Purification and biochemical characterization of membrane-bound neutral ceramidase from camel brain (Camelus dromedarius)

Shahanas Chathoth, Faisal Thayyullathil, Alaa Galadari, Mahendra Patel, Sehamuddin Galadari

Research output: Contribution to journalArticle

Abstract

Ceramidases cleave the N-acyl linkages of ceramide to generate sphingosine and its subsequent product sphingosine-1-phosphate (S1P). Ceramide and S1P are important bioactive lipids, and ceramidases are important in regulating the availability of these lipids. In this study, we report the purification and characterization of camel brain neutral ceramidase (CBCDase). The novel CBCDase was purified from camel brain using sequential chromatography of DEAE-Sepharose, Phenyl-Sepharose, Superdex, and Mono Q column. The Mono Q fractions containing ceramidase activity were used for enzyme characterization. The purified CBCDase showed a single band corresponding to a molecular weight of ~ 100 kDa, displaying classical Michaelis-Menten kinetics, with maximum enzymatic activity at pH 7.0. Deglycosylation of the enzyme yields an apparent molecular weight of ~80 kDa. The purified CBCDase was inhibited by Zn2+ and Cu2+, while Ca2+ stimulates the activity. Phosphatidic acid, phosphatidylserine and phosphatidylcholine completely inhibited enzyme activity at low concentrations. Thiol-containing compounds inhibited the CBCDase activity. Among the nucleotides, ADP, UMP, and TMP inhibited the enzyme activity at low concentrations, whereas, ATP inhibited the activity at higher concentrations only. The CBCDase catalysed both ceramide hydrolysis and reverse CDase reactions. For the first time, we have purified to apparent homogeneity of a ~100 kDa nCDase from camel brain.

Original languageEnglish (US)
Pages (from-to)54-66
Number of pages13
JournalInternational Journal of Biochemistry and Molecular Biology
Volume4
Issue number1
StatePublished - 2013

Keywords

  • Ceramidase
  • Characterization
  • Chromatography
  • Glycosylation
  • Purification
  • pH optimum

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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