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, Makoto Ito

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)3462-3468
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number5
DOIs
Publication statusPublished - Feb 4 2000

Fingerprint

Neutral Ceramidase
Ceramides
Liver
Purification
Ceramidases
Enzymes
Alkaline Ceramidase
Proteins
Galactosylceramides
Sulfoglycosphingolipids
Acyl Coenzyme A
Sphingosine
Gene encoding
Dictyostelium
Sphingomyelins
Molecular mass
Fungi
Pseudomonas aeruginosa
Polysaccharides
Amino Acid Sequence

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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. / Tani, Motohiro; Okino, Nozomu; Mitsutake, Susumu; Tanigawa, Tetsuo; Izu, Hiroyuki; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 275, No. 5, 04.02.2000, p. 3462-3468.

Research output: Contribution to journalArticle

@article{dd6d6e2eb46940a3a104593ff6792579,
title = "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",
abstract = "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.",
author = "Motohiro Tani and Nozomu Okino and Susumu Mitsutake and Tetsuo Tanigawa and Hiroyuki Izu and Makoto Ito",
year = "2000",
month = "2",
day = "4",
doi = "10.1074/jbc.275.5.3462",
language = "English",
volume = "275",
pages = "3462--3468",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "5",

}

TY - JOUR

T1 - 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

AU - Tani, Motohiro

AU - Okino, Nozomu

AU - Mitsutake, Susumu

AU - Tanigawa, Tetsuo

AU - Izu, Hiroyuki

AU - Ito, Makoto

PY - 2000/2/4

Y1 - 2000/2/4

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034602962&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034602962&partnerID=8YFLogxK

U2 - 10.1074/jbc.275.5.3462

DO - 10.1074/jbc.275.5.3462

M3 - Article

C2 - 10652340

AN - SCOPUS:0034602962

VL - 275

SP - 3462

EP - 3468

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 5

ER -