Enzymatic synthesis of omega-amino-ceramide: preparation of a sensitive fluorescent substrate for ceramidase.
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 omega-amino-fatty acid by using the condensation reaction of the enzyme. omega-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 omega 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 omega-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 omega-aminododecanoic acids. The 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled N-dodecanoylsphingosine was easily prepared from the omega-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.
Tani
M
M
Faculty of Agriculture, Kyushu University, 6-10-1, Hakozaki, Fukuoka, Higashi-ku, 812-8581, Japan.
Kita
K
K
Komori
H
H
Nakagawa
T
T
Ito
M
M
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D055572:Ceramidases; D002518:Ceramides / Q000138:chemical synthesis*; D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D012680:Sensitivity and Specificity; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D002518:Ceramides; D005456:Fluorescent Dyes; C115938:omega-amino-ceramide; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase; D055572:Ceramidases