16
Biochimica @ Elsevier
el Biophysics
Scientific
Acta,
Publishing
431 (1976) 16-21 Company, Amsterdam
- Printed
in The Netherlands
BRA 56749
SYNTHESIS
JOANNE
L. GELLERMAN,
The Hormel
(Received
OF ANACARDIC
Institute,
October
lst,
ACIDS IN SEEDS OF GINKGO BZLOBA
WAYNE H. ANDERSON
University
of Minnesota,
Austin,
and HERMANN Minn.
55912
SCHLENK (U.S.A.)
1975)
Summary Anacardic (6alkylsalicylic) acids and common lipids are efficiently synthesized by immature seeds of Ginkgo biloba. The seeds were incubated with 14C!labeled acetic, malonic and palmitoleic acids, glucose, and other potential precursors. Levels of 14C in common lipids and in anacardic acids, and the distribution of 14C in anacardic acids were determined. The results show that the salicylic moiety is synthesized by a polyketide pathway via malonic acid. The chain moiety for anacardic acid synthesis is in a different state of activation and/or site than chains that are used for synthesis of the common lipids. Labeled shikimic acid did not contribute 14C to anacardic acids, nor to other lipids, and palmitoleic acid was incorporated only into common lipids.
Introduction Anacardic acids are 6-alkylsalicylic acids in which the alkyl chains are saturated or olefinic straight chains of 13, 15 or 17 C atoms [l-3]. Unlike the common fatty acids, anacardic acids occur only as free acids and are found only in a few plants, namely in Anacardiaceae and in Ginkgo biloba [4]. In anacardic acid biosynthesis, it is likely that the alkyl chains derive from fatty acids [5] and this assumption is supported by the position of double bonds in them [6]. A polyketide pathway has been suggested for the synthesis of the salicylic moiety [ 5,7] and some evidence in support of this was brought forward by experiments in this laboratory [ 8,9]. Infusion of [ l-14C] - and [ 2-14C] acetic acid into Ginkgo plants yielded radioactive lipids. A small part of the 14C was found in anacardic acids. Chemical degradation of these revealed considerable randomization but an alternating pattern of labeling was apparent. In addition, it was found that 14C!/C equivalent in the salicylic part of the molecule was more than twice that in the chain. The level of 14C in ring C-6 showed that this C atom originated with synthesis of the chain rather than the ring.
17
A more efficient system of anacardic acid biosynthesis has now been found. “C-labeled acetic acid into anacardic Immature Ginkgo seeds incorporated acids much more efficiently than the whole plant and with less randomization. This enabled us to test also malonic and p~mitoleic acids and other potential precursors, including shikimic acid, for incorporation into the salicylic as well as the chain moiety. Materials and Methods Ginkgo plants about 1 m high were obtained from previous planting close to The Hormel Institute. Seeds were obtained from trees at the University of Northern Iowa, Cedar Falls, Iowa. Methods for analysis of anacardic acids in plant tissues were as previously described 161. Gas liquid chromatography was carried out on OV-1, 5% on Gas Chrom Q, 100/120 mesh (Applied Science Laboratory, Inc., U.S.A.) at 234°C. Radioactivity of lipids was measured in toluene-based scintillation fluid (Packard Instrument Co., U.S.A.) and of CO, in Hyamine with such fluid added [9] . Compounds labeled with 14C were obtained ~ommerci~ly. For incubations, acetic and hexanoic acids were used as Na salts and fatty acids as NH, salts. All substrates, 2-100 PC, were dissolved in 100 ~1 distilled water for each set of incubations. These were carried out with immature Ginkgo seeds of approximately 1 g, harvested in late June, and with seeds of approximately 8 g, harvested in mid August. The seeds were kept at 3°C until use, within 4 h after harvest, or for the specified storage times. The peduncle was removed from the seed so that a small cavity was left at the point of attachment, to which the radioactive solution was added. In each experiment, four seeds were mounted on cubes of Styrofoam and placed in a humidified culture dish with transparent cover. They were kept at 25” C under 400 foot-candles illumination. Water was added as radioactive solutions were taken up. Incubations were terminated by immersing the seeds into chloroformimethanol, 2 : 1, by vol., and the lipids were obtained by two extractions with this solvent in an Omnimixer. The combined extracts were shaken with 0.2 vol. of 0.9% aqueous NaCl and the lipids were recovered from the chloroform phase. Anarcardic acids were separated from other lipids by thin layer chromatography. Samples of 70 mg were streaked on Silica Gel H (E. Merck A.G., G.F.R.), 1 mm thick on 20 X 20 cm plates and developed in chloroform/methanol/10 M NH,OH, 65 : 30 : 4. The band of anacardic acids, RF approximately 0.75, was located by its fluorescence under ultraviolet light. Anacardic acids were extracted with diethyl ether saturated with aqueous concentrated HCl. The solution was washed to neutrality and the ether removed under vacuum. Common lipids were extracted from the silica gel with chloroform and methanol. The major portion of each anacardic acid fraction was converted to the methyl ether of methyl anacardate with excess diazomethane [ 61. The dimethyl derivatives were purified by thin layer chromatograms developed in petroleum ether (b.p. 60_70“C)/diethyl ether/acetic acid, 80 : 20 : 1. They were located by their fluorescence in ultraviolet light and recovered by extraction with chloroform. The radiopurity of the anacardic acids from the first chromatog-
18
raphic fractionation was calculated from the ratio of 14C in pure dimethyl derivatives and in contaminants as separated in the second chromatogram. The radioactivity of the anacardic carboxyl group was determined by counting 14C0, released by thermal decarboxylation of the acids [9]. For determination of the radioactivity in the alkyl chains, the olefinic double bonds of the dimethyl derivatives were hydrogenated. Subsequent oxidation with CrO, in acetic acid yielded a mixture of fatty acids which essentially represents the alkyl chains [ 91, Their radioactivity was measured and that of the salicylic moiety was obtained by subtraction. The dimethyl derivatives of anacardic acids were fractionated according to their olefinic unsaturation by chromatography on glass fiber paper impregnated with Si02 (Gelman Instrument Co., U.S.A.) and AgN03 [lo]. The developing solvent was petroleum ether (b.p. 30--6O”C)/diethyl ether/acetic acid, 90 : 10 : 1. The bands were detected in ultraviolet light after spraying with dichlorofluorescein. The materials were recovered by extraction with chloroform and aliquots were counted for radioactivity. The fractions were also analyz$d by gas liquid chromatography for chain length composition and monounsaturated fractions were ozonized to determine the position of the double bonds [6 J . Results and Discussion
Anacardic acids were found in all tissues of Ginkgo biloba except the embryo, and relative to other lipids, highest levels were found in the outer fleshy layer of mature seeds and in whole developing seeds (Table I). Anacardic acids with monounsaturated alkyl chains were prominent in all tissues (see footnote in Table I for anacardic acid composition of young seeds). Accordingly, further investigations focused on seeds. However, seeds harvested in the middle of August incorporated only 0.9% of 14C offered as sodium acetate or malonic acid into anacardic acids and 10-34s into other lipids.
TABLE
I
OCCURRENCE
OF ANACARDIC
ACIDS
IN GINKGO
_ Tissue
Total lipids % of fresh weight
Anacardic acids % of total lipids
Leaves Spur shoots Tips Stem i Bark + Wood Roots Mature seed ( (October)
3.0 2.5 3.0 5.0 0.9 0.8 3.0 0.5 2.0 3.0-3.4
20 20 20 10 5 15 50 25 0 60-75***
Cambium
outer layer* Shell** Embryo
Whole developing (June-August)
seed
* Outer fleshy layer of integument. ** Stony layer of integument. * * * Approximate composition, according 13 : 0, with small amounts
of 15 : 0. 17
to alkvl chains, 70% : 1 (n-5) and 17 : 2.
15
:1
(n-7).
12%
17 : 1 (n-7).
10%
19
Apparently synthesis of common lipids was predominant for the maturing seed while only small amounts of anacardic acids were synthesized during this period of growth. After storage of seeds for 12 weeks at 3”C, incorporation from [ 2-14C] malonic acid was lowered to 0.2% in anacardic acids, but was raised to 50% in other lipids. Immature seeds, harvested during the second half of June, proved to be more efficient for synthesis of anacardic acids. Young seeds incorporated 5-12% of 14C offered as sodium acetate or malonic acid into anacardic acids, and 1219% into other lipids. After 3 weeks storage at 3”C, incorporation of 14C decreased twice as much for anacardic acids than for other lipids. The results listed in Table II are from seeds harvested in late June and incubated within 4 h after harvest. Incorporations into anacardic acids were highest from [ l-14C] acetate and reached values of lO--12% after 12 h. This was the approximate period needed for uptake of the radioactive solutions by the seeds and longer incubation did not markedly change the radioactive levels of anacardic acids from acetate. ~eve~heless, a standard incubation time of 72 h was chosen for compa~son with other precursors. Incubations for 72 h with [ 2-14C] acetate, [ 2-14C] malonic acid, [ 1-14C] hexanoate and [U-‘4C] glucose yielded 5-6% 14C in anacardic acids. However, these substrates gave greatly different levels of 14C in other lipids, decreasing from malonic acid, acetate, hexanoate to glucose. Malonic acid was the best precur-
TABLE II INCORPORATION SEEDS Harvest
AND DISTRIBUTION
Precursor
Incubation (h)
OF “C
IN ANACARDIC ACIDS OF IMMATURE GINKGO
Percent of offered 14c
14C in anacardic acids
Anacardic Other lipids acids
Ring/ Chain*
[I-14c1acetate [l-l 4Cl acetate [l-’ 4CJacetate [l-14C]acetate
6 12 24 12
6.6 10.5 11.7 12.0
13.0 18.3 18.6 14.4
4.2 5.3 4.8 4.0
II
[l-14C]acetate [Z-l 4c1 acetate 12-14C1 malonate 11-*4C1 hexanoate
12 72 72 72
12.0 5.6 6.0 5.6
11.7 12.1 18.9 5.3
5.4 3.4 43.3 16.5
III
IU-I4Clglucose [l-’ 4Cl gIucose f2-‘%I ducose 13-I 4 Cl glucose [6-14Cl~lucose
72 72 72 72 72
5.5 5.1 5.8 3.2 6.0
2.7 4.3 5.3 2.6 5.4
1.2 1.2 1.2 0.8 1.1
I
[l-14c11aurate ~l-14Clpalmitoleate
72 72
1.0 0.5
20.6*** 30.4***
I
-
Carboxyl**
108.9 7.8 1.6 107.9 -
* Ratio of “C/C equiv. in ring to * 4 C/C equiv. in chain. Ring CB is measured with the chain as fatty acid and its “C/C equiv. is at the level of I 4C/C equiv. of the chain [Sf . ** Percent of theoretical value for even labefing of alternate carbons in the saIicyIic moiety. *** Corrected by subtraction of radioactivity in free fatty acids.
20
sor for the common lipids. However, the ratios of 14C in ring/chain show that it was also by far the best precursor for the salicylic moiety and very poor for the chains of anacardic acids. The efficiency of ring synthesis decreased from malonic acid in the sequence, hexanoate, acetates and glucose. Radioactivities in the anacardic carboxyl groups indicate that they originated from carboxyl groups of acetic and malonic acids as is expected for a polyketide synthesis. Randomization of 14C was between 1 and 9% in reference to alternate labeling of the ring. It was lowest from malonic acid which strongly suggests that the salicylic moiety was formed from acetate via malonate. High radioactivity in the anacardic carboxyl group and ring was also found from [ l-14C] hexanoate. The synthesis apparently proceeded via degradation to acetate. Consistent with this, the randomization in the carboxyl group was equal to that from both acetates as extraneous substrates. The high value for 14C in ring/chain and the low value of 14C in other lipids also indicated that hexanoate as such was not an efficient precursor for chain synthesis. The Cz units in de novo form were apparently more readily available for synthesis of the salicylic acid moiety than for the chain. [U-14C] glucose was the only substrate from which labeling was markedly higher in anacardic acids than in other lipids. The ratios of 14C in ring and chain were similar with all labeled glucoses (Table II). Acetate derived from metabolism of glucose apparently was equally available for synthesis of the salicylic and the chain moietes of anacardic acids, but was less suitable than exogenous acetate for synthesis of chains in common lipids. Very little 14C from laurate and palmitoleate was found in anacardic acids, although seeds of this harvest synthesized them at the usual levels from acetate. The fatty acids were accessible for synthesis of other lipids and, from palmitoleate, 20% of the incorporated activity was in longer chain and more highly unsaturated acids while palmitic acid was of low relative specific activity. Palmitoleic acid has the olefinic (n-7) structure that is predominant in anacardic acids of the tissue (Table I, footnote ***), but as an exogenous substrate it did not serve directly for synthesis of anacardic acids. The minor amount of radioactivity found in them problably entered via acetate. The relative specific activities of anacardic acids with olefinic chains were not higher than of those with saturated chains. Seeds incubated with [ G-14C] shikimic acid or [ U-14C] phenylalanine failed to incorporate 14C into anacardic acids. However, 3.8% of 14C from the latter substrate was found in the common lipids. Incorporation of these compounds into salicylic acid by mycobacteria was indicative of the smkimic acid pathway [ 111 ; whereas their non-incorporation into 6-methylsalicylic acid, was in accord with the polyketide pathway established for the latter [ 121. The experiments described here show that the salicylic moiety of anacardic acids in synthesized, as it has been shown for 6-methylsalicylic acid [ 13,141 by a polyketide mechanism which involves malonate. Comparison of radioactivities from different precursors in anacardic alkyl and salicylic moieties, and in common lipids, indicates that the Ginkgo seed synthesizes acyl chains of two different states of activation and possibly in different subcellular sites. One serves as precursor for the alkyl chain of anacardic acids and occurs mainly in the young seed. This synthetic system does not convert exogenous fatty acids
21
into anacardic acids and it is rather unstable in storage. The acyl chains of the other state of activation or site are precursors for common lipids but not for anacardic acids. This system incorporates exogenous fatty acids and predominates in the mature seed where it is very stable in storage. Acknowledgments The investigation was supported in part by U.S. Public Health Service Grant AM 05165 from the National Institute of Health; U.S. Public Health Service Grant HL 08214 from the Program Project Grant Branch, Extramural Programs, National Heart Institute; and by The Hormel Foundation. We are thankful to Dr. D.D. Smith for providing Ginkgo seeds. References
6 I a 9 10 11 12 13 14
Backer. H.J. and Hawk, N.H. (1941) Rec. Trav. Chim. Pays-Bas 60, 661-677 Izzo. P.T. and Dawson, C.R. (1949) J. Org. Chem. 14,1039-1047 Paul, V.J. and Yeddanapalli. L.M. (1954) Nature 174, 604 Boekenoogen, H.A. (1967) Chem. Ind. (London) 387-397 Geissman. T.A. and Grout, D.H.G. (1969) Organic Chemistry of Secondary Plant Metabolism, p. 109, Freeman, Cooper and Company. San Francisco Gellerman, J.L. and Schlenk, H. (1968) Anal. Chem. 40. 739-743 Birch, A.J, (1957) Progress Chemistry Organic Natural Products (Zechmeister, L., ed), Vol. 14, pp. 186-216. Springer Verlag. Vienna Gellerman, J.L. and Schlenk, H. (1969) Lipids 4. 484487 Gellerman, J.L., Anderson, W.H. and Schlenk. H. (1974) Lipids 9, 722-725 Graff. G., Marcel, Y.L. and Holman, R.T. (1969) J. Chromatogr. Sci. 7. 298-299 Hudson. A.T. and Bentley, R. (1970) Biochemistry 9,3984-3987 Hudson, A.T., Campbell, I.M. and Bentley, R. (1970) Biochemistry 9.3988-3992 Bu’Lock, J.D. and Smalley, H.M. (1961) Proc. Chem. Sot. 209-211 Dimroth, P., Walter, H. and Lynen, F. (1970) Eur. J. tiiochem. 13, 98-110
Biochimica @ Elsevier
el Biophysics
Scientific
Acta,
Publishing
431 (1976) 16-21 Company, Amsterdam
- Printed
in The Netherlands
BRA 56749
SYNTHESIS
JOANNE
L. GELLERMAN,
The Hormel
(Received
OF ANACARDIC
Institute,
October
lst,
ACIDS IN SEEDS OF GINKGO BZLOBA
WAYNE H. ANDERSON
University
of Minnesota,
Austin,
and HERMANN Minn.
55912
SCHLENK (U.S.A.)
1975)
Summary Anacardic (6alkylsalicylic) acids and common lipids are efficiently synthesized by immature seeds of Ginkgo biloba. The seeds were incubated with 14C!labeled acetic, malonic and palmitoleic acids, glucose, and other potential precursors. Levels of 14C in common lipids and in anacardic acids, and the distribution of 14C in anacardic acids were determined. The results show that the salicylic moiety is synthesized by a polyketide pathway via malonic acid. The chain moiety for anacardic acid synthesis is in a different state of activation and/or site than chains that are used for synthesis of the common lipids. Labeled shikimic acid did not contribute 14C to anacardic acids, nor to other lipids, and palmitoleic acid was incorporated only into common lipids.
Introduction Anacardic acids are 6-alkylsalicylic acids in which the alkyl chains are saturated or olefinic straight chains of 13, 15 or 17 C atoms [l-3]. Unlike the common fatty acids, anacardic acids occur only as free acids and are found only in a few plants, namely in Anacardiaceae and in Ginkgo biloba [4]. In anacardic acid biosynthesis, it is likely that the alkyl chains derive from fatty acids [5] and this assumption is supported by the position of double bonds in them [6]. A polyketide pathway has been suggested for the synthesis of the salicylic moiety [ 5,7] and some evidence in support of this was brought forward by experiments in this laboratory [ 8,9]. Infusion of [ l-14C] - and [ 2-14C] acetic acid into Ginkgo plants yielded radioactive lipids. A small part of the 14C was found in anacardic acids. Chemical degradation of these revealed considerable randomization but an alternating pattern of labeling was apparent. In addition, it was found that 14C!/C equivalent in the salicylic part of the molecule was more than twice that in the chain. The level of 14C in ring C-6 showed that this C atom originated with synthesis of the chain rather than the ring.
17
A more efficient system of anacardic acid biosynthesis has now been found. “C-labeled acetic acid into anacardic Immature Ginkgo seeds incorporated acids much more efficiently than the whole plant and with less randomization. This enabled us to test also malonic and p~mitoleic acids and other potential precursors, including shikimic acid, for incorporation into the salicylic as well as the chain moiety. Materials and Methods Ginkgo plants about 1 m high were obtained from previous planting close to The Hormel Institute. Seeds were obtained from trees at the University of Northern Iowa, Cedar Falls, Iowa. Methods for analysis of anacardic acids in plant tissues were as previously described 161. Gas liquid chromatography was carried out on OV-1, 5% on Gas Chrom Q, 100/120 mesh (Applied Science Laboratory, Inc., U.S.A.) at 234°C. Radioactivity of lipids was measured in toluene-based scintillation fluid (Packard Instrument Co., U.S.A.) and of CO, in Hyamine with such fluid added [9] . Compounds labeled with 14C were obtained ~ommerci~ly. For incubations, acetic and hexanoic acids were used as Na salts and fatty acids as NH, salts. All substrates, 2-100 PC, were dissolved in 100 ~1 distilled water for each set of incubations. These were carried out with immature Ginkgo seeds of approximately 1 g, harvested in late June, and with seeds of approximately 8 g, harvested in mid August. The seeds were kept at 3°C until use, within 4 h after harvest, or for the specified storage times. The peduncle was removed from the seed so that a small cavity was left at the point of attachment, to which the radioactive solution was added. In each experiment, four seeds were mounted on cubes of Styrofoam and placed in a humidified culture dish with transparent cover. They were kept at 25” C under 400 foot-candles illumination. Water was added as radioactive solutions were taken up. Incubations were terminated by immersing the seeds into chloroformimethanol, 2 : 1, by vol., and the lipids were obtained by two extractions with this solvent in an Omnimixer. The combined extracts were shaken with 0.2 vol. of 0.9% aqueous NaCl and the lipids were recovered from the chloroform phase. Anarcardic acids were separated from other lipids by thin layer chromatography. Samples of 70 mg were streaked on Silica Gel H (E. Merck A.G., G.F.R.), 1 mm thick on 20 X 20 cm plates and developed in chloroform/methanol/10 M NH,OH, 65 : 30 : 4. The band of anacardic acids, RF approximately 0.75, was located by its fluorescence under ultraviolet light. Anacardic acids were extracted with diethyl ether saturated with aqueous concentrated HCl. The solution was washed to neutrality and the ether removed under vacuum. Common lipids were extracted from the silica gel with chloroform and methanol. The major portion of each anacardic acid fraction was converted to the methyl ether of methyl anacardate with excess diazomethane [ 61. The dimethyl derivatives were purified by thin layer chromatograms developed in petroleum ether (b.p. 60_70“C)/diethyl ether/acetic acid, 80 : 20 : 1. They were located by their fluorescence in ultraviolet light and recovered by extraction with chloroform. The radiopurity of the anacardic acids from the first chromatog-
18
raphic fractionation was calculated from the ratio of 14C in pure dimethyl derivatives and in contaminants as separated in the second chromatogram. The radioactivity of the anacardic carboxyl group was determined by counting 14C0, released by thermal decarboxylation of the acids [9]. For determination of the radioactivity in the alkyl chains, the olefinic double bonds of the dimethyl derivatives were hydrogenated. Subsequent oxidation with CrO, in acetic acid yielded a mixture of fatty acids which essentially represents the alkyl chains [ 91, Their radioactivity was measured and that of the salicylic moiety was obtained by subtraction. The dimethyl derivatives of anacardic acids were fractionated according to their olefinic unsaturation by chromatography on glass fiber paper impregnated with Si02 (Gelman Instrument Co., U.S.A.) and AgN03 [lo]. The developing solvent was petroleum ether (b.p. 30--6O”C)/diethyl ether/acetic acid, 90 : 10 : 1. The bands were detected in ultraviolet light after spraying with dichlorofluorescein. The materials were recovered by extraction with chloroform and aliquots were counted for radioactivity. The fractions were also analyz$d by gas liquid chromatography for chain length composition and monounsaturated fractions were ozonized to determine the position of the double bonds [6 J . Results and Discussion
Anacardic acids were found in all tissues of Ginkgo biloba except the embryo, and relative to other lipids, highest levels were found in the outer fleshy layer of mature seeds and in whole developing seeds (Table I). Anacardic acids with monounsaturated alkyl chains were prominent in all tissues (see footnote in Table I for anacardic acid composition of young seeds). Accordingly, further investigations focused on seeds. However, seeds harvested in the middle of August incorporated only 0.9% of 14C offered as sodium acetate or malonic acid into anacardic acids and 10-34s into other lipids.
TABLE
I
OCCURRENCE
OF ANACARDIC
ACIDS
IN GINKGO
_ Tissue
Total lipids % of fresh weight
Anacardic acids % of total lipids
Leaves Spur shoots Tips Stem i Bark + Wood Roots Mature seed ( (October)
3.0 2.5 3.0 5.0 0.9 0.8 3.0 0.5 2.0 3.0-3.4
20 20 20 10 5 15 50 25 0 60-75***
Cambium
outer layer* Shell** Embryo
Whole developing (June-August)
seed
* Outer fleshy layer of integument. ** Stony layer of integument. * * * Approximate composition, according 13 : 0, with small amounts
of 15 : 0. 17
to alkvl chains, 70% : 1 (n-5) and 17 : 2.
15
:1
(n-7).
12%
17 : 1 (n-7).
10%
19
Apparently synthesis of common lipids was predominant for the maturing seed while only small amounts of anacardic acids were synthesized during this period of growth. After storage of seeds for 12 weeks at 3”C, incorporation from [ 2-14C] malonic acid was lowered to 0.2% in anacardic acids, but was raised to 50% in other lipids. Immature seeds, harvested during the second half of June, proved to be more efficient for synthesis of anacardic acids. Young seeds incorporated 5-12% of 14C offered as sodium acetate or malonic acid into anacardic acids, and 1219% into other lipids. After 3 weeks storage at 3”C, incorporation of 14C decreased twice as much for anacardic acids than for other lipids. The results listed in Table II are from seeds harvested in late June and incubated within 4 h after harvest. Incorporations into anacardic acids were highest from [ l-14C] acetate and reached values of lO--12% after 12 h. This was the approximate period needed for uptake of the radioactive solutions by the seeds and longer incubation did not markedly change the radioactive levels of anacardic acids from acetate. ~eve~heless, a standard incubation time of 72 h was chosen for compa~son with other precursors. Incubations for 72 h with [ 2-14C] acetate, [ 2-14C] malonic acid, [ 1-14C] hexanoate and [U-‘4C] glucose yielded 5-6% 14C in anacardic acids. However, these substrates gave greatly different levels of 14C in other lipids, decreasing from malonic acid, acetate, hexanoate to glucose. Malonic acid was the best precur-
TABLE II INCORPORATION SEEDS Harvest
AND DISTRIBUTION
Precursor
Incubation (h)
OF “C
IN ANACARDIC ACIDS OF IMMATURE GINKGO
Percent of offered 14c
14C in anacardic acids
Anacardic Other lipids acids
Ring/ Chain*
[I-14c1acetate [l-l 4Cl acetate [l-’ 4CJacetate [l-14C]acetate
6 12 24 12
6.6 10.5 11.7 12.0
13.0 18.3 18.6 14.4
4.2 5.3 4.8 4.0
II
[l-14C]acetate [Z-l 4c1 acetate 12-14C1 malonate 11-*4C1 hexanoate
12 72 72 72
12.0 5.6 6.0 5.6
11.7 12.1 18.9 5.3
5.4 3.4 43.3 16.5
III
IU-I4Clglucose [l-’ 4Cl gIucose f2-‘%I ducose 13-I 4 Cl glucose [6-14Cl~lucose
72 72 72 72 72
5.5 5.1 5.8 3.2 6.0
2.7 4.3 5.3 2.6 5.4
1.2 1.2 1.2 0.8 1.1
I
[l-14c11aurate ~l-14Clpalmitoleate
72 72
1.0 0.5
20.6*** 30.4***
I
-
Carboxyl**
108.9 7.8 1.6 107.9 -
* Ratio of “C/C equiv. in ring to * 4 C/C equiv. in chain. Ring CB is measured with the chain as fatty acid and its “C/C equiv. is at the level of I 4C/C equiv. of the chain [Sf . ** Percent of theoretical value for even labefing of alternate carbons in the saIicyIic moiety. *** Corrected by subtraction of radioactivity in free fatty acids.
20
sor for the common lipids. However, the ratios of 14C in ring/chain show that it was also by far the best precursor for the salicylic moiety and very poor for the chains of anacardic acids. The efficiency of ring synthesis decreased from malonic acid in the sequence, hexanoate, acetates and glucose. Radioactivities in the anacardic carboxyl groups indicate that they originated from carboxyl groups of acetic and malonic acids as is expected for a polyketide synthesis. Randomization of 14C was between 1 and 9% in reference to alternate labeling of the ring. It was lowest from malonic acid which strongly suggests that the salicylic moiety was formed from acetate via malonate. High radioactivity in the anacardic carboxyl group and ring was also found from [ l-14C] hexanoate. The synthesis apparently proceeded via degradation to acetate. Consistent with this, the randomization in the carboxyl group was equal to that from both acetates as extraneous substrates. The high value for 14C in ring/chain and the low value of 14C in other lipids also indicated that hexanoate as such was not an efficient precursor for chain synthesis. The Cz units in de novo form were apparently more readily available for synthesis of the salicylic acid moiety than for the chain. [U-14C] glucose was the only substrate from which labeling was markedly higher in anacardic acids than in other lipids. The ratios of 14C in ring and chain were similar with all labeled glucoses (Table II). Acetate derived from metabolism of glucose apparently was equally available for synthesis of the salicylic and the chain moietes of anacardic acids, but was less suitable than exogenous acetate for synthesis of chains in common lipids. Very little 14C from laurate and palmitoleate was found in anacardic acids, although seeds of this harvest synthesized them at the usual levels from acetate. The fatty acids were accessible for synthesis of other lipids and, from palmitoleate, 20% of the incorporated activity was in longer chain and more highly unsaturated acids while palmitic acid was of low relative specific activity. Palmitoleic acid has the olefinic (n-7) structure that is predominant in anacardic acids of the tissue (Table I, footnote ***), but as an exogenous substrate it did not serve directly for synthesis of anacardic acids. The minor amount of radioactivity found in them problably entered via acetate. The relative specific activities of anacardic acids with olefinic chains were not higher than of those with saturated chains. Seeds incubated with [ G-14C] shikimic acid or [ U-14C] phenylalanine failed to incorporate 14C into anacardic acids. However, 3.8% of 14C from the latter substrate was found in the common lipids. Incorporation of these compounds into salicylic acid by mycobacteria was indicative of the smkimic acid pathway [ 111 ; whereas their non-incorporation into 6-methylsalicylic acid, was in accord with the polyketide pathway established for the latter [ 121. The experiments described here show that the salicylic moiety of anacardic acids in synthesized, as it has been shown for 6-methylsalicylic acid [ 13,141 by a polyketide mechanism which involves malonate. Comparison of radioactivities from different precursors in anacardic alkyl and salicylic moieties, and in common lipids, indicates that the Ginkgo seed synthesizes acyl chains of two different states of activation and possibly in different subcellular sites. One serves as precursor for the alkyl chain of anacardic acids and occurs mainly in the young seed. This synthetic system does not convert exogenous fatty acids
21
into anacardic acids and it is rather unstable in storage. The acyl chains of the other state of activation or site are precursors for common lipids but not for anacardic acids. This system incorporates exogenous fatty acids and predominates in the mature seed where it is very stable in storage. Acknowledgments The investigation was supported in part by U.S. Public Health Service Grant AM 05165 from the National Institute of Health; U.S. Public Health Service Grant HL 08214 from the Program Project Grant Branch, Extramural Programs, National Heart Institute; and by The Hormel Foundation. We are thankful to Dr. D.D. Smith for providing Ginkgo seeds. References
6 I a 9 10 11 12 13 14
Backer. H.J. and Hawk, N.H. (1941) Rec. Trav. Chim. Pays-Bas 60, 661-677 Izzo. P.T. and Dawson, C.R. (1949) J. Org. Chem. 14,1039-1047 Paul, V.J. and Yeddanapalli. L.M. (1954) Nature 174, 604 Boekenoogen, H.A. (1967) Chem. Ind. (London) 387-397 Geissman. T.A. and Grout, D.H.G. (1969) Organic Chemistry of Secondary Plant Metabolism, p. 109, Freeman, Cooper and Company. San Francisco Gellerman, J.L. and Schlenk, H. (1968) Anal. Chem. 40. 739-743 Birch, A.J, (1957) Progress Chemistry Organic Natural Products (Zechmeister, L., ed), Vol. 14, pp. 186-216. Springer Verlag. Vienna Gellerman, J.L. and Schlenk, H. (1969) Lipids 4. 484487 Gellerman, J.L., Anderson, W.H. and Schlenk. H. (1974) Lipids 9, 722-725 Graff. G., Marcel, Y.L. and Holman, R.T. (1969) J. Chromatogr. Sci. 7. 298-299 Hudson. A.T. and Bentley, R. (1970) Biochemistry 9,3984-3987 Hudson, A.T., Campbell, I.M. and Bentley, R. (1970) Biochemistry 9.3988-3992 Bu’Lock, J.D. and Smalley, H.M. (1961) Proc. Chem. Sot. 209-211 Dimroth, P., Walter, H. and Lynen, F. (1970) Eur. J. tiiochem. 13, 98-110
