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J. Biol. Chem., Vol. 276, Issue 20, 16758-16766, May 18, 2001
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From the Department of Biochemistry and Molecular Biology, Medical
University of South Carolina, South Carolina 29425
We have previously purified a membrane-bound
ceramidase from rat brain and recently cloned the human homologue. We
also observed that the same enzyme is able to catalyze the reverse
reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited
Michaelis-Menten kinetics; however, the enzyme did not utilize
palmitoyl-CoA as substrate. Also, the activity was not inhibited
in vitro and in cells by fumonisin B1,
an inhibitor of the CoA-dependent ceramide synthase. The
enzyme showed a narrow pH optimum in the neutral range, and there was
very low activity in the alkaline range. Substrate specificity studies
were performed, and the enzyme showed the highest activity with
D-erythro-sphingosine (Km
of 0.16 mol %, and Vmax of 0.3 µmol/min/mg),
but D-erythro-dihydrosphingosine and the three
unnatural stereoisomers of sphingosine were poor substrates. The
specificity for the fatty acid was also studied, and the highest
activity was observed for myristic acid with a Km
of 1.7 mol % and a Vmax of 0.63 µmol/min/mg.
Kinetic studies were performed to investigate the mechanism of the
reaction, and Lineweaver-Burk plots indicated a sequential mechanism.
Two competitive inhibitors of the two substrates were identified,
L-erythro-sphingosine and myristaldehyde, and
inhibition studies indicated that the reaction followed a random
sequential mechanism. The effect of lipids were also tested. Most of
these lipids showed moderate inhibition, whereas the effects of
phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin
stimulated ceramidase activity. These results define the biochemical
characteristics of this reverse activity. The results are discussed in
view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.
Biochemical Characterization of the Reverse Activity of Rat
Brain Ceramidase
A CoA-INDEPENDENT AND FUMONISIN B1-INSENSITIVE
CERAMIDE SYNTHASE*
*
This work was supported by National Institutes of Health
Grant GM-43825.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Medical University of South Carolina, 171 Ashley
Ave., Charleston, SC 29425. Tel.: 843-876-5217; Fax: 843-792-4322;
E-mail: hannun@musc.edu.
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