The kinetic and structural changes of the mitochondrial F1-ATPase with temperature

Vol.

136.

May

14,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1986

THE KINETIC

AND STRUCTURAL CHANGES OF THE MITOCHONDRIAL WITH TEMPERATURE

A. Baracca*, *Istituto

G. CuratolaO,

G. Parenti

Castelli**

Fl -ATPase

and G. Solaini**

ed Orto

Botanico, Universita' di Bologna, Via 40126 Bologna, Italy di Biochimica, Universitl di Ancona, Via Monte Ancona, Italy di Chimica Biologica, Universita' di Bologna, 48, 40126 Bologna, Italy

'Istituto **Istituto

Received

891-898

Pages

March

17,

Irnerio

42,

D'Ago,

60100

Via

Irnerio

1986

Mitochondrial Fl-ATPase shows a break in the Arrhenius plot with an increase of the activation energy below 17"C, this may imply that the F1 -ATPase undergoes a conformational change at this temperature. Further, a structural change of the Fl-ATPase is indicated by analysis of the intrinsic fluorescence at 307 nm between 33 and 11°C and also by evaluation of the circular dichroism spectra of the enzyme 'at temperatures below and above the temperature corresponding to the discontinuity of the Arrhenius plot. It is therefore suggested that F1 -ATPase exists in two temperature dependent conformational states to which different catalytic properties may be assigned. @ 1986 Academic Press, Inc.

The

terminal

enzyme

of

oxidative

adenosine-5'-triphosphatase of

synthesis complex,

number properties properties Recent dependence biphasic

(ATP-synthase)

ATP

from

F1 -ATPase,

membrane,

exhibits of

studies of

ADP

which

be

have

been

undertaken

enzyme and possibly structure our

of

the activity

with

activation

from

activity.

a reversible

catalyzes

The soluble

removed

the hydrolytic

this

from

can

is

which

and phosphate.

only

and molecular studies

phosphorylation

part

of this

the mitochondrial In the

to

the

last

determine

the correlation

years

a

the kinetic between

these

(l-3).

laboratory

have

of isolated energies

shown that

the temperature

olygomycin-sensitive increasing

below

ATPase

is

20-25

'C.

0006-291X/86 891

All

Copyright 0 1986 rights qf reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol.

136,

No. 3, 1986

BIOCHEMICAL

Conformational changes

changes

(1,Z).

It

conformational state

lipids

disagreement

of

using other

Fl -ATPase

suggested

observations

by

in

Arrhenius

hand other

(8-10)

that

these

the

soluble the

present

enzyme

ultraviolet

MATERIALS

is directly

region.

conformational content,

and

The

change,

above the

both

This

several

suggestion

of

and

in the physical

authors,

plots

kinetic

was not who

Fl

in

did

not

catalyzed

ATP

reported

breaks

temperatures

were

in Arrhenius

and Kerimov

due to conformational

et al.

changes

plot (11) within

of temperature.

paper

Fl -ATPase

authors

breaks

that

(4-7).

at different

the enzyme as a function In

enzyme.

COMMUNICATIONS

to the kinetic

to modifications

the

Fl

RESEARCH

suggested

are associated

solubilized

BIOPHYSICAL

to be associated

tentatively

discontinuities

the

reported

surrounding

with

hydrolysis On

was

changes

of

observe

were

AND

critical

we report analyzed by

a study

through its

show that

of

fluorescence

of

dichroism

pattern

Fl undergoes

accompanied

temperature

the conformation

intrinsic

circular

results probably

the

where

by

decrease

in the

a reversible of a-helix

of 18°C.

AND METHODS

The mitochondrial Fl -ATPase was purified from bovine heart mitochondria isolated according to Smith (12), essentially following the method of Penin et al. (13). Protein was determined by the biuret method (14) in the presence of 1% deoxycholate or according to Lowry et al. (15). ATPase activity was measured at pH 7.5 using an ATP-regenerating system as follow: the reaction mixture (1 ml> contained 25 mM Trislacetate, 25 mM KOH, 0.3 M sucrose, 5mM MgC12, 200 /uM NADH, 1.5 mM phosphoenolpyruvate, 10 units of lactate dehydrogenase and 7 units of pyruvate kinase (Sigma). were finally 4 Pg enzyme and 40 ,nl ATP from a 0.1 M stock solution added. The decrease of optical density at 340 nm was followed on a Zeiss PMQ III spectrophotometer equipped with a Servogor recorderer and a thermostating system. For the determination of tyrosine fluorescence 1.2 nmol of the isolated enzyme were added to 1 ml of 40 mM Tris/S04 , pH 8 and the

892

Vol.

136,

No.

BIOCHEMICAL

3, 1986

fluorescence spectra excitation at 276 with a thermostating

were nm in system.

The circular dichroism J-500A spectropolarimeter cuvettes of 0.1 cm path at each temperture. A temperatures; some of temperature, others from the legend to table I.

AND

recorded a Jasco

BIOPHYSICAL

RESEARCH

between 290 and FP-550 spectrofluorimeter

COMMUNICATIONS

450

nm after equipped

spectra were taken at 9 and 30°C on a Jasco equipped with a thermostating system, using length. Samples were prepared for the scanning number of these samples was analyzed at both them were taken from the lower to the higher the higher to the lower. Details are given in

RESULTS AND DISCUSSION To

determine

the

enzyme

The

specific

were

higher

tested were

was

isolated

to a highly of

than

(8 "C)

the

purified

after

heart

form

protein

bands

SDS-polyacrylamide

gel

-ATPase, to (13).

used in our

at the

characteristic

Fl

according

enzyme preparations

10 /lmol/min/mg

and the five

of beef

lowest

study

temperature

of the Fl-ATPase

electrophoresis

(16)

separation

shown).

An

ATP

-regenerating

interference plot

(by

of

mM)

ATP

in

range 18°C;

however

intersecting

lines,

this

than

and Dorgan

4.2 Kcallmol et

al.

lies

range, above

it;

respectively. (10)

have

between

and quite

temperature below

(4

activation

energies

are with

very

similar

those

of the

range

the lower rises

in agreement

893

of all

of hydrolysis

data

(9).

a

plots

17-21'C;

calculted These

concentration

can be seen intersecting

the Arrhenius we

so no product

1 shows the Arrhenius

substrate lines

the rate the

the assay,

Fig.

saturating

considering

which

in

18).

Two straight

assayed,

temperature

temperature

(17,

at the

8-36*C.

preparations

was used

occurred

hydrolysis

the

about

system

ADP)

Fl -ATPase

of

temperature-dependence

activity

detected

(not

at

the

of limit

faster

with

were

21.9

to those

of Harris

of

et al.

Vol.

136,

BIOCHEMICAL

No. 3, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

t, 0

3.2

3.3

3.4

3.5

3.6

T-K) x1000

Fig.

1: Arrhenlus plot of the rate of ATP hydrolysis ATPase activity was measured according to under the materials and methods section.

Among

the

Arrhenius step

plots

which

change

Little

is

on

easily

(22).

the be

Fig. and

maximum increase the

centered

or

the

most

widely

shape

of 307

present

excitation (not

quantum

are

shown).

yield the

nm,

which

emission

spectra

if

rather 894

tryptophan fluorescence which

of the

It

that,

temperature,

present

in

Fl

also

of the enzyme subunits

the

appears

mitochondrial

residues,

at 276 run, where

are is

used

the

temperature.

of one only

in all

at the lower

spectra

with

of tyrosine

limiting

we analyzed

of

presence

in the

conformational

possibility,

aminoacid

the fluorescence

33 OC after

of

the

hand a number

detected,

breaks

in the rate

fluorescence

lack

absorption

this

intrinsic

other

a change

the

fluorescence

the

2 shows

at

explore intrinsic

about

to

is

of

by F1-ATPase. The details reported

by a temperature-dependent

To

enzyme

(20-22),

studies;

in

enzyme.

(19)

reactions

be induced

the

due

interpretations enzymic

known

-ATPase,

11

the of

residue

for

could

of

variation

can

possible

the

with

similar

enzyme at

enzyme shows its we except

a large

no major

changes

an emission to the

maximum

fluorescence

Vol.

136,

No. 3, 1986

Fig.

maximum a

high

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

2: Fluorescence spectra at 276 nm excitation wavelength F1-ATPase in 40 mM Tris/S04, pH 8 at 11 (solid line) (dashed line).

of

free

tyrosine

percentage

in water

of the

tyrosyl

centered

of 1.2 PM and 33°C

at 303 nm (23),

residues

COMMUNICATIONS

indicating

in the hydrophilic

domain

of

the enzyme complex.

as

By

plotting

the

a

function

of

linearly

from

9

fluorescence

intensity

temperature to

19"C,

but

(fig.3),

of the endogenous it

no further

is

found

that

decrease

occurs

30

T ‘C

it

tyrosine decreases up to 24'C;

I 0

10

20

Fig. 3: Temperature-dependence of the intrinsic tyrosine fluorescence of intensity at the emission purified F1-ATPase. The fluorescence maximum of 307 nm is reported in arbitrary units. 895

Vol.

136,

No. 3, 1986

above

the latter

a

smaller

temperature

slope

behaviour

of

temperatures plots

than

the

in the

tyrosine

included

kinetic

To

the

decreases

temperature

range

of of

to

which

the

tyrosine

we

compared

again

range.

This

but

with

anomalous

the Fl-ATPase

the breaks

suggesting

reaction,

COMMUNICATIONS

occurs

at

in the Arrhenius

a correlation

between

the reaction fluorescence

and its can

be

(21).

confirm

this

of

Fl -ATPase

spectra difference

between

225 run, where

a-helix

lower

RESEARCH

of the enzyme catalyzing

properties

associated

BIOPHYSICAL

fluorescence

in

properties

structural

AND

the fluorescence

of the ATP-hydrolytic

the

and

BIOCHEMICAL

and

percentages

the of

ellipticities

at

possibility

ran at 9 and 30°C (fig.4).

the

two

curves,

b-forms

structure

a-helix

of proteins

0

200

in

220

is

an obvious between

associated are present

calculated

reported

dichroism

evident

ellipticities

content, Inn,

There

particularly

the maximum negative

208

the circular

from

table

I,

240

260

by

the

205

to the (24).

The

negative

the method

of

h(nm)

Fig.

4:

Typical circular dichroism (---) and 30°C ( -----). preincubated for 10 min at Tris/S04, pH 8. The spectra at a rate of 20 nmlmin.

spectra of purified F1-ATPase at 9°C The enzyme (0.175 mg/ml) was the preset temperatures in 40 mM were recorded between 195 and 260 nm

896

Vol.

136,

No. 3. 1986

BIOCHEMICAL

TABLE I:

AND

ELLIPTICITY

208 nm

T("3

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

VALUES OF Fl-ATPase

221 nm

224 nm

crOSSO"er

[O] x 10m3 deg . cm2 . dmol-'

(A. nm)

9

14.9 -+ 0.5*

12.3 -+ 0.3"

10.6 -+ 0.3+

198.6

30

13.6 -+ 0.5*

11.2 -+ 0.7*

9.9 +- 0.62

198.2

ellipticities The molar were obtained from the mean of two spectra recorded on one sample and at each temperature six samples were examined. The experiment was repeated at least three times with different enzyme preparations. Values are means +- SD *P< O.Ol;fP< 0.05. The ellipticities were calculated in degrees . centimeter squared per decimal on the basis of a mean molecular weight per aminoacid of 115.

Greenfield 30 OC

and respectively,

temperature The undergoes at

(251,

results

are

of

this

investigation

temperatures.

The change

temperature

kinetics

range.

changes

appear

membrane

(3),

membrane

sector

consequent

Since

is

changes

17-21"C,

-+ 1.7 at 9°C and

o-helix

suggest

hydrolytic

in

the F,Fl-ATPase by changes that

activity

in lipid

Fl-ATPase of o-helix

the observed in

in situ

lipid-dependent

may be transmitted

that

for

the

plot.

decrease

may be responsible

ATP

above

the Arrhenius

with

of

suggested

of ATPase

in

strongly

at

to be modulated it

of

to the break-point

change

the

-+ 1.8 and 33.2 decrease

a

a conformational

in

37.7

indicating

corresponding

higher

changes

with

Fasman

the same

the kinetic

composition changes

to the catalytic

of the in

the

portion

in activity.

ACKNOWLEDGEMENTS We thank Prof. G. Lenaz for stimulating discussion. Professors B. Samori' and P. Biscarini for helpful advises during the use of the spectropolarimeter are acknowledged. This work was supported by M.P.I. and C.N.R.. 897

Vol.

136,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. Curatola G., Fiorini R.M., Solaini G., Baracca A., Parenti Castelli G. and Lenaz G., (1983) FEBS Lett. 155 , 131-134. 2. Parenti Castelli G., Baracca A., Fato R. and Rabbi A. (1983) Biochem. Biophys. Res. Commun. 111 , 336-372. Castelli G. and Lenaz G. (1984) J. 3. Solaini G., Baracca A., Parenti 16 , 391-406. Bioenerg. Biomembr. 4. Clark D.D. and Schuster S.M. (1980), J. Bioenerg. Biomembr. 12 , 369-378. 5. Melnick R.L., Hanson R.M. and Morris H.P. (1977) Cancer. Res. 21 , 4395-4399. Biophys. Acta 649 , 550-556. 6. Ahlers J. (1981) Biochim. 127-128. 7. Solaini G. and Bertoli E. (1981) FEBS Lett. 132, 8. Gomez-Puyou, M.T., Gomez Puyou A. and Gerban J. (1978) Arch. Biochem. 187 , 72-77. Biophys. 9. Harris D.A., Dall-Larsen T. and Klungsoyr L. (1981) Biochim. Biophys. Acta 635 , 412-428. Urbauer J.L. and Schuster S.M.(1984) J. Biol. Chem. 259 , 10. Dorgan L.J., 2816-2821. 11. Kerimov T.M., Mil'grom Y.M., Kozlov I.A. and Ruuge E.K. (1978) Biokhimiya 43 , 1525-4399. 12. Smith A.L. in: Estabrook R.W., Pullman M.E. eds "Methods in Enzymology" Academic Press, New York, (1967) g , 81-86. 13. Penin F., Godinot C., Gautheron D.C. (1979) Biochim. Biophys. Acta 548 , 63-71. C.J. and David M.M.(1949) J. Biol. Chem. 177 , 14. Gornall A.G., Bardawill 751-758. 15. Lowry O.H., Rosenbrough N.J., Farr A.L. and Randall R.J. (1951) J. Biol. Chem. 193, 265-275. 16. Knowles A.F. and Penefsky H.S. (1972) J. Biol. Chem. 247 , 6617-6623. 17. Harris D.A., Gomez-Fernandes J.C., Klungsoyr L. and Radda G.K. (1978) Biochim. Biophys. Acta. 504 , 364-383. 18. Grubmeyer C. and Penefsky H.S. (1981) J. Biol. Chem. 256, 3728-3734. 19. Dixon M. and Webb E.C. (1964) Enzymes, Longmans, London. 20. Knowles A.F. and Penefsky H.S. (1972) J. Biol. Chem. 247, 6624-6630. 21. Fernandez-Belda F., Ternel J.A. and Gomez-Fernandez J.C. (1985) Int. J. Biochem. 11 , 223-228. 22. Walker J.E., Fearnley I.M., Gay N.J., Gibson B.W., Northrop F.D., Powell S.J., Runswick M.J., Saraste M. and Tybulewicz V.L.J. (1985) J. Mol. Biol. 677-701. 184, 23. Teale F.W.J. and Weber G. (1957) Biochem. J. 65 , 476-482. 24. Adler A.S., Greenfield N.S. and Fasman G.D. in: Hirs C.H.W. and Timasheff S.N. eds "Methods in Enzymology" Academic Press, New York and London (1973) 2 , 675-735. 25. Greenfield N. and Fasman G.D. (1969) Biochemistry S , 4108-4116.

898