Energy coupling in lysolecithin-treated submitochondrial particles

Vol.69,No.3,

1976

BIOCHEMICAL

ENERGY

AND BIOPHYSICAL

COUPLING

IN

LYSOLECITHIN-TREATED

SUBMITOCHONDRIAL H.

D.

Komai,

R.

J.

Hunter,

H.

Institute

for

February

R.

Enzyme

University

Received

PARTICLES

Southard,

of

Madison,

RESEARCH COMMUNICATIONS

A.

Haworth

and

0.

E.

Green

Research

Wisconsin

Wisconsin

53706

5,1976

SUMMARY Lysolecithin, at a level of 1.2 mg per mg protein, completely suppressed active transport of K and energized H uptake in ETPH, as well as eliminating vesicular profiles as determined by electron microscopic examination of positively stained ultrathin sections. Nonetheless, lysolecithin-treated ETP showed essentially complete uncoupler-stimulated retention of two coupled functions, n ! mely, ATPase activity and uncoupler-stimulated NADH oxidase activity and exchange activity and phosphorylation partial retention of ATP-Pi indicate that the capacity coupled to NADH+oxidation. Thes$ results to generate a H gradient or a K gradient is not a prerequisite for energy coupling and that the basic postulate of the chemiosmotic hypothesis (1) is therefore invalid. INTRODUCTION In that of

communications

lysolecithin the

major

inner

membrane in

retention not

of

in

of

that our

earlier

laboratory,

generated

retained

studies the

for

energy

coupling,

retention

of

capability

was

capacity it coupling

we

have

non-vesicular

coupling

If

which

this

ETP'

coupling.

essential

conditions

from

treatment

limitation

the is

previous

partial

to

generate

is

be essentially

fragments (2,3).

the

should

reported

possible

The

character an

ion to

complete

ABBREVIATIONS m-ClCCP

= carbonylcyanide

m-chlorophenylhydrazone

FCCP

= carbonylcyanide

p-trifluoromethoxyphenylhydrazone

TTFB

= 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole

FOOTNOTE

Copyright All rights

1

Submitochondrial particles prepared by sonication of heavy beef heart mitochondria in a medium 0.25 M in sucrose and 10 mM in Tris-HCl, pH 7.8.

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

695

of gradient find in

Vol.69,

such our

No. 3, 1976

treated

BIOCHEMICAL

particles.

studies

with

coupling

was

The

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

present

communication

lysolecithin-treated

examined

under

generating

a H+ and

a Kt

MATERIALS

AND METHODS

ETPH conditions

gradient

in is

is

(rather which

completely

a report

than the

ETP)

capacity

of in

which

for

eliminated.

Heavy beef heart mitochondria were prepared according to the method of Hatefi and Lester (4), and electron transport particles (ETP ) were prepared essentially according to the method of Hansen and Smith (5). ETP (protein concentration 20-40 mg/ml) in a medium 0.25 M in sucrose, YO mM in Tris-HCl, pH 7.8, 1 mM in ATP, 1 mM in potassium succinate, and 1 mM in MgCl was stored frozen at -20". Lysolecithin treatment was carried ou 2' at 0" for 30 minutes at a protein concentration of lo-15 mg/ml. Lysolecithin dissolved in a medium 0.25 M in sucrose and 10 mM+in Tris-HCl, pH 7.8 was used in For energized H uptake most experiments. measurement, lysolecithin was dissolved in a medium 0.25 M in sucrose and 1 mM in Tris-HCl (pH 7.8) and the pH was adjusted to 7.1 with KOH to minimize the amount of buffer introduced with the sample into the reaction mixture. ATPase activity was measured essentially according to the (6) except that the reaction method described by Tzagoloff et aZ. mixture was 0.25 M in sucrose, 20 mM in Tris-HCl, pH 8.5, 10 mM in ATP, and 1 mM in MgCl and contained 0.2 mg/ml of particles. NADH oxidase activity was 4 etermined at 30' using a Beckman Oxygen Analyzer in a reaction mixture (4 ml) which was 0.25 M in sucrose, 10 mM in Tris-HCl, pH 7.4, 10 mM in KCl, 1 mM in NADH and contained 0.5 mg particles/ml. ATP-P. exchange activity was measured as described earlier (2). Oxidatlve phosphorylation was measured as described previously (3), except that the NADH concentration was 1 mM. Cytochrome oxidase activity was measured polarographically at 30" in a medium 0.25 M in sucrose, 10 mM in Tris-HCl, pH 7.4, 10 mM in KCl, 20 mM in potassium ascorbate, and 10 uM in cytochrome c. NADH dehydrogenase activity was measured essentially according to the method of King and Howard 47) with potassium ferricyanide as the electron acceptor. Energized H uptake was measured essentially according to the method described by Southard et aZ. (8). Electron micrographs of ultrathin sections were obtained according to the method described previously Protein concentration was determined by the Biuret method (9), (2). using bovine serum albumin as the standard. Lysolecithin from egg lecithin (Sigma, Grade I) was routinely used. FCCP was obtained from Pierce, and durohydroquinone from the Nigericin and TTFB were gifts of Dr. Henry A. K & K Laboratories. Lardy and Dr. Brian Beechey respectively. RESULTS Figure

1 shows

ultrathin was

section

generated

protein electron

for

a representative of

by

ETPH

exposing

30 minutes

micrograph

at of

and

electron of

lysoETPH

ETPH

to

0".

The

lysoETPH

1.2

is

696

loss

micrograph

of

LysoETPH

respectively.

mg of of

complete,

lysolecithin vesicular indicating

an

per profiles that

mg in

the

lysoETPH

Vol.69,No.3,1976

FIGURE

1

Electron and (B)

BIOCHEMICAL

micrographs lysoETP,,.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Of ultrathin Magnification:

697

section x 51,000.

of:

(A)

ETP,,

Vol.69,No.3,1976

BIOCHEMICAL

AND BIOPHYSICAL

Lysokithin

FIGURE

is

2

(mg/mg

RESEARCH COMMUNICATIONS

protein)

Effect of lysolecithin treatment on energized H+ uptake of ETP ETP was incubated with the levels of lysolecithin as Y'nd icat k!d for 30 minutes at 0; prior to assay. The reacti on mixture for energized H uptake measurement (10 ml ) was 0.25 M in sucrose; 1 mM in Tris-HCl, pH 7.1, 10 mM in KNO 1 mM in durohydroquinone, and contained 12.5 pg catajise/ml; and 1.8 mg particles/ml. Energization of particles was effected by generation of 0 in the anaerobic reaction mi+ture by the addition of 2.6 pmol$s of H 0 Energized H uptake and the corresponding rate of d$r$hydroquinone oxidation (in the absence of FCCP) were recorded simultaneous?y, and the rate of durohydroquinone oxidation in the presence of FCCP (6 PM) was determined separately. Uncoupler-stimulated durohydroquinone oxidase activity was obtained by subtracting the rate in the absence of FCCP Durohydroquinone from the rate in the presence of FCCP. oxidase activity (in the absence of FCCP) of ETP treated with 1.2 mg of lysolecithin per mg protein was 5 !iJ nmoles per min per mg (24% of the ac$ivity of untreated ETPH). 0 -0 - Energized H uptake A -----A - Uncoupler-stimulated durohydroquinone oxidase activity

indistinguishable

absence

of

from

electron

1ysoETP

(3)

microscopically

in

respect

to

detectable

closed

the

complete vesicular

structure. Energized ETPH,

and

a level that

of the

lysolecithin,

H+ uptake

the 1.2 loss

was

inhibited

H+ uptake

was

mg per

mg protein

of

energized

as

FCCP

still

by

completely

H'

stimulated

698

treatment

suppressed

(Figure uptake

lysolecithin

2).

was

not

by Figure

due

durohydroquinone

to

of

lysolecithin 2 also

at shows

uncoupling oxidase

by

Vol.69,No.3,1976

FIGURE

3

activity A similar

treated

ETPH result

carried

out of The

was

in

with

combination

of

respiratory

control

by

constitutes

evidence 3 shows

The

gradients

uptake

when

by

on the stimulation ionophores.

(1

pg/ml)

energized

per

Ht

of

+ nigericin

lysolecithin

medium

are

of

K+ gradient the

uptake with

the

of K+ was

alternative

gradients, measurements

seems

nigericin

mg protein. measurement

KC1

of

by

abolished

the

unlikely described

of

a H'

treatment

by

(50

was

mM)

in

and

in

view

above. 699

of

presence

the

release

nigericin the

the

and

that

results

of

gradient. stimulation

indicating

during

of

ionophores

Kt

lysolecithin

namely, but

the

eliminates

was generated

lysoETPH,

the

ionophores,

possibility, in

(10);

action

presence

ETPH

in

formation

synergistic

a KC gradient

generated

and

lysolecithin

activity

transport t a H nor

lysoETPH.

the

mg of

valinomycin

for

that

NADH oxidase

neither

treatment ETP

a valinomycin-containing

H+ and

active

1.2

obtained

both

of

of

RESEARCH COh, hUNlCATlONS

KN03.

K+ abolishes

Figure

AND BIOPHYSICAL

Effect of lysolecithin NADH oxidase activity

of

place

BIOCHEMICAL

that

that

energization Ht fails of

and to

of Kt abolish

energized

H'

BIOCHEMICAL

Vol.69,No.3,1976

TABLE I.

Effect

of ionophores

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on ATP-Pi

exchange

and oxidative

phosphorylation

Oxidative

Phosphorylation

Rate of Pi ATP-Pi

exchange

esterification

Particles

Addition

(nmoles/min.mg)

ETPN

None

216

540

1.45

Val

190

325

1.42

Nig

169

547

1.43

0

109

0.18

None

50

48

1.09

Val

4%

42

0.88

Nig

22

50

1.08

Val + Nig

32

40

0.85

Val + Nig lysoETP,,

LysoETP,,

= ETP,, treated

with

(nmoles/min.mg)

1.2 mg of lysolecithin

-p/o

per mg protein

for

30 minutes

at 0". Val

= Valinomycin

Oxidative

and

is

Kt

in

yet

can

ETPH

by

but

ETPH, exchange

in

not and

way ETPH.

suppress

inducing

protein);

of The

ATP-Pi cyclic

with

the

combination

transport

(0.5

pg/mg protein).

NADH (1 mM) as the substrate.

demonstrating

exchange

shows

of

or of

synergistic

phosphorylation

by

generation

a

and

phosphorylation

Table

I shows

inhibition the

of

valinomycin

oxidative

K'.

the

lysoETPH, oxidative

Nig = Nigericin

was measured

another

gradient

nigericin

pg/mg

phosphorylation

There Ht

(2.5

that of

combination

of

ATP-Pi the

ionophores. As can was

essentially

the

same

results required for

be

seen

in

Figure

completely

retained

in

ETPH.

The

with

FCCP.

activity were

obtained

for

maximal

stimulation

and

ETP,,

lysoETPH

4,

for

uncoupler-stimulated in

lysoETPH

uncoupler

used

Since of

respectively,

700

the

ATPase the

ATPase with was

reference TTFB,

but

concentration activity comparison

activity

of was

not should

to similar uncoupler the

same be

Vol.69,No.3,

FIGURE

made of

4

of

the

TTFB

the exact

uncoupler

respectively.

(the

difference

absence

of ETPH

of

since

of

least inasmuch activities activity

in

of

lysoETPH

uncoupler

as

for 20%

were when

of

ETPH

either

varying

and

1ysoETPH.

particle,

regardless

was

the

uncoupler

not

due

activity

of

of

about

any

of

the

inactivation

in

the

was

about

by

is of

supernatant

other

than

as

the

105,000

the found loss

of

the

inacti-

presence

of

activity fully

as

accounts, in

at

lysoETPH,

cytochrome no

of

coupled

complexes

had

lyso-

treatment,

in

25% of that

at

that the

NADH oxidase and

701

and

in

However,

corresponding

dissociation of

and

50% of

three

activity

lysolecithin

lysoETPH

centrifuged

ETPH

presence

measured

the

NADH dehydrogenase

of

factor

ETPH

1ysoETPH 50% of

the

used.

to

of

NADH oxidase

lysoETPH

The

lysoETPH

activity

of

ETPH.

found

concentrations

NADH oxidase

NADH oxidase

untreated part,

of

in

was

also

active

with

activity

activity

was

of

uncoupler-stimulated

uncoupler)

maximum

The

the

the

NADH oxidase

the

ETPH.

on The

an

activity

response

effect

between

in

vation

that

the

regardless

coupling

of ATPase ETP - 1ysdETPH

RESEARCH COMMUNICATIONS

concentration.

uncouplers

ETPH

AND BIOPHYSICAL

-

uncoupler

5 shows

different

an

stimulation 0 -0 A ------A

maximal

Figure

with

BIOCHEMICAL

1976

oxidase

NADH oxidase

x g for

30 minutes.

Vol.69,No.3,1976

FIGURE

BIOCHEMICAL

5

Uncoupler stimulation and lysoETPH O0 A -----A

AND BIOPHYSICAL

of

RESEARCH COMMUNICATIONS

NADH oxidase

activity

of

ETPH

ETP lystiETPH

-

DISCUSSION The that

results

ETPH

minutes

of

treated

at

gradient,

0"

with

in

showed

the

ATPase

activity.

This may

partial

(see

pation

of retention

Table an

and

by by

of

ATP-Pi

I).

It

uncoupler of

coupling

the

ion

exchange

is

be

in

that

lysoETPH.

702

lysoETPH

namely,

energy

that

are

used.

LysoETPH

activity

and

for

take

uncoupler-

NADH oxidase

a happenstance

a precondition

30

a Kt

to

Yet

functions,

gradients

methods

cannot

a H+ and

uncoupler-stimulated possibility

for

Kt transport

of

coupled

indicate

mg protein

structure.

the

the

paper

generating

inability

two

this per

of

bilayer

of

driven

retention

the

in

lysolecithin

incapable

intact

activity

accurately

lation

full

of

precludes

be

mg of

to

retention

stimulated

determined

due

absence

described

was

likely

complete

lysoETPH

1.2

(lysoETPH)

most

place

experiments

coupling too

the

Electron

small

also oxidative that

in to

showed phosphory-

the

partici-

demonstration transfer

be

of (or

ATP

Vol. 69, No. 3, 1976

hydrolysis)

results

uptake

of

Ht

(11).

If

the

aqueous the

on

obtained

in

process

in not

oxidative

the

ETPH 3 but

rather

fact

that

not

much

lower

side

is

not

exposed

H+ is

transfer of

(or

an

low,

the the

as

mobility

P/O

than

of

ratio

that

protons

ETPH

lysoETPH,

would anion

mobility

the

ATP-Pi

be

of

rate-limiting exchange.

It

is

which

is

reduced

in

lyso-

which

is

reduced

in

lysoETPH.

with

is

in

lipophilic the

is

in

such

(measured of

(a

this

and

external

hydrolysis)

that

that

the

and

membrane

hindered

uncoupler

and

efficiency

to

ATP

suggesting

side inner

sterically

phosphorylation

coupling

The

mitochondrial

of

is

intracristal

the

We are

lysoETPH

the

of

presence H+).

H+ on

side

electron

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

release

of

transport within

release

matrix

the

rate

protons

thus

the

or

only can

in

intracristal

phase

maximum

that

BIOCHEMICAL

NADH)

fully

for

lysoETPH

compatible

with

was

this

interpretation. Since

it

generate to

appear

a transmembrane

the

ion

would

loss

of

gradient

fundamental

postulate

Ht

gradient

is

has

now

been

ion

coupling,

cannot

the

that

it be

the of

to

elimination

gradient

as

necessarily

the

in

force

be

invalid.

to

thank

in

the

capacity

lysoETPH

in

chemiosmotic force

of

follows

driving

driving

shown

the

does

that

not

lead

a transmembrane

energy

coupling.

hypothesis

energy

to

(1)

coupling.

The is

This

that

the

postulate

ACKNOWLEDGEMENTS The Walent were

authors for

wish

their

excellent

generously

Robert

A.

of

a Wellcome

in

part

by

is

Program

by

grateful

Research Project

Janette

technical

provided

Haworth

Ms.

Travel

assistance.

Oscar to

Rudkin

Mayer the

Wellcome

Grant. GM-12847

This of

703

and

the

and Meat

Company Trustees investigation NIGMS.

of

Ms.

Jane

by-products Madison. for

the was

award supported

BIOCHEMICAL

Vol.69,No.3,1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Mitchell, Komai, Res. Hunter, Res. Hatefi, Hansen, Tzagoloff, Chem., King, Vol. Press, Southard, Chem., Gornall,T Chem., Montal, 201. Mitchell,

P. (1966) Biol. Re V ., g: 445. H., Hunter, D. R. and Takahashi, Y. (1973) Biochem. Biophys. Commun., 53: 82. D. R., Emai, H. and Haworth, R. A. (1974) Biochem. Biophys. Commun., 56: 647. Y. and Ester, R. L. (1958) Biochim. Biophys. Acta, 27: 83. M. and Smith, A. L. ( 1964) Biochim. Biophys. Acta, 81: 214. A., Byington, K. H . and Mac Lennan, D. H. (1968)J. Biol. 243: 2405. T.E. and Howard, R. L. (1967) in: "Methods in Enzymology," X, R. W. Estabrook and M. E. Pullman, eds., p. 275, Academic New York, London J. H., Penniston, J. T. and Green, D. E. (1973) J. Biol. 248: 3546. G., Bardwell, C. J. and David, M. M. (1949) J. Biol. 177: 751. M., Chance, B. and Lee, C.-P. (1970) J. Membrane Biol., 2: P.

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J.

(1965)

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