| http://purl.uniprot.org/citations/16571104 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
| http://purl.uniprot.org/citations/16571104 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
| http://purl.uniprot.org/citations/16571104 | http://www.w3.org/2000/01/rdf-schema#comment | "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 K(m) and V(max) of Des2 for the substrate N-octanoylsphinganine were 35 microM and 40 nmol x h(-1) x mg of protein(-1) respectively. The K(m) of the hydroxylase for mb5 was 0.8 microM. 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."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.org/dc/terms/identifier | "doi:10.1042/bj20051938"xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.org/dc/terms/identifier | "doi:10.1042/bj20051938"xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Miyazaki M."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Miyazaki M."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Suzuki A."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Suzuki A."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Yubisui T."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Yubisui T."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Kozutsumi Y."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Kozutsumi Y."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Enomoto A."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Enomoto A."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Omae F."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/author | "Omae F."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/date | "2006"xsd:gYear |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/date | "2006"xsd:gYear |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/name | "Biochem. J."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/name | "Biochem. J."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/pages | "289-295"xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/pages | "289-295"xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/title | "Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5."xsd:string |
| http://purl.uniprot.org/citations/16571104 | http://purl.uniprot.org/core/title | "Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5."xsd:string |