Evidence for a new biosynthetic pathway of sphingomyelin in SV 40 transformed mouse cells

BIOCHEMICAL

Vol. 47, No. 6, 1972

EVIDENCE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FOR A NEW BIOSYNTHETIC

PATHWAY OF SPHINGOMYELIN

IN

SV 40 TRANSFORMED MOUSE CELLS H.Diringer,

W.D.Marggraf,

Max-Planck-Institut

Received

fiir

M.A.Koch,

and F.A.Agderer

Virusforschung,

74 Tiibingen,

West-Germany.

May 8, 1972

SUMMARY

Sphingomyelin metabolism has been studied in logarithmically growing SV 40 transformed mouse fibroblasts.32P-phosphate and 3H-choline pulse chase experiments indicate that sphingomyelin is synthesized by an immediate transfer of phosphorylcholine from lecithin to sphingosine or N-acylsphingosine.

The biosynthesis

of the

two choline

containing

phospholipids,

have been reported

to be reactions

of

lecithin

and sphingomyelin,

cytidine

diphosphate

glyceride

and with

this

has been proved to be an important mechanism in 1 cells whereas in the case of sphingomyelin there is

activated

choline

N-acylsphingosine

(CDP-choline) respectively.

with For

diacyl

lecithin

pathway

mammalian only

weak evidence

Kennedy' which

such a reaction

investigated

was able

CDP-choline. all

for

an enzyme

to catalyze They obtained

naturally

occuring

An alternate

pathway

the

mechanism.

system

prepared

reaction

has been

threo exist

reported

system

where

sphingosylphosphorylcholine

and yielded

both

forms

of sphingomyelin.

this

communication

of

sphingomyelin

@ 1972. by Academic

liver

in

was acylated

that

the

biosynthesis

a route

different

of

those

reported

Press, Inc.

although

3,4 . erythro-form et al. 5 studying

evidence

follows

and

the

we present

1345 Copyright

rat

sphingomyelin

by Brady

an enzyme

In

from

and

of N-acylsphingosine

exclusively

sphingolipids

Scribney

when

BIOCHEMICAL

Vol. 47, No. 6, 1972

investigated likely

in

the

sine

logarithmically

transfer

growing

mammalian

of phosphorylcholine

or N-acylsphingosine

biosynthesis

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

in this

the

main,

cells.

from

lecithin

possibly

the

Most to sphingo-

only

way of

system.

For our

experiments we used a line of SV 40 transformed . which can be grown in suspension (STU 51A-232B)

cells

maintained

in

fresh

medium.

under

the

the

conditions

composition

the

used.

according

by appropriate

time

of this

of

Neither

lipid

the

unpublished

cells

the

phospholipid

with

was about

as determined

we performed

12 hours

content by the

is dependent

range

and can be

dilution

the

class

to Bartlett'

within

culture

(Diringer

growth

The doubling

rus content of

logarithmic

mouse

on the our

nor phospho-

cell

density

experiments

observations).

experiments with 32 P-phosphate showed that incorporation rate of 32 P into sphingomyelin was significantly retarded when compared to the 32 P incorporation into lecithin. Preliminary

These

pulse

findings

two lipids from

the

indicated either different turnover rates of these 32 or a P precursor of sphingomyelin which is different

CDP-choline.

turned

To decide

to pulse

between these two alternatives with 32 P-phosphate and tritiated

experiments

we

choline

tJH-methyl) following the kinetics during incorporation and chase. The 32 P chase was performed by a replacement of the phosphate to prevent

buffered

medium.

a possible

The chase

interference

of

with

'H-choline the

was omitted

logarithmic

growth

conditions. Cells

were grown

500 ml of growth calf

serum

in

and 1 mCi of about

in suspension medium

the

5% CO2 in

containing

presence

3H-choline air

with

starting

of

4.5

10% of heat

5 mCi of carrier

(17 Ci/m rapid

with

mol) stirring

1346

under

x lo*

cells

in

inactivated fetal 32 free P-phosphate

an atmosphere

in a 1 1 flask.

of After

Vol. 47, No. 6, 1972

12 hours

cells

4.0

Logarithmic

growth

hemocytometer

were

taken.

cellular

This

cell

in

and sphingomyelin

visualized

was removed

by centrifugation

was counted

in

counting

in

25 ml aliquots

to Folch

and the al. 7 .

et

by two dimensional

which

allows

the

(to be published).

Bartlett6.

of

thin-

clear

separation

The spots

individual

the

containing

were

lipids

data

radioactivity

bound

are plotted

logarithmically

according

given

in

this

to cellular

sphingomyelin calculated on the basis of diluted 32 P and 3H counts of lecithin and sphingomyelin experiment

versus

material

The phos-

was determined

radioactivity

Bray's

supernatant

Tri-Carb-Scintillation-Counter.

The absolute represent

lipid

a

between

by centrifugation

and the

a Packard

content

cation

dilution.

intervals

time

concentration

by autoradiography, scrapped off and eluted with 8 mixture containing 10% of water. The carrier

scintillation

phorus

a cell

was kept

separated

a system

give

by cell

according

were

250 ml of warm

to a 3 fold

concentration

were extracted

chromatography

to

was controlled

were harvested

phospholipids

of lecithin

meilium

At given

The cells

Individual

washed with

corresponds

of cells

cells/ml.

lipids

layer

in growth

and the

- 2.0 x lo6

0.4

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were centrifuged,

and resuspended x lo 5 cells/ml.

medium

of

the

BIOCHEMICAL

to

communi-

lecithin

and

aliquots. of a typical time

(Figure

l).It

can be seen that after a period of rapid incorporation the curves of the 32 P as well as of the 3H uptake run about paralell to one another

in

each

lipid

activity

must

however,

the

those

sphingomyelin

labels in This

of

of

be incorporated incorporation

lecithin

sphingomyelin becomes

indicating

more

curves it

clear

of lecithin

becomes

steadily both

that in both cases the radioas 32 P-phosphoryl-3 H-choline. When,

labels

evident

decrease appear

when the

shortly

after

to increase

absolute 1347

are compared with that the 32 P and 3H the

chase

further.

radioactivities

are

while

Vol. 47, No. 6,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

.’

.’

m-.-m,. **-*d.\

;p

.

1 105

I II

/

I/

.

-.---I

--•-*-

/ :

-2.-m-.

.’

.I

A

,106

‘/ I

i

-0-d

.-t Z 5 K E ,o

q // 0 I

I

I

Chase

1 36

I

4

8

12

16

hours

Figure

of

in

lecithin

labels that

a linear

lecithin

after

of cell

24

26

32

lrol -t

growth

Semilogarithmic plot of incorporation of 32 P-phosphate (circles) and 3H-choline (squares) into lecithin (e,m ) and sphingomyelin (0 ,0 ) during logarithmic growth of STU 51A-232B mouse cells. Pulse period: O-12 hours.

1

plotted

20

versus

and sphingomyelin is

the

reached

chase.

(see Figure lecithin

phorylcholine

scale

3 hours

precursor

true

From this

and sphingomyelin pools.

Figure

2a the

are compared. The maximum and that of 32 P-sphingomyelin

The same holds 2b).

In

time.

the

for

observation must

have

When the

1348

maxima

it different

prolonged

32 P labels 32p,

of

20 hours

of the

3H

can be concluded immediate

phos-

incorporation

of

BIOCHEMICAL

Vol. 47, No. 6, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a)

1I

1.5106 10 12

16

20

hours

Figure

24

26

32

cd cell growth

reaction

into of

sphingomyelin

phosphorylcholine

as discussed

by Kennedy'

one would

slope

radioactivity

curves

of the

radioactivity

obviously

not

the

Compartementation unlikely,

under

given

the pool

of

2.4

26

of cell

32

growth

CDP-choline since

growth

expect

36

-

result

begins

from

into

a distinct

of the

at that

to decrease.

the

lecithin

change

of sphingomyelin

time

This

the

is

pool

of sphingomyelin

two cell

cycles.

Such a big

phosphoryl-3H-choline

in the

as common precursor doubling

conditions

of lecithin

throughout

would

incorporation

lecithin

precursor

myelin

20

is

case. of

be rather

cursor

16

hours

32 P-phosphoryl-3H-choline

when the

12

Linear plot of (a) incorporation of 32 P-phosphate into lecjthin (0) and sphingomyelin (01 and (b) incorporation of H-choline into lecithin (m) and sphingomyelin (0) during logarithmic growth of STU 51A-232B mouse cells. Pulse period: O-12 hours.

2

reversed

10

36

-

time

was about

exhausted

precursor

experiment

lo fold is 1349

enough pool

to

cells

The pre-

3 hours

whereas

to

for

which

higher

lecithin.

mouse

12 hours.

within

is big

about

of the

appears

last

nearly 3zp,

contains

amounts Therefore

the

than

sphingo-

a transfer

Vol. 47, No. 6, 1972

Table

1

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

32 P-phosphate of 3H-choline to lecithin and sphingomyelin of STU 51A-232B mouse cells.

Ratios of absolute counts incorporated into cellular during logarithmic growth Pulse period: O-12 hours.

Quotient of of 3H/32 P-ratios Lecithin /Sphingomyelin

3 H/ 32 P-ratios of Lecithin Sphingomyelin

hours of cell growth

7.00 8.50 10.00 11.00 11.75 13.00 14.00 17.00 23.50 27.00

75.3 72.9 68.9 67.8 65.5 63.7 59.5 56.5 54.6 57.0

of phosphorylcholine

80.4 76.0 73.3 73.3 71.1 69.3 64.7 62.9 59.2 58.6

from

0.94 0.96 0.94 0.92 0.92 0.92 0.92 0.90 0.92 0.97

lecithin

to the

free

sphingosine can be suggested. To prove this 3 H/ 32 P ratios of lecithin and of sphingomyelin in table

1 for

various

times.

to 32 P-phosphate

at any time

Identical

two lipids

in

about

constant.

shortly

after

the

constant

the

Since the

the

chase

quotient

of

interpreted only in terms 32 P-phosphoryl-3H-choline %hen

lecithin

specific Zilversmit relationships:

is

the

between and the

while the

pools

which

listed

both

quotient

are nearly

of the in

precursor

of

lipids

must

1350

of 'HTcholine

7 and 27 hours

predicted activity

ratios

lecithin

were increasing lipids

are

for

the

can be long

living

unknown.

sphingomyelin obey

is

were decreasing

Other

such as lecithin

specific

are

ratios

cooperation.

have been the

the

those in sphingomyelin 3 H/ 32 P ratios of both

of direct

of

we calculated

the

radioactivities

immediate

radioactivities et al. 9 which at first

As expected

or N-acylated

the criteria

of

precursor-product

of the

precursor

will

Vol. 47, No. 6, 1972

be greater the

than

product.

precursor

of the

The point

activity

criteria

state

that

and product

specific the

BIOCHEMICAL

lecithin

cross

of Zilversmit In

Figure

the

limits

myelin

result

precursor myelin)

before

that

lecithin

is

one.

The participation

same moment

versus

error

in

from

a precursor

the

that curves

for

which

the

method

of Bartlett6

in Figure

cross

growth.

3, i.e.

of both the al. 9 . An

when the

the

its

than

et

product curve (sphingolo,11 maximum , would mean but

one immediate

not

the

immediate

precursor

would

b) /‘\* I/

hairs

Figure

3

\1

of16cell

&owOf-

Plots of specific radioactivities of 32 P-phosphate (a), and 3H-choline (b) incorporated into lecithin (I) and into sphingomyelin (0). Deviations indicate the absolute error. 1351

of of

the

case of sphingo-

determination

of sphingomyelin

of more

of cell

of Zilversmit

plots

attains

time

chemical

criteria

will

product

which

by the

of the

(lecithin) the

be the

than

activity/time

were plotted

predominantly

curve

specific

less

is a maximum. According to Reiner et al. 9 are not restricted to steady 32 3 the specific P and 3H activities of

of absolute

interpretation

the

will

small amounts of phospholipids 32 P and the 3H plots obey the extreme

subsequently

product

and sphingomyelin

Within

product,

at which

of the

systems.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lo

BIOCHEMICAL

Vol. 47, No. 6, 1972

not

the criteria by Reiner lo . It

fit

is

cells

evident, is

of

sine.

The biosynthetic

other

investigators

biosynthesis ments

that

sphingomyelin

by a sofar

of phosphorylcholine

from

might

the

growing

immediate

to sphingosine of

sphingomyelin

in the enzyme

in

observed

contribute

of sphingomyelin

to demonstrate

not

lecithin

pathways 2,5

et al. 9 as has been emphasized

Zilversmit

therefore,

synthesized

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity

only cell

transfer

or N-acylsphingoas discussed

to a minor

system in

mammalian

a cell

part

to the

used by us.

Experi-

extract

progress. REFERENCES 1. 2. 3. 2 4: 8. 9. lo. 11.

Kennedy, E.P., Fed&ration Proc., 16, 847 (1957). Scribney, M., and Kennedy, E.P., J.Biol.Chem., 233, 1315 (1958). Carter, H.E., Fujino Y., J.Biol.Chem., 221, 879 (1956). Groom, V., Scribney, M., J.Lipid Res., 6, 220 (1965). Brady, P.O., Bradley, R.M., Young, O.M., Kaller,H., J.Biol.Chem., 240, PC 3693 (1965). Bartlett, G.R., J.Biol.Chem., 234, 466 (1959). Folch, J., Lees, M., Sloane Stanley, G.H., J.Biol.Chem., 226,497 (1957). Bray, G.A., Anal.Biochem.,l, 279 (1960). Zilversmit, D.B., Entenman, C., and Fishler, M.C., J.Gen.Physiol., 26, 325 (1943) . Reiner, J.M., Arch.Biochem.Biophys., 46, 53 (1953). Radin, N.S., Nucleonics, I, 51 (1947).

1352

by

are

in