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J. Biol. Chem., Vol. 278, Issue 32, 29948-29953, August 8, 2003

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The Reverse Activity of Human Acid Ceramidase^*

Nozomu Okino   ¶, Xingxuan He , Shimon Gatt ||, Konrad Sandhoff **, Makoto Ito  and Edward H. Schuchman  

From the Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, the Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan, the ^||Department of Biochemistry, Hebrew University-Hadassah School of Medicine, Jerusalem 91120, Israel, and the ^**Kekule Institut für Organische Chemie und Biochemie der Friedrich-Wilhelms Universität, D-53121 Bonn, Germany

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 5.5, as compared with 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 K_m and V_max values toward sphingosine were 23.75 µM and 208.3 pmol/µg/h, respectively, whereas for lauric acid they were 73.76 µM and 232.5 pmol/µg/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 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.

Received for publication, March 31, 2003 , and in revised form, May 14, 2003.

^* This work was supported in part by National Institutes of Health Grant R01 DK54830, Grant 6-FY-00-241 from the March of Dimes Birth Defects Foundation, and Grant 5R03 TW 01372 from the Fogarty International Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science.

To whom correspondence should be addressed: Dept. of Human Genetics, Box 1498, Mount Sinai School of Medicine, 1425 Madison Ave., Rm. 14-20A, New York, NY 10029. Tel.: 212-659-6711; Fax: 212-849-2447; E-mail: Edward.Schuchman{at}mssm.edu.

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