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Structural elucidation of sphingoethanolamine and its distribution in aquatic animals
211
SHORT COMM~TNICATIONS
BBA 531.54 Structural
elucidation of sphingoethanolamine
and its distribution
in
aquatic animals The possible occurrence of sphingoethanolamine
in nature was first indicated
in 1958 by CARTER, SMITH AND JONES' while studying the egg yolk sphingolipids. However, they found later that it was a glyceryl ether phospholipid. CRONE AND BRIDGES~in 1963 reported the presence of sphingoethanolamine in the ether-insoluble lipids from a housefly, MUSCC~ domestica, although a study of its isolation and precise structure was not described. On the other hand, we previously reported3 the isolation of sphingoethanolam~e from a pond snail, ~~~~ro~~~ long&+-a, analytical data and some physical properties were determined, and in the acid hydrolysates the presence of spbingosine and phosphorylethanolamine was observed on thin-layer chromatogram and infrared spectrum. In this communication we present: (I) data showing that phosphorylethanolamine, an amino residue of sphingoethanolamine, is linked through the primary hydroxyl group of the first carbon of sphingosine; (2) data on the distribution of sphingoethanolamine in some species of aquatic animals. Nearly 40 mg of highly purified sphingoethanolamine were isolated from 5.5 kg of the viscera of the snail shellfish according to the procedures previously reported by the present authors3. Ux~d~~~~edegradation of s~~~ngo~~~~olarn~~ew.d ~d~~~~~~a~~on of rn~r~s~~~acid
and serine as $finaEproducts. Sphingoethanolamine
used in this experiment
gave the
expected theoretical values for palmitoyl-sphingosyl-phosphorylethanolamine and infrared spectrum gave all the absorption peaks expected for this molecule (Fig. I). IO mg of the sample lipid were oxidized according to the method of SIMON AND ROUSER*. The lipid was dissolved in 0.3 ml of formic acid with 0.1 ml of hydrogen peroxide (60%), placed in the dark at 37” for IZ h, and then kept at o0 for 12 h. After
Fig. I. Infrared spectrum of sphingoethanolamine pellet 0.16 mm thick).
from the pond snail shellfish (I.=J~/;, in RBr, Biochim.
Bioph~ys. Ada,
152 (1968) 211-213
212
SHORT COMMUNICATIONS
the removal of excess formic acid under reduced pressure at 40”, 0.4 ml of I M NaOH was added to the residue and the mixture was stirred in the dark at 37” for 20 h. By the addition of the appropriate amount of 5 M H,SO, the pH was brought to 1.3 and the resulting suspension was dried in wwuo at 40”. The residue was dissolved in 0.5 ml of water, brought to pH 5.4 with Na,HPO,, and 0.5 ml of 0.2 M NaIO, was added. This mixture was shaken in the dark at 37” for x5 h and then extracted twice with ether to obtain the lipophilic product. The ether extract was dried over Na,SO,. The solvent was removed and the residue was dissolved in I ml of glacial acetic acid containing 0.1 ml of a saturated aqueous solution of CrO,. Oxidation was carried out at 50” for 3 h, I ml of water was added and the solution was extracted twice with 0.8 ml of n-hexane. The hexane-soluble portion was methylated with 8% methanolic HCl, and identified by gas chromatography according to the method previously reported5. Consequently, myristic acid was detected and identified by comparison with the authentic sample of methyl myristate. Traces of other acids (lauric 3) were detected. On the other hand, the ether-insoluble substance formed in the above experiment was brought to pH 2.0 with 80% H3P04, and treated with ethylene glycol to destroy the excess periodate. The resulting mixture was immediately oxidized with excess KMnO., at 40’ for 30 min. After decolourization with NaHSO,, the solution was refluxed with I ml of 12 M HCl for 5 h and then cooled in an ice box overnight. The suspension was filtered and the filtrate was distilled under reduced pressure to remove HCl. The residue was extracted twice with n-butanol, and the extracts were evaporated. The resulting residue was dissolved in 0.2 ml of water and assayed on an automatic amino acid analyzer fitted with a column (0.6 cm x IZO cm) of Amberlite CG-120 with 0.2 N citrate buffer (pH 3.25) at 60”. As shown in Fig. 2, two peaks were obtained. The first peak corresponded to phosphorylethanolamine and the second peak corresponded to serine. It appeared that the phosphate moiety of sphingoethanolamine yielded phosphorylethanolamine.
Fig. 2. Elution pattern of phosphorylethanolamine Mitamura automatic amino acid analyzer 4-220-III. Biochim.
Biof&ys.
Acta,
152 (rg68)
211-213
and serine from sphingoethanolamine, The details are described in the text.
on a
213
SHORT COMMUNICATIONS
Distribution of sphingoethanolamine. 6 species of bivalves, 13 species of snail shellfish, 2 spieces of cephalopoda, 2 species of crustacea and 4 species of fish were obtained from Lake Biwa, which is a fresh-water lake, and from the Bay of Ise, which is part of the Pacific Ocean. From fresh tissues of the materials (about I kg each) the sphingolipid fractions (120 mg to 300 mg) were prepared on a smaller scale than befores. The distribution of sphingoethanolamine among these lipid fractions was analyzed by thin-layer chromatography and infrared spectroscopy, and sphingoethanolamine could be only found in snail shellfish, Heterogen Zongispira, Sinotaia histrica, Semisulcospira belzsoni (fresh-water species) and Batillaria multiformis (sea-water species). These facts suggest that sphingoethanolamine is x-(O-phosphorylethanolamine)-N-acyl sphingosine and seems to occur only in a limited number of snail shellfish species which belong to Mesogastropoda. Department of Chemistry, Faculty of Liberal Arts,
TARO HORI MUTSUMI SUGITA IKUKOARAKAWA
Shiga University, Otsu, Shiga (Japan) I
z 3 4 5 6
H. E. CARTER, D. B. SMITH AND D. N. JONES, J. Biol. Chem., 232 (1958) 681. H. D. CRONE AND R. G. BRIDGES, Biochem. J.. 8g (1963) II. T. HORI, 0. ITASAKA, H. IHOUE, M. GAMO AND I. ARAKAWA. Japan. J. Exptl. Med., 36 (1966) 85. G. SIMON AND G. ROUSER, Lipids, 2 (1967) 55. T. HORI, 0. ITASAKA, H. INOUE AND M. AKAI, Japan. J. Exptl. Med., 35 (1965) 81. T. HORI, I. ARAKAWA AND M. SUGITA, J, Biochem., 62 (1967) 67,
Received September nsnd, 1967 Biochim.
E-Oxidation of a p-methyl-substituted mitochondria
Biophys.
Acta,
152 (1968) 211-213
fatty acid in guinea-pig liver
Until recently only a few data were available concerning the metabolism of @methyl-substituted fatty acids. Such a methyl group represents a metabolic hindrance for ordinary /&oxidation. This hindrance can be bypassed by an initial o-oxidation followed by p-oxidations from the w-endl. In the metabolism of isovaleric acid in the leucine pathway a fixation of CO, to the /?-methyl group occurs before a further degradation takes place. A CO, fixation mechanism is also involved in the degradation of farnesoic acid in bacteriaz. Experiments with 3,6-dimethyloctanoic acid, which neither can be directly p-oxidized, nor B-oxidized after an initial w-oxidation, demonstrated that there must exist in man an alternative pathway, different from o-oxidation and the isovaleric acid pathway, for the degradation of /?-methyl-substituted fatty acids3. Independently in 3 different laboratories, this degradation was shown by studies in v~vo*~~to be an Biochim.
Biophys.
Acta,
152 (1968) 213-216
SHORT COMM~TNICATIONS
BBA 531.54 Structural
elucidation of sphingoethanolamine
and its distribution
in
aquatic animals The possible occurrence of sphingoethanolamine
in nature was first indicated
in 1958 by CARTER, SMITH AND JONES' while studying the egg yolk sphingolipids. However, they found later that it was a glyceryl ether phospholipid. CRONE AND BRIDGES~in 1963 reported the presence of sphingoethanolamine in the ether-insoluble lipids from a housefly, MUSCC~ domestica, although a study of its isolation and precise structure was not described. On the other hand, we previously reported3 the isolation of sphingoethanolam~e from a pond snail, ~~~~ro~~~ long&+-a, analytical data and some physical properties were determined, and in the acid hydrolysates the presence of spbingosine and phosphorylethanolamine was observed on thin-layer chromatogram and infrared spectrum. In this communication we present: (I) data showing that phosphorylethanolamine, an amino residue of sphingoethanolamine, is linked through the primary hydroxyl group of the first carbon of sphingosine; (2) data on the distribution of sphingoethanolamine in some species of aquatic animals. Nearly 40 mg of highly purified sphingoethanolamine were isolated from 5.5 kg of the viscera of the snail shellfish according to the procedures previously reported by the present authors3. Ux~d~~~~edegradation of s~~~ngo~~~~olarn~~ew.d ~d~~~~~~a~~on of rn~r~s~~~acid
and serine as $finaEproducts. Sphingoethanolamine
used in this experiment
gave the
expected theoretical values for palmitoyl-sphingosyl-phosphorylethanolamine and infrared spectrum gave all the absorption peaks expected for this molecule (Fig. I). IO mg of the sample lipid were oxidized according to the method of SIMON AND ROUSER*. The lipid was dissolved in 0.3 ml of formic acid with 0.1 ml of hydrogen peroxide (60%), placed in the dark at 37” for IZ h, and then kept at o0 for 12 h. After
Fig. I. Infrared spectrum of sphingoethanolamine pellet 0.16 mm thick).
from the pond snail shellfish (I.=J~/;, in RBr, Biochim.
Bioph~ys. Ada,
152 (1968) 211-213
212
SHORT COMMUNICATIONS
the removal of excess formic acid under reduced pressure at 40”, 0.4 ml of I M NaOH was added to the residue and the mixture was stirred in the dark at 37” for 20 h. By the addition of the appropriate amount of 5 M H,SO, the pH was brought to 1.3 and the resulting suspension was dried in wwuo at 40”. The residue was dissolved in 0.5 ml of water, brought to pH 5.4 with Na,HPO,, and 0.5 ml of 0.2 M NaIO, was added. This mixture was shaken in the dark at 37” for x5 h and then extracted twice with ether to obtain the lipophilic product. The ether extract was dried over Na,SO,. The solvent was removed and the residue was dissolved in I ml of glacial acetic acid containing 0.1 ml of a saturated aqueous solution of CrO,. Oxidation was carried out at 50” for 3 h, I ml of water was added and the solution was extracted twice with 0.8 ml of n-hexane. The hexane-soluble portion was methylated with 8% methanolic HCl, and identified by gas chromatography according to the method previously reported5. Consequently, myristic acid was detected and identified by comparison with the authentic sample of methyl myristate. Traces of other acids (lauric 3) were detected. On the other hand, the ether-insoluble substance formed in the above experiment was brought to pH 2.0 with 80% H3P04, and treated with ethylene glycol to destroy the excess periodate. The resulting mixture was immediately oxidized with excess KMnO., at 40’ for 30 min. After decolourization with NaHSO,, the solution was refluxed with I ml of 12 M HCl for 5 h and then cooled in an ice box overnight. The suspension was filtered and the filtrate was distilled under reduced pressure to remove HCl. The residue was extracted twice with n-butanol, and the extracts were evaporated. The resulting residue was dissolved in 0.2 ml of water and assayed on an automatic amino acid analyzer fitted with a column (0.6 cm x IZO cm) of Amberlite CG-120 with 0.2 N citrate buffer (pH 3.25) at 60”. As shown in Fig. 2, two peaks were obtained. The first peak corresponded to phosphorylethanolamine and the second peak corresponded to serine. It appeared that the phosphate moiety of sphingoethanolamine yielded phosphorylethanolamine.
Fig. 2. Elution pattern of phosphorylethanolamine Mitamura automatic amino acid analyzer 4-220-III. Biochim.
Biof&ys.
Acta,
152 (rg68)
211-213
and serine from sphingoethanolamine, The details are described in the text.
on a
213
SHORT COMMUNICATIONS
Distribution of sphingoethanolamine. 6 species of bivalves, 13 species of snail shellfish, 2 spieces of cephalopoda, 2 species of crustacea and 4 species of fish were obtained from Lake Biwa, which is a fresh-water lake, and from the Bay of Ise, which is part of the Pacific Ocean. From fresh tissues of the materials (about I kg each) the sphingolipid fractions (120 mg to 300 mg) were prepared on a smaller scale than befores. The distribution of sphingoethanolamine among these lipid fractions was analyzed by thin-layer chromatography and infrared spectroscopy, and sphingoethanolamine could be only found in snail shellfish, Heterogen Zongispira, Sinotaia histrica, Semisulcospira belzsoni (fresh-water species) and Batillaria multiformis (sea-water species). These facts suggest that sphingoethanolamine is x-(O-phosphorylethanolamine)-N-acyl sphingosine and seems to occur only in a limited number of snail shellfish species which belong to Mesogastropoda. Department of Chemistry, Faculty of Liberal Arts,
TARO HORI MUTSUMI SUGITA IKUKOARAKAWA
Shiga University, Otsu, Shiga (Japan) I
z 3 4 5 6
H. E. CARTER, D. B. SMITH AND D. N. JONES, J. Biol. Chem., 232 (1958) 681. H. D. CRONE AND R. G. BRIDGES, Biochem. J.. 8g (1963) II. T. HORI, 0. ITASAKA, H. IHOUE, M. GAMO AND I. ARAKAWA. Japan. J. Exptl. Med., 36 (1966) 85. G. SIMON AND G. ROUSER, Lipids, 2 (1967) 55. T. HORI, 0. ITASAKA, H. INOUE AND M. AKAI, Japan. J. Exptl. Med., 35 (1965) 81. T. HORI, I. ARAKAWA AND M. SUGITA, J, Biochem., 62 (1967) 67,
Received September nsnd, 1967 Biochim.
E-Oxidation of a p-methyl-substituted mitochondria
Biophys.
Acta,
152 (1968) 211-213
fatty acid in guinea-pig liver
Until recently only a few data were available concerning the metabolism of @methyl-substituted fatty acids. Such a methyl group represents a metabolic hindrance for ordinary /&oxidation. This hindrance can be bypassed by an initial o-oxidation followed by p-oxidations from the w-endl. In the metabolism of isovaleric acid in the leucine pathway a fixation of CO, to the /?-methyl group occurs before a further degradation takes place. A CO, fixation mechanism is also involved in the degradation of farnesoic acid in bacteriaz. Experiments with 3,6-dimethyloctanoic acid, which neither can be directly p-oxidized, nor B-oxidized after an initial w-oxidation, demonstrated that there must exist in man an alternative pathway, different from o-oxidation and the isovaleric acid pathway, for the degradation of /?-methyl-substituted fatty acids3. Independently in 3 different laboratories, this degradation was shown by studies in v~vo*~~to be an Biochim.
Biophys.
Acta,
152 (1968) 213-216