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

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

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38 Citations (Scopus)

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

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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