Kinetic characterization of mammalian ceramide synthases: Determination of Km values towards sphinganine

Ceramide is a key metabolite in the pathway of sphingolipid biosynthesis. In mammals, ceramide is synthesized by N-acylation of a sphingoid long-chain base by a family of ceramide synthases (CerS), each of which displays a high specificity towards acyl CoAs of different chain lengths. We now optimize a previously-described assay for measuring CerS activity for use upon over-expression of mammalian CerS, and using these conditions, establish the Km value of each CerS towards sphinganine. Remarkably, the Km values towards sphinganine are all similar, ranging from 2 to 5 μM, even for CerS proteins that are able to use more than one acyl CoA for ceramide synthesis (i.e. CerS4). The availability of this assay will permit further accurate characterization of the kinetic parameters of mammalian CerS proteins.