Pool sizes and metabolism of CDPcholine and CDPethanolamine in skeletal muscle

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

POOL SIZES AND METABOLISM

OF CDPCHOLINE AND

CDPETRANOLAMINE IN SKELETAL MUSCLE F.A.

SBAMGAR* AND F.D.

The Russell University Received

Grimwade

of Melbourne,

March

School

Parkville,

COLLINS** of Biochemistry, Victoria

3052

(Australia)

28,1977

SUMMARY

1. The pool sizes of CDPcholine and CDPethanolamine muscle were measured by isotopic dilution and found 1.5 umole/lOO g muscle respectively.

in skeletal to be 0.7 and

2. The specific radioactivities of phosphorylcholine derived from CDPcholine and of phosphorylethanolamine derived from CDPethanolamine determined 3 h after the administration of [3*P] orthophosphate were found to be 9.1% and 3.2% of the specific radioactivity of the serum inorganic phosphate respectively. 3. The3specific radioactivity ortho [ P] phosphate has also study during a 24 h period.

Measurement species

of the

[3'4]

liver

and muscle

of phosphatidylcholine in

liver

activity

the average five

the ratio

*Present

is

times

3 h after

is

in liver of ortho

of specific

of the average

[ 1,2 ] [32~]

pattern

of 32P

radioactivity

of phosphatidylethanolamine.

phosphatidylethanolamine that

out

incorporation

in the ratio

to that

of molecular

the injection in the

of

whilst

has a specific phosphatidylcholine,

radioin muscle

0.3.

address:

Parkville,

radioactivities

has been carried

The main difference

phosphate. between

specific

of phosphoglycerides

1 h, and in muscle

of muscle Pi after an injection been determined in a time-course

Victoria

Department 3052,

of Physiology,

University

of Melbourne,

Australia.

** Deceased

Copyright 0 1977 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

550 ISSN 0006-291X

Vol. 76, No. 2, 1977

BIOCHEMICAL

The aim of the present the specific

radioactivity

phosphoglycerides the muscle establish

and the measured phosphate

MATERIALS

[3], diet food were were time

in skeletal

moieties

muscle. during

a precursor-product muscle

was to measure

of CDPbases

Pi was determined whether

study

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P i as well

of phosphoglycerides

involved

the in

The specific

pool

radioactivity in order

relationship

between

could

and

the biosynthesis

a 24 h period

as between

size

the muscle

of of

to serum P

i

Pi and the

be demonstrated.

AND METHODS

Male Buffalo rats were placed on either a commercial cubes diet a safflower seed oil containing diet [4], or a saturated fat [4], three weeks after birth and allowed continuous access to Five rats and water. The animals were 6 months old when used. The rats used in each experiment and their tissues were pooled. injected with ortho [32~] phosphate [3,4] and an incorporation of 3 h was allowed.

CDPcholine (methyl-14C), specific radioactivity 25 mCi/mmol, was purchased from New En land Nuclear Co., 575 Albany Street, Boston, specific radioMass., U.S.A. and CDP (2- H4C) ethan-l-ol-2-amine, activity 28 mCi/mmol, from the Radiochemical Centre, Amer.&am, Bucks., England. The isotopes were each diluted with the respective CDPbase to a specific radio-, (Sigma Chemical Co., St. Louis, MO., U.S.A.) activity of 0.73 mCi/mmol for CDPcholine and 0.91 mCi/mmol for CDPethanolamine. Various amounts of either CDPbase were added to the aqueous extracts containing the phosphoglyceride precursors for pool size determination. The purity of the CDPbases used was checked by paper chromatography and found to be greater than 97%. Muscle and serum Pi were determined in pooled samples of five rats as described by Berenblum and Chain [5]. The pooled muscles were extracted with cold (-16') 67% ethanol [6]and the residue subjected to lipid extraction. The 67% extract was purified [7] and chromatographed on Dowex AGl-X4 (200-400 mesh, chloride form, Bio Rad Laboratories, 32nd and Griffin, Richmond, Calif., U.S.A. [8]. column eluate.

A fraction into the

collector scintillation

was modified to collect eluates vials, each vial containing

from the 10 ml

All vials were examined for radioactivity and absorbance at 280 nm and the identification of CDPbases was facilitated by chromatographing a standard mixture of phosphorylcholine, phosphorylethanolamine, CDPcholine and CDPethanolamine under the same conditions (Fig. 1). The column chromatography method employed did not separate CDPbases from dCDPbases. Fractions chromatographed

containing CDPbases were dried on Whatman 3 MM paper (9) for

551

and pooled residues 16 h. The purified

bases

Vol. 76, No. 2, 1977

BIOCHEMICAL

FRACTION

AND BIOPHYSICAL

NUMBER

RESEARCH COMMUNICATIONS

(IO ml/fraction)

Fig. 1. Column chromatography of a 67% ethanol extract of leg muscles of five rats which had been injected with [32P i C) was orthophosphate 3 h before death. CDPcholine (methyl-l added as an internal standard. a) distribution 0f ~32~1. absorbance at 280 nm. c) distribution of [l4C] radio(b) activity. d) distribution of phosphate of a standard mixture of 0.5 mg phosphorylcholine, 0.5 mg phosphorylethanolamine, 0.25 mg CDPcholine (methyl-14C) containing approx. 100,000 dpm) and 0.25 mg CDPethanolamine chromatographed under the same conditions. The broken line shows the increase in the concentration of formic acid during the elution, which began from fraction 15.

were eluted with 0.01 M formic acid by centrifugation [lo] and hydrolyzed with 1 M HCl for 1 h at 100'. After evaporation of HCl, the residues were chromatographed for 16 h for separation of phosphorylcholine [9] and for 46 h for separation of phosphorylethanolamine (11). Cytidine-containing at 280 nm (Emax= 13000

comp_pundzlwere quantified at pH 2) whilst .cm l.m01

by their absorbance phosphate esters

were detected under uv light after spraying with acid molybdate spray [ll] andquantified as described by Itaya and Ui [12]. Using these CDPmethods the following molar cytidine/phosphorus were obtained: choline 1:1.92, CDPethanolamine 1:1.94 and CMP 1:0.94 and 1:0.96 respectively. Radioactivity was measured as previously described [3,4]. RESULTS CDPbases The pool in control

rats

size

of CDPcholine

and in rats

in skeletal

deficient

muscle

in essential

552

was determined fatty

acids

[4],

CDPbase

size/lOOg

Pool

muscle

relative specific radioof any phosphoglyceride

percentage radioactivity

dpmlnmol

dpmlnmol

g urn01

dpm/umol

pm01

dpmfpmol

pm01

Unit

of the specific of serum Pi*

x 10 -3

x 10 -3

x 10 -3

x 10 -3

(82.8)

(1.13)

(0.352)

1.94

9.1

25.1

(4.2)**

(13.2)

(78.5)

148 (1391)

91.2

0.69

181 (102)

623 (655)

0.448

1619

0.782

CDPcholine

0.62

3.2

12.8

105

62.2

1.5

106

820

0.24

2018

1.1

CDPethanolamine

,”

,,

,,I

”

.”

.,

,,.

product [3]

moieties five rats injected AGl-X4 and fractions may be slightly results of an ex-

* 592,000 dpm/pmol for control rats and 1,770,OOO dpm/umol for rats deficient in essential fatty acids ** 1-oleoyl-2-linoleoyl phosphatidylcholine (1-oleoyl-2-palmitoleogrl phosphatidylcholine [4]is the probable of phosphorylcholine moiety of CDPcholine as 1-palmitoyl/oleoyl-2-docosahexaenoyl. phosphatidylethanolamine is the product of the phosphorylethanolamine moiety of CDPethanolamine.

Highest activity

radioactivity derived from

Relative specific of phosphorylbase CDPbase

specific radioactivity CDPbase

radioactivity

0f

(32P)

(14C)

(14C)

Relative specific of CDPbase

Initial (32~)

Specific radioactivity of isolated CDPbase

weight

Muscle

Specific radioactivity of isolated CDPbase

Isolated

Added CDPbase Specific radioactivities of added CDP base

Parameter

Pool sizes of CDPbases and relative specific radioactivitiesof phosphorylbase Table 1. derived from3yPbases. CDPbases were extracted with 67% ethanol from pooled leg muscles or with ortho [ P] phosphate 3 h before sacrifice. The extract was chromatographed on Dowex containing CDPbases were purified by paper chromatography. The pools of CDPbases obtained smaller because they may have included small amounts of dCDPbases. Figures in brackets are periment with rats deficient in essential fatty acids.

5..3 oz .! -a Y

Vol. 76, No. 2, 1977

BIOCHEMICAL

3, 13

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

12

HOURS

OF

24

INCORPORATION

Fig. 2. Specific radioactivities of serum (W) and muscle (n------d)Pi as a function of time. Similar measurements for the phosphatidylcholine fraction (a-0) possessing the highest specific radioactivity [3] are shown for comparison. The sera and tissues of five rats were pooled for samples obtained after 1, 3, 6, 12 and 24 h of incorporation. Other samples originated from a single rat.

and it

was found

to be 0.7

pmol and 1.1 nmol per

respectively.

The relative

specific

choline

was 8.1% for

control

acid

moiety deficient

diet

and the moiety

Muscle

size

[3].

relative derived

in Table

and

of CDPethanolamine The pool

size

specific

radioactivity

from

of the phosphoryl-

13.2% for

essential

was determined

was found

fatty

in

rats

fed a

to be 1.5 vmol/lOO

g muscle

of the phosphorylethanolamine

CDPethanolamine

was 3.2%.

plot

specific

The results

are

listed

1. Pi

The semilogarithmic and muscle

Pi as well

(possessing incorporation curves

radioactivity

rats.

The pool cubes

100 g muscle

results

the highest time

is

of the

as that

of a phosphatidylcholine

specific shown

radioactivity

radioactivity)

in Fig.

in an intersection

2. time

fraction as a function

An extrapolation of approx.

554

of serum

70 h.

of

of the Pi

BIOCHEMICAL

Vol. 76, No. 2, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION The incorporation involves

the

following

phosphorylbases glyceride

molecules

the

from

CDPcholine

essential

in

Pi in both

control

rats

than

that

precursor-product essential

pool

pool

is

demonstrated muscle

is

involved

bound

cellular muscle

of muscle

[14,15]

Both

findings

muscle

Pi as a mandatory

findings a small

and therefore

slowly

Any scheme of

is

that

the

compartment It

of th

has been P i in

1131 as an intermediate

in phosphoglyceride

1161.

exist.

of intracellular

proteins

deficient

P, and a

biosynthesis.

fraction

a

in rats

of muscle

and only

CDPcholine

is

not available

biosynthesis. into

the

support

as a

Moreover,

intracellular

extra-

space

the concept

of

of heterogeneity

P i'

The pool that

activity

from

of phosphorylcholine

of our

in phosphoglyceride

P seems to penetrate cells

that

includes

heterogeneous

to myofibrollar

precursor

However

no longer

reason

a considerable

in myosin-ATPase phosphate

muscle

a possible

of Pi is

that

could

into

Hence,

than

of

and therefore

radioactivity

was higher

incorporation

measured

specific

relationship

intermediate.

this

the

CDPcholine

precursor-product phosphate

acids

in

3 h of incorporation.

rats

is possible.

derived

in essential

derived

Pi in control

relationship fatty

from

was 10% after

found

radioactivity

deficient

of phosphorylcholine

of muscle

We have

deficient

specific

and those

results)

radioactivity

CDPbases.

was 8.1% and in rats The relative

(unpublished

Pi-+

of phosphorylcholine

13.2%.

The specific

derived

rats

phosphoglycerides

of CDPbases + phosphovia

acids

acids

was lower

moieties

radioactivity

control

muscle

Pi + serum Pi * muscle

synthesized

specific

fatty

the muscle

in

directly

relative

into

intermediates:

+ phosphorylbase

that

fatty

of orthophosphate

of liver.

of CDPcholine

size

of CDPcholine Some of the

in liver

(Table

in skeletal

discrepancy

muscle

in data

2) can be accounted

555

is

approx.

regarding for

l/10

the amount

by methodology

of

BIOCHEMICAL

Vol. 76, No. 2, 1977

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2

Pool size 1OOg liver).

of CDPcholine

and CDPethanolamine

in rat

CDPcholine

Source Sundler

[17]

Sundler

et al [18]

Trewhella and Collins (unpublished results)

10.4

Wilgram

et al

Kennedy

and Weiss

Schneider

[ZO]

5.0

[7] [19]

* Based on 10 g wet weight

However, cold

of muscle

CDPethanolamine

0.87 4.8

we used

pmol/

2.4*

Shamgar and Collins (unpublished results)

[18].

liver

in relation

[-16'1

the

5.3

3.4

4.3

of liver.

to the amounts

67% ethanol

and hence

10.4

for

likelihood

of CDPbases

the homogenization of enzymic

in muscle, and extraction

changes

in

the amount

of

CDPbases was reduced. The pool

size

of CDPethanolamine

the corresponding The actual

pool pools

they

may have

to less

than

l/20

considerably whereas

3 times

that

in muscle

dCDPbases.

corresponding

radioactivities

higher

the reverse

radioactivity

included

of the

than is

is

approx.

l/3-1/2

of

liver.

of CDPbases

because

The specific

in

in muscle

true

for

be slightly

The liver

CDPbases

of muscle

those

could

dCDPbases

of the phosphorylcholine

phosphatidylcholines

[19,20]. moiety

of the phosphorylethanolamine

In muscle, of CDPcholine

moiety

556

amount

1201.

of phosphatidylethanolamines liver

smaller

are [3,4] the

specific

is

approx.

of CDPethanolamine.

Vol. 76, No. 2, 1977

A possible

cause

BIOCHEMICAL

for

the higher

phosphatidylcholines could i.e.

high

the phosphorylcholine The recent

explain

the are

presence

of the back

the

much lower

radioactivities

those

specific moiety

that

cholines

cannot

with

demonstrations

fact

possibility

specific

compared

be the relatively

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of phosphatidylethanolamines

radioactivity

specific

reactions

radioactivities

those

reaction

of their

of their

[17,21]

formation

could

of liver

precursors.

phosphatidylAlthough

has not been demonstrated

of some CDPcholine

precursor,

of CDPcholine.

of the back

than

of muscle

the

in muscle,

from phosphatidylcholine

be excluded.

ACKNOWLEDGEMENTS

Financial Research

support

Grant.

and Dr M.T.

was received

F.A.

Shamgar

McQuillan

for

from

a University

is greatly

assistance

indebted

with

of Melbourne to Prof.

A.J.

Day

proof-reading.

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Trewhella,

Acta 2.

Trewhella,

Acta 3. 4.

M.A.

and Collins,

F.A.

and Collins,

F.A. I.

6.

Bjornstad,

P. and Bremer,

8.

Biochim.

Bio&s.

F.D.

(1973)

Biochim.

Biuphys.

Biochim. Biophys.

P.D.

(1975).

F.D.

(1975).

and Collins,

Eerenblum,

Wilgram,

Biol.

(1973)

Biochim. Biophye.

409, 116-127.

5.

7.

F.D.

409, 104-115.

Shamgar,

Acta

and Collins,

296, 51-61.

Shamgar,

Acta

M.A.

296, 34-50.

G.F.,

and Chain,

Holoway,

Biochem. J.

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and Kennedy,

H.,

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E.P.

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Hurlbert,

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J. BioZ. Chem. 209, 23-39. 9.

Schneider,

W.C. and Rotterham,

J.

(1958).

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557

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the

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10. Edstrom, R.D. (1968).

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Anal. Biochem. 26, 204-205.

11. Hanes, C.S. and Isherwood, F.A. K. and Ui, M. (1966).

(1949) Nature, 164, 1107-1112.

12.

Itaya,

13.

Cheesman,D.F. and Whitehead, A. (1969) Nature, 221, 736-739.

CZin. Chim. Acta 14, 361-366.

14. Tokiva, T. and Tonomura, Y. (1965) J. Biochem. 0'07cyo) 57, 616-626. 15.

Tonomura, Y. and Kanazawa, T. (1965) J. BioZ. Chem. 240, 4110-4112.

16. Manery, J.F. and Bale, W.F. (1941) Am. J. PhysioZ. 132, 215-231. 17.

Sundler, R. and Akesson, B. (1975) Biochem. J. 146, 309-315.

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Sundler, R., Arvidson, G. and Akesson, B. (1972) Biochim. Biophys. Acta 280, 559-568.

19.

Kennedy, E.P. and Weiss, S.B. (1956) J. BioZ. Chem. 222, 193-214.

20.

Schneider, W.C. (1971) J. Nat. Cancer Inst.

21.

Yanoh, H. and Ohno, K. (1973) Biochim. Biophys. A&Z 306, 203-217.

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