期刊
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
卷 74, 期 2, 页码 316-321出版社
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.90645
关键词
ceramide; ceramidase; sphingosine; Mycobacterium tuberculosis; Pseudomonas aeruginosa
类别
资金
- Ministry of Education, Culture, Sports, Science, and Technology, Japan [19770086]
- Grants-in-Aid for Scientific Research [19770086] Funding Source: KAKEN
Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was expressed in Escherichia coli and purified by Ni-Sepharose and gelfiltration. The purified recombinant enzyme showed a single band and a molecular weight estimated to be 71 kDa on SDS-PAGE. It had a pH optimum at 8.0-9.0 and quite broad specificity for various Cers. Of the Cers of different fatty acid moieties tested, those composed of C6-C24 fatty acids were well hydrolyzed, and Cers with mono unsaturated fatty acids were much more hydrolyzed than those with saturated fatty acids. Using N-dodecanoyl-7-nitrobenz-2-oxa-1,3-4-diazole (NBD)-D-erythro-sphingosine (C12-NBD-Cer) as substrates, the reaction followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values for C12-NBD-Cer were 98.7 mu M and 21.1 pmol/min respectively. The purified enzyme also catalyzed the synthesis of Cer in vitro, using NBD-labeled dodecanoic acid and Sph as substrates.
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