Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli

BIOCHEMICAL

Vol. 58, No. 1, 1974

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OXIDATIVE PHOSPHORYLATION AND PROTON TRANSLOCATION IN MEMBRANE VESICLES PREPARED FROM ESCHERICHIACOLI Elliot Section

Received

March

L. Hertzberg

of Biochemistry, Cornell University,

8,

and Peter Molecular Ithaca,

C. Hinkle

and Cell Biology New York 14850

1974 SUMMARY

Membrane vesicles have been prepared from E. cali which are capable of catalyzing oxidative phosphorylation with several physiological substrates. Phosphorylation efficiencies of 0.62 + .06 moles ATP formed per gram-atom oxygen consumed have been observed with NADH as substrate. The phosphorylation is sensitive to uncouplers, fulfilling an essential requirement for the study of oxidative phosphorylation in cell-free systems. Since the vesicles also cataeither during respiration or ATP hydrolysis, uncoupler sensitive uptake lyze, of protons, they are inverted with respect to whole bacteria and allow the study of additional parameters relating to energy coupling.

The discovery phorylation linked

(1) has resulted functions

to secondary tion

in this

the

systems

ciencies

(3),

because

of the

(9),

uncoupled

in an inverted

membrane

In this

paper

E. coZi

which

variety

of (13),

substrates. the

vesicles

the

from the

In contrast catalyze

of

E. coZi

sensitive to proton

the

uptake 178

but

(2),

(lo),

authors

studying has also

proton not

of a particulate phosphorylation extrusion of protons

mutants

in cell-

or lack

of relatively the

reduc-

phosphorylation obtained

oxidation

energy-

confined

uncoupled

of oxidative

one report

preparation

uncoupler

0 1974 by Academic Press, Inc. in any form reserved.

of reproduction

is

by the

phosof

have been

generally

literature,

preparation

studies

study

transhydrogenase

measurement

There

in oxidative in the

transport

The possibility

we describe

catalyzes

as the

nucleotide

in the (12).

these

low efficiencies

(11).

of phosphorylation sensitivity

direct

pyridine

to uncouplers

uncoupler

such

or substrate

the

deficient

of interest

However,

processes,

most part,

omitted

sensitivity

Copyright All ri&t.r

bacterium.

energy-linked

For

has been

cells

in a resurgence

of NAD+ by succinate

(4-8).

free

of Escherichicz coZi

of mutants

high failed

of effito explore

translocation been pursued. fraction

from

of ADP with

observed in response

with

a

whole to energiza-

BIOCHEMICAL

Vol. 58, No. 1, 1974

tion

by either

respiration

AND BIOPHYSICAL

or ATP, as predicted

RESEARCH COMMUNICATIONS

for

an inverted

preparation.

METHODS E. coZi Cells

were

wick

W1655 was obtained grown

Fermentor,

NH4C1,

Cells the

batch-wise,

either

in a defined

minimal

3 mM MgC12,

to 20 J.lg/ml. were

pH 7.0,

50 mM KPi, by a single

passage

4500+500

pounds

trifugation

at

centrifuge.

39,000

x g for

Vesicles

pended

rotor

by gentle

sucrose,

suspended

are

two weeks

activity

for

at

Oxidative vesicle

1 mg/ml

fatty

free

* Abbreviations used: chlorophenylhydrazone;

Cell

in liquid

suspension Cells

rotor

of a Sorvall

removed

210,000

5 mg protein

10% (v/v)

at by cen-

x g for

two

was resus250 mM

methanol,

was finally

Vesicles

and remain

broken

RC-2B

per ml in

The pellet

nitrogen

were

in

43398-A)

The pellet

and thawing,

in

50 mM KPi,

(Model

at

same buffer.

15 mM.

were

5 mM MgC12,

in the

methionine

cells

ultracentrifuge.

prepared

stable

stabilizes

re-

for

virtually

by at all

two months. was measured mixture

by adding

containing

mM EDTA, 32 mM glucose,

acid

with

pH 7.0.

Pressure

repeated.

9 mM

by centrifugation twice

in a SS-34

sulfonate,

Storage

to a reaction

0.3

of

to approximately

to freezing

phosphorylation

protein

2 mM MgS04,

least

at a concentration

by centrifugation L-2

50 mg/ml

at -70'.

with

and unbroken

centrifugation

stable

supplemented

1 r&l DTT*,

minutes

sedimented

to approximately

procedure

least

ten

of a Beckman

and the

of 30 mM KPi,

and washed

French

Debris

10 mM morpholinopropane

University.

or in a New Bruns-

and harvested

methanol,

homogenization

1 mM DTT, pH 7.5,

this

were

Yale

to a 20% (wet weight/volume)

an Aminco

pressure.

in a 50 Ti

phase

10% (v/v)

total

Center,

consisting

was glycerol,

was resuspended

through

Stock

medium,

and resuspended

5 mM MgC12,

Coli

1 uM Ens04 , pH 7.0,

source

pellet

the

in an incubator-shaker,

to mid-exponential

The cell

5 mM MgC12,

hours

1 nM FeS04,

The carbon grown

cold.

from

10.0

BSA, 20 mM [32P]-KPi,

BSA, bovine serum DTT, dithiothreitol.

179

approximately

250 mM sucrose, units/ml pH 7.4,

albumin;

hexokinase, at a specific

CCCP, carbonyl

1 mg of 5 mM Tris-Cl, 1 mM ATP, activity

cyanide

m-

BIOCHEMICAL

Vol. 58, No. 1, 1974

of

50-100

cpm per

nanomole,

NADH as substrate, units

alcohol

thirty

the

in a 1.2 ml oxygraph

reaction

mixture

dehydrogenase

(Sigma,

substrate

in amounts

seconds,

AND BIOPHYSICAL

reaction

was generally

occurred,

usually

in about

three

mented

added

to

by centrifugation, Respiration

method

of Mitchell

Thayer

and Hinkle

and Moyle (17).

to nitrogen-bubbled equilibration

proton

150 mM KCl,

proton

translocation

pH 6.15

in

order

pulses

was measured to prevent

acid.

net

acid

or base

withdrawn was sedi-

(14).

was measured pH meter

by the

pulse

described

2-5 mg of protein

by

was added

was initiated

of proton

standardized,

in a medium

After

anaerobiosis

of air-saturated

Amounts of

10

tables

Protein

analyzed

a recording

quantities

ethanol.

with

was then

experiments

microliter

by calibration

ml aliquot

5 mM MgC12 and respiration

5 mM MgC12 or oxygen-saturated estimated

using

of the until

translocation

(15,16)

With

with

to proceed

phosphate

In respiration

by injecting

legends

10% trichloroacetic

and ATP driven

25".

supplemented

in the

A 0.5

and esterified

at

and 10 ~1 95% ethanol.

allowed

minutes.

0.5 ml cold

maintained

was previously

indicated

and the

and rapidly

cell

Type A-7011)

was added

RESEARCH COMMUNICATIONS

150 mM KCl,

translocation

anaerobic

of 150 mM KCl, formation

after

from

HCl.

were ATP-driven

5 mM MgC12 at the

hydrolysis

of

ATP (17). Protein were

was measured

obtained

from

by the

biuret

procedure

(18).

CCCP and valinomycin

Sigma.

RESULTS AND DISCUSSION As shown the

in Table

conditions

following

described.

removal

particulate

The bulk

of unbroken

fraction.

and consists

1, approximately

contaminated

by cell

unlikely

to interfere

with

2 shows

cells

packed

fraction

Table

of

the

the

is

decanted This

material. wall

debris

breakage

respiratory

and large

The remainder

of poorly

25% cell

debris

was achieved activity

is

with

recovered

associated the

procedure

and ribosomes.

under

with

supematant

probably However,

the fractions,

yields these

a are

our measurements.

respiratory

rates

and phosphorylation

180

efficiencies

with

BIOCHEMICAL

Vol. 58, No. 1, 1974

TABLE 1.

Cell

Breakage

AND BIOPHYSICAL

and Fractionation

Volume ml

Protein mgfml

Yield total mg

34.6

36.4

1259

29,000 x g Supernatant

19

16.2

308

210,000 x g Supernatant

18.8

11.4

214

9.5

3.2

Fraction Cell

Suspension

210,000 Wash

x g

Particles

1.2

47

39

RESEARCH COMMUNICATIONS

of Respiratory Specific units/mg

Activity protein

Activity Total

Activity units

9.4

11,700

248

Respiration was measured utilizing the NADH regenerating system described in the text in the presence of 167 uM NAD+. The medium was 250 mM sucrose, 5 mM MgC12, 20 mM RPi, pH 7.4. Units are expressed in ng atoms oxygen consumed per minute.

TABLE 2.

Oxidative

Specific unitsjmg

Substrate

IUD+(Alcohol genase

Phosphorylation Activity protein

in Respiratory

Vesicles

Oxygen Uptake ng atoms 0

Pi Esterified nmoles Pi

p,o

dehydro-

limiting) + CCCP

66 68

634 658

463 0

.73 0

NAD+ (Excess alcohol dehydrogenase) + CCCP

172 202

624 658

353 11

-57 .02

+ CCCP

212 230

627 672

239 3

.38 0

+ CCCP

86 66

620 658

278 1

.45 0

+ CCCP

30 30

277 249

152 14

.55 .06

a-glycerolphosphate D-lactate Succinate

Oxidative phosphorylation was measured as described in the text. NAD+ was added to 167 uM. The level of alcohol dehydrogenase required for limitation of the respiratory rate due to the regenerating system was determined empiriAll other substrates were used at 12.5 mM. CCCP was used at 83 uM. cally. Units are expressed in ng atoms oxygen consumed per minute.

181

Vol. 58, No. 1,1974

various

substrates.

for

low

levels

ute

per mg protein).

ciencies

were

Omission

of

indicate

Since

the

two sites there

is

between

Figures

The uptake whole

semi-logarithmic a ratio

of

observed.

limiting

phosphorythe

oxida-

in 94% uncoupling.

P/O. (19)

(data

generally

for

not

shown)

utilizes

only

determining

flavoproteins suggesting

NADH and the

total

electron

and cytochromes

The data

that

point

of

are

there

in Table

are

2 also

entry

and a-glycerolphosphate,

After

protons

per

of NADH (15),

that

correcting of the

the

for

driven

suggest

of reducing

and another

value is

apparently

was 1.38. expected about

versus

Since for half

182

with

of the time

a stoichiometry

of the

sites

respiration

respect

electrode

(20)

of oxygen

two coupling

in

respectively.

inverted

time

gram-atom

translocation

pulses,

are

response

pH change per

proton

oxygen

vesicles

the

observed

translocated

oxygen

respiration and saturating

indicates

The uncorrected

translocated

oxidation

with

plot 1.70

from

sub-

and oxygen.

of protons cell.

between

D-lactate

1 and 2 demonstrate

preparation,

BSA was omitted

technique

strain.

the

linked cases,

are obtained,

in this

site

succinate,

point

flavoprotein In all

nucleotide

values

of coupling

one coupling

from this

high

on

coup-

of Hempfling

quinones,

based

that

significant

out

pyridine

.06,

NADH

is

of the

This

f

the

pre-

in a 15% decrease

cells.

in

effi-

fourteen

and succinate.

by the method

per min-

dehydrogenase,

the

If

0.66

corrected

coupling

limiting

4.2 pM CCCP resulted

of reduced

somewhat

It of

CCCP.

nanomoles

of NADH, higher

alcohol

that

has been

(2-3

was rate

+ .06.

than

mixture,

in whole

levels

phosphate

a mean P/O of

excess

uncoupler

of reduced

considered,

equivalents

tons

2.5

levels

actually

the

carried

a P/O of

flow.

this

With

D-lactate

BSA resulted

initial

that

showed

assay

Experiments

not

Such a system

to the

phosphorylation

case

dehydrogenase

of NADH was higher

was sensitive

tive

in the

alcohol

a-glycerolphosphate,

RESEARCH COMMUNICATIONS

incorporation

a mean P/O of 0.62

efficiency

lation

the

if

preparations.

exhibited

strates,

of inorganic

Interestingly,

obtained

different

ling

The esterification

system.

parations

AND BIOPHYSICAL

of respiration-independent

regenerating four

BIOCHEMICAL

by a

(not

shown),

consumed of

to

was four

linked is not

pro-

to the coupled

Vol. 58, No. 1, 1974

BIOCHEMICAL

AND BIOPHYSICAL

I

0

0

I

RESEARCH COMMUNICATIONS

@6.02,

2

Time 1mid

Time (mini

Figure 1. Respiration-driven proton translocation in respiratory vesicles. A 0.6 ml anaerobic pH cell containing 2.2 mg vesicle protein was used. Valinomycin (0.1 up> was present during the assay. The substrate was 167 uM NAD+ in the presence of the regenerating system described earlier. At the time indicated by the arrow, 10 ul of air-saturated 150 mM KCl, 5 mM MgC12, corresponding to 4.7 ng atoms of oxygen, was added. Where indicated, CCCP was present at 16.7 uM.

Figure 2. Respiration-driven The experiment was performed that 2.65 mg vesicle protein of oxygen-saturated ethanol.

proton translocation in respiratory vesicles. as described in the legend for Figure 1 except was used, and the oxygen pulse consisted of 5 ul

+CCCP

6.13 -

6.151 0

I Time

2

3

(mid

Figure 3. ATP-driven proton translocation in respiratory vesicles. 2.65 mg vesicle protein suspended in 0.6 ml 150 mM KCl, 2 mM MgC12 was placed in the pH cell. Valinomycin (0.1 ug) was also present during the assay. At the time indicated by the arrow 15 nmoles ATP, pH 6.15 were added. Where indicated, CCCP was present at 16.7 uM.

in this

preparation.

with

flavin-linked

one,

consistent Figure

Preliminary substrates with

indicates

the above

3 demonstrates

measurements

of the

proton-to-oxygen

a' proton-to-oxygen

ratio

ratio approaching

interpretation.

ATP-driven,

uncoupler

183

sensitive

uptake

of protons

Vol. 58, No. 1, 1974

in

these

warrant

BIOCHEMICAL

vesicles.

Since

coupling

a detailed

analysis

of

AND BIOPHYSICAL

efficiencies

the

proton

were

RESEARCH COMMUNICATIONS

low,

the

to ATP stoichiometry

data

do not

yet

(17).

ACKNOWLEDGEMENTS The authors for

would

many helpful

HL 11483

from

like

suggestions. the

National

to thank This Institutes

Career

Development

Award

GM-22427

Health

Predoctoral

Traineeship.

Professors work

A. Jane

was supported

of Health. and E.L.H.

Gibson

and Efraim

by research

P.C.H.

of a National

is the

Racker

grant recipient

Institutes

of of

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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