Purification and characterization of a novel ceramidase from pseudomonas aeruginosa

Nozomu Okino, Motohiro Tani, Shuhei Imayama, Makoto Ito

Research output: Contribution to journalArticle

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Abstract

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2- oxa-1,3-4-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:l) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent K(m) of 139 μM, and a V(max) of 5.3 μmol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.

Original languageEnglish
Pages (from-to)14368-14373
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number23
DOIs
Publication statusPublished - Jun 5 1998

Fingerprint

Ceramidases
Pseudomonas aeruginosa
Ceramides
Purification
Enzymes
lauric acid
phytosphingosine
Galactosylceramides
Sulfoglycosphingolipids
Silver Staining
Sphingosine
Mammals
Sphingomyelins
Molecular mass
Atopic Dermatitis
Pseudomonas
Electrophoresis
Silver
Polyacrylamide Gel Electrophoresis
Bacteria

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and characterization of a novel ceramidase from pseudomonas aeruginosa. / Okino, Nozomu; Tani, Motohiro; Imayama, Shuhei; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 273, No. 23, 05.06.1998, p. 14368-14373.

Research output: Contribution to journalArticle

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