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Enzymatic conversion of labeled ketodihydrosphingosine to sphingosine
Biochimicvlet BiophysiicLI Acta, 296 (1973) 457-460 6 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA Report BBA 51160
Enzymatic conversion of labeled ketodihydrosphingosine to sphingosine
MASUO NAKANO and YASUHIKO FUJINO Depkmenf
of Agricultuml C%emistry,Obibiro Zootechnical Unhrersity,Obihiro, Hokkaido (Japan)
(Received January 2nd, 1973)
SUMMARY
It was demonstrated that sphingosine (l $d.ihydroxy-2-amino+octadecene) was derived from ketodihydrosphingosine (1 hydroxy-2-amino-3-oxo-octadecane) in rat liver particulates. The findings suggest that ketodihydrosphingosine may convert directly to sphingosine in the system, probably by an isomerization mechanism.
It has been reported in the enzyme system obtained from Jf. cifem’1y2,rat liver3 and mouse brain4 that dihydrosphingosine is formed v& ketodihydrosphingosine by the enzymatic condensation of palmitoyl-CoA with serine. The biosynthetic pathway of sphingosine, however, is somewhat complicated and several suggestions have been proposed up to now. Mari et al.‘, employing an enzyme system obtained from H. ciferri, recently postulated that 2-rmns-hexadecenoyl_CoA would be the original long-chain fragment in the formation of sphingosine. Stoffel et al.6 have demonstrated that dihydrosphingosine is the precursor of sphingosine in experiments in viva with rat. We recently noted a possibility that ketosphingosine, enzymatically derived from ketodihydrosphingosine by dehydrogenation79s, might convert to sphingosine. The present paper describes experiments which indicate that sphingosine may be formed by isomerization of ketodihydrosphingosine in rat liver particulates. We previously observed that the enzymatic condensation of palmitoylCoA with serine in rat liver particulates produces not only ketodihydrosphingosine but also sphingosine under the conditions without NADPH (ref. 3). This could be explained by the isomeric conversion of ketodihydrosphingosine to sphingosine. Radioactive ketodihydrosphingosine was enxymatically synthesized from palmitoyl-CoA and [3-14C]serine according to the procedure previously described’, and purified with silicic acid column chromatography. The product of ketodihydrosphingosine was not contaminated with ketosphingosine.
458
BBA REWRT
The-enzyme system was prepared from the liver of lCday-old rats according to the procedure of Brady et al. 9. The tissue was homogenized with 4 vol. of 0.25 M sucrose. The homogenate was centrifuged at 8600 X g for 12 min and the microsomal fraction was obtained by sedimentation of the supernatant liquid at 105 000 X g for 30 min. The particulate fraction was suspended in 0.1 M potassium phosphate buffer (pH 7.6), equal to 0.2 vol. of the original homogenate. [ 1-14C]Ketodihydrosphingosine and the rat liver particulates were mixed and incubated at 37 “C for 2 h. At the end of incubation a small amount of authentic preparations of sphingosine base, ceramide, cerebroside and sphingomyelin were added as carrier sphingolipid. Total lipid was extracted several times with chloroform-methanol (2: 1, v/v). The total lipid obtained was applied to a silica gel G plate and developed with chloroform-methanol-2 M NH40H (40: 10: 1, v/v/v) lo and analyzed with radio-thinlayer chromatography and autoradiography. As seen in Fig. 1, with the rat liver particulates the radioactivity was found only at the sphingosine spot, but not at the dihydrosphingosine spot, after incubation of the lipids whereas no radioactivity was detected in the control experiment. _( ._
I
radioscanning
9 f
e
Origin
i;i1 Radio-thin-layer
d c b a .....
.. 1
2 chromatogram
1
Thin-layer
2
chromatogram
1
2
Autoradioqam
Fig. 1. Enzymatic conversion of [ l-‘4C] ketodiydrosphingosine to labeled sphingosine. Each tube contained 50 @moles of potassium phosphate buffer (pH 7.6), 4580 cpm of [l-W] ketodiiydrosphingosine, 2 mg of Triton X-100 and 0.2 ml of particulate fraction prepared from rat liver homogenates in a fmal volume of 0.5 ml. The tubes were incubated at 37 “C for 2 h. Four tubes were combined for the analysis. A silica gel G plate was developed with chloroform-methanol-2 M NH,OH (40: 10: 1, v/v/v), detected with iodine vapor and examined by radio-8canning or radio-printing. 1. Lipids incubated with boiled enzyme (control). 2. Lipids incubated with enzyme. a, sphingomyelin; b, phoqrhatidylcholine; c, ceramide diexoside; d, pho8phatidylethanolamine; e, cerebroside; f, dihydrosphingosine; g, sphingosine; h, ketodihydmsphingo8ine; i, ceramide; j, neutral lipids.
The radioactivity at the sphingosine spot in Fig. 1 was analyzed with a liquidscintillation spectrometer. It was recognized that about 6% of the original radioactive ketodihydrosphingosine converted to sphingosine, as given in Table I. The spot material corresponding to sphingosine was scraped from the plate, converted to the dinitrophenyl derivative” and developed with chloroform-methanol
BBAREPORT
459
TABLE1 RATEOF ENZYMATICCONVERSION OF KETODIHYDRO[ 1-t’C]SPHINGOSINE TO LABELED SPHINGOSINE Condition
Ketodihydrosphingosine @pm)
Sphingosine synthesized (cpm)
Rate I%)
Withboiled enzyme Withenzyme
4580 4580
0 283
0 6.2
(90: 10, v/v) on a borate-impregnated silica gel G plate to be analyzed with radio-thinlayer chromatography and autoradiography. As depicted in Fig. 2, the radioactivity was discerned only at the sphingosine area. The radioactive sphingosine fraction in Fig. 1 was added to non-radioactive sphingosine as carrier and acetylated” to obtain triacetylsphingosine in a yield of 92.2%. Approximately 85% of the original radioactivity was found to be recovered in the preparation of triacetylsphingosine.
Front
-I 0
radioscanning
l 0
Origin 1 Radio-thin-layer chromatogram
radio-
0
...o._ 1
Thin-layer
-printing
l
.
Oo ....“~._..O
. ..
...”
I
2 3 chromatogram
,.I.. .. . . . ..-, i...:
1
Autoradiogram
Fig. 2. Confiiation of labeled sphingosine as dinitrophcgylderivative.A silicagel G-borate plate wasdevelopedwith chloroform-methanol (90:10, v/v) and’detectedby radio-scanningor radioprinting. 1. Dinitrophenylsphingorine derivedfrom labeledsphin8osinein the incubationmixture (Fig. 1- 21.2. DinitrophenyIdihydro@rIn8osine (standard).3. Dinitrophenylsphingosine (standard). These data suggest that ketodihydrosphingosine converted directly to sphingosine in the rat liver particulates probably by isomerization mechanism, although the proportion might be considerably low. This pathway may be one, among the several possible biosynthetic pathways for sphingosine. Further studies, however, are required for determining if the isomerization mechanism for the formation of sphingosine actually does occur in vivo.
460
BBA REPORT
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
Braun, P.E. and Snell, E.E. (1968)J. Biol. Chem. 243,3775-3783 Stoffel, W., LeKhn, D. and Sticht, G. (1968) Z. Physiol. Chem. 349,664-670 Fujino, Y. and Nakano, M. (1971) Agr. Biol. Chem. 35,40-46 Braun, P.E., Morell, P. and Radht, N.S. (197O)J. Biol. Chem. 245,335-341 Mart, S.J.D., Brady, R.N. and Snell, E.E. (1971) Arch. B&hem. Biopbys. 143,553-565 Stoffel, W,Asaman, G. and Bister, K. (1971) Z. Physiol. Chem. 352,1531-1544 Fujhto, Y. and Nakano, M. (1971) Agr. BioL Chem. 35,885-889 Fujino, Y. and Nakano, M. (1971) Biochim. Biophys. Acta 239; 273-279 Brady, R.O., Bradley, R.M., Young, O.M. and Kaller, H. (1965)J. BioL Chem. 240, PC3693-3694 Sambasivarao, K. and McCluer, R.H. (1963) J. Lipid Res. 4,106-108 Karlason, K. (1960) Nature 188,312-313 Carter, H.E. and Greenwood, F.L. (1952) J. Biol. CAem. 199,283-288
BBA Report BBA 51160
Enzymatic conversion of labeled ketodihydrosphingosine to sphingosine
MASUO NAKANO and YASUHIKO FUJINO Depkmenf
of Agricultuml C%emistry,Obibiro Zootechnical Unhrersity,Obihiro, Hokkaido (Japan)
(Received January 2nd, 1973)
SUMMARY
It was demonstrated that sphingosine (l $d.ihydroxy-2-amino+octadecene) was derived from ketodihydrosphingosine (1 hydroxy-2-amino-3-oxo-octadecane) in rat liver particulates. The findings suggest that ketodihydrosphingosine may convert directly to sphingosine in the system, probably by an isomerization mechanism.
It has been reported in the enzyme system obtained from Jf. cifem’1y2,rat liver3 and mouse brain4 that dihydrosphingosine is formed v& ketodihydrosphingosine by the enzymatic condensation of palmitoyl-CoA with serine. The biosynthetic pathway of sphingosine, however, is somewhat complicated and several suggestions have been proposed up to now. Mari et al.‘, employing an enzyme system obtained from H. ciferri, recently postulated that 2-rmns-hexadecenoyl_CoA would be the original long-chain fragment in the formation of sphingosine. Stoffel et al.6 have demonstrated that dihydrosphingosine is the precursor of sphingosine in experiments in viva with rat. We recently noted a possibility that ketosphingosine, enzymatically derived from ketodihydrosphingosine by dehydrogenation79s, might convert to sphingosine. The present paper describes experiments which indicate that sphingosine may be formed by isomerization of ketodihydrosphingosine in rat liver particulates. We previously observed that the enzymatic condensation of palmitoylCoA with serine in rat liver particulates produces not only ketodihydrosphingosine but also sphingosine under the conditions without NADPH (ref. 3). This could be explained by the isomeric conversion of ketodihydrosphingosine to sphingosine. Radioactive ketodihydrosphingosine was enxymatically synthesized from palmitoyl-CoA and [3-14C]serine according to the procedure previously described’, and purified with silicic acid column chromatography. The product of ketodihydrosphingosine was not contaminated with ketosphingosine.
458
BBA REWRT
The-enzyme system was prepared from the liver of lCday-old rats according to the procedure of Brady et al. 9. The tissue was homogenized with 4 vol. of 0.25 M sucrose. The homogenate was centrifuged at 8600 X g for 12 min and the microsomal fraction was obtained by sedimentation of the supernatant liquid at 105 000 X g for 30 min. The particulate fraction was suspended in 0.1 M potassium phosphate buffer (pH 7.6), equal to 0.2 vol. of the original homogenate. [ 1-14C]Ketodihydrosphingosine and the rat liver particulates were mixed and incubated at 37 “C for 2 h. At the end of incubation a small amount of authentic preparations of sphingosine base, ceramide, cerebroside and sphingomyelin were added as carrier sphingolipid. Total lipid was extracted several times with chloroform-methanol (2: 1, v/v). The total lipid obtained was applied to a silica gel G plate and developed with chloroform-methanol-2 M NH40H (40: 10: 1, v/v/v) lo and analyzed with radio-thinlayer chromatography and autoradiography. As seen in Fig. 1, with the rat liver particulates the radioactivity was found only at the sphingosine spot, but not at the dihydrosphingosine spot, after incubation of the lipids whereas no radioactivity was detected in the control experiment. _( ._
I
radioscanning
9 f
e
Origin
i;i1 Radio-thin-layer
d c b a .....
.. 1
2 chromatogram
1
Thin-layer
2
chromatogram
1
2
Autoradioqam
Fig. 1. Enzymatic conversion of [ l-‘4C] ketodiydrosphingosine to labeled sphingosine. Each tube contained 50 @moles of potassium phosphate buffer (pH 7.6), 4580 cpm of [l-W] ketodiiydrosphingosine, 2 mg of Triton X-100 and 0.2 ml of particulate fraction prepared from rat liver homogenates in a fmal volume of 0.5 ml. The tubes were incubated at 37 “C for 2 h. Four tubes were combined for the analysis. A silica gel G plate was developed with chloroform-methanol-2 M NH,OH (40: 10: 1, v/v/v), detected with iodine vapor and examined by radio-8canning or radio-printing. 1. Lipids incubated with boiled enzyme (control). 2. Lipids incubated with enzyme. a, sphingomyelin; b, phoqrhatidylcholine; c, ceramide diexoside; d, pho8phatidylethanolamine; e, cerebroside; f, dihydrosphingosine; g, sphingosine; h, ketodihydmsphingo8ine; i, ceramide; j, neutral lipids.
The radioactivity at the sphingosine spot in Fig. 1 was analyzed with a liquidscintillation spectrometer. It was recognized that about 6% of the original radioactive ketodihydrosphingosine converted to sphingosine, as given in Table I. The spot material corresponding to sphingosine was scraped from the plate, converted to the dinitrophenyl derivative” and developed with chloroform-methanol
BBAREPORT
459
TABLE1 RATEOF ENZYMATICCONVERSION OF KETODIHYDRO[ 1-t’C]SPHINGOSINE TO LABELED SPHINGOSINE Condition
Ketodihydrosphingosine @pm)
Sphingosine synthesized (cpm)
Rate I%)
Withboiled enzyme Withenzyme
4580 4580
0 283
0 6.2
(90: 10, v/v) on a borate-impregnated silica gel G plate to be analyzed with radio-thinlayer chromatography and autoradiography. As depicted in Fig. 2, the radioactivity was discerned only at the sphingosine area. The radioactive sphingosine fraction in Fig. 1 was added to non-radioactive sphingosine as carrier and acetylated” to obtain triacetylsphingosine in a yield of 92.2%. Approximately 85% of the original radioactivity was found to be recovered in the preparation of triacetylsphingosine.
Front
-I 0
radioscanning
l 0
Origin 1 Radio-thin-layer chromatogram
radio-
0
...o._ 1
Thin-layer
-printing
l
.
Oo ....“~._..O
. ..
...”
I
2 3 chromatogram
,.I.. .. . . . ..-, i...:
1
Autoradiogram
Fig. 2. Confiiation of labeled sphingosine as dinitrophcgylderivative.A silicagel G-borate plate wasdevelopedwith chloroform-methanol (90:10, v/v) and’detectedby radio-scanningor radioprinting. 1. Dinitrophenylsphingorine derivedfrom labeledsphin8osinein the incubationmixture (Fig. 1- 21.2. DinitrophenyIdihydro@rIn8osine (standard).3. Dinitrophenylsphingosine (standard). These data suggest that ketodihydrosphingosine converted directly to sphingosine in the rat liver particulates probably by isomerization mechanism, although the proportion might be considerably low. This pathway may be one, among the several possible biosynthetic pathways for sphingosine. Further studies, however, are required for determining if the isomerization mechanism for the formation of sphingosine actually does occur in vivo.
460
BBA REPORT
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
Braun, P.E. and Snell, E.E. (1968)J. Biol. Chem. 243,3775-3783 Stoffel, W., LeKhn, D. and Sticht, G. (1968) Z. Physiol. Chem. 349,664-670 Fujino, Y. and Nakano, M. (1971) Agr. Biol. Chem. 35,40-46 Braun, P.E., Morell, P. and Radht, N.S. (197O)J. Biol. Chem. 245,335-341 Mart, S.J.D., Brady, R.N. and Snell, E.E. (1971) Arch. B&hem. Biopbys. 143,553-565 Stoffel, W,Asaman, G. and Bister, K. (1971) Z. Physiol. Chem. 352,1531-1544 Fujhto, Y. and Nakano, M. (1971) Agr. BioL Chem. 35,885-889 Fujino, Y. and Nakano, M. (1971) Biochim. Biophys. Acta 239; 273-279 Brady, R.O., Bradley, R.M., Young, O.M. and Kaller, H. (1965)J. BioL Chem. 240, PC3693-3694 Sambasivarao, K. and McCluer, R.H. (1963) J. Lipid Res. 4,106-108 Karlason, K. (1960) Nature 188,312-313 Carter, H.E. and Greenwood, F.L. (1952) J. Biol. CAem. 199,283-288