Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG–Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent Km and Vmax of Des2 for the substrate N-octanoylsphinganine were 35 μM and 40 nmol·h−1·mg of protein−1 respectively. The Km of the hydroxylase for mb5 was 0.8 μM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.
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Research Article|
June 28 2006
Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5
Ayako Enomoto;
Ayako Enomoto
* Sphingolipid Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan
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Fumio Omae;
Fumio Omae
* Sphingolipid Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan
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Masao Miyazaki;
Masao Miyazaki
* Sphingolipid Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan
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Yasunori Kozutsumi;
Yasunori Kozutsumi
† Glyco-chain Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan
‡ Department of Signal Transductions, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan
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Toshitsugu Yubisui;
Toshitsugu Yubisui
§ Department of Biochemistry, Okayama University of Science, Okayama 700-0005, Japan
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Akemi Suzuki
Akemi Suzuki
1
* Sphingolipid Expression Laboratory, Frontier Research System, RIKEN, Wako 351-0198, Japan
1 To whom correspondence should be addressed (email aksuzuki@riken.jp).
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Publisher: Portland Press Ltd
Received:
December 06 2005
Revision Received:
March 17 2006
Accepted:
March 29 2006
Accepted Manuscript online:
March 29 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 397 (2): 289–295.
Article history
Received:
December 06 2005
Revision Received:
March 17 2006
Accepted:
March 29 2006
Accepted Manuscript online:
March 29 2006
Citation
Ayako Enomoto, Fumio Omae, Masao Miyazaki, Yasunori Kozutsumi, Toshitsugu Yubisui, Akemi Suzuki; Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5. Biochem J 15 July 2006; 397 (2): 289–295. doi: https://doi.org/10.1042/BJ20051938
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