α-Hydroxylation of lignoceroyl-CoA by a cyanide-sensitive oxygenase in rat brain microsomes

Abstract

1. 1. The enzymatic mechanism of α-hydroxylation of lignoceroyl-CoA, an intermediate in the synthesis of hydroxyceramide, was studied. In the presence of NADPH, sphingosine and microsomes from 20-day-old rat brain, 14C from [1- 14C]lignoceroyl-CoA was incorporated into hydroxyceramide. 2. 2. Activity was linear with time (up to 40 min) and with protein (up to 0.8 mg). The apparent K m for lignoceroyl-CoA was about 10 μM. 3. 3. NADPH was a more efficient electron donor than NADH. Oxygen was required for activity, which increased linearly up to 20% O 2. 4. 4. In 5 and 10% oxygen, the reaction was inhibited by 0.1 mM cyanide and by electron transfer chain inhibitors, cytochrome c, ferricyanide, menadione, and p-chloromercuriphenyl sulphonate; CO and SKF-525A had no effect. Moreover none of the inhibitors affected the formation of hydroxyceramide. 5. 5. Lignoceroyl-CoA α-hydroxylase appears to be an oxygenase requiring NADPH and oxygen, which involves cyanide-sensitive enzyme.