Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

Vol.

73,

No.

3,

BIOCHEMICAL

1976

DEPENDENCE

AND

OF ESCHERI

BIOPHYSICAL

CHIA

COLI

TRANSHYDROGENASE AND

Raymond

ITS

L.

Department

of Cell 20 Staniford Department of

Received SUMMARY

September :

PYRI DINE

NUCLEOTIDE

TO N-ETHYLMALEIMIDE

Robert

J.

Fisher

and

D.

Boston Biomedical Boston, Massachusetts Chemistry, Harvard

Physiology, Street, Biological

COMMUNICATIONS

ON PHOSPHOLIPIDS

SENSITIVITY

Houghton,

RESEARCH

Rao

Sanadi

Research 02114 Medical

Institute and School

22,1976

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of Escherichia coli by means of deoxycholate extraction and DEAE-cellulose chromatogr* in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phospha tidy1 ethanolamine, the major phospholipid in the outer membrane of E. coli,was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibi ted by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition.

INTRODUCTION:

dria

The

pyridine

and

Escherichia

several has,

however,

the

partial

and

its of

and

the

AND coli

by

strain

Attempts

mitochondrial

enzyme

high

sensitivity (2). k.

phospholipids.

NAD(P)+ by

coli

the

heart

have

been

enzyme

NAD(P)+

to

mi tochonmade

by

Purification to

communication

We also

transhydrogenase various

this

beef

(l-4).

of In

a lipid-depleted

inhibition

METHODS

unpurified.

ipases

purified

membrane-bound against

the

phosphol

transhydrogenasesfrom

yet the

by

of

reactivation

MATERIALS

Copyright All rights

hampered

salts

as

isolate

purification

protection

g.

are

to

been

, bi le

NAD(P)’

coli

laboratories

sol vents

vi ty

nucleotide

organic we

report

transhydrogenase

report

the

sensiti-

N-ethylmaleimide

and

nucleotides.

: ~6

(a

proline

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

auxotroph

751

derived

from

ATCC

96377)

was

grown

Vol.

73, No.

on

a minimal

piratory

3, 1976

BIOCHEMICAL

salts

medium

particles

previously

were

reported

buffer

pH

medium.

7.8

(5)

acetyl

respiratory

were

first combined

The

1 ipid

under

NAD+

was

N.Y.

in

TDE

centrifugation RESULTS

AND

Partial

Purification

from

membrane

deoxycholate

pH

the

at

80,OOOxg

Triton

followed

8.0

with

a

with

100

in

added the

linear

obtained

described

by

for

and

reduction

to

dryness

beef

100%

heart

by

acetone.

under

vacuum.

@:I)

and

stored

used

car-

without

fur-

Concentrates,

(7).

al.

Wood-

Water-dispersed

phospholipids

Suspensions

nitrogen.

Particles

phosphatidyl

and

Associated

sonicating

obtained

mitochondrial

Pa.

et

of

(6).

Hager

with

as

isolation

were

then

Belfonte,

Kagawa,

the

glycerol,

and

from

obtained

5

for

were

clarified

by

30 minutes.

to

DE52

100

to

a final

cold,

the

a final

(TDE-T

salt

of

the

was

was

same

reservoi

of column

The

100

spun

at

0.5%

and

mM

in

chamber 0.5

752

with

the

1M KC1 After

for

against

in with

min-

TDE.

Triton

was

applied

TDE containing 100

Further

ml4 NaCl.

and

stirring

thirty

solution

contained 500

to

(w/v).

180,DOOxg

washed NaCl.

made

0.5%

overnight

was

mixing

r TDE-T

TDE was

pre-equilibrated

column

made The

in

of

dialyzed

concentration

gradient. ,

mg/ml)

concentration

mixture

0.5). the

6-8

ml,

DEAE-cellulose

ml

mM NaCl

(50

supernatant

X-100 by

was

as

the

and

phosphatidyl

Inc.,

used by

cell

mM Tris-sulfate

chloroform-methanol

&

Supelco

of

was

Cunningham

in

50

phospholipids

evaporated

lecithin

preparation

added

a Whatman

and

plant

presence

resulting

was

by

L.

were

in

minutes The

0.5%

as

viz.,

coli

Res-

DISCUSSION:

The

X-100

redissolved

preparations

minutes

utes.

then

purified

r.

source.

pressure

a French

(TDE)

COMMUNICATIONS

carbon

measured

@:I)

I tered

sole

in

was

acetone:water fi

the

1 mM EDTA

Crude

were

RESEARCH

modification,

described

Asolectin and

as

breakage

and

Purified

purification.

glucose

one

(5).

as

purchased

BIOPHYSICAL

activity

cardiolipin, were

IO

for

NADPH

at-20°C.

phospholipid

to

by

extracts extract

diolipin

for

except

with

ethanolamine,

side,

after

particles

nitrogen

ther

prepared

1 mM DTT

extracted

The

30mM

transhydrogenase

pyridine

from

using

containing

NAD(P)+

AND

ml

elution 250

ml Figure

of

TDE-T was

TDE-T

0.5 achieved

0.5

1 shows

a

BIOCHEMICAL

Vol. 73, No. 3, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2 r

EFFLUENT Figure

1 -

(mls)

Elution profile of NAD(P)+ transhydrogenase activity lose in the presence of Triton X-100. Fractions lected and (50X) samples assayed for enzyme activity in the Methods section.

TABLE --

Typical

Purification

Fraction Membranes Deoxycholate

Extract

DE-52 Fraction Cholate -

f

Assayed

typical

in

elution

0.25-0.30 added stirred was

after (NH4)2

the

to

resuspended

4°C

of

Bf

Active

and in

Specific Activity pmoles/min x m9

-

Total Phosphorus nmol es/mg Protein

% Yield

358

483

1.35

184

374

2.03

77

-

125

13 -6”’

26

o-3

9.2

profile

1% followed at

Totai Units pnoles/ min

Transhydrogenase

100

2.4

SO4

presence

M salt.

1

of

Total Protein mg

from DEAE-celluml ) were colas described

(10

by

5.

protein

and

fractions solid

and

at dialyzed

ipids.

enzyme

were

ammonium

centrifuged TDE

co1 i phosphol

activity,

combined, sulfate

30,000x9 1 to

753

and (250

for 2 hours

which

potassium

mg/ml).

15 minutes, against

was

the

eluted

in

cholate

pH 8.0

The

mixture

was

The

precipitate

same

buffer.

Vol. 73, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

E. coli

ETHANOLAMINE I 0

i IO

I 20

nmoles

Figure

This

subsequent

the

This

by

Reactivation

giving

by

The

activity

2).

A

crude

was

had is

I i ttle reported

of

the

k.

coli

a

low

1 ipid-depleted lipid

outline

activity

of

effect, be

effective

stimulated

which other

constitutes i.e.,

could

13.6 pmoles/min

cardiolipin any

fraction

fraction

of

than which

to

has

was is

used

shown

0.44-0.93

for in

xw

umoles/min

phospholipids.

-coli

concentration

ethanolamine,

A purification

which

activity

E.

transhydrogenase,

in

Phospholipids

a maximal

pholipid lower

I 50

Pi

NAD(P)+

experiments.

preparation

stimulated

(Fig.

Phospholipid

lipid-depleted

reactivation 1.

was

I 40

2 - Reactivation of NAD(P)+ transhydrogenase by various phospho? ipids. Protein (10 pg) was preincubated in 500 pmoles phosphate buffer pH 6.5 in the presence of 500 nmoles NADPH and 6 pmoles KCN and varying amounts of phosphol ipids. After 5 min prelncubation, the assay medium was made to 3 ml with water and 500 nmoles acetyl pyridine NAD+ added to initiate the reaction. The figures given brackets are the maximal activity in Umoles/min x mg. The basal activity was 0.72 umoles/min x mg.

preparation,

Table

I 30

phosphol 80% of

only

a 2in

to

the 3-fold’

reconstituting

5

The

inner

stimulation. lipid-depleted

754

most

ten-fold

phosphol

Is-fold,

effective

Surprisingly coli

by

almost

activity

ipid.

restored

activity

x mg.

stimulated

be

phosat

a much

phosphatidylmembrane

phospholipids,

Phosphatidylethanolamine DCCD-sensitive

ipids

BIOCHEMICAL

Vol. 73, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

No NEM xr

NEM t NADP

I

NEM t NADPii

I 80

I 0

I 160

0

I

240

320

MINUTES

Fi - gure

3 -

ATPase of

-E.

Time dependent inactivation NAD(P)+ transhydrogenase by N-ethylmaleimide E. coli membranes (0.8 mg/ml) in 0.2 M potassium phosphate buffer FH 7.0 were incubated at 25’C with excess NEM (10 mM) in the presence of various nucleotides (10 mg/ml). Al iquots (25 A) were removed at The first the designated times and assayed for enzyme activity. order rate constant k for the initial declines in activity were 26, m-m 46, and o-o 63 min-1 (x 104). x-x 0, A-A9, +-+ Control activity was 1.63 umo\es/min x mg.

in

F.

col

s,was

trations

than

comparable

pared

heart to

inhibition the

beef with

as

-coli may

heart

beef

heart

mitochondria

Beef

heart

cardiolipin

ipin.

also

restoring

activity

Asolectin

was

stimulated

cardiolipin, be

absent

from but

also

the at

inner

membrane

higher

effective

concenin

the

same

lecithin.

to

cardiolipin. fatty

acids

1 ipid-depleted

or

the

The

(9)

the

but

related

cyclopropane

An analagous

in

cardiol

cardiolipin g.

a phospholipid

effective

bacterial range

Beef

in

Lecithin,

partially

concentration

This

(8).

at

higher

different latter in

the

found

side

chains

combinations

of

755

this

of

g.

transhydrogenase

activated lipid

by with

low

was

acid

unsaturated

be

at

concentrations

has

of

to

activity

fatty

preparation was

enzyme

inhibitory.

side

chains

fatty

acids

&

levels,

cardiol isolated

phosphatidylcholine. phosphatidylethano-

present as

comipin. from

Vol. 73, No. 3,1976

lamine

or

phosphatidylcholine

stimulated of

the

cardiol

with

--coli

glycerol

or

During

the

initial

into

the

a sensitive on to

obtained

if

the

two

not

exceed

of

NAD+

-SH

in

NADPH

into

the

ated

the

rate

of

more

fully

glyoxal found

that

NAD+

of

observed the

The

stimulation

not

the

reaction.

that

with

phosphotidyl

did

the

ized”.

activity,

of

observed and

found

each

half-maximal

Vm for

“solubil

the

enzyme

affect

enzyme,

aithough

Incorporation

implicating

showed

(Fig.

present gave

an

instead

of

the

of

both

of

protecting

enzyme

presence

to

NEM.

protection,

NAD+

and

that

the

inhibition

the

the

presence

NADP+

sites.

against

was

form

of

inhibition of

did functionally

lncorporat

inhibition

reduced

indeed

A combination

and

suggest

the

was

against

exposure

results

indicating

the

Protection

increased The

vicinity

that

3).

during

5 hours.

the

the

was

obtained

enzyme,

arginyl

residues

enzyme the

at

the

nucleotide

transhydrogenase

has

Similar

butanedione.

in or

was

gave

the

was

that

by of

NEM,

the

acceler-

enzyme

with

the

was

more

presence

.E,. &

results

of

(data to

NADPH,

but

al ready

with

enzyme

susceptible

binding been

not

and It

shown). by in

an

of

the

imp1 icated

butanedione

modification

protected

sites

(10)

phenylwas

again

butanedione

additive

or manner

NADP+. data

ion

-SH.

with

phenylglyoxal by

the

mi tochondrial

were

when

ipid,

cardiolipin

only

to

glycerol,

cardiolipin

it

stabilize

inactivation,

presence

studies

labile

(NEM)

system,

exposed

membrane-bound from

in

but

procedures

more to

after

its and

NAO’

isolation

nucleotides even

to

phospholipids

pyridine

NADP+

that

similar

phospholipid

phosphatidyl

-co1 i phosphol

due

also

range

.-E. -coli

and

, suggesting

membrane-bound

or

groups

-*E

coli

was

with

mainly

N-ethylmaleimide

40%

The

--E.

served

the

oxidized

active

is

sulfhydryl

Studies

a concentration

cardiolipln

nmole

acetyl

medium

Phosphatidylglycero]

in

crude

I

membrane,

the

sensitive

coli

phospholipid

NADPH

inhibitory.

activity

the

roughly

Km for

in

I.

The

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

stimulation

of

content.

stable DTT

enzyme

515% wItB

- E.

the

a

Half-maximal

comprise

crude

were

10 nmoles,

stimulation

of

r.

ipin.

roughly

which

BIOCHEMICAL

presented

indicate

that

the

756

E.

col _-

i

transhydrogenase

is

phospho-

has

Vol.

73, No.

lipid-dependent

and

glycerol

and

enzyme

which

in

both

the

r.

the

is

best

This

was

by

inhibited

the

coli

inner

Studies

with the

specific

NEM

g.

vicinity

coli of

BIOPHYSICAL

by

is

in

low

contrast

mitochondrial

lipid

membrane)

AND

reactivated

cardiolipin.

cases

residues, in

BIOCHEMICAL

3,1976

suggest enzyme

may

the

nucleotide

was

supported

in to

that

levels

of

to

beef

the

E.

cardiolipin

present

appeared

RESEARCH

in

the

parent

produce

to

phosphatidyl-

heart

mitochondrial

high

highest the

However,

levels. (mitochondrial

or

activity.

presence

functionally

binding

CC

membrane

the

addition

contain

at

COMMUNICATIONS

of

active

arginyl

sulfhydryl

residues

sites.

ACKNOWLEDGEMENTS

This Science

3. 4. 5.

by

Grant

No.

PCM75-03532AOl

from

the

National

Foundation.

REFERENCES

I. 2.

research

:

Kaufman, B. and Kaplan, Kaplan, N.O., Colowick, l-15. Rydstrom, J.D., Hoek, Commun. 60, 448-455. Rydstrom, J.O., Kanner, Commun. 67, 831-839. Houghton, R. L., Fisher,

N.O. S.P. J.B. N. R.J.

(1961)

and

J. Neufeld,

Biol. E.F.

Chem. 236, (195rJ.

2133-2139. Biol. Chem.

205,

and

Hundal,

T.

(1974)

Biochem.

Biophys.

Res.

and

Racker,

E.

(1975)

Biochem.

Biophys.

Res.

and

D.R.

Sanadi

(1975)

Biochim.

Biophys.

396, 17-23. 6. Ii: 9. IO.

Cunningham, C.C. and Hager, L.P. Kagawa, Y. and Racker, E. (1971) Peter, H.W. and Ahlers, J. (1975) Rydstrom, J., Hoek, J-B., Ericson, Biophys. Acta 430, 419-425. Djavadi-Ohaniance, L. and Hatefi,

(1971) J. Biol. Arch. B.G. Y.

757

J.

Biol. Chem. 246, 1575-1582. Chem. 246, 5477-5487. Biochem.-i%ophys. 170, 169-178. and Hundal, T. (19m Biochim.

(1975)

J.

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250,

9397-9403.

Acta