Publications

• The reverse activity of human acid ceramidase.

  Okino N., He X., Gatt S., Sandhoff K., Ito M., Schuchman E.H.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 278:29948-29953(2003) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Substrate specificity, membrane topology, and activity regulation of human alkaline ceramidase 2 (ACER2).

  Sun W., Jin J., Xu R., Hu W., Szulc Z.M., Bielawski J., Obeid L.M., Mao C.

  Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltayd ... >> More

  Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation. << Less

  J. Biol. Chem. 285:8995-9007(2010) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase.

  El Bawab S., Birbes H., Roddy P., Szulc Z.M., Bielawska A., Hannun Y.A.

  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 stu ... >> More

  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 micromol/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 micromol/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. << Less

  J. Biol. Chem. 276:16758-16766(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.