[14C]ceramide synthesis by sphingolipid ceramide N-deacylase: new assay for ceramidase activity detection

Sphingolipid ceramide N-deacylase (SCDase) is an enzyme which hydrolyzes the N-acyl linkage between fatty acid and sphingosine in ceramide of various glycosphingolipids and sphingomyelin. Recently the enzyme was found to catalyze the hydrolysis and its reverse reaction under different conditions. We report here an innovative method for synthesis of radioisotope-labeled ceramide with high specific activity using the reverse hydrolysis reaction of SCDase. More than 80% of free fatty acid was transferred to sphingosine when 1 nmol [14C]stearic acid and 2 nmol sphingosine were incubated with 5 microU SCDase at 37 degrees C for 20 h in 10 microliters of 25 mM phosphate buffer, pH 7.0, containing 0.3% Triton X-100. Free [14C]fatty acid and sphingosine were easily separated from synthesized [14C]ceramide by using a Sep-Pak Plus Silica and a Sep-Pak CM cartridge, respectively. We also developed a sensitive assay method for ceramidase using the [14C]ceramide prepared. The method consists of separation of the [14C]fatty acid released from [14C]ceramide by the action of enzyme on thin-layer chromatography followed by analysis and quantification with an imaging analyzer (BAS1000). This method was sensitive and qualitative and enabled the detection of ceramidase activity in invertebrates for the first time as well as in several human cancer cell lines.