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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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.
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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.
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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.
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Effect of Reducing Agents and Nucleotides--
We had shown that
reducing agents dithiothreitol and
-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.
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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.
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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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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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.