Enzymatic synthesis of ω-amino-ceramide: Preparation of a sensitive fluorescent substrate for ceramidase

Motohiro Tani, Katsuhiro Kita, Hironobu Komori, Tetsuto Nakagawa, Makoto Ito

Research output: Contribution to journalArticle

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Abstract

Sphingolipid ceramide N-deacylase catalyzes the reversible reactions in which the N-acyl linkage of ceramides of various sphingolipids is hydrolyzed or synthesized under different conditions. We report here a new method for preparation of ceramide containing ω-amino-fatty acid by using the condensation reaction of the enzyme. ω-Aminododecanoic acids were efficiently condensed by the enzyme to sphingosine in 25 mM glycine-NaOH buffer, pH 10, containing 0.3% Triton X-100 when the amino residue at the ω position of the fatty acid was blocked with trifluoroacetate. The reaction product was purified sequentially from the reaction mixture on a C18 reversed-phase column and Sep-Pak Plus Silica and Sep-Pak QMA cartridges with an overall yield of 80% and determined to be ω-aminododecanoylsphingosine by thin-layer chromatography and fast atom bombardment-mass spectrometry analyses after removing the block of trifluoroacetate by alkaline treatment. The enzyme can also be applied successfully to the synthesis of various glycosphingolipids and sphingomyelin containing ω-aminododecanoic acids. The 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled N-dodecanoylsphingosine was easily prepared from the ω-amino-ceramide by coupling with NBD-fluoride. This fluorescent ceramide was found to be hydrolyzed by ceramidase of B16 melanoma cells much faster than NBD-labeled N-hexanoylsphingosine in vitro as well as in vivo, indicating that the former is an excellent substrate for the assay of ceramidase.

Original languageEnglish
Pages (from-to)183-188
Number of pages6
JournalAnalytical Biochemistry
Volume263
Issue number2
DOIs
Publication statusPublished - Oct 15 1998

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Ceramidases
Ceramides
Trifluoroacetic Acid
Substrates
Enzymes
Fatty Acids
Fast Atom Bombardment Mass Spectrometry
Thin layer chromatography
Glycosphingolipids
Experimental Melanomas
Sphingolipids
Sphingosine
Acids
Condensation reactions
Sphingomyelins
Octoxynol
Thin Layer Chromatography
Fluorides
Reaction products
Silicon Dioxide

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Enzymatic synthesis of ω-amino-ceramide : Preparation of a sensitive fluorescent substrate for ceramidase. / Tani, Motohiro; Kita, Katsuhiro; Komori, Hironobu; Nakagawa, Tetsuto; Ito, Makoto.

In: Analytical Biochemistry, Vol. 263, No. 2, 15.10.1998, p. 183-188.

Research output: Contribution to journalArticle

Tani, Motohiro ; Kita, Katsuhiro ; Komori, Hironobu ; Nakagawa, Tetsuto ; Ito, Makoto. / Enzymatic synthesis of ω-amino-ceramide : Preparation of a sensitive fluorescent substrate for ceramidase. In: Analytical Biochemistry. 1998 ; Vol. 263, No. 2. pp. 183-188.
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