Int J Biochem Mol Biol 2013;4(1):54-66

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

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

Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain, UAE
Received March 10, 2013; Accepted March 26, 2013; Epub March 31, 2013; Published April 15, 2013

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.
(IJBMB1303002).

Keywords: Ceramidase, characterization, chromatography, glycosylation, pH optimum, purification

Address correspondence to: Sehamuddin Galadari, Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health
Sciences, UAE University, P.O. Box 17666, Al Ain, UAE. Phone: +97137137507; Fax: +97137672033; E-mail: sehamuddin@uaeu.ac.ae
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