Atractyloside-sensitive translocation of phosphonic acid analogues of adenine nucleotides in mitochondria

BIOCHEMICAL

Vol. 30, No. 5, 1968

ATRACTYLOSIDE-SENSITIVE ANALOGUES

Duke

with

February

P.V.

the technical

nucleotides

nucleotides

the

of adenine

study

membranes

(Bruni

; Brierley

and Pfaff,

Vignais

assistance

Moureau

1966

nucleotide aal.,

reactions

involving

be shown of which tool

mitochondria nucleotides

for since

; D&e

some

nucleotides

acid

the study

of adenine

are

rapidly

taken

1965

analogues

may

of adenine et al.

normal it may

be overcome

with

internal

paper

nucleotides,

(1963,

dependent

reactions

546

from

. In the present

reaction

: AOPCP : Adenosine-5’ AOPOPCP : 5’ -adenylyl AOPCPOP : Adenosine-5’

with

of mitochondrial

nucleotides

or transphosphorylation

loaded

limitation

exchanged

; Klingenberg

as an advantage,

aspects

by Myers

possible

and Green,

different

analogues

described

made

1965 ; Brierley

a This

nucleotide

functions

mitochondrial

and Vignais,

often

has been

they

they

across

not metabolically

to investigate

phosphonic

many

1966) o Mitochondria

has been

adenine

to elucidate

etal.,

by an atractyloside-sensitive

to phosphorylation

Abbreviations

are

adenine

that

; Pfaff

1965

this

by the use of adequate

a valuable

269

B.P. France.

~ In particular,

and Duke,

~ Although a limitation

useful

translocation

1964

nucleotides

constitute

been

in mitochondria

; Vignais

adenine

synthesis

have

and O’Brien,

mitochondria

it will

IN MITOCHONDRIA

ACID

5, 1968

of adenine

labelled

NUCLEOTIDES

de Biochimie, Centre d’Etudes Nuclkaires, de MBdecine et Pharmacie, 38 - Grenoble,

Radioactive

1965

OF PHOSPHONIC

and

of M.

Received

TRANSLOCATION

OF ADENINE

E,D.

Laboratoire et Faculte

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1965), reactions adenine

but do not participate within

mitochondria.

-methylenediphosphonate methylenediphosphonate -methylenediphosphono-P2-phosphate

the are in

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

Materials

and Methods

“Commissariat

: (32P)-orthophosphate

b 1’Energie

Bioresearch,

Inc..

AOPOPCP

Atomique,

The

following

and AOPCPOP

Saclay”,

analogues

(ATP

was obtained 14 ( C)-ADP : AOPCP

analogues),

were

from

the

from

(ADP

Schwarz

analogue),

purchased

from

Miles

and Co.

Adenosine

- 5’ -0-P-CH2-P-OH 1 OH

(AOPCP) bH

Adenosine

- 5’

Adenosine

Fi - 5’ -0-P-CH2-P-O-r-OH

(AOPOPCP)

OH

Rat liver Digitonin

mitochondria

particles

(1958).

AMP

Adam

(1963) Tests

adenine HCl,

by the method

1 mM

Mitochondria as follows

: rat

KCI-Tris-EDTA

loaded liver

centrifuge

mitochondrial

0.25

M sucrose

dria

were

pellet and

resuspended

and

sedimentation

(1963). and added

KCl,

be referred

being

of

Trautschold

20 mM

Tris-

to as KCl-Tris-

by centrifugation

force

for

5 min.

attained

within

time

mitochondria

for

at

30 sec.

45 sec.. with

(14C) -a d enine

mitochondria

mitochondria

The

ended

and Lehninger

intramitochondrial

will

(1955).

by the method

of Lamprecht

was

supplemented

of incubation,

by Devlin

enzymaticaIly

medium

the mean

as being

to Hogeboom

out at 0” in 110 mM

; this

period

according

between

carried

EDTA

conditions,

calculated

determined

the exchange were

(AOPCPOP)

as described

and ATP

IO 000 x g, the maximum

was

isolated

prepared

incubation

these

OH

were

pH 7.3,

under

bH

and ADP

to measure

The

R

were

were

nucleotides

EDTA.

0 II

(15 mg/ml) with

were

0.25

in 0.25

mM

centrifuged

was washed

centrifugation.

nucleotides

three After

M sucrose.

547

were

were

prepared

incubated

at 0” in

(14C)-ADP

; after

for

at 10 000 x g-

times the third

10 min.

by resuspension washing,

20 min.

in

mitochon-

;

BIOCHEMICAL

Vol. 30, No. 5, 1968

Results

: The

reactivity

and of AOPOPCP been

tested

adenine

with

(Adam,

or ATP

analogues

or hexokinas these

cerate

1963), are

o While

is inhibitory.

of either

only

very

none

of the analogues

of these

acid

analogues

reported

which

that

AOPOPCP

was

reaction

(Elesher

et al.,

1960)

et al.,

1960)

unable

for

with

this

extends

or ATP

phosphoglyenzyme

AMP, lack

and

ATP

ADP

findings

in hexokinase

in actomyosin (Simon

and

of reactivity

previous

to replace

phosphorylase

pyruvate

can be estimated

. The

inhibitory

1963).

of ADP

to measure

here

and was

and polynucleotide

slowly

analogues

of

kinase,

the reaction

however

assays

adenylate

is not a substrate

of phosphonic showed

for

has

Trautschold,

for

do not inhibit

but it is possible

in the presence

and

on the one hand

on the other

used

not substrates

Therefore

tests

of ATP

(La m p recht

reacts

of ADP

commonly

AOPOPCP

AOPCPOP

by enzymatic ATP

enzymes

e, but they

enzymes

kinase,

AOPCP

as an analogue as analogues

different

nucleotides

The ADP kinase

of AOPCP

and AOPCPOP

towards

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Moos

and Myers,

1961)

systems. The dria

comparative

in adenine

content

nucleotides

(AMP

then

washed

with

previous

t ADP with

upon

remaining

inside

AOPCP

Since

The

given

in Table

absorbing

release

t AMP

absorbance I.

AOPCP

cannot

nucleotides

Mitochondria with

of internal

which

saline

after

medium

absorbance

ADP adenine

or with

data

AOPCP.

nucleotides

548

but of of

with by perchloric

the amount

result

addition

upon

neutralization

of whether This

upon

incubated

extracted

that

independent

released

spectrophoto-

after

indicate

suggesting

incorporation

been

were

t ATP

the amount

determined have

On the

enzymatically,

at 260 mu,

at 260 mp of the extracts

The

with

be assayed

and

1965).

t ADP

has been

was

ADP

is in agreement

decreased

present

t ATP)

with

; this

of AMP

as ADP

nucleotides

and Vignais,

the sum

nucleotides

at 260 mp is the same,

incubated

(D&e

adenine

t ADP

washed

not vary

1965),

of mitochon-

The adenine

incubated

is substantially

adenine

and then

does

on the content I.

of mitochondria

coefficient

at 260 n-q.

acid.

in Table

of AOPCP,

extinction

(AOPCP

metrically

is shown

et al.,

of internal

intramitochondrial AOPCP

and AOPCP

mitochondria

of AOPCP.

has the same

of ADP

medium

(Pfaff

addition

that a fraction

were

+ ATP)

a saline

data

contrary,

addition

effect

is of compounds

the mitochondria suggests of AOPCP

that

the

is compen-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

Table

I :

Effect of ADP and AOPCP on the content and the distribution of adenine nucleotides in mitochondria. I

Time

Addition

Adenine

nucleotides

AMP

found

(mpmoles)

Absorbance at 260 mp

ADP

ATP

Sum

Zero

None

280

250

60

590

0.750

20 min.

None

260

270

60

590

0.760

20 min.

ADP

290

300

60

650

0.770

20 min.

AOPCP

220

140

40

400

0.760

Mitochondria (48 mg of protein) were incubated at 0’ in KCl-Tris-EDTA (cf. Materials and Methods). Final volume : 5.5 ml. Additions were either Incubation was stopped by centrifuga0.44 mM ADP or 0.36 mM AOPCP. tion ; the mitochondrial pellet was washed with KCl-Tris -EDTA and extracted with 2 ml of 2 o 5 N perchloric acid. Adenine nucleotides were determined enzymatically on the neutralized extract (cf. Materials and Methods) and absorbance was taken at 260 mp after a six-fold dilution.

sated

by the uptake

A direct from

demonstration

(14C)-adenine

either

ADP

inhibited

of the release

of (

the latter

of internal

shown

of internal

may

be taken

adenine

nucleotides

are

exchanged

results

were

when

inhibition

as an indication

mitochondria

or AOPOPCP.

549

external were

induced

et.,

net leakage with

of

is much

(Vignais

adenine

whereas

the

by atractyloside induced that

AOPCP. incubated

by

internal Similar with

more 1967)

of internal

external

nucleotides

with

addition release

by atractyloside

(14C)-adenine

nucleotides

by ADP,

previously

The

of AOPCP

nucleotide

between

inhibited

addition

obtained

1. The

to distinguish

being

C)-adenine upon

the one induced

is atractyloside-insensitive.

of the release

AOPCPOP

in Fig.

than

and exchange

14

mitochondria

It was

allows

nucleotides

nucleotides, former

slower

by atractyloside.

adenine

of AOPCP.

is given

although

atractyloside

amount

nucleotide-loaded

or AOPCP

by AOPCP,

that

of an equal

( 14C)-

Vol. 30, No. 5, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

WITHOUT ATRACTYLOSIDE

WITH ATRACTYLOSIDE

60

30

0

MINUTES

14 nucleotides with - Exchange of intramitochondrial ( C)-adenine external ADP or AOPCP. Mitochondria were previously loaded with (14C)-adenine nucleotides, as described in Materials and Methods. 19 mg of mitochondria containing 110 mumoles of (14C)-adenine nucleotides were incubated at 0” in KClTris-EDTA. Additions were either 0.23 mM ADP or 0.23 mM AOPCP ; atractyloside, when present, was 23 PM. Final volume 2.2 ml. Incubation was stopped by centrifugation and the amount of (f4C)-adenine nucleotides present in the supernatant fluid was measured by liquid, scintillation counting. Fig.1

the

Since

AOPCP

and AOPOPCP

same

atractyloside-sensitive

interesting

to test

acceptor

in oxidative

hydrolyzed order

in the

to diminish

carried

out with

on mitochondria, particles

remains

unaltered.

some

AOPCP

translocated

process AOPCP

same

manner

replace

as ATP

and ATP, ADP

particles

AOPOPCP

factors,

obtained

is also

not hydrolyzed.

in the preparation

550

does

be

mitochondria. this

by action

II, ATP is rapidly -I-+ with Mg , whereas

Furthermore,

it was

could

in Table

supplemented

by

as phosphate

by uncoupled

of permeability

submitochondrial

contaminant

mitochondria

and if AOPOPCP

the interference

AOPCPOP

into

as ADP

could

phosphorylation

As shown

digitonin

hydrolysis.

whether

are

In study

of digitonin

hydrolyzed

by

AOPOPCP

not inhibit

However, of AOPCPOP,

was

ATP

owing the

to

BIOCHEMICAL

Vol. 30, No. 5, 1968

Table

II :

Reactivity towards

Oxidative

of phosphonic acid reactions of oxidative

analogues of ADP phosphorylation.

phosphorylation

None

70

ADP AOPCP ADP + AOPCP

P Acetoac.

0

and ATP

ATP-as

I

32p. 1 esteriiied (mpmoles)

Acetoacetate formed (mpmoles)

Additions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pi formed (pmoles,

Substrate

0

e

I ATP

2.4

0.14

AOPOPCP

0.0

0.82

ATP + AOPOPCP

1.9

130

100

0.77

70

12

110

90

I

Conditions : Oxidative phosphorylation : the reaction medium contained : 82 mM KCl, 15 mM Tris-HCl, 8.3 mM phosphate labelled with 32P, 18 mM 8-hydroxybutyrate, 4 mg/ml BSA and digitonin particles (2.7 mg of protein). ADP and AOPCP were L .5 mM. Final volume : 2.6 ml ; temperature 28” ~ ed after 5 min, by addition of 0.2 ml of 30% trichloraIncubation was sto R Piesterified cetic acid (w/v). was determined by the method of Nielsen and Lehninger (1955), acetoacetate according to Walker (1964). ATP-ase : digitonin particles (1.2 mg of protein) were incubated for 5 min. at 28” in the following medium : 100 mM KCl, 10 mM Tris-HCl, pH 7.3, 5 mM Mg C12. Additions were either 5.2 mM ATP or 5.2 mM AOPOPCP or both. Final volume was 1.9 ml. Incubation was stopped by the addition of 0.2 ml of 30% trichloracetic acid. Inorganic phosphate was determined as described by Fiske and Subbarow (1925).

significance AOPCP

of this is a poor

oxidized

result phosphate

by digitonin

than ADP

cannot

(8 times).

be assessed.

acceptor

On the other

: when

particles,

AOPCP

Separation

of ADP,

hand,

8-hydroxybutyrate

is phosphorylated ATP,

is much

AOPCP,

slower

and AOPCPOP

by paper chromatography (D&e, 1967) revealed that the small amount 32 P)-phosphate esterified in the presence of AOPCP was actually of ( present does

in AOPCP032P. not apparently

Furthermore, interiere

with

experiment

not presented

here

of rat liver

mitochondria

showed

the respiration

of mitochondria

data

in Table

the phosphorylation

and bearing that ADP previously

551

II show oi ADP.

on the respiratory is still

capable

loaded

with

that AOPCP Another control

to stimulate

AOPCP.

The

1

1

I 1

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 30, No. 5, 1968

same

experiment

to Mattoon

extended

and Balcavage

In conclusion, analogues

of ADP

and AOPOPCP

are

observations

is kept

acid constant

nucleotides our

analogues while

are

very

Such

to study

exchanged

into

mitochondria,

slowly,

if at all.

acid with

AOPCP

mitochondria of total

of enzymatically

decreased.

D

an atractyloside-sensitive

the amount

the amount

according

the phosphonic

rapidly

to prepare

in which

are

through

only

, it is possible

results

which

translocated

metabolized

prepared

identical

nucleotides

easily

is strikingly

laboratory

gave

respectively,

adenine Although

mitochondria

and AOPOPCP

and ATP

translocation.

phosphonic

(1967),

AOPCP

intramitochondrial

above

to yeast

the compartmentation

on the

loaded

with

adenine

reactive

a system

Based

adenine

is now

of adenine

nucleotides

being

used

nucleotides

in in

mitochondria. Experiments Pfaff,

recently

1966),

(Kemp

the specificity

of the

ADP

by the

is caused

nucleotides experiments apply

and Groot, overall

at the level reported

to the phosphonic

reported

in the literature 1967),

process

specificity

acid

indicate

of oxidative

membrane that this

analogues

and

led to the conclusion

for

for

the adenine

of mitochondria. conclusion

of adenine

that

phosphorylation

of the translocation

of the inner here

have

(Klingenberg

does

The not

nucleotides.

Acknowledgments : This investigation was supported by research grants from the “Centre National de la Recherche Scientifique” (RCP 21), the “For&&ion de la Recherche Mkdicale” and the “Dkl&gation G&krale a la Recherche Scientifique et Technique” (Contrat no 67-00-625),

REFERENCES Adam, H. (1963), in Bergmeyer, H.U., Methods of Enzymatic Analysis, Academic Press, New York, p.573. Brierley, G. and Green, D.E. (1965), Proc. Natl. Acad. Sci. U.S., 53, 73. Brierley, G. and O’Brien, R.L. (1965), J. Biol. Chem., 240, 4532. Bruni, A., Luciani, S. and Contessa, A.R. (1964), Nature, 201, 1219. Delvin, T.M. and Lehninger, A.L. (1958), J. Biol. Chem., 233, 1586. Duke, E .D. and Vignais, P.V. (1965), Biochim. Biophys o Acta, 107, 184. Duke, E.D. (1967), Bull. Sot. Chim. Biol. (submitted). Fiske, S.H. and Subbarow, Y. (1925), J. Biol. Chem., 66, 375. Oester, T.Y. and Myers, T.C. (1960), Nature, 185, 772. Flesher, J.W., Hogeboom, G. (1955) in Colowick, S.P. and Kaplan, N.O., Methods in Enzymology, Academic Press, New York, , Vol.1, p. 16.

552

Vol. 30, No. 5, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A. and Groot, G.S.P. (1967) Biochim. Biophys. Acta, 143, 628. Kemp, Jr., Klingenberg, M. and Pfaff, E. (1966), in Tager, J.M., Papa, S., Quagliariello, E. and Skater, E. C. (Editors), Regulation of Metabolic Processes in Mitochondria, Elsevier Publ. Co., Amsterdam, B. B .A. Library, Vo1.7, p. 180. Lamprecht, W. and Trautschold, I. (1963), in Bergmeyer, H.U., Methods of Enzymatic Analysis, Academic Press, New York, p. 543. Mattoon, J-R. and Balcavage, W.X. (1967), in Colowick, S.P. and Kaplan, N. 0.) Methods in Enzymology, Academic Press, New York, Vol. x, p. 135. Moos, C., Alpert, N.R. andMyers, T.C. (1960), Arch. Biochem. Biophys ., 88, 183. Myers, T. C . , Nakamura, K. and Flesher, J.W. (1963), J. Am. Chem. SW., 85, 3292. Nakamura, K. and Danielzahed, A.B. (1965), J. Org. Myers, T.C., Chem., 0, 1517. Nielsen, S.O. and Lehninger, A.L. (1955), J. Biol. Chem., 215, 555. Pfaff, E., Klingenberg, M. and Heldt, H.W. (1965), Biochim. Biophys. Acta, 104, 312. Simon, L.N. and Myers, T.C. (1961), Biochim. Biophys. Acta, 51, 178. Vignais, P.V. and Duke, E .D. (1966), Bull. Sot. Chim. Biol., 48, 1169. Vignais, P.V., D&e, E.D. and Huet, J. (196i’), manuscript in preparation. Walker, P.G. (1964), Biochem. J., z, 699.

553