From the
Ceramidase (CDase) catalyzes the hydrolysis of ceramides to
yield sphingosine and fatty acid. In this paper, two forms of
membrane-bound alkaline ceramidase, have been, for the first time,
purified from guinea pig epidermis by chromatography on DEAE-cellulose,
phenyl-Superose, HCA-hyroxyapatite, isoelectric focusing, Mono Q, and
TSK-3000SW column. One species (CDase-I) migrated upon
SDS-polyacrylamide gel electrophoresis as a single band with an
apparent molecular mass of 60 kDa; the other (CDase-II) was only
partially purified with apparent M
Ceramidase (EC 3.5.1.23) is an enzyme which catalyzes the
hydrolysis of ceramide to yield sphingosine and fatty acid and is
widely distributed in animal tissues such as brain, kidney, and spleen
(1-5). However, the biological role of ceramidase in epidermal
sphingolipid metabolism remains unclear because of no success in its
purification to homogeneity. The substrate ceramide constitutes the
core structure of several sphingolipids such as sphingomyelin,
gangliosides, and sulfatides which are reported to play essential roles
in cell proliferation and
differentiation
(6, 7, 8) . Ceramide serves a
central role in sphingolipid metabolism and is involved as a modulator
in cell growth and vitamin D
The cholate extract
was directly applied to a DEAE-cellulose column (Fig. 1). At this
stage, the elution profile exhibited two peaks of CDase activities. One
appeared as the initial pass-through fraction (Fraction I), while the
other activity lied between 0 and 0.2 M NaCl gradient
(Fraction II). More than 85% of the total CDase activity was recovered
in these two fractions. Fraction I contained about 75% of the total
activity. These two highly active fraction were designated as CDase-I
and -II, respectively. This DEAE-cellulose column chromatography was
efficient in separating and purifying these CDase species,
demonstrating 141- and 34-fold increase in the specific activity for
CDase-I and -II, respectively (). Each active fraction was
subsequently eluted through a phenyl-Superose column after
supplementing with NaCl to 3 M (data not shown). The CDase was
bound to the hydrophobic column and eluted by decreasing NaCl gradient
from 2 to 0 M, demonstrating 97-fold increase in the specific
activity, whereas CDase-II was not bound to this hydrophobic column
without any resulting increase in the specific activity ().
This led us to omit this procedure for the purification of CDase-II.
Sphingolipid metabolism is a key cellular event involved in
the regulation of cell proliferation and differentiation in many
tissues (6-8). Ceramide is involved as a structural and
functional component in the sphingolipid metabolism. As evidence is
accumulating that ceramide plays an important role as an intracellular
effector molecule (32, 33), enzymes that regulate metabolism of
ceramide stand as potential regulators of ceramide levels and
consequently ceramide-mediated function. Among them, the process of
ceramide breakdown mediated by CDase seems important in controlling the
cellular level of a series of sphingolipid metabolites. Recent evidence
suggests that cellular levels of ceramides are deeply associated with
stimulation of the phosphorylation of several kinases, the activity of
protein kinase, the levels of the c-myc protooncogene, the
activity of phospholipase A
Our present studies are the first to describe the
extensive purification of an enzyme utilizing ceramide in mammalian
tissues. We demonstrated that at least two CDase species with different
molecular masses and enzymatic properties are present in guinea pig
skin. For the first time, of the two, one designated herein as CDase-I,
was purified to apparent homogeneity in mammalian tissues
(Fig. 4B). CDase activity was first described by
Gatt
(1) , who characterized and partially purified the enzyme
from rat brain. Although there are a few reports of the partial
purification of membrane-bound CDase isozymes from human
spleen
(5, 39, 40) , the enzymatic and regulatory
properties of CDase remain largely unknown. Al et al. have
reported an acid CDase activity partially isolated from human spleen,
which has an molecular mass of about 100 kDa. On the other hand,
Nilsson
(3) described a neutral CDase in human small intestine.
Sugita et al. (4) reported an alkaline CDase activity in the
human cerebellum. However, the molecular mass of these CDases was
unknown, and then biochemical properties of these CDase proteins did
not coincide with those of epidermal isozymes. Therefore, the two CDase
species purified from guinea pig epidermis in the present study appear
to be unique for enzymological and biological aspects of ceramide
metabolism.
In epidermal tissue, Wertz and Downing
(27) reported only the existence of enzymatic ceramide
hydrolytic activity in porcine epidermis, but the examination of
enzymatic properties was not sufficient to clarify the biochemical
properties because the enzymatic characterization, including the pH
profile or the K
Our study revealed
that major CDases present in the guinea pig epidermis are alkaline
CDases with the optimal pH of 7.0-9.0, while this is the case for
the stratum corneum, the upper layers of the skin. On the other hand,
the surface pH of mammalian skin is maintained in acidic side with
human skin being at pH 4.2-5.6
(45) and that of guinea pig
skin shows pH 5.0-6.5. It is, therefore, likely that the
epidermal alkaline CDase present in the stratum corneum does not
function effectively in normal physiological conditions. However, under
some barrier-disturbed conditions where living layer (having alkaline
side of pH) can get in contact with the stratum corneum, CDase may
perform the function to degrade ceramides. On the other hand, Anderson
(46) has reported that the surface pH of some dermatitis, for example
atopic dermatitis or ichthyosis, is observed on alkaline
side
(46) . Although the alteration of surface pH may relate with
the depression of barrier function or the abnormality of lipid
metabolism in these disorders, they would provide a physiological basis
for activating CDase, leading to the deficiency in the mass of
ceramides and the abnormalities in keratinization process due to the
loss of balance between ceramides and sphingosine.
In conclusion,
our present study elucidated the biochemical properties of the
epidermal CDases which regulate the quantity of sphingolipid
metabolites involved as regulatory molecules in cell growth and
differentiation during epidermal keratinization process. The regulatory
features of epidermal CDases through the enzyme-associated sphingolipid
metabolites are suggestive of the notion that sphingolipid metabolism
undergoes the strictly controlled regulations in epidermal tissue
through the product feedback and precursor suppression mechanisms.
Based upon the presence of CDase species in epidermal tissue, it would
be of importance to define the participation of each CDase species in
the regulation of cell growth or keratinization in the epidermis, and
further characterization of each is required for clarifying their roles
in regulating sphingolipid metabolism.
The ceramidase activities at each step were determined
using [1-
The CDase activities were compared at pH 4.5 and 9.0 among
palmitoylsphingosine, linoleoylsphingosine, and oleoylsphingosine as
substrates. The activity was expressed as disintegrations/min (dpm) of
each released radioactive free fatty acid by subtracting background
disintegrations/min (dpm) (approximately 10,000 disintegrations/min
(dpm)). The percent value represents relative activity for each
substrate when the hydrolysis value for palmitoylsphingosine is taken
as 100%. Results are expressed as means of two separate experiments.
of about
148,000 estimated by gel filtration. The specific activities of the two
species increased by 1.130- (for CDase-I) and 400-fold (for CDase-II)
over the original tissue extract. The activity of both enzymes for
ceramide species decreased in the order of linoleoyl > oleoyl >
palmitoylsphingosine. The optimal pH for enzyme activity was
approximately 7.0-9.0 for CDase-I and 7.5-8.5 for CDase-II.
Interestingly, both enzymes were inhibited by the reaction product
sphingosine with a concentration for half-maximal inhibition
(ID
) of 100-130 µM, compared to the
apparent kinetic parameters with CDase-I (K
= 90 µM, V
=
0.62 unit) and CDase-II (K
= 140
µM, V
= 0.50 units). Some
lipids, such as phosphatidylcholine and sphingomyelin, are also
inhibitory with IC
values of 50-250 µM,
suggesting well controlled CDase activity by sphingolipid metabolites.
These studies begin to elucidate a regulatory mechanism for the balance
of the ratio of ceramide/sphingosine which can serve as an
intracellular effector molecule in epidermis.
-induced
differentiation
(9, 10) . Ceramide is recently implicated
as an inducer of programmed cell death
(11) . In epidermal
tissue, ceramide is an important determinant for the permeability
barrier and water reservoir of the upper most layers of the
epidermis
(12, 13, 14, 15) , the stratum
corneum which is directly exposed to outer environmental circumstances.
In addition to a physiological role, ceramide is documented as a
stimulator for the phosphorylation of epidermal growth factor receptor
in epidermoid carcinoma cells
(16) . On the other hand, the
reaction product sphingosine is an endogenous inhibitor of protein
kinase C-mediated biochemical reaction in several cells or
tissues
(17, 18, 19) . Due to the importance of
protein kinase C action in a wide range of cellular events, sphingosine
has been implicated as an essential biological mediator in cellular
responses to extracellular signals as well as in sustained biological
phenomena such as tumorigenesis, cell growth, and
differentiation
(20, 21, 22, 23) . In
mammalian epidermis, where ceramide is a major end product of epidermal
differentiation, namely the keratinization process, the balance of
sphingosine and ceramide may serve a control mechanism for the
regulation of epidermal growth and differentiation through the varied
functions of ceramidase
(24, 25, 26) . Despite
the involvement of ceramidase in the essential hydrolysis of
sphingolipids as well as in the control process of epidermal
keratinization, little is known about its biochemical properties
because of the lack of purification studies. Although Wertz and Downing
(27) have recently demonstrated ceramide hydrolytic activity in
fractions derived from pig epidermis, the control mechanism of the
hydrolysis has remained unclear. Thus, detailed enzymological studies
are required for characterization of the physiological function of the
enzyme. Based upon the importance of the ceramidases in understanding
the regulatory mechanism of sphingolipid metabolism in the epidermis,
in this study we describe the purification of two ceramidase species
from guinea pig epidermis and their biochemical properties.
Materials
DEAE-cellulose was purchased from Whatman BioSystems Ltd.
Phenyl-Superose (HR 5/5) and Mono Q (HR 5/5 and HR 10/10) columns were
purchased from Pharmacia LKB Biotechnology Inc. TSK-3000SW was obtained
from Toyo Soda Industries, LTD. and
[1-C]palmitic acid was purchased from Amersham
International plc (Bucks, United Kingdom). Sphingosine and
phospholipids were obtained from Funakoshi Chemical Company (Tokyo). Preparation of
[1-
C]Palmitoylsphingosine-Thionyl chloride
was refluxed with [1-
C]palmitic acid in
petroleum to produce [1-
C]palmitoylsphingosine.
The palmitoyl chloride was condensed with sphingosine benzoate
according to the method of Shapiro and Flowers
(28) . The
condensation product was subjected to mild alkaline hydrolysis with
0.15 ml of sodium acetate (50%) and 120 µl of tetrahydrofuran for
90 min at 25 °C. The reaction was terminated with 2 ml of
chloroform/methanol (2:1) and 0.4 ml of water. The mixtures were vortex
mixed for 1 min and centrifuged for 5 min at 2,000
g.
The upper phase was aspirated, and the lower organic phase was washed
once with 1.3 ml of methanol:water (1:1), then evaporated under
nitrogen gas. The crude product was isolated by preparative silicic
acid column chromatography with a mobile phase of chloroform/methanol
(9:1). The extract was dried under nitrogen gas, then 0.5 ml of 0.5
N methanol-saturated NaOH was added, and the mixture was
incubated at 37 °C for 2 h. The reaction was terminated by the
addition of 1 ml of chloroform, followed by 0.3 ml of water, and
centrifugation for 5 min at 2,000
g. The lower phase
was washed once with 0.75 ml of methanol/water (1:1). The upper phase
was aspirated and the lower phase was evaporated under nitrogen gas.
The crude ceramide was applied on a thin layer chromatography Silica
Gel G plate and developed with chloroform/methanol/acetic acid
(94:1:5). The R
value of
[1-
C]palmitoylsphingosine showed 0.25 in this
condition, on the other hand, unreacted
[1-
C]palmitic acid was R
= 0.95 and sphingosine was located in the origin.
Furthermore, the preparations of
[1-
C]linoleoylsphingosine and
[1-
C]oleoylsphingosine were performed under
almost same condition of
[1-
C]palmitoylsphingosine. The radiochemical
purity of the synthetic ceramides checked with a radioscanner was at
least 97%, respectively.
Preparation of Membrane Fraction
Epidermal sheets were
peeled from the guinea pig skin (Wister species, male) at 4-5
weeks of age after incubation with 1000 units/ml dispase at 4 °C
overnight. Briefly, after washing with phosphate-buffered saline,
epidermal sheet scissored off chips was resuspended in a 5-fold volume
of 60 mM phosphate buffer, pH 7.4, containing 0.25 M
sucrose, 1 mM EDTA, 1 mM EGTA, and 0.5 mM
phenylmethylsulfonyl fluoride (buffer A), then homogenized on ice for a
total of 3 min with 30-s bursts of a Polytron (Kinematica AG,
Littau/Luzern, Switzerland). After removing unbroken epidermal tissues
by centrifugation at 800 g for 10 min, the supernatant
fraction was centrifuged at 10,000
g for 30 min. The
pellet was resuspended in buffer A and stored frozen at -135
°C until a sufficient amount was obtained for purification. The
collected pellets (about 750 mg/65 ml of protein) were thawed,
resuspended in a 3-fold volume of buffer A, and extracted with an equal
volume of buffer A containing 1.0% sodium cholate. The effect of sodium
cholate on extraction and stability of ceramidase activity from the
particle is mostly not affected (see ``Results''). After 2 h
at 4 °C, the suspension was sedimented by centrifugation at 18,000
g for 30 min to obtain a cholate extract for
subsequent purification.
Purification of Membrane-bound Ceramidase
All
procedures were carried out at 4 °C unless otherwise indicated.
DEAE-cellulose Column Chromatography
The cholate
extract was applied to a DEAE-cellulose column (3.2 18 cm)
equilibrated with 60 mM phosphate buffer, pH 7.4, containing 1
mM EDTA, 1 mM EGTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5% sodium cholate (buffer B). The
column was washed with 600 ml of the same buffer, then eluted with a
linear concentration gradient of NaCl from 0 to 1.0 M in 700
ml of buffer B. The flow-through (Fraction I) and eluted fractions
(Fraction II) were collected in 8-ml portions. The active fractions
were pooled and concentrated to about 40 ml using a hollow fiber (Mini
Modules HC, Asahi Kasei Co.; exclusion limit: 13,000 daltons). The two
ceramidase (CDase)
(
)
activities of flow-through
(Fraction I) and eluted fractions (Fraction II) were designated as
CDase-I and CDase-II.
Phenyl-Superose Column Chromatography
Fraction I
from the DEAE-cellulose column was supplemented with NaCl to 3
M and applied to a phenyl-Superose packed column (HR 10/10;
1.0 10 cm, Pharmacia) equilibrated with buffer B containing 2.0
M NaCl. The proteins were eluted at a flow rate of 3.0 ml/min
with successively decreasing NaCl gradients from 2.0 M to 0
M for 30 min (using total volume of 90 ml) under a high
resolution liquid chromatography Bio-Dimension System (Bio-Rad).
Fractions of 0.5 ml were collected. The active fractions were pooled
and concentrated to about 3.0 ml using a hollow fiber. The concentrated
solutions were dialyzed twice for at least 20 h against 2 liters of 50
mM Tris-HCl buffer, pH 7.4 (25 °C), containing 0.5
mM phenylmethylsulfonyl fluoride and 0.5% sodium cholate
(buffer C).
HCA®-Hydroxyapatite Column
Chromatography
Aliquots (about 4 ml) of CDase-I and -II
obtained from the phenyl-Superose and the DEAE-cellulose columns,
respectively, were applied to HCA-hydroxyapatite packed columns (P-4001
+ A-7610, 4 10 mm, Koken Co., LTD, Japan) equilibrated
with buffer C. The proteins were eluted at a flow rate of 1.0 ml/min
with successively increasing phosphate buffer, pH 7.4, gradients from 0
to 125 mM for 30 min, from 125 to 250 mM for 5 min
(using total volum of 40 ml), then 250 mM for 5 min using the
HRLC Bio-Dimension System. Fractions of 0.5 ml were collected. CDase-I
and -II were eluted between 70-90 and 200-250 mM,
respectively.
Isoelectric Focusing
The solutions obtained from
the HCA-hydroxyapatite column were dialyzed for at least 20 h against
10 mM phosphate buffer, pH 7.4, containing 0.5% CHAPS,
respectively. Three milliliters of dialyzed solutions were diluted with
52 ml of 1% Bio-Lyte ampholytes, pH 3.0-10.0. All solutions were
separately applied to a preparative isoelectric focusing (IEF) chamber
(Rotofor Cell, Bio-Rad). The power supply was set to 12 watts constant
power and the run proceeded for 4.5 h at 4 °C. The focusing chamber
is divided into 20 discrete compartments, each of which was collected
simultaneously into 20 tubes at the end of the run. The pH of all
fractions was measured and neutralized for measuring the CDase
activities. The active fractions were pooled and were dialyzed twice
for at least 20 h against 2 liters of buffer B containing 200
mM NaCl. The dialyzed enzyme solutions were concentrated to
about 0.4 ml in a Centricon 10 (Amicon, Millipore Corporation).
TSK-3000SW Column Chromatography
Aliquots (about
0.4 ml) of CDase-I and -II obtained from IEF were applied to TSK-3000SW
columns (0.6 60 cm) equilibrated with buffer B containing 200
mM NaCl. The enzymes were eluted with the same buffer at a
flow rate 1.0 ml/min (using total volum of 50 ml), and 0.2-ml fractions
were collected. The active fractions were pooled and concentrated to
about 0.15 ml using a Centricon 10 (Amicon) and stored at -135
°C.
Assay of CDase Activity
CDase activity was assayed
by measuring the amount of radioactive palmitic acid from
[1-C[palmitoylsphingosine as described
previously
(1, 27) . The standard reaction mixture (final
volume, 200 µl) contained 125 mM Tris-HCl buffer, pH 9.0
(at 37 °C), 0.75 µCi of
[1-
C[palmitoylsphingosine, 100 µg of Tween
20, 250 µg of Triton X-100, and enzyme solution. To examine the
effect of pH on purified CDase activity, enzyme solutions were dialyzed
twice for at least 20 h against 2 liters of 125 mM acetate (pH
3.0-6.0), phosphate (pH 6.0-8.0), or borate buffers (pH
9.0-11.0), and 0.5% Triton X-100. On the other hand, to examine
the effects of various lipids or cations on purified CDase activity,
each agent was added to the standard reaction mixture. The reaction
mixture was incubated for 60 min at 37 °C and terminated by the
addition of 50 µl of carrier palmitic acid, followed by 3.0 ml of
Dole's reagent (2-propanol/heptane, 1 N NaOH =
40:10:1)
(29) . Heptane, 1.8 ml, and 1.6 ml of water were then
added. The mixtures were vortex mixed for 1 min and centrifuged for 5
min at 2,000
g. The upper phase was carefully
aspirated, and the under-phase was washed twice with 2 ml of heptane.
Thereafter, 1 ml of 1 N H
SO
and 2.4 ml
of heptane was added, and the mixture was vortex-mixed for 1 min, then
centrifuged for 10 min at 2,000
g. A 1-ml portion of
the upper phase was transferred to a vial and mixed with scintillation
fluid. The radioactivity was determined in a liquid scintillation
counter. When required, the upper phase was evaporated under nitrogen
gas, then residual lipids were dissolved in 20 µl of
chloroform/methanol (6:1) and analyzed by thin layer chromatography as
described previously
(30) . One unit of CDase activity was
defined as the amount of enzyme which produced 1 nmol of palmitic
acid/min under the described conditions.
Other Methods
Analytical SDS-polyacrylamide gel
electrophoresis (PAGE) was performed by the method of Laemmli
(31) in a linear polyacrylamide gradient of 8-16%. The
protein concentration was determined using a Bio-Rad protein assay kits
with bovine serum albumin as the standard.
Purification of Two Ceramidase Activities
Prior
to purification experiments, the subcellular distribution of CDase
within epidermal cells was examined. About 65% of the total CDase
activity in the guinea pig epidermis homogenate was found in the 10,000
g particulate fraction, and the specific activity was
the highest in this fraction. Therefore, we purified the CDase from the
particulate fraction of guinea pig epidermis. In other studies, Triton
X-100 has been shown to enhance the activity of CDase obtained from
human spleen
(5) , and sodium cholate increased that derived from
the rat brain
(1) . Therefore, we determined the effect of
detergents on extraction and stability of CDase from the particulate
fraction by using sodium cholate, Triton X-100, Brij 35, Nonidet P-40,
and CHAPS. Sodium cholate, Triton X-100, and Nonidet P-40 at a
concentration of 0.5% markedly enhanced the extraction and stimulated,
though to a different extent, the palmitoylsphingosine hydrolyzing
activity. Sodium cholate enhanced the CDase activity about 4-fold over
original 10,000
g particulate fraction. In contrast,
Brij 35 and CHAPS were not effective at the same concentration. During
solubilization of the particulate fraction by detergents, about
40-60% of CDase activity was lost in the presence of 0.5% Triton
X-100 at 4 °C for 48 h, whereas 90-95% of CDase activity
retained in the presence of 0.5% sodium cholate (data not shown). Based
on this experiment, we used 0.5% sodium cholate upon the extraction and
purification of CDase. In the extraction step for 2 h with 0.5% sodium
cholate, about 90% of the activity was solubilized with about 50% of
the total protein in the particulate fraction.
Figure 1:
Elution profiles on
DEAE-cellulose column of CDase-I and -II. The cholate extract was
applied to a DEAE-cellulose column, and the column was washed and
eluted as described under ``Experimental Procedures.'' CDase
activities were measured in the presence of 0.5% sodium cholate.
CDase-I and -II activities, indicated by a bar, were pooled.
-, absorbance at 280 nm; ,
, CDase
activities.
These CDase isozymes were separately purified by successive
chromatography upon HCA-hydroxyapatite, Mono Q, IEF, and TSK-3000SW
under the HRLC Bio-Dimension System. Upon HCA-hydroxyapatite, the
activity of CDase-I was detected in the fractions through 80 to 100
mM phosphate buffer, pH 7.4, while CDase-II was eluted at 250
mM of the same phosphate buffer (Fig. 2). The elution
pattern for the two enzyme species was the same for both pH 4.5 and 9.0
at which the activity was assayed, suggesting that both represent
alkaline ceramidases. This chromatographic process provided 447- and
17-fold purification index for CDase-I and -II, respectively
(). On IEF, the pI value of CDase-I and -II was 6.8 and
7.2, respectively (Fig. 3). Both enzyme species were not
inhibited by ampholytes that were used in the IEF. The specific
activity following this chromatographic process showed 820- and
162-fold increases for CDase-I and -II as compared to the original
fraction (). During the above successive chromatographic
process, a split or a reduction in the molecular weight of CDases was
not detected. Activity of CDase-II was relatively unstable with lower
recovery, whereas CDase-I was stable during repeated chromatography.
The extent of purification from the original membrane estimated from
the final specific activity following TSK-3000SW gel chromatography was
1,130-fold for CDase-I and 400-fold for CDase-II (). The
apparent molecular weight of these two CDase species as estimated on
the final gel chromatography was 150,000 (CDase-I) and 62,000
(CDase-II) (Fig. 4A). These fractions were separated on
SDS-PAGE, demonstrating that CDase-I migrated as a single band
(Fig. 4B). On the other hand, CDase-II still migrated as
five bands on SDS-PAGE (data not shown), indicating that it was
partially purified. The results of the purification of CDase species
are summarized in .
Figure 2:
Elution profiles on HCA-hydroxyapatite
columns of CDase-I and -II. The dialysate from phenyl-Superose
(CDase-I) or DEAE-cellulose (CDase-II) were
separately applied to HCA-hydroxyapatite columns and eluted as
described under ``Experimental Procedures.'' Samples were as
follows: A, the concentrated solutions which were eluted
fraction from phenyl-Superose, followed by dialysis against buffer C.
B, the NaCl-eluted fraction (Fraction II) of DEAE-cellulose.
-, absorbance at 280 nm; ,
, CDase
activities.
Figure 3:
Isoelectric focusing of CDase species. The
CDase species were isoelectrically focused with a power supply set to
12 watts constant power and run for 4.5 h at 4 °C. The 20
compartments were collected simultaneously in 20 tubes at the end of
the run, neutralized, and assayed as described under
``Experimental Procedures.'' , pH;
, CDase
activities.
Figure 4:
Molecular mass determination of the two
CDase species at the final step of gel filtration and
SDS-polyacrylamide gel electrophoresis of CDase-I. The apparent
molecular masses of both CDase species were determined at the final
step of purification by gel filtration through TSK-3000SW. The protein
standards were 440,000 (ferritin), 232,000 (catalase), 158,000
(aldolase), 67,000 (bovin serum albumin), and 45,000 (ovalbumin).
Protein bands were visualized by means of Coomassie Blue staining.
Details are described under ``Experimental Procedures.''
CDase-I was obtained by chromatography on a TSK-3000SW column (7 µg
of protein). Molecular mass markers; 200,000, myosin; 93,000,
phosphorylase b; 66,000, bovine serum albumin; 45,000,
ovalbumin; 30,000 carbonic anhydrase; 20,000, soybean trypsin
inhibitor.
Substrate Specificity of CDase Species
The
CDase activities were compared at pH 4.5 and 9.0 among
palmitoylsphingosine, linoleoylsphingosine, and oleoylsphingosine as
the substrate in the presence of 0.5% sodium cholate. The activity of
both enzymes for ceramide species was in the order of linoleoyl
oleoyl
palmitoylsphingosines at pH 4.5 or 9.0 ().
Effects of Phospholipids
The CDase activities were
markedly suppressed by either phosphatidylcholine (PC) (porcine brain
or egg origin) or sphingomyelin (SM) (porcine brain or egg origin) in
the presence of 0.5% sodium cholate at pH 7.4 (Fig. 5). When the
final purified enzymes were dialyzed for 24 h against 50 mM
Tris-HCl buffer, pH 7.4, containing 0.5% CHAPS, the similar inhibitory
effect of SM or PC on these isozyme activities remained (data not
shown). Half-maximal inhibition (IC) by PC (egg yolk) was
about 50 µM (CDase-I; Fig. 5A) and 125
µM (CDase-II; Fig. 5C). Furthermore, the
IC
for SM (egg yolk) was about 350 µM
(CDase-I; Fig. 5B) and 700 µM (CDase-II;
Fig. 5D). The activities of CDase-I and -II were reduced
by only 5-15% in the presence of 1 mM PE, PI, or PG
(Fig. 5, A and C)). Thus, phosphatidylglycerol
(PG) (brain), phosphatidylinositol (PI) (soybean), or
phophosphatidylethanolamine (PE) (egg) were not effective
(Fig. 5, A and C). Neither caldiolipin (brain)
nor 1,2-diacylglycerol (brain) had any significant inhibitory effect.
(Fig. 5, B and D).
Figure 5:
Effect of lipids on CDase activities. The
purified species (each approximately 0.09 µg of protein) were
assayed in the presence or absence of lipids, which were sonicated
separately and added to the reaction mixture before the assay as
described under ``Experimental Procedures.'' The control
values are expressed as 100% for specific activity of 1.20 nmol/min
(CDase-I) and 0.97 nmol/min (CDase-II). Results are
expressed as means of three separate experiments. PI,
; PE,
; PG,
; PS,
;
PA,
; PC-b,
; PC-e,
;
diacylglycerol (DG),
; caldiolipin (CAL),
▾; GC,
; BRAIN EXT, +;
dimyristorylphosphatidylcholine (DMPC),
;
SM-b,
; SM-e,
or
.
Effects of Lysophospholipids
Fig. 6
shows
the effect of five lysophospholipids on the activities of both
isozymes. Lysophosphatidic acid (lysoPA) was inhibitory to
CDase-I but not CDase-II. Lysophosphatidylethanolamine
(lysoPE), lysophosphtidylcholines (lysoPC), or
lysophosphatidylserine (lysoPS) did not affect the enzyme
activity.
Figure 6:
Effects of lysophospholipids on CDase
activities. The CDase species (approximately 0.10 µg of protein
each) were assayed in the presence or absence of lysolipids, which were
sonicated separately and added to the reaction mixture before the
assay. The control values are expressed as 100% for specific activity
of 1.32 nmol/min (CDase-I) and 1.08 nmol/min
(CDase-II). The reaction proceeded as described under
``Experimental Procedures.'' Results are expressed as means
of three separate experiments. LysoPC-e, ; lysoPC-b,
;
lysoPE,
; lysoPS,
; lysoPA,
.
Effects of Sphingosine
The effects of sphingosine
upon the CDases were examined (Fig. 7). The addition of
0.05-1 mM of sphingosine (bovine brain) significantly
suppressed the catalytic activities of both CDase-I and -II. The two
species had almost the same sensitivity to sphingosine with an
IC of about 100 µM (CDase-I) and 130
µM (CDase-II), respectively. The mode of product
inhibition by sphingosine in palmitoylsphingosine hydrolysis by these
two CDase species was competitive (Fig. 8, A and
B). When palmitoylsphingosine was used as the substrate, the
apparent Kinetic inhibitory parameters
(K
) determined were about 260
µM (CDase-I; K
= 90
µM, V
= 0.62 unit) and 225
µM (CDase-II; K
= 140
µM, V
= 0.50 unit).
Figure 7:
Effects of sphingosine on CDase
activities. The CDase species were assayed in the presence of varying
concentrations of sphingosine (bovine brain), which was sonicated
separately and added to the reaction mixture before the assay. The
reaction proceeded as described under ``Experimental
Procedures.'' , CDase-I activity.
, CDase-II
activity. Results are expressed as means from three separate
experiments.
Figure 8:
Effects of sphingosine on CDase activities
according to double-reciprocal Lineweaver-Burk plot analysis. The
ceramide hydrolysis activities of CDase species were measured in the
presence () or absence (
) of varying concentrations of
sphingosine (bovine brain), which was sonicated separately and added to
the reaction mixture before the assay. The reaction proceeded as
described under ``Experimental Procedures.'' A,
Lineweaver-Burk plots of CDase-I. B, Lineweaver-Burk plots of
CDase-II. Results are expressed as means of three separate
experiments.
Effects of pH
When palmitoylsphingosine was used
as the substrate, the highest activities were observed at pH
7.0-10.0 and 8.0-9.0, respectively, in the presence of 0.5%
sodium cholate (Fig. 9). Even when the each finally purified
enzyme solution was dialyzed for 24 h against 50 mM Tris-HCl
buffer, pH 7.4, containing 0.5% CHAPS, the pH optima of each isozyme
was not affected (data not shown).
Figure 9:
Effects of pH on CDase activities. Assays
contained 125 mM acetate (, pH 3.0-6.0), or
phosphate (
, pH 6.0-8.0), Tris-HCl (
, pH
8.0-10.0), or borate buffers (
, pH 9.0-11.0). The
reaction proceeded as described under ``Experimental
Procedures.'' Results are expressed as means of two separate
experiments.
, and prostagalandin release
(17-19), resulting in a significant modification of several
cellular functions. Thus, ceramide analogs exert specific and potent
antiproliferative effects in HL-60 cells at concentrations as low as
1-10 µM, mimicking the action of tumor necrosis
factor
, interleukin-1
, 25-dihydroxyvitamin D
,
and
-interferon on HL-60 cells
(32) . They are also active
against other leukemia cells, malignant cells in tissue culture, and
normal fibroblasts in logarithmic phase of growth (for review, see Ref.
34). In myeloid, lymphoid cells, and fibroblasts, ceramide analogs
caused early, potent, and specific internucleosomal DNA fragmentation,
a hallmark of apoptosis
(35) , suggesting that ceramide mediates
the effects of tumor necrosis factor
on programmed cell death and
participate in other events of apoptosis, as evidenced by the fact that
ceramide levels are significantly increased in HIV-infected T
lymphocytes undergoing apoptosis. Other studies raise the possibility
that ceramide may function as a regulator of protein trafficking in
which C
-ceramide inhibits secretion of vesicular stomatitis
virus glycoprotein from infected Chinese hamster ovary cells (36).
Studies in human fibroblasts suggest that ceramide may play a role in
modulation of immune function and inflammatory response by modulating
secretion of prostaglandin E
in response to the action of
interleukin-1
(37) . On the other hand, the hydrolysis product,
sphingosine is also known to affect a wide variety of biological
activities
(38) . Sphingosine may exert many of its biological
effects through its ability to inhibit protein kinase C activity. In
addition, a role for sphingosine in PI turnover has been suggested in
which it stimulates PI hydrolysis by stimulating phospholipase C
activity and may represent a pathway by which it may exert some of its
many effects upon cell function. Based upon evidence that sphingosine
and ceramide play an important role in such fundamental biological
processes as cell proliferation, differentiation, receptor function,
and oncogenesis, the biological analysis and the enzymatic regulation
mechanisms of ceramide hydrolysis and sphingosine producing enzyme,
CDases should provide insight into the role of sphingolipid metabolites
as the ultimate endogenous control molecules for maintaining tissue
homeostasis.
for ceramide, revealed
the possible additive or mixed effects of two or more CDase species. In
this study, we biochemically and kinetically characterized two
membrane-bound CDase species as the terminal enzyme of sphingolipid
metabolism. The specific activity of ceramide hydrolysis in guinea pig
epidermis homogenate (5.05 units/mg protein) was higher than that of
brain (3.24 units/mg protein), suggesting that CDase activity has an
important role in epidermal metabolism, because in addition to the
cellular action as biochemical effector molecules, ceramides themselves
are the principal differentiation products in epidermis and the major
constituents of the stratum corneum lipids which serve the epidermal
barrier against water loss
(12) . Furthermore, the long chain
base sphingosine is a potent endogenous inhibitor of protein kinase C
in vitro with the ability to inhibit the proliferation of
germitive epidermal cells
(17, 18, 19) . The
injured stratum corneum may facilitate the mutal contact between its
main lipid constituent, ceramides, and living epidermal cells, probably
leading to the stimulation of reproduction of keratinocytes, as
evidenced by our preliminary study in that in contrast to the
inhibitory effect of short chain base alkyl acyl
sphingosines
(41) , authentic ceramides elicit a marked
stimulation of the proliferation of human keratinocytes (data not
shown). Because our study demonstrated that a high specific activity of
CDase is also localized in the stratum corneum, the uppermost layer of
the epidermis, it is conceivable that under such injured conditions,
the function of CDase in the stratum corneum becomes crucial in
reducing the biochemical activity of the ceramide by hydrolyzing it and
subsequently in abrogating the activated cellular events by liberating
sphingosine. The fact that two CDase species in guinea pig epidermis
were suppressed by the sphingosine (Fig. 7) suggests that
ceramide hydrolysis liberating free sphingosine could also provide a
feedback mechanism for regulating ceramide breakdown, supporting the
notion that CDase activities are controlled by the balance of substrate
degradation and production. Mass contents of ceramides exist at least
50-100-fold greater than those of sphingosine bases in mammalian
epidermis, and Wertz et al.(42, 43, 44) have recently reported that ceramides serve about 40% of
the total lipid in the stratum corneum, while sphingosine bases account
for about 0.44% in same portion
(42, 43, 44) ,
indicating that control metabolism underlying the well regulated
balance of ceramide/sphingosine may be the important physiological
phenomena in epidermal keratinaization process. Our study also
demonstrated that several lipids, in particular SM and PC, markedly
inhibited the purified enzyme activities in the presence of sodium
cholate (Fig. 5, A-D). SM is metabolized by SMase to
yield ceramides and PC in the upper subcorneal layers where ceramides
are essential components to subsequently form bilayer of lipids in the
intercellular spaces between stratum corneum cells for serving barrier
and water-holding function. Therefore, the inhibitory effects by SM and
PC can be viewed as precursor suppression for preventing ceramides from
prior degradation, although we could not conclude whether or not these
lipids affected the enzymes or the ceramides.
Table:
Purification of CDase isozymes from guinea pig
epidermis
C]palmitoylsphingosine as a substrate
as described under ``Experimental Procedures.''
Table:
Substrate specificity of CDase species
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.