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. 1997 Oct 1;327(Pt 1):125–132. doi: 10.1042/bj3270125

Conversion of dihydroceramide into ceramide: involvement of a desaturase.

L Geeraert 1, G P Mannaerts 1, P P van Veldhoven 1
PMCID: PMC1218771  PMID: 9355743

Abstract

Ceramide has been suggested to be a potent bioactive lipid involved in cell growth, differentiation and apoptosis. Its precursor, dihydroceramide, does not affect these processes. The truncated dihydroceramide analogues N-hexanoyl-[4,5-3H]-d-erythro-sphinganine and N-[1-14C]-hexanoyl-d-erythro-sphinganine were used to study the conversion of dihydroceramide into ceramide by rat hepatocytes. The formation of tritiated water after the addition of the tritiated substrate to intact and permeabilized rat hepatocytes was followed to measure enzyme activity. Desaturation was severely depressed in permeabilized hepatocytes, suggesting loss of cofactors. Of a variety of cofactors tested in the permeabilized cells, NADPH appeared to be stimulatory, pointing to the involvement of a desaturase. In agreement with this, the addition of inhibitors and redox effectors known to affect Delta9-stearoyl-CoA desaturase and Delta1-plasmanyl-ethanolamine desaturase to intact cells resulted in severe inhibition of the desaturation. When added to permeabilized cells fortified with NADPH, these compounds counteracted the NADPH stimulation. The enzyme system was further studied in broken cells. On cell fractionation, the activity was recovered in the microsomal fraction. The results indicate that the conversion of dihydroceramide into ceramide is ctalysed by a desaturase and not by a dehydrogenase or an oxidase as was generally believed.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. BRADY R. O., KOVAL G. J. The enzymatic synthesis of sphingosine. J Biol Chem. 1958 Jul;233(1):26–31. [PubMed] [Google Scholar]
  3. Ballou L. R., Laulederkind S. J., Rosloniec E. F., Raghow R. Ceramide signalling and the immune response. Biochim Biophys Acta. 1996 Jun 11;1301(3):273–287. doi: 10.1016/0005-2760(96)00004-5. [DOI] [PubMed] [Google Scholar]
  4. Bielawska A., Crane H. M., Liotta D., Obeid L. M., Hannun Y. A. Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide. J Biol Chem. 1993 Dec 15;268(35):26226–26232. [PubMed] [Google Scholar]
  5. Braun P. E., Morell P., Radin N. S. Synthesis of C18- and C20-dihydrosphingosines, ketodihydrosphingosines, and ceramides by microsomal preparations from mouse brain. J Biol Chem. 1970 Jan 25;245(2):335–341. [PubMed] [Google Scholar]
  6. Chen R. F. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967 Jan 25;242(2):173–181. [PubMed] [Google Scholar]
  7. Clough R. C., Barnum S. R., Jaworski J. G. Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate. Anal Biochem. 1989 Jan;176(1):82–84. doi: 10.1016/0003-2697(89)90276-5. [DOI] [PubMed] [Google Scholar]
  8. Coste H., Martel M. B., Azzar G., Got R. UDPglucose-ceramide glucosyltransferase from porcine submaxillary glands is associated with the Golgi apparatus. Biochim Biophys Acta. 1985 Mar 28;814(1):1–7. doi: 10.1016/0005-2736(85)90412-2. [DOI] [PubMed] [Google Scholar]
  9. Croes K., Casteels M., De Hoffmann E., Mannaerts G. P., Van Veldhoven P. P. alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methylacyl-CoA intermediate. Eur J Biochem. 1996 Sep 15;240(3):674–683. doi: 10.1111/j.1432-1033.1996.0674h.x. [DOI] [PubMed] [Google Scholar]
  10. De Ceuster P., Mannaerts G. P., Van Veldhoven P. P. Identification and subcellular localization of sphinganine-phosphatases in rat liver. Biochem J. 1995 Oct 1;311(Pt 1):139–146. doi: 10.1042/bj3110139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujino Y., Nakano M. Enzymatic conversion of labeled ketodihydrosphingosine to ketosphingosine in rat liver particulates. Biochim Biophys Acta. 1971 Jul 13;239(2):273–279. [PubMed] [Google Scholar]
  12. Futerman A. H., Stieger B., Hubbard A. L., Pagano R. E. Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus. J Biol Chem. 1990 May 25;265(15):8650–8657. [PubMed] [Google Scholar]
  13. Füssle R., Bhakdi S., Sziegoleit A., Tranum-Jensen J., Kranz T., Wellensiek H. J. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. doi: 10.1083/jcb.91.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harshman S., Sugg N., Cassidy P. Preparation and purification of staphylococcal alpha toxin. Methods Enzymol. 1988;165:3–7. doi: 10.1016/s0076-6879(88)65004-x. [DOI] [PubMed] [Google Scholar]
  15. Holloway P. W., Katz J. T. A requirement for cytochrome b 5 in microsomal stearyl coenzyme A desaturation. Biochemistry. 1972 Sep 26;11(20):3689–3696. doi: 10.1021/bi00770a005. [DOI] [PubMed] [Google Scholar]
  16. Holloway P. W., Wakil S. J. Requirement for reduced diphosphopyridine nucleotide-cytochrome b5 reductase in stearly coenzyme A desaturation. J Biol Chem. 1970 Apr 10;245(7):1862–1865. [PubMed] [Google Scholar]
  17. Kolesnick R. Signal transduction through the sphingomyelin pathway. Mol Chem Neuropathol. 1994 Feb-Apr;21(2-3):287–297. doi: 10.1007/BF02815356. [DOI] [PubMed] [Google Scholar]
  18. Lee T. C., Wykle R. L., Blank M. L., Snyder F. Dietary control of stearyl CoA and alkylacylglycerophosphorylethanolamine desaturases in tumor. Biochem Biophys Res Commun. 1973 Dec 10;55(3):574–579. doi: 10.1016/0006-291x(73)91182-0. [DOI] [PubMed] [Google Scholar]
  19. Lipsky N. G., Pagano R. E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol. 1985 Jan;100(1):27–34. doi: 10.1083/jcb.100.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mandon E. C., Ehses I., Rother J., van Echten G., Sandhoff K. Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver. J Biol Chem. 1992 Jun 5;267(16):11144–11148. [PubMed] [Google Scholar]
  21. Merrill A. H., Jr, Schmelz E. M., Wang E., Schroeder J. J., Dillehay D. L., Riley R. T. Role of dietary sphingolipids and inhibitors of sphingolipid metabolism in cancer and other diseases. J Nutr. 1995 Jun;125(6 Suppl):1677S–1682S. doi: 10.1093/jn/125.suppl_6.1677S. [DOI] [PubMed] [Google Scholar]
  22. Merrill A. H., Jr, Wang E. Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s). J Biol Chem. 1986 Mar 15;261(8):3764–3769. [PubMed] [Google Scholar]
  23. Merrill A. H., Jr, van Echten G., Wang E., Sandhoff K. Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J Biol Chem. 1993 Dec 25;268(36):27299–27306. [PubMed] [Google Scholar]
  24. Obeid L. M., Hannun Y. A. Ceramide: a stress signal and mediator of growth suppression and apoptosis. J Cell Biochem. 1995 Jun;58(2):191–198. doi: 10.1002/jcb.240580208. [DOI] [PubMed] [Google Scholar]
  25. Obeid L. M., Linardic C. M., Karolak L. A., Hannun Y. A. Programmed cell death induced by ceramide. Science. 1993 Mar 19;259(5102):1769–1771. doi: 10.1126/science.8456305. [DOI] [PubMed] [Google Scholar]
  26. Ong D. E., Brady R. N. In vivo studies on the introduction of the 4-t-double bond of the sphingenine moiety of rat brain ceramides. J Biol Chem. 1973 Jun 10;248(11):3884–3888. [PubMed] [Google Scholar]
  27. Oshino N., Imai Y., Sato R. Electron-transfer mechanism associated with fatty acid desaturation catalyzed by liver microsomes. Biochim Biophys Acta. 1966 Oct 17;128(1):13–27. doi: 10.1016/0926-6593(66)90137-8. [DOI] [PubMed] [Google Scholar]
  28. Pagano R. E., Martin O. C. A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells. Biochemistry. 1988 Jun 14;27(12):4439–4445. doi: 10.1021/bi00412a034. [DOI] [PubMed] [Google Scholar]
  29. Pagano R. E., Sepanski M. A., Martin O. C. Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J Cell Biol. 1989 Nov;109(5):2067–2079. doi: 10.1083/jcb.109.5.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Paltauf F., Holasek A. Enzymatic synthesis of plasmalogens. Characterization of the 1-O-alkyl-2-acyl-8n-glycero-3-phosphorylethanolamine desaturase from mucosa of hamster small intestine. J Biol Chem. 1973 Mar 10;248(5):1609–1615. [PubMed] [Google Scholar]
  31. Paltauf F. Plasmalogen biosynthesis in a cell-free system. Enzymic desaturation of 1-O-alkyl (2-acyl) glycerophosphoryl ethanolamine. FEBS Lett. 1972 Jan 15;20(1):79–82. doi: 10.1016/0014-5793(72)80021-8. [DOI] [PubMed] [Google Scholar]
  32. Polito A. J., Sweeley C. C. Stereochemistry of the hydroxylation in 4-hydroxysphinganine formation and the steric course of hydrogen elimination in sphing-4-enine biosynthesis. J Biol Chem. 1971 Jul 10;246(13):4178–4187. [PubMed] [Google Scholar]
  33. Rhead W. J., Hall C. L., Tanaka K. Novel tritium release assays for isovaleryl-CoA and butyryl-CoA dehydrogenases. J Biol Chem. 1981 Feb 25;256(4):1616–1624. [PubMed] [Google Scholar]
  34. Ridgway N. D., Merriam D. L. Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells. Biochim Biophys Acta. 1995 Apr 28;1256(1):57–70. doi: 10.1016/0005-2760(95)00010-a. [DOI] [PubMed] [Google Scholar]
  35. Rother J., van Echten G., Schwarzmann G., Sandhoff K. Biosynthesis of sphingolipids: dihydroceramide and not sphinganine is desaturated by cultured cells. Biochem Biophys Res Commun. 1992 Nov 30;189(1):14–20. doi: 10.1016/0006-291x(92)91518-u. [DOI] [PubMed] [Google Scholar]
  36. Schwarzmann G. A simple and novel method for tritium labeling of gangliosides and other sphingolipids. Biochim Biophys Acta. 1978 Apr 28;529(1):106–114. doi: 10.1016/0005-2760(78)90108-x. [DOI] [PubMed] [Google Scholar]
  37. Sreekrishna K., Joshi V. C. Inhibition of the microsomal stearoyl coenyme A desaturation by divalent copper and its chelates. Biochim Biophys Acta. 1980 Aug 11;619(2):267–273. doi: 10.1016/0005-2760(80)90075-2. [DOI] [PubMed] [Google Scholar]
  38. Stals H. K., Mannaerts G. P., Declercq P. E. Factors influencing triacylglycerol synthesis in permeabilized rat hepatocytes. Biochem J. 1992 May 1;283(Pt 3):719–725. doi: 10.1042/bj2830719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stoffel W., Assmann G., Bister K. Metabolism of sphingosine bases. XVII. Stereospecificities in the introduction of the 4t-double bond into sphinganine yielding 4t-sphingenine (sphingosine). Hoppe Seylers Z Physiol Chem. 1971 Nov;352(11):1531–1544. doi: 10.1515/bchm2.1971.352.2.1531. [DOI] [PubMed] [Google Scholar]
  40. Stoffel W., Bister K. Studies on the desaturation of sphinganine. Ceramide and sphingomyelin metabolism in the rat and in BHK 21 cells in tissue culture. Hoppe Seylers Z Physiol Chem. 1974 Aug;355(8):911–923. doi: 10.1515/bchm2.1974.355.2.911. [DOI] [PubMed] [Google Scholar]
  41. Stoffel W., LeKim D., Sticht G. Metabolism of sphingosine bases. IV. 2-Amino-1-hydroxyoctadecane-3-one (3-oxodihydrosphingosine), the common intermediate in the biosynthesis of dihydrospingosine and sphingosine and in the degradation of dihydrosphingosine. Hoppe Seylers Z Physiol Chem. 1967 Dec;348(12):1570–1574. doi: 10.1515/bchm2.1967.348.1.1570. [DOI] [PubMed] [Google Scholar]
  42. Strittmatter P., Spatz L., Corcoran D., Rogers M. J., Setlow B., Redline R. Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4565–4569. doi: 10.1073/pnas.71.11.4565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Van Veldhoven P. P., Bishop W. R., Bell R. M. Enzymatic quantification of sphingosine in the picomole range in cultured cells. Anal Biochem. 1989 Nov 15;183(1):177–189. doi: 10.1016/0003-2697(89)90186-3. [DOI] [PubMed] [Google Scholar]
  44. Van Veldhoven P. P., Mannaerts G. P. Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver. J Biol Chem. 1991 Jul 5;266(19):12502–12507. [PubMed] [Google Scholar]
  45. Walter V. P., Sweeney K., Morré D. J. Neutral lipid precursors for gangliosides are not formed by rat liver homogenates or by purified cell fractions. Biochim Biophys Acta. 1983 Feb 7;750(2):346–352. doi: 10.1016/0005-2760(83)90039-5. [DOI] [PubMed] [Google Scholar]
  46. Wang E., Norred W. P., Bacon C. W., Riley R. T., Merrill A. H., Jr Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J Biol Chem. 1991 Aug 5;266(22):14486–14490. [PubMed] [Google Scholar]
  47. Zahlten R. N., Stratman F. W. The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique. Arch Biochem Biophys. 1974 Aug;163(2):600–608. doi: 10.1016/0003-9861(74)90519-0. [DOI] [PubMed] [Google Scholar]
  48. van 't Hof W., van Meer G. Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane. J Cell Biol. 1990 Sep;111(3):977–986. doi: 10.1083/jcb.111.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]

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