Warfarin and vitamin K accelerate protein and glycoprotein synthesis in isolated rat liver mitochondria in vitro

Vol. 41, No. 2, 1970

BIOCHEMICAL

WARFARIN AND VITAMIN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

K ACCELERATE PROTEIN AND GLYCOPROTEIN

SYNTHESIS IN ISOLATED RAT LIVER MITOCHONDRIA --IN VITRO1 Ralph

.I. Bemacki

and H. Bruce

Bosmann'

Department

of Pharmacology and Toxicology University of Rochester School of Medicine and Dentistry Rochester, New York 14620

Received

September

18, 1970

Summary: Protein and glycoprotein synthesis is accelerated by vitamin K1 and warfarin in isolated rat liver mitochondria and slightly accelerated in rat liver microsomes and brain mitochondria At 2 x 10s3 M vitamin KI, --in vitro. protein synthesis increased 5 fold and glycoprotein synthesis 7 fold in isolated rat liver mitochondria. There is no concomitant increase in RNA and DNA synthesis in the isolated rat liver mitochondria with vitamin K1 or warfarin. Cycloheximide has no effect on this increase in mitochondrial protein and glycoprotein synthesis in the rat liver mitochondria but chloramphenicol Conversely, L5178Y mouse leukemia cell completely abolishes the increase. macromolecular synthesis is greatly inhibited by vitamin K1 and warfarin. Earlier

workers

have

somes may be associated factors

--in vitro

synthesis, dence showed vitro

level

a molecular

brain sodium

mitochondria warfarin

Medical

(3,7)

the liver

of vitamin

with

liver

mitochondria

of prothrombin

Studies

(l-3).

of protein and rat

involving have been

K is

vitamin reported

(4) but (5).

of vitamin

and glycoprotein

K.

synthesis

by vitamin

clotting

K in protein

of prothrombin

the addition

microsomes

and other

contradictory

inhibition

or micro-

the

Recent

evi-

reports

biosynthesis The present in

rat

liver

K1 (AquamephytonR)

3 report and and

(CoumadinR>.

1 Supported in part No. l-Pll-GM-15190 No. 5-TOl-GM-00032 2 HBB is a Research General

either

the production

of cycloheximide

and --in vivo

shows an acceleration

that

of ATP generation, action

the reversal (6)

with

and --in vivo

at the

for

thought

Cancer Society, by Grant No. P529 from the American from the National Institutes of Health and Training from the National Institutes of Health. Career Sciences.

Development

Awardee

498

of The National

Institute

Grant Grant of

BIOCHEMICAL

Vol. 41, No. 2, 1970

Mitochondria (8-lo),

lipids

proteins for

are (11,

(14,

capable 12),

the mitochondria's

provide

glycolipids

of other

type

the intact

cell

have

shown

cycloheximide

glycoprotein and that

later

synthesis chloramphenicol but

findings

the present

initiated

and brain

mitochondrial

Materials

and Methods

Schneider were

liver

and Hogeboom

prepared

to the

140,000

described

diphosphate

determinations

and glycoprotein

Results

and Discussion mitochondria

of the rat

synthesis synthesis.

of protein

in These

K1 on rat

liver

and glycoprotein.

of the method

(19).

Rat liver

of Schneider

and Kuff

by the addition

of

microsomes (23).

pH 5

of 1 M acetic

acid

were

prepared

by a modification

of the

(14). 14

l-fucose-3H

Nuclear

Protein

Liver

or

synthesis,

of vitamin

described

precipitated

glucose-

(15 Ci/mMole)

from New England Protein

were

protein

protein

21)

protein

by a modification

of the method

mitochondria

10 Ci/Mole),

thymidine-3H

isolated

as previously

separately

previously

(sip. ac.

synthesis

(20,

x g supernatant.

Intraneural

Uridine

(22)

were

by a modification

enzymes prepared

method

mitochondria

mitochondrial

on the effects

and microsomal

studies

and glycoprotein

on microsomal

or

or enzyme)

Recent

microsomal

protein

the formation

glycoprotein,

liver

liver

synthesized

the mitochondria

for

prothrombin.

inhibit

inhibit

study

that

acids

and glyco-

are presumably

proteins,

e.g.

has no effect

(14-18)

inconceivable

(e.g.

does inhibit

of nucleic

proteins

or glycoprotein

does not

does

14),

is not

of protein

liver.mitochondria

The rat

it

secretes,

but

synthesis

macromolecules

macromolecules

that

that

(13,

these

own use,

some precursor

activation

of the autonomous

Although

19).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C (sp.

(sp.

ac.

ac. 14

and l-leucine-

200 Ci/Mole),

10 Ci/Mole), C (sp.

ac.

glucosamineuridine-3H

14C

(10 Ci/mMole),

250 Ci/Mole)

were

purchased

Corp. were synthesis

showed

made by the procedure was determined

the greatest

499

of Lowry

as previously

acceleration

--et al. described

of protein

(22). (14,

and glyco-

19).

cn 8

:

(1)

6.8

a.5

Kl K1 Kl K K1 K;

42.2 31.2 22.1 16.0 6.9

100 pg/ml cycloheximide 250 wg/gl cyclobaximide 2 x lo- M Warfarin +lOO pg/ml cycloheximide 2 x 10-3 Warfarin +250 vg/ml cycloheximide 2 x 10-3M Vitamin Kl +lOO vg/ml cycloheximide 2 x 10-3M Vitamin Kl +250 vg/ml cycloheximide

100 pg/ml chloramphenicol 260 l.lg!!jl chloramphenicol 2 x 10 M Warfarin +lOO pg/ml chloramphenicol 2 x lo-3M Warfarin +250 ug/ml chloramphenicol 2 x 10m3M Vitamin K1 +lOO pg/ml chloramphenicol 2 x 10-3M Vitamin K1 +250 pg/ml chloramphenicol

10m3M Vitamin 10-3M Vitamin 10% Vitamin 10-5MVitamin 10m6M Vitamin 10-7M Vitamin

40.2 24.4 18.3 17.1 14.2

x x x x x x

+2 +l +2 +2 +2 +2

2.3 2.6 2.3

_-_ _-_ -_-

14.0 40.9 42.3

26.1 25.9 41.0 40.3

glucosamina-14c+,

2.5

---

14.1

8.5

(2)

2.2 1.8

-__ -__

6.8 6.9

a.4

,

5.1

---

19.0

4.9

L-laucine-14CB

5.1

-_-

21.0

6.2

4.7

--_

6.0

2.9

4.8

---

7.0

4.4

4.4 4.5

-__ m-v

5.3 5.0

5.2 4.6 4.4 4.4 4.4 4.4

4.9 4.6 4.4 4.4 4.4 4.4

4.4 0.3 0.2

15.1 12.3 9.2 4.5 2.7 2.7

6.0 5.0 4.0 2.7 2.7 2.7

2.7 0.2 0.1

(3)

Liver Protein (1)

2.4 2.1

14.0 9.6 8.0 6.8 6.8 6.8

25.9 14.1 10.5 8.8 6.4 8.4

6.8 0.2 0.2

(2j

Mitochondria Glvcoprotein

+2 x 10w3M Warfarin 1 x 10m3M Warfarin 2 x 10m4MWarfarin 2 x 10e5M Warfarin 2 x 10m6M Warfarin 2 x 10w7M Warfarin

Liver Protein (1)

(3)

ULIP-glucose-%++

9.0

9.6

4.2

4.5

4.5 4.0

12.4

12.3

5.6

5.7

5.6 5.8

12.4 8.2 5.9 5.6 5.6 5.6

5.6 5.6 5.6 5.6 5.6 5.6

5.6 0.3 0.4

(2)

Microsanas Glycoprotein

---

-__ _--

___--

-__

___ e-v

---

2.2 2.0 1.4 1.2 1.2 1.2

1.2 1.2 1.2 1.2 1.2 1.2

1.2 0.1 0.1

(2)

Mitochondria Glycoprotein

---

28.0 21.0 18.0 14.0 12.0 12.0

12.0 12.0 12.0 12.0 12.0 12.0

12.0 0.6 0.5

Brain Protein (1)

Acceleration of incorporation of leucine- 14C and monosaccharide-l4 C into protein and glycoprotein isolated mitochondria and microsanes. Data are given a8 pmoles/mg protein incorporated.

8.4 0.4 0.2

1.

Control "0" time Boil Control

Synthesis: Precursor:

System

Table

by

Vol. 41, No. 2, 1970

Table

1.

BIOCHEMICAL

In each instance in the designated 1 hour at 37'C.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the mitochondria or microsomes were incubated system with the indicated labeled compound for

*The medium contained 10 mM MgC12, 5 mM sodium phosphate (pH 7.6), 50 mM Tris (hydroxymethyl) amino methane (pH 7.6), 5 mM phosphoenol pyruvate, 20 ug of pyruvic kinase, 2 mM adenosine triphosphate, 2 mM EDTA, 22.5 mg/ail of a complete amino acid mixture minus leucine, 10 ~.rl of the labelled compound, 4 to 10 mg of mitochondrial or microsomal protein, and 0.154 M KC1 to a final volumecf 240 ~1. The liver microsomal assays also contained guanosine triphosphate (0.2 mM) and approximately 2 mg of pH 5 enzymes to a final volume of 300 V.I. {IUniforml labeled L-leucine-lk (0.2 uCi). +Uniformly J C (0.2 uCi). labeled glucosamine%niformly labeled uridine diphosphate glucose-14C (0.2 uCi). Dashed lines indicate the experiment was not performed.

protein

synthesis

increase

in

(Table

the incorporation 14

in glucosamineThese min

increases

tion

only

brain

increase

in both

mitochondria.

Thus

synthesis

with

vitamin

which

K, also

mitochondria trations

of vitamin

incorporation No effects

but has little

indicate thesis

that caused

or warfarin with

high

presumably

protein

as great;

increase

occurred inhibits

with

in the liver mitochondrial

on microsomal

the inhibition

of isolated

50 percent of vitamin

cannot

of the

protein liver

Kl was obtained

501

of

to similar

increase

liver concen14C

in l-leucine-

incorporation

occurred.

or brain

mitochondria.

and glycoprotein

mitochondrial substantially

of glucosaminein

mitochondria.

inhibitor

synthesis.

be reversed

response

liver

compared

microsomes protein

rat

rat

in rat

a 3-fold

and in

synthesis

in monosaccharide

effect

but

only

C incorporation

and glycoprotein

as a competitive

warfarin

of vita-

C incorporation

protein

and glycoprotein

The increases

1).

by chloramphenicol

levels

in vivo

14 14

in the isolated

acts

was found.

14 C incorpora-

l-leucine-

glucosamine-

Kl accelerates

increase

glycoprotein

concentrations

C and glucosamine-

acceleration

and a 2-fold

Chloramphenicol (20)

the vitamin

K are not

of warfarin

14

higher

Kl a 5-fold

and a 7-fold

into

microsomal

microsomal

l-leucine-

accelerated

(Table

curve;

in liver

in liver

protein

indorporation

increase

the greatest

Warfarin,

14 C into

a dose response

increase

At 2 x 10 -3 M vitamin

Kl.

of l-leucine-

a slight

and a 2-fold

a 2-fold

vitamin

C and DDP-glucose-14C

followed

showed

5

1) with

the presence

14

synthesis

The present

data

protein

syn-

by vitamin

C incorporation of chloramphenicol

Kl

Vol. 41, No. 2, 1970

(Table

1).

No effect

was observed poration

BIOCHEMICAL

with

of glucosamine-l4

acceleration K

inhibit

microsomal

sonml (Table

1

vitamin

at concentrations

in rat

liver

2-fold

synthesis

in microsomal

to 250 pg/uil

synthesis

These

and glycoprotein 14

or glycoprotein

increase

of 100 pgfml

or glycoprotein

of glycosamine-

protein

incor-

Kl was unaffected.

mitochondria.

protein

acceleration

found

C incorporation

effect

warfarin

and

with

concentrations synthesis

does not

of cycloheximide

and depress by vitamin

the micro-

K1 by 50 percent

1). The time

vitamin

microsomal and the

C with

of protein

vitamin

liver

chloramphenicol

Cycloheximide the

on rat

AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

course

of acceleration

K, and warfarin

of protein

in isolated

rat

and glycoprotein

liver

mitochondria 4.0

4.0

0

20

40

synthesis

was observed

by along

1

60

TIME (MIN)

TIME (MN) 0 CONTROL 0 WARFARIN A VITAMIN K,

Figure 1. Time Course of Acceleration of Protein and Glycoprotein Synthesis by Vitamin KI and Warfarin in Isolated Rat Liver Mitochondria. Data are p moles/m protein. Warfarin and vitamin KI final concentrations were 2 x lo- s M. The same system for macromolecular synthesis was employed as described in Table 1. Concentrations of labelled compounds were as given in Table 1 for leucine-14C and glucosamine-Ik. 0.5 $1 of uridine-3H and 0.5 uCi of thymidine-3H were added to the complete system for RNA and DNA synthesis. Macromolecular bound radioactivity was determined as described in Materials and Methods. 502

Vol. 41, No. 2,197O

with

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the effect

of vitamin

DNA synthesis isolated

(Fig.

rat

as the fashion

mitochondria

thereafter

cells,

K

10 percent

horse

The cells,

harvested

the

synthesis

Table

2.

radioactively

x x x x

10m2M 10m3M 10w3M 10m3M

Warfarin Warfarin Warfarin Warfarin

1 5 2 1

x x x x

10m2M 10m3M 10w3M 10e3M

Vitamin Vitamin Vitamin Vitamin

2.

Kl Kl Kl Kl

K 1 occurred

in as early

in a more or less

cells

linear

mouse leukemic

grown

in Fischer

were

culture

in L5178Y

by high

as previously

growth,

were

precursor,

The results

in Table

K 1'

was inhibited In general

concentration medium

described

resuspended

that

by concentrations

the vitamin

(25,

in Fischer

in the presence 2 indicate

plus 26). medium

or absence RNA, DNA, of 10 -2 to

K1 was more toxic

at

Effect of Warfarin and Vitamin Kl on Macromolecular Data are pmoles/mg protein. in L5178Y Cells.

Dridine-H3

Control

RNA and

14 C incorporation

inhibited

labeled

synthesis

or vitamin

rrecursor

Table

K 1'

that

was severely

in logarithmic

Inhibitory Synthesis

1 5 2 1

2 indicate

L5178Y

and glycoprotein of warfarin

mitochondrial

and vitamin

and occurred

serum in suspension

or vitamin

liver

C and glucosamine-

by warfarin

in Table

and warfarin.

required

of warfarin protein

1

on rat

1).

presented

of vitamin

14

of incubation

(Fig.

macromolecular

plus

Leucine-

20 minutes

The data

10-3

1).

liver

first

Kl and warfarin

Thymidine-H3

Leucine-14C

Fucose-H3

12.4

101

40

4.0

7..6 5.8 7.9 11.9

9 12 68 101

16 20 25 36

2.1 2.6 3.7 4.1

0.4 0.5 1.6 2.3

4 5 8 16

3 5 12 12

1.6 1.7 1.8 2.4

Inhibitory effect of warfarin and vitamin Kl on macromolecular synthesis in L5178Y cells. Data are expressed as p moles/mg protein. Logarithmic L5178Y cells (lo5 cells/ml) were incubated at 37'C for 60 minutes in Fischer medium plus the indicated labeled compound to a final volume of 0.5 ml. The amount and specific activity of each radioactive compound was: leucine-14C (0.3 uC ), l-fucose-3H 4 H (1.0 $i). uridine-3H (1.0 uCi) and thymidineMacro(0.5 IQ>, molecular bound radioactivity was determined as given in the Materials and Methods. 503

Vol. 41, No. 2, 1970

equimolar

BIOCHEMICAL

concentrations

The results warfarin

presented

accelerate

thesis

warfarin.

cient)

rat

liver

synthesis

this

are

of great

autonomy,

the effects assaying

significantly to the vitamin

that

vitamin

to the

mechanism

were

vehicles

protein due to vitamin for

the

from

the controls.

K. and warfarin

but

not

not

Kl at 2 x 10 7 fold; of vitamin

-3

the

Thus the

DNA or RNA syn-

results

effect K defi-

M increases

pro-

implications

K action,

of

mitochon-

synthesis.

K and warfarin

instance

analogue

a vitamin

and glycoprotein

two compounds

In every

Kl and its

K1 had a greater (i.e.

synthesis

in each of the systems. differ

synthesis

in a normal

and glycoprotein

herein

vitamin

vitamin

system,

and extracytoplasmic

the suspending

Aquamephyton

remains

importance

reported

that

mitochondria;

mitochondrial 5 fold

indicate

and glycoprotein

liver

The fact

tein

drial

rat

the warfarin.

herein

protein

in isolated

than

than

AND BlOPl-!YSlCAL RESEARCH COMMUNICATIONS

That

was shown by

and photo-inactivated these

assays

reported

did are

not due

molecules.

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504

Vol. 41, No. 2, 1970

20. 21. 22. 23. 24. 25. 26.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R. A. Submitted. Bosmann, H. B., and Winston, 941 abs (1970). Winston, R. A., and Bosmann, H. B., Fed. Proc. 2, Schneider, W. C. and Hogeboom, G. H., J. Biol. Chem. 183, 123 (1950). Schneider, W. C. and Kuff, E. L., J. Biol. Chem. 244, 4843 (1969). Lowry, 0. H., Rosebrough, J., Farr, A. A., and Randall, R. J., 3. Biol. Chem. 193, 266 (1951). Bosmann, H. B., and Bemacki, R., J. Exp. Cell Res., in press. Bemacki, R. J., and Bosmann, H. B., Fed. Proc. 2, 783 (abstract) (1970).

505