Restoration of oxidative phosphorylation in ‘non-phosphorylating’ submitochondrial particles by oligomycin

BIOCHEMICAL

Vol. 18, No. 4, 1965

RESTORATION OF OXIDATIVE

AND BIOPHYSICAL

PHOSPHORYLATION IN 'NON-PHOSPHORYLATING

SUBMITOCHONDRIAL PARTICLES Chuan-pu Wenner-Gren

Institute,

RESEARCH COMMUNICATIONS

BY OLIGOMYCIN

LeeX and Lars University

Ernster

of Stockholm,

Stockholm,

Sweden

Received December 30, 1964 In a previous

paper

non-phosphorylating* carry

out

with

ATP as the

of respiratory

assayed these but

of

under

suitable

et al.,

1964a)

driven

by aerobically

lating

particles.

amount

of oligomycin

hydrogenaee

of the

finding

suggested

(Fig. %ellow

2):

that

Closer

required

that

activity oligomycin

an inhibition of the

Jane

of the Coffin

can also

Child6

may exhibit

523

19581.

observation

(Fig.

for

Medical

the

of the

trans-

maximal

in-

preparation.

a high-energy for

reaction

1) that

stimulation required

Fund

known

in non-phosphory-

two effects

of

factors*,

transhydrogenase

revealed

that

of

et at.,

previous

same particle

Memorial

of the

intermediates

maximal

when

restoration

(Lardy

the

process,

or 'coupling

amounts

effect

hydrolysis

the

enzymes

by the

oxidative

of this

be shown,

than

of the

perform

reversal

stimulated

to achieve

makes them

we

oligomycin

of this

what

paper

added

was much smaller

ATPase

and that

are still

In this

high-energy

study

particles

with

ADP to ATP.

appropriate

oligomycin

than

primary

was prompted

generated

reaction

hibition

of

more

from

as the

phosphorylation,

investigation

these

can

energy

As will

addition

of oxidative The present

as well

heart

to transfer

particles

no externally

by the

that

of ability

such

beef

so-called

intermediates

energy-coupling,

conditions.

requires

is achieved

inhibitor

ADP to ATP,

from

high-energy

phosphorylate

that

shown that

transhydrogenation

was concluded

lack

to

evidence

activities

(Lee

the

intermediates

phosphorylation

nucleotide

chain-linked is

to present

It

we have

preparations

generated

of energy.

'non-phosphorylating*

wish

1964a)

al.,

pyridine

aerobically

source

high-energy

et

submitochondrial

energy-dependent

efficiently

capable

(Lee

on the

This particles

intermediate, Research

1963-65.

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

I 0.4

Fig.

~1.

0.8

1.2 1.6 Irg oligcmycin/mg

2.0 2.4 protein

28’

312

Effect

NADP+ reduction bv NADH. ATPase was assayed in a reaction mixture consisting of 160 mM sucrose, 50 mM Tris-acetate buffer, pH 7.5, 4 mM (prepared in the presence of 2 mhI EDTA, pi-l 8.6) conMgso # particles tain 4 ng 0.2 mg protein, and oligomycin as indicated. Reaction was started by the addition of 5 mM ATP. Final volume, 1 ml, temp., 30°C, time of incubation, 5 min. The reaction mixture for the transhydrogenase assay consisted of 180 mM sucrose, 50 mh! Tris-acetate buffer, 10 mM MgSD , 0.2 mM NADH, 0.15 mhf NADP+, PH 7.5, 3.3 pM rotenone, 1 mM oxidized glutathione, an amoun 4 of glutathione reductose capable of oxidizing 0.5 umoles of NADPHfmin., particles containing 0.6 mg protein and oligomycin as indicated. Reaction was started by the addition of 5 mM succinate. Final volume, 3 ml, temp., 30°C.

succinate

ascorbate

f

I

energy-ckpendent transhydrogenast. ion translocation

high)m oligomycin

[valinomycin]

ATP

Fig. 2. resoiratorv coupling transfer

Hvoothetic scheme of the energy-transfer svstem linked to the chain. C , C , C denote electron carriers at the energyI and X denote hypothetic energy sites of the'resgira s ory chain; carriers (for further definitions, see Ernster and Lee, 1964). u bars indicate site of action of inhibitors; been bar indicates site of stimulation by 2,4-dinitrophenol (DNP). 524

Vol. 18; No. 4, 1965

I-X,

at

BIOCHEMICAL

low concentrations

action

of

the

that

(or

its

presented

below

As shown

in Fig.

only

tion,

in accordance

a very

considerable

occurred

tion,

which

stimulated

the

this

low rate

of

with

previous

prepared

results

only

a certain

within

ATP-supported

amount

reduction

rate

of NAD+ reduction

instead stimulating

of

being

virtually

oligomycin

exceeded

of

1964a).

ATP induced

The reaction

of

(Fig.

concentra-

Oligomycin

needed

for

or ascorbate

constant

as was the This 525

also

+ N,N,lj'N'-

analogous

noticed

by succinate

concentrations.

was

to the

Stimulation of ATPFig. 3. -suooorted succinate-linked + NAD reduction bv oliPomvcin The reaction mixture consisted of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7.5, 1.6 mM KCN, 10 mM hlgso , 0.2 mM NAD+, 5 mM succ 4 nate and particles (prepared in the presence of 2 mM KDTA, pH 8.5) containing 0.75 mg protein. Other additions: 3 mM ATP, 0.15 pg oligomycin, 1.6 mk'malonate, and 1.6 /.tM rotenone& Final volume 3 ml, temp., 30 C.

was also

that

a

the

oligomycin 4).

in a fashion

or rotQncnQ

It

When

As anticipated,

range

KDTA exhi-

NAD+ reduc-

of NAD+ by ascorbate

(TMPDI,

NAD+ reduction. of oligomycin

presence

was added,

was inhibited

The

case.

et al.,

protein

it

particles.

NAD+ reduction.

t-nahate

the

(Lee

higher

oxidative

succinate-linked

and rotenone.

reaction

the

inter-

correct,

enhance

in the

ATP-supported

-tetramethyl-D-phenylenediamine

succinate-linked

is

by malonate

the

were

might

indeed

of succinate-linked

stimulation

ATP, at

in non-phosphorylating

3, particles

inhibited

above

interpretation

per mg of particle

rate

of the

Pi and ADP, to yield

this

reversal)

RESEARCH COMMUNICATIONS

and an inhibition

of oligomycin

show that

pg of oligomycin

completely

If

low concentrations

bited

0.2

with

of oligomycin.

phosphorylation data

of oligomycin;

same intermediate

concentrations follows

AND BIOPHYSICAL

maximal

shown)

that,

stimulation,

+ 'IMPD decreased case

is

(not

at or below

consistent

with

when the

with

tine,

maximally the

assumption

Vol. 18, No. 4, 1965

that,

with

becomes

BIOCHEMICAL

increasing

concentrations

increasingly

sequently,

limited

more and

AND BIOPHYSICAL

Similar

though

less

eC Begg,

19591,

whereas

of oligomycin,

by the

dependent

more

striking

RESEARCH COMMUNICATIONS

generation on the

effects

were

octylguanidine

of

I-X

equilibrium

from

with

(II):

1963)

was

temp.,

added

Final

as expected

0P

start

volume,

in replacing

succinate-linked In Fig.

(McMurray was in-

Effect of varvirv amounts Fig. 5. of oliaomvcin on oxidative phosphorvlation with different substraThe reaction mixture conIXS. sisted of 180 mM sucrose, 50 mM Tris-acetate buffer, pH 7.5, 15 mM NIgso , 3 mh4 ATP, 15 mM glu2 mM 74 units hexokinase, ;"ns (1.2 x lo6 cpdpmole), partfcles (prepared in the presence of 2 mM EDTA, pH 8.6) containing 0.6 mg protein and oligomycin as The srbstrate was indicated. either 0.2 mM NAD , 160 mM ethanol and 16 pg alcohol dehydrogenase; or 5 mM succinate; or 5 mM succinate, 3 c~s antimycin A and 0.2 mM TMP$. Final volume, 3.1 ml, temp., 30 C. 0 consumption was measured with a2Clark electrode and the esterification of P was determined by the isotope d f stribution method (Lindberg & Ernster, 1955).

the

3 ml,

30°C.

effective

the

to

reaction.

ATPase

on ATPase and on the ATP-suooorted reduction of NADf bv succinate and bv ascorbate + TMPD. Assay system for ATPase was the same as in The reaction mixture for the Fig. 1. ATP-supported reduction of NAD by succinate and by ascorbate + TMPD consisted of 180 mM sucrose, 1.6 mM KCN, 10 mM MgSO , 0.2 mM NADf, particles (prepare d in the presence of 2 mM EDTA, pli 8.9) containing 0.6 mg protein and oligomycin as indicated+ In the case of succinate-linked NAD reduction, 5 mM succinate was present; and in the zase of ascorbate + TMPD-linked NAD reduction, 2.5 mM ascorbate and 0.2 mM TMPD were present. 3 mM ATP

latter

NAD+ (ATP)

oliPomvcin

reaction.

ATP, and con-

valinomycin

0.3

(1): Succ. -

of NAD+ reduction

of the

observed

(Pressman,

rate

the

oligomycin,

and even

abolished

the

oligomycin-induced

NAD+ reduction. 5,

phosphorylating

data

are summarized capacity

of

the

concerning particles 526

the

effect

prepared

of oligomycin in the

presence

on of

Vol. 18, No. 4, 1965

BIOCHEMICAL

EDTA.

Low concentrations

either

NADH or succinate

the

case

less

system

where

restored

marked,

also

pronounced.

rise

or ascorbate

the

absence

partially

by DNP.

These

preliminary

control,

or,

in other

taking

place

at the

level

Lee,

19641,

utilization mitochondria For

(or

whereas

the

for

translocation

ion

effect

(Klingenberg, obtaining

the

of 8.5

a subatahtial succinate-linked

effects the

to 9.0.

capacity

(cf.

A, oligowas less

an enhancement

case

of the

succinate-

did

not

affect

respir-

phosphorylation

maximally.

the either

respiration,

when the

exb*bit

Fig.

is

2).

by preventing

hydrolysis

a good only

extent

a slow

formation;

of of respir-

hydrolysis

DNP most probably

C-I

cf.

acts Ernster

of Kg++ may be due to an enhancement , analogous

was

by Kg++ + Pi + ADP, or

that,

there

which

to that

found

& of

with

intact

above,

it

by

I-X

1963).

the

to maintain

inhibited

that

1964b3).

phosphorylation-stimulating

particles

words,

al.,

the

4 and 5).

Figs.

stimulated

suggest

the

over

this

causing

oligomycin

and completely

of C-I

hydrolysis

essential range

greatly

by oligomycin,

this

that

findings

atory

promoting

above,

et

but

as in the

(cf.

A and then

of antimycin uptake,

in

in the

transfer

(Lee

oligomycin

Pi and ADP, however,

by Mg",

blocked

of

same range

in concentrations

of oligomycin

restored

is

amount

employed

of Mg++,

concentrations

in phosphate

4-fold

obtained

by antimycin

presence

with

A significant,

was also

chain

in the

uptake

was about

of electron

respiratory

the

phosphate

succinate.

blocked

NAD+ reduction

conditions

when added

In the

the

was within

+ TMPD-linked

Under ation

cases,

of

a by-pass

to an increase

In all

of phosphorylation

I-X

establishing of the

the

uptake

was first

+ TMPD as substrate,

gave

case

in phosphate

site

RESEARCH COMMUNICATIONS

The enhancement in the

oxidation

energy-coupling

mycin

3-fold

increase

succinate

ascorbate

enhanced

as substrate.

by ThiPD, thus

second

oligomycin

of NADH, and about

though

With

of

AND BIOPHYSICAL

of

oligomycin

pH of the

KDTA-containing

At pH values for

both

NAD+ reduction

below

oxidative even

as described

8.5,

the

particles

phosphorylation

when assayed

527

sonicating

in the

was found

medium

within

may retain

and ATP-supported absence

of oligo-

Vol. 18, No. 4, 1965

mycin; less is

BIOCHEMICAL

at pH values pronounced,

lar

coupling;

(which Haas,

1964)

have

it

is

*non-phosphorylating'

for

the

earlier

(Lee

Of great

recently

and the

three

*site

specific*

act

operation these

oligomycin

of

a missing

are

This work has been We thank Miss Ulla-Britta

present

respiratory

1964a1,

but

for

three

the

and associates

present

sites

Racker of Green

phosphorylation

in the

in the

but

particles.

latter rather

as

results

coupling

factors'

the

cold-labile

& Conover, et

al.

of oxi-

respiratory

present

primarily

factors

species,

presence

of the

of soluble

the

so-called

process

of the

(cf.

that

reduction.

in the

complete

implications

laboratories,

oxida-

energy-coupling,

coupling

mode of action

enzyme

energy-

that

prepared

for

implies

respiratory

results

only

coupling

heart-muscle

NADf

particles

the

factors,

one already

relationships

the

presimi-

no oligomycin-stimulated

succinate-linked from

can restore

as do these

by restituting

for

in different

of Racker

particles

capacity

the

F 1'

that

the

revealed

in the

in principally

possesses

may be the

called

resulted

Keilin-Hartree

involving

studied

have

prepared

with

al.,

of

particles

becomes

phosphorylation

those

not

importance

of oligomycin

whereas

so far

ATPase,

fact

with 1963)

evident

et

interpretation widely

of oxidative

capacity,

phosphorylation

the

stimulation

submitochondrial

of EDTA possess

chain.

as the

or ATP-supported

In conclusion,

dative

effect

effects,

phosphorylation

concluded

stimulating

& Conover,

likewise

RESEARCH COMMUNICATIONS

the

experiments

(Racker

much weaker

preparation

tive

Preliminary

of ammonia but

9.0,

especially

concerned.

sence

above

AND BIOPHYSICAL

1963),

(1963).

same type

The

of

most probably

do not

by facilitating

the

Attempts

to clarify

in progress. supported by a grant from Samuelsson for excellent

the Swedish Cancer Society. technical assistance.

REFERENCES Ernster, L,, and Lee, C.P. (1964) Ann. Rev. Biochem., 3, 729. Green, D.E., Beyer, R.E., Hansen, M,, Smith, A.L., and Webster, G. (1963) Federation Proc., 22, 1460. Haas, D-W, (1964) Biochim. Biophys. Acta, &$ 543. Klingenberg, M. (1963) &: a) Funktionelle und morphologische Organisation der Zelle (P. Karlson, ed., Springer-Verlag, Heidelberg) p. 236; b) Energy-linked Functions of Mitochondria (B. Chance, ed., Academic Press, New York) p. 247. 528

Vol. 18, No. 4, 1965

BIOCHEMICAL

AND 8lOPHYSlCAL

Lardy,

RESEARCH COMMJNlCATlONS

H.A., Johnson, D., and McMurray, W.C. (1958) Arch. Biochem. Biophys., 78, 587, Lee, C.P., Azzone, G.F., and Ernster, L. (1964al Nature, 201, 152. L. (1964b) u An International K., and Ernster, Lee, C.P., Nordenbrand, Symposium on Oxidases and Related Oxidation-Reduction Systems (T-E, King, H.S. Mason, and M. Morrison, eds., Wiley & Sons, New York) in press. Lindberg, O., and Ernster, L. (1955) Meth. Biochem. Anal., 3, 1. McMurray, W.C., and Begg,R.W. (1959) Arch. Biochem. Biophys., 84, 546. Pressman, B.C. (19631 J. Biol. Chem., 238, 401. Racker, E., and Conover, T.E. (1963) Federation Proc,, 22, 1088.

529