The reverse activity of human acid ceramidase.
An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.
Okino
Nozomu
N
Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA.
He
Xingxuan
X
Gatt
Shimon
S
Sandhoff
Konrad
K
Ito
Makoto
M
Schuchman
Edward H
EH
D055573:Acid Ceramidase; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002412:Cations; D002478:Cells, Cultured; D055572:Ceramidases; D003001:Cloning, Molecular; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D037341:Fumonisins / Q000494:pharmacology; D002517:Galactosylgalactosylglucosylceramidase / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000494:pharmacology; D050356:Lipid Metabolism; D008214:Lymphocytes / Q000378:metabolism; D016464:Lysosomal Storage Diseases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism*; D013109:Sphingomyelins / Q000737:chemistry; D013110:Sphingosine / Q000737:chemistry; D013379:Substrate Specificity; D015032:Zinc
D002412:Cations; D003902:Detergents; D037341:Fumonisins; D007850:Lauric Acids; D011994:Recombinant Proteins; D013107:Sphingolipids; D013109:Sphingomyelins; C030358:lauric acid; C056933:fumonisin B1; D012965:Sodium Chloride; D002517:Galactosylgalactosylglucosylceramidase; D000581:Amidohydrolases; C526693:ASAH1 protein, human; D055573:Acid Ceramidase; D055572:Ceramidases; D015032:Zinc; D013110:Sphingosine