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The synthesis of long-chain fatty acids from acetate in flax, Linum usitatissimum L
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
64, 1-5 (1956)
The Synthesis of Long-Chain Fatty Acids from Acetate L.l.2 in Flax, Linum usitatissimum Walter P. Gibble and Edwin B. Kurtz, From the Department
of Botany, College of Agricullure, Tucson, Received
Jr.
University
of Arizonu,
Arizona
April
12, 1056
Numerous studies have sho\rn that acetate is the precursor of fatty acids in animals (I), but there is litt,le evidence that higher plant,s make fatty acids by multiple condensation of acetate. Kurtz (2) showed t,hat’ i?z vifro cult’ures of developing flax fruits produce more fat when acetate is supplied as the substrate, and New-comb and Stumpf (3) shelved that slices of peanut cotyledons synt,hesize radioactive long-chain fatty acids from acetnt’c labeled with carbon-14. The object of the present study was to determine whether a higher plant condenses acetate “head-totail” to form long-chain fatt,y acids. METIIODH
Flas (Linum usitatissimunz L., var. Punjab) was grown in the greenhouse. Fruits of the same age in days were obtained by tagging the peduncle of each Hower with a small piece of colored pressure-sensitive tape (Scotch tape) at the time of flowering. Fruits were harvested by cutting the peduncle about 1 cm. belon the fruit and immediately immersing them in distilled \vater. The total lipide con tent of seeds from fruits obtained in this manner was shown to vary by less than 2% (1). The system used for in oitro culture of the fruits was similar to the one used by Kurtz (2). The complete culture system is as follows [experimental support for the procedure may be found elsewhere (4)] : Light, 300-100 ft.-candles from white fluorescent tubes. Temperature, IS-25°C. ’ This study was supported by U. S. Atomic Energy Commission AT(ll-1).262, Supplement No. 1, and was submitted by the senior Graduat,e College, University of Arizona, in partial fulfillment of the for the Ph.D. degree. ? Arizona Agricultural Experiment Station Technical Paper So.
Contract So. author to the requirements 3S*.
2
\V.\LTEli
1’.
GIBl3LE
AND
EDWIN
I3.
KIJRTZ,
JR.
Composition of C’ulture Solution, 2940 mg. potassium acetate, 100 mg. indoleacet,ic acid, and 5 pg. biotin/l. of 0.1 ill K2HP04-KH2PC4 buffer, pH 6.5. Aerntion, vigorously with humidified air. Pltcnt kfnteriul, fruits usually 12 or 13 days old at beginning of culture, and 50-75 fruits/100 ml. culture solution. Culture Durution, 48 or 72 hr. In preliminary experiments it was noted that many fruits become infiltrated with culture solution and sink to the bottom of the culture. Such fruits do not continue to synt,hesize lipides. To overcome this, each fruit was dipped in molten (55-60°C.) parafhn prior to being placed in the culture solution. The paraffin coating prevented the displacement of air from the large fruit locules. The pedurlcle of the fruit. was not coated Tvith par&in so that the culture solution could bc translocated through t8he peduncle into the fruit. At the end of the cult,ure period the fruits lvere placed in ice water and the paraffin was readily removed. Fruits cultured in this manner remained green, appeared healthy, and synthesized fat. After each culture the fruit,s were dried for 12 hr. at 80°C. The seeds \vcre sepa rated from the fruit debris, counted, and extracted for 2 hr. with petroleum ether (b.p. 30-60°C.) in a micro-Soshlet cxt~ractor. The results are expressed as milligrams lipide/lOO seeds (2). sodium acetate ~vas used. In all experiments involving Cll, carboxyl-labeled All det’erminations of radioactivity were made by plating the sample onto a 25-mm. diameter copper plan&et and counting in a Tracerlab windowless flop counter. Results are expressed as counts per minute (counts/mm.) above backgroun(I.
As shown in Table I, acetate does promote synthesis of lipides by paraffin-coated de\-eloping flax fruits wltjured in t&o. That this increased yield of lipide is due to condensation of awtate suppkl by the medium is shown in Table II in which the culture inedium was supplemented with acet*ate-1-C’“. In order to determine whether the whole acetate molecule is condensed into long-chain fatty acids, the radioactiae fatty acids obtained front in vitro cultures containing acetate-l-C4 were separated and stepwiw TABLE The Effect of Acetute OIL the Yield of Lipides Initial
age of fruits days
10
Culture
duration
of Flux
Fruits
Substrate
hr.
Cultured
in vitro
Lipid&00
seeds nig. 20.0
0
72
None 2940 mg. K acotatc;‘l.
0 4s JS
Xone 2940 mg. Ii acetate/l.
72
13
I
25, s
30.6 35.5 45,s 49 c
The Effect of Concentration Lipides Synthesized
of Acetate-l-Cl4 on the Radioactioify by Flax Fruits Cultured in vitro
o.F
Initial age of fruits. 13 d:~.:-s;culture duration, 48 hr. Lipides/lOO seedi Ya acetate-l-C”/l. mp.
cowzls/min.
13:l
2SO 900
26.S 107.2
1220
Oleic
Initial Iv-eight Total weight, after dilution with nonratlionctive stearic acid Yield of fat,tj- acid after remowl of: csrbon X0. 1 carbon
No. 2
carbon No. 3 carbon No. 4
76s i GIO,z 1 310 / 18
312 6
Linoleic
2!1S 20.3 s
5:it I -1 1
(1The 610 mg. obtained after removal of carbons 1 ant1 2 was tiiluted with nonradioactive palmit,ic acid to n total weight of 1086 mg.
TABLE
IV
Specijk Activitya of the Benzoic Acid Products Obtained from thr Stepwise Degradation of the Long-Chain Fatty Acids rind of the Residual Fatty Acids after Degradation
Values t,ake into account, the dilutions with nonrsdioactivc acids during degradation (see Table III). Benzoic acid product, counts/ncin./g. Position of degraded carbon in fatty acid
Stearic-palmitis Oleic J,inoleic Linolenic a Counts/minute/gram.
Residual fatty acid rou~tts/min./g.
1
7
3
.I
11,010 11,868 7,949
841 1,992
15,512
2,033
Original fatty acid fraction
2,565
f:Lt 1)
3,558 “0,133
1,038
67 63X
3.588
X,41,! -~-
tionation, hydrogenation, degradation, and counting procedures may be found elsewhere (5). Results of the stepmise degradation of carbons 1 ant1 2 of oleic acid, carbons 1, 2, and 3 of both linoleic and linolenic acids, and carbons l-4 of the stearic-palmitic acid mixture show a much greater distribution of Cl4 in the first and third carbons than in the evell-numbered positions (Table I\:). Inasmuch as t’he residual acids remaining after the degradations were also radioactive, it, appears that most if not, all of the carbon atoms in each species of long-chain fatty acid of flax fruits result from the multiple coudensstion of ac&te molecules. Whereas the specific activity of t,he even-numbered carbons \V:IS in all cases less than that of the odd-nurnbered carbons, the occurrence of some radioactivity suggests either slight contamination of the benzoio acid as a result, of the degradation process, or that somt acct,atc WLIS metabolized before t,he atoms were incorporatctl illto fatty acids. In :I similar study of animal biosynthesis of fa,tty a(& by Dauben (,t nl. (G), t,he even-numbered carbons were also slightly radioactive. In neither of these studies has it been possible to establish whether the isotope distribution in these carbons resulted from contamination or aretatr metabolism, although t,he latter appears most likely.
An in vitro system was produced in which developing flax fruits ut’ilizc acetate-l-C’”
to synthesize
degradation of the individual
long-chain
fatty
acids. Upon
separation
and
fatty acids synthesized in citro, the greatest)
F.\TTY
ACID
SYNTHESIS
FROM
ACETATE
F $1
radioactivity was found in the odd-numbered carbon atoms. These results show t’hat’ palmitic, stearic, oleic, linoleic, and linolenic acids are each formed in flax by multiple condensation of acetate.
1. L., AND BROWS, G. W. JR., in “Chemical Pathways of Metabolism” (I>. 31. Greenberg, cd.), vol. 1, p. 277-347. Academic Press, New York. 1954. Iicwrz, E. B., JR., Ph.D. thesis, California Institute of Technology, 1951. NEWCOMB, E. H., ASD STUMPF, P. Ii., .I. Biol. Chenz. 200, 233 (1953). GIBELE, W. I’., Ph.D. thesis, University of Arizonz, 1955. Gmnm, W. I’., KURTZ, E. B., JR., AND Iimux, A. E., .I. Am. Oil Chonists’ Sot. 33, 66 (1956). DAUBEN, IV. G., HOERGER, E., ASI) PETERSEN, J. W., ./. APZ. Chenc. Sot. 75, 2347 (1953).
1. CHAIKOFP,
2. 3. 4. 5. 6.
OF BIOCHEMISTRY
AND
BIOPHYSICS
64, 1-5 (1956)
The Synthesis of Long-Chain Fatty Acids from Acetate L.l.2 in Flax, Linum usitatissimum Walter P. Gibble and Edwin B. Kurtz, From the Department
of Botany, College of Agricullure, Tucson, Received
Jr.
University
of Arizonu,
Arizona
April
12, 1056
Numerous studies have sho\rn that acetate is the precursor of fatty acids in animals (I), but there is litt,le evidence that higher plant,s make fatty acids by multiple condensation of acetate. Kurtz (2) showed t,hat’ i?z vifro cult’ures of developing flax fruits produce more fat when acetate is supplied as the substrate, and New-comb and Stumpf (3) shelved that slices of peanut cotyledons synt,hesize radioactive long-chain fatty acids from acetnt’c labeled with carbon-14. The object of the present study was to determine whether a higher plant condenses acetate “head-totail” to form long-chain fatt,y acids. METIIODH
Flas (Linum usitatissimunz L., var. Punjab) was grown in the greenhouse. Fruits of the same age in days were obtained by tagging the peduncle of each Hower with a small piece of colored pressure-sensitive tape (Scotch tape) at the time of flowering. Fruits were harvested by cutting the peduncle about 1 cm. belon the fruit and immediately immersing them in distilled \vater. The total lipide con tent of seeds from fruits obtained in this manner was shown to vary by less than 2% (1). The system used for in oitro culture of the fruits was similar to the one used by Kurtz (2). The complete culture system is as follows [experimental support for the procedure may be found elsewhere (4)] : Light, 300-100 ft.-candles from white fluorescent tubes. Temperature, IS-25°C. ’ This study was supported by U. S. Atomic Energy Commission AT(ll-1).262, Supplement No. 1, and was submitted by the senior Graduat,e College, University of Arizona, in partial fulfillment of the for the Ph.D. degree. ? Arizona Agricultural Experiment Station Technical Paper So.
Contract So. author to the requirements 3S*.
2
\V.\LTEli
1’.
GIBl3LE
AND
EDWIN
I3.
KIJRTZ,
JR.
Composition of C’ulture Solution, 2940 mg. potassium acetate, 100 mg. indoleacet,ic acid, and 5 pg. biotin/l. of 0.1 ill K2HP04-KH2PC4 buffer, pH 6.5. Aerntion, vigorously with humidified air. Pltcnt kfnteriul, fruits usually 12 or 13 days old at beginning of culture, and 50-75 fruits/100 ml. culture solution. Culture Durution, 48 or 72 hr. In preliminary experiments it was noted that many fruits become infiltrated with culture solution and sink to the bottom of the culture. Such fruits do not continue to synt,hesize lipides. To overcome this, each fruit was dipped in molten (55-60°C.) parafhn prior to being placed in the culture solution. The paraffin coating prevented the displacement of air from the large fruit locules. The pedurlcle of the fruit. was not coated Tvith par&in so that the culture solution could bc translocated through t8he peduncle into the fruit. At the end of the cult,ure period the fruits lvere placed in ice water and the paraffin was readily removed. Fruits cultured in this manner remained green, appeared healthy, and synthesized fat. After each culture the fruit,s were dried for 12 hr. at 80°C. The seeds \vcre sepa rated from the fruit debris, counted, and extracted for 2 hr. with petroleum ether (b.p. 30-60°C.) in a micro-Soshlet cxt~ractor. The results are expressed as milligrams lipide/lOO seeds (2). sodium acetate ~vas used. In all experiments involving Cll, carboxyl-labeled All det’erminations of radioactivity were made by plating the sample onto a 25-mm. diameter copper plan&et and counting in a Tracerlab windowless flop counter. Results are expressed as counts per minute (counts/mm.) above backgroun(I.
As shown in Table I, acetate does promote synthesis of lipides by paraffin-coated de\-eloping flax fruits wltjured in t&o. That this increased yield of lipide is due to condensation of awtate suppkl by the medium is shown in Table II in which the culture inedium was supplemented with acet*ate-1-C’“. In order to determine whether the whole acetate molecule is condensed into long-chain fatty acids, the radioactiae fatty acids obtained front in vitro cultures containing acetate-l-C4 were separated and stepwiw TABLE The Effect of Acetute OIL the Yield of Lipides Initial
age of fruits days
10
Culture
duration
of Flux
Fruits
Substrate
hr.
Cultured
in vitro
Lipid&00
seeds nig. 20.0
0
72
None 2940 mg. K acotatc;‘l.
0 4s JS
Xone 2940 mg. Ii acetate/l.
72
13
I
25, s
30.6 35.5 45,s 49 c
The Effect of Concentration Lipides Synthesized
of Acetate-l-Cl4 on the Radioactioify by Flax Fruits Cultured in vitro
o.F
Initial age of fruits. 13 d:~.:-s;culture duration, 48 hr. Lipides/lOO seedi Ya acetate-l-C”/l. mp.
cowzls/min.
13:l
2SO 900
26.S 107.2
1220
Oleic
Initial Iv-eight Total weight, after dilution with nonratlionctive stearic acid Yield of fat,tj- acid after remowl of: csrbon X0. 1 carbon
No. 2
carbon No. 3 carbon No. 4
76s i GIO,z 1 310 / 18
312 6
Linoleic
2!1S 20.3 s
5:it I -1 1
(1The 610 mg. obtained after removal of carbons 1 ant1 2 was tiiluted with nonradioactive palmit,ic acid to n total weight of 1086 mg.
TABLE
IV
Specijk Activitya of the Benzoic Acid Products Obtained from thr Stepwise Degradation of the Long-Chain Fatty Acids rind of the Residual Fatty Acids after Degradation
Values t,ake into account, the dilutions with nonrsdioactivc acids during degradation (see Table III). Benzoic acid product, counts/ncin./g. Position of degraded carbon in fatty acid
Stearic-palmitis Oleic J,inoleic Linolenic a Counts/minute/gram.
Residual fatty acid rou~tts/min./g.
1
7
3
.I
11,010 11,868 7,949
841 1,992
15,512
2,033
Original fatty acid fraction
2,565
f:Lt 1)
3,558 “0,133
1,038
67 63X
3.588
X,41,! -~-
tionation, hydrogenation, degradation, and counting procedures may be found elsewhere (5). Results of the stepmise degradation of carbons 1 ant1 2 of oleic acid, carbons 1, 2, and 3 of both linoleic and linolenic acids, and carbons l-4 of the stearic-palmitic acid mixture show a much greater distribution of Cl4 in the first and third carbons than in the evell-numbered positions (Table I\:). Inasmuch as t’he residual acids remaining after the degradations were also radioactive, it, appears that most if not, all of the carbon atoms in each species of long-chain fatty acid of flax fruits result from the multiple coudensstion of ac&te molecules. Whereas the specific activity of t,he even-numbered carbons \V:IS in all cases less than that of the odd-nurnbered carbons, the occurrence of some radioactivity suggests either slight contamination of the benzoio acid as a result, of the degradation process, or that somt acct,atc WLIS metabolized before t,he atoms were incorporatctl illto fatty acids. In :I similar study of animal biosynthesis of fa,tty a(& by Dauben (,t nl. (G), t,he even-numbered carbons were also slightly radioactive. In neither of these studies has it been possible to establish whether the isotope distribution in these carbons resulted from contamination or aretatr metabolism, although t,he latter appears most likely.
An in vitro system was produced in which developing flax fruits ut’ilizc acetate-l-C’”
to synthesize
degradation of the individual
long-chain
fatty
acids. Upon
separation
and
fatty acids synthesized in citro, the greatest)
F.\TTY
ACID
SYNTHESIS
FROM
ACETATE
F $1
radioactivity was found in the odd-numbered carbon atoms. These results show t’hat’ palmitic, stearic, oleic, linoleic, and linolenic acids are each formed in flax by multiple condensation of acetate.
1. L., AND BROWS, G. W. JR., in “Chemical Pathways of Metabolism” (I>. 31. Greenberg, cd.), vol. 1, p. 277-347. Academic Press, New York. 1954. Iicwrz, E. B., JR., Ph.D. thesis, California Institute of Technology, 1951. NEWCOMB, E. H., ASD STUMPF, P. Ii., .I. Biol. Chenz. 200, 233 (1953). GIBELE, W. I’., Ph.D. thesis, University of Arizonz, 1955. Gmnm, W. I’., KURTZ, E. B., JR., AND Iimux, A. E., .I. Am. Oil Chonists’ Sot. 33, 66 (1956). DAUBEN, IV. G., HOERGER, E., ASI) PETERSEN, J. W., ./. APZ. Chenc. Sot. 75, 2347 (1953).
1. CHAIKOFP,
2. 3. 4. 5. 6.