Biochemical Characterization of the Reverse Activity of Rat Brain Ceramidase

A CoA-INDEPENDENT AND FUMONISIN B1-INSENSITIVE CERAMIDE SYNTHASE*

Samer El Bawab, Helene Birbes, Patrick Roddy, Zdzislaw M. Szulc, Alicja Bielawska, and Yusuf A. HannunDagger

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, South Carolina 29425

Received for publication, October 12, 2000, and in revised form, February 7, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingolipid metabolites are now recognized as important components in signal transduction, not only in mammalian cells, but also in yeast, where they are implicated in heat stress responses. Ceramide (Cer)1 is one of these sphingolipid metabolites, and it has been shown to play a role in apoptosis, cell cycle arrest, and differentiation (for recent reviews, see Refs. 1-3).

Ceramidases (CDase) are enzymes that cleave the N-acyl linkage of ceramide into sphingosine (SPH) and free fatty acid. CDases may exert important functions in the regulation of its substrate Cer or in the regulation of its immediate product SPH or the downstream metabolite sphingosine 1-phosphate (S1P). Current understanding indicates that the major pathway for the formation of sphingosine is via the degradation of ceramide and not from the de novo pathway (4, 5). This suggests that CDases are the key enzymes to regulate levels of SPH and/or S1P. Indeed, several reports have shown the involvement of ceramidases in the regulation of Cer, SPH, and/or S1P levels in agonist-mediated cell responses. Activation of ceramidases leading to an increase of SPH and/or S1P levels and to responses associated with these lipids has been shown in rat glomerular mesangial cells stimulated with platelet-derived growth factor (6), in rat hepatocytes stimulated with low concentrations of interleukin 1 (7), in rat mesangial cells stimulated with nitric oxide donors (8), and in vascular smooth muscle cells treated with oxidized low density lipoprotein (9). On the other hand, studies using inhibitors of CDases (N-oleoylethanolamine and D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol) have also shown that inhibition of these enzymes causes an elevation in the endogenous level of ceramide, which is either sufficient to inhibit growth or augments the effects of other inducers of growth arrest (10, 11). Taken together, these observations underscore the potential importance of CDases and their roles in different process such as apoptosis and proliferation.

In addition, recent studies on ceramidases have revealed the complex nature of these enzymes. In the original report on ceramidase, Gatt (12) proposed that a single protein catalyzes the hydrolysis of ceramide (ceramidase activity) and the reverse reaction through a CoA-independent mechanism (ceramide synthase). This intriguing observation was recently confirmed by two groups for ceramidases isolated from yeast and from mouse (13, 14). We have purified and characterized a rat brain membrane-bound ceramidase (15), and we recently cloned the human isoform and found that this isoform is localized to mitochondria (16). Further studies of this enzyme also revealed that this rat brain enzyme catalyzes the reverse reaction of ceramide synthesis (16).

To understand this enzyme, in this study, the biochemical characteristics and mechanism of action of this reverse activity were investigated. Furthermore, labeling experiments indicated that this reverse activity may account for a portion of ceramide synthesis in cells, which is not inhibitable by fumonisin B1. Biochemical characterization experiments showed specificity for the substrates and that the reaction follows a random sequential mechanism. They also suggest a possible differential regulation of the enzyme's two activities (ceramidase and reverse activity) by the intracellular pH and by the presence of cardiolipin and/or phosphatidic acid.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Frozen rat brains were purchased from Pel-Freez Biologicals (Rogers, AK). Bradford protein assay was from Bio-Rad. BCA protein assay and Triton X-100 were from Pierce. [3H]Palmitoyl-CoA was from American Radiolabeled Chemicals. Lipids were from Avanti Polar Lipids. TLC plates were from Merck (Darmestad).

Cell Culture-- Human embryonic kidney 293 cells overexpressing empty vector (pcDNA3.1/His) or vector containing human mitochondrial ceramidase (16) were cultured in minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 100 µg/ml Geneticin.

Lipid Synthesis-- [3H]C16-Cer, [3H]D-erythro-SPH, and [3H]D-erythro-dihydrosphingosine were synthesized as described previously (17, 18). Ceramides with various chain length, SPH, and dihydrosphingosine were synthesized as described (19).

Protein Purification-- The purification of the protein was carried out as described previously (15). Briefly, the enzyme was extracted from the 10,000 × g pellet with Triton X-100. The Triton X-100 extract was then applied to Q-Sepharose anion exchange chromatography, followed by blue-Sepharose, phenyl-Sepharose, and MonoS cation exchange chromatography. Using this protocol of purification, the specific activity was increased ~20,000-fold, and the protein on SDS-polyacrylamide gel electrophoresis silver staining appeared as a single band in the first fractions of the last column MonoS.

CDase Assay-- CDase activity was measured in a Triton X-100/Cer mixed micelle assay as described previously (15).

Reverse CDase Assay-- Reverse CDase activity was performed using the purified protein. Briefly, the substrates [3H]palmitic acid and SPH were first dried. The dried mixture was then resuspended by sonication in 100 µl of 200 mM Hepes buffer (pH 7) containing 0.4% of Triton X-100, and the appropriate amount of enzyme in 100 µl volume was then added. The final Triton X-100 concentration in the assay was 0.2%. The reaction was terminated by adding 2 ml of Dole solution (isopropyl alcohol/heptane/1 N NaOH, 4:1:0.1), followed by 1 ml of water and 1 ml of heptane. Under these conditions the unreacted free fatty acid remains in the aqueous/alcoholic phase. After centrifugation, the upper phase was collected, and the lower phase was washed one more time with 2 ml of heptane. The heptane phases containing the product [3H]Cer were combined and counted in liquid scintillation.

Ceramide Synthase Assay-- Ceramide synthase (CoA-dependent) activity was assayed using rat brain microsomes as described (20). Briefly, the assay mixture (100 µl) contained 25 mM Tris buffer (pH 7.4), 0.5 mM dithiothreitol, 10 µM [3H]dihydrosphingosine, 200 µM palmitoyl-CoA, and 150 µg of protein. After 30 min of incubation, the reaction was stopped by the addition of 1 ml of methanol, 0.5 ml of chloroform, and unlabeled dihydro-C16-Cer as carrier. 1 ml of chloroform and 3 ml of water were then added, and the mixture was vortexed. The aqueous layer was then discarded, and the chloroform layer was dried and applied on TLC, and lipids were separated using the solvent mixture ethyl acetate:isooctane:acetic acid (50/50/10, v/v). The dihydroceramide band was then scraped and counted.

Protein Assay-- Protein concentration was determined using the Bradford assay or the BCA assay in samples containing Triton X-100.

Substrate Specificity and Kinetics of the Reverse Activity-- In experiments studying the SPH specificity, the assay contained [3H]palmitic acid at saturating concentration of 16 mol %; the assay in these experiments was performed as described above. In experiments studying the fatty acid chain specificity, [3H]SPH was used in the assay instead of [3H]palmitic acid at a saturating concentration of 3 mol %. At the end of the incubation, lipids were extracted and applied on TLC to separate the labeled Cer formed.

Mechanism of the Reverse Activity-- Initial velocity studies were performed by varying concentrations of SPH at several fixed concentrations of [3H]palmitic acid. Lineweaver-Burk plots were then generated. Secondary plots were next generated by replotting the slopes and the y intercepts of the lines as a function of 1/[palmitic acid]. A random sequential mechanism follows the equation, v = VmaxAB/(KiaKb + KaB + KbA AB), where A and B represent the concentrations of substrates SPH and palmitic acid, respectively, and Kia represents the dissociation constant of the enzyme-A complex. The values of KSPH, Kpalmitic acid, V, and Kia can be determined from the slopes and y intercepts of the secondary plots as described (21).

Inhibition studies were performed by varying the concentrations of SPH or palmitic acid in the presence or absence of increasing concentrations of the inhibitor as described (22).

Labeling Experiments-- Cells plated in 100-mm culture dishes were labeled with 1 µM [3H]SPH (1 µCi/ml) for different times. Lipids were extracted by the method of Bligh and Dyer and separated on TLC using the solvent ethyl acetate:isooctane:acetic acid (50/50/10; v/v).

Other Procedures-- All experiments were performed two or three times (unless indicated) on the enzyme obtained from the single-band fractions of the MonoS column (15). When reducing agents were tested, the enzyme was preincubated with these agents for 2 min prior to the assay. When lipid effects were tested, lipids were dried with the substrate, and the mixture was resuspended with Hepes buffer containing Triton X-100 at a final concentration of 0.2%. All linear regression plots were performed using the Cricket Graph V3 program.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reverse Activity of Rat Brain Ceramidase-- Further investigation of the ceramidase purified from rat brain revealed that the enzyme also catalyzes the reverse reaction. At first we studied the substrate requirement for this reverse activity. Fig. 1a shows that the enzyme catalyzes the condensation of SPH and palmitic acid into C16-Cer. The enzyme failed to form Cer when palmitoyl-CoA was used as a substrate, indicating that the enzyme acts through a CoA-independent mechanism. Similar observations were first described by Gatt (12) using semi-purified ceramidase, and more recently for phytoceramidase from yeast (13) and from mouse liver (14), but interestingly, a dihydroceramidase from yeast did not display significant reverse activity (23). Furthermore, fumonisin B1 (FB1), an anti-fungal, is known to inhibit the CoA-dependent Cer synthase activity (24). As shown in Fig. 1b, FB1 inhibited the CoA-dependent ceramide synthase activity of rat brain microsomes but failed to inhibit the reverse CDase activity purified from rat brain. These results clearly indicate that the two activities represent different enzymes and further attest to the specificity of fumonisin B1.


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Fig. 1.   Fatty acyl-CoA independence and FB1 resistance of ceramidase reverse activity. a, ceramidase reverse activity was assayed in the presence of saturating concentrations of SPH (3 mol %, 100 µM) and increasing concentrations of [3H]palmitic acid or [3H]palmitoyl CoA (1.5, 3, and 6 mol %; 50, 100, and 200 µM). At the end of the incubation, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate [3H]Cer from substrates using the system ethyl acetate:isooctane:acetic acid (50/50/10, v/v). The ceramide standard is shown in lane 1. b, effect of FB1 on reverse CDase activity and on the CoA-dependent ceramide synthase activity. For reverse CDase activity, the purified protein (see "Experimental Procedures") was used, for the CoA-dependent ceramide synthase activity in the assay rat brain microsomes were used.

To study the enzyme, we developed an assay with the purified protein as described under "Experimental Procedures." In this assay, product is separated by a basic Dole extraction, and the activity is linear with time and protein up to 100 µg in the assay (not shown). Having in our hands a reliable assay, we next characterized and investigated the biochemistry of this enzyme activity to gain insight into its physiological role.

pH Optimum-- The purified enzyme showed reverse activity in a narrow pH spectrum (Fig. 2), distinct from the ceramidase activity, which showed a broad pH range from 5.5 to 10 (15). There was very low activity in the alkaline range (pH > 8) and in the acidic range (pH < 5), and the optimum activity was observed at pH 6.5-7. These results indicate that the ceramidase activity is neutral/alkaline whereas the reverse activity is strictly neutral.


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Fig. 2.   pH dependence of reverse CDase activity. Reverse CDase activity was performed by the Dole extraction as described under "Experimental Procedures" using [3H]palmitic acid. The final Triton X-100 concentration was 0.2%. The pH was adjusted by the addition of the indicated buffers at a final concentration of 100 mM. At the end of the incubation, the pH within each tube was adjusted to pH 9 using Tris 1 M.

Effect of Cations-- The addition of MgCl2, MnCl2, CaCl2, and LiCl was without any effect on the reverse activity (Fig. 3a). ZnCl2 and CuCl2 inhibited the enzyme, and total inhibition was observed at around 1 mM. In addition, EDTA up to 10 mM did not show any effect on the reverse activity. These results clearly indicate that the enzyme is totally independent of cations for stimulation of activity.


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Fig. 3.   Effects of cations and reducing agents on reverse CDase activity. Reverse CDase activity was assayed using SPH at 0.6 mol % (20 µM) and [3H]palmitic acid at 3 mol % (100 µM). a, effect of cations; b, effect of reducing agents.

Effect of Reducing Agents and Nucleotides-- We had shown that reducing agents dithiothreitol and beta -mercaptoethanol inhibited ceramidase activity. When tested on the reverse activity, similar effects were observed (Fig. 3b). Also, ATP up to 10 mM did not affect the activity (not shown).

Substrate Specificity of the Reverse Activity-- First we studied the specificity for SPH. Sphingosine harbors two chiral centers and therefore exhibits four stereoisomers, only one of which, the D-erythro (2S,3R) is known to exist naturally. As shown in Fig. 4a, the enzyme showed Michaelis-Menten kinetics when D-erythro-SPH was used as substrate. There was very low activity when D-threo, L-threo, or L-erythro-SPH isomers were used, showing a high specificity for the naturally occurring substrate. In addition, there was very low activity with D-erythro-dihydrosphingosine, a sphingolipid metabolite in the de novo pathway (5), which differs from D-erythro-SPH in lacking the 4-5 trans double bond. Next, we studied the effect of the fatty acid chain. Fig. 4b shows the kinetic curves when various fatty acids were used, and Table I represents the apparent Km and Vmax values deduced from the double-reciprocal plots of each fatty acid. The Km values for the fatty acids were all comparable and within a range of 1.1 to 2.2 mol %, but the Vmax values were more variable, ranging from 0.08 to 0.63 µmol/min/mg. Thus, as judged by the Vmax/Km ratio, the enzyme showed the highest synthesis rate with myristic acid (highest Vmax/Km ratio).


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Fig. 4.   Substrate specificity of reverse CDase activity. The specificity for substrates, SPH, and fatty acids was examined. a, specificity for SPH. The assay was performed in the presence of a saturating concentration of [3H]palmitic acid (15 mol %) and the indicated increasing concentrations of sphingoid bases. b, specificity for the fatty acid chain. The assay was performed in the presence of a saturating concentration of [3H]SPH (3 mol %) and the indicated increasing concentrations of fatty acids. The Dole extraction could not be used here because labeled SPH is used in the assay. Thus, at the end of the assay, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate the substrates from the product [3H]Cer. The spots corresponding to Cers were then scraped, and the radioactivity was quantified using liquid scintillation counting.

                              
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Table I
Apparent Km and Vmax values of various fatty acids
The results are obtained from the Lineweaver-Burk plots of the data in Fig. 4b.

Kinetics of the Reverse Activity-- To study the kinetic mechanism of the enzyme, the reverse activity was measured as a function of varying concentrations of SPH (0.19-1.56 mol %) at five fixed concentrations of [3H]palmitic acid (0.39-6.2 mol %). The Lineweaver-Burk plots of the data were linear and thus followed Michaelis-Menten kinetics (Fig. 5a). The plots intersected to the left side of the ordinates, indicating a sequential kinetic mechanism (21).


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Fig. 5.   Kinetics of the reverse reaction. The mechanism of the reaction was studied by varying the concentrations of SPH at five fixed concentrations of [3H]palmitic acid. At the end of the assay, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate the substrates from the product [3H]Cer. The spots corresponding to Cers were then scraped, and the radioactivity was quantified using liquid scintillation counting. a, Lineweaver-Burk plots of the data. The intersection of the plots on the left side of the ordinates is indicative of a sequential mechanism. b, secondary plots generated from the slopes and y intercepts of the primary plots. The slope and the y intercept of the Slope plot represent (Kia.Kpalmitate)/V and KSPH/V, respectively. The slope and y intercept of the y intercept plot represent Kpalmitate/V and 1/V, respectively (21). Experiments were performed two times and results are representative of one experiment.

To distinguish between ordered sequential mechanism and random sequential mechanism, inhibition studies were performed (22). First, L-erythro-SPH was used as a dead-end inhibitor to perform these studies. When the reverse activity was measured as a function of SPH concentrations in the absence or presence of L-erythro-SPH, double-reciprocal plots of the data reflected typical competitive inhibition (Fig. 6a). On the other hand, L-erythro-SPH showed a noncompetitive inhibition pattern when studied as a function of palmitic acid concentration (Fig. 6b). Next, we screened several compounds to find another inhibitor for the second substrate, palmitic acid. Hexadecanol, palmitic acid methyl ester, palmitaldehyde, and myristaldehyde were among the products tested for inhibition. Both of the aldehyde compounds showed inhibition (~50% at 2.5-3 mol %). Therefore, myristaldehyde was used for the following experiments. When SPH concentrations were varied, a noncompetitive pattern was observed (Fig. 6c), and when varying the concentrations of palmitic acid, a competitive inhibition was observed (Fig. 6d). These results indicate that the reverse activity follows a random-sequential mechanism (22). In this mechanism association and dissociation of both SPH and fatty acid are fast, and there is no obligate order binding of the substrates. Secondary plots were next generated (Fig. 5b), and the kinetic constants obtained from the slopes and y intercepts of these plots are presented in Table II (22). The Km for SPH was 0.16 mol %, and the Km for palmitic acid was 2.1 mol %. These values were close to the apparent Km values obtained in previous and independent analysis for SPH (0.27 mol %, Fig. 4a) and for palmitate (2.2 mol %, Table I), suggesting that the binding of the first substrate does not affect the binding of the second substrate.


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Fig. 6.   Mechanism of the reverse reaction. Inhibition studies were performed to distinguish between ordered and random mechanism. a and b, competitive and noncompetitive inhibition of the reverse reaction with L-erythro-SPH when varying the concentration of SPH and palmitic acid, respectively. c and d, competitive and noncompetitive inhibition of the reverse reaction with myristaldehyde when varying the concentration of palmitic acid and SPH, respectively. Experiments were performed two times, and results are representative of one experiment. At the end of the assay, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate the substrates from the product [3H]Cer. The spots corresponding to Cers were then scraped, and the radioactivity was quantified using liquid scintillation counting.

                              
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Table II
Kinetic parameters of the reverse reaction
The data were obtained from the secondary plots shown in Fig. 5b.

Effect of Lipids-- The effects of various sphingolipids and phospholipids on the reverse activity were investigated. These lipids were added at the indicated mol % concentration with the substrates. Fig. 7a shows that sphingomyelin inhibited the reverse activity with half-maximal inhibition at around 5 mol %. Cerobrosides were less effective. C16-Cer, the product of the reaction, showed moderate inhibition, with 50% inhibition at 10 mol %.


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Fig. 7.   Effect of lipids on reverse CDase activity. Reverse CDase activity was performed by the Dole extraction as described under "Experimental Procedures" using [3H]palmitic acid. Lipids were dried with the substrates, and the mixture was resuspended with reaction buffer containing Triton X-100 at a final concentration of 0.2%. a, effects of sphingolipids; b, effects of phospholipids.

Next we studied the effect of various phospholipids and lysophospholipids (Fig. 7b). Lysophosphatidic acid was without any effect. At 10 mol %, phosphatidylcholine, phosphatidylserine (PS), phosphatidylglycerol, and lysophosphatidylcholine had moderate inhibition of the activity, with maximum inhibition of around 25-50%. Very interestingly, phosphatidic acid (PA) and cardiolipin (CL) inhibited totally the reverse activity and at lower concentrations (2.5-5 mol %).

Because the human isoform was found to be localized to mitochondria (16) and because CL is a major lipid of mitochondrial membranes, this effect of CL could be of physiological relevance. Thus, we further investigated the effect of this lipid on the enzyme. Surprisingly, and as shown in Fig. 8a, CL stimulated ceramidase activity within the same range of concentrations that inhibited the reverse activity, with a 2.5-fold increase at 8-10 mol %. These intriguing observations were further investigated to confirm these results. First, the stimulatory effect on ceramidase activity was independent of the pH of the reaction, because the increase of the ceramidase activity was still observed when the reaction was performed at pH 7 (data not shown). Second, because of the negative charges, CL could interfere with the assay extraction. To answer this, at the end of the incubation, the reaction media were dried, total lipids were applied and separated on TLC, and the Cer band was scraped and counted. Results shown in Fig. 8b indicate that CL still inhibited the reverse activity and activated the ceramidase activity, indicating that CL did not interfere with lipid extraction. Third, CL could inhibit the reverse activity by acting as a donor of fatty acid. To exclude this possibility, the assay was performed in the presence of [3H]SPH and increasing concentrations of CL. Total lipids were then extracted and applied on TLC, and the Cer band was scraped and counted. There was no formation of Cer under these conditions (not shown), indicating that the enzyme uses only free fatty acid, and that CL was not used as a fatty acid donor in a transacylase reaction. Next, the mechanism of activation and inhibition by CL was investigated. Fig. 9a shows that CL increased the Vmax of the ceramidase reaction. When varying the concentration of SPH, a competitive type of inhibition of the reverse activity was observed, and when varying the concentration of palmitic acid, CL showed a noncompetitive type of inhibition (Fig. 9b). Those results disclose specific and different effects of CL on ceramidase activity and the reverse activity.


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Fig. 8.   Effect of cardiolipin on the enzyme. a, the effect of cardiolipin was tested on ceramidase activity. The assay was performed using [3H]C16-Cer as described under "Experimental Procedures." b, effect of cardiolipin on ceramidase and reverse ceramidase activities. At the end of the incubation, the reaction was stopped by the addition of chloroform/methanol. The mixture was then dried, resuspended in chloroform/methanol, and applied on TLC to separate the substrates from the product. Labeled products were then scraped, and the radioactivity was quantified by liquid scintillation counting.


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Fig. 9.   Mechanism of CL activation and inhibition. The effect of CL on CDase and reverse CDase activities was determined as a function of varying concentrations of the substrates. a, effect of CL on the Vmax of ceramidase activity. The assay was performed using [3H]C16-Cer as described under "Experimental Procedures." b, effect of CL on the reverse reaction. At the end of the assay, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate the substrates from the product [3H]Cer. The spots corresponding to Cers were then scraped, and the radioactivity was quantified using liquid scintillation counting. When studied as a function of SPH concentrations, CL decreased the km value, and when studied as a function of palmitic acid concentrations, CL decreased the Vmax.

Ceramidase and Reverse Ceramidase Activities in the Presence of all Substrates-- Double-labeling experiments were performed to study the direction of the reaction in the presence of all substrates, and this, in the absence or presence of increasing concentrations of CL. Each substrate, [3H]C16-Cer, SPH, and [14C]palmitic acid, was added at its Km value (1.3 mol % for Cer, 0.16 mol % for SPH, and 2.2 mol % for palmitic acid), Km/3 or 3 × Km. As shown in Fig. 10 (a and b), the enzyme catalyzed both activities, ceramidase (monitored by the release of [3H]palmitic acid) and reverse CDase activity (monitored by the formation of [14C]Cer). In addition, both activities followed saturation curves, and their ratio was close to unity (Fig. 10c). In the presence of increasing concentrations of CL, as observed before, CDase activity was stimulated while the reverse activity was inhibited, and this in a dose-dependent manner. In the presence of 8 mol % CL, and at the Km values, the ratio between the two activities was about 10. Thus, the interaction with CL may play a role in the balance of the reaction.


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Fig. 10.   Effect of cardiolipin on the equilibrium of the hydrolysis and synthesis of ceramide. The enzyme was incubated in the presence of SPH, [14C]palmitic acid, as well as [3H]C16-Cer. The substrates were added at three concentrations corresponding to the Km value, Km/3, and 3 × Km, in the presence or absence of increasing concentrations of cardiolipin. The reaction was stopped after 1 h of incubation by the addition of chloroform/methanol. The mixture was dried, and lipids were separated on TLC. The bands corresponding to free palmitic acid and to Cer were scraped, and the radioactivity associated within each band ([3H] + [14C]) was measured by liquid scintillation counting. Results are expressed as specific activities of Cer produced (reverse ceramidase reaction) and palmitic acid released (ceramidase activity). Experiments were performed two times.

Reverse CDase Activity in 293 Cells Overexpressing the Human Homologue-- To investigate whether this reverse activity can work in cells and not only in vitro, we established 293 cells overexpressing the human ceramidase homologue (the specific activity in control cells was 0.15 nmol/h/mg and in overexpressing cells 1.5 nmol/h/mg) and performed labeling studies using [3H]SPH in the presence or absence of 30 µM FB1. Fig. 11 shows in control cells an increase of the formation of [3H]Cer over time, this increase was higher in overexpressing cells, indicating that this reverse activity can enhance ceramide synthesis in cells. Next, we studied the effect of FB1 on [3H]Cer synthesis in control and overexpressing cells. As shown in Fig. 11, in control cells the synthesis of ceramide was severely diminished when cells were preincubated for 2 h with 30 µM FB1 before labeling. This reduction is probably caused by the inhibition of the CoA-dependent ceramide synthase activity. In overexpressing cells, a decrease of [3H]Cer levels was also observed, but [3H]Cer levels remained higher over time than control cells. Therefore, the observed higher levels in overexpressing cells, which are not inhibited by FB1, probably derives from the reverse ceramidase activity.


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Fig. 11.   Synthesis of ceramide in HEK 293 cells overexpressing the human homologue. a, stable 293 cells overexpressing empty vector (ceramidase specific activity in these cells is 0.15 nmol/h/mg) or the human ceramidase (ceramidase specific activity in these cells is 1.5 nmol/h/mg) were treated with 1 µM [3H]SPH for the indicated times in the presence or absence of 30 µM FB1. At the end of the incubation, lipids were extracted and separated on TLC. Equal amounts of lipid phosphorous were loaded on each lane. The results are representative of two separate experiments. Lane 1 represents Cer standard. b, the ceramide bands at 60 min were scraped from the plate and counted. Shown are the FB1 inhibitable activity [(dpm - FB1) - (dpm + FB1)] and the FB1 resistant activity.

To estimate the contribution of this reverse activity, ceramide bands at 60 min were scraped and counted. Results shown in Fig. 11b indicate that in control cells ~40% of total Cer synthesis is resistant to inhibition by FB1. This activity may include, at least as a component, the reverse activity of this ceramidase, which is not inhibited by FB1 and therefore would be included in the FB1-resistant column. Overexpressing CDase resulted in a significant increase (~30%) in FB1-resistant Cer synthesis.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have characterized the reverse activity of a purified rat brain membrane-bound ceramidase. The results indicate that the enzyme would act through a CoA-independent mechanism to function as a ceramide synthase. Substrate specificity studies showed that the enzyme uses the D-erythro-SPH isomer, whereas dihydrosphingosine was a poor substrate. In addition, fumonisin B1 did not affect this reverse activity in vitro and did not inhibit the synthesis of Cer catalyzed by this enzyme in cells. In contrast, FB1 inhibited the CoA-dependent ceramide synthase of rat brain microsomes. These results indicate that this activity is distinct from the CoA-dependent ceramide synthase activity that is present in the ER and which is thought to be responsible for the major de novo synthesis of ceramide (26). Examination of the specificity for the second substrate (fatty acid) showed that the enzyme has close affinity for all fatty acids tested, but the rate of synthesis with myristic acid was the highest.

Biochemical characterization of the enzyme revealed many common characteristics that were also observed for a homologue enzyme from Pseudomonas aeruginosa (27). On the other hand, some differences were also observed. In this study, we showed that the reverse CDase activity was independent of cations, whereas the reverse activity of ceramidase from P. aeruginosa was shown to require calcium (27, 28). This difference in cofactor requirement is not clear at present but could be related to species differences. Also, it is possible that the pathogenic bacterial enzyme functions in the extracellular milieu, which contains approximately 2 mM calcium. The enzyme was inhibited by reducing agents, as we have reported previously for ceramidase activity (15). It is not clear how these agents act on the enzyme.

Kinetic studies showed that the reverse activity followed a random sequential mechanism. The Km value for the ceramidase activity for C16-Cer was determined previously to be 1.3 mol % (Table II). Here we found a Km value of 0.16 mol % for the reverse activity of SPH. Thus, the enzyme showed higher affinity for SPH than for Cer, whereas the Vmax values were in the opposite direction (see Table II). Thus, the kinetic results do not favor any direction of the reaction (in particular, when taking into account that in cells Cer levels are known to be on the order of 10-fold higher than those of SPH). Similar observations were obtained from experiments at equilibrium (Fig. 10c). The availability of free fatty acids could be a limiting step for this reaction, because fatty acids are probably mainly present as fatty acyl-CoAs. On the other hand, our results showed that cells that overexpress the human ceramidase homologue are able to synthesize ceramide at a higher rate than control cells in a FB1-insensitive manner. Thus, it is possible that the enzyme may have access to a pool of free fatty acids even though this pool is in limited amounts.

Saposins are sphingolipid activator proteins that are required for the activities of many sphingolipid enzymes (29), and recently, saposins have been found to be cofactors for acid ceramidase activity (30). An interaction with saposins was not studied here, but this could be a factor that may regulate the direction of the reaction, in particular saposins may turn on the ceramidase activity as it has been shown for acid ceramidase activity.

Another important feature to arise from this study is that the activity of this reverse reaction was optimal in the neutral range, and the enzyme showed very low activity, if any, in the alkaline range in contrast to the ceramidase activity, which had a very broad pH optimum ranging from 7 to 10. These observations raise one possible scenario of regulation of the enzyme activities. For instance, the intracellular pH fluctuations may affect whether the enzyme works in one direction (i.e. ceramidase) or in the reverse direction (i.e. CoA-independent ceramide synthase). For example, an increase in the intracellular pH in cells would result in turning on the alkaline ceramidase activity and turning off the reverse activity, resulting in a decrease in Cer levels. This question becomes more relevant when taking into account the localization of the human homologue to mitochondria, where fluctuations of the pH occur. Furthermore, it is known that acidification and alkalinization occur, respectively, during apoptosis (31) and growth factor stimulation (32). It is tempting to speculate that, under these processes, ceramidase activity is modulated and in different directions.

An interaction with PA and/or CL would be another scenario of regulation arising from this study. Two lines of evidence suggested that the effects of CL on ceramidase activity and on the reverse activity proceed through different mechanisms. First, the effect of CL was only cooperative (with a Hill number of 2, data not shown) on the reverse activity and not on the ceramidase activity (Hill number of 1, data not shown). Second, CL affected the affinity of the enzyme for SPH and not for Cer. Third, although PS, PA, and CL all stimulated the ceramidase activity, only PA and CL inhibited the reverse reaction potently. From our results, at least three possibilities emerge explaining the mechanism of action of CL. First, CL acts through SPH sequestration. However, the observation that anionic lipids other than CL, such as lysoPA and PS, either did not inhibit or inhibited very moderately the reverse activity argues strongly against this possibility. Second, the enzyme may have different sites for Cer and for SPH, and CL acts separately on each site. This possibility would explain why the affinity of the enzyme for SPH is decreased, whereas the affinity for Cer is not (CL increased the Vmax of ceramidase activity), in the presence of CL. On the other hand, the competitive inhibition of SPH toward the ceramidase (15) activity is not in favor of this hypothesis, because it suggests (but does not prove) direct interaction of SPH with the Cer site (which is also inferred from the role of SPH as product of the ceramidase reaction). Third, the enzyme may harbor the same active site for SPH and for Cer, and CL acts on a regulatory domain as an allosteric regulator. In this case, in the presence of CL, the affinity of the enzyme for SPH is decreased, and this would result in a release of SPH from the active site and would drive ceramidase activity (removing the inhibition by SPH). Another observation that supports this hypothesis is that SPH inhibits ceramidase activity very potently, with a Ki value of 0.012 mol % (15). Thus, under these circumstances (and assuming that the enzyme acts on Cer concentration around the Km value (1.3 mol %, Table II), the ceramidase activity will be hindered because of the inhibition by SPH. Thus, in this hypothesis CL would act by removing the inhibition caused by SPH, the product of ceramidase reaction.

The potent effects of CL and PA raise distinct possibilities for these interactions in the regulation of ceramidase and ceramide synthase activity. Cardiolipin is known to be a major lipid of the mitochondrial membrane, and it is thought to be enriched in the inner mitochondrial membrane. At present, it is not clear whether the enzyme is associated with the inner or the outer mitochondrial membrane and whether the interaction with CL in vitro occurs in cells, but the interaction of the enzyme with CL could regulate its activity. According to this scenario, when the enzyme associates with CL, it will function predominantly as a ceramidase and cause a drop in mitochondrial Cer. When the enzyme is excluded from interaction with CL, it may then function as a ceramide synthase driving an increase in the levels of Cer.

PA could be another putative regulator of the enzyme activities. PA regulates the enzyme in vitro in a manner similar to CL. More is known about the regulation of PA metabolism (but not in mitochondria), and PA levels are elevated by the action of either phospholipase D or diacylglycerol kinase. Therefore, it is possible that the action of either of these enzymes could result in enhanced ceramidase activity with a drop in Cer levels. PA has been shown to modulate several enzyme activities, including raf kinase (33), protein phosphatase 1 (34), and cyclic nucleotide phosphodiesterases (25); however, little is known about structural domains required for interaction with PA, except for a putative domain defined in raf kinase (33).

In conclusion, the reverse activity of the enzyme may operate in cells where it may account for fumonisin-insensitive ceramide synthesis. The activity appears to follow a random sequential mechanism and may be affected by the intracellular pH and by an interaction with the anionic phospholipids PA and CL.

    FOOTNOTES

* 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.

Dagger 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.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009331200

    ABBREVIATIONS

The abbreviations used are: Cer, ceramide; CDase, ceramidase; PA, phosphatidic acid; PS, phosphatidylserine; SM, sphingomyelin; SPH, sphingosine; S1P, sphingosine 1-phosphate; CL, cardiolipin; FB1, fumonisin B1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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