Effect of ion-transporting antibiotics on the energy-linked reactions of submitochondrial particles

Vol. 34, No. 1 t 1969

5lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF ION-TRANSPORTING ENERGY-LINKED

ANTIBIOTICS

ON THE

REACTIONS OF SUBMITOCHONDRIAL PARTICLES*

M. Montal,

B. Chance,

C. P. Lee,

and A. Azzi

Johnson Research Foundation School of Medicine University of Pennsylvania Philadelphia, Pennsylvania 19104

Received December 2, 1968

Certain natural

antibiotics

have the property

and artificial

elucidation

membranes

of the relationship

mechanisms

in mitochondria

are still

capable

and are

(2)

coupling

and energy

disruption

energy system

and ion

as a tool

and chromatophores

by sonic

a more elementary

energy

reported

of the valinomycin

the

derived

phenomena

transport

in the

coupling (3,4,5).

of mitochondria

coupling

in which

across

and oxidative to understand

across

the

inner

the mito-

membrane. The data

actions

chloroplasts

particles

between

chondrial

membrane

of respiratory-chain-linked

phosphorylation, relationship

in

ion movements

They have been utilized

between (1)

Submitochondrial

(1).

of activating

in this

and nigericin

of submitochondrial

current

hypotheses

paper type

show that strongly

affect

The results

particles. of energy

ion-transporting

coupling

antibiotics

the energy-linked are discussed

in oxidative

re-

in terms

of

and photosynthetic

phosphorylation. MATERIALS AND METHODS EDTA particles described

previously

derived (6).

from

beef

heart

Oxygen consumption

*Supported by the National Institutes F05 TW-01291), the Jane Coffin Childs the National Institute for Scientific

mitochondria

were

prepared

was measured

polarographically

of Health (GM 12202, 1 K4 GM 38822, Memorial Fund for Medical Research, Research (Mexico).

104

as

and and

Vol.34,No.1,1969

with

BIOCHEMICAL

a Clark

hydrogenation were

--et al. Dr.

(7)

assayed

responses

electrode.

oxygen

(IO),

to Ernster

measured

nucleotide

trans-

of NAD+ by succinate

The energy-linked

and Lee.

by Chance

Valinomycin

of this

pyridine

reduction

as described

respectively.

B. C. Pressman

RESEARCH COMMUNICATIONS

The energy-linked

and the ATP-supported

according

were

ANDBIOPHYSICAL

and Mela

and nigericin

(8)

BTB* and ANS

(9)

and by Azzi,

kindly

were

supplied

by

laboratory. RESULTS

Lee and Ernster

(11)

of respiration

in E-SMP,

the presence

of oligomycin

have shown which there

supported

energy-linked

reduction

of NAD+ by succinate,

Fig.

1, the

release

as a function

the

control.

system

50%, in

carrier

question,

comparison these

other

pyridine

2 shows

antibiotics

nigericin

alone

in

alone

the presence

in

induced

transfer

alone

the

acting

re?eased

about

reactions

were

of the

In order

reaction.

of k about

50% of

and/or

of respiration

by

The question

arises

of the electron

trans-

to elucidate

this

studied.

antibiotics

to 95% inhibition the presence

level

plotted

combination

although

alone.

at the

is

control,

stimulation

by either

process.

of K+ inhibits

control

uncouplers,

the oligomycin

in a further

are

the effect

leads

other

In

by uncouplers.

of FCCP to the valinomycin-

transhydrogenase

antibiotics

while

resulted

chain

and of the energy-linked inhibited

and nigericin

addition

energy-linked

nucleotide

of both

released

in

respiratory

respiratory

Like

to that

or the energy

Figure

are also

an inhibition

furthermore,

of the

reaction

of the oligomycin-induced

Furthermore,

as to whether fer

a stimulation

which

had no effect,

nigericin-treated about

is

induces

by uncouplers;

transhydrogenase

and nigericin alone

oligomycin

released

of KC1 concentration.

of valinomycin va'linowcin

is

that

on the energy-linked As can be seen,

of the

rate

There

presence

of NADPH formation,

has no effect, 50%.

the

is

and valinomycin no effect

of the

'Abbreviations used: BTB, bromthymol blue, 3,3" dibromothymolsulfonphthalein; E-SMP, EOTA submitochondrial particles; ANS, N-arylaminonaphthalene sulfonate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

105

Vol. 34, No. 1, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.

. IO

0

I

I

I

I

I

50

30

KCLmM

Figure 1. Effect of valinomycin and nigericin on the oligomycin-induced respiratory control as a function of KC1 concentration. The reaction mixture contained 0.1 mg protein per ml of E-SMP, 0.25 M sucrose, 20 mM Tris-HCl, pH 7.4, 0.6 pM valinomycin, and 0.06 uM nigericin. Final volume, 3 ml. Temperature, 30" C.

0

I

2 Min

3

4

Effect of valinomycin and nigericin on the energy-linked The reaction mixture contained pnuc!eotlde transhyd rogenatlon. 1 mg protein per ml of t-SMP 0.25 M sucrose, 0.05 M Tris-HCl, pH 7.4, 0105 M KCl, 0.5 ug per ml of oiigomycin, 3.3 pM rotenone, 135 uM NADH, 5 mM succinate. Final volume, 10 ml. Temperature, 30' C. The reaction was started by the addition of 180 pM NADP. Assays were done according to the method of Lee and Ernster (7).

Vol. 34, No. 1, 1969

antibiotics

BIOCHEMICAL

on the

non energy-linked

BTB (9,12), indicators

effect

of the

and ANS (upper oxidation

cycles

are

duration. bited;

is

The effect

by succinate

the of the

antibiotics

studied.

100% of the

only

the

3c shows that

the

and 50% shorter

in

to that

induced

by FCCP under

alone

in the

on the Valinomycin

reaction,

and valinomycin

cycle

presence about

is

and nigericin

in

alone

similar

50%. reduction

nigericin

95% inhi-

of Kt (not

energy-linked

whereas

of

in the presence

the

only

alone

in the presence

the

of NAD+ presence

in

the pres-

of Kt

50%.

366+560mp Fluorescence InCEOSe

t 618-700mp Absorbance 1 Increase IObpM

NADH

IOQM

NADH

-d

t

I IOOpM

oligomygin,

25' C.

(cl6sec

Nigerian

ID

366+560mp Fluorescence lncreose 618;7OOmp Absorbonce Increase

NADH

IpM Vahomycin

Effect sr;;po~

70nM

of nigericin and valinomycin on the energy-linked BTB 0 9 mg protein per ml of E-SMP in 0.3 M mannitolris-Hi1 pH 7.4 supplemented with 0.5 ug per ml of ! PM BTB, aid 10 uM'ANS. Final volume, 1 ml. Temperature,

107

3

trace)

cycle

of valinomycin,

BTB and ANS responses

ence of Kt had no effect, inhibited

was added. in amplitude

of valinomycin

was also

of KC1 inhibited

comparable

as a control,

as

Figure

BTB (lower

of NADH oxidation

30% larger

the addition is

to inhibit

a cycle

to act

particles.

energy-linked

3Ashows,

70 nM nigericin

inhibition

The effect

on the

3Bis

now approximately

have been shown

of submitochondrial

responses.

In 3l& after this

ANS (lo),

antibiotics

thereafter,

conditions. shown)

trace)

state

of 100 uM NADH.

of 20 njrl KCl;

reaction.

and more recently,

of the energized

shows the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

VoL34,No.

1,1969

BIOCHEMICAL AND BIOPHYSICALRESEARCH

The well-documented Nat

(1,13)

by the

reactions

in a medium

was partially

specificity strictly

of valinomycin

is demonstrated

energy-linked Nigericin

specificity

(1).

effective

The effect

dependent

complete

lack

in which

on the presence

to K+ rather

of inhibition

the

than

of all

according

on addition

of both

to

the

Kt has been replaced

in a Nat medium,

observed

COMMUNICATIONS

by Na+.

to its

known

antibiotics

was

of K+.

DISCUSSION

Previous (tentatively during

studies identified

coupled

tt of Ca inconsistent

the (9)

and direct

drial

evidence

of a cation to the Ht uptake

supported

inward

in submitochondrial

of the

Ht movements

(18);

the cation

movement particles

described

is by

pumps seem to

here

afford

indirect

in submitochondrial

of electron

particles

between

transfer

evidence

that (9,18)

monovalent

and energy

the cation may be Kt,

cation

conservation

movements in submitochon-

particles.

an uncoupled of the

system

Kt/Ht

maximal

rate

and K' of the

in fact on the

to an influence

rate

of electron

rate

of oxygen

of the

it

is evident

Kt or low Ht concentration

is

a requirement

in

environment

Due to the

transport.

by nigericin,

promoted

ionic

uptake

that

the presence for

having

effect

of

a

of respiration.

The effect valinonlycin

chain

exchange a high

by nigericin points

respiratory

of either

linked

opposite

and Moyle

of an interaction

The stimulation

known

(14)

direction

reported

Ht movements

and the process

movement

and Racker

inward

requirement

directions.

The results for

the

The succinate

and Mitchell

in opposite

required

Cat+) (9).

by Loyter

with

and Mela

operate

with

respiration

observed

Chance

have considered

of valinomycin

and nigericin

reactions

in

and K+ and the synergistic the presence

in submitochondrial

of Kt,

particles,

inhibiting suggests

several as well

energya close

Vol. 34, No. 1, 1969

interaction

BIOCHEMICAL

between

ion

One mechanism activated

outward

movements

is

based

movement

the transhydrogenase,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and energy

conservation.

upon the above

of K' which

the BTB-ANS

is

mentioned

valinomycin-

responsible

responses

for

and the

the inhibition

reversal

of

of electron

transfer. The combined energy

dissipating

nigericin

ion

would

the activity presence

effects

of valinomycin

movement

be to restore

across the

and nigericin the membrane,

Kt content

as proposed

for

in which

inside

a cyclic

the effect

of

depleted

by

the vesicle

of a pump or of a membrane potential,

of valinomycin,

suggest

that operates

mitochondria

(1)

the

effect

in the

and chromatophores

(3,5,16,17). An additional

explanation

carrying

antibiotics

constant

of the membrane,

case,

uncoupling

binding

of ionic

inhibitory conformational

effect

is

that

is to transport

would species

which

result

are not

into

required

by cyclic

regions for

transport,

indicated

and nigericin

This

of the

ion

of low dielectric

energy

in such an environment.

of valinomycin changes

ions

combined

coupling. but is

In this

due to the simple supported

in the presence

of k

by the on the

by ANS and BTB.

SUMMARY

The effects of ion-transporting antibiotics on the energy-linked reactions of submitochondrial particles were studied. It was found that valinomycin and nigericin in the presence of K' led to the release of the oligomycin-induced respiratory control, and to a virtually complete inhibition of the four energy-linked reactions: pyridine nucleotide transhydrogenation, BTB and ANS responses, and reversal of electron transfer. Valinomycin alone in the presence of K+ did not release the oligomycin-induced control, but inhibited the energy-linked reactions by approximately 50%. Nigericin alone in the presence of Kt stimulated the oligomycin-inhibited respiration by approximately 50%, but did not affect the energy-linked reactions. The results are discussed in terms of current hypotheses of energy coupling in oxidative and photosynthetic phosphorylation.

109

Vol. 34, No. 1, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS

The authors are indebted discuss-ions, and to Miss Birgitta

to Dr. B. C. Pressman for stimulating Johansson for valuable assistance.

REFERENCES

1. 2. 3. 4. 5. 6. L: 1:: ;:: 13.

ii: 16. 17. 18.

Pressman, B. C., Harris, E. J., Jagger, W. S., and Johnson, J. H. Proc. Natl. Acad. Sci., g, 1949 (1967). A. Shavit, N., Dilley, R. A., and San Pietro, Biochemistry, 7-, 2356 (1968). Thore, A., Keister, D. L., Shavit, N., and San Pietro, A. Biochemistry, 7, 3499 (1968). in press. Nishimura, M., and Pressman, B. C. Biochemistry, A. R., and Von Stedingk, L. V. Jackson, J. B., Crofts, European J. Biochem., a, 41 (1968). lo, 543 (1967). Lee, C. P., and Ernster, L. Methods in Enzymol., Biophys. Acta, 81, 187 (1964). Lee, C. P., and Ernster, L. Biochim. l& 729 (1967). Ernster, L., and Lee, C. P. Methods in Enzymol., Chance, B., and Mela, L. J. Biol. Chem., 242, 830 (1967). Aizi, A., Chance, B., Lee, C. P., and Radda, G. K. Proc. Natl. Acad. Sci., in press. Lee, C. P., and Ernster, L. BBA Library 7, 218 (1966). D. R. Arch. Biochem. Biophys., 126, 722 Kurup, C. K. R., and Sanadi, (1968). In Biochemistry of Mitochondria Chappell, J. B., and Haarhoff, K. (E. C. Slater, 2. Kaniuga, and L. Wojtczak, Eds.) London: Academic Press, 1967. p. 75. Trans. N. Y. Acad. Sci., in press. Loyter, A., and Racker, E. Lardy, H. A., Graven, S. N., and Estrada-O., S. Fed. Proc., 26, 1355 (1967). Coupling in Oxidative and Photosynthetic Mitchell, P. Chemiosmotic Phosphorylation. Glynn Research, Bodmin, Cornwall, 1966. Mitchell, P. Chemiosmotic Coupling and Energy Transduction. Glynn Research, Bodmin, Cornwall, 1968. Mitchell, P., and Moyle, J. Nature, 208, 1205 (1965).

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