Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized.
We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.
Tani
M
M
Department of Bioscience, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Shiga 520-2134, Japan.
Okino
N
N
Mitsutake
S
S
Tanigawa
T
T
Izu
H
H
Ito
M
M
D055574:Alkaline Ceramidase; D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism*; D004795:Enzyme Stability; D005260:Female; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D016415:Sequence Alignment; D013379:Substrate Specificity
D002518:Ceramides; D000581:Amidohydrolases; C526750:ACER1 protein, human; C526755:ACER2 protein, human; C526744:ASAH2 protein, human; C526753:Acer1 protein, mouse; D055574:Alkaline Ceramidase; C526747:Asah2 protein, mouse; D055572:Ceramidases; D055576:Neutral Ceramidase