Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney

Susumu Mitsutake, Motohiro Tani, Nozomu Okino, Kaoru Mori, Sachiyo Ichinose, Akira Omori, Hiroshi Iida, Takashi Nakamura, Makoto Ito

Research output: Contribution to journalArticle

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Abstract

Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229-11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that the N-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside.

Original languageEnglish
Pages (from-to)26249-26259
Number of pages11
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
Publication statusPublished - Jul 13 2001

Fingerprint

Neutral Ceramidase
Ceramidases
Cloning
Molecular Cloning
Purification
Rats
Glycosylation
Kidney
Thawing
Liver
Enzymes
Membranes
G(M1) Ganglioside
Tunicamycin
Proteins
HEK293 Cells
Endosomes
Enzyme activity
Endoplasmic Reticulum
Organelles

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney. / Mitsutake, Susumu; Tani, Motohiro; Okino, Nozomu; Mori, Kaoru; Ichinose, Sachiyo; Omori, Akira; Iida, Hiroshi; Nakamura, Takashi; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 26249-26259.

Research output: Contribution to journalArticle

Mitsutake, Susumu ; Tani, Motohiro ; Okino, Nozomu ; Mori, Kaoru ; Ichinose, Sachiyo ; Omori, Akira ; Iida, Hiroshi ; Nakamura, Takashi ; Ito, Makoto. / Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 28. pp. 26249-26259.
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