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.