Fat Metabolism in Higher Plants
XXVII. Synthesis of Long-Chain Fatty Acids by Preparations
of Hordeum Vulgare L. and other Graminae'
2
J. C. Hawke" and P. K. Stumpf
Department of Biochemistry and Biophysics, University of California, Davis, California
In general, the leaf lipids contain high proportiolls of oleic, linoleic, linolenic, and palmitic acids
(9). However, seedling tissues of a number of
plant species, e.g. sorghum. flax, maize. barley are
able to utilize acetate-C14 for the synthesis of appreciable amounts of the C20-C2 long chain fatty acids.
The saturated fatty acids in this range of carbon numbers are minor components (approx. 0.5-2.0 %) of
most plant lipids although in some cases individual
long chain fatty acids comprise an appreciable proportion of the total fatty acids. e.g. the nut oil of
Lop/hira alata is reported to contain 14.2 % behenic
acid (17). The fatty acids with 26 or more carbon
atoms are considered to be constituents of monoester
waxes rather than glycerolipids.
The C20-C24 fatty acids are relatively abundant
ill the cerebrosides of the mammalian brain and nerv-ouIs systemii. The biosvynthesis of lignoceric from
acetate-I-C'4 lhas been investigated in rats (5. 7).
The data of Futilco anld Mea(l (5) is consistenit with
the de lnovo synithesis of this fatty acid from acetate.
However, Hajra anid Radini (7) and \Vakil (31)
have presented evidence supporting a miiechanism of
clhaini elongation.
Sinlce uniformii Hordeuimt zvulgaris seedlings were
readily obtained in large quanitity this tissue was selected in the present work to study the incorporation
of C14-label from acetate-i-C'4 into long chain fatty
acids although preliminary experiments will be cited
to show that this capability is not restricted to this
particular tissue. A preliminary account of this work
has been presented elsewhere (8).
Seedling tissues of the following members of the
Gr-au'iniae family were used in these experiments:
Hordemn zulgare, L. (var. California mariout), Triticunm aestiviutmn L. (var. Ramona 50), Aventa sativa
L. (Curt), Zea wi1ays, L. (var. De Kolb 895), Lalium
perennne, L. Seeds were germinated in the dark at
room temperature on vermiculite which had been
saturated with distilled water, either in open trays or
in sealed Mason jars. When open trays were used,
the seeds were dusted with Semesan fungicide.
Since seedlings grown in open-i trays for 6 to /7 days
are contaminated vith bacteria. all later work was
carried out with seedlings grown in Mason iars
under sterile conditionis. MIason jars with the
required quantity of moistened vermiculite were
sterilized for two 45-minute periods at 24-hour intervals. Seeds which had been dipped in 95 % ethanol
and immersed on 0.5 % sodium hypochlorite for 30
minutes were transferred to the jars within a transfer cabinet. The lids of the Mason jars were provided with a one-inch hole, which was plugged with
cotton before sterilization of the jars. To check the
procedure, unwashed shoots of 7-day-old seedlings
growln under these conditions ^vere immersed in a
nutrient medium for 3 days at room temperatuire.
Fuingal growth occurred at the point of attachmenit
of the shoot to the seed in approximatelv 10 % of the
shoots but nlo bacterial growth vas observed. To re(lt1ce the possibility of fuilgal contamination about
oile-half inich of the shoot was left attached to the
seedv
wheni the shoots were lharvested. The remaininig
slhoot miiaterial was waslhed several timiies vith distilled
\-ater to remove vermiiiculite particles.
For the incubation of tissue with acetate-1-C'4
cross-sectional slices approximiiately 1 mm thick were
cut by hand with a sharp razor blade and imllmediately immersed in the appropriate 0.1 M buffer.
To prepare plastid and chloroplast fractions, barley tissue was ground at 5° in a mortar with sterile
sanid and Nvith 2 volumes of a solution of 0.5 At sucrose, 0.01 M ascorbate, 0.01 WI NaCl, 0.01 M K2HPO. 0.001 Ni mercaptoethanol, and 0.001 AI EDTA.
The homogenate (pH 6.35-6.5) was then squeezed
through 4 layers of cheesecloth, centrifugedl in a
RC-2 Sorvall refrigerated centrifuge at 400 X g for
1 minute and the supernatant fraction centrifuged at
4000 X g for 10 minutes. The precipitate was resuspended in the homogenizing medium and recentrifuged at 4000 X g to sediment the plastid alndl chloroplast fractions. The fractions were resuspended in
0.25 M sucrose and 0.1 At potassiuml)phosphate buffer
at pH 7.4 and used promptly. Protein was determined according to the procedure of Gornall et al.
Received May 14, 1965.
2 Tihis work was supported in part by grants-aid from
NIH GM10132-03 an(d NSF G14823.
3 Present address: Department of Chemistry and Biochemistry, Massey University of Manawatu, Palmerston
North, New Zealand.
A similar procedure was used to prepare subcellular fractions of barley tissue to investigate the
distribution of labeled fatty acids following incubatioll of the tissue with acetate-I-C'4. In this ecxperiment a particulate fraction, consisting of plastids
and mitochondria. was .sedimented at 14.000: X g.
Materials and Methods
I
(6).
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1023
Copyright © 1965 American Society of Plant Biologists. All rights reserved.
12PLANT PHYSIOLOGY
1024
The supernatanit fraction was thenl centrifuged at
1 10,000 X g for 60 minlutes to give a microsomiial
sedimlenlt and a supernatanlt fractionl freed of mlicrosomles. The labeled fatty aci(ds were (letermiiined in
the l)articulate, imicrosomal al(l sul)ernatant fractiols.
together wvith the total cell debris obtained after
homogenlization. The supernatalnt was evaporated to
dryness in a rotaryr evaporator and(I resuslpended in a
smliall volume of water.
C Uhicals (a)1d( Sutbstra tcs. The following comiipounds w-ere obtaine(d froim commiiiiercial sources:
ATR. CoA, DPNH. I'P-. GSH. glulcose-6-p from
Sigmla Chemlical Conmpanyv sodiunm acetate- 1 -C1 4
(2 ttc/0.05 /mole) froml N\ew England(I Nuclear Corporation.
Itic1lbationI of Tissuecs au1(1 SuIbcellmal(1- I'ractioiis.
Unless otherwise stated the incubation of tissue wvith
suibstrate x%vas in the proportionls 1.0 g sliced tissue
(fr wvt). 200 ,umoles potassium phosplhate at pH 7.4.
and 50 umoles KHCO3 in a total volume of 2.0 ml.
W\hole shoots wvere incubated wvith acetate-1-C'4
with the cut ends immersed in the incubationi mixture.
The shoots were cut un(ler water imnle(liatelv before
the incubation. A streanm of warm air was blown
over the leaves to assist the uptake of soltitioil.
Fatty acid svnthetase activity was testedl in clhloroplasts and l)lastid fractionis witlh reactioni miiixtuires
similar to those (lescril)ed for avocado (25. 27). The
reactioni systemi containied the following cofactors
(Mmnioles of eaclh): ATP, 5.0; CoA, 0.15: DPNH.
0.25; TPN-. 0.15: glutcose-6-), 0.25: GSH. 5.0:
MnCl,, 1.0; KHCO:,. 15.0; an( Tris-HCI huffer.
100.0. Incubationis were carrie(l otut at 300 uin(ler
aerobic conditions. with shaking.
Toalysis of I$u,(ldogciious and(i Labcled IT(atty Acids.
Total lipids w-ere extracted with AMeOfl :CHCl, according to the mlethodI of Bligh anid Dyer (2). The
comiiponent lipids w ere fractionated into polar and
nonl)olar lipids by preparative thin-layer chromiiatography (TLC). Silica gel G (E. Merck anid Conipany, Germany ) was spread on glass plates by the
method of Lees and )e MAulria (14 ) ain(I activated
for 2 hours at 1100 before uise. The lipids were applied as a streak at the origin together with spots of
the aj)propriate stanidards (15).
Three solvent mlixtures were used in the following sequience to separate the major classes of lipid:
A) toluene ethyl acetate ethanol ( 10 :5 :5, by volume).
B) hexane:diethvl ether:acetic acid (70:30:1, by volume) and C ) hexane :benzene ( 1:1. by volume).
Solvent mixture (A) was used to isolate the phospholipids and other polar lipids (RF. 0-0.05), digalactosvlglycerides (RJ,. 0.2) and monogalactosylglycerides (RF 0.6). The remiaining neuitral lipids
chromlatograplhed at or near the solvent front (RF.
0.8-1.0) and these were rechromatographed uising
solvent (B) to isolate sterols (RF,, 0.4) and triglycerides (RF," 0.8). 'I'he sterol esters and waxes
wvhich chromatographedI at the solvent front in solveint (BI were rechromatographled in solv-ent (C) to
g-ive RF's of 0.6 and 0.45 respectively. After spaycling the plates lightly with 2',7',-dichlorofluorescein
(0.2 % in ethaniol) the lipidls or lipid fractionis were
recovered fromii the chromllatographic p)lates l\- remnoving the appropriate area of silica gel anld extracting the lipid with sol-ent of suitable p)olarity unltil n1o
further radioactivity was remioved. Sterol esters.
elutedI by usinig 1 %
methaniol in diethvl ether and galactolipids anid phosp)holipids with chloroform :nmethanol :xvater (30 :1 :
0.01. bv volumle). Recoveries of radioactivity ov-er
the entire separatioll were 60 to 70 %.
.Scparatioiz and(i Ana(ly-sis of Falttly Aci(ls. Eitlher
lipid fractions, whole tissue or subcellular fractions
were saponlified with an equal volumie of I.; % metlhanolic KOH for 2 to 3lhours at 80Q. An e(lqul volunie of methanolic KGH was also uised to stol) reactions at the end of ani incubation lerio(l. After
saponification, approxiniatelv onie-half of the metlhanol was evaporated andl the samlples adjtusted to pH
1.0 with 6 N HCI. The fatty acidls were extractedl
2 timiies with aplproximatelv equial -olumiles of chloroforimi andl the extracts washed with 1 % acetic aci(l
in water, dried over anlhvdrous so(litum sutlfate aln(d
evaporatedl at 80(. 'The final I to 2 mil of chloroformii was eval)orate(l either witlh a strealmi of niitrogen or in v-actlo. Tlle fatty aci(ls w-ere then conlxverted to mnethyl esters with freshly -l)rel)ared (liazowax esters and triglycerides were
methaine.
In preliminary ex)erinlents, uinsapoiiifie(l lipid
was extracte(l undi(ler alkaline coniditionis With diethvl
ether prior to extractiol with chlloroforinx. but this
step was omitted when it was fotund that very little
labeled miiaterial was removed and that gas-li(jtuid
chromiiatograpllic patterlls \-ere the same with and
without remiioval of unsal)onifie(d lipid.
Detcrininiatioau of Radioacti-vity. Fatty acids and
lipid fractions wvere couniited in toluene containinig
0.6 % 2.5-diphenvloxazole (PPO) and 0.05 % 1.4bis-2- (5r-p)helnyloxazoly1 ) -benizenie (POPOP) with a
Packardl liquiid scintillation spectromieter.
.Yeparatioii of lieth vl Esters by, Thin -Lave-rcChroinatographl v (TLC). The radioactive fatty
acids syinthesized by the incubation of tisstues and
subcelltular fractions with acetate-l-C'4 contained apl)roximately 10 % hydroxvl fatty acids. mainly the
,8-hydroxyl derivatives of laturate and mvristate.
Sinlce the hvdroxyl esters initerfere with the gasliqtuid chromiiatographic separations of the fatty
esters, they were removed by preparative TLC oln
activated silica gel G using hexane :diethv\l ether
(6 :4. by volume) as the eluting solvent (16).
For (legradative studies and as a method of identification. methyl esters were fturther selparated into
saturated,. milonoenle, dielne anid trielne comiponents by
TLC on AgNO3-impregnated silica gel G (18). The
solvent system was hexanie :diethyl ether (6:4, by
volume).
Gas Chraiiiatograpllic amid Radioclicemical Aiialsis. A gas-liquidl clhromatograph (GLC) (Aero-
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HAWKE AND STUMPF-LONG CIIAIN FATTY ACID Bl0SY_.ThESIIS
graph, Model A-9OP2) fitted with a thermal conductivity detector was used routinely to separate
methyl esters of the fatty acids. A GLC with a hydrogen flame ionization detector (Aerograph, Model
Hy-Fi 600) was used in the quantitative analysis of
the high molecular weight fatty acids (C24-C30).
Two columns were used to separate and characterize
the methyl esters. A) A 5 foot X 0.25 inch column
packed with 12 % diethylene glycol succinate
(DEGS) on Anakrom P (60/70 mesh) held at 1650
and at a flow rate of 60 ml He/minute. B) A 5
foot X 0.125 inch column packed with 5 % methyl
silicone (SE-30) oin Anakrom P (60/70 mesh) and
held at 2400 and a flow rate of 50 ml He/minute.
The products of oxidation of unsaturated fatty
acids and methyl esters were also separated on the
DEGS column. Esters of monocarboxylic acids (C,Cl1) were chromatographed at 1400 and the esters
of dicarboxylic acids and their semialdehydes and the
aldehydes (C7-C11) at 1600.
Radiochemical analysis of methyl esters and their
oxidation products was accomplished by passing the
effluent from the GLC (MIodel A-9OP2) through a
proportional radioactivity detector (Nuclear-Chicago
Biospan Miodel 4998) at 2500. Areas under the
mass and radioactivity peaks for each fatty acid were
measured by planimetry and the values obtained used
for further calculations. A NIH standard (D) consisting of 14:0. 16:0, 16:1. 18:0 and 18:1 (11.8, 23.6,
6.8. 13.0 and 44.6 % by wt) gave relative peak areas
of 12.7. 24.6. 6.5. 13.1 and 43.1 % respectively on the
chromatogrami witlh the thermiial conductivitv detector.
Good agreemenit between relative peak areas onl
the radiochromatogramii and the liquid-scintillation
miethod was obtained for mixtures of radioactive
fatty acids. The percentage radioactivity of a mixture of C14-labeled methyl esters of myristic. palmitic.
stearic, arachidic, behenic and lignoceric acids were
respectively 8.4, 23.2, 22.6. 10.3, 16.8 and 18.7 % by
the peak area method and 9.2, 23.3, 18.8, 10.7, 17.3
and 20.7 % by liquid scintillation counting of the
inidividual fatty esters after they had been collected
in the effluent gas stream of the GLC. Purified
methyl esters for degradative studies were obtained
by collecting fractions from the GLC with the proportional counter detached. Samples were collected
in drying tubes loosely packed with glass wool which
was moistened with methanol (11).
Cheiiiical Modificationi anid Identification of Radioactize Fattyi Acids. The endogenous and C14labeled fatty acids were identified by their chromatographic behavior using the carbon number method of
Woodford and Van Gent (32). Methyl esters were
chromatographed before and after A) catalytic hydrogenation in a Parr pressure reaction apparatus,
B) bromination (12). C) catalytic cleavage (13),
and D) permanganate-periodate oxidation (29).
Degradation1 of Fatty Acids. Ozonolysis. The
method of Stein and Nicolaides (26) was used to degrade the C14-labeled monoenes. A fine stream of
12
1025
ozone from a Welshbach ozone generator was passed
into a solution of the methyl esters in dichloromethane for approxinmately 5 seconds at -60°. The
ozonides were cleaved reductively wvith triphenyl
phosphine and used directly for gas-liquid chromiiatographic and radiochemical analysis.
Decarboxylatiot. The C 4-labeled saturate(d fatty
acids (C14-C06) were decarboxylated in 25 ml Erlenmeyer flasks equipped with a cenlter wvell accordinig
to the miiethod of Brady (3).
Results
Incorporation of Acetate i11to Fatty, Acids by
Seedling Tissues of the Gramiiae FamiliZy. C'4labeled acetate was incorporated to the extent of 3.0
to 21.4 % into fatty acids by 3 to 7-day-old seedling
tissues of the 5 species of the Gramtinae family investigated (table I). Although etiolated tissue was
used in the experiments cited in table I, similar levels
of incorporation into total fatty acids were found for
green tissue of similar age which had been exposed
to light for 12 to 48 hours before incubationi with
acetate- 1-C14.
A study of the nature of the C'4-labeled fatty
acids synthesized from acetate-l-C14 showed that an
appreciable proportion (43-55 %) of the total radioactivity was arachidic, behenic anid lignoceric acids
(table II). Labeled cerotic acid was also presenit
(approx. 10 % of the total activity) but the long retentioni times for this fatty acidc made the routine
determiiination inconvenient anid the data has niot beenl
incltuded in the calculations. Table III slhows that of
the enidogenous fatty acids l)almitic acid is quantitativeely the most important saturated fatty acid and
linoleic anid linolenic the most abundant unisatuirated
fatty acids. The analysis of Loliumii pcrcnn1le seedling
tissues given in table Ill shows somewhat higher proportions of palmitic acid and lower proportions of
linolenic acid than reported for mature leaf tissue
(24, 23). The saturated long chain fatty acids comprise a comparativelv small proportion of the total
endogenious fatty acids.
In the more detailed studies with Hordenim vitigare L, similar levels of incorporation of acetate into
fatty acids was obser-ed in the range pH 6.7 to 8.0
using either potassium phosphate or Tris-HCl buffer.
Potassium phosphate buffer at pH 7.4 was used in
most experiments with barley tissue (200 ,umoles/
1.0 g fr wt). Bicarbonate was found to stimulate
incorporation slightly and was added in most experiments.
Figure 1 shows a typical GLC-C14 monitored tracing of the fatty acids synthesized by tissue slices from
barley seedlings which had been grown aseptically in
the dark. The long chaini fatty acids were characterized as fl-saturate(l fatty acids froml carboll lnumiiber calculations and cochronmatography of methyl
esters writh authentic compounds on DEGS and SE30 columns. This characterization followed isolationi
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1026
PLANT PHYSIOLOGY
Table 1. Incorporation of Acetate-l-C'4 into Lonog Chiain latty Acids
1by Etiolated Secdlinig Tissuie of the Graminae laini1y
Conditions of incubation: 2.6 g tissue slices (fr wt), 400 umoles phosphate buffer at pH 7.4, 1.50 ,umoles acetate1-C"4 (8 ,c), total volume 4.0 ml, 6 hours at 30°. 1.0 g tissue (fr wt) = 75 to 85 mg dry wt. Total lipid, mg/g
fresh weight: barley, 6.7; wheat, 6.3 oat, 7.0; maize, 6.1; ryegrass. 7.0.
Days after
Hordc un
sown'1110
(Barley)
3
5
6
7
8.3
10.4
Incorporationi of label into fatty acids, %
Zea
Avcna
(Oat)
(Maize)
(\Vheat)
...
10.5
4.3
6.8
...
7.9
3.0
...
5.7
4.1
5.6
4.1
Lolinini
(Ryegrass )
7TriticumZ1
10.2, 9.9
Table
21.4
7.9
11. S'nthecsis of Fatt.Acids fromi.Acetate-i-(C
by Seedling Tissiue of the Graminae Famiiily
Conl(litionis of incubation: 2.0 g (fr wt) tissue slices incubated for 4 hours at 30° with 79.0 mntmoles acetate-i-C'4
(4 ,c), 200 ,umoles phosphate at pH 7.4, total volume, 2.0 ml. Seedlings grown in dark for 5 days.
Tissue
Height of
seedlings
at harvest
(iinches)
Horde:im
Triticion
A4vncoa
3
2
Zca.
11/2
Lolinn,,
*
Total
fatty
acids
12:0
14:0
5450
5690
4900
5050
8780
87
273
137
333
140
72
205
206
Incorporation of acetate ( Aimoles)
Individual fatty acids
16:1
18:0
18:1 18:2 18:3
16:0
174
746
1030
1362
777
1880
111
280
*
..
.
..
-
..
.
..
.
762
1030
681
1222
1185
467
682
235
167
351
190
87
...
.
.
..
20:0
22:0
24:0
348
546
510
445
694
970
1030
1540
894
848
1440
3065
921
555
1185
None detected.
Table lll. Fatty .tcid Comiposi.tioni of the Lipids of tlc Lcaf Tlissule of Gramllinac
Seedlings Grown. for S Daias ini the Dark
Tissue
Wt lipid
g fr wt)* *
(mg/1.0
Component fatty acids (mole % of total)
12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 22:0 24:0 26:0 30:00
2.1
1.4
0.9
0.5 22.4
2.8
4.8 34.5 20.2 2.5 2.9
2.1 2.9
Hordeum
8.5
*
*
3.8
1.0 38.1
1.9
Tritictumt
3.8
5.7 22.9 22.8 *
5.0
*
*
11.3
1.4 28.9
1.4
1.4
7.0
7.7 33.1 14.8 *
Av4ea
*
*
*
0.7 26.9
*
*
Zea
6.4
3.4
3.4
5.1 47.1 10.1 *
3.3
*
*
*
12.5
2.3
6.2 23.5 12.6 *
1.6 39.0
2.3
Loliiini
7.5
* Not determined quantitatively. The amounts present in Tritic',,,:, Avenia, Zea and Lolinin tissue was of the same
order as that for Hordeunm.
1.0 g tissue (fr wt) = 75 to 85 mgm dry weight.
Table IV. Distribution of the Label ii Fatty .4cids Synthresized froi,i Acetate-i-C'4
by Etiolated Barley Tissue Prepared froiii Seedlings Grown for 3 to 10 Days
Conditions of incubation: 1.0 g (fr wt) tissue slices incubated for 4 hours at 300 with 44.6 m,tmoles acetate-i-C'4
(2 Ac), 200 ALmoles phosphate at pH 7.4, 30 ,moles KHCO, total volume 2.0 ml.
Days
after
planting
3
.5
10
Height c3f
seedling s
lharvesst
(inclies)
V2-1 Y
at
2-3
5-7
Total
fatty
acids
5560
4380
1870
12:0
..8
84
70
61
14:0
106
58
61
Incorporation of acetate (nm,umoles)
Individual fatty acids
16:0 16:1 18:0 18:1 18:2 18:3 20:0
585
600
378
78
140
29(
484
613
150
239
376
219
133
153
36
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
...
350
70
17
280
105
22:0
24:0
961
780
176
2535
1240
368
1027
HANW'KE AND STUMPF-LONG CHIAIN FATTY ACID BIOSYNTTHESIS
FIG. 1. A GLC-C14 monitored chromatogram of the
methyl esters of the fatty acids from tissue slices prepared from etiolated barley tissue. DEGS column at
1600. (Upper):C14-labeled fatty acids synthesized from
acetate-1-C14. Reaction conditions: 2.0 g (fr wt) tissue
slices 0.089 Amole acetate (4 uc). 200 umoles potassium
phosphate buffer at pH 7.35, 30 /Amoles KHCO3, total
volume, 4.0 ml. 4 hour incubation at 300. (Lower)
Constituenit fatty acids determined by a thermal conductivitv detector.
of the saturated fatty esters by TLC on AgNO./silica
gel. Bromination. hydrogenation, reductive cleavage
and permanganate-periodate oxidation neither altered
the chroniatographic constants nor eliminated the
peaks.
XVhen tissue slices prepared from barley seedlings
grown for 3 to 10 days were used as experimental
material, the incorporation of acetate-i-C'4 into fatty
acids during a 4-hour incubation decreased with increased age of the tissue (fig 2).
Using 3-day-old tissue, over 60 % of the total
label incorporated into fatty acids (12-13 %) were
C90:0 to C24:0 fatty acids, whereas the same fatty acids
comprised about 35 % of the total label incorporated
(4-5 %) by 10-day-old tissue (table IV). For later
experiments 6 to 7-day-old seedlings provided a satis-
factory compromise between the amounit of tissue
available and the extent of fatty acid synthesis from
acetate.
The rate of fatty acid svnthesis at 300 (fig 3)
shows an initial rapid rate of incorporationi of label
and a gradual leveling off up to 9 hours. The incorporation of label into fatty acids with 20 or more carbon atoms did not increase after about 3 hours.
These results could be interpreted as being indicative
of either cessation of synthesis or an equilibrium between ,8-oxidation and synthesis. As demonstrated
in table V in addition there appears to be an initial
lag in the synithesis of unsaturated acids. The ratio
of 18:0/18:1 synthesized is 2.50, 2.21, 0.36 at 0.5. 3.0
and 9.1 hours respectively. During this period the
amounts of stearic acid reached a maximum at 3 to
4 hours and then decreased, while the amount of
palmitic acid synthesized increased steadily over this
period.
The results of 2 experiments presented in table
VI demonstrates 2 further aspects of the utilization
10
E
S
8
E
5
a-
0~iSs |
0 F3
4
5
'
_
^
^
.^
6
7
8
9
10
TIME AFTER PLANTING (DAYS)
FIG. 2. In1corporation of acetate-i-C'4 into fatty acids
by tissue slices prepared from barley seedlings grown
for 3 to 10 days. Reaction conditions: 1 g (fr wt) tissue slices, 200 umoles potassium phosphate buffer at pH
7.4, 30 ,moles KHCO,, 0.045 Amole acetate-i-C'4 (2,tc),
2.0 ml total volume, 4 hours at 30° with shaking.
Table V. Effect of Tinme of Incuibation on the Fatty Acids Synzthesi2ed fromii Acetate-i-C14
by Etiolated Barley Tissue Prepared fromii Seedlings Grozen for 6 Days
Reaction mixture: 2.0 g (fr wt) tissue slices, 400 ,&moles phosphate at pH 7.35, 30 Amoles KHCO3, 89.2 mutmoles
acetate-i-C'4 (4 ,kc), 4.0 ml total volume.
Time of
incubation
(hr at 30°)
0.5
1.0
2.3
3.0
5.5
9.1
Total fatty
acids
1200
2170
3930
5530
5480
6790
12:0
38
41
79
171
175
421
14:0
19
41
79
116
132
285
16:0
154
289
420
868
1008
1398
Incorporation of acetate (m,tmoles)
Individ(al fatty acids
16:1
...
...
193
348
876
1100
18:0
192
393
534
636
394
279
18:1
77
83
153
288
394
767
18:2
29
41
153
171
88
143
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
20:0
106
206
263
459
482
767
22:0
211
456
610
747
570
700
24:0
374
620
1446
1726
1361
630
PLANT P H YSIOLOGY
1028S
w
3 - "o4
i-;
III
0
;Da
4
LA
0
Q
0
, m
o
Z
m
w
z
0
0
U
cL
>
I
0
0.
0
-
24
z
REACTION TIME (hr.) AT 30
FIG. 3. Effect of time of incubation on the biosyn-
tlhesis of fatty acids by barley tissue from acetate-i-C'4.
Total fatty acids (C,0-C24). Long chain fatty acids
(C.,2, 4). Reaction conditionis at 300 2.0 g (fr wt)
tissue slices prepared from established barley (6 day),
200 /Lmoles phosphate buffer at pH 7.35, 0.089 gLmole
acetate-I-C'4 (4 Ac), 30 ,umoles KHCO3, total volume,
4.0 ml.
of acetate for the synthesis of fatty acids. In a comparison of green and etiolated tissue, it was found
that the amoount of C.,0:, to C.)6:0 fatty acids synthesized fromii acetate wass essenitially the same. FHowever greenl tisstue. in conitrast to etiolated tissue. utilize(d acetate-I_-C1 for ani appreciable sx-litliesis of C,,
iiisatirate(l fatty- acids notably (lieiie ai)li trienle.
Greeni excised slloots which were allowed to take
tl) acetate throtuglh tlle cut enlds, synthesized the salmle
fatty acids as tissue slices prepared from tlle samiie
batch of seedlings (expt 2, table VI). About onlefiftlh of the fatty acids synlithesized hb whole shoots
were in the ranige C-,, ,-26:0. As shownl in table
V, the distribution of label varies witlh timne of inctubation and consequently the differenices in detail
between the fatty acids synthesized 1b the tissue slice
aind the whole tissue systems w-hiclh represelnt differclit timiie relatiolnships shouild not be emiiphasize(l.
\V hen it was found that long clhaini fatty acids arc
synthesized under a variety of conditionls it \-as of
interest to determine both the association of these
fatty acids w-ith tissue fractions anid the distribution
of label between the Imlainl lipid classes in the tissule.
Preparations of plartictlilate (chloroplasts and iniito-
clhoiidria), imiicrosomlal anld microsomiial-free
supernatant fractions from etiolated barlev tissue were incubated with acetate-i -CI4. Analvsis for labele(d
fatty acids in these (lifferenlt cell fractions slhowede
that radioactive fatty acids w-ith more thani 20 carbonl atoms were not found in the supernatant fraction1 but w-ere constituelnts of the particulate anid microsomal fractions (fig 4). Tlhe residue fromz griniding the tissue in sanid (cell debris) contains cell wall
imiaterial, unlbroken cells and entrapped particulate
anid microsomal material anld as would be expected.
the distributionl of label was similar to that obtainle(d
from whole tissue in other experimenits. Although
the distribution of labeled fatty acids is very similar
in the cell del)ris and the particulate fraction, the
microsomal fraction containied mluch less of the unsaturated fatty acids relative to the saturated acids.
Of interest, the fatty acids associated with the supernatanit fraction were only C,0 and C1. fatty acid. No
lon,g chain saturated fatty acids \-ere foundl ill this
fraction.
Table VII shows the distributionl of radioactive
fattv aci(ls ill the lipid classes of etiolated barley tis-
suie after inicubationi with acetate-I-C'4. \ wi(le
variety of fattv acids were labeled in bothl neutral and
lpola1r lipids. However. tllere \-ere clean-cutt (liffer-
ences in the extent to wlhiclh the individual fatty acids
were incorporated into these 2 imiaini classes of lipids.
The saturated fatty aci(ds, wvIhich inlcluded the fatty
atci(ls in the range CQ0 0, .,2, were concentrated in
the. neutral lipids whereas the p)olar lipids coiltain(le
large proportions of monoenes w-hich w,vere labeled.
Some palmitate was also inlcorporated into the l)olar
lipids but only small amounts of the higher imiolectular
weight saturated fatty acids were lpresent. The fatty
acids of the wax esters were analyzed separately be-
Table VI. Inicorporation of .Aclctate-1-( '4 into Fatty' Aciids by, (;recc (1(td Etiolaitc(I l' i.ssle
Slices an(d by E.rci6ed Green. Shoots of Barley Seed1ings
ilncubation conditions: Tissue slice experiments as in table V. Shoot tissue experimenlt. 2.0 g tissue, 80 MUmioles
potassium phosphate at pH 7.4, 89.2 m,umoles acetate-l-C4 (4 uc), total volume, 0.6 mnl.
Incorporationl of acetate-l-C'4 (m,tmolole-s)
Total
fatty
Tissue
Expt
acids
10:0 12:0
14:0
16:0
16:1
Individual fatty acids
18:0 18:1 18:2 18:3
20 :0
22:0
24:0
26:0
Green
slices
1
9.2
0.08
0.12
0.12
1.14
0.08
1.70
1.43
0.73
0.28
0.57
1.06
1.49
0.40
Etiolated
slices
Green
slices
I
:5.77
...
0.16
0.12
0.72
0.49
0.29
0.35
.....
0.32
0.58
2.19
0.55
11.33
0.07
0.33
0.36
1.92
0.26
1.12
1.75
1.22
0.43
0.62
1.25
1.31
0.69
8.25
0.04
0.15
0.21
2.15
1.28
0.50
0.96
0.43
0.35
0.28
0.62
0.54
0.74
2**
Excise(d
shoots
*
2
4 hours incubation.
5 hours incubation.
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
HIA\WKE AND STUMIPF
0
3
E
2
LONG CIAIN FA.:TTY- ACID BIOSYNTHES-S1S
102'9
160
20;
0~~~~~~~1:
Z
2240
0 1420
I4:1
C'4- LABELLED FATTY ACIDS CI4LABELLED FATTY ACIDS
II(;. 4. Distributionl of radioactive fatty acids in cell
fraction1s of etiolated barley tissue (6 days old) l)repared
after incubation of sliced tissue with acetate-i-C'4. Reaction conditions: 20 g (fr wt) tissue slices, 8.0 mmoles
p)otassium phosphate buffer at pH 7.4, 1.24 ,umole acetate1-C'4 (60 c). Total volume 50 ml. Incubated for 6
hours at 300
1.
cause of the usual associationi of lonig clhaini fatty
acids with these comiipounlds. As seen in table V7 II
only a smiiall total incorporationi of acetate-i-C'4 was
foutlnd in this lipid fractioll the fatty acids fromii Cl,
to C24 beillg fairly- unliformly distributed. Further
work oni the nieutral lipids fractioni shiowe(d tllat the
trigly cerides miionlogalactosvlglvceri(les and (ligalactosylglycerides conitainied appreciable amilotulnts of labeled
fattv acids but the distribution of label in the com11pollent fatty acids in each of these classes of lipid
was simiiilar to that of the total neutral lipid fraction.
In order to obtaini iniformationi oni the miiechanism
of biosynithesis of the saturated loing chaini fatty acids
the radioactive fatty acids w-hich lha(l been isolate(d
l)v- GLC w-ere decarboxvlated anid the radioactivity
of the CO, measured. For both etiolated and greeni
tissue the percentage of the total radioactivity in the
carboxyl carboni of palmitate NN'as alpproximatelv
19 % (fig 5). In contrast. the carboxyl carbon of
stearic acid accounted for approximately 60 % anid
43 % of the total radioactivity in etiolated and greeln
tissue respectively. The contribution of the C'4COOH to the total radioactivity of the C20 to C.,:
fatty acids decreased almliost linearly with inicreased
carbon lunuber.
Table V-II. L)istrimution of Radioactivc 1 atty Acids in tflc Lipid C ltsse.v
of Etioloted( Bacrlc1' Tissuec
Conditions of inicubationl: 8.0 - (fr wt) tissue slices prepared frollm seedlings grown for 7 days in tlle (lark. 310
uimoles acetate-i-C'4 (62 Ac). in 3.0 mmoles phosphate buffer at pH 7.4, total volulme 30 ml, 6 hours at 300.
Total
fatty acids
31.0
0.9
18.9
11.2
IncorPoration of acetate-i-C'14 ( Ulll01eS )
Individual fatty aci(ds
12:0
14:0
16:0
16:1
lipid fractioi
0.49
0.73
8.36
6.11
Total
0.02
0.02
0.10
0.03
\\.ax esters
0.47
2.18
Neutral lipids*
0.70
5.67
...
3.90
2.59
0.01
Polar lipids**
The approxiimate contribution (g,wmoles) of the differenlt classes x-as
monogalactosylglyceride (9:0), digalactosylglyceride (4.5).
Mainly phospholipids.
18:0
1.97
0.28
1.19
0.50
18:1
6.48
0.03
2.6,
3.80
20:0
2.09
0.19
1.7
0.20
22:(0
1.81
0.11
1.5
0.20
24:0
2.96
0.12
2.84
...
sterol esters (0.5). triglycerides (6.0)
Table VIII. Double Bont-d Position of Moniocan cs Suttlit(silzed lvNoarl'v
Tissute Slices froii Acctatc-1-( 14
Coniditionis of incubationi: 2.0 (fr wt) tissue slices prepared from seedlings groNvin for 7 dlays (24 hours in light),
400 umoles phosphate buffer at pH 7.4, 100 ,umoles KHCO., 89.0 m,umole acetate (4 ,ic), 4.0 ml total volume. 4 hours
incubationi at 300.
Monoene
16:1
18 :1
Amount
% of total
fatty acids
synthesized
22.4
22:4
Labeled fragments produced
by oxidation of isolated
monoene with ozone
Heptylaldehyde
Adipic semialdehydle
Azelaic semialdehyde
Heptylaldehyde
Nonylaldehyde
Undecylaldehyde
Adipic semialdehyde
Suberic seemialdehy(le
Azelaic semialdehyde
lUndecanedioic seniialdehl-de
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
Amount of fragmlient
(% of radioactivity in
oxidized products)
12.4
5.0
82.6
4.6
17.6
1.5
2.3
3.8
35.1
35.1
10()30
PLANT PHYSIOLOGY
FATTY ACIDS FROM
strate that hiigh proportioiis of the total label inicorporated from acetate-i-C14 inito long chain fatty
acids bv seedling tissues of the
family are
in the loinger chiaini fatty acids of the n-satur)present
ate(l series in the range C .... Althollglh labeled
AAND
GREEN
-j
|\
60
0
Grainiinae
TISSUE
I-
cerotic (26 :0) w-as the highest miolecenlar- weight
fattv acid recorded in these experimiienits it is possible
0
that hiigher molecular xveiglht fatty acids are syUnthesized also. The re(quiremienits for the quantitatioll
of the GLC-C14 mionitored data dictated use of
z
0
_
I
I \
m 401
/
/
S
chroimatographic conlditiolns whlich
\
ate
-/
\~
0
cm
r
1/
for the detection of
of
zt/gare L. the age of the tissue is
imlportant factor in so far as the activity anid thle
type of fattv acid synthesis is conicerned. Biosvnlthetic activit-y and also the proportions of the longer
chain fatty acids Nas highest in 3-day-old barley tissue buit the capacity for synthesis was still appreciable in 10-day-old seedliings, wAhich was the oldest
tissuie used. How-ever the time of incubation further
weight
proportions
were
fattv acids
and silnce for most
44 to 6-houlr synlthesized
incutbationsx were chlosen. thle
exlleriments wh-}lich
-b
of lof
synthesizing ability
ages slholld nlot be overemlplhasized.
similar range of fatty acids {was syvitllesizedl by
both green and etiolated tissule. In greeln tissuie.
however. there is a greater total sylnthesis of tinsaturate(l fatty acidls and also greater uinsaturated/
saturated ratios. Tl'is is conisistenit with earlier o0)servatioins that light stimiiulates the svyntlhesis of unIn Hordeutui
X
20
an
0
0
4
16
18
20
22
24
26
influences
CARBON NUMBER OF FATTY ACID
Crl)oxvl labeling of tlle saturate(d fattv
of
the
acids
iz*ll
from acetate-1-0' ' y.ytissue
iz<e(l acetate-i-Ctissue slices
froiii
slicesp)repared
I)repare(l
comparative
5.
svNltliesi
tiolate(d
hiugs. Siee
froinn
b
*
*
et
text
for
anid greie
cond(1itionls.
*
---- @barlev seed-
,
fraction
nonoene
m
the C16
is palmitoleic (i.e. A9 16:1
smiiall amount of A( 16:1). The C18
a
monoene
Inolysis and cleav-age gave equal proportions
of rad lioactive C, and C,, semialdehydes. Correspoindiinj fragments from themethyl end of the chaini
xvould be the C, and C, aldehvdes which were also
MIinor aimiounits of radioactive fragmiienits
present
x-hich would correspond to Al, A, A8. 18:1 fatty
acids vere also detected.
;empts
Att
to synthesize fatty acids with 20 ornmore
from acetate using cell-free prepacarl)on
rations wvere uinsuccessful. Preliminary experiments
showec I that the plastid or chloroplast fractions were
the ml ost active synlthesizilng systems. Palmitate.
stearat e and oleate x-ere the principal fatty acids
svnthe~ sized. Further investigations on the fatty
ynthetase activity of plastid and chloroplast
acid
will be reported elsewhere.
fractioIus
Onl ozC
g
t.
atomls
s
Discussion
iscussion
-Alt bougth the biosyntlhesis of the longer chain
from acetate units has been refattv a cids
(C.21,_24)
ported in animals, the nervous and brain tissue in
particnitlar (5,. 7). theutilization of acetate by plants
for th biosynlthesis of this class of fatt acid does
a GLCnot all)I)ear to have beeiiinestigated. Using- (
'}C onitor svstem it has been possible todemiionMl
high molecular
tissues
vary1vii1
.\
'he distribution of label in the cleavage prodlthe ozoni(les obtained fromii the isolated C,
anid C, ii monoenes is shown in table VIII. Most of
lucts 0o
x-ith
iinappropri-
~does niot include cerotic acid.
e
u
/
z
l
8
were
radioactivity peaks
COIilpoundls with large retenition volunles anid for the same
reason some of the comparative data reported here
x
fatty acids in higher plants (20. 28) and(
photosyntheticnmicroorganismiis (4, 22).
satuirate(d
in
A comparison of the types of fatty acids
in the en(logenous lipids of barley seedlings and
found(l
those
acetate-C14 discloses a
svnthesized de novo from
Thus while palmitate andlinoleic
sharl) difference.
linoleniic are theprincipal comiiponents in theendogenous lipids, the de novo synithesized acids are
and
primarily
palmlitic,
stearic and oleic and the long chain
fatty acids archidic, behenic
and
lignoceric and ceroThe results reported in this paper suggest
timie of incubation as well as age of the seedling
tissue are important factors. In somemaniner the
final lipid composition of a given tissue is remarkedly
constant, as indicated in tableIII.
Although the tissue slice technique has been used
routinely in the present work, a similar ranlge of C14-
acid.
that
tic
labeled
fattv acids
svnthesized from acetatetiunw\-asconditions,
namiiely when whole
their cUt enlds.
der more physiological
excised shoots took ul) substrate fromii
Thlus the biosynthesis of these fatty aci(ds islnot ail
artifact of the tissue slice technique.
The presence of labeled long clhaini fatty acids ini
thel)articulate fractioni prepare(l fromii tissule p-revotisly incubated xvith acetate-i-C'4is conisistent\xith
the knoxv-in locationi of enzymnes controllingthe caliin
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
1031
HAWKE AND STUMPF-LONG CIIAIN FATTY ACID BIOSYNThIESIS
lengtlheninig of fatty acid substrates in animal tissnes.
Wakil and coworkers (30) showed that the elongation of palmityl CoA to stearate was a function of
the mitochondrial fraction of rat liver cells. More
recently, the mitochondrial fraction has been used to
demonstrate a similar elongation of arachidyl anid
behenyl CoA to behenic, lignoceric acids respectively
(31). TPNH, DPNH, and the CoA esters of the
fatty acids were requirements for the reactions.
The long chain fatty acids synthesized under the
conditions described are incorporated into a number
of classes of lipids. Despite the usual association of
galactolipids with highly unsaturated fatty acids, particularly those with 18 carbon atoms, it was found
that this group of lipids contained an appreciable part
of the total n-saturated fatty acids synthesized from
acetate. While the total radioactive fatty acids synthesized is very small on a molar basis, it is interesting to note that the saturated fatty acids are incorporated into galactolipids in view of the association of
this class of lipid with highly unsaturated fatty acids
and with the membranous structure of chloroplasts
(1). Only a small proportion of the total labeled
fatty acids appeared in the wax ester fraction. The
nature of the alcohol moiety was not investigated but
the fatty acids were mainly saturated, of which the
C.,,,24 fatty acids formed the major part. However,
this only formed a small proportion of the total
C90_24 fatty acids incorporated into tissue lipids. In
contrast the polar lipids, which would have been
mainly phosopholipid in nature, contained predominantly labeled monoene with much less of the labeled
saturated constituents. For this reason the polar
fraction was not further characterized.
Rosenberg (21) found that etiolated cells of Euglenta gracilis accumiiulated a waxy ester fraction which
contained unusually large amounts of fatty acids of
intermediate chain length. There was no evidence
for the synthesis of substantial amounts of these fatty
acids by etiolated barlev tissue.
With acetate-l-C14 as substrate if the C14-label
was uniformly distributed along the fatty acid chain
at the odd numbered carbons, the radioactivity at the
-COOH for the fatty acids 14:0, 16:0, 18:0. 20:0,
22:0, 24:0 and 26:0 would be 14.3, 12.5, 11.1, 10.0.
9.1, 8.3 and 7.7 % respectively. Of the determined
values reported here, only those for myristate and
palmitate are consistent with a pattern of uniform labeling. The saturated fatty acids with even carbon
numbers from 18:0 to 26:0 are presumeably synthesized from 16:0 of relatively low specific activity
and acetate of high specific activity. With each successive addition of a radioactive acetate moietv the
proportion of the label residing at the -COOH of
the molecule would be expected to decrease in a manner similar to that found experimentally (fig 5). A
relatively short incubation period was used in the
present experiments. In experiments with rats (7)
the distribution of label was more uniform as the
time of incubation was increased. Hajra and Radin
(7) reasoned that with increased time the specific
activity of the acetate available for synthesis decreases while the available 16:0 is of relativelv high
radioactivity.
Likewise the biosynthesis of the fatty acids fromii
18:0 to 26:0 studied here can be considered to involve 2 steps, A) the synthesis of palmitate as the
primary fatty acid and B) chain lengthening of palmitate by addition of C9 moieties, probably acetate,
viz.
C2
C2
C2
C2
C.
16:0 - 18:0 -, 20:0 -+ 22:0 -* 24:0 -- 26:0.
For some time vaccenic acid has been regarded as
being largely confined to certain microorganisms.
However, there have been earlier reports (e.g. Morton and Todd (19) of minor amounts of vaccenic
acid occurring in animal tissues. Holloway and Wakil (10) recently observed that 20 to 50 % of the
total 18:1 fatty acids of rat liver cells was vaccenic
and they have presented evidence that palmitoleyl
CoA was the precursor. In barley tissue appreciable
proportions of the labeled 18:1 was vaccenic. 50 %
in the experiment cited (table VIII) although the
value was frequently of the order of 30 % of the
total 18:1. Direct evidence for palmitoleic acid being a precursor of vaccenic was not obtained but it
was noted that when labeling of palmitoleic was high
the vaccenic/oleic ratios were also high. It was
further noted that excised whole shoots give large
amounts of labeled palmitoleic. Since all the work
presented in this paper on the appearance of vaccenic
acid has been with sterile tissue, we feel confident
that the results can not be ascribed to bacterial contamination.
Summary
The incorporation of acetate-1-C'4 into the long
chain fatty acids is examined in the seedling tissues
of a number of the Graminae family. A novel feature of the synthesis is that a large proportion of the
total fatty acids are normal saturated fatty acids in
the range Coo to C26.
Further experiments with seedling tissues of Hordeuni vulgare L. shows that the capacity to synthesize these longer chain fatty acids is greatest with
3-day-old tissue. The synthesis of these fatty acids
occurs rapidly during the first 2 to 3 hours of incubation and then levels off. The capacity to synthesize C,0 to C2, fatty acids from acetate-l-C'4 is
shared by etiolated and green tissue slices and by
whole excised shoots.
The C14-labeled long chain fatty acids are incorporated into the particulate fraction of the cell anid
are absent from the supernatant fraction after centrifuging at 110,000 X g. Moreover, the labeled
long-chain fatty acids are found to predominate in
the nonpolar lipids, including the mono- and digalactosyl lipids. Only a little occurs in the wax
esters. The polar lipids contain predominantly the
monoene fatty acids synthesized from acetate.
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Copyright © 1965 American Society of Plant Biologists. All rights reserved.
10)32
PLA NT PHYSIOLOGY
The distribution of label in the 2() :0 to 26:0 fatty
actlds is coinsistenlt with the svnltlhesis occurring via
the primary fatty acid palmitate by the successive ad(lition of 2-carhbon moieties, probably acetate.
An additiolnal nlovel featture is the synthesis of up
to 50 % of the labeled 18:1 fatty acid as vaccenic.
Palmitoleic aci(l is also svithesized fromii acetate.
Acknowledgments
TIhe authors are grateful to Mr. Kenneth Kimble of
the Department of Planit Pathology of this University for
instruction an(l assist-nce in the techni(que for growing
seedlings under sterile coniditions. The seeds for this
study were providled by Dr. Charles Schaller of the
I)epartment of Agrononmy of this Universitv.
Literature Cited
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Ann. Rev. Plant Physiol. 15: 1-16.
2. BIGIIH, E. G. AND W. J. DYER. 1959. A rapid
method of total liDid extraction and purif:cationl.
Can. J. Biochemn. Physiol. 37: 911-17.
3. BRADY, R. O., R. M. BRADLEY, AND E. G. TRAMS.
1960. Biosynithesis of fatty acids. I. Studies
with enzymles from liver. J. Biol. Chem. 235:
3093-98.
4. ERWIN, J. AND K. BRiocti. 1964. Biosynthesis of
unsaturated fatty acids in microorganisms. Science 143: 1006-12.
5. FuIco, A. T. AND J. F. MEAD. 1961. The synthesis of lignoceric, cerebronic and nervonic acids.
J. Biol. Chem. 236: 2416-20.
6. GORNAii., A. G., C. J. BARDAWNII.I., AND M. M.
DAVII). 1949. Determination of serunm proteins
by means of the hiuiret reaction. J. Biol. Chem.
177: 751-66.
7. HAJRA, A. K. ANI) N. S. RADIN. 1963. BiosyInthesis of odd and eveni-numnbered cerebroside fatty
acids: Evidence for two routes. Biochimi. Biophys.
Acta 70 97-99.
8. HAWKE, J. C. AND P. K. STUTMPF. 1965. Syntlhesis
of behenic, lignoceric and cerotic acids by preparations of Hordennmii 71' i1are L. tissue. Fed. Proc.
24: 290.
9. HILDITCH, T. P. AND P. N. ATILITA-MS. 1964. The
chemical constitution of natural fats. Chapman
and Hall, Ltd. London, p 180.
10. HOLLOWAY, P. W. AND S. J. WAKIL. 1964. Synthesis of fatty acids in animal tissues II. The
occurrence and biosynthesis of cis-vaccemic acid.
J. Biol. Chemii. 239: 2489-95.
11. JAMES, A. T. 1960. Qualitative and quantitative
determination of the fatty acids by gas-liquidl
chronmatography. Metlh. of Biochem. Anal. 8: 159.
1 2. J AM-Es, A. 1'. A N1) \. J. P. MARTl-IN. 1956. Gasliquid c4hromatography. The separation and idlentification of the methyl esters of saturated anid
unsatlurate(l aci(ls from forrmic acid to ni-octa(lecanoic acid. Biochemii. J. 63: 144-52.
13. KANASHIRO, T. ANDI A. G. MARR. 1961. Cis-9,10methylene hexadecanoic acid from the phospholipids of Fscheric-hia coli. J. Biol. Chem. 236:
2615-19.
14. LEES, T. M. AND P. J. DE MURIA. 1962. A simple
metho(x for the preparation o)f thiln layer chroma-
tography plates. J. Chromatog. 8: 108-09.
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