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Occurence and identification of cis-vaccenic acid in Leuconostoc mesenteroides
208
SHORT COMMUNICATIONS
sc 53008
Occurrence and identification of cis-vaccenic acid in Leuconostoc mesenteroides The occurrence of An-octadecenoic acid to the virtual exclusion of the dg-isomer has been reported in a number of bacteria of the order Eubacteriales 1. These organisms possess a non-oxidative pathway of unsaturated-fatty-acid biosynthesis which leads to the formation of this isomer 2. It differs from the oxidative pathway preseDt in other types of microorganisms and in higher plants and animals which leads to the formation of the Zig-isomer (oleic acid). The configuration of the acid present in Lactobacillus arabinosus a, L. casei 4, a Group-C Streptococcus s and Agrobaclerium (Phytomonas) tumeJ'aciens 6 has been determined as cis-An-octadecenoic acid (cis-vaccenic acid) by chemical means by HOFMANN and coworkers. The cis-configuration has been also assigned to the Aaa-octadecenoic acid in Escherichia coli on the basis of its infrared spectrum 7. On examination of the f a t t y acids of Leuconostoc mesenteroides by gasliquid chromategraphy a prominent peak corresponding to a singly unsaturated C18 acid was obtained which accounted for approx. 75 % of the total fatty acids (Fig. xA). Only a small amount of palmitic acid and only trace amounts of palmitoleic and stearic acids were present (Fig. xA). The material gave rise to stearic acid on hydrogenation (Fig. IB) and has been identified as cis-vaccenic acid by oxidation to a lowmelting form of dihydroxystearic acid characteristic of cis-octadecenoic acids and further oxidation to I,XX-undecanedioic acid. The presence of such a large proportion of cis-vaccenic acid in such a readily grown organism which contains 3 - 4 % of total f a t t y acids s offers a convenient source of the compound. L. mesenteroides P-6o (ATCC 8o42 ) was grown on a synthetic medium as previously described 8. I g of lyophylized whole cells was refluxed with ioo nfl of 2 N HC1 for 18 tl and the hydrolysate extracted 4 times with 5o-ml portions of ether, the first portion being used first to rinse out the reflux condenser. The ether extract was evaporated to dryness and the residue extracted 4 times with 5-ml portions of light petroleum (b.p. 3o-6o°). The light-petroleum extract was evaporated to dryness and the resulting residue methylated by distilling in an excess of ethereal diazomethane. The methylation mixture was evaporated to dryness. The residue was dried further in a vacuum desiccator and dissolved in absolute ether to give I ml of solution for gas chromatography. Hydrogenation of the fatty acids was carried out by dissolving the residue from the light-petroleum extract in to ml of glacial acetic acid, adding IOO mg of platinic oxide catalyst, and hydrogenating at I a t m of H 2 until the uptake of gas had ceased. The catalyst was filtered off, the filtrate evaporated to dryness in vacuo and the residue methylated as described above. The f a t t y acids before and after hydrogenation were examined by gas chromatography (Figs. IA and IB). The initial peak after the solvent peak was indistinguishable from methyl levulinate when the two were chromatographed together. After hydrogenation a new peak indistinguishable from y-valerolactone or methyl laurate was observed. A sample of levulinic acid carried through the hydrogenation and methylation procedure also gave rise to the same peak. Pure 7-valerolactone was synthesized by the method of SCHUETTE AND SAH9 and could not be distinguished from methyl laurate when the two compounds were simultaneously injected for gas chromatography. 48.2 mg of f a t t y acids from 2.66 g ofL. mesenteroides P-6o whole cells were oxidized by the method of SWERN et al. 1° by dissolving the material in 0.25 ml formic acid and Biochim. Biophys. Acta, 84 (1964) 2o8-21o
209
SHORT COMMUNICATIONS
I(A)-BEFORE HYDROGENATION
a
z_ J > hi -3
I-,-
Z W
.J
8 I
I(B)-AFTER HYDROGENATION
.>,
i ) Fig. i. Gas-liquid chromatography of the fatty acids from the acid hydrolysis of L. mesenteroides. A, before hydrogenation. B, after hydrogenation. Performed on an aerograph, Model A-IIo-C (Wilkins Instrument and Research, Inc., Walnut Creek, Calif.), equipped with a io ft × 0.25 in column of butanediol succinate (2o% on firebrick) which was operated at 213° and 5° ml/min of He flow. adding 0.05 m l 3 0 % H~Ov After I h at room t e m p e r a t u r e several ml of water were added a n d the m i x t u r e extracted with ether. The ether extract was e v a p o r a t e d to dryness a n d the residue dissolved in I ml of I N NaOH. After I h the m i x t u r e was acidified with HC1, whereupon a crystalline precipitate was obtained. The acidified solution including the precipitate was extracted with ether, the ether extract evaporated to dryness, a n d the solid residue washed with light petroleum. The light-petroleuminsoluble residue was recrystallized from an a c e t o n e - l i g h t petroleum m i x t u r e (I : 2, v/v) to give 13. 4 m g of crystals m e l t i n g at 92.5-95.5 °. According to the very thorough Biochim. Biophys. Acta, 84 (x964) 2o8-2Io
210
SHORT COMMUNICATIONS
study of HUBERn the melting point corresponds to that of a dihydroxy acid from a cis-octadecenoic acid rather than from a trans-octadecenoic acid. The dihydroxy acid was dissolved in 1.5 ml ethanol and a solution of 35 mg HIO4 in I ml water added. After 4 h at room temperature IO ml of water were added and the reaction mixture extracted with ether. The ether extract was evaporated to dryness and the residual material steam-distilled in a micro apparatus. To the non-steam-volatile material was added o.i ml cone. H~S04. The solution was placed in a 5 °0 bath and powdered K M n Q added until a pink color persisted. The reaction mixture was decolorized with NaHSOs, and extracted with ether. The ether was evaporated to dryness and the residue recrystallized from aqueous ethanol to give 3.7 mg of crystals melting at lO7-1o9 °. The isolated acid did not depress the melting point of/, i I-undecanedioic acid (obtained from Professor J. CASON) but did depress the melting point of azelaic acid. The steam-volatile fraction had the odor of heptaldehyde, the other expected cleavage product of cis-vaccenic acid, but this was not further identified. This work was supported (in part) by a grant (E-3134) from the United States Public Health Service.
Department of Biochemistry, University of California, Berkeley, Calif. (U.S.A.)
MIYOSHI IKAWA*
I G. SCHEUERBRANDT AND K . BLOCH, jr. Biol. Chem., 2 3 7 (1962) 2064. 2 G. SCHEUERBRANDT, H . GOLDFINE, P. E. BARONOWSKI AND K . BLOCH, .7. Biol.
Chem., 236
(1961) PC7o. a 4 s 6
K. K, I(. K. T. 8 M. 9 H.
HOFMANN, R . A. LUCAS AND S. M. SAX, J. Biol. Chem., 195 (1952) 473. HOFMANN AND S. M. SAX, dr. Biol. Chem., 205 (1953) 55. HOFMANN AND F . TAUSSIG, J. Biol. Chem., 213 (1955) 4 1 5 . HOFMANN AND F. TAUSSIG, J. Biol. Chem., 2 1 3 ( I 9 5 5 ) 4 2 5 . I~ANESHIRO AND A. G. MARR, J. Biol. Chem., 236 (1961) 2615. IKAWA, J. Bacteriol., 85 (1963) 772. A. SCHUETTE AND P . T. SAIl, J. Am. Chem. Soc., 48 (1926) 3163. SWERN, G. N. BILLEN, T. W. FINDLEYAND J. T. SCANLAN,,[. z ~ . Chem.
z0 D. 1786. 11 W. F. HUBER, J. Am. Chem. Soe., 73 (1951) 2730.
Sot., 67 (1945)
Received December 9th, 1963 * Present address: Department of Biochemistry, University of New Hampshire, Durham, N.H. (U.S.A.).
Biochim. Biophys. Acla, 84 (1964) 2o8-21o
sc 53oo6
The action of galactose oxidase on several sphingoglycolipids The enzyme, galactose oxidase, isolated from Polyporus circinagus (more recently this organism has been identified as Dactylium dendroides 2) oxidizes the terminal position ofgalactose. The substrate for this enzyme need not be the free hexose since it has been demonstrated that various galactosides are attacked to varying degrees 1. AGRANOFF et al. ~ demonstrated that the galactose present in galactose cerebroside is quantitative-
Biochim. Biophys. Acta, 84 (1964) 21o-212
SHORT COMMUNICATIONS
sc 53008
Occurrence and identification of cis-vaccenic acid in Leuconostoc mesenteroides The occurrence of An-octadecenoic acid to the virtual exclusion of the dg-isomer has been reported in a number of bacteria of the order Eubacteriales 1. These organisms possess a non-oxidative pathway of unsaturated-fatty-acid biosynthesis which leads to the formation of this isomer 2. It differs from the oxidative pathway preseDt in other types of microorganisms and in higher plants and animals which leads to the formation of the Zig-isomer (oleic acid). The configuration of the acid present in Lactobacillus arabinosus a, L. casei 4, a Group-C Streptococcus s and Agrobaclerium (Phytomonas) tumeJ'aciens 6 has been determined as cis-An-octadecenoic acid (cis-vaccenic acid) by chemical means by HOFMANN and coworkers. The cis-configuration has been also assigned to the Aaa-octadecenoic acid in Escherichia coli on the basis of its infrared spectrum 7. On examination of the f a t t y acids of Leuconostoc mesenteroides by gasliquid chromategraphy a prominent peak corresponding to a singly unsaturated C18 acid was obtained which accounted for approx. 75 % of the total fatty acids (Fig. xA). Only a small amount of palmitic acid and only trace amounts of palmitoleic and stearic acids were present (Fig. xA). The material gave rise to stearic acid on hydrogenation (Fig. IB) and has been identified as cis-vaccenic acid by oxidation to a lowmelting form of dihydroxystearic acid characteristic of cis-octadecenoic acids and further oxidation to I,XX-undecanedioic acid. The presence of such a large proportion of cis-vaccenic acid in such a readily grown organism which contains 3 - 4 % of total f a t t y acids s offers a convenient source of the compound. L. mesenteroides P-6o (ATCC 8o42 ) was grown on a synthetic medium as previously described 8. I g of lyophylized whole cells was refluxed with ioo nfl of 2 N HC1 for 18 tl and the hydrolysate extracted 4 times with 5o-ml portions of ether, the first portion being used first to rinse out the reflux condenser. The ether extract was evaporated to dryness and the residue extracted 4 times with 5-ml portions of light petroleum (b.p. 3o-6o°). The light-petroleum extract was evaporated to dryness and the resulting residue methylated by distilling in an excess of ethereal diazomethane. The methylation mixture was evaporated to dryness. The residue was dried further in a vacuum desiccator and dissolved in absolute ether to give I ml of solution for gas chromatography. Hydrogenation of the fatty acids was carried out by dissolving the residue from the light-petroleum extract in to ml of glacial acetic acid, adding IOO mg of platinic oxide catalyst, and hydrogenating at I a t m of H 2 until the uptake of gas had ceased. The catalyst was filtered off, the filtrate evaporated to dryness in vacuo and the residue methylated as described above. The f a t t y acids before and after hydrogenation were examined by gas chromatography (Figs. IA and IB). The initial peak after the solvent peak was indistinguishable from methyl levulinate when the two were chromatographed together. After hydrogenation a new peak indistinguishable from y-valerolactone or methyl laurate was observed. A sample of levulinic acid carried through the hydrogenation and methylation procedure also gave rise to the same peak. Pure 7-valerolactone was synthesized by the method of SCHUETTE AND SAH9 and could not be distinguished from methyl laurate when the two compounds were simultaneously injected for gas chromatography. 48.2 mg of f a t t y acids from 2.66 g ofL. mesenteroides P-6o whole cells were oxidized by the method of SWERN et al. 1° by dissolving the material in 0.25 ml formic acid and Biochim. Biophys. Acta, 84 (1964) 2o8-21o
209
SHORT COMMUNICATIONS
I(A)-BEFORE HYDROGENATION
a
z_ J > hi -3
I-,-
Z W
.J
8 I
I(B)-AFTER HYDROGENATION
.>,
i ) Fig. i. Gas-liquid chromatography of the fatty acids from the acid hydrolysis of L. mesenteroides. A, before hydrogenation. B, after hydrogenation. Performed on an aerograph, Model A-IIo-C (Wilkins Instrument and Research, Inc., Walnut Creek, Calif.), equipped with a io ft × 0.25 in column of butanediol succinate (2o% on firebrick) which was operated at 213° and 5° ml/min of He flow. adding 0.05 m l 3 0 % H~Ov After I h at room t e m p e r a t u r e several ml of water were added a n d the m i x t u r e extracted with ether. The ether extract was e v a p o r a t e d to dryness a n d the residue dissolved in I ml of I N NaOH. After I h the m i x t u r e was acidified with HC1, whereupon a crystalline precipitate was obtained. The acidified solution including the precipitate was extracted with ether, the ether extract evaporated to dryness, a n d the solid residue washed with light petroleum. The light-petroleuminsoluble residue was recrystallized from an a c e t o n e - l i g h t petroleum m i x t u r e (I : 2, v/v) to give 13. 4 m g of crystals m e l t i n g at 92.5-95.5 °. According to the very thorough Biochim. Biophys. Acta, 84 (x964) 2o8-2Io
210
SHORT COMMUNICATIONS
study of HUBERn the melting point corresponds to that of a dihydroxy acid from a cis-octadecenoic acid rather than from a trans-octadecenoic acid. The dihydroxy acid was dissolved in 1.5 ml ethanol and a solution of 35 mg HIO4 in I ml water added. After 4 h at room temperature IO ml of water were added and the reaction mixture extracted with ether. The ether extract was evaporated to dryness and the residual material steam-distilled in a micro apparatus. To the non-steam-volatile material was added o.i ml cone. H~S04. The solution was placed in a 5 °0 bath and powdered K M n Q added until a pink color persisted. The reaction mixture was decolorized with NaHSOs, and extracted with ether. The ether was evaporated to dryness and the residue recrystallized from aqueous ethanol to give 3.7 mg of crystals melting at lO7-1o9 °. The isolated acid did not depress the melting point of/, i I-undecanedioic acid (obtained from Professor J. CASON) but did depress the melting point of azelaic acid. The steam-volatile fraction had the odor of heptaldehyde, the other expected cleavage product of cis-vaccenic acid, but this was not further identified. This work was supported (in part) by a grant (E-3134) from the United States Public Health Service.
Department of Biochemistry, University of California, Berkeley, Calif. (U.S.A.)
MIYOSHI IKAWA*
I G. SCHEUERBRANDT AND K . BLOCH, jr. Biol. Chem., 2 3 7 (1962) 2064. 2 G. SCHEUERBRANDT, H . GOLDFINE, P. E. BARONOWSKI AND K . BLOCH, .7. Biol.
Chem., 236
(1961) PC7o. a 4 s 6
K. K, I(. K. T. 8 M. 9 H.
HOFMANN, R . A. LUCAS AND S. M. SAX, J. Biol. Chem., 195 (1952) 473. HOFMANN AND S. M. SAX, dr. Biol. Chem., 205 (1953) 55. HOFMANN AND F . TAUSSIG, J. Biol. Chem., 213 (1955) 4 1 5 . HOFMANN AND F. TAUSSIG, J. Biol. Chem., 2 1 3 ( I 9 5 5 ) 4 2 5 . I~ANESHIRO AND A. G. MARR, J. Biol. Chem., 236 (1961) 2615. IKAWA, J. Bacteriol., 85 (1963) 772. A. SCHUETTE AND P . T. SAIl, J. Am. Chem. Soc., 48 (1926) 3163. SWERN, G. N. BILLEN, T. W. FINDLEYAND J. T. SCANLAN,,[. z ~ . Chem.
z0 D. 1786. 11 W. F. HUBER, J. Am. Chem. Soe., 73 (1951) 2730.
Sot., 67 (1945)
Received December 9th, 1963 * Present address: Department of Biochemistry, University of New Hampshire, Durham, N.H. (U.S.A.).
Biochim. Biophys. Acla, 84 (1964) 2o8-21o
sc 53oo6
The action of galactose oxidase on several sphingoglycolipids The enzyme, galactose oxidase, isolated from Polyporus circinagus (more recently this organism has been identified as Dactylium dendroides 2) oxidizes the terminal position ofgalactose. The substrate for this enzyme need not be the free hexose since it has been demonstrated that various galactosides are attacked to varying degrees 1. AGRANOFF et al. ~ demonstrated that the galactose present in galactose cerebroside is quantitative-
Biochim. Biophys. Acta, 84 (1964) 21o-212