JBC, Vol. 270, Issue 19, 11098-11102, May, 1995
Purification, characterization, and biosynthesis of human acid ceramidase
K Bernardo, R Hurwitz, T Zenk, RJ Desnick, K Ferlinz, EH Schuchman and K Sandhoff
Institut fur Organische Chemie und Biochemie, Universitat Bonn, Germany.
Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal
enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty
acid. Its inherited deficiency causes ceramide accumulation in Farber's
disease. The enzyme was purified to apparent homogeneity from human urine
by sequential chromatography on octyl- Sepharose, concanavalin A-Sepharose,
blue-Sepharose, and DEAE- cellulose. The final preparation, which was
enriched approximately 4450- fold over the starting material, resulted in a
polypeptide of approximately 50 kDa and could be reduced into two subunits
of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of
the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced
the molecular mass of the beta subunit to approximately 30-35 and
approximately 27 kDa, respectively. In contrast, the molecular mass of the
alpha subunit was unchanged. The purified enzyme had an apparent Km of 149
microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate.
Polyclonal antibodies were raised against the purified urinary enzyme and
used to investigate the biosynthesis of acid ceramidase.
Immunoprecipitation studies on metabolically labeled skin fibroblasts
indicated that both subunits arose from a single precursor of approximately
55 kDa. A minor portion of newly synthesized acid ceramidase was secreted
into the medium as a monomeric 47-kDa protein, indicating that generation
of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal
compartments.
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