Inhibition of mammary gland acetyl CoA carboxylase by fatty acids

Vol.

13, No.

BIOCHEMICAL

4, 1963

lNHll3ITlON

OF

MAMMARY

GLAND

ACETYL

H. The

Received

Ben

September

It rat

has

mammary

cessation

inhibition

by

addition

concept

(Levy,

hoary

extracts (Levy,

residue was

the

following

weaning

acid of of

quantities

The

and

upon

the

ACIDS

of

rat

this

was

activity

in

Chicago,

acetyl

clear

of

and

inguinal

Sprague-Dawley

rats

rat

entirely

in

the

x g;

solution

CoA

mammary 15-20

nature

of

carboxylase.

glands

days

none

(Levy, the

acetyl

in by

elucidate

of

this

inhibited

105,000

supernatant

inhibition

abdominal

at

for

carboxylase

located

to

The produced

support

markedly

attempts

1963b).

extracts

CoA

milk

rapid

l963a;

mananary

was

lactating

the

provides

the

the

the

toward

milk

enzyme

in

(Levy,

of

mechanism

primiparous

rat

centrifuging or

milk

contribute

in

describes

The

of

Examination

inhibitory

layer

Experimental. from

University

ensues

of

that

communication

inhibitor

excised

synthesis

1963b).

fat

may

acid

small

o bt ained the

accumulation

which

revealed

This

l

BY FATTY

Levy*

synthesis fatty

1963a;

(Rlo5)

19636)

CARBOXYLASE

for Cancer Research, Chicago, Illinois

that

fatty

in

Richard

suggested

1963b).

found

CoA

3, 1963

been

of

the

Laboratory

gland

strong

milk

May

AND BIOPHYSICAL RESEARCH COAVvtUNlCATlONS

were

post-partum.

All

. subsequent were

operations

homogenized

potassium removal extracts minutes. solution

*

present

were in

a Waring

phosphate, of

most

were

the

further

(Spzo)

address:

blendor

pH 7.3, of

Crude

performed

CoA

dissolving

Biological Bacteriology New York.

approximately

for

1 minute

containing and

7.0

debris

clarified

acetyl by

fat

at

by

by

centrifuging

carboxylase

was

the

protein,

rrH

4 with

glands of

0.1

p-mercaptoethanol.

After

at

at prepared

2000

20,000

x

from

the

x g,

g for

precipitated

Research Laboratories, and Botany, Syracuse

267

The

2 volumes

centrifugation

which

.

Department University,

the

30

supernatant upon

adding

of Syracuse,

M

Vol.

13, No.

ammonium 6.5

sulfate

20,000

x g,

20,000

the

7.0

or

x

The

The

enzyme

and

Christian

mg of

ATP;

4 umoles of

of

)

15 minutes

counted

in

centrifuged

plated

on

ethanol-sodium

acetate

OR-17,

Thiolesters

1961).

methanolic

sodium product

over

identical

Phares,

90% to

of that

ml

off

and

visualized

a glacial

1952).

Acids were

malonic

located radioactive of

acid.

of

the The

268

with

super-

dryness,

and

product in

and the

alkaline

specific

was sample; activity

identity hydrolysis system

with

spot

an Circular

for

aid

of

was

Laboratories

visualized

ml

clear

acid-ether-water

applied

0.1

protein

the to

its

0.15 After

nitroprusside

the

of

precipitated

Support

with

was

and

chromatography

1957).

ware

reaction

halted

Pabst

acetic

1.4

controls.

reaction

chromatographing

in

the

aliquot

with

(Stadtman,

pH

10 umoles

2 N KCl;

from

(1963).

incubation

mc/m~l)~

was

paper

al

kept

Warburg

The

(0.075

an

if

and

ml.

from

at

phosphate,

evaporated

(modified

by

of

containing

of

by

--et

noted};

0.6

The

CoA

radioactivity of

0.2

malonyl

A single

the

reaction

counter.

spots

scanner.

the

gas-flow

were

1960)

omitted

planchets,

obtained

radioactive

chromatogram for

was

was

weeks

prior

where of

centrifuging

with

potassium

NaHC1403

steel

hydroxide

(Vagelos, and

37’,

system

of

same

recentrifuging

method

a 15 minute

this

and

Vagelos

the

a solution of

by

of

pH

preparations several

that

umoles

of

CoA

were

as

for

a volume

umoles

followed

thawing

(except

ml

by

enzyme

by to

40

at

identified

and

on

subjected

25

a windowless

tentatively

green

based

Acetyl

acid,

solution

(Dennison

was

0.4

for

natant

product

stable

in

MnCl 2;

perchlorate

the

was

phosphate against

clarified

gave

citrate

adding

M potassium

freezing,

enzyme

potassium

CoA.

by

RESEARCH COMMUNICATIONS

dialyzing

was

determined

was

acetyl

5 N perchloric

of

or

containing

by

incubation

and

g,

B -mercaptoethanol,

initiated

umoles

enzyme

protein,

a solution

then

0.05 and

procedures

procedure

(1941)

of

x two

The

of

in

dialyzed

last

assay

10 umoles

saturation,

105,000

The

(l-7

37 a in

umoles

The

activity.

frozen.

of

AND BIOPHYSKAL

mM p-mercaptoethanol,

at

g-

highest

6.5;

50%

3 hours.

for

at

to

containing

buffer

at

BIOCHEMICAL

4, 1963

of

bromocresol a paper

found

which its of

Rf

accounted was

NaHC1!03

BfOCHEMfCAL

Vol. 13, No. 4, 1963 was

determined

by

containing

extracted

indicated

rat

milk);

from

tardy

planchets in

liquid

identified

acids

acids

of

(Table

the

the

greater

experiment,

of

was

therefore

myristic

in

presence The

acids however,

solution

Table

potent

inhibitors

renders

the

more

inhibition compared

The

effective

in

seen to

with

of

myristic

and

lb,

of

acetyl

subjected acids

of

and carboxylase

fatty

acids, upon

that this

enzyme.

may

palmitic

by

acids

it long-chain

with

these

experiment

which

the

acetyl

maintaining

in

bound

capric,

found

although

were

CoA

acids

1 shows

rat

was

palmitic

used

respect,

of

extract

then

inhibition

this

ml

following

in

procedure

experiment

the

difficulty lack

(0.1

and

fatty

examined.

significance.

proved

free

when

Rlo5

unesterified of

the

milk

(1959)

of

one

extracts

myristic,

certain

that

acetone

of

in

was

evaporation).

rat

al

inhibition

clarified

(acetone

by

of

lauric,

effect

were

20.5

in

esters

the

29.0

removed

et

milk

incorporated

milk);

Stoffel

the

were

(control);

rat

rat

Thus,

02

extract

that

milk.

C

14

acids

acetone of

the

doubtful 1)

fatty

retention

emphasized,

fatty

found

time:

carboxylase

be

of

their

in

and

solvent

procedure

from

particles

lauric,

Rlo5;

suggested

result

ml

methyl

This

might

of

an

temperature,

43.9

made:

chr~atography, by

stearic.

of

0.1

(1956)

the

room

from

activity.

from

When

by

gas

2

account

acids quantities

in

for this

added

were

let-. The

and

steel

counting

fraction

inhibitory

were

(Rlo5

microsomes.

methylated

the

at

quantities

quantity

and

1 iver

of

additions

same

Pressman

smal

on

and

R105

acetone

following

23.9

the

with most

the

the

the

aliquots

drying,

a twice-washed

times

contained

experiment

must

hydroxide,

When

five

extract

CoA

barium

diluted

RESEARCH COMMUNfCATfONS

counter. Results.

to

suitably

excess

gas-flow

to

plating

AND BIOPHYSICAL

Lardy

stimulation

(1956)

C1402

incorporation.

using

King’s

of does

modification

not

latent

ATPase

seem

ATPase

to was

(King,

be

the

cause

assayed 1932)

by of

269

fatty

for

measuring the

method

observed the

by

inhibition phosphate of

Fiske

Pressman of

liberation and

BIOCHEMICAL

Vol. 13, No. 4, 1963 Subbarow but

(1925)

under

substituting effect

either

in

enzyme

preparations

the

acid

upon

absence

used for

or

in

swelling

is

free

acetyl

CoA

carboxylase

(which

had

no

No ATPase

was

detected,

0.5

of

of

RESEARCH COMMUNICATIONS

buffer

presence

mM caprate.

mitochondria,

involved.

Biotin

Inhibition Acid

of

Experiment vmoles C 14

Added

not

the

upon

protect

against

Acetyl

I

CoA

Carboxylase

la*

by

Experiment

O2

wmoles incorporated

Fatty

Acids

1 b*

I4 C

Experiment

O2

2*

wmoles C incorporated

%

14

O2 %

incorporated

%

43.9

100

43.9

100

70.1

100

69.2 69.7

98 99

44.4

63 1.6

None

48.3

110

46.9

107

Caproi

c

45.6

104

46.8

106

Capryl

ic

43.9

100

29.8

68

Butyr

Since

no effect

did

assay,

inhibition.

TABLE

Fatty

the

activity).

the

were

in

phosphate

carboxylase

used

mitochondrial fatty

conditions

tris-acetate

substantial

AND BIOPHYSICAL

ic

Capric

7.57 0.88

17 2.0

0.59

1.10

1.3

0.96

1.4

0.67

1.0

Myristic

17.1 38.6

39 88

31.6

72

Palmitic

41.7

95

40.7

93

46.8

67

Stearic

46.5

106

47.6

109

68.7

98

Laur

*

ic

Concentration of fatty acids, if completely dissolved (see text): experiment la, 0.5 nJt; lb, 5.0 mM; 2, 2.0 mM. Fresh Sp20 was used for experiments la and lb; the same enzyme, kept frozen for 1 week, thawed, and clarified by centrifuging at 20,000 x g was used in In experiments la and lb, fatty acids were added to experiment 2. the incubation tubes as petroleum ether solutions, the solvent evaporated, and the other components then added. In experiment 2, the acids were added as potassium sa?ts in aqueous solution and were completely clear when added. Subsequent addition of the other reactior. components led to some precipitation of the long chain fatty acids.

That

the

inhibition

activation

of

carboxylic

acids

is

suggested

by

acetyl

by

CoA (Martin

the

fatty

acids

carboxylase and

experiments

by

Vagelos,

may

involve

citrate

or

1962;

shown

270

in

Table

Waite 2.

interference other and In

diWakil, experiment

with and

tri-

ig62), 1,

BIOCHEMICAL

Vol. 13, No. 4, 1963

high

concentrations

effect with

of

of

caprate.

citrate

or

inhibition.

malonate

but,

as

able,

gave

partly,

3 show

2 and considerable

these

by

acids

Martin

were

of

Caprate

and

less

Activatinq

Activator

A.

the

preincubation

and

Vagelos

enzyme

caprate

also

provided

(1962)

enzyme

inhibitory of

against

DL-malate

and

Waite

activators.

2

Acids

No

overcome

on

Acetyl

CoA

incorporated, B.

Caprate

Carboxylase

wmo1e.s

0.5

mM Caprate

8x

100

A

umoles/ml

1

None Citrate,

5 ::

i

that

effective

C1402 Experiment * No.

to

and

TABLE Effects

RESEARCH COMMUNICATIONS

protection

d -ketoglutarate, demonstrated

(1962),

Wakil

were

Experiments

Guccinate,

protection and

citrate

AND BIOPHYSICAL

1.26 38.9 46.' 48.7

6.77 13.0 '6.'

'7 28 33

2

None Citrate,10 Malonate,lO

O.S! '2.8 2.72

0.80 4.08 0.56

'57 32 2'

3

None Citrate,10 Malonate,lO

0.85 20.2 4.34

0.56 '4.8 3.17

66

::

In experiment 1 both citrate and caprate were present in the prior incubation. In experiment 2 caprate was present in the prior incubation and citrate or malonate were added with substrates, whereas in experiment 3 the activator was present in the prior incubation and caprate was added with substrates. The same enzyme was used for experiments 2 and 3; a different preparation was employed for experiment I.

These activation The fatty

data of

acetyl

specificity acid

suggest

of

CoA this

synthesis

Acknowledqement. Mr.

Laurence

E.

that

effect by

fatty

carboxylase

milk

of

by and

is

Gas-liquid Frazier

certain

the

its

under

di-

acids

may

and

tricarboxylic

relationship

to

interfere

with acids.

the

inhibition

of

investigation. chromatography

Department

271

of

was Pathology,

kindly University

performed

by of

Vol. 13, No. 4, 1963

BIOCHEMICAL

Excellent

Chicago.

technical

Supported

Harrington. Service

and

the

AND BIOPHYSICAL

assistance

by grants

American

was

from

Cancer

the

RESEARCH COMMUNICATIONS

provided

United

by

Mr.

States

Richard

Public

W-

Health

Society. REFERENCES

Dennison, Fiske,

F. C.

W.,

H.,

Jr.,

and

and

Phares,

Subbarow,

Y.,

King,

E.

J.,

Biochem.

Levy,

H.

R.,

Federation

Proc.

Levy,

H.

R.,

Manuscript

in

Martin,

D.

Pressman,

458 Stadtman, in

B.,

and

B. C., (1956).

J.,

in

Stoffel,

W.,

Vagelos,

P.

R.,

Vagelos, m,

P.

R., Alberts, (1963).

Waite, Warburg,

Chu,

Lardy,

S. Vol.

F., J.

P.

Biol.

Biol.

292

Anal.

Chem.,

Chem.

66,

3, 375

1628

(1932).

363 (1963a)

, 22,

-

(1963b).

P.

R.,

J.

H.

A.,

Biochim.

Ahrens,

Biol.

Chem., et

m,

1787

Biophys.

E.

Chem.,

u,

A.

W.,

and

J.,

J.

Biol.

H.,

Jr.,

Anal.

346

(1960).

Martin,

D.

B.,

(1962).

Acta,

2l,

Chem.,

J.

Biol.

Methods 1957, p. 2,

307

Chem.,

533 M.,

and O.,

Waki and

1,

Christian,

S.

W.

(1952).

(1925).

Colowick and N. 0. Kaplan (Editors), Academic Press Inc., New York,

III, and

J.

F.,

preparation

Vagelos, and

E. R., Enzymology,

26,

E.

Chem.,

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272

Z.,

m 310,

2750

384

(1962). (1941)

*

931. (1959).