52] Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and ω-aminoceramide

Abstract

Publisher Summary This chapter isolates a novel enzyme that hydrolyzes the N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids and tentatively names it “sphingolipid ceramide N-deacylase” (SCDase). SCDase is found to catalyze the hydrolysis and its reverse reaction as well, under different conditions. Under acidic conditions including a high concentration of Triton X-100, SCDase catalyzes the hydrolysis reaction efficiently. In contrast, at neutral pH with a low concentration of the detergent, the reverse reaction tends to be favored. It is possible therefore to utilize the desired reaction of SCDase if the conditions are changed. This chapter describes the synthesis of [ 14 C]ceramide, [ 14 C]glycosphingolipids, and ω-aminoceramide by the reverse hydrolysis (condensation) reaction of SCDase. Because the specificity of the enzyme is very specific to the amino group of sphingosine bases, no by-products are generated during the reaction. The fact that a wide range of sphingolipid species and sample amounts (from the nanomole to millimole level) can be used is significantly advantageous.