- Email: [email protected]
Fat metabolism in higher plants
BIOCHIMICA ET BIOPHYSICA ACTA
FAT METABOLISM XXII.
ENZYMIC
PARTICULATE
S. F. YANG* Department
IN HIGHER
SYNTHESIS
P.
PLANTS
OF I+HYDROXY-II-EICOSENOIC
PREPARATIONS
AND
27
OF AVOCADO
ACID
BY
MESOCARP
K. STUMPF
of Biochemistry
and Biophysics,
University
of California.
Davis.
Galif. (U.S.A
.)
(Received March 3rd. 1964)
SUMMARY I. The polar acidformedbyincubating [I-Wlacetyl-CoA with sonically solubilized enzyme from avocado particulate particle was identified as x4-hydroxy-rreicosenoate with most of the radioactivity associated on the carboxyl carbon. 2. r+Hydroxy-II-eicosenoate is synthesized by incubating ricinoleate and acetyl-CoA with avocado particulate particle. Cofactors include ATP, TPNH, and DPNH. HCO; is not required and malonyl-CoA is inactive. This elongation system appears to be specific for long-chain hydroxy fatty acids.
INTRODUCTION
In 1962 BARRON AND STUMPF~ described the formation of along-chain polar fatty acid when acetyl-CoA was incubated with either intact mitochondrial particles isolated from avocado mesocarp or with extracts derived from these particles after brief exposure to intense sonication. Malonyl-CoA was not converted to this polar acid. In this paper we shall present evidence concerning the formation and characterization of this polar acid and will discuss its possible function. EXPERIMENTAL PROCEDURES
The particulate fraction was isolated from avocado mesocarp as previously described’. For the preparation of sonically solubilized enzyme, the particulate fraction, suspended in 15-20 ml of sucrose-phosphate buffer was sonicated with the Biosonik Probe sonicator (Bronwill Scientific Co., Rochester, N.Y.) at the maximum output for a total of 10-15 set at 5-see intervals to minimize heating. The Probe was immersed in an ice bath before each interval of sonication and the test tube containing the suspension was surrounded with ice to dissipate the heat. The sonicated material was * Present address : Department of Pharmacology. New York University School of Medicine, New York City, N.Y. Biochim.
Biophys.
Ada,
98 (1965) q-35
S. F. YANG
28
AND P. K. STUMPF
centrifuged at 12000 x g for 20 min, the pellet was discarded and the supernatant fluid was termed the “sonically solubilized enzyme”. The assay system was the same as previously employed”. Either 0.3 ml of the particulate fraction or I ml of the sonically solubtied enzyme was used. The extraction procedure, the counting techniques, the isolation of the reaction products and the methods of reversed-phase paper chromatography have been described in the previous paper8. Polar and non-polar acids were separated as methyl esters by silicic acid thinlayer chromatography with hexane-diethyl ether (6: 4, v/v) as the developing system*_ By this technique methyl esters of various types of fatty acids could be separated into classes according to their functional groups. Esters were visualized by spraying the thin-layer chromatography plates lightly with 0.05% 2,7dichlorofluorescein in 50% ethanol and examining under ultraviolet light. The resolved esters were then eluted from the silicic acid layer by transferring the gel to a centrifuge tube and then extracting three times with diethyl ether. The ether extracts were evaporated to dryness under a stream of N, and redissolved in light petroleum. For the purpose of routine counting, the fatty acid ester areas in the thin-layer chromatography plates were scraped directly into a scintillation vial containing 5 ml of toluene-phosphor solution and counted in a Packard liquid scintillation spectrometer. The SCHMIDTdecarboxylation reaction was employed as modified by GOLDFINEAND BLOCH~. [I-14C]Acetyl-CoA was synthesized from [I-X]acetic anhydride by the method of SIMONAND SHEMIN~. [2-%]Malonyl-CoA was prepared by the method of EGGERER AND LYNEN~. A-l
B-I
fr_.___A. KP
16:O t 16:l
16:O
180
180
POLAR
ACID
A-2
POLAR ACID \
Fig. I. Reaction mixture contained: 200 pmoles potassium phosphate buffer (pH 7.4), IO pmoles ATP, 2 ymoles MnCl,, 0.15 pmole TPN, 0.5 pmole Glc-6-P, 0.25 pmole, [r-W]acetyl-CoA (7.10~ counts/min), and 0.3 ml of particulate particle containing 1.5 mg of protein or I ml of sonically solubilized particulate enzyme containing I .8 mg of protein, in a total volume of I .5 ml. Reaction mixture was incubated at 37” for 1.5 h. The siliconized paper strips were first developed with 85% acetic acid and scanned with a NuclearChicago strip counter (A-I and B-I). The same paper strips were then developed with acetic acid30% H,O,-formic acid (7: I : I, v/v) and scanned (A-2 and B-2). InPanels A-I and A-2 a particulate preparation was used and in Panels B-I and B-2 sonically solubilized enzyme was used. Biochim.
Biophys.
Acta, 98 (1965) q-35
I&IYDROXY-II-EICOSENOIC
ACID SYNTHESIS
29
RESULTS
When [r-ldC]acetyl-CoA is incubated with the particulate fraction, palmitic, oleic and stearic acids are the predominant products with a polar acid as a minor component. With the sonically solubilized enzyme, the major product is a polar acid which migrates to the front when a reversed-phase paper chromatogram is developed with 85% acetic acid. These results are illustrated in Fig. I. In another series of experiments, sonically solub~zed enzyme is incubated with either [r-X]acetyl-CoA, [r-r*C]acetyl-CoA plus COI, or with [r-l*C]malonyl-CoA and the products are analyzed by reversed-phase paper chromatography. As summarized in Table I, when acetyl-CoA is the sole substrate the major product is thepolaracid; TABLE I SUBSTRATE
SPECIFICITY
FOR
THE
SYNTHESIS
OF
POLAR
ACID
BY
TEE
SONXCALLY
SOLUBILXZED
ENZYME
Reaction mixtures contained 160 mpmoles of [I-W)acetyl-CoA (300 ooo countslmin) or 200 mpmoles of [a-W]malonyl-CoA (120 ooo counts/min). Cofactors and the reaction conditions were the same as described in Fig. I except that 30 pmoles of HCO,- were added where indicated. Substrate
TOM incor#w-at&m
Acetyl-CoA Acetyl-CoA + HCO,Malonyl-CoA
2.48 6.44 16.4
Distribnrtion of label in acid ~m~rno~s~ ., ~ Pahitate P&Y acid Stearate 0.10
1.84 IO.9
0.34 2.30 5.2
2.04 2.30 0.3
when CO, is introduced, appro~at~y a s-fold st~ulation in acetyl-CoA incorporation is noted with a marked increase in non-polar fatty acids but no noticeable increase in the polar acid. With malonyl-CoA as the substrate, little if any polar acid is synthesized but large amounts of pahnitic and stearic acids are formed. These data would suggest that acetyl-CoA and not malonyl-CoA is involved in the synthesis of the polar compound, in agreement with the earlier results of BARRON AND STUMPF~. The identi~cation of this polar acid was thus undertaken.
The following results indicate that the polar acid is a Cl0 monohydroxymonounsaturated fatty acid with the radioactive carbon exclusively associated with its carboxyl carbon. (a) Free fatty acids were obtained by saponifying the total lipid isolated from a reaction mixture in which [r-l*C]acetyl-CoA was incubated with a sonically solubilized enzyme. The reaction mixture was saponified, acidified, and the free fatty acid extracted with ether. The free fatty acids were chromatographed on siliconized paper strips with 85% acetic acid as the developing solvent. After locating the polar acid by scanning the developed paper strips with a qr Nuclear-Chicago Actigraph II strip counter, the polar acid was eluted with methanol and spotted on silica gel G thin-layer plates and developed with hexane-ether (IO: 6, v/v) as solvent. All the radioactivity remained at the origin. The radioactive compound was eluted from the silica gel G by extracting with diethyl ether, methylated with diazomethane, and rechromatographed ~iochim. Biophys. Acta, 98
(IQ@)
27-35
S. F. YANG AND P. K. STUMPF
30
with the same solvents on silica gel G thin-layer chromatography plates. The radioactivity now moved to an area where the methyl esters of monohydroxymonounsaturated fatty acids migrate {RF 0.50). This result indicated that the material is a carboxylic acid with a polar functional group (hydroxy or keto). (b) No change in RF value occurred when the material was reduced with NaBHI and rechromatographed on thin-layer chromatography plates. This result indicated the absence of a keto function since the conversion of a keto (less polar) to a hydroxy (more polar) function would cause a decrease in the RF value in a silica gel G thin-layer chromato~aphy system. (c) The polar ester was eluted off the thin-layer chromatography plate with diethyl ether and subjected to gas-liquid chromatography at 231’ on an Apiezon-M column 2.5 ft long, and 0.25 in in diameter. Most of the radioactivity appeared with a retention time equivalent to a methyl ester of a fatty acid with 21.6 carbons. This retention time corresponded precisely to that of the methyl ester of authentic x4hydroxy-r r-eicosenoic acid. (d) When the polar acid was exposed to bromine? and then chromatographed as in (c), the radioactivity was retained on the column. After catalytic reduction with 10% Pd on charcoal at 40 lb H, pressure at room temperature for 2 h, the polar compound was again chromatographed on an Apiezon-M column and the retention time was now equivalent to a methyl ester of a fatty acid with 22.1 carbon atoms. This retention time corresponds precisely with a methyl ester of rq-hydroxyeicosanoic acid and strongly suggests the presence of a single double-bond system. (e) The polar acid was refluxed with HI (2 ml) and red phosphorus (200 mg) in propionic acid (I ml) for 6 h. Under these conditions hydroxy and double-bond functions are fully reduced. After dilution with water, the acid was extracted with diethyl ether, washed with aqueous NaHSO,, methylated and chromato~aphed on thin-layer chromatography with hexane-ether as solvent. The resulting acid now moved to the solvent front indicating the complete reduction of the polar function. This reduced radioactive acid was then subjected to gas-liquid chromatography on an Apiezon-M column, and the appearance of radioactivity in the effluent vapors corresponded precisely to the retention time of a methyl ester of eicosanoic acid. These results indicate that the polar acid is a monohydrox~onounsaturated fatty acid with 20 C atoms. (f) The position of unsaturation was determined by permanganate-periodate oxidation of the polar acids 8. When the products were extracted, methylated and chromatographed on thin-layer chromatography, all the radioactivity appeared at the area of the dimethyl ester of a dicarboxylic acid (RF 0.68). Essentially no radioactivity could be recovered in the region of a monohydroxydic~boxylate ester (RF 0.35), the monohydroxymonocarboxylate ester (RF 0.50) or a non-polar fatty acid ester (RF 0.95). When chromatographed on an Apiezon-L column at 210’ ,the radioactivity was associated with the peak of authentic dimethylundecanedioate. Thus the double bond is located between C-II and C-12, the hydroxy group of the polar acid must be located between C-13 and C-20, and all the radioactivity is located between C-I and C-XI. (g) The position of the hydroxy group was indicated by chromic acid oxidation. After catalytic hydrogenation, the radioactive polar acid was dissolved in I ml of glacial acetic acid along with 2 mg each of rz-hydroxystearic acid and rq-hydroxyBiochim.Siop&s. Acta, 98
(1~65)
27-35
IL+-HYDROXY-II-EICOSENOIC
31
ACID SYNTHESIS
rr-eicosanoic acid as carriers and 30 mg of chromic acid. The mixture was heated to 80” for z h. The oxidation products were extracted with ether, methylated and subjected to gas-liquid chromatography in an Apiezon-M column. Because of unavoidable over-oxidation, the radioactivity could not be recovered in a single peak but was associated with a series of dicarboxylic acids ranging from C,, to lower homologues. Since homologues higher than C,, were not observed and since all the radioactivity was associated with the C,,-dicarboxylic acid and its lower homologues, the results suggest that the position of the hydroxy function is on C-14. Control experiments with known I2-hydroxystearic and 14-hydroxyeicosanoic acids confirmed this pattern of oxidation products. (h) The polar ester co-chromatographed with known methyl x4-hydroxy-IIeicosenoate on both polar (Craig) and non-polar (Apiezon M) columns by gas-liquid chromatography. (i) Decarboxylation of the polar acid was carried out by the SCHMIDT reaction as described previously’. Preliminary experiments with known ro-hydroxy-[II-X] palmitic acid showed that essentially no radioactivity was released as CO, indicating that under the prescribed conditions this method was applicable to a hydroxy fatty acid. The yield of CO, and fatty amine from both polar and non-polar fatty acids is shown in Table II. The data indicate that at least 80% of the total radioactivity of TABLE YIELD
II OF
CO,
AND
FATTY
AMINE
BY
SCHMIDT
DE~ARBOX~LATJON
Polar and non-polar fatty acids synthesized under the conditions described in Fig. I B were separated by paper chromatography, and a portion of the eluate was subjected to decarboxylation’s’ after catalytic hydrogenation. Non-polar acid contained about 85% of palmitic and 15% of steak acids. Fully amine (countslmin)
Acid
CO, (countslmin)
Polar Non-polar
920
JJO
200
JO20
CO, : fatty
amine
J:o.Iz
1:5.1
the polar acid is associated with the carboxyl carbon whereas the ratio of CO,: amine of non-polar fatty acids isolated from reaction mixtures is close to that expected from synthesis de novo. We therefore conclude that the polar acid is x4-hydroxy-[I-*F]-II-eicosenoic acid which is formed by the addition of [r-*C]acetyl-CoA to a preexisting acceptor presumably Iz-hydroxy-g-octadecenoic acid according to the series of reactions: x2-hydroxy-g-octadecenoyl-CoA
+ acetyl-CoA
DPNH TPNH_ Iq-hydroxy-1
I-eicosenoyl-CoA
+ CoA
Enzymic
synthesis of rq-hydroxy-rr-eicosenoic acid With the discovery that the chemical structure of the polar compound is 14hydroxy-x1-eicosenoic acid, it became of interest to elucidate further the mechanism of its biosynthesis. In Table III is a series of experiments designed to shed some light on the nature of the long-chain acceptor of [r-“Clacetyl-CoA. In these experiments the particulate fraction is employed since there is little if any polar acid formed in the absence of added long-chain polar acid as the C, acceptor. The data demonstrate that Biochim.
Biophys.
Acla, 98 (1965)
27-35
S. F. YANG AND P. K. STUMPF
32 TABLE
III
ELONGATION
OF HYDROXY
FATTY
ACID
BY
AVOCADO
PARTICULATE
ENZYME
Reaction mixture contained IOO mpmoles of [I-W]acetyl-CoA (350 ooo countslmin) or 200 rnpmoles of [2-Wjmalonyl-CoA (IZO ooo counts/mm) and 0.3 ml of particulate preparation containing 2.0 mg (Expt. A) and 1.6 mg (Expt. B) of protein. Cofactors and the reaction conditions were the same as described in Fig. I except that HCO,- was added only where indicated. Fatty acids added are all 0.5 pmole except otherwise indicated. Non-polar and polar acids were separated by thinlayer chromatography as methyl ester@. Total incorporation (mpmoles)
Addition
Substrate
A. Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
12-hydroxystearate ricinoleate stearate oleate
Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
HCO,HCO,HCO,HCO,-
Malonyl-Cob Malonyl-CoA Malonyl-CoA Malonyl-CoA
12-hydroxystearate ricinoleate stearate
B. Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
+ 12hydroxystearate + ricinoleate + stearate
3-hydroxylaurate 3-hydroxydecanoate ricinoleate (I @mole) ricinoleate (0.5 pmole) ricinoleate (0.2 pmole)
Non-polar acid (m~moles)
Polar acid (mfimoles)
3.12 4.04 4.00 2.48 2.25
3.04 3.00 2.00 2.44 2.20
0.08 1.04 2.00 0.04 0.05
14.94 8.60 9.00 8.40
14.84 7.68 7.00 8.32
0.10 0.92 2.00 0.08
16.2 15.6 14.0 14.5
16.2 15.6 14.0 14.5
0 0 0 0
2.8 2.3 2.9 3.0 4.5 6.0
2.8 2.3 2.9 1.0 I.7 2.7
0 0 0 2.0 2.8 3.3
rz-hydroxystearic and ricinoleic acid (rz-hydroxy-g-octadecenoic acid) are elongated by the addition of [rJ4C]acetyl-CoA. Both stearic and oleic acids are inactive. HCO,is not required in this reaction. Furthermore malonyl-CoA is inactive as a donor. DL-3-hydroxydecanoic and DL-3-hydroxylauric acid are inactive as C, acceptors. Of TABLE
IV
EFFECT OF COFACTORSON ELONGATIONOF RICINOLEATE WITH
ACETYL-COA
BY
PARTICULATE
PREPARATION
Reaction mixture contained 100 mpmoles of [I-Xlacetyl-CoA (350 ooo countslmin) and 0.5 pmole of ricinoleate. Cofactors and the reaction conditions were the same as described in Fig. 1. FMN was 20 mpmoles. &factor Acetyl-CoA % omitted OYadded incorfiorated non-polar polar (mymoles) Complete + FMN - ATP - Mn*+ - TPN+, Glc-6-P - DPNH - Ricinoleate
4.1 4.1 0.2 3.7 I.9 2.6 2.9
Bdochim. Bio$+vs. Acta. 98 (1965) 27-35
15 17 14 16 20 98
85 83 86 84 80 2
-
x4-HYDROXY-II-EICOSENOIC
33
ACID SYNTHESIS
interest is the observation that I pmole of ricinoleic acid is somewhat inhibitory. At this concentration 3 mpmoles of acetyl-CoA are incorporated whereas at a concentration of 0.2 holes of ricinoleic acid, 6 mwoles of acetyl-CoA are accepted under the same conditions. The formation of the polar acid from ricinoleic acidcan occur under anaerobic as well as aerobic conditions. Some cofactor requirements are listed in Table IV. ATP, TPN+, glucose 6-phosphate, and DPNH are essential for the elongation of the polar acid. These cofactors were also essential for the formation of non-polar acids in the absence of an added acceptor. TABLE
V
REACTION PRODUCTS PARTICULATE ENZYME
PRODUCED
BY
INCUBATING
Reactions were carried out as described describedby YANGANDSTUMPP? Fraction
Neutral lipid Free fatty acid Phospholipid Long-chain acyl-CoA
in Table
AND
%
7000 4500 1500
[I-I%]ACETYL-COA
IV and separation
Distribuiion
Total Counls/ min
0
RICINOLEATE
Non-polar
followed
of label acid
WITH
the procedures
1% Polar acid
Counts/ min
yO
Countsf min
y0
54 35
1960 ‘350
52 36
5040 3x50
55 34
12 0
450 0
I2 0
1050 0
xx 0
Table V summarizes the data concerning the distribution of the polar acid in different classes of lipids in the reaction mixture. Most of the newly synthesized acid is associated with neutral lipids. Chemical identification of the polar product formed by incubating ricinoleate and [I-W}acetyl-CoA with the particulate fraction was carried out as follows. (a) Free fatty acids isolated from the reaction mixture after saponification were esterified and then subjected to thin-layer chromatography. A radioactive region corresponded with the monohydroxy acid region. The methyl esters were eluted with ether and were used for subsequent analyses. (b) The polar ester co-chromatographed with known methyl r+hydroxy-IIeicosenoate both on stabilized diethylene glycol succinate-polyester column and on an Apiezon-M column by gas-liquid chromatography. (c) Permanganate-periodate oxidation8 yielded 95% of the total radioactivity in the dicarboxylic acid which was separated from reaction products as a dimethyl ester on thin-layer chromatography. The dimethyl ester was identified as undecanedioic acid by gas-liquid chromatography of the dimethyl ester on Apiezon-M column. (d).%iMIDT decarboxylation of the catalytically hydrogenated polar acid showed essentially all the radioactivity with the carboxyl carbon of the acid. These results indicate that the radioactive polar acid was formed by the addition of [I-W)acetyl-CoA to the carboxyl end of ricinoleic acid. Under the same condition no elongation reaction occurs with steak or oleic acids as examined by gas-liquid chromatography. BiocLim. Biophys.
Acta, 98 (1965) 27-35
S. F. YANG AND P. K. STUMPF
34 DISCUSSION
It is somewhat difficult to evaluate the physiological role of this elongation reaction. The Cs addition appears to be specific for long-chain hydroxy acids since under the same conditions no elongation could be observed with either stearic, palmitic, myristic, 3-hydroxydecanoic or 3-hydroxylauric acids. Neither r4-hydroxy-rr-eicosenoic acid nor r2-hydroxy-o-octadecenoic acid (ricinoleic acid) is a constituent of avocado lipids. However the former is the major constituent of the seed oil of the genus LesquereUa* and the latter is the main fatty acid component of castor bean seeds. Whatever the role of the elongation reaction, it may be a fortuitous assay system which permits detection of the presence of trace amounts of r2-hydroxy-o-octadecenoic acid. This acid, as a possible intermediate in the synthesis of linoleic acid, exists in too low a concentration to be detected by the usual methods. Although linoleic acid constitutes IS?; of the total fatty acid in the avocado, biosynthesis of this acid in avocado cell-free systems has not been successful. We therefore suggest the following sequence : fatty acid syntlvet.w system
acetyl-CoA
-+
+ CO, --0,
[I z-hydroxy-goctadecenoic
acid]
Labile: inactive in preparations
aoetyl-CoA --s-----f DPr;H, TPNH
I)-hydroxy-II-eicosenoicacid
enzyme
& linoleic
acid
Presumably the C,, polar acid, unable to be converted to linoleic acid because of the absence of a specific enzyme system, will react with acetyl-CoA to give the elongation product. Since the C,, polar acid has no radioactivity associated with it except in its carboxyl carbon, its immediate acceptor is not synthesized de %OUOfrom acetylCoA but evidently occurs in trace amounts in the lipid components of the crude enzyme extract. A knowledge of the mechanism of formation of linoleic acid would place in correct perspective the function of this elongation reaction. However it should be noted that BLOCH et uZ.10 have shown with intact yeast cells that [*H]ricinoleic acid does not serve as a substitute for the formation of linoleic acid. The yeast cell will rapidly acetylate the hydroxyl groups to the corresponding acetoxy derivatives which simply accumulate and do not undergo further alterationsiO. ACKNOWLEDGEMENTS
This work was supported in part by grants from the National Science Foundation (G-14823) and National Institute of Health (GM-10132-o1). We are indebted to Dr. P. OYEKATR for preparing a generous supply of [2-r%]malonyl-CoA. We are also indebted to Dr. T. H. APPLEWRITE for a sample of pure methyl r4-hydroxy-xr-eicosenoate and to Dr. A. G. MARR for samples of DL-phydroxydecanoicand -1auric acids.
Biochim.
Biophys. Acta, 98 (x965) 27-35
I+HYDROXY-II-EICOSENOIC
ACID SYNTHESIS
35
REFERENCES I E. J. BARRON AND P. K. STUMPF, J. Biol. Chem., 237 (1962) PC 613. 2 S. F. YANG AND P. K. STUYPF, Biochim. Biophys. Acta, g8(Ig63)Ig. 3 H. K. MANGOLD, J. Am. Oil Chemists’ SOL, 38 (1961) 708. 4 H. GOLDFINE AND K. BLOCH, J. Biol. Gem., 236 (1961) 2596. 5 E. J. SIMONAND D. SHEMIN, J. Am. Chem. Sot., 75 (1953) 2520. 6 H. EGGERER AND F. LYNEN, Biochem. Z., 335 (1962) 540. 7 A. T. JAMES, Methods Biochem. Anal., 8 (1960) I. 8 E. VON RUDOLF, Can J. Chem., 34 (1956) 4131. g K. L. MIKOLAJCZAK. F. R. EARLE AND L. A. WOLFF, J. Am. Oil Chemists’ Soc., 3g (1962) 78. IO R J. LIGHT, W. J. LENNARZ AND K. BLOCH, 1. Biol. Chem., 237 (1961) 1793.
Biochim.
Biophys.
Acta,
g8 (1965) 27-35
FAT METABOLISM XXII.
ENZYMIC
PARTICULATE
S. F. YANG* Department
IN HIGHER
SYNTHESIS
P.
PLANTS
OF I+HYDROXY-II-EICOSENOIC
PREPARATIONS
AND
27
OF AVOCADO
ACID
BY
MESOCARP
K. STUMPF
of Biochemistry
and Biophysics,
University
of California.
Davis.
Galif. (U.S.A
.)
(Received March 3rd. 1964)
SUMMARY I. The polar acidformedbyincubating [I-Wlacetyl-CoA with sonically solubilized enzyme from avocado particulate particle was identified as x4-hydroxy-rreicosenoate with most of the radioactivity associated on the carboxyl carbon. 2. r+Hydroxy-II-eicosenoate is synthesized by incubating ricinoleate and acetyl-CoA with avocado particulate particle. Cofactors include ATP, TPNH, and DPNH. HCO; is not required and malonyl-CoA is inactive. This elongation system appears to be specific for long-chain hydroxy fatty acids.
INTRODUCTION
In 1962 BARRON AND STUMPF~ described the formation of along-chain polar fatty acid when acetyl-CoA was incubated with either intact mitochondrial particles isolated from avocado mesocarp or with extracts derived from these particles after brief exposure to intense sonication. Malonyl-CoA was not converted to this polar acid. In this paper we shall present evidence concerning the formation and characterization of this polar acid and will discuss its possible function. EXPERIMENTAL PROCEDURES
The particulate fraction was isolated from avocado mesocarp as previously described’. For the preparation of sonically solubilized enzyme, the particulate fraction, suspended in 15-20 ml of sucrose-phosphate buffer was sonicated with the Biosonik Probe sonicator (Bronwill Scientific Co., Rochester, N.Y.) at the maximum output for a total of 10-15 set at 5-see intervals to minimize heating. The Probe was immersed in an ice bath before each interval of sonication and the test tube containing the suspension was surrounded with ice to dissipate the heat. The sonicated material was * Present address : Department of Pharmacology. New York University School of Medicine, New York City, N.Y. Biochim.
Biophys.
Ada,
98 (1965) q-35
S. F. YANG
28
AND P. K. STUMPF
centrifuged at 12000 x g for 20 min, the pellet was discarded and the supernatant fluid was termed the “sonically solubilized enzyme”. The assay system was the same as previously employed”. Either 0.3 ml of the particulate fraction or I ml of the sonically solubtied enzyme was used. The extraction procedure, the counting techniques, the isolation of the reaction products and the methods of reversed-phase paper chromatography have been described in the previous paper8. Polar and non-polar acids were separated as methyl esters by silicic acid thinlayer chromatography with hexane-diethyl ether (6: 4, v/v) as the developing system*_ By this technique methyl esters of various types of fatty acids could be separated into classes according to their functional groups. Esters were visualized by spraying the thin-layer chromatography plates lightly with 0.05% 2,7dichlorofluorescein in 50% ethanol and examining under ultraviolet light. The resolved esters were then eluted from the silicic acid layer by transferring the gel to a centrifuge tube and then extracting three times with diethyl ether. The ether extracts were evaporated to dryness under a stream of N, and redissolved in light petroleum. For the purpose of routine counting, the fatty acid ester areas in the thin-layer chromatography plates were scraped directly into a scintillation vial containing 5 ml of toluene-phosphor solution and counted in a Packard liquid scintillation spectrometer. The SCHMIDTdecarboxylation reaction was employed as modified by GOLDFINEAND BLOCH~. [I-14C]Acetyl-CoA was synthesized from [I-X]acetic anhydride by the method of SIMONAND SHEMIN~. [2-%]Malonyl-CoA was prepared by the method of EGGERER AND LYNEN~. A-l
B-I
fr_.___A. KP
16:O t 16:l
16:O
180
180
POLAR
ACID
A-2
POLAR ACID \
Fig. I. Reaction mixture contained: 200 pmoles potassium phosphate buffer (pH 7.4), IO pmoles ATP, 2 ymoles MnCl,, 0.15 pmole TPN, 0.5 pmole Glc-6-P, 0.25 pmole, [r-W]acetyl-CoA (7.10~ counts/min), and 0.3 ml of particulate particle containing 1.5 mg of protein or I ml of sonically solubilized particulate enzyme containing I .8 mg of protein, in a total volume of I .5 ml. Reaction mixture was incubated at 37” for 1.5 h. The siliconized paper strips were first developed with 85% acetic acid and scanned with a NuclearChicago strip counter (A-I and B-I). The same paper strips were then developed with acetic acid30% H,O,-formic acid (7: I : I, v/v) and scanned (A-2 and B-2). InPanels A-I and A-2 a particulate preparation was used and in Panels B-I and B-2 sonically solubilized enzyme was used. Biochim.
Biophys.
Acta, 98 (1965) q-35
I&IYDROXY-II-EICOSENOIC
ACID SYNTHESIS
29
RESULTS
When [r-ldC]acetyl-CoA is incubated with the particulate fraction, palmitic, oleic and stearic acids are the predominant products with a polar acid as a minor component. With the sonically solubilized enzyme, the major product is a polar acid which migrates to the front when a reversed-phase paper chromatogram is developed with 85% acetic acid. These results are illustrated in Fig. I. In another series of experiments, sonically solub~zed enzyme is incubated with either [r-X]acetyl-CoA, [r-r*C]acetyl-CoA plus COI, or with [r-l*C]malonyl-CoA and the products are analyzed by reversed-phase paper chromatography. As summarized in Table I, when acetyl-CoA is the sole substrate the major product is thepolaracid; TABLE I SUBSTRATE
SPECIFICITY
FOR
THE
SYNTHESIS
OF
POLAR
ACID
BY
TEE
SONXCALLY
SOLUBILXZED
ENZYME
Reaction mixtures contained 160 mpmoles of [I-W)acetyl-CoA (300 ooo countslmin) or 200 mpmoles of [a-W]malonyl-CoA (120 ooo counts/min). Cofactors and the reaction conditions were the same as described in Fig. I except that 30 pmoles of HCO,- were added where indicated. Substrate
TOM incor#w-at&m
Acetyl-CoA Acetyl-CoA + HCO,Malonyl-CoA
2.48 6.44 16.4
Distribnrtion of label in acid ~m~rno~s~ ., ~ Pahitate P&Y acid Stearate 0.10
1.84 IO.9
0.34 2.30 5.2
2.04 2.30 0.3
when CO, is introduced, appro~at~y a s-fold st~ulation in acetyl-CoA incorporation is noted with a marked increase in non-polar fatty acids but no noticeable increase in the polar acid. With malonyl-CoA as the substrate, little if any polar acid is synthesized but large amounts of pahnitic and stearic acids are formed. These data would suggest that acetyl-CoA and not malonyl-CoA is involved in the synthesis of the polar compound, in agreement with the earlier results of BARRON AND STUMPF~. The identi~cation of this polar acid was thus undertaken.
The following results indicate that the polar acid is a Cl0 monohydroxymonounsaturated fatty acid with the radioactive carbon exclusively associated with its carboxyl carbon. (a) Free fatty acids were obtained by saponifying the total lipid isolated from a reaction mixture in which [r-l*C]acetyl-CoA was incubated with a sonically solubilized enzyme. The reaction mixture was saponified, acidified, and the free fatty acid extracted with ether. The free fatty acids were chromatographed on siliconized paper strips with 85% acetic acid as the developing solvent. After locating the polar acid by scanning the developed paper strips with a qr Nuclear-Chicago Actigraph II strip counter, the polar acid was eluted with methanol and spotted on silica gel G thin-layer plates and developed with hexane-ether (IO: 6, v/v) as solvent. All the radioactivity remained at the origin. The radioactive compound was eluted from the silica gel G by extracting with diethyl ether, methylated with diazomethane, and rechromatographed ~iochim. Biophys. Acta, 98
(IQ@)
27-35
S. F. YANG AND P. K. STUMPF
30
with the same solvents on silica gel G thin-layer chromatography plates. The radioactivity now moved to an area where the methyl esters of monohydroxymonounsaturated fatty acids migrate {RF 0.50). This result indicated that the material is a carboxylic acid with a polar functional group (hydroxy or keto). (b) No change in RF value occurred when the material was reduced with NaBHI and rechromatographed on thin-layer chromatography plates. This result indicated the absence of a keto function since the conversion of a keto (less polar) to a hydroxy (more polar) function would cause a decrease in the RF value in a silica gel G thin-layer chromato~aphy system. (c) The polar ester was eluted off the thin-layer chromatography plate with diethyl ether and subjected to gas-liquid chromatography at 231’ on an Apiezon-M column 2.5 ft long, and 0.25 in in diameter. Most of the radioactivity appeared with a retention time equivalent to a methyl ester of a fatty acid with 21.6 carbons. This retention time corresponded precisely to that of the methyl ester of authentic x4hydroxy-r r-eicosenoic acid. (d) When the polar acid was exposed to bromine? and then chromatographed as in (c), the radioactivity was retained on the column. After catalytic reduction with 10% Pd on charcoal at 40 lb H, pressure at room temperature for 2 h, the polar compound was again chromatographed on an Apiezon-M column and the retention time was now equivalent to a methyl ester of a fatty acid with 22.1 carbon atoms. This retention time corresponds precisely with a methyl ester of rq-hydroxyeicosanoic acid and strongly suggests the presence of a single double-bond system. (e) The polar acid was refluxed with HI (2 ml) and red phosphorus (200 mg) in propionic acid (I ml) for 6 h. Under these conditions hydroxy and double-bond functions are fully reduced. After dilution with water, the acid was extracted with diethyl ether, washed with aqueous NaHSO,, methylated and chromato~aphed on thin-layer chromatography with hexane-ether as solvent. The resulting acid now moved to the solvent front indicating the complete reduction of the polar function. This reduced radioactive acid was then subjected to gas-liquid chromatography on an Apiezon-M column, and the appearance of radioactivity in the effluent vapors corresponded precisely to the retention time of a methyl ester of eicosanoic acid. These results indicate that the polar acid is a monohydrox~onounsaturated fatty acid with 20 C atoms. (f) The position of unsaturation was determined by permanganate-periodate oxidation of the polar acids 8. When the products were extracted, methylated and chromatographed on thin-layer chromatography, all the radioactivity appeared at the area of the dimethyl ester of a dicarboxylic acid (RF 0.68). Essentially no radioactivity could be recovered in the region of a monohydroxydic~boxylate ester (RF 0.35), the monohydroxymonocarboxylate ester (RF 0.50) or a non-polar fatty acid ester (RF 0.95). When chromatographed on an Apiezon-L column at 210’ ,the radioactivity was associated with the peak of authentic dimethylundecanedioate. Thus the double bond is located between C-II and C-12, the hydroxy group of the polar acid must be located between C-13 and C-20, and all the radioactivity is located between C-I and C-XI. (g) The position of the hydroxy group was indicated by chromic acid oxidation. After catalytic hydrogenation, the radioactive polar acid was dissolved in I ml of glacial acetic acid along with 2 mg each of rz-hydroxystearic acid and rq-hydroxyBiochim.Siop&s. Acta, 98
(1~65)
27-35
IL+-HYDROXY-II-EICOSENOIC
31
ACID SYNTHESIS
rr-eicosanoic acid as carriers and 30 mg of chromic acid. The mixture was heated to 80” for z h. The oxidation products were extracted with ether, methylated and subjected to gas-liquid chromatography in an Apiezon-M column. Because of unavoidable over-oxidation, the radioactivity could not be recovered in a single peak but was associated with a series of dicarboxylic acids ranging from C,, to lower homologues. Since homologues higher than C,, were not observed and since all the radioactivity was associated with the C,,-dicarboxylic acid and its lower homologues, the results suggest that the position of the hydroxy function is on C-14. Control experiments with known I2-hydroxystearic and 14-hydroxyeicosanoic acids confirmed this pattern of oxidation products. (h) The polar ester co-chromatographed with known methyl x4-hydroxy-IIeicosenoate on both polar (Craig) and non-polar (Apiezon M) columns by gas-liquid chromatography. (i) Decarboxylation of the polar acid was carried out by the SCHMIDT reaction as described previously’. Preliminary experiments with known ro-hydroxy-[II-X] palmitic acid showed that essentially no radioactivity was released as CO, indicating that under the prescribed conditions this method was applicable to a hydroxy fatty acid. The yield of CO, and fatty amine from both polar and non-polar fatty acids is shown in Table II. The data indicate that at least 80% of the total radioactivity of TABLE YIELD
II OF
CO,
AND
FATTY
AMINE
BY
SCHMIDT
DE~ARBOX~LATJON
Polar and non-polar fatty acids synthesized under the conditions described in Fig. I B were separated by paper chromatography, and a portion of the eluate was subjected to decarboxylation’s’ after catalytic hydrogenation. Non-polar acid contained about 85% of palmitic and 15% of steak acids. Fully amine (countslmin)
Acid
CO, (countslmin)
Polar Non-polar
920
JJO
200
JO20
CO, : fatty
amine
J:o.Iz
1:5.1
the polar acid is associated with the carboxyl carbon whereas the ratio of CO,: amine of non-polar fatty acids isolated from reaction mixtures is close to that expected from synthesis de novo. We therefore conclude that the polar acid is x4-hydroxy-[I-*F]-II-eicosenoic acid which is formed by the addition of [r-*C]acetyl-CoA to a preexisting acceptor presumably Iz-hydroxy-g-octadecenoic acid according to the series of reactions: x2-hydroxy-g-octadecenoyl-CoA
+ acetyl-CoA
DPNH TPNH_ Iq-hydroxy-1
I-eicosenoyl-CoA
+ CoA
Enzymic
synthesis of rq-hydroxy-rr-eicosenoic acid With the discovery that the chemical structure of the polar compound is 14hydroxy-x1-eicosenoic acid, it became of interest to elucidate further the mechanism of its biosynthesis. In Table III is a series of experiments designed to shed some light on the nature of the long-chain acceptor of [r-“Clacetyl-CoA. In these experiments the particulate fraction is employed since there is little if any polar acid formed in the absence of added long-chain polar acid as the C, acceptor. The data demonstrate that Biochim.
Biophys.
Acla, 98 (1965)
27-35
S. F. YANG AND P. K. STUMPF
32 TABLE
III
ELONGATION
OF HYDROXY
FATTY
ACID
BY
AVOCADO
PARTICULATE
ENZYME
Reaction mixture contained IOO mpmoles of [I-W]acetyl-CoA (350 ooo countslmin) or 200 rnpmoles of [2-Wjmalonyl-CoA (IZO ooo counts/mm) and 0.3 ml of particulate preparation containing 2.0 mg (Expt. A) and 1.6 mg (Expt. B) of protein. Cofactors and the reaction conditions were the same as described in Fig. I except that HCO,- was added only where indicated. Fatty acids added are all 0.5 pmole except otherwise indicated. Non-polar and polar acids were separated by thinlayer chromatography as methyl ester@. Total incorporation (mpmoles)
Addition
Substrate
A. Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
12-hydroxystearate ricinoleate stearate oleate
Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
HCO,HCO,HCO,HCO,-
Malonyl-Cob Malonyl-CoA Malonyl-CoA Malonyl-CoA
12-hydroxystearate ricinoleate stearate
B. Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA Acetyl-CoA
+ 12hydroxystearate + ricinoleate + stearate
3-hydroxylaurate 3-hydroxydecanoate ricinoleate (I @mole) ricinoleate (0.5 pmole) ricinoleate (0.2 pmole)
Non-polar acid (m~moles)
Polar acid (mfimoles)
3.12 4.04 4.00 2.48 2.25
3.04 3.00 2.00 2.44 2.20
0.08 1.04 2.00 0.04 0.05
14.94 8.60 9.00 8.40
14.84 7.68 7.00 8.32
0.10 0.92 2.00 0.08
16.2 15.6 14.0 14.5
16.2 15.6 14.0 14.5
0 0 0 0
2.8 2.3 2.9 3.0 4.5 6.0
2.8 2.3 2.9 1.0 I.7 2.7
0 0 0 2.0 2.8 3.3
rz-hydroxystearic and ricinoleic acid (rz-hydroxy-g-octadecenoic acid) are elongated by the addition of [rJ4C]acetyl-CoA. Both stearic and oleic acids are inactive. HCO,is not required in this reaction. Furthermore malonyl-CoA is inactive as a donor. DL-3-hydroxydecanoic and DL-3-hydroxylauric acid are inactive as C, acceptors. Of TABLE
IV
EFFECT OF COFACTORSON ELONGATIONOF RICINOLEATE WITH
ACETYL-COA
BY
PARTICULATE
PREPARATION
Reaction mixture contained 100 mpmoles of [I-Xlacetyl-CoA (350 ooo countslmin) and 0.5 pmole of ricinoleate. Cofactors and the reaction conditions were the same as described in Fig. 1. FMN was 20 mpmoles. &factor Acetyl-CoA % omitted OYadded incorfiorated non-polar polar (mymoles) Complete + FMN - ATP - Mn*+ - TPN+, Glc-6-P - DPNH - Ricinoleate
4.1 4.1 0.2 3.7 I.9 2.6 2.9
Bdochim. Bio$+vs. Acta. 98 (1965) 27-35
15 17 14 16 20 98
85 83 86 84 80 2
-
x4-HYDROXY-II-EICOSENOIC
33
ACID SYNTHESIS
interest is the observation that I pmole of ricinoleic acid is somewhat inhibitory. At this concentration 3 mpmoles of acetyl-CoA are incorporated whereas at a concentration of 0.2 holes of ricinoleic acid, 6 mwoles of acetyl-CoA are accepted under the same conditions. The formation of the polar acid from ricinoleic acidcan occur under anaerobic as well as aerobic conditions. Some cofactor requirements are listed in Table IV. ATP, TPN+, glucose 6-phosphate, and DPNH are essential for the elongation of the polar acid. These cofactors were also essential for the formation of non-polar acids in the absence of an added acceptor. TABLE
V
REACTION PRODUCTS PARTICULATE ENZYME
PRODUCED
BY
INCUBATING
Reactions were carried out as described describedby YANGANDSTUMPP? Fraction
Neutral lipid Free fatty acid Phospholipid Long-chain acyl-CoA
in Table
AND
%
7000 4500 1500
[I-I%]ACETYL-COA
IV and separation
Distribuiion
Total Counls/ min
0
RICINOLEATE
Non-polar
followed
of label acid
WITH
the procedures
1% Polar acid
Counts/ min
yO
Countsf min
y0
54 35
1960 ‘350
52 36
5040 3x50
55 34
12 0
450 0
I2 0
1050 0
xx 0
Table V summarizes the data concerning the distribution of the polar acid in different classes of lipids in the reaction mixture. Most of the newly synthesized acid is associated with neutral lipids. Chemical identification of the polar product formed by incubating ricinoleate and [I-W}acetyl-CoA with the particulate fraction was carried out as follows. (a) Free fatty acids isolated from the reaction mixture after saponification were esterified and then subjected to thin-layer chromatography. A radioactive region corresponded with the monohydroxy acid region. The methyl esters were eluted with ether and were used for subsequent analyses. (b) The polar ester co-chromatographed with known methyl r+hydroxy-IIeicosenoate both on stabilized diethylene glycol succinate-polyester column and on an Apiezon-M column by gas-liquid chromatography. (c) Permanganate-periodate oxidation8 yielded 95% of the total radioactivity in the dicarboxylic acid which was separated from reaction products as a dimethyl ester on thin-layer chromatography. The dimethyl ester was identified as undecanedioic acid by gas-liquid chromatography of the dimethyl ester on Apiezon-M column. (d).%iMIDT decarboxylation of the catalytically hydrogenated polar acid showed essentially all the radioactivity with the carboxyl carbon of the acid. These results indicate that the radioactive polar acid was formed by the addition of [I-W)acetyl-CoA to the carboxyl end of ricinoleic acid. Under the same condition no elongation reaction occurs with steak or oleic acids as examined by gas-liquid chromatography. BiocLim. Biophys.
Acta, 98 (1965) 27-35
S. F. YANG AND P. K. STUMPF
34 DISCUSSION
It is somewhat difficult to evaluate the physiological role of this elongation reaction. The Cs addition appears to be specific for long-chain hydroxy acids since under the same conditions no elongation could be observed with either stearic, palmitic, myristic, 3-hydroxydecanoic or 3-hydroxylauric acids. Neither r4-hydroxy-rr-eicosenoic acid nor r2-hydroxy-o-octadecenoic acid (ricinoleic acid) is a constituent of avocado lipids. However the former is the major constituent of the seed oil of the genus LesquereUa* and the latter is the main fatty acid component of castor bean seeds. Whatever the role of the elongation reaction, it may be a fortuitous assay system which permits detection of the presence of trace amounts of r2-hydroxy-o-octadecenoic acid. This acid, as a possible intermediate in the synthesis of linoleic acid, exists in too low a concentration to be detected by the usual methods. Although linoleic acid constitutes IS?; of the total fatty acid in the avocado, biosynthesis of this acid in avocado cell-free systems has not been successful. We therefore suggest the following sequence : fatty acid syntlvet.w system
acetyl-CoA
-+
+ CO, --0,
[I z-hydroxy-goctadecenoic
acid]
Labile: inactive in preparations
aoetyl-CoA --s-----f DPr;H, TPNH
I)-hydroxy-II-eicosenoicacid
enzyme
& linoleic
acid
Presumably the C,, polar acid, unable to be converted to linoleic acid because of the absence of a specific enzyme system, will react with acetyl-CoA to give the elongation product. Since the C,, polar acid has no radioactivity associated with it except in its carboxyl carbon, its immediate acceptor is not synthesized de %OUOfrom acetylCoA but evidently occurs in trace amounts in the lipid components of the crude enzyme extract. A knowledge of the mechanism of formation of linoleic acid would place in correct perspective the function of this elongation reaction. However it should be noted that BLOCH et uZ.10 have shown with intact yeast cells that [*H]ricinoleic acid does not serve as a substitute for the formation of linoleic acid. The yeast cell will rapidly acetylate the hydroxyl groups to the corresponding acetoxy derivatives which simply accumulate and do not undergo further alterationsiO. ACKNOWLEDGEMENTS
This work was supported in part by grants from the National Science Foundation (G-14823) and National Institute of Health (GM-10132-o1). We are indebted to Dr. P. OYEKATR for preparing a generous supply of [2-r%]malonyl-CoA. We are also indebted to Dr. T. H. APPLEWRITE for a sample of pure methyl r4-hydroxy-xr-eicosenoate and to Dr. A. G. MARR for samples of DL-phydroxydecanoicand -1auric acids.
Biochim.
Biophys. Acta, 98 (x965) 27-35
I+HYDROXY-II-EICOSENOIC
ACID SYNTHESIS
35
REFERENCES I E. J. BARRON AND P. K. STUMPF, J. Biol. Chem., 237 (1962) PC 613. 2 S. F. YANG AND P. K. STUYPF, Biochim. Biophys. Acta, g8(Ig63)Ig. 3 H. K. MANGOLD, J. Am. Oil Chemists’ SOL, 38 (1961) 708. 4 H. GOLDFINE AND K. BLOCH, J. Biol. Gem., 236 (1961) 2596. 5 E. J. SIMONAND D. SHEMIN, J. Am. Chem. Sot., 75 (1953) 2520. 6 H. EGGERER AND F. LYNEN, Biochem. Z., 335 (1962) 540. 7 A. T. JAMES, Methods Biochem. Anal., 8 (1960) I. 8 E. VON RUDOLF, Can J. Chem., 34 (1956) 4131. g K. L. MIKOLAJCZAK. F. R. EARLE AND L. A. WOLFF, J. Am. Oil Chemists’ Soc., 3g (1962) 78. IO R J. LIGHT, W. J. LENNARZ AND K. BLOCH, 1. Biol. Chem., 237 (1961) 1793.
Biochim.
Biophys.
Acta,
g8 (1965) 27-35