Glycerophosphatide biogenesis: I. Subcellular localization of cytidine triphosphate: Phosphatidic acid cytidyl transferase

Glycerophosphatide biogenesis: I. Subcellular localization of cytidine triphosphate: Phosphatidic acid cytidyl transferase

‘Vol. 40, No. 4, 1970 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS GLYCEROPHOSPHATIDE BIOGENESIS: CYTIDINE TRIPHOSPHATE: I. PHOSPHATIDIC ...

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‘Vol. 40, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GLYCEROPHOSPHATIDE BIOGENESIS: CYTIDINE

TRIPHOSPHATE:

I.

PHOSPHATIDIC

M. L. Vorbeck Department School

SUBCELLULAR LOCALIZATION

OF

ACID CYTIDYL TRANSFERASE

and A. P. Martin

of Pathology,

of Medicine,

University

Columbia,

of Missouri

Missouri

65201

Received July 8, 1970 SUMMARY phosphatidic The subcellular localization of cytidine triphosphate: acid cytidyl transferase (CTP: PA cytidyl transferase) has been investigated. Rat liver and bovine heart homogenates were fractionated by differential centrifugation and the composition of the fractions evaluated by marker enzymes and In both rat liver and bovine heart preparations, the electron microscopy. highest relative specific activity was associated with the mitochondrial fraction and closely paralleled the distribution pattern for succinoxidase, a mitochondrial marker. INTRODUCTION Cytidine cytidyl

triphosphate:

transferase)

phosphatidic

catalyzes

CTP + Phosphatidic CDP-diglycerides vated

forms

glycerol

(S),

tute

coli the

and possibly

other

In view

nucleotide of the

biosynthesis, cellular to evaluate

central

of the

actual

tissues,

phosphatidyl

(6) , and cardiolipin biological

role

Marker

composition

(CTP:

PA

systems, for

the

biogenesis

901

and electron subcellular

been

glycerol

identified (3,4)

and

and phosphatidyl glycerol

CDP-diglycerides

was undertaken

of the

have

acti-

(6),

(7) in Escherichia

of CDP-diglycerides

enzymes

(1).

can be considered

phosphatidyl

in animal (51,

investigation

formation.

which

+ PP

liponucleotides

(2),

(3,4)

intermediate

the present site

These

ethanolamine phosphate

sole

nucleotides

inositol

glycerol

transferase

CDP-Diglyceride

acids.

phosphatidyl

phosphatidyl

e

phosphate

cytidyl

reaction:

lipid-soluble

of phosphatidyl

phosphatidyl

In 5.

acid

of phosphatidic

as precursors

serine

are

the

acid

coli.

may consti-

of glycerophosphatides in glycerophosphatide to determine microscopy fractions.

its were

intraused

Vol. 40, No. 4, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

MATERIALS AND METHODS

[+3HJCytidine purchased

from

5'-triphosphate,

Schwarz

BioResearch

phosphate,

disodium,

proteinase

from Enzyme

sodium

salt,

pared lipase

from

egg yolk

D (Calbiochem.

liver

prepared tained

from

Corp.,

were

fractions

of the

several

minutes

was trimmed

meat grinder.

Fifty

sucrose

containing

(Hepes)

pH 7.6

4N KOH.

(S-H buffer).

pared

mince

from

the

minutes half

the

bottom

original

supernatant

10,000 fluid

male

Sprague-Dawley

(10).

animals

phospho-

heart. animals

heart

were

Hearts

were

and placed

tissue

and ground

washed

twice

in

in

ob-

in ice. a precooled

200 ml cold

0.25M

from

death

examination

of the

homogenate of the

tube.

volme

x g for B.

of the The extent

homogenate.

of S-H buffer

20 minutes. The firmly

using

of cell

breakage

with

The fluffy

902

pink

was

of the

was monitored were

g values

pre-

computed

at 750 x g for

was rehomogenized to obtain

supernatant layer

of

ball-type

fractions

was centrifuged

with

volume

preparation

Subcellular

was combined

pellet

a final

a Dounce

until

The pellet

7.6 by addition

The suspension

and recentrifuged

packed

to

to

centrifugation

A collected.

fluid

added.

animal

The homogenate

fluid

at 7.4

homogenized

by differential

acid

in S-H buffer

S-H buffer)

and then

one hour.

The supernatant at

(10 mg/ml

than

and supernatant

fraction. fuged

the

using

from

of the

was suspended

20 minutes

was less

from

or pre-

and bovine

of Brierley

were

Corp.)

of bovine

acid,

Ill.

liver

The pH was maintained

Time elapsed

by microscopic

tissue

Phosphatidic

rat

Homogenates

death

and Nagarse

0.Ol.M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

at 0 C for

homogenate

(8).

and connective

g of ground

The washed

homogenizer.

Calif.)

procedure after

of fat

200 ml and one ml Nagarse stirred

(9).

5'-tri-

Rockford,

Chemical

prepared

cytidine MO.,

N.Y.

Corp.,

from

was

Louis,

(Sigma

prepared were

by modification

St.

Chemical

Ci/mmole)

N.Y.;

New York,

choline

previously

Each heart

Corp.,

Pierce

as described

within

Corp.,

Los Angeles,

fractions

subcellular

(150-2OOg)

of

Development

(13.5

Orangeburg,

Chemical

phosphatidyl

Subcellular Rat

Inc.,

Sigma

was obtained

from

tetralithium

A

the

was resuspended

in onenuclear

and centri-

was removed

10

with

in S-H buffer

Vol. 40, No. 4, 1970

BIOCHEMICAL

and centrifuged

at

10,000

fluffy

layer

pellet

was resuspended

was removed

The combined 105,000 nated

taining acid, in

1 hour.

pmole

100 pmoles a final (11)

and the

10 minutes.

ml.

(11)

complished

tained

lipid

from

applied

Dr.

of

were

TriCarb following

at the

scintillation

benzene,

determined

as disintegrations Relative

per

specific

to percent Purity heart

Marker

enzyme

described

minute

(12)

addition

for

by solvent else-

g;

ratio

(11). (ob-

The remaining in a Packard medium

1,4-bis

of the

[2-(5-phenyloxaof counting

and the

an Olivetti

was ac-

radioactivity

The efficiency

as the

shaker

CDP-didecanin

a scintillation

procedures

was computed

of the

paper

was determined

1000 ml.

MgC12

as CDP-diglyceride

CDP-didecanin.

4.0

(DPM) using

nmoles

to be described

95% of the

using

con-

results

Programma

was

computed

101 computer.

of the percent

total

DPM

protein.

of the

bovine

the

standardization

activity

total

than

2,+diphenyloxazole,

external

the

authentic

Radioactivity

50 mg; and toluene,

using

assay

with

spectrameter

composition:

zoly)]

origin.

and 2.5

on formaldehyde-treated

with

fraction.

mixture

was measured

lipid

and greater migrated

cytosol

250 ug phosphatidic

after

lipid

3H-Labelled

was co-chromatographed

found

Ci/mole),

at

and desig-

at 37 C in a gyratory

disc

chromatography

chromatogram

liquid

out

filter

the

500 pg protein,

3H-CTP into

of the

B.W. Agranoff)

to the

counts

carried

fraction.

was centrifuged

in an incubation

The MgC12 was added

or by a modified

by descending

The labelled

300 to

packed

mitochondrial

in S-H buffer fluid

0.5

and any

The firmly

layer

supematant

activity

incubation

The identification

fluffy

was assayed

pH 7.2,

The incorporation

extraction where.

and the

(specific

of 0.25

the

fluid

B. the

was resuspended

transferase

Tris-KC1

volume

containing

fraction

3H-CTP

supematant

and designated

The pellet

PA cytidyl

0.5

protein

fluid

The supernatant

with

in S-H buffer

microsomal

CTP:

20 minutes.

and combined

supematant

x g for the

g for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

individual

was evaluated assays

previously

for (9).

subcellular by marker

evaluation Alkaline

enzyme of the

fractions assays

both

and electron

subcellular

phosphatase,

903

from

assayed

fractions

rat

liver

and

microscopy. were

by modification

as of

Vol. 4Q, No. 4, 1970

the

procedure

marker

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Neumann

for

the bovine

culated

as the

Protein

was determined

procedure

and Van Breedendaal

heart

ratio

of the

for

and lead

electron

(12)

microscopy

Samples

were

were

and viewed

specific

activity

to percent

total

of the

fixed

washed

calorimetric

in the

in sodium

of ethyl

Ultrathin

as a microsomal was calprotein. micro-Folin

(14).

concentrations

in Epon 812.

citrate

activity

and MacPherson

in progressive

and embedded

total

by a modification

1% osmium tetroxide. hydrated

percent

was used

Relative

subfractions.

of Heidelberqer

Samples

(13),

sections

in a Hitachi

cold

cacodylate

alcohol, were

for

buffer,

with

using de-

and propylene

stained

IS electron

1 hour

oxide

uranyl

acetate

microscope.

RESULTS AND DISCUSSION In the

present

fractionated (rat

designation

presents

only),

of the

various

liver

with

are

representative

The data

of

shaw that transferase

associated 92% of

with the

total

pattern

sent

the

subfractions, ferase

average the

was observed

activity

specific

activity

fraction.

for is

CTP:

heart

PA cytidyl

of

The results

the

CTP:

PA

homogenate)

is

fraction

contained

relationship

transferase

homogenates

is

transferase given

of five

separate

fractionation

highest

relative

specific

in the

of

this

The close

PA cytidyl

activity

fractionations.

of the

Moreover,

1

between

and succinoxidase,

a

apparent.

of CTP:

of bovine

specific

the

Figure

enzymes.

separate

"inter-

composition

transferase

three

of the homogenate.

The distribution fractions

the

mitochondrial

marker,

from

relative

times

activity

distribution

mitochondrial

highest

(5.3 the

obtained

actual

microscopy.

marker

been

Although

their

CTP: PA cytidyl of the

have

mitochondrial,

fractions.

and electron

pattern

homogenates

nuclear,

was arbitrary,

of the

those

the

into

enzymes

distribution

heart

and cytosol

fractions

comparison the

and bovine

microsomal,

by use of marker

a direct

rat

liver

centrifugation

liver

was determined

cytidyl

rat

by differential

mediate"

the

study,

mitochondrial

in Table

1.

among subThe values

experiments. activity

fraction.

904

activity

for

As with

reprerat

CTP:

PA cytidyl

The activity

in this

liver trans-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 40, No. 4, 1970

f

I

;

: I

t

8

t

1 I

:CTP:PA

4

:

cvtidrl

!

f

I

0

100

SO

Percent

Protein

PA cytidyl transferase and marker patterns for CITP: Figure 1. Distribution enzymes among subfractions of rat liver homogenate (nuclear, N; mitochondrial, I; microsomal, M; and cytosol, C). Conditions were as IQ; "intermediate", described in the text. fraction distribution

was 6.9

times

parallels

the that

specific of

the

activity mitochondrial 905

of the

homogenate

marker

and the

succinoxidase.

Vol. 40, No. 4, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Relative Subfraction

Specific

CTP: PA cytidyl transferase

Activity

Succinoxidase

Alkaline Phosphatase

Nuclear

1.1

1.2

1.0

Mitochondrial

6.9

6.3

1.1

Microsomal

1.6

1.8

10.2

cytoso1

0.1

0

0.3

Table 1. Relative specific activities of CTP: PA cytidyl transferase, succinoxidase (mitochondrial marker enzyme), and alkaline phosphatase (microsomal marker enzyme) in subfractions of bovine heart homogenates. Conditions were as described in the text.

Figure

2 is

an electron

prepared

from

membrane

and most have

active

a bovine

mitochondria

few orthodox other

heart

as described forms

(11)

and bovine using

using

chick

brain

preparations.

pig

liver,

of the

localization with ration

the

seen.

localization is

Whether

ration

staining

by Hackenbrock

heart

guinea

fraction.

The mitochondria

densely

are

mitochondrial

fraction

show a distinct

matrix

that

and others

The preparation

outer

is typical

(15,16,17).

of

Very

is relatively

free

of

elements.

The mitochondrial liver

of a typical

homogenate.

a condensed,

or swollen

cellular

micrograph

in agreement

reported

this

is

the

of CDP-diglyceride

the

synthesis

into

transferase

results

in rat

of Petzold

and Agranoff

contrary,

Carter

and Kennedy

activity

in the

microsomal

greatest

remains

Zborowski

of glycerol-3-phosphate

PA cytidyl

On the

fractions

of

with

due to a species

subcellular

observations

of CTP:

difference

or differences

to be determined. shown by our

and Wojtczak

(19)

glycerophosphatides

in prepa-

The mitochondrial

data

that

(18)

also

is

compatible

CTP stimulated by isolated

rat

incorpoliver

mitochondria. The role investigated

of mitochondria

in glycerophosphatide

in many laboratories

in the

synthesis

isolated

liver

(19,20,21,22,23).

of many glycerophosphatides microsomal

fractions

is known

(231,

906

conflicting

biosynthesis Although

has been the

final

to be carried evidence

on the

stage

out by ability

of

Vol. 40, No. 4, 1970

BIOCHEMICAL AND BlOPHYSlCAL

RESEARCH COMMUNlCATlOtiS

Figure 2. Electron micrograph of a freshly isolated mitochondrial fraction obtained from a bovine heart homogenate. The preparation was fixed with osmium tetroxide and stained with uranyl acetate and lead citrate. x 20,000

isolated

mitochondria

(20,241.

The role

phosphatide other

to synthesize of CTP:

biogenesis

subcellular

PA cytidyl

as well

fractions

glycerophosphatides

is

as its

transferase role

currently

has been in mitochondrial

in glycerophosphatide under

reported glycerosynthesis

in

investigation.

This work was supqzted by a grant-in-aid from the American Ackncwledgements: Heart Association. Electron microscopy was performed by P. L. French and the prints prepared by D. D. Thompson, Department of Pathology. The computer program for calculation of the scintillation counting data was written by E. F. Malewski of the Department of Pathology. The technical assistance of L. S. Erhart is gratefully acknavledged.

907

Vol. 40, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Agranoff, B.W., Bradley, R.M., and Brady, R.O. J. Biol. Chem. 233, 1720 (1958). H. and Kennedy, E.P. 3. Biol. Chem. 235, 1303 (1960). Paulus, J. Biol. Kiyasu, J.Y., Pieringer, R.A., Paulus, H., and Kennedy, E.P. Cher. 238, 2293 (1963). StanacerN.Z., Stuhne-Sekalec, L., Brookes, K.B., and Davidson, J.B. Biochim. Biophys. Acta 176, 653 (1969). Kanfer, J.N. and Kennedy, E.P. J. Biol. Chem. 239, 1720 (1964). Chang, Y.Y. and Kennedy, E.P. J. Lipid Res. 8, 447 (1967). Stanacev, N.Z., Chang, Y.Y. and Kennedy, E.P. J. Biol. Chem. 242, 3018 (1967). in METHODS OF ENZYMOLOGY, Editor J.M. Kates, M. and Sastry, P.S., Lowenstein, Vol 14, Academic Press, N.Y. 1969, p. 197. Martin, A.P., Halter-man, D.R., Vorbeck, M.L., Kuo, M.C. and Lucas, F.V. In press (1970). Brierley, G.P. J. Biol. Chem. 242, 1115 (1967). Petzold, G.L. and Agranoff, B.W.J. Biol. Chem. 242, 1187 (1967). Unpublished work. Vorbeck, M.L. Neumann, H. and Van Breedendaal, M. Clin. Chim. Acta 2, 183 (1967). Heidelberger, M. and MacPherson, C.F.C. Science 97, 405; -98, 63 (1943). J. Cell Biol. Hackenbrock, C.R. 30, 269 (1966). J. Cell Biol. Hackenbrock, C.R. 37, 345 (1968). Green, D-E., Asai, J., Harris, R.A., and Penniston, J.T. Arch. Biochem. Biophys. 125, 684 (1968). Carter, Jx and Kennedy, E.P. J. Lipid Res. 7, 678 (1966). Zborowski, J. and Wojtczak, L. Biochim. Biophys. Acta 187, 73 (1969). Stoffel, W. and Schiefer, H.G. 2. Physiol. Chem. 349, 1017 (1968). McMurray, W.C. and DawSon, R.M.C. Biochem. J. 112, 91 (1969). Sarzala, M.G., Van Golde, L.M.G., de Kruyff, B., and Van Deenen, L.L.M. Biochim. Biophy. Acta 202, 106 (1970). Jungalwala, F.B. and D&on, R.M.C. Eur. J. Biochem. 12, 399 (1970). G.V. Biochim. Biophys. Acta 202, 91 (1970). Fang, M. and Marinetti,

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