Oxidative phosphorylation and respiratory control in lysolecithin treated electron transport particles

Vol. 56,No.3,

BIOCHEMICAL

1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OXIDATIVE PHOSPHORYLATION AND RESPIRATORY CONTROL IN LYSOLECITHIN TREATED ELECTRON TRANSPORT PARTICLES Douglas

R. Hunter,

Hirochika

Komai

and Robert

A. Haworth

Institute for Enzyme Research University of Wisconsin Madison, Wisconsin 53706

Received

December

17,197s

SUMMARY: Lysolecithin treatment of electron transport particles (ETP) generated sicular fragments of membrane that can catalyze oxidative phosphorylation. Electron m,icrographs of ultrathin sections of lysolecithin treated ETP were devoid of circular patterns characteristic of closed vesicular structures, No synergistic uncoupling of oxidative phosphorylation by valinomycin plus nigeritin in the presence of K+ was observed in such fragments of membrane, which remained sensitive to classical uncouplers and to oligomycin. Preceding total destruction of closed vesicular structure, lysolecithin caused a drastic alteration in the membrane as evidenced by a greatly diminished effect of the ionophores in releasing respiratory control. In a previous non-vesicular

fragments

electron

transport

exchange

reaction

brief

communication

activity

(1).

The small of

ETP retained cation

this

fragments

lysolecithin

however,

low

NADH oxidase

we klish

to

measurable

report

and respiratory

the

control

results

sensitive obtained

we found

activity.

of experiments

ATP-32Pi

not

be measured.

lysolecithin In the

treated present

on oxidative

in uncentrifuged,lysolecithin

a

NADH oxidase

could

that

of

after

ETP had negligible

to NADH oxidation

shown that

treatment

uncoupler

of membrane

treated

coupled

we have

by lysolecithin

can catalyze

to centrifugation, but

laboratory,

generated

(ETP)

and phosphorylation Prior

tion

of membrane

particles

centrifugation

from

communi-

phosphoryla-

treated

ETP.

MATERIALS AND METHODS ETP were at

30°C using

control, 7.4), reaction.

the

prepared

as described

a Beckman reaction

10 mM; KCl,

oxygen

mixture

For

analyzer. (4 ml)

10 mM; particles

Lysolecithin,

earlier

30 mg/ml

contained (0.5

Oxygen

the

determination

sucrose,

mg/mg)

in water,

(1).

uptake

0.25

of respiratory M; Tris-HCl

and NADH, 1 mM to

was added

to the

was measured

start

reaction

(pH the flask

BIOCHEMICAL

‘401.56, No. 3, 1974

Figure

after

1.

0.5

noted, t?ve

minutes.

Then,

after

solutions

phosphorylation

10 mM;'MgC12,

(pH 7.4),

4 mM (ca.

mixture

esterification

aliquots

from

4 x lo5

removal

of

described

earlier

(1).

method

(Sigma) zone

was used. (FCCP) were

used were

inorganic

earlier

from

at intervals

(1).

sections

Lysolecithin

gift

from

comercial

phosphate

Dr.

M; Tris-HCl

additions

32P i

containing

5 mM; hexokinase,

20 units/mg;

as indicated.

was measured

Phos-

by withdrawing the

esterified

Radioactivity

was measured

by the method

of Lowry

were

obtained

prepared

and carbonylcyanide

The oxida-

0.25

and determining

(2).

ultrathin

added.

sucrose,

oxidation

was determined

of

Nigericin

obtained

and other

phosphate

Protein

a generous

glucose,

otherwise

were

contained:

substrate

mixture

micrographs

described

(5 ml)

mg/ml

to

reaction

32P after

Electron

0.4

unless

or ionophores

CPM/pmole);

coupled

the

minute,

3 mM; ADP, 1 mM; potassium

NADH, 2 mM; and particles, phate

one additional

of uncouplers

assay

(pH 7.4),

the

RESEARCH COMMUNICATIONS

Time course of the inactivation of NADH oxidase activity by lysolecithin. The figure is a composite of traces obtained from several experiments in which the time intervals between addition of lysolecithin (1 mg/mg protein) and that of FCCP (3 x 1O-7 M) were varied.

alcoholic

(3).

AND BIOPHYSICAL

et al --*

according

from

as

to

egg lecithin

e-trifluoromethoxyphenylhydra-

Henry

A. Lardy.

All

other

chemicals

sources.

RESULTS AND DISCUSSION The NADH oxidase inhibited the

reaction

activity

by lysolecithin mixture,

(4). the

rate

of

submitochondrial

Ten seconds

after

of NADH oxidation

648

particles addition dropped

is of

to

strongly

lysolecithin

30% of

the

to rate

in

Vol.56,No.3,

1974

BIOCHEMICAL

AND BIOPHYSICAL

0.25

0.5

LYSOLECITHIN

Figure

the

Effect of of ETP to described

2.

absence

Figure

of

1).

an uncoupler,

upon addition of

untreated

of For the

in a medium

measurement 0.25

M in

mg/mg protein)at

vesicular

structure.

was about

0.06

'less

than

phorylation

that

control

0°C for

30 minutes

catalyzed

to

by lysolecithin

ETP

ionophores, Concentrations

as FCCP in control

complete

9% of

upon

the

in Table

with

ETP (10 mg/ml) lysolecithin

destruction

lysolecithin activity

phosphorylation

As can be seen

649

(pH 7.8)

of such

of oxidative

treated

we treated

insure

activity

or approximately

ETP.

ETP to the

respiratory

phosphorylation,

and 10 mM in Tris-HCl

of untreated

50%

treated

as effectively

in releasing

decrease for

by lysolecithin.

sucrose

efficiency

not

2).

The NADH oxidase

the

of

of

was an increased

ETP to FCCP did

sensitivity

(see

presence

ETP and lysolecithin

respiratory

Figure

result

activity was

level

of FCCP required

decreased

ineffective

this in the

and the of

the

of oxidative

vmole/min.mg,

Nevertheless,

slowly,

was greatly

(see

from

of NADH oxidation

untreated

contrast,

became

lysolecithin

slowly

The concentration

released

ETP rapidly

very

The sensitivity

In sharp

that

rate

of both

and nigericin,

of ionophores

ETP.

the

much more

index."

respiration

10m7 M.

valinorqycin

addition

hand,

of lysolecithin.

stimulation

(1.2

other

control

was about

and decreased

FCCP, decreased

"respiratory

0.75

(mg/mgETP)

lysolecithin on the sensitivity of NADH oxidase FCCP and to ionophores. Experimental procedure in Materials and Methods.

lysolecithin,

On the

RESEARCH COMMUNICATIONS

I,

ETP was sensitive

of

closed

treated

ETP

of untreated was only oxidative

slightly phos-

to uncouplers

Vol.56,No.3,

Figure

1974

3.

BIOCHEMICAL

hand,

lysolecithin there

RESEARCH COMMUNICATIONS

Electron micrographs of positively stained, ultrathin sections of (3a) ETP, (3b) lysolecithin treated ETP (1 minute, 30°C at 1 mg lysolecithin/mg protein) and (3~) lysolecithin treated ETP (30 minutes, 0°C at 1.2 mg lysolecithin/mg protein).

and to oligomycin. with

AND BIOPHYSICAL

Note for

that

30 minutes

was no synergistic

the

rate

of NADH oxidation

was still inhibition

enhanced

by uncouplers.

by valinomycin

650

catalyzed

plus

by ETP treated

On the other nigericin in the

Vol. 56, No..~, 1974

TABLE I.

BIOCHEMICAL

E:ffect of Catalyzed

AND BIOPHYSICAL

Uncouplers and Oligomycin by Lysolecithin-Treated

RESEARCH COMMUNICAT!ONS

on Oxidative ETP.

Phosphorylation

Initial rate of NADH oxidation (units/mg)

Addition None

p/o

0.06

0.49

0.17

0.05

0.18

0.07

S13 (4.0 x 1O-7 M)

0.24

0.01

Oligomycin

0.05

0.00

DNP (1.6

x 1O-4 M)

FCCP (2.4

x 1O-7 M)

(0.8

pg/ml)

-Abbreviations:

presence

DNP = 2,4-dinitrophenol; methoxyphenylhydrazone; 4'-nitrosalicylanilide.

of

K' of the

by nigericin

alone

and valinomycin negligible

together.)

of hexokinase

At caused

tion effect of

after

the

ultrathin

showed phase

two phases

major

sections disruption

was considerably

change

of in the

both

inhibition

nigericin

organic

phosphorus

substrate,

indicating

that

32Pi

incorporation

reaction.

The results substrate.

in the

structure

rapid

phase,

lysolecithin absence

in releasing after of

with

of 32 Pi into

NADH as the

The first,

ionophores

(The

was

the

amount

due to adenylate contained

Similar

in Table

results

were

as substrate.

of

addition

NADH oxidation of

with

II).

Table

inhibition

to eliminate exchange

obtained

by lysolecithin.

of

same as the

an oxidizable

durohydroquinone

least

10 seconds

of

and ATP-32Pi

with

ETP (see

Incorporation

absence

II were

obtained

the

used was sufficient

reaction

and Table

treated

was almost

in the

kinase

lysolecithin

FCCP = carbonylcyanide e-trifluoroS1s = 5-chloro-3-tertbutyl-2'-chloro-

1 minute vesicular

slower

to

and functions which

takes

and greatly

treatment

structure

ETP with

(Figures total

651

of

disruption

ETP were

within

about

by inhibidiminished

Electron

respiratory-control.

and involved

place

ETP, was characterized

of an uncoupler

of

of

micrographs

lysolecithin

3a and 3b). of closed

The second vesicular

I

Vol. 56,No.3,

TABLE II.

1974

BIOCHEMICAL

Effect

of

Ionophores

AND BIOPHYSICAL

on Oxidative

RESEARCH COMMUNICATIONS

Phosphorylation.

Particles

Addition

p/o

ETP

None

0.59

Val

0.53

Nig

0.45

Val + Nig

0.13

None

0.49

Val

0.42

Nig

0.30

Val + Nig

0.28

Lysolecithin-treated

Val

ETP

= Valinomycin

structure

(1 ug/ml);

as indicated

lysolecithin,

by the

in the

Two important ments

described

membrane dative

the

is

presence

lipid very

different of

bilayers, likely

effect

through

rather

than

clusion

is

Kt. did

that

presence

paper

by the

phosphorylation

uncoupler

of

not

(I)

mechanism

diminish

interaction by transporting by the

uncouplers with

of

ETP with

and oxidative

synergistic

inhibition

the

of experi-

vesicular (5)

mechanism of

a protein

structure is

uncoupling

sensitivity such

not

component

through

results

of studies

essential

by the

the

lipid

for

ionophores

a reagent of

of the

inner oxi-

by a classical

that

disrupts

ETP to FCCP, it

appears

as FCCP exert

protons

results

of uncoupling

to lysolecithin, the

of

from

hypothesis

from

exposure

patterns

absence

closed

the

Since

no circular

can be drawn

and (II) the

of treatment

Kt.

that are:

pg/ml).

30 minutes

complete

chemiosmotic

classical

supported

showed

(0.2

After

showed

conclusions

in this

demanded

3c.

micrographs

measurements

ionophores

= Nigericin

in Figure

electron

phosphorylation

Nig

their

of the bilayers

on respiratory

in

uncoupling

inner

membrane (7).

control

This

(6) con-

in cyto-

Vol. 56, No. 3, 1974

chrome

oxidase

vesicles

oxidase

and lipid,

control

while

effective

BIOCHEMICAL

(8).

the

In this

ionophores

classical

due to the

system

were

uncouplers

presumably

AND BIOPHYSICAL

that

very

in the absence

RESEARCH COMMUNICATIONS

contained

effective absence

of

the

purified

cytochrome

in releasing of

valinomycin

protein

respiratory were

responsible

much less

for

uncoupler

binding. ACKNOWLEDGEMENTS The authors helpful

wish

discussions

grateful Grant.

to

to express

during

the Wellcome

We a're grateful

Meat by-prodwcts tigation Sciences,

were

course

Trustees

for

to Ms. J. generously

was supported Program

the

Project

in part Grant

their

thanks of this the

Hurley supplied

by the

work.

award for

to Dr.

Robert

E. Green

for

A. Haworth

is

of a Wellcome

her expert

by Oscar

National

David

Mayer

Institute

Research

technical and Co. of General

his

Travel

assistance. This

inves-

Medical

GM-21847. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8.

Biophys. Res. Commun., Y., Biochem. Komai, H., Hunter, D. R. and Takahashi, 53, 1 (1973). Endberg, 0. and Ernster, L. in Glick, D. (ed.) Methods in Biochemical Analysis& Interscience, New York (1956) p. 1. A. L. and Randall, R. J., J. Biol. Lowry, 0. H., Rosenbrough, N. J., Farr, Chem ., x32, 265 (1951). Honjo, I. and Ozawa, K., Biochim. Biophys. Acta, 162, 624 (1968). Coupling and Energy Transduction, Glynn Research Mitchell, P., Chemiosmotic Bodmin, Cornwall (1968). W. G., Davis, K. A. and You, K. S., Ann. N.Y. Acad. Hatefi, Y., Hanstein, 277 (in press). Sci., E. J., Wiechmann, A.M.C.A., and Van Dam, K., Bakker, II. P., Van Den Heuvel, Biochim. Biophys. Acta, 292, 78 (1973). Hunter, D. R. and Capaldi,R. A., manuscript in preparation.

653