RGD Reference Report - Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain. - Rat Genome Database

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

Authors: El Bawab, S  Bielawska, A  Hannun, Y A 
Citation: El Bawab S, etal., J Biol Chem. 1999 Sep 24;274(39):27948-55.
RGD ID: 13838800
Pubmed: PMID:10488143   (View Abstract at PubMed)

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 C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle 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 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/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 IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
ceramide catabolic process involved_inIDA 13838800PMID:10488143UniProt 
sphingosine biosynthetic process involved_inIDA 13838800PMID:10488143UniProt 

Molecular Function
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
N-acylsphingosine amidohydrolase activity enablesIDA 13838800PMID:10488143UniProt 

Objects Annotated

Genes (Rattus norvegicus)
Asah2  (N-acylsphingosine amidohydrolase 2)


Additional Information