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J Biol Chem, Vol. 275, Issue 5, 3462-3468, February 4, 2000

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^*

Motohiro Tani, Nozomu Okino, Susumu Mitsutake, Tetsuo Tanigawa^§, Hiroyuki Izu^§, and Makoto Ito^¶

From the  Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581 and the ^§ Biotechnology Research Laboratories of Takara Shuzo Co., Ltd., Seta 3-4-1, Otsu, Shiga 520-2134, Japan

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 C16-¹⁴C-ceramide were 22.3 µM and 29.1 µmol/min/mg and 72.4 µM and 3.6 µmol/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.

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^* This work was supported in part by a grant-in-aid for Scientific Research (B) from the Ministry of Education, Science, and Culture of Japan (09460051).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) U82513.

^¶ To whom correspondence should be addressed. Tel.: 81-92-642-2900; Fax: 81-92-642-2907; E-mail: makotoi@agr.kyushu-u.ac.jp.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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