Purification and characterization of a second type of neutral ceramidase from rat brain

A second more hydrophobic form of rat brain ceramidase

Faisal Thayyullathil, Shahanas Chathoth, Abdulkader Hago, Mahendra Patel, Zdzislaw M. Szulc, Yusuf Hannun, Sehamuddin Galadari

    Research output: Contribution to journalArticle

    Abstract

    Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200 pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q, the specific activity of the enzyme increased by ~ 15,000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110 kDa) than the previously purified (90 kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca2+ and is inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg2+ and Fe3+, while it is not affected by Mn2+. EDTA was somewhat inhibitory at a 20 mM concentration.

    Original languageEnglish (US)
    Pages (from-to)242-252
    Number of pages11
    JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    Volume1811
    Issue number4
    DOIs
    StatePublished - Apr 1 2011

    Fingerprint

    Neutral Ceramidase
    Ceramidases
    Brain
    Enzymes
    Pyrimidine Nucleotides
    Thymidine Monophosphate
    Uridine Monophosphate
    Sphingosine
    Ceramides
    Octoxynol
    Glycosylation
    Nonesterified Fatty Acids
    Edetic Acid
    Sepharose
    Polysaccharides
    Polyacrylamide Gel Electrophoresis
    Hydrolysis
    Nucleotides
    Molecular Weight
    Metals

    Keywords

    • Ceramidase
    • Ceramide
    • Chromatography
    • Glycosidase F
    • Sphingosine

    ASJC Scopus subject areas

    • Molecular Biology
    • Cell Biology

    Cite this

    Purification and characterization of a second type of neutral ceramidase from rat brain : A second more hydrophobic form of rat brain ceramidase. / Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Szulc, Zdzislaw M.; Hannun, Yusuf; Galadari, Sehamuddin.

    In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, Vol. 1811, No. 4, 01.04.2011, p. 242-252.

    Research output: Contribution to journalArticle

    Thayyullathil, Faisal ; Chathoth, Shahanas ; Hago, Abdulkader ; Patel, Mahendra ; Szulc, Zdzislaw M. ; Hannun, Yusuf ; Galadari, Sehamuddin. / Purification and characterization of a second type of neutral ceramidase from rat brain : A second more hydrophobic form of rat brain ceramidase. In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 2011 ; Vol. 1811, No. 4. pp. 242-252.
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    AU - Patel, Mahendra

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