Biosynthesis of spin-labelled phospholipids. Enzymatic incorporation of spin-labelled stearic acid into phosphatidic acid

Vol. 46, No. 1, 1972

BIOCHEMICAL

BIOSYNTHESIS ENZYMATIC

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF SPIN-LABELLED

INCORPORATION

PHOSPHOLIPIDS.

OF SPIN-LABELLED

INTO PHOSPHATIDIC N.Z.

Stanacev

STEARIC

ACID

ACID

and L. Stuhne-Sekalec

Department of Pathological Faculty of Medicine, University

Chemistry, of Toronto, Canada

Banting Institute, Toronto 181, Ontario

and S. Schreier-Muccillo* Biochemistry

Received

October

Laboratory, Ottawa,

and Ian

National Ontario,

C.P.

Smith

Research Council Canada KlA OR6

of Canada

18, 1971

SUMMARY Guinea pig liver microsomes were tested for their capability to catalyze the incorporation of spin-labelled stearic acid into A system utilizing sn-glycero-3-phosphate-2-'4C, phospholipids. and microsomal fractions isolated from 12-doxyl-stearic acid, identified as phosphatiguinea pig liver yielded a phospholipid, These experiments die acid containing 14C and spin label. established the capability of a mammalian enzyme system to utilize spin-labelled stearic acid in the biosynthesis of a phospholipid. INTRODUCTION Recent systems study

have

Although *

spin-labelled

of the it

Canada-Brazil de Quimica Sao Paulo,

the

has been

steroids

shown that

membranes

degree

acids

Press, Inc.

the

(1,2). have

been

crassa

(j-10). (11)

Visiting Scientist; Permanent address: da Universidade de Sao Paulo, Caixa Postal Brazil.

0 1972 by Academic

in

of organization

of membranes

Neurospora

114 Copyright

approach

and fatty

of the

components

to biological

of the

of biological

indicators lipid

probes

usefulness

organization

to be sensitive

and fluidity

of spin-labelled

demonstrated

of molecular

In particular, found

applications

and

Instituto 20780,

Vol. 46, No. 1,1972

Mycoplasma for

the

BIOCHEMICAL

laidlawii formation

of a mammalian

of

can utilize

lipid(s), system

into

phosphatides

is

We report

here

microsomes

is a key

in

for

the

the

2.5

umoles

of 8.04

x lo5

dimethyloxazolidine referred fied,

derivative

buffer

(pH 7.0),

umoles

of L-cysteine,

of Triton-X100

and 0.5

as described

according

ml.

The reaction

mixture

labelled

lipid

The purification obtained

by silicic

purification acid

column

a specific

of the

50.0

ymoles

umoles 0.1

35.9

nmoles/mg

of phosphate 10.0

of CoA, 2.0

microsomes

was incubated

incubation

puri-

of NaF,

mg of protein)

of the

henceforth

prepared,

umole

liver

N-oxyl-4',4'-

acid,

(3);

(prepared

in

a final for

and isolation protein

mg

30 minutes

of

lipids

of the biosyn-

was obtained. and characterization

was carried

Partial

(15),

with

2),

pig

7.1

isolated,

Fig.

of ATP,

ml of guinea

to Bligh-Dyer

thesized

umoles

termination

contained:

(13)

(see

50.0

and containing

After

phospholipids.

acid

pmoles

of MgC12,

(13)

of 1.2

at 230.

4.0

which

as favorable

of 12-keto-stearic acid

umoles

fatty

described

(14)

2.0

as described

6.0

liver

acid,

(prepared,

previously

to as 12-doxyl-stearic

volume

to that

dpm/umole);

and characterized

sub-

AND DISCUSSION

of sn-glycero-3-phosphate-2-'4C

activity

ability

pig

of complex

of phosphatidic

as described

guinea

as phosphatidic

similar

synthesis

and characterized

the

for

of a spin-labelled

biosynthesis

system,

enzymatic

that

incorporation

EXPERIMENTAL The incubation

acid

a spin-labelled

evidence

identified

intermediary

fatty

lacking.

the

a phospholipid,

evidence

incorporate

experimental

can catalyze

into

to

RESEARCH COMMLJNKATIONS

a spin-labelled

conclusive

enzyme

strate

acid

(12)

AND BIOPHYSKAL

out

of labelled

lipid

thus

as follows: of biosynthesized chromatography.

115

lipid(s) After

washing

was achieved with

chloro-

Vol. 46, No. 1,1972

form,

acidic

When 2.5 jected

lipid

umoles

of the

labelled

linear

partially

compound

-

chloroform:methanol purified

(1:l).

labelled

chromatography

exhibiting

lipid

was sub-

as described

paramagnetism

of NH ,,-acetate

- -----

E

with

column

gradient

600

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was eluted

to DEAE-cellulose

a single with

BIOCHEMICAL

as shown in

(14),

was eluted Fig.

1.

The

WIN LABEL

A L 4 ,4OO? 8 ? 8 v, 200 Y

0

IO

20

30 FRACTION

40

50

55

Figure 1. DEAE-cellulose column chromatography of spin-labelled, obtained by a large scale preparation. The 14C-labelled lipid, preparation of labelled lipid and the chromatographic procedures are described in the text.

recovery

of the

eluted

compound

impregnated (16),

lipase gave

(SG-81)

acid

D). in

Mild

97.7% yield

spot

was 93.2%.

to thin-layer

(obtained

in

(representing

alkaline

from

a water

egg-yolk

soluble

These "C

(14)

solvent

more

than

was converted

the

of acid systems

90% of

standard

lecithin

with

of the

labelled

product

experiments

116

four

from

hydrolysis

An aliquot and silicic

indistinguishable

as 3-glycerophosphate. glycero-3-phosphate-2-

column

chromatography

a radioactive

radioactivity)

phosphatidic

the

when subjected

paper

gave

applied

14C from

-of phosphocompound

which

was identified

established

that

with

the

microsomal

sn-

Vol. 46, No. 1, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r

n

INCORPORATED INTO GUINEA PIG LIVER MICROSOMES

Figure 2. (A) ESR spectra of buffer solutions of guinea pig liver microsomes biosynthetically spin-labelled in the lipid portion with 12-doxyl-stearic acid. (B) Control (with heat treated guinea pig liver microsomes). enzyme

system

stearic

acid. Electron

instrument

resonance

are

shown in

The ESR spectrum tive

of rapid,

with

a correlation

time

2(A)

Figs.

Spectra

and

to be less

3(A)

of the

isotropic, of approximately

after

is of

obvious spin

1% of the

amount

12-doxyl-stearic extracted

(B).

spin-labelled

117

It

phospholipid and

washed

treatment,

amount than

E3

of the

(B).

a minimal

spin-labelled

12-doxyl-

on a Varian

by heat

Spectra

of the

effectively

(17).

only

estimated

purified

were taken

in Figs.

biosynthetically.

and of the

containing

inactivated

processes

can be retained,

also

described

compared

by non-enzymatic

microsomes

spectra

techniques

are

incorporated acid

spin

acid,

and of microsomes

incorporation,

label

phosphatidic

using

microsomes,

that

into

motion

microsomes

is

of the

spin

3 x lo-'

seconds.

indicalabel

from

Vol. 46, No. 1,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IZ-DOXYL

STEARIC ACID

CHROMATOGRAPHED PHOSPHOLIPIDS

B

of chloroform:methanol:water Figure 3. ESR spectra volume) solutions of: (A) 12-doxyl-stearic acid, 10D4 M. (B) Intact spin-labelled lipid purified on the column (Fig. 1, fraction 38).

(1:2:0.8

by

DEAE-cellulose

CONCLUSION Data first

presented

time

can serve tidic that

acid the

fatty

spin-labelled

as the

substrate

nature of

Studies

communication

for

the

system.

is

in

are designed

regarding

acid

as precursor

the

application

in the

acid) of phospha-

Moreover,

they

demonstrated

label

into

more

of spin-labelled of

a substrate

to the

to gain

biosynthesis

have

complex

of

enzymatic

experimental phosphatidic

phospholipids

in

membranes. ACKNOWLEDGEMENTS

This Research

study Council

was supported of

Canada

in part and the 118

the

formation

acceptable

system.

for

enzymatic

a perturbation

progress

established

(12-doxyl-stearic

of a nitroxy-spin

a mammalian

detail

have

fatty-acid

a mammalian

introduction

catalysis

this

that

in

acid

biological

in

by grants

Ontario

Heart

from

the

Foundation

Medical to

Vol. 46, No. 1,1972

N.Z.

BIOCHEMICAL

Stanacev,

National

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and by a National

de Pesquisas

fellowship

Research

Council-Conselho

to S. Schreier-Muccillo.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9.

10. -11.

McConnell, H.M. and McFarland, B.G., Quart. Rev. Biophysics, 3, 91 (1970). Applications of Electron Spin Smith, I.C.P., in "Biological Resonance", edited by H. Swartz, J.R. Bolton and D. Borg, J. Wiley & Sons (1971), in press. Waggoner, A.S., Kingzett, T.J., Rottschaefer, S., Griffith, O.H. and Keith, A.D., Chem. Phys. Lipids, 3, 245 (1969). Libertini, L.J., Waggoner, A.S., Jost, P.C. and Griffith, O.H., Proc. Natl. Acad. Sci. U.S., 6& 13 (1969). Hsia, J.C., Schneider, H. and Smith, I.C.P., Biochim. Biophys. Acta, 202, 399 (1970). Butler, K.W., Dugas, H., Smith, I.C.P. and Schneider, H., Biochem. Biophys. Res. Commun., 9, 770 (1970). I.C.P. and Schneider, H., Biochim. Butler, K.W., Smith, Biophys. Acta, 219, 514 (1970). Hsia, J.C., Schneider, H. and Smith, I.C.P., Chem. Phys. Lipids, 4, 238 (1970). Hsia, J.C., Schneider, H. and Smith, I.C.P., Can. J. Biochem., ‘I&

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