title: Purification and biochemical characterization of membrane-bound epidermal ceramidases from guinea pig skin.
abstract: Ceramidase (CDase) catalyzes the hydrolysis of ceramides to yield sphingosine and fatty acid. In this paper, two forms of membrane-bound alkaline ceramidase, have been, for the first time, purified from guinea pig epidermis by chromatography on DEAE-cellulose, phenyl-Superose, HCA-hyroxyapatite, isoelectric focusing, Mono Q, and TSK-3000SW column. One species (CDase-I) migrated upon SDS-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 60 kDa; the other (CDase-II) was only partially purified with apparent M(r) of about 148,000 estimated by gel filtration. The specific activities of the two species increased by 1.130- (for CDase-I) and 400-fold (for CDase-II) over the original tissue extract. The activity of both enzymes for ceramide species decreased in the order of linoleoyl > oleoyl > palmitoylsphingosine. The optimal pH for enzyme activity was approximately 7.0-9.0 for CDase-I and 7.5-8.5 for CDase-II. Interestingly, both enzymes were inhibited by the reaction product sphingosine with a concentration for half-maximal inhibition (ID50) of 100-130 microM, compared to the apparent kinetic parameters with CDase-I (Km = 90 microM, Vmax = 0.62 unit) and CDase-II (Km = 140 microM, Vmax = 0.50 units). Some lipids, such as phosphatidylcholine and sphingomyelin, are also inhibitory with IC50 values of 50-250 microM, suggesting well controlled CDase activity by sphingolipid metabolites. These studies begin to elucidate a regulatory mechanism for the balance of the ratio of ceramide/sphingosine which can serve as an intracellular effector molecule in epidermis.
authors.affiliation: Institute for Fundamental Research, Kao Corporation, Tochigi, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Yada
authors.forename: K
authors.initials: K
authors.lastname: Higuchi
authors.forename: G
authors.initials: G
authors.lastname: Imokawa
mesh_terms: D000581:Amidohydrolases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism*; D004817:Epidermis / Q000201:enzymology*; D006168:Guinea Pigs; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology; D012867:Skin / Q000201:enzymology*; D013109:Sphingomyelins / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D002518:Ceramides; D007527:Isoenzymes; D008246:Lysophospholipids; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D000581:Amidohydrolases; D055572:Ceramidases