Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C₁₆- ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelie assay was developed, and using Triton X- 100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 μmol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 μmol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC₅₀ of 5-10 μM. These results indicate that the purified enzyme is a novel ceramidase.