.7 mg protein) was incubated at room temperature for 20 minutes in a total volume of 0.25 ml with additions as indicated. PFK activity was assayed with 20 ~'1 aliquots of the incubated mixtures in a total volume of 2 ml in the presence of excess aldolase, triose phosphate isomerase, and L-4 glycerophosphate dehydrogenase (Boehringer), 0.1 mM NADH, 5 mM ethanethiol, 50 mM imidazole, pH 7.5, 5 mM MgCl 0.2 mM fructose-6-P (obtained by treating glucose-6-P wi 4' h glucose phosphate isomerase until equilibrium), ahd 0.05 mM or 0.5 mM ATP, as indicated. The reaction was started by the addition of the preincubated extract and followed at room temperature by the decrease in optical density at 340 without fructose-6-P were carried out in parallel, m,u o Blanks Activity is expressed as n;umoles of substrate transformed per minute per mg protein.

Extract

Preincubation

Undialyzed

20

mM NaF

20

mM NaF

enzyme", indicates

0,l

M EDTA

None

64

16

86

70

153 17

70

12

niM NaF

74

12

20 20

mM NaF and 1 mM ATP-Mg mM NaF, 1 mM ATP-Mg, and 2 mM cyclic 3',5'-AMP

65

30

67

89

above

successful as

mM ATP

20

The the

and

PFK activity mM ATP 0,5

0.05

None

Dialyzed

by

additions

has

separation

shown

that

hypothesis

the

in

Table

presumed

of II.

partially

PFK

from

The

fluoride

phosphatase 245

been

an

confirmed

"activating requirement

catalyzing

the

Vol. 15, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Separation

of

PFK

RESEARCH COMMUNICATIONS

II

and

its

"activating"

enzyme

A crude extract prepared as in Table I was fractionated with ammonium sulfate, The fraction precipitated between 40 and 60% saturation was dissolved in 10 mM magnesium acetate, 1 mM ethanethiol, 0.1 mM EDTA, adjusted to pH 7.5, and dialyzed against the same medium. The dialyzed preparation was adjusted to pH 6 and fractionated with acetone between collected between 5 and 15% and +2 and -8Q C. The fractions between 35 and 5046 acetone were dissolved in 25 mM potassium phosphate, pH 6.5, 1 mM ethanethiol, 0.1 mM EDTA. Their protein content was 58 and 18 mg per ml, respectively0 0.05 ml aliquots of the 5-15 fraction were incubated at room temperature for 15 minutes in a total volume of 0,5 ml containing 10 mM potassium phosphate, pH 6.5, 5 mM MgC12, 1 mM ethanethiol, and other additions as indicated. After this incubation, PFK activity was assayed in 5 to 50 ul aliquots with 0,5 mM fructose-6-P and 1 mM ATP as indicated in Table I

Preincubation

additions

PFK

activity

None 0.1

11 ml

35-50

fraction

11

0.1 ml 35-50 fraction, and 0.1 mM cyclic

20 3',5'-AMP

Complete

system,

minus

Complete Complete

system, system,

minus plus

Complete

system,

minus

reverse

conversion

above

35-50

220

fraction

15

ATP-Mg 0.1

13 11

M EDTA

NaF

still

9

accompanies

one

or

both

of

the

enzymes. The

inhibition because

mM NaF, 1 mM ATP-Mg, ("complete system")

by of

(Krebs

and

effect

of

the

two ATP,

may

of be

resemblance

Fischer, the

forms

1962;

concentration

insensitive

PFK, designated to

the

as

ATP

246

a and

muscle

Parmeggiani of

and

on

sensitive

to

b respectively

phosphorylase

system

and

Morgan,

1962).

the

activity

of

The these

Vol. 15, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

ATP

1.

Fig.

Effect

of

the

, mM

concentration of

RESEARCH COMMUNICATIONS

of

PFK a and

ATP

on

the

activity

2

A 40-60 ammonium sulfate fraction of yeast extract was dissolved in 25 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, and dialyzed against the 1 mM ethanethiol, and 5 mM MgC12, same medium. To obtain the 2 form, this preparation was incubated with 1 mM ATP-Mg, 0.1 mM cyclic 3',5'-AMP, 20 mM NaF, 5 mM MgC12, and 10 mM potassium phosphate, pH 6.5, at room temperature for 15 minutes. The 2 form is the same preparation similarly incubated with MgC12 and phosphate After these treatments PFK activity was assayed in only. aliquots of 5 pl for PFK 2 and up to 50 ~1 for PFK &, as described in Table I with 0.5 m&l fructose-6-P and ATP as indicated in the figure. Ilr , PFK 2; 0 , PFK 20

two of

forms

of

yeast

1964)

PFK also

PFK to

pH 7.5

at

inhibition

disappears

when

Preliminary resting cant

yeast

3 Inhibition the

physiological

by

citrate

converted

yeast

the

a form

PFK range.

by

in

Fig. (M.L. to

the

in are

ATP

247

present

decreases

3

1.

Sensitivity

Salas

et

that the

al.,

2 form.

indicate

essentially

occurs of

of

shown

observations

PFK

proportions

is

although

b form, in

with

growing

the

in

signifiyeast.

pH in

Vol. 15, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION A form can of

make

possible

high

PFK

insensitive

a high

be

ing

yeast.

of

important

PFX

for

parallelisms

and

muscle

citrate

can

Garland

et

1963)

1964).

A simultaneous

tissue

would

be

the

presence

This

possibility

processes

regulatory

mechanisms

are

phosphorylase

efficient

for

In

(M.

both

muscle,

1963; Salas

enzymes

a rapid

grow-

between

Bowman, 5

of

in

striking. and

"activation"

very

inhibition

biosynthetic

(Parmeggiani

and

in

endproducts.

in

PFK

endproduct

rate

phosphorylase

inhibit al.,

the

certain

The

to

b-1 y colytic

concentrations

may

yeast

of

et

al.,

in

a given

mobilization

of

glycogen.

3',5'-AMP

Cyclic It

yeast. changes with

has in

the

(Madman

and

in efficiency

in

concentration an

as

certain

been

coli,

been

The

a chemical tissues

is

E.

external

1963).

act

yet

have

of

Sutherland, could

adjustment

cal

its

not

identified

availability

3',5'-AMP

maximal

been

has

a problem

of

where found

source

in of

relation energy that

for

phosphorylase considerable

in

marked

possibility signal

of

estimated

the

cyclic

parallel and

PFK

physiologi-

interest, REFERENCES

Garland, P.B., Randle, P.J., and Newsholme, E.A., Nature, aoo, 169 (1963). Krebs, E.G., and Fischer, E.H., in F.F. Nord (Editor), Advances in Enzymology, Vol. 2, Interscience Publishers, New York, 1962, p. 263. Inc., and Sutherland, E.W., Federation Proc., 22, Makman, R.S., 470 (1963). Mansour, T.E., J. Biol. Chem., e, 2285 (1963). Mansour, T.E., and Mansour, J.M., J. Biol. Chem., 122, 629 (1962). Parmeggiani, A., and Bowman, R.H., Biochem. and Biophys. Research Communs., 12, 268 (1963).

248

to

Vol. 15, No. 3, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

parmeggiani, A., and Morgan, H.E., Biochem. and Biophys. Research Communs., 2, 2.52 (1962). passonneau, J.V., and Lowry, O.H., Biochem. and Biophys. Research Communs., 9, 372 (1963). Salas, M., Vifiuela, E., and Sols, A., unpublished results. Salas, M.L., Vikela, E., Salas, M., and Sols, A., unpublished results. Vifiuela, E., Salas, M.L., and Sols, A., Biochem. and Biophys. Research Communs., 12, 140 (1.963).

249