Substrate specificity and some other enzymatic properties of dihydroceramide desaturase (ceramide synthase) in fetal rat skin.
Dihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4,5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2,3-dicyano-5,6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3,3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.
Mikami
T
T
Departments of Chemistry and Biochemistry, Sapporo Medical University School of Medicine, S1 W17 Chuo-ku, Sapporo 060-8556, Japan.
Kashiwagi
M
M
Tsuchihashi
K
K
Akino
T
T
Gasa
S
S
D000818:Animals; D002518:Ceramides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004622:Embryo, Mammalian / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012867:Skin / Q000196:embryology / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014316:Tritium
D002518:Ceramides; D005227:Fatty Acids; D014316:Tritium; D010088:Oxidoreductases; C108138:dihydroceramide desaturase