Biosynthesis of α-linolenic acid by disrupted spinach chloroplasts

BIOCHEMICAL

Vol. 51, No. 2,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

BIOSYNTHESISOF a-LINOLENIC ACID BY DISRUPTED SPINACUCULOROPIASTS1

Bruce S. Jacobson, C. Gamini Kannangara and P. K. Stumpf Department of Biochemistry and Biophysics University of California Davis, California 95616 Received February 9, 1973 SUMMARY A disrupted spinach chloroplast preparation readily synthesized [14C]olinolenate from [2-14C]ace te under anaerobic conditions. It can be shown by degradation data that [ El Cloleate is not a precursor of [14C]linolenate and that cis 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [14C]linolenate.

INTRODUCTION For someyears, widence has been submitted suggesting a sequential desaturation and finally

of stearic

acid (as an activated

to a-linolenic

acid (1).

described which convert stearyl CoA to linoleyl a-linolenic

acid from linoleic

to oleic,

linoleic

While a number of enzymes have been

CoA to oleyl

CoA (S-7), the direct

derivative)

CoA (2-4) and in turn oleyl

demonstration of the formation of

acid by an enzyme preparation has not been

made. Recently,

it was observed that intact

ach leaves synthesized oleic, (8).

In addition,

without affecting

linoleic

cyanide inhibited a-linolenic

formed only palmitic

?his

and linolenic

from immature spin-

acids from f14C]acetate

the formation of oleic and linoleic

acid synthesis.

acids

Disrupted chloroplasts

and stearic acids under similar conditions.

this communication, we shall report system that readily

chloroplasts

In

on a modified disrupted chloroplast

forms "C-a-linolenic

acid from [2-14C]acetate.

Evi-

work was supported by NSFGrant GB-19733Xand NIH Grant GM-19213-01.

Copyright @ 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved.

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Vol. 51, No. 2, 1973

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dence would suggest that the precursor of a-linolenic hexadecatrienoic

acid and not oleic acid or linoleic

acid is cis 7,10,13acid.

MATEKIALSANDMETHODS Intact market.

chloroolasts

The isolation

were isolated from spinach purchased at the local mediumconsisted of 600 mMsorbitol,

60 mMNaHC03,

10 mMK2HPO4, 1 mMMgC12, 0.1% Na ascorbate, and 100 mM methyl) methyl glycine and was adjusted to pH 7.9 with NaOHat room temperature.

Mature spinach leaves were chopped in a blender with an equal

volume of isolation

medium. The mixture was filtered

and the resultant chloroplasts

filtrate

for 45 set at 3600~. The pelleted

were brought to 1 mgiml of chlorophyll

volume of isolation chloroplasts

centrifuged

through miracloth

with an appropriate

medium. At this stage there were about 70-80% intact

as determined by phase contrast microscopy.

suspension was then disrupted

The chloroplast

in a French pressure cell at 15,000 psi

to yield a pressate of stroma mixed with small fragments of lamellae.

All

the above procedures were conducted at O". The reaction

mixture for acetate incorporation

pressate (600 ug chlorophyll)

consisted of 0.6 ml

and in umoles: NADP, 0.5; NASH, 0.5; glucose-

6-phosphate, 4.0; ATP, 2.0; dithiothreitol,

1.0; coenzyme A, 0.2; 0.15

units glucose6-phosphate dehydrogenase and 0.5 mg Escherichia coli ACP; the final

volume was 0.8 ml.

The amount of [2- 14Clacetate in the reaction

mixture was 1 uc (22.4 UC per umole).

The reactions were run in the dark

at 13O for 1 hr and stopped by addition Lipid extraction,

methyl transesterification

chromatogranhy and scintillation described (8). stearic

Nicolaidis

(9) and reductive

, gas-liquid

and thin layer

counting were conducted as previously

Chemical a-oxidation

acid by Pt02/H2 reduction)

of Harris --et al.

of 0.2 ml 10 N H2S04.

of linolenic

acid (first

converted to

was carried out according to the procedure ozonolysis by the method of Stein and

(10).

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BIOCHEMICAL

Vol. 51, No. 2,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION While lenic

the most prevalent

acid,

its

synthesis

by disrupted,

fragmented

demonstrated.

However,

immature

spinach

appropriate

the synthesis

spinach

chloroplasts.

[Z-l4 Clacetate pressates,

able

at 0'.

of labeled to detect

when pressates synthesized

were all

the

The following ic acid (1)

formed

fatty

synthesis for

fatty

observed

acids

As illustrated with

from a trace the pressformation,

al-

for

In

high

percentage

we have never fractions

alone,

i.e.

x g the supernatant

1 except

the structure

been

linolenic

and origin

acid.

of a-linolen-

pressate.

in Figure authentic

To date

in Figure

from

incorporation.

90 min at 100,000

elucidates

by the chloroplast

18:3

important

by stromal

to

With different

varied

greater

the

from mature

of time

Clacetate

acids.

Under

synthesized

1.

acid

were

centrifuged

evidence

graphs

[Z-

of protein

acid

acids

the length

14

(8).

from

was possible

in Figure

favored

in total

unsaturated linolenic

of fatty

upon

heen previously

by pressates

a-linolenic

storage

concentrations

acids

depicted

to depend

Longer

it

a-lino-

acids

chloroplasts acid

above,

pattern

of labeled

was a decrease

high

a-linolenic

fatty

is

intact

is fatty

has not

that

described

A typical

and appeared

there

addition,

synthesize

of unsaturated

the percentage

ate was stored

yields

did

chloroplasts

polyunsaturated

preparations

has been shown

by the pressate

to 30 percent

though

it

in spinach

of other

chloroplast

conditions

observe

acid

and that

leaves

isolating

fatty

1,

[14C]methyl

methyl

a-linolenate

a-linolenate

co-chromato-

on a 10 foot

HI-EFF-ZBP

column. (2)

The [14C]product nated

silica

Therefore, (3)

co-chromatographed G TLC plates

the

The TLC purified methyl

a-linolenate

with

[14 Clproduct

on 7% silver authenic

contained

[14C]product

again

on a HI-EFF-ZBP

489

methyl three

nitrate a-linolenate.

double

co-chromatographed column.

impreg-

bonds. with

Vol. 51, No. 2, 1973

(4)

After

catalytic

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AND BIOPHYSICAL RESEARCH COMMUNiCATIONS

reduction with Pt02/H2, the [14C]product co-

chromatographed tith

methyl stearate.

Thus the [14C]product

had a Cl8 chain length. (5)

Reductive ozonolysis of the [14C]product gave a single [14C]peak coincidental that the first

with methyl azelate semialdehyde, thus establishing double bond in relation

was in the 9,lO position. ["Cl

to the carboxylate

carbon

Furthermore, over 90%of the total

was associated with the methyl azelate semialdehyde peak.

The short chain aldehydes and malonic dialdehyde had little any [14C] incorporation. been related

Since this low recwery

to the volatility

Figure 1.

might have

of the products, further

.

INJI

if

degrada-

llON

Gas liquid chromatography pattern of methyl esters of fatty acids synthesized by spinach chloroplast pressates from [2-14C]Chromatography was performed on a 10 ft chromosorb 15% acetate. HI-EF'F-2BP column at 180“. (A) Palmitate, (B) Stearate, (C) Hexadecatrienoate, (D) Oleate, (E) Linoleate, (P) Linolenate.

tion was carried out by chemical a-oxidation

of the fully

reduced

[14C]product , namely [14C]stearate . (6)

As summarized in Table I, chemical a-oxidation

490

of the [14]stearate

BIOCHEMICAL

Vol. 51, No. 2,1973

Table I.

Localization

AND BIOPHYSICAL

of [14C]label

oleate synthesised from [2-

RESEARCH COMMUNICATIONS

in the carbon chain of linolenate

14 Clacetate by chloroplast

pective acids were purified

pressates.

and

The res-

by AgND3-TLCand reduced to stearate with

Pt02/H2 and subjected to chemical a-oxidation

with KIMnO (9).

Products of chemical a-oxidation Specific activity (DPM/ug)

f14ClProduct

Rxperiment

18:0

17:o

16:0

15:o

Linolenate

1.

1.58

1.54

0.12

n.d.

2.

4.04

3.90

0.27

n.d.

1.

2.08

1.99

1.67

1.71

ohate

*

*

n.d. - not detected.

revealed that the [ [2-14C]acetate, ate.

14 Cl derived originally

was essentially

In sharp contrast,

catalytic

from the substrate,

located on the Cl7 of [14C]stear-

the [14C]stearate which was obtained by

reduction from [L4C]oleate synthesized by the chloro-

plast pressate had its

[14C]1abel distributed

along the Cl8

hydrocarbon chain.

Reductive ozonolysis of [14C]oleate obtained

from [2-14C]acetate

by the pressate system had 55%of its

[14C]

in n-nonanal and 45% in the methyl azelatesemialdehyde peak. Clearlv,

then, the chloroplast

pressate had synthesized a-linolenic

by a chain elongation of one C2 unit to a pre-existing ably cis 7,10,13-hexadecatrienoic

acid, an acid

present

precursor,

acid

presum-

in spinach chloro-

plasts. To analyze this possibility

further,

of anaerobic conditions

advantage was taken of the effects

(N2 phase),CN anion, FMN, and FAD on the biosynthesis of oleic acid by chloroplasts (8,11,12). Table 11 clearly shows that under

491

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Table II. fatty

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pattern of incorporation

of radioactivity

into long chain

acids from [14C]acetate by pressates of chloroplasts

under different

gas phases.

Incubating condition

Total DPM in lipids

16:O

Percent Radioactivity 18:O 18:l 18:3

air

22,170

29.5

26.8

13.0

30.7

O2

22,380

30.5

24.4

24.3

20.9

N2

17,760

35.3

35.3

0

29.4

Effect

Table III. activity

of inhibitors

into long chain fatty

on the pattern of incorporation

of radio-

14 acids from [ Clacetate by pressates of

chloroplasts.

Incubating Condition

Total DPM in lipid

16:0

Percent Radioactivity 17:o 18:0 18:l

18:3

control

522,200

18.5

4.8

46.8

26.3

2.9

0.1 mMKCN

595,500

12.8

5.7

75.1

4.3

1.4

0.1 mMFMN

529,100

16.8

4.0

74.2

0.4

4.0

0.1 mMFAD

512,100

16.7

3.3

76.0

0.4

2.5

aerobic conditons, (9,12,15),

[14C]acetate was readily

incorporated

while under anaerobic conditions,

ceased but the synthesis of [ 14C]18:3(9,12,15) III

summarizes the data on the effect

of unsaturated fatty inhibited Finally

acids.

oleate biosynthesis,

into 18:1(g) and 18:3

the synthesis of [14C]oleate was not inhibited.

Table

of Cd, PMN, and FAD on the synthesis

Again, whereas these three compoundsmarkedly no decrease in linolenate

synthesis occurred.

it should be pointed out that in Figure 1, no formation of linoleic

492

BIOCHEMICAL

Vol. 51, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acid, a possible precursor of linolenic In conclusion,

the chloroplast

acid,was observed.

pressate contains all

the enzymes re-

quired for the flow of [14C]acetate into long chain acids including biosynthesis of oleate and linolenate. be inhibited

the

Since the formation of oleate

under conditions where no linolenate

inhibition

can

was observed,

since the degradation data fully ing a striking linolenate, the rupted

pressate

difference

support the inhibition studies by reveal14 in the distribution of [ C] in oleate and in

and since there was no indication

of linoleic

system, we conclude that a-linolenate

chloroplast

hexadecatrienoic

acid synthesis by

is formed by the dis-

system by the elongation of endogeneous&

7,10,13-

acid which remains unlabeled under these conditions.

Palmitate cannot be considered as the Cl6 precursor since under anaerobic conditions, chain (12).

double bond systems cannot be introduced into the hydrocarbon Whether or not this sequence of biosynthesis of linolenate

occurs in the plant cell remains for further These data do explain,

however,

and Stumpf (8) with intact

the

investigations

observations madeearlier

to determine. by Kannangara

chloroplasts. ACKNOWLEDGNENTS

The authors wish to acknowledge the helpful discussion of Dr. Jan Jaworski and technical assistance of Mrs. Barbara Clover. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

Hitchcock, C. and Nichols, B.W., Plant Lipid Biochemistry. Academic Press, NewYork (1971). Holloway, P.W. and Katz, J.T., Biochem. ll, 3689 (1972). Oshino, N., Imai, Y. and Sato, R., Biochim. Biophys. Acta 128, 13 (1966). Nagai, J. and Bloch, K., J. Biol. Chem.243, 4626 (1968). 360 (1972). Vijay, I.K. and Stumpf, P.K., J. Biol. Chem.H, Baker, N. and Lynen, F., Eur. J. Biochem. l9, 200 (1971). Ben Abdelkader, A., Cherif, A., Demandre, C. and Mazliak, P., Eur. J. Biochem. 32, 155 (1973). Kannangara, C.G. and Stumpf, P.K., Arch. Biochem. Biophys. 3, 414 (1972). Harris, R.V., Harris, P. and James, A.T., Biochim. Biophys. Acta 106 465 (1965). Stein, R.A. and Nicolaides, N., J. Lipid Res. 2, 476 (1962). Given, C.V. and Stumpf, P.K., Plant Physiol. 2, 510 (1971). Stumnf, P.K. and James, A.T., Biochim. Biophys. Acta 70, 20 (1963).

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