The reversible activation of mitochondrial ATPase by high pH

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

77-KPEVEPSIBLEACTIVATION OF IIITOCilONDIZI.4L ATPase by IIIGH pH A. Fonyo*, L. Szende and Ilona ilezei Experimental &sear&

Dapartment, Universitv Bud&est, I-Iunga~.

Yedical School,

ReceivedJanuary 25, 1966 no general theories of oxidative The high-energy intermediate review:

phosphorylation

theory,

exist presently.

formulated seine 15 years ago (for

Lehninger, 1964) and the recent "chemi-osmotic-coupling"

(Mitchell,

1961; Yiitchell

and %yle,

1965).

hjjrpothesis

The two theories differ

in

the postulated role of ATPase. In the high-energy intermediate theory ATPase is supposedto function hydrolytic

only under artificial

uncouplers; (Penefsky et al. hypothesis, function,

in one of the transfer

tne "vectorial"

reactions and is

conditions (as in the presence of 1960).

In the chemi-osmotic-coupling

ATPase has a postulated central role,

driven by proton translocation

through the mitochondrial

membrane,would result

in the synthesis of ATP.

of importance to by,

what conditions favor the ATP-hydrolysis

may 'W the mechanismfor tne activation Activation

of mitochondrial

by %anson (1956) and later present paner, the similaritv

For botn theories

and what

ATPasebv increased p?I ;%lasfirst

confimd

it is

of the hydrolysis. *nor-ted

by ?TIers and Slater (1957). In fne

of this r3II ion activation

caused @I the uncoupler 2,4-dinitmphenol =erimental

Its

to the activation

(LNP) has been studied.

Procedure,

lhe experiments were Frformed

on 2 ws

of preparations:

intact

rat

heart mitochondria isolated in 0,25 M sucrose, and rat heart mitochondrial ik Pwsent address: Pediatric Research I!epa&nent, Baltimore, Maryland, U.S.A.

511

University

of Maryland,

Vol. 22, No. 5, 1966

fragments,

BIOCHEMICAL

The latter

muscle preparation

was a modification

of the Keilin and Hartree heart

as adapted for rat heart and performed in the cold: the

heart was washedwith distilled buffer,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

water and subsequentl;l r,rith 0,02 M phosphate

homogenized in 0,02 M phosphate buffer

genizer,

centrifuged

p!I 7,2 in an all glass homo-

at 600 g for 20 min.; the suparnatant was centrifuged

at 8500 ,g for 15 min., the sediment washed in 0,25 M sucrose, recentrifuged at 20,000 g for 15 min. and finally preparation

shoded only a slight

Vith succinate the P:O ratio could be activated

by IW,

susnendedin 0,25 M sucrose. capacity to oxidatively

was about 0,l.

induced ATPase was about one third

ATPase activity 50 mMtris-Cl

buffer,

mitochondria 0,15 mg mitochondr&l

of this IXJP

heart mitochondria.

results.

5 mMATP, in the case of intact

protein

The concentration

and in the case of fragments,

of DJP employed was 0,2 ml-i. The

incubation time was 10 min. and ATPase activity basis of inorganic phosphate liberated protein/l0

ATPase

was determined at 30° in a mediumthat contained

or tris-acetate

0,20 mg protein.

activity

that of intact

Both types of preparations yielded similar

phosphorylate.

)Jevertheless, its

although the specific

This

was calculated on the

and expressed asmle

p/m,g

min.

Results. Tine ATPase of both intact

mitochondria and fragments was activated

by raising the pH; this activation In Fig. 1 the buffer

took place in the absence of Mg2+ ions.

above pH 9.5 with tris,

but the activation

1-propanol-Cl and Annonia-Cl buffers. 'Ihe actitition

No measurementcouldbe.

used was tris-Cl.

performed

was similar with 2-amino-2-methyl-

The activity

decreased above pH 9.5.

also occurred in the absence of added buffer when the pH

of the reaction mixture was adjusted to pH 9.0 - 9.5 by the addition of NaOH. Tnis "high pH activated to the ATPase activated by high concentrations

ATPase" was inmany respects quite similar

at pH 7.4 by CNP. They were similarly of sucrose (Fig.21,

512

inhibited

oligomycin (Fig.31 and by aging

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2-

I,

5

Fig.1.

6

7 PH

9

6

Effect of -pII on ATPase activity mitochondrial fraqkents.

A

7

of heart muscle

pMol I’

%5cx--Jo mM SUCROSE

Fig. 2.

Effect

of sucrose on ATPase of heart muscle pH

0

:

pH

7,4,

5

mMMg2+;* -

513

7,4,

0,2 mMDIP; pH 9,5, 5 ti

Ng2+.

Vol. 22, No. 5, 1966

BIOCHEMICAL

A/d01

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P

41

OLIGOMYCIN Arc)/ml.

Fig. 3.

Effect

of oligomycin A on ATPase of heart

LO

at

(Fig.4).

37

inhibit.

pH 7,4, 5 m?lb@+;*-.

The non-ionic

muscle

pH 9,5, 5 mMMg2'

detergent TRITON-X-100 and azide also

This is in sharp contrast with the behavior of the ;4g2+ activated

ATPase, which is inhibited or by aging.

very slightly

if at all by sucrme, TRITOSX-100

These data suggest that the high pH activated

different

fxm the Mg2+ activated

identical

with the enzyme or system which responds to DIP,

The activating

effect

ATPase and may be closely related

to or

of high pH is to a large degree reversible

Part A is a control,

shows the activity

pH 9,5 and the effect

of neutralization

no difference

ATPase is

(Fig.5).

in the presence of 0.2 m?4DIJPat of a sample to pH 7,4

exists between the activities.

at

2,s

min.

-

In part B of this figure

the

lowest line represents the ATPase at pH 7,4, the upper line at pH 9,s.

At

514

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-

60 min

Fig. 4. Effect of aging on high pH activated and DNPactivated ATPase heart muscle mitochondrial fragments preincubated in 0.25 M sucrose pH 7,4. ATPase measured at: xv pH 7,4;I-+I-]pH 9,5, AA pH 7,4 0.2 mMDNP -

6 5 4 3 2 '

t l!!Lk 2'30' 5'

w--' 7’30’

IO'

2'30"5'

7’30”

IO'

Fig. 5. Reversibility of high pH activated ATPase heart muscle mitochondrial fragments. A: pH 9,5, 0,2 mMDNP At the arrow HCl was added to bring the pH to 7,4 B: x-x pH 7,4;1-1 -l-lpH 9,s; At the arrow HCI wa?.added tg bring the pH to 7,4

515

Vol. 22, No. 5, 1966

BIOCHEMICAL

the arrows a titrated

axxnt

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of HCl yas added in a very

reaction mediumat pri 9,5 to bring the pH to 7,4. the rate of ATP-splitting after

neutralization

decreased significantly,

does not precisely

that the reversibility

parallel

of the activation

After

VOGUE to the

small

neutralization

Tne fact that the rate the control rate,

suggests

is about 60 per cent complete.

Ihe apparent l$,, values for ATP differed

at 7,4 (in the presence of IMP)

and at 9,5: at pH 7,4 the apparent Kmwas of the order of 10-4 3 and at pH 9,s it was 6,4. 10e3 M. This sugests that the lmorehighly ionized ATP molecule may be less tightly

bound to the enzyme at the hipjer

ph.

Discussion. Those enzymes which play a role in ATP synthesis can catalyze hydrolysis under certain artificial

conditions such as in the presence of the usual un-

couplers of ox&dative phosphorylation strated whether the high pH itself There are several different the activation

or by high pH. It remains to be demon-

acts as a reversible theoretical

of ATP hydrolysis

possibilities

at high pH,

uncoupler, which might explain

The degree of ionization

of

substrates and products at pH 7,4 and ~1-19,5 may markedly influence the catalytic

activity

of the enzyme, ATP which is more highly ionized at pH 9,5

may be more susceptible to enzyrnic hydrolysis possibilities

than ATP at pH 7,4.

These

are weakened but not ruled out by the I$, data which indicates

that the less ionized species is the preferred substrate for hydrolysis, Another possibility reversibly

is that the structure

at high pII.

create an active and favoring

Tne ionization

of a dissociable group might

center causing a change in the catalytic

reaction with water,

uncouplers may operate bv a similar et al. 1964).

of the enzyme is altered

An alternative

a possible explanation,

ma&anism

It has been suggested that some group-ionizing

mechanism(Bovell

view, the chemi-osmotic theory,

According to its newest formulation

Moyle, 1965; Jagendorf, pers. ccn-m.1protons are translocated mitochondrially

during electron transport.

516

also gives (Mitchell extra-

The proton gradient frmn

and

Vol. 22, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

outside to inside tnus created, coupled with a reversible, oriented,

spatially-

membranelocated ATPase is the cause of ATP svnthesis.

One

might expect, that diminishing or reversing the proton gradient by increasing the external hydrolysis.

pH might direct

AT&se in the direction

This rmrk is consistent with such a mechanism, but does not

rule out a possibility

of alterations

in enzyme structure,

Xle authors wish to express appreciation A. Jagendorf and B.C. Layne for helpful of the manuscript, gratefully

of

lhe expert technical

to Drs. S.P. Bessman,

discussions during the preparation assistance of I%s. S. Lip&h is

acknowledged.

References Bovell, C.R., Packer, L,, and Schonba~, S.R., Arch. Biochem. Biophys, UN, 458, 1964, Lehni:nSer, A.L.,

The I%tochondrion.

'?J.A. Benjamin, NewYork - Pmsterdan, 1964.

Mitchell,

P., Nature, 191, 144, 1961.

fitchell,

P., and Hoyle,J.,

Xyers, D.K., and Slater,

Nature, 208, 147, 1965.

E.C., Biochem. J. 67, 559, 1357.

Penefsky, H.S., Pullman, M.E., Datta, A., and Racker, E., J. Biol. 3330, 1960. Swanson, iq.A., &xhim.

Biophys. Acta, 20, 85, 1956.

517

Chem,235,