Inhibition of yeast pyruvate carboxylase by L-aspartate and oxaloacetate

Inhibition of yeast pyruvate carboxylase by L-aspartate and oxaloacetate

Vol. 24, No. 5, 1966 BIOCHEMICAL INHIBITION OF BY YEAST Palacian, Instituto pyruvate M. Losada Spain and (Fuller -*et al baker’s yeast...

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Vol. 24, No. 5, 1966

BIOCHEMICAL

INHIBITION

OF

BY

YEAST

Palacian,

Instituto

pyruvate

M.

Losada

Spain

and

(Fuller

-*et al

baker’s

yeast

1966)

are

activated

niser

(Bloom

(Seubert

Keech,

and

and

; the rat

9 19611,

(Ruiz-Amil

Johnson,

Remberger,

1962)

and

1961)

are

Phosphoenolpyruvate murium

(Theodore

and

(Amarasingham,

1959;

valent

to that

acetate

and

1965;

Maeba

also

and

carboxylase described

from (Maeba This

inhibited the

Ashworth

of yeast are

by two

,

Sanwal,

and

carboxylase by

Sanwal,

communication

by

tvphicoli

a function

synthesis

(C&novas

L-aspartate

acetyl-CoA.

Salmonella

h ave

Th e inhibition

by

Asoerqihus

Escherichia

in the

acetyl-CoA

1965).

Salmonella

from and

1964)

citronellolis at all

9 1965)

et al -0

pyruvate activated

1964)

acetyl-

Benedict,

from

affected

way

Seubert, and

Pseudomonas

not

same

photosynthetic

Cooper

carboxylase

Englesberg

in the

and

enzymes

ions, have

absolutely

(Henning

the

magnesium

from

9 1965;

of

sources

requires

liver

acetyl-CoA;

and

affected

enzymes

-*et al

carboxylation

biological

not

enzyme

I9631

by

are

liver

direct

of ATP different

activities

avian

the

presence from

their

The

(Utter

catalyzes

in the

but

acetyl-CoA.

tween

and

de Fisiologia Celular Cefular, C. S. I. C. , Madrid,

carboxylases

described,

bacteria

is

OXALOACETATE

Torrontegui,

carboxylase

Several

and

CARBOXYLASE

July 18, 1966

to oxaloacetate

-CoA

de

Section Biologia

de

pyruvate

by

AND

G.

Pyruvate

been

PYRUVATE

L-ASPARTATE

E.

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

equiof oxalo-

and

Kornberg,

of phosphoenolpyruvate has

been

recently

1965). shows

L-aspartate,

a fact

that

carboxylating

enzymes.

644

that

yeast

increases Oxaloacetate

pyruvate the

carboxylase

similarities , the

reaction

be-

,

BIOCHEMICAL

Vol. 24, No. 5, 1966

product

of pyruvate

pyruvate

viously by

carboxylation,

carboxylase

preparation

METHODS

- The

used

work

in this

carried

out

either

(Ruiz-Amil

-*et al

CO2-fixing

reaction,

employed, would genase

reaction

oxalate

being

reaction

then

in

10 min,

amount

both

was

On

and

activity

with

possibility

the

data was

that

to pyruvate

in Table independent

and

thus

1,

right

I that of the

corresponded

oxaloacetate

hand, ( Fig.

involved

1 . From

is

am-

possible

in

fumarate

1,

would

inhibit

in the

assay,

inhibitory

and

practically

any

L-aspartate

Appropriate

Lineweaver

Fig.

carboxylase

by

concentration

to trap

shown

have

a competitive

645

The

maximal

As

to the

the

dehydrogenase

inhibition

the

the

dehydrogenase

not

to pyruvate.

L-aspartate

reaction

also

bicarbonate, other

respect

presented

The

enzyme.

of NADH,

L-malate,

did

.carbo-

to inhibit

pyruvate

citrate, mM

added dehydro-

1965).

the

the

was

pyruvate

absence

-

inhibited

of 2.5

of the

assay,

DISCUSSION

oxaloacetate

dehydrogenase

the

of the

, L-glutamate,

noncompetitive

-aspartate and

malate

at a concentration

malate from

enough

conditions

the

malate

contained

AND

the

the

fraction the

on

oxaloacetate

coenzyme,

et.,

RESULTS

excluded

the

Ruiz-Amil

under

on the

in the

were method

NADH-malate

to proceed

affecting

assays

method

the

difficulty,

without

determined

radioactive

radioactive

this

pre-

of oxaloacetate

auxiliary

with

was

the

effect

1964;

D-aspartate

effect

the

fraction

Enzyme

or

conditions,

together

present.

succinate

yeast

carboxylase

Protein

et.,

L-aspartate

to inhibit

sulfate

(1941).

of the

avoid

of oxaloacetate

activity.

study

by the

allowed

of pyruvate

sulfate

1965 1.

usual

to

added,

(Losada

monium

the trapped

was

ammonium

Christian

To

In order

carboxylation

the

a modification under

system.

found

pyruvate

spectrophotometric

1965).

immediately

xylase

and

the

,

since, be

been

yeast

et.,

of Warburg by

baker’s was

(Ruiz-Amil

method

has also

activity.

described

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

controls the

coupled

It is

apparent

effect

of L-

of ATP, noncompetitive inhibitor and

M&+ type.

with Burk

respect plots

BIOCHEMICAL

Vol. 24, No. 5, 1966

( Fig.

1) , Ki

L-aspartate

values and

for

of 1.9

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mM

and

oxaloacetate,

0.22

mM

respectively.

were

deduced

Fig.

2 shows

for the

Figure 1, left. Noncompetitive inhibition of pyruvate carboxylase by L-aspartate. The assays were carried out spectrophotometrically at room temperature by measuring optical density changes at 340 rnp, Each cuvette contained in a final volume of 3 ml: 300 ,,umoles of Tris-HCl (pH 7.6) ; 30 ,umoles of MgC12; 15 ,umoles of ATP; 45 ,umoles of KHC03; 0.4 ,u moles of NADH; 0.36 mg of protein (ammonium sulfate fraction) ; sodium pyruvate and L-aspartate as indica ted.

carboxylase Figure 1, right. Competitive inhibition of pyruvate GO oxaloacetate. Enzyme assays were carried out by measuring 2 fixation. A total volume of 2.0 ml contained: 200 ,umoles of Tris-HCl H 7.6) ; 20 moles of MgC12; 10 ,umoles of ATP; 38 ,umoles of KH i$ CO3 (50 /UC r ; 0.24 mg of protein (ammonium sulfate fraction) sodium pyruvate and oxaloacetate as indicated. After the pyruvate carboxylase reaction proceeded for 15 min at 30°, aliquots of 0.5 ml were pipetted into tubes containing, in 0.2 ml, 1 ,umole of NADH and Ruiz-Amil 0.7 ,nmoles of oxalate (Losada aal. , 1964; --et al , 1965). The malate dehydrogenase reaction was then allowed to proceed for 10 min at 30°, at which time 0.1 ml of 50 % trichloroacetic acid was added. After centrifugation , the radioactivity was measured in the supernatant.

ratios

of pyruvate

at different

carboxylase

concentrations

activities of this

aminoacid.

646

with

and

without

L-aspartate

;

Vol. 24, No. 5, 1966

BIOCHEMICAL

Maeba hibition

and

AND BIOPHYSICAL RESEARCH COhtMUNlCATlONS

Sanwal

(19651

of phosphoenolpyruvate

-aspartate

is

mammalian

pointed

carboxylase

a property

pyruvate

have

that

from

distinguishes

this

. Our

carboxylase

results

out

that

the

in-

by

L-

Salmonella enzyme

from

the

indicate

that

yeast

[L- ASPARTATE] mM Figure 2. Inhibition of pyruvate carboxylase activity at different concentrations of L-aspartate. Enzyme assays were carried out by measuring 14C02 fixation. The reaction mixture contained in a final volume of 2.0 ml: 200 ,umoles of Tris-HCl (pH 7.6) ; 20 ,umoles of ,umoles of ATP; 20 ,umoles of sodium pyruvate; 30,umoles &Cl2 i1JO of KH CO3 (20 ,uCl; 0.6 ,umoles of NADH; 0.24 mg of protein (ammonium sulfate fraction1 ; L-aspartate as indicated. After incubation for 30 min at 30°, 2.0 ml of 50 % trichloroacetic acid was added and the radioactivity was measured in the supernatant after centrifugation.

pyruvate

carboxylase

Salmonella,

is

, Iike

inhibited

Since ation

pathway

can

carboxylase leading of pyruvate

sidered

as

inhibition The the

an

regulation

be

effect of the

removed

for

be

as the

by

end-product.

activity

reaction

oxaloacetate

product level

647

first

within

greater. carboxyl-

enzyme

of the the

may The

of the

is

of aspartate,

aspartate

is

from

by the

Therefore,

by

carboxylase

Ki

synthesis

activity

inhibition

the

the

regarded

its

formed

to aspartate.

carboxylase

by

carboxylase although

oxaloacetate

pyruvate

allosteric

of pyruvate

inhibitory

L-aspartate, the

may

from

hibition

by

in yeast

of pyruvate

pyruvate

phosphoenolpyruvate

be

be

the

cell.

con-

oxaloacetate

competitive

may

in-

of interest

type. in

and the window

ed

mixture Geiger

Enzyme in a final

2

1

AT

but

Complete

but

Complete

mM

3 mM

KHC03

Mg ++

ATP

KHC03

mM

15 mM

3.5

0.25

System

CONCENTRATIONS

L-ASPARTATE

OF

No

THE

31.2

15.6

10.2

14.3

5.6

11.3

14CC2

x

REACTION

10 -3/

9.7

6.1

4.0

7.6

2.4

4.9

min

69

61

61

47

57

57

%

Inhibition

ACTIVITY COMPONENTS

L-aspartate (3 mM)

fixed

CARBOXYLASE

counts

L-aspartate

OF

PYRUVATE

assays volume

14 CO -fixation. The complete system contaitl were carried out by measuring of MgCl ; 5,umoles of 2.0 ml: 200 ,umoles of Tris-HCl (p ?-I 7.6) ; 20 ,umoles sodium pyruvate; 66 /umoIes of KH14C03 (55 PC in experiment 21, and 5.5 1 ,umoIe of NADH; 6 rmoles of L-aspartate; 0.36 mg of protein (ammonium . After incubation for 10 min at 30°, 0.2 ml of 50 % trichloroacetic acid was added was centrifugated. The radioactivity was measured in the supernatant with a thin counter in experiment 1 and with a gas flow Geiger counter in experiment 2.

but

Complete

Complete

but

BY

Complete

Complete

DIFFERENT

I - INHIBITION

Experiment

Table

Vol. 24, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES Amarasingham,

C. R. , Federation

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J.M., 94,

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and

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W.

M.

M. , G. de Torrontegui, J.Biol.Chem. and M. Losada,

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W.,

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T. S. , and M.F., O.,

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W.

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