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(Received for publication, November 21, 1996, and in revised form, March 25, 1997)
From the Departments of Medicine and Cell Biology, Duke University
Medical Center, Durham, North Carolina 27710
ABSTRACT Sphingomyelin hydrolysis through the activation
of sphingomyelinases has become a potentially important signaling
pathway with the product ceramide implicated in the regulation of cell growth, differentiation, apoptosis, and inflammatory responses. However, little is known about the regulation of sphingomyelinases. In
this study, we show that the magnesium-dependent, neutral
pH-optimum and membrane-associated sphingomyelinase (N-SMase) is
inhibited, in a dose-dependent manner, by glutathione (GSH)
at physiological concentrations with a greater than 95% inhibition
observed at 5 mM GSH. The inhibitory effect of GSH
was reproduced by INTRODUCTION Sphingolipids have emerged as important components of signal
transduction pathways involved in a variety of cellular processes. This
is best illustrated in the case of the sphingomyelin
(SM)1 cycle (1), which has become a
cornerstone in studying the role of sphingomyelin, ceramide, and other
related products in cellular regulation. In this pathway, a number of
extracellular agents such as tumor necrosis factor Although the ceramide generated in response to the action of
extracellular agents appears to derive from the hydrolysis of sphingomyelin, not much is known about the mechanisms regulating the
activity of the involved SMases. To date, at least five types of SMases
have been identified. An acidic sphingomyelinase (A-SMase) was first
discovered and found to have an optimum pH at 5.0 (10). Although the
acidic sphingomyelinase has been primarily found to reside in the
lysosomes, it has also been detected as a soluble form in cytosol and
extracellular media (11). This enzyme has been cloned, and it is
deficient in Neimann-Pick cells (12). It is also suggested to play a
role in radiation-induced apoptosis (13) and has been described to be
activated by Fas (14) and tumor necrosis factor- In ongoing studies on characterizing N-SMase, we discovered that this
enzyme was inhibited in vitro by physiologically relevant concentrations of glutathione (GSH) and it was activated in cells depleted of GSH. Glutathione, a tripeptide
(L- EXPERIMENTAL PROCEDURES Materials
Molt-4 human leukemia cells were obtained from ATCC (Rockville,
MD). RPMI 1640 and fetal calf serum were purchased from Life Technologies, Inc. Glutathione and related compounds were from Sigma.
Leukotrienes were from Cayman Chemicals (Ann Arbor, MI). Stripped rat
brains were from Pel-Freez Biologicals (Rogers, AR). DEAE-Sepharose was
from Pharmacia Biotech Inc.
[N-methyl-14C]Sphingomyelin was
synthesized as described previously (19).
Methods
Molt-4 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum at 37 °C in a humidified
environment with 5% CO2. Cells were seeded at a density of
2.5 × 105/ml and grown to near confluence (1.5 × 106/ml) before use.
Molt-4 cells
(2.5 × 109) were washed three times with
phosphate-buffered saline and then homogenized with a Teflon pestle
glass homogenizer in lysis buffer (25 mM Tris-HCl, pH 7.4, 2 mM EDTA, 5 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml each of chymostatin,
leupeptin, antipain, and pepstatin A). The post-nuclear homogenate was
centrifuged for 1 h at 100,000 × g, and the
pellet was dissolved in 1% Triton X-100 in lysis buffer. The Triton
X-100-soluble fraction was loaded onto a DEAE-Sepharose column (1 × 10 cm) equilibrated in buffer A (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1 mM
phenylmethylsulfonyl fluoride). After washing off the unbound proteins,
the enzymes were eluted by stepping to 1 M NaCl in buffer A, followed by a gradient of Triton X-100 from 0-1% in buffer A. Fractions (4 ml) were collected and assayed for activities of N-SMase
and A-SMase. The recovery of the N-SMase over the DEAE-Sepharose column
was typically greater than 85%. For isolating SMases from rat brain, 4 rat brains were used and the procedure was the same as that for Molt-4
cells as described above. All procedures were performed at 4 °C.
The activity of both N-SMase and A-SMase were
determined using a mixed micelle assay system (19). For determining
N-SMase activity, enzyme preparation (5-10 µl, 0.5-1 µg of
protein) diluted to 50 µl with 20 mM Tris-HCl, pH 7.4, was mixed with 10 nmol of [14C]sphingomyelin (100,000 dpm) dissolved in 0.05% Triton X-100 in 50 µl of 100 mM
Tris-HCl, pH 7.4, containing 5 mM magnesium chloride. The
reaction proceeded for 30 min at 37 °C and was then terminated by
the addition of 1.5 ml of chloroform:methanol (2:1, v/v) followed by
0.2 ml of H2O. After phase separation, a portion of the
upper phase was removed and the radioactivity determined by liquid
scintillation counting. A-SMase activity was determined using
[14C]sphingomyelin resuspended in 50 µl of 100 mM sodium acetate, pH 5.0. The identity of the product in
the upper phase was confirmed by thin layer chromatography as described
(19).
Molt-4
cells were seeded at 2.5 × 105/ml in complete medium,
rested for 24 h, and then treated with or without 250 µM L-buthionine-(SR)-sulfoximine (BSO) for various time intervals. Afterward, cells were pelleted and
total cellular lipids extracted by the method of Bligh and Dyer (49).
Ceramide content was determined using a modified diacylglycerol kinase
assay as described previously (7, 8, 17). SM level was measured as
described previously (8). Briefly, cellular lipids were separated by
TLC using a solvent system of chloroform/methanol/acetic acid/water
(50:30:8:5). The sphingomyelin spots were visualized by iodine
staining, scraped, and measured for phosphate. Sphingomyelin was
quantitated using an external standard and was normalized to total
phosphate.
Intracellular GSH content
was determined by the Griffith (50) modification of Tietze's enzymatic
procedure (51). Briefly, cells (2 × 107) were
resuspended in 400 µl of 10% 5-sulfosalicylic acid. The acid-precipitated proteins were pelleted by centrifugation at 4 °C
for 10 min at 2000 × g. To determine the GSH content,
aliquots of the acid-soluble supernatant were mixed with 125 mM sodium phosphate, pH 7.5, 6.3 mM EDTA, 0.21 mM NADPH, and 0.6 mM
5,5-dithiobis(2-nitrobenzoic acid) in a total volume of 1 ml. Upon
addition of glutathione reductase, the increase in absorption at 412 nm
was monitored and used to determine the amount of GSH in the sample by
comparison to a reference curve generated with known amounts of GSH
standard. Protein content was determined by the Bradford dye binding
assay (52) using bovine serum albumin as standard.
The formation of GSH fragments,
RESULTS As part of the biochemical characterization of N-SMase, we
examined the effect of reducing agents on the in vitro
activity of the enzyme. To this end, we partially purified the N-SMase from the membranes of Molt-4 cells which have very little cellular acidic SMase activity. On DEAE-Sepharose column, three peaks of SMase
activity were resolved (see Fig. 4A). Peak I (flow-through) contained predominantly A-SMase and peak II (salt elution) had a small
quantity of A-SMase. Peak III (detergent elution) had the highest
activity of N-SMase (4 times that of peak II) and no A-SMase activity.
The N-SMase in peak III was typically 50-100-fold purified over
post-nuclear homogenate, and the N-SMase in peak II was 10-20-fold
purified. Therefore, the N-SMase in peak III was used for this study.
The partially purified N-SMase was preincubated with the reducing
agents of interest, and this was followed by incubation with 10 mol%
of radiolabeled sphingomyelin substrate in a Triton X-100 mixed micelle
assay in the presence of 5 mM magnesium. The radiolabeled
product formed after a 30-min incubation with substrate was separated
by organic solvent extraction and its quantity determined by liquid
scintillation counting. The reaction was linear within the range of the
amount of enzyme protein, concentration of substrate, and incubation
time used in this study. As shown in Fig. 1,
preincubation of N-SMase for 15 min at 37 °C with concentrations of
GSH ranging from 1 to 20 mM resulted in an inhibition of
the N-SMase within a very narrow GSH concentration range. Little
inhibition was detected at concentrations of GSH below 2 mM, whereas GSH above 4 mM gave rise typically
to a 90-100% inhibition of N-SMase (Fig. 1A).
Dithiothreitol or
The inhibition by GSH of the N-SMase was enhanced by preincubation of
GSH with the enzyme. When no preincubation time was allowed, GSH at 5 mM inhibited the enzyme activity by 30% (Fig. 1B). When the enzyme was preincubated with GSH for time
periods ranging from 5 to 15 min, the degree of inhibition
progressively increased such that preincubation with 5 mM
GSH for 5, 10, and 15 min resulted in a 70, 90, and 100% inhibition,
respectively (Fig. 1B). Preincubation of the enzyme with GSH
for time periods longer than 15 min did not result in further
inhibition (data not shown). Interestingly, for preincubation time
intervals longer than 5 min, similar degree of inhibition was observed
for GSH concentrations between 3 and 5 mM (Fig.
1B). To rule out effects of preincubation on stability of
the enzyme or GSH, the following experiments were performed. First,
preincubation of N-SMase, in the absence of GSH, for up to 20 min at
37 °C prior to the initiation of activity assay (30 min) did not
affect the enzyme activity (data not shown). Second, thin layer
chromatography analysis of GSH (5 mM) following incubation
with N-SMase preparation for up to 60 min at 37 °C under the
conditions identical to SMase assay did not indicate any metabolism of
GSH to either We next examined the specificity for GSH inhibition of the N-SMase by
testing the effects on the enzyme activity of GSH related compounds. As
shown in Fig. 2, glutamic acid, glutamine, and glycine at concentrations up to 10 mM were totally ineffective in
inhibiting the enzyme (Fig. 2A). Interestingly, cysteine
exhibited a triphasic mode of action for its effect on the N-SMase. An
initial inhibitory phase was observed with concentrations of cysteine
from 0.05 to 1 mM, and this was followed by a stimulatory
phase for concentrations between 1 and 2.5 mM. At
concentrations starting at 2.5 mM and up to 10 mM, cysteine was inhibitory, almost completely coinciding with that of GSH (Fig. 2B).
When the enzyme was preincubated with the GSH fragment
cysteinyl-glycine, a biphasic mode of action was observed. At
concentrations ranging from 0.01 to 1 mM, cysteinyl-glycine
was partially inhibitory of the enzyme, which was followed by reversal
of its inhibition at concentrations from 1 to 10 mM,
closely resembling the initial two phases for cysteine (Fig.
2C). In contrast to cysteine, at concentrations beyond 2 mM, cysteinyl-glycine did not show further inhibition
toward N-SMase. The effect of another fragment of GSH, Since DTT and Because leukotrienes are GSH-modified bioactive lipid molecules
important in cellular regulations, we also examined their effects on
the activity of the N-SMase in vitro. As shown in Fig. 2F, amounts of leukotrienes C4 and
D4 well above physiologically relevant concentrations had
no effect on the activity of the enzyme. Taken together, these results
suggested that the minimal requirement for inhibiting the N-SMase was
Next, we examined the interaction of GSH with the substrate
sphingomyelin and the activator magnesium. To study the interaction of
GSH with sphingomyelin, enzyme was preincubated with 0-2.5 mM GSH followed by incubation with 1-20 mol% of
sphingomyelin. As shown in Fig. 3A,
concentrations of GSH ranging from 1.5 to 2.5 mM gave rise
to similar degrees of inhibition over a broad range of substrate
concentrations. Because of the steep nature of the inhibitory effect of
GSH on the N-SMase, we could not construct reliable double-reciprocal
plots for these studies. However, since increasing concentrations of
substrate did not overcome inhibition by GSH, the mode of inhibition
did not appear to be of the competitive type. Similarly, when the
enzyme was preincubated with 0-2.5 mM GSH followed by
incubation with 10 mol% of sphingomyelin in the presence of 0-10
mM magnesium, a sharp inhibition was observed between 1.5 and 2 mM GSH over a broad range of magnesium
concentrations. Again, the inhibition did not appear to be of a
competitive nature (Fig. 3B).
We next investigated whether the inhibitory effect of GSH was specific
to the N-SMase. To this end, we resolved, on a DEAE-Sepharose column
the A-SMase and N-SMase prepared from detergent-solubilized rat brain
homogenate which contains substantial amounts of both enzymes. As shown
in Fig. 4A, the majority of the A-SMase was found in peak I (flow-through) with a small amount in peak II, while
peaks II and III contained approximately 35% and 65% of the N-SMase
activity, respectively. The N-SMase in peak III was more pure than that
in peak II and was essentially free of A-SMase activity (Fig.
4A). Therefore, we compared the effect of GSH on the
activity of enzyme preparations from peak I (A-SMase) and peak III
(N-SMase). When preincubated with 1-10 mM GSH, N-SMase from rat brain was inhibited by GSH with a complete inhibition at 2 mM GSH, while DTT was totally ineffective (Fig.
4B). In contrast, GSH up to 10 mM had little
effect on the activity of A-SMase from rat brain, while DTT
significantly abolished the activity of the acid SMase (Fig.
4C) as reported previously (24). Similar results for GSH and
DTT were obtained for purified human placenta A-SMase of commercial
source (data not shown).
Finally, we analyzed the effect of the levels of GSH in intact cells on
the hydrolysis of SM and the generation of ceramide. Cultured Molt-4
cells were treated with 250 µM BSO for 12-48 h, and the
total intracellular GSH content was determined. As shown in Fig.
5A, treatment of cells with BSO resulted in a
time-dependent decrease in the intracellular level of GSH.
The GSH level of the BSO-treated Molt-4 cells dropped to 18 and 9% of
that of untreated cells at 24 and 48 h post-treatment,
respectively. When ceramide content in the BSO-treated cells was
examined, an increase in the cellular ceramide level was observed as
early as 12 h post-treatment of cells with BSO (Fig.
5B). The ceramide level at 24 and 48 h after BSO
treatment was significantly higher than that of untreated cells (68%
and 102% over that of corresponding control, respectively). A 32%
decrease in SM content was observed in cells treated with 250 µM BSO for 48 h (Fig. 5C), indicating
that the ceramide generated was due to the activation of a
sphingomyelinase.
DISCUSSION In this study we demonstrate that glutathione at physiologically
relevant concentrations inhibits the neutral
magnesium-dependent sphingomyelinase but not the acidic
sphingomyelinase and that depletion of cellular GSH results in the
hydrolysis of SM and generation of ceramide. The inhibition of the
neutral magnesium-dependent sphingomyelinase does not
appear to be a general characteristic of thiol-containing reducing
agents since dithiothreitol and This structural specificity in inhibiting N-SMase suggests that GSH may
function as a specific allosteric regulator of N-SMase. However, since
partially purified sphingomyelinases were used in this study, we cannot
rule out the possibility of an indirect mechanism of action for GSH
inhibition of the N-SMase. The precise mechanism responsible for GSH
inhibition of the N-SMase remains to be elucidated.
Our results raise a number of physiologic implications. Current
estimation of intracellular concentrations of free GSH in most cell
types suggests a range between 1 and 10 mM (25, 26). In
hepatic cells, the lower limit of intracellular GSH level is believed
to be around 3 mM and the higher limit 20 mM.
This leads us to speculate that under resting and stable conditions,
the N-SMase in the cells exists as an inactive enzyme in the presence of GSH. Depletion of GSH from the cell may then relieve the inhibition by GSH, resulting in activation of N-SMase.
Depletion of GSH has been described for several agents such as
oxidative and alkylating agents, tumor necrosis factor- Since activation of N-SMase leads to the generation of ceramide,
changes in GSH levels may influence ceramide levels through N-SMase. In
turn, this direct biochemical coupling may tie-in the various
regulators and modulators of the redox state of the cell (and of GSH
levels) with ceramide-mediated cellular responses. These include cell
differentiation (5), apoptosis (35), cell cycle arrest (8), Rb
dephosphorylation (36), activation of a protein phosphatase (37) and a
protein kinase (38), inhibition of cellular protein kinase C- As a corollary to this hypothesis, N-SMase in the cells may remain
inactive until GSH is depleted. This would argue against the
possibility that under physiological conditions N-SMase can be
activated without the depletion of GSH. Alternatively, a mechanism may
exist for activating N-SMase by overriding the inhibition by GSH.
While the intracellular GSH levels are between 1 and 20 mM,
the level of free intracellular cysteine is believed to be in the high
micromolar range. In studying the uptake of radiolabeled L-[35S]cysteine by human fibroblasts, Bannai
and Kitamura (42) estimated that the intracellular concentration of
cysteine was approximately 0.1 mM. In another study by Lu
et al. (43), the ratio of total hepatic GSH to cysteine was
found to be around 42 to 1. Combined with the fact that the
intracellular GSH in hepatocytes is 3-20 mM and that
intracellular cysteine transported from outside of the cell is rapidly
metabolized (26), the actual level of intracellular cysteine is
probably very low and in the micromolar range (75-500 µM). Therefore, the inhibition by cysteine of N-SMase
observed in vitro may not be very relevant physiologically,
but cannot be discounted at this point. Interestingly, depletion of
both GSH and cysteine has been reported in several studies
(44-46).
Similarly, the in vitro inhibition of N-SMase by the
oxidized glutathione, GSSG, observed in this study may not have much physiological relevance. GSSG is formed as a result of the reaction of
GSH with reactive oxygen species. Compared with GSH, GSSG exists in
cells at a much lower concentration (20-40 µM) due to
the activity of glutathione reductase, which favors the formation of
GSH (23, 47). In this study, GSSG was found to be approximately 3 times more effective than GSH in inhibiting N-SMase, whereas in cells the
ratio of GSH/GSSG is generally considered to be in the range of
100-500. Therefore, in cells under normal conditions N-SMase is more
likely to be inhibited by GSH, but not GSSG.
In conclusion, inhibition of N-SMase by GSH as shown in this study
serves as a direct link between two important areas involved in the
regulation of mammalian stress responses, the oxidative stress, and the
sphingomyelin/ceramide cycle. A diverse array of agents and factors
initiate the hydrolysis of sphingomyelin and the generation of ceramide
leading to cell cycle arrest and apoptosis. In addition, many of these
same agents and factors cause the depletion of GSH in the course of
inducing apoptosis. Further investigation is required to understand the
relationship between GSH and sphingomyelinases at the cellular
level.
FOOTNOTES ACKNOWLEDGEMENT We thank Dr. Lina M. Obeid for critical review
of this manuscript.
REFERENCES
Volume 272, Number 26,
Issue of June 27, 1997
pp. 16281-16287
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
-glutamyl-cysteine, but not the cysteinyl-glycine
fragment of GSH. The S-modified GSH analogs were as
effective as GSH in inhibiting the N-SMase. On the other hand, neither
dithiothreitol nor 
-mercaptoethanol had any effect on the N-SMase,
suggesting that the sulfhydryl in GSH is not required for inhibition of
N-SMase. GSH had no effect on the acid pH-optimum SMase, whereas
dithiothreitol inhibited the acid SMase. These results suggest that in
cells the N-SMase is inactive in the presence of physiological
concentrations of GSH (1-20 mM). Finally, treatment of
cultured Molt-4 cells with the GSH synthesis inhibitor,
L-buthionine-(SR)-sulfoximine, resulted in a
time-dependent depletion of GSH, accompanied by an
increased hydrolysis of sphingomyelin and production of ceramide. Since GSH depletion is observed in a variety of cells in the process of
cellular injury and apoptosis, these studies suggest that depletion of
GSH may be an important mechanism in activation of N-SMase. This
mechanism may therefore bring together the fields of oxidative stress
and signaling through products of sphingomyelin hydrolysis.
(2),
1,25-dihydrovitamin D3 (1), interleukin 1 (3, 4), nerve
growth factor (5), chemotherapeutic agents (6, 7), and serum
deprivation (8) results in activation of sphingomyelinases (SMases),
which causes the hydrolysis of sphingomyelin, leading to the generation
of ceramide. In turn, ceramide has been suggested to play important roles in differentiation, cell cycle arrest, apoptosis, inflammation, and the regulation of eukaryotic stress responses (9).
(15). A neutral
pH-optimum and magnesium-dependent SMase (N-SMase) has also
been described (16). This enzyme is associated with the plasma
membrane. It is found to be activated in response to tumor necrosis
factor-, Fas, Ara-C, and serum deprivation, and its activation
appears to be closely related to growth suppression and apoptosis (6,
8, 17, 18). Recently, a cytosolic magnesium-independent neutral SMase
was partially purified from HL-60 cells following treatment with
vitamin D3 (19). In addition, a zinc-dependent
acidic SMase was detected in sera from a variety of species (20), and
recent studies suggest that it is derived from the lysosomal A-SMase
(21). Finally, an alkaline pH-optimum SMase has been described in
intestinal cells (22).
-glutamyl-L-cysteinyl-glycine), is the
most abundant nonprotein thiol-containing small molecule in mammalian
cells. It plays a critical role in cellular defense against oxidative
stress by inactivating free radials, reactive oxygen species, and a
variety of cytotoxic electrophiles including alkylating agents. GSH
functions as an antioxidant by directly reacting with free radicals,
conjugating with various agents to form S-substituted
adducts nonenzymatically or through the action of GSH
S-transferase, and serves as a substrate for GSH peroxidase (23). Under normal conditions, cells maintain a high level of intracellular GSH (1-20 mM). In this study, we report on
the effects of GSH on N-SMase. The implications of inhibition of
N-SMase by GSH will be discussed.
-glutamyl-cysteine and cysteinyl-glycine, was examined by incubating
N-SMase preparation with GSH for a given time, and an aliquot of the
mixture was separated on a silica thin layer chromatographic plate
developed with a solvent system of 1-butanol, pyridine, and water
(1:1:1, v/v) as reported by Tate and Meister (48). The compounds were
identified by spraying with ninhydrin and their migration compared with
authentic standards.
-mercaptoethanol at concentrations ranging from 1 to 20 mM were completely ineffective in inhibiting the
N-SMase (Fig. 1A). Taken together, these results demonstrate
that physiological concentrations of GSH are inhibitory to the neutral
magnesium-dependent sphingomyelinase and that this inhibitory property of GSH does not appear to be a general property of
thiol-containing reducing agents.
Fig. 4.
Differential effects of GSH and DTT on
N-SMase and A-SMase. A, DEAE chromatography of rat brain
SMases. Rat brain particulate fraction was solubilized in Triton X-100
and loaded onto a DEAE-Sepharose column (1 × 10 cm). Unbound
proteins were washed off, and the column was first eluted with 1 M NaCl and then with 1% Triton X-100 as described under
"Experimental Procedures." Fractions were collected and assayed for
A-SMase and N-SMase activities. One unit of enzyme refers to the amount
required to hydrolyze 1 nmol of SM at 37 °C in 1 h.
B, effects of GSH and DTT on rat brain N-SMase.
DEAE-purified N-SMase from rat brain (peak III) was preincubated at
37 °C for 15 min with 0-10 mM GSH or DTT. C,
effects of GSH and DTT on rat brain A-SMase. DEAE-purified A-SMase from
rat brain (peak I) was preincubated at 37 °C for 15 min with 0-10
mM GSH or DTT. Results in B and C are
average of duplicate determinations and are representative of two
separate experiments.
[View Larger Version of this Image (15K GIF file)]
Fig. 1.
Inhibition of N-SMase by GSH. A,
effect of reducing agents on N-SMase activity. DEAE-Sepharose-purified
N-SMase (1 µg of protein) from Molt-4 cells was preincubated for 15 min at 37 °C with the indicated concentrations of GSH, DTT, or
-mercaptoethanol (-ME) in 50 µl of 20 mM
Tris-HCl, pH 7.5, followed by incubation for 30 min at 37 °C with 10 mol% [14C]SM in a Triton X-100 (0.05% final
concentration) mixed micelle containing 100 mM Tris-HCl, pH
7.5, and 5 mM MgCl2. The reaction product was
extracted and radioactivity determined as described under
"Experimental Procedures." Data are mean ± S.D. from three separate experiments performed in duplicate. B, time course
for GSH inhibition of N-SMase. DEAE-purified Molt-4 cell N-SMase was preincubated at 37 °C with 0-5 mM GSH for 0-15 min
prior to the initiation of activity assay. Results are mean of
duplicate determinations and are representative of two separate
experiments.
[View Larger Version of this Image (19K GIF file)]
-glutamyl-cysteine or cysteinyl-glycine (data not
shown). To study the reversibility of GSH inhibition of N-SMase, enzyme
was preincubated with 3 mM GSH for 15 min at 37 °C and
then assayed for SMase activity or diluted 5 times to achieve a final
concentration of GSH of 0.6 mM before activity assay.
N-SMase activity (standardized based on equal amount of enzyme) was
completely recovered after diluting out GSH. These results show that
inhibition is reversible, and that the enhancement of inhibition by
preincubation is not a result of metabolism of GSH or an irreversible
change in the enzyme.
Fig. 2.
Effect of GSH and related analogs on N-SMase.
A, effect of GSH-related amino acids on N-SMase.
DEAE-purified N-SMase was preincubated for 15 min at 37 °C with
0-10 mM GSH or the indicated amino acids. B,
comparison of the effect of GSH and cysteine on N-SMase. DEAE-purified
N-SMase was preincubated for 15 min at 37 °C with 0-10
mM GSH or cysteine. C, comparison of the effect of cysteine and GSH fragments on N-SMase. DEAE-purified N-SMase was
preincubated for 15 min at 37 °C with 0-10 mM cysteine,
cysteinyl-glycine (Cys-Gly), or -glutamyl-cysteine
(-Glu-Cys). D, effect of thiol-modified GSH on
N-SMase. DEAE-purified N-SMase was preincubated for 15 min at 37 °C
with 0-10 mM S-methyl GSH or S-ethyl
GSH. E, comparison of the effect of GSH and GSSG on N-SMase.
DEAE-purified N-SMase was preincubated for 15 min at 37 °C with 0-5
mM GSH or GSSG. F, effect of leukotrienes on
N-SMase. DEAE-purified N-SMase was preincubated for 15 min at 37 °C
with 0-100 µM leukotriene C4 (LTC4) or leukotriene D4
(LTD4). Results in A and F are
averages of duplicate determinations and are representative of two
separate experiments. Results in B and C-E are
mean ± S.D. of four and three separate experiments, respectively,
each performed in duplicate.
[View Larger Version of this Image (27K GIF file)]
-glutamyl-cysteine, on the enzyme activity was very similar to that
of GSH except that -glutamyl-cysteine was slightly more potent such
that the range for total inhibition was from 1-3 mM for
this fragment (Fig. 2C; 2-4 mM for GSH). These
results showed that the minimal configuration of GSH required for
inhibition resided in the -glutamyl-cysteine segment of the
molecule, which faithfully reproduced the effects of GSH on
N-SMase.
-mercaptoethanol did not inhibit the enzyme, we next
addressed the requirement for the free sulfhydryl group on GSH. For
these experiments, we used oxidized GSH, GSSG, and the thiol-modified
GSH molecules, S-methyl GSH and S-ethyl GSH. Both
S-methyl GSH and S-ethyl GSH were equally potent
as GSH in inhibiting the enzyme (Fig. 2D). Surprisingly,
oxidized glutathione GSSG was more effective than GSH in inhibiting the
enzyme, with an apparent EC50 of between 0.5 and 1 mM compared with 2.5 mM for GSH (Fig.
2D). The greater potency of GSSG did not seem to arise from
spontaneous reduction of GSSG to two molecules of GSH. First, GSSG was
more than twice as potent as GSH. Second, the inhibition of N-SMase
activity by GSSG was not altered in an oxidizing environment created by
co-incubation with H2O2 (100 µM).
In this case, the EC50 remained at 0.5 mM.
-glutamyl-cysteine and that the free sulfhydryl group on the GSH
molecule was not essential for the inhibitory effect.
Fig. 3.
A, effect of substrate concentration on
GSH inhibition of N-SMase. N-SMase was preincubated at 37 °C for 15 min with 0, 1.5, 2, or 2.5 mM GSH, followed by incubation
with 1-20 mol% substrate (SM) and 5 mM MgCl2.
B, effect of MgCl2 concentration on GSH
inhibition of N-SMase. N-SMase was preincubated at 37 °C for 15 min
with 0, 1.5, 2, or 2.5 mM GSH, followed by incubation with
20 mol% substrate (SM) in the presence of 0-10 mM
MgCl2. Results in A and B are
averages of duplicate determinations and are representative of two
separate experiments.
[View Larger Version of this Image (21K GIF file)]
Fig. 5.
A, effect of BSO on intracellular GSH
levels in Molt-4 cells. Cells were treated with 250 µM
BSO for 12-48 h and their intracellular GSH levels determined as
described under "Experimental Procedures." Results are mean ± S.D. of triplicate determinations from one representative experiment.
Similar results were obtained in two other experiments. B,
effect of BSO on ceramide levels. Molt-4 cells were treated with 250 µM BSO for 12, 24, or 48 h. Total cellular lipids
were extracted and ceramide content determined as described under
"Experimental Procedures." Results are mean ± S.D. of
triplicate determinations from two experiments (n = 6).
C, effect of BSO on SM levels. Molt-4 cells were treated
with 250 µM BSO for 48 h. Total cellular lipids were
extracted and SM content determined as described under "Experimental
Procedures." Results are mean ± S.D. of triplicate
determinations from one representative experiment. Similar results were
obtained in two other experiments.
[View Larger Version of this Image (17K GIF file)]
-mercaptoethanol were ineffective.
Moreover, the free sulfhydryl of GSH is not required since inhibition
was still observed with the thiol-modified GSHs, S-ethyl and
S-methyl GSH, as well as with oxidized glutathione, GSSG.
Studies with analogs and fragments of GSH suggest that the structural
requirements for inhibition reside in the -glutamyl-cysteine moiety
of GSH.
, and Fas in
various cell types (27). These include lung epithelial cells exposed to
high doses of O2 (28), the leukemic cell lines HL-60, U937,
and K562 exposed to the alkylating agent melphalan (29), and U937 cells
treated with hydrogen peroxide and protein synthesis inhibitors (30).
In addition to depletion, GSH levels may be reduced by extrusion from
the cell as has been observed with Fas, which causes a rapid efflux of
GSH from cells. Cells with reduced GSH levels either undergo apoptosis
or become more sensitive to various death-inducing agents (29, 31, 32). For example, Kain and associates reported that depletion of GSH in a
neural cell line committed it to apoptosis (33). Restoring and/or
maintaining the intracellular GSH content through increased synthesis
or addition of exogenous GSH or GSH methyl esters appears to protect
cells from damage/death otherwise inflicted by the cytotoxic agents. In
fact, Chiba et al. reported that a Fas-resistant human T
cell line reverted to Fas sensitivity upon inhibition of its GSH
synthesis capacity (34).
(39),
phospholipase D (40), and cellular senescence (41). Generation of
ceramide has been attributed to the activation of N-SMase in studies
with serum starvation (8), Fas-induced apoptosis (17), and cellular senescence studies (41). The fact that GSH depletion is frequently observed in most of these situations suggests that the levels of
cellular GSH regulates, at least in part, ceramide generation. We show
that, in Molt-4 cells, the GSH synthesis inhibitor BSO not only, as
predicted, depletes the GSH content of these cells, but also causes the
hydrolysis of SM and generation of ceramide. These data demonstrate
that GSH may indeed play a role in regulating SM hydrolysis and
ceramide generation in cells.
Recipient of an individual National Research Service Award.
§
To whom correspondence should be addressed: Box 3355, DUMC, Durham,
NC 27710. Tel.: 919-684-2449; Fax: 919-681-8253.
1
The abbreviations used are: SM, sphingomyelin;
SMase, sphingomyelinase; N-SMase, neutral
magnesium-dependent SMase; A-SMase, acid SMase; DTT,
dithiothreitol; BSO,
L-buthionine-(SR)-sulfoximine.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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