The experimental approach to deplete cellular glycosphingolipids (GSLs) with the specific inhibitors of glycosphingolipid biosynthesis has the potential to identify functions of endogenous GSLs. Most GSLs are derived from glucosylceramide (GlcCer), which is synthesized from ceramide and UDP-glucose. The sequential addition of further monosaccharide and sialic acid to GlcCer results in a complex family of structures, such as the ganglio, globo, isoglobo, neolacto and lacto series.
It has been demonstrated by Inokuchi and Radin1) that an analog of ceramide, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, Fig. 1) inhibits UDP-glucose:N-acylsphingosine glucosyltransferase (EC. 2.4.1.80), which produces GlcCer. Kinetic analysis showed that D-PDMP acted by mixed competition against ceramide with IC50 and Ki values of 5 μM and 0.7 μM, respectively. It should be noted that PDMP does not inhibit UDP-galactose:N-acylsphingosine galactosyltransferase and beta-glucocerebrosidase. As shown in Fig. 1, the stereospecific action of PDMP was demonstrated very clearly, since only one of the four PDMP isomers, D-threo isomer (1R, 2R), was able to inhibit GlcCer synthesis. |
| Category | Biosynthesis & Metabolism |
| Protocol Name | |
Authors
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Inokuchi, Jin-ichi
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University
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| KeyWords |
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Reagents
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D-PDMP : (D-threo(1R,2R)-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) |
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Specification: D-PDMP・HCl・H2O
- Molecular Weight: 445
- Molecular Formula: C23H41ClN2O4
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Physical Appearance: White solid (m.p. 146–148°C) |
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Instruments
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Conventional cell culture system and apparatus |
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| Methods |
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1. |
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Dissolve D-PDMP in H2O to make 4 mM solution. |
Comment 0
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Filter through 0.22 μM membrane filter and store at 4°C as a stock solution. |
Comment 0
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| 3) |
Add the PDMP stock solution to culture medium. |
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| 5) |
Analyze GSLs and cellular functions. |
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| Notes |
- Dissolve PDMP at 40°C with sonication bath.
- 4mM PDMP stck solution will be stable at refrigerator at least one month.
- The final concentration of PDMP will be 5–40 μM dependent on cell type and treatment periods
- The outcome of the experiments should be considered both due to depletion of GSLs and accumulation of ceramide and precursor sphingoid base2). Contribution of endgeneous GSLs can be assessed by the additon of glucosylceramide liposome3) or gangliosides4).
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| Figure & Legends |
Figure & Legends 
Fig. 1. Structure of D-PDMP and its action in GSL metabolism
This is the pre-peer reviewed version of the following article: Inokuchi J. et al. "Effects of D-threo-PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells." J Cell Physiol. 1989, 141(3):573–83. which has been published in final form at DOI: 10.1002/jcp.1041410316. |
| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2015-01-15 13:57:38 |
- Inokuchi, J., and Radin, N.S. (1987) Preparation of Active Isomer of 1-Phenyl-2- Decanoylamino-3-Morpholino-1-Propanol, Inhibitor of murine glucocerebroside synthetase. J. Lipid Res. 28, 565–571 [PMID : 2955067]
- Felding-Hetbermann, B., Igarashi, Y., Fenderson, B.A., Park, L.S., Radin, N.S., Inokuchi, J., Strassmann, G., Handa, K., and Hakomori, S. (1990) A ceramide analogue inhibits T cell proliferative response through inhibition of glycosphingolipid synthesis and enhancement of N,N,-dimethylsphingosine synthesis. Biochemistry. 29, 6314–6322 [PMID : 2207076]
- Inokuchi J, Momosaki K, Shimeno H, Nagamatsu A, Radin NS (1989) Effect of D-threo-PDMP, an inhibitor of Glucosylceramide Synthetase, on Expression of Cell Surface Glycolipid Antigen and Binding to Adhesive Proteins by B16 Melanoma Cells. J. Cell. Physiol. 141, 573–583 [PMID : 2531751]
- Mizutani, A., Kuroda, Y., Muramoto, K., Kobayashi, K., Yamagishi, K., and Inokuchi, J. (1996) Effects of Glucosylceramide Synthase Inhibitor and Ganglioside GQ1b on Synchronous Oscillations of Intracellular Ca2+ in Cultured Cortical Neurons. Biochem. Biophys. Res. Commun. 222, 494–498 [PMID : 8670233]
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