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

We have purified a membrane bound ceramidase 22,300-fold to apparent homoge neity. The purification scheme included Triton X-100 extraction of membrane s followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS colu mn chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reduci ng conditions and 95 kDa by chromatography on Superose 12. Using C-16-ceram ide as substrate, the enzyme showed a broad pH optimum in the neutral to al kaline range. A mixed micelle assay was developed, and using Triton X-100/c eramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kin etics, with a K-m of 1.29 mol % and a V-max of 4.4 mu mol/min/mg. When dihy droceramide was used as substrate, these values were 3.84 mol % and 1.2 mu mol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides p referentially. The activity of the purified ceramidase did not require cati ons, and it was inhibited by reducing agents. Phosphatidylcholine and sphin gomyelin were without effect on the enzyme activity, whereas phosphatidic a cid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acte d as a competitive inhibitor with an IC50 of 5-10 mu M. These results indic ate that the purified enzyme is a novel ceramidase.