CHARACTERIZATION OF CERAMIDE SYNTHESIS - A DIHYDROCERAMIDE DESATURASEINTRODUCES THE 4,5-TRANS-DOUBLE BOND OF SPHINGOSINE AT THE LEVEL OF DIHYDROCERAMIDE

Ceramide (N-acylsphingosine) biosynthesis has been proposed to involve introduction of the 4,5-trans-double bond of sphingosine after synthe sis of dihydroceramide (i.e. N-acylsphinganine). For the first time, t he in vitro conversion of dihydroceramide to ceramide has been demonst rated using rat liver microsomes and N-[1-C-14]octanoyl-D-erythro-sphi nganine (st-H,Cer) and either NADH or NADPH as co-substrate; the appar ent K-m values for st-H,Cer and NADH were 340 and 120 mu M, respective ly. Molecular oxygen is required for enzymatic activity, and cyanide, divalent copper, as well as antibodies raised against cytochrome b(5) are inhibitory, which suggests that this enzyme should be named dihydr oceramide desaturase based on these similarities with the mechanism of Delta(9)-desaturase (stearoyl-CoA desaturase). Factors that influence d the activity of dihydroceramide desaturase include the alkyl chain l ength of the sphingoid base (in the order C-18 > C-12 > C-8) and fatty acid (C-8 > C-18); the stereochemistry of the sphingoid base (D-eryth ro- > L-threo-dihydroceramides); the nature of the headgroup, with the highest activity with dihydroceramide, but some (similar to 20%) acti vity with dihydrosphingomyelin (activity was not detected with dihydro glucosylceramide, however); and the ability to utilize alternative red uctants (ascorbic acid could substitute for a reduced pyridine nucleot ide, but was inhibitory at higher concentrations). Dihydroceramide des aturase was inhibited by dithiothreitol, which suggests that it might be possible to alter ceramide synthesis by varying the thiol status of hepatocytes, Consistent with this hypothesis, when rat hepatocytes we re cultured in varying concentrations of N-acetylcysteine (5 and 10 mM ), there was a decrease in the relative incorporation of [C-14]serine into [C-14]ceramide. These studies have conclusively established the p athway of ceramide synthesis via desaturation of dihydroceramide and h ave uncovered several properties of this reaction that warrant further consideration for their relevance to both sphingolipid metabolism and signaling.