The reversible inhibition of succinoxidase by naphthoquinones

BIOCHEMICAL

Vol. 2, No. 1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Jan. 1960

THE REVERSIBLE INHIBITION OF SUCCINOXIDASE BY NAPHTHOGUI~NES David

Hendlin

and Thomas M. Cook

Merck Sharp & Dohme Research Laboratories Division of Merck & Co., Inc., Rahway, New Jersey Received

December

Ball,

1959

14,

and Cooper

Anfinsen,

(1947)

+alkylnaphthoqui

nones,

which

their

effect

on respiration

inhibitory

They pinpointed between recent

discovery

for

1957)

were

communication

inhibitory

effect

succinoxidase

system

of

(Crane,

of beef

antimalarial heart

also

between

transport

to

inhibit

chain

(Potter

as a

Hatefi,

Lester,

possibility

which

transport

2 and 2 in

l952),

that

demonstrate

compounds.

cytochromes

and Reif,

(CoOlo),

naphthoquinones

electron

by Co010 and related

The

system.

Co010 site.

studies

(ETP) can be reversed known

the

the

describes the

010

investigate at

to a site

transport

coenzyme

exert

succinoxidase.

compounds

electron

inhibiting

2-hydroxy-

activity,

by inhibiting

transport

us to

that

antimalarial

of these

the

electron

prompted

The present the

action

of a benzoquinone,

naphthoquinones

that

of

possess

2 and c in

essential

and Widmer,

the

locus

cytochromes

cofactor

the

the

demonstrated

is

not

on

particles Antimycin

the

electron

so antagonized.

METHODS The preparation previously

(Crane,

of these Crane,

particles Widmer,

unextracted

of

ETP from

Glenn,

and Green,

was determined Lester,

bovine

and Hatefi

heart

1956).

manometrically (1959).

enzyme was used and no phospholipid 71

has been

described

Succinoxidase according In the

present

was added

activity to studies to the

A,

Vol. 2, No. 1

reaction

BIOCHEMICAL

vessel.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The tests

were conducted

1 mg of enzyme protein

containing 4

-I

260-

in a Warburg

in a total

A.

volume

vessel

of

1.5 ml.

B

I

‘)VNINHI8ITED ,’ CONTROL 1

220-

Jan. 1960

UNlNHl8lTED CONTROL

l

l80E e

l40-

zs k

IOO-

-I

0 Figure

IO

20

I

0

30

I

.

I

IO

.

,

20

30

MINUTES 1.

Reversal

of

Naphthoquindne

Inhibition

A. Inhibition by 2-hydroxy-3-(3’,7’-dimethyl) octyl-1,4-naphthoquinone. . (1) 1 pg naphthoquinone tipped into vessel. (2) 300 pg of indicated reversing agents tipped into vessel. B. Inhibition by 2-hydroxy-3-cyclohexylpropyl-1,4-naphthoquinone . (3) 10 pg naphthoquinone tipped into ve8sel. (4) 300 Pg CoQlg tipped into vessel. RESULTS The addition to

of

the l?TP system

‘the analogs

tested,

most potent. narrow

strongly

J-substituted inhibited

Fifty

2-hydroxynaphthoquinonss”

succinoxidase

the 3-(3’,7’-dimethyloctyl)

The inhibition

range.

* We wish available

several

to thank Prof. to us.

derivative

was proportional

per cent

inhibition

L. Fieser 72

for

activity.

Ofwas the

to the dose over occurred

making

these

with

0.5 Pg

compounds

a

Vol. 2, No. 1

BIOCHEMICAL

(1.6x10-9

moles)

erable

degree

reaction

and 1.0 pg caused

of protection

was obtained

studies

was reproducible

min Kl,

Complete

was obtained

and several

with

CoQlo analogs

methyl-21

butenyl),

The oxygen

uptake

in antimycin

A considCo010 in the

be reversed

not

Similar

reversal

of the

side

arm.

were obtained

occurred.

subsequent

to the This

6-(3’-

homolog

of CoQlO).

system,

compounds.

stopped

A toxicity

Higher

levels

by tipping could

not

of the naphtho-

CoQlo supplementation.

with

another

analog,

When 10 kg of complete

inhibition

addition

6-heptyl,

some of these

Antimycin

3-cyclohexylpropyl-l,&-naphthoquinone. pound were added

vita-

naphthoquinone

be completely

by further

be obtained

of Co010 hydroquinone

with

analogs.

effect

a-tocopherol,

6-farnesyl,

could

be reversed

results

as with

obtained

by CoQlO or its

could

as well

could

and the monoethoxy

so restored the

the protective

and reversal

(6-phytyl,

results

A from

that

the diacetate

6-methyl,

1A shows the

quinone

protection

of CoQlo analogs

and squalene.

inhibition

dase

inhibition.

by including

demonstrated

and that

a variety

Figure

complete

vessel.

More detailed

with

Jan. 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibition

was completely

of 300 pg Co010 (Fig.

2-hydroxythis

of

com-

succinoxf-

reversed

by the

18).

DISCUSSION Crane,

Widmer,

CoQlO functions area which If

Lester,

between

cytochromes

is sensitive

either

of these

be expected

that

inhibitors their reverse

nones but

not

antimycin

that

they

inhibit

acts

toxicity

indeed

naphthoquinones

(1959)

demonstrated

2 and 2.

This

to the naphthoquinones

Co010 did

of

and Hatefi

the

and antimycin at the

be

inhibitory

A.

is the

same

and antingrcin

at the Co010 site,

could

that

reversed effect

it

would

by CoQlO.

of the naphthoqui-

This

may indicate

that:

(a)

A act

at different

loci,

or (b)

same locus’but 73

that

A.

antimycin

the

A is more

Vol. 2, No. 1

BIOCHEMICAL

tenaciously

bound

The ability

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

the

enzyme.

of several

show no CoQLG activity), also sis

to reverse that

naphthoquinone

is apparent

be relieved

CoQLG analogs a-tocopherol,

naphthoquinones

It

act

quinones

findings

are reminiscent

of

and Wiss

(1958).

It

possible

is

1956;

(Mahler, around

the

blocked

of antimycin for

the

Weber,

of

obtained

If

such

for

ETP. “by-pass”

diacetates

their

electrons

then

the

A. of Co010

lipophilic

the naphthoquinone

site

to account

to antimycin

in

Gloor, the

transferring

must be different

may reside

They may act by displacing

by Weber,

is the case,

and the

squalene

can These

as an electron

1958)

and Wiss,

toxicity

isooctane-extracted

act

the “by-passe

Kl hydroquinones

on the hypothe-

requirement

of

the quinones

Gloor,

of

Kl

substances.

a similar

A and naphthoquinone

The activity vitamin

the results

reaction.

sensitivity

doubt

the naphthoquinone

E reductase

that

and vitamin

casts

or lipophilic

They observed

of cytochrome

squalene

which

those

at the CoQlD locus.

our data

from

(including

toxicity

by either

restoration

Jan. 1960

from

and

properties. the

lipoprotein

complex. It

is

evident

of naphthoquinones ies

should

sequence

that

the

will

require

be directed between

elucidation

at the

of

further

the

exact

site

Such stu-

investigation.

individual

of action

steps

in the

O.,

J. Biol.

Chem.,

D. E.,

Biochim.

et Bio-

reaction

k and 2.

cytochromes

REFERENCES Ball,

Anfinsen, 257 (1947).

E. G.,

9, Crane,

F. L.,

phys. Crane,

Glenn,

Acta,

22,

C. B.,

and Cooper,

J. L., and Green, 475 (1956).

F. L., Hatefi, Y., et Biophys. Acta, a,

Lester, R. L., 220 (1957). 74

and Widmer,

C.,

Biochim.

Vol. 2, No. 1

Crane,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

F. L., Widrner, C., Lester, R. L., et Biophys. Acta, 2, 476 (1959).

Mahler,

H. R.,

Potter,

V. R. and Reif,

Weberll;SBIyloor,

Advances

U.,

in Eneymol, A. E.,

&

J. Biol.

and Wiss,

75

O.,

Helv.

and Hatefl,

Jan. 1960

Y.,

Biochim.

223 (1956). Chem., Chim.

l&, Acta,

287 (1952). &l,

1046