Effect of atractyloside on mitochondrial protein synthesis

BIOCHEMICAL

Vol. 33, No. 4, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF ATRACTYLOSIDE

ON MITOCHONDRIAL

PROTEIN

SYNTHESIS Sharad V. Gangal and Samuel P. Bessman Department of Pharmacology, University California Medical School, Los Angeles,

of Southern California

Received October 17, 1968

Bruni inhibits

and Azzone

the

mitochondrial

phosphorylated

chain

Klingenberg

and Pfaff

the

that

inner

ported site

exchange

both

liver

level.

and Vignais

diffusion

level

about

and Duee (1966)

the

Winkler

carrier

system

effects

of high

carrier

in

translocation et al

atractyloside is

of the

More recently

on a catalytic

brings

mitochondria

ATP from

of

(1968)

sup-

sensitive

an obligatory

binding

catalytic

specifically

located

in the

membrane.

oside

on two energy

chondria:

requiring

citrulline

insensitive

of added

evidence

that in fatty

(1967).

atractyloside acid

The present atractyloside

in

in the

absence

fatty

orthophosphate,

and Van Den Bergh

on the

concentrations

processes

synthesis

and dinitrophenol

steps

substrate

the

atractyloside

of external at the

acts

that

that

both

directions.

Stimulatory

absence

(1966)

which

hypothesis

in rat

inner

and at the

membrane

the

formed

atractyloside

ADP and ATP in

concluded

formation

compounds

respiratory

proposed

(1964)

acid have

Allman inhibits

of

rat

liver

oxidation

(1966)

several

mito-

of added in

been reported

et al

atractyl-

ADP the

by Charles

presented of the

component

effect

of

oxidation. communication

reports

incorporation

675

the

of 1-leucine

Cl4

into

Vol. 33, No. 4, 1968

protein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by isolated

muscle

sarcosomes

oxidative

phosphorylation

of excess

of phosphoenolpyruvate

or external

with

0.25

4 times

with

was the

same as described

variations at

the

under

in

30°C the

acetic

the

acid

scribed were

(Hammel

source.

dissolved

counting

in

and protein The ATPase

the

5 percent

for

de-

precipitates

al.,

in

liberated method

dissolved

scintillation et.

system

by the

trichloro-

as previously

was determined

was determined

incubation

were washed,

(Lowry

phosphate

for

one hour

and used

generating

medium

except

The washed

1964).

acid

activity

The inorganic

cubation

with

determination

of ATP (2 mM) with 1960).

After

and rewashed

formic

muscle

and washed

(1965)

The TCA precipitates

and Bessman,

breast

The incubation

by Roodyn

in NaOH and reprecipitated

system.

conditions

was stopped

(TCA).

presence

generating

pigeon

medium.

energy

reaction

from

aseptic

isolation

either

METHODS

were prepared

m sucrose,

by

ATP in the

linked

EXPERIMENTAL Sarcosomes

supported

1951).

the

presence

(Pullman

et.

after

15 min.

of Chen et,

al.,

al.,

in(1956).

RESULTS Table mycin

I shows the

on leucine

Cl4 acid

ATP + generating

system

of leucine

causes into

of atractyloside the

system

slightly stimulatory

of atractyloside

incorporation

by o-ketoglutaric

Atractyloside

effects

(crkg)

by mitochondria

+ AMP, succinate

and without

increase

supported

markedly

by oxidative

when ATP and generating effect

of atractyloside

676

+ AMP, energy

in the

system

effect

by oligomycin

phosphorylation was added.

was observed

source.

incorporation

The stimulatory

protein.

was diminished

supported

any external

a significant

mitochondrial

and oligo-

in

and only The whether

or

BIOCHEMICAL

Vol. 33, No. 4, 1968

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I EFFECT OF ATRACTYLOSIDE ON LEUCINE Cl4 INCORPORATION ISOLATED SKELETAL MUSCLE MITOCHONDRIA

System

Per Cent Incorporation

Addition

2 kg + AMP

Succinate

Oligomycin Atractyloside Atractyloside

5 pgs 5 F.IM + Oligomycin

Oligomycin Atractyloside Atractyloside

5 Figs 5 uM + Oligomycin

Oligomycin Atractyloside Atractyloside

5 ugs 5 uM + Oligomycin

+ AMP

BY

100 93

170 129 100 26 258 43

ATP + PEP + PK

100

50 139 129

No Substrate or Nucleotide Atractyloside

100 195

5 PM

The incubation medium contained in 1 ml, 0.1 M sucrose, 0.04 M Kcl, 1.3 mM EDTA, 0.02 M nicotinamide, 0.016 M KH2P04, 0.5 mM NAD, 5 mM MgSO4, 50 pgs synthetic amino acid mix, minus leucine, 2-3 mgs mitochondrial protein, pH 7.4. The energy source was either a-ketoglutaric acid 10 mM or succinate 10 mM, and AMP 1 mM; or ATP 2 mM phosphoenolpyruvate (PEP) 5 mM, pyruvate kinase (PK) 100 pgs.

not

the

before

mitochondria the

addition

Table

II

that

the

concentration

fact,

incubated

with

effect

on leucine-C

not

stimulatory of

of different

decreased

stimulation

energy

source

the

concentrations

14 incorporation

effect

atractyloside

seem to affect

the

atractyloside.

by akg + AMP or ATP + generating

noted

did

of

shows the

of atractyloside either

were

supported

system.

increased up to

5 pm.

incorporation of incorporation

677

with

It

was

increasing

Further

increase

significantly,

in

was observed

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 33, No. 4, 1968

TABLE II EFFECT OF DIFFERENT CONCENTRATIONS OF ATRACTYLOSIDE ON LEUCINE Cl4 INCORPORATION INTO PROTEINS BY SKELETAL MUSCLE MITOCHONDRIA System Atractyloside Con. (PM)

a-kg + AMP (sp.act.1

0.0 1 2.5 5 25

ATP + PEP +PK ( sp. act.)

704 732 1164 1816 1855

The incubations Table I.

were performed

in

the

618 661 675 1057 704

medium

described

under

TABLE III EFFECTS OF VARIOUS CONCENTRATIONS OF ATRACTYLOSIDE ON LEUCINE Cl4 INCORPORATION INTO PROTEIN AND ATPase ACTIVITY OF MITOCHONDRIA

Atractyloside Concentration (PM) 0 1 2.5 5 25

887 883 1100 1387 914

71.3 67.5 43.8 40.1 34.5

Complete system as described in ATPase activities and atractyloside the same batch of mitochondria.

at higher ternal

concentration

+ AMP was the

energy III

text. effects

of atractyloside

ATP was used as the

Table

ATPase Activity Pi ugs Liberated/l5 min.

(ATP + PEP + PK) Sp. Act.

energy

source

were

studied

(25 PM) when exbut

not

when akg

source.

shows the

effect

678

of various

concentrations

on

BIOCHEMICAL

Vol, 33, No. 4, 1968

of atractyloside

on leucine

Cl4 incorporation

No correlation

activity.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of ATPase activity

was observed

and stimulation

poration.

At higher

inhibition

of ATPase occurred

increase

in stimulation

between

of

concentrations

and ATPase the

inhibition

14 incorC

leucine

(25 PM) where maximum

there

was either

or a decreased

no significant

stimulation

was

observed.

DISCUSSION In

intact

phosphate

mitochondria

acceptor-stimulated

and ATP-ADP exchange This

effect

sequence

phosphorylated energy

chondrial

the

high

source

internally

has been shown to

it

is

supported

The reports

atory

to

amino acid

energy

1964).

energy the

hypothetical

the energy

either

the

and Lehninger, for

by phosphate substantial

1966) in-

of atractyloside

incorporation

from oxidative

in mito-

requirement

stimulated the

Although

phosphorylation

ATP (Wheeldon

for

transfer

and ADP.

of oxidative

account

to be

incorporation

the known action

in acceptor,

stimulation by mitochondria,

phosphorylation

(ctkg +

+ AMP). by Winkler

of the nucleotide diffusion

between

of amino acid

by energy

AMP or succinate

of the

intermediate

the respiration

difficult

step

reaction

satisfy

With

by atractyloside

uptake

the

1963) or external

corporation.

and Azzone,

has not been established

generated

preventing

(Bruni

the

the Pi-ATP,

has been postulated

energy

for

protein

(Kroon,

reactions

with

involving

inhibits

respiration,

of atractyloside

due to interference

the

atractyloside

process

et al

(1968)

by mitochondria resulting

679

suggest occurs

in a constant

that

the

by an obliginternal

Vol. 33, No. 4, 1968

pool

size

uptake

irrespective

system

affinity the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

being

inner

from

is

of

specific

and Lehninger,

1968).

cubation

medium

nucleotide

The stimulation

thus

of atractyloside

mitochondria. have found

that

maintained

in

the the

appearance to

endogenous

ADP at

mitochondrial

atractyloside

may however in mitochondria

suggest

acid

in the

intermediates atractyloside. one or more

presence

possibility

can go on in the

intramitochondrially which

Alternatively of the

Heldt,

inhibits

680

of

1967). into

of atractyloside protein

in the

processes

synthesis

of ATP per -se

high

atractyloside

ATP requiring

has also

incorporation

absence

accumulate

it

strongly

that

generated would

(40,g);

phosphorylation

of amino

(1967) is not

the 1967,

effect

the

Atractyloside

(Bruni,

the

inside

medium.

partially

protein

to the

ATP level

although

The stimulation

the membrane.

level

of atractyloside

30°C

and

of ATP in the

intramitochondrial

inhibit

in-

inhibition

and Van Den Bergh

of ATP in the

been found

in the

loss

presence

falls

membrane

concentration

its

Charles

the

the

may be adduced

increasing

However

"carrier"

from

through

the

on

different

prevents

the

its

may enter observed

thereby

continually

upon

in preventing

medium

binding

reverse

depending

of the

on the

of 1 or 2 mM nucleotide

by atractyloside a part

at a site

nucleotide

may partially

and the

of atractyloside

is

by its

This

nucleotides

nucleotides

of the

The presence

concentration.

The action

or the release

(Winkler

using

adenine

and atractyloside

binding

the

for

of mitochondria,

used by adenine

mOleCUle

outer

external

ADP>ATP>AMP.

membrane

that

its

energy

phosphate

presence might in

the

of

block mito-

BIOCHEMICAL

Vol. 33, No. 4, 1968

chondria

such as fatty

compete for however,

energy

acid

with

the

would postulate

ATP which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oxidation

which

incorporating

an increase

has not been found

may otherwise system.

This,

in intramitochondrial

(Charles

and Van Den Bergh,

1967).

ACKNOWLEDGEMENTS We are very his

generous

by National Heart

Institute

grateful

gift

to Dr.

Albert This

of atractyloside.

Science

Foundation

L.

grant

Lehninger

study

was supported

number GB 4153;

number 5 Ti HE 5536-07;

for

Natl.

and NINDB grant

number

5 R01 NB 01365-11. REFERENCES

Allman, D.W., Galzigna, L., McCaman, R.E., and Green, D.E., Arch. Biochem. Biophys. 117, 413 (1966) Bruni, A., in J.M. Tager, S. Papa, E. Quagliariello and E.C. Slater (Editors) Regulation of Metabolic Processes in Mitochondria, Vol. 7, B.B.A. Library. American Elsevier Publishing Company Inc., New York, P. 548 (1966) Bruni, A. and Azzone, G.F., Biochim. Biophys. Acta. 93, 462 (1964). Charles, R. and Van Den Bergh, S.G., Biochim. Biophys. Acta., 131, 393 (1967) Chen, P.S., Jr., Toribara, T.Y. and Warner, H., Anal. Chem. 28, 1756 (1956) Hammel, C.L. and Bessman, S.P., J. Biol. Chem., 230, 2228 (1964) Heldt, H.W., in J.M. Tager; S. Papa; E. Quagliariello and E.C. Slater (Editors), Regulation of Metabolic Processes in Mitochondria, Vol. 7, B.B.A. Library, American Elsevier Publishing Co., Inc., New York; P. 547 (1966) Klingenberg, M. and Pfaff, E., in J.M. Tager, S. Papa, E. Quagliariello and E.C. Slater (Editors), Regulation of Metabolic Processes in Mitochondria, Vol. 7, B.B.A. Library, American Elsevier Publishing Company, Inc., New York; P. 180, 1966 Kroon, A.M., Biochim. Biophys. Acta. 72, 391 (1963) Lowry, O.H., Rosenbrough, .J., Farr,T.A. and Randall, R.J., J. Biol. Chem. 193, 265 (1~51) Pullman, M.E., Penefsky, H.S., Datta, A. and Racker, E., #J. Biol, Chem. 235? 3322 (1960) Roodyn, D.B., Biochem. J. 97, 782 (1965) Vignais, P.V. and Duee, E.C., Bull. Sot. Chim. Biol., 5, 1169, (1966) Wheeldon, L.W. and Lehninger, A.L. Biochemistry, 2, 3533 (1966) Winkler, H.H., Bygrave, F.L. and Lehninger, A.L., J. Biol. Chem. 243, 20 (1968) Ekler, H.H. and Lehninger, A.L., J. Biol. Chem. E, 3000 (1968)

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