Sphingosine kinase catalyzes the formation of the
bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays
important roles in numerous physiological processes, including growth,
survival, and motility. We have purified rat kidney sphingosine kinase
6 × 105-fold to apparent homogeneity. The
purification procedure involved ammonium sulfate precipitation followed
by chromatography on an anion exchange column. Partially purified
sphingosine kinase was found to be stabilized by the presence of high
salt, and thus, a scheme was developed to purify sphingosine kinase
using sequential dye-ligand chromatography steps (since the enzyme
bound to these matrices even in the presence of salt) followed by
EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase
bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration,
the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and
Superdex 75 gel filtration columns. Purified sphingosine kinase has an
apparent molecular mass of ~49 kDa under denaturing conditions on
SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is
a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the
phosphorylation of phosphatidylinositol, diacylglycerol, ceramide,
DL-threo-dihydrosphingosine, or
N,N-dimethylsphingosine. However, the latter
two sphingolipids were potent competitive inhibitors. With sphingosine
as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed
Michaelis-Menten kinetics, with Km values of 5 and
93 µM for sphingosine and ATP, respectively. This study
provides the basis for molecular characterization of a key enzyme in
sphingolipid signaling.
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INTRODUCTION |
Sphingolipid metabolites, such as ceramide, sphingosine, and
sphingosine 1-phosphate
(SPP),1 are members of a
novel class of lipid second messengers (1-4). Ceramide is an
important regulatory component of stress responses and programmed
cell death, known as apoptosis (2, 5, 6). In contrast, we have
implicated a further metabolite of ceramide, SPP, as a second messenger
in cellular proliferation and survival induced by platelet-derived
growth factor, nerve growth factor, and serum (7-9). Previously, we
showed that SPP protects cells from apoptosis resulting from elevations
of ceramide (7, 9) and proposed that the dynamic balance between levels
of the sphingolipid metabolites (ceramide and SPP) and consequent
regulation of opposing signaling pathways is an important factor that
determines whether a cell survives or dies (7). Recently, we
demonstrated that this ceramide/SPP rheostat is an evolutionarily
conserved stress regulatory mechanism influencing growth and survival
of yeast (10). A variety of stress stimuli, including Fas ligand, tumor necrosis factor-
, interleukin-1, growth factor withdrawal,
anticancer drugs, oxidative stress, heat shock, and ionizing radiation,
stimulate sphingomyelinase, leading to increased ceramide levels (2, 5,
6), whereas platelet-derived growth factor and other growth factors
stimulate ceramidase and sphingosine kinase and elevate SPP levels (3,
4, 8, 11). Progress in determining the importance of these sphingolipid
metabolites has been hampered because most of the relevant metabolic
enzymes have not yet been purified or cloned.
The level of SPP in cells is low and determined by the relative
contributions of its formation, mediated by sphingosine kinase (12),
and its degradation, catalyzed by an endoplasmic reticulum pyridoxal
phosphate-dependent lyase and specific phosphatases (10,
13, 14). SPP was initially described as an intermediate in the
degradation of long-chain sphingoid bases (15). However, the roles of
SPP in cellular proliferation, survival, and other cellular responses
(reviewed in Ref. 16), as well as the observations that SPP triggers
novel signal transduction pathways of calcium mobilization (17, 18),
activation of phospholipase D (19), and the Raf/MKK/ERK signaling
cascade (20, 21), suggest that the importance of sphingosine kinase is
not restricted to the catabolism of sphingolipids as was originally
proposed nearly 20 years ago (22).
Sphingosine kinase is a ubiquitous enzyme found in yeast (22);
Tetrahymena pyriformis (23); rat liver, kidney and brain (24, 25); bovine brain (26); and human and porcine platelets (25, 27).
Although it is known that sphingosine kinase activity increases in
response to certain growth-promoting agents, such as platelet-derived
growth factor (8, 28, 29), phorbol esters (3, 7, 30), the B subunit of
cholera toxin (31), and nerve growth factor (9), little is yet known
about the properties or mechanism of regulation of sphingosine kinase.
Thus, purification and characterization of sphingosine kinase are
important goals to gain understanding of its physiological roles. We
have now succeeded in purifying sphingosine kinase from rat kidneys by
>6 × 105-fold to apparent homogeneity.
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EXPERIMENTAL PROCEDURES |
Materials--
Frozen rat kidneys were purchased from Pel-Freez
Biologicals (Rogers, AK). DEAE-cellulose (DE52) was purchased from
Whatman. Dye-ligand gel chromatography medium (blue 3GA and green A)
and Centriprep, Centricon, and Microcon sample concentrators were from
Amicon, Inc. (Beverly, MA). The prepacked hydroxylapatite column was
purchased from Tonen Chemical Co. (Tokyo, Japan). EAH-Sepharose, calmodulin-Sepharose 4B, prepacked Mono Q, and Superdex 75 columns and
silver staining reagents were purchased from Pharmacia Biotech (Uppsala, Sweden). Alkyl-agaroses were from Sigma.
[32P]ATP was from ICN (Costa Mesa, CA). Sphingosine was
obtained from Matreya, Inc. (Pleasant Gap, PA). Coomassie Plus protein reagent was from Pierce. Molecular mass markers for polyacrylamide gels
and the gel electrophoresis apparatus were from Bio-Rad.
Assay of Sphingosine Kinase Activity--
Sphingosine kinase
activity was determined as described previously with minor
modifications (12). Samples (up to 40 µg) and 10 µl of 1 mM sphingosine (dissolved in 5% Triton X-100) were mixed
with buffer A (20 mM Tris (pH 7.4), 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM
sodium orthovanadate, 40 mM
-glycerophosphate, 15 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 mM
phenylmethylsulfonyl fluoride, and 0.5 mM
4-deoxypyridoxine) in a total volume of 190 µl, and reactions were
started by addition of 10 µl of [32P]ATP (10 µCi, 20 mM) containing MgCl2 (200 mM) and
incubated for 5 or 15 min at 37 °C. Reactions were terminated by
addition of 20 µl of 1 N HCl followed by 0.8 ml of
chloroform/methanol/HCl (100:200:1, v/v). After vigorous vortexing, 240 µl of chloroform and 240 µl of 2 M KCl were added, and
phases were separated by centrifugation. The labeled lipids in the
organic phase were resolved by TLC on Silica Gel G60 with
1-butanol/ethanol/acetic acid/water (80:20:10:20, v/v) and visualized
by autoradiography. The radioactive spots corresponding to authentic
SPP were identified as described (32), scraped from the plates, and
counted in a scintillation counter or, alternatively, quantified with a
Molecular Dynamics Storm PhosphorImager. Sphingosine kinase specific
activity is expressed as pmol of SPP formed per min (unit)/mg of
protein. At each purification step, linearity of the enzymatic reaction with time of incubation and protein concentration was observed.
Extraction and Ammonium Sulfate Fractionation--
Frozen rat
kidneys (0.5 kg) were thawed in 0.5 liter of cold 20 mM
Tris (pH 7.4) containing 20% glycerol, 1 mM
dithiothreitol, 1 mM EDTA, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.5 µg/ml soybean trypsin inhibitor, and 0.2 mM phenylmethylsulfonyl fluoride (buffer B), decapsulated,
transferred to fresh buffer (2 ml/g), minced, and then homogenized in a
blender. After centrifugation at 5000 × g for 15 min,
the supernatant fraction was filtered through glass wool, centrifuged
at 20,000 × g for 1 h, filtered through glass
wool again, and centrifuged at 100,000 × g for 90 min.
This supernatant was then centrifuged at 100,000 × g
for 1 h to obtain the cytosolic fraction, which was fractionated
by precipitation with ammonium sulfate. The 25-45% ammonium sulfate precipitate was resuspended in 150 ml of buffer B and dialyzed overnight against the same buffer.
DEAE-cellulose Chromatography--
The dialysate was clarified
by centrifugation for 15 min at 100,000 × g and
applied overnight at a flow rate of 70 ml/h to a DEAE-cellulose column
(5-cm diameter, 500-ml bed volume) equilibrated with buffer B. After
washing with buffer B, proteins were eluted with a linear gradient to
0.5 M NaCl in the same buffer at a flow rate of 250 ml/h.
Sphingosine kinase activity was determined on aliquots of each
fraction, and peak activity fractions were pooled.
Dye-Ligand Affinity Chromatography on Green A- and Blue
A-Sepharose Columns--
The pooled DEAE fractions were applied to a
green A-Sepharose column (5-cm diameter, 200-250-ml bed volume)
equilibrated with buffer B containing 0.2-0.22 M NaCl at a
flow rate of 120 ml/h. After the sample was applied, the flow rate was
increased to 300 ml/h, and stepwise elution was performed with 3 bed
volumes of buffer B containing 0.2 and 0.4 M NaCl,
respectively, and then 6 bed volumes of buffer B containing 1 M NaCl. The 1 M NaCl fraction contained most of
the sphingosine kinase activity and was diluted 1:1 with buffer B and
applied at 120 ml/h to a blue A-Sepharose column (5-cm diameter, 125-ml
bed volume) equilibrated with buffer B containing 0.5 M
NaCl. The blue A-Sepharose was washed at 250 ml/h with 3 bed volumes of
buffer B containing 0.5 M NaCl and with 3 bed volumes of
buffer B containing 0.7 M NaCl, and the sphingosine kinase
activity was eluted with 6 bed volumes of buffer B containing 2 M NaCl.
EAH-Sepharose Chromatography--
Half of the 2 M
NaCl fraction was concentrated 100-fold in Centricon Plus-20
concentrators (Mr 10,000 cutoff) and then
dialyzed against buffer B. The dialysate was centrifuged to remove
precipitated proteins and, after addition of Triton X-100 to a final
concentration of 0.05%, was loaded onto a 20-ml EAH column
pre-equilibrated with buffer B containing 0.05% Triton X-100. The EAH
column was washed stepwise with 60-ml fractions of buffer B and 0.05%
Triton X-100 containing 0, 30, 150, 200, and 600 mM NaCl.
Most of the sphingosine kinase activity was eluted with 150 mM NaCl.
Affinity Chromatography on Calmodulin-Sepharose
4B--
CaCl2 was added to the EAH fraction to a final
concentration of 4 mM and immediately applied to a
calmodulin-Sepharose 4B column (12 ml) pre-equilibrated with buffer B
without EDTA and containing 100 mM NaCl, 0.05% Triton
X-100, 2 mM CaCl2, and 10% sucrose. The column
was washed successively with 30 ml of equilibration buffer and 60 ml of
the same buffer containing 2 mM EGTA. Sphingosine kinase
activity was then eluted with 30 ml of equilibration buffer containing
2 mM EGTA and 1 M NaCl, and five fractions of 6 ml each were collected. The sphingosine kinase-containing fractions (fractions 2 and 3) were concentrated in a Centriprep-10 concentrator, diluted with buffer B containing 0.05% Triton X-100 and 10% sucrose, and reconcentrated until the final salt concentration was 50 mM or less.
Hydroxylapatite Chromatography--
The concentrated
calmodulin-Sepharose fraction was injected onto a hydroxylapatite
column equilibrated with buffer B containing 0.05% Triton X-100, 10%
sucrose, and 25 mM potassium phosphate and was eluted at a
flow rate of 0.4 ml/min with a linear gradient of 0.025-0.5
M potassium phosphate in the same buffer (Waters HPLC
system). Fractions (0.4 ml) were collected in tubes containing 100 µl
of 5 M NaCl, and sphingosine kinase activity was
determined.
Mono Q Anion Exchange Chromatography--
The pooled
hydroxylapatite fraction was concentrated and desalted using
Microcon-10 concentrators and injected onto a Mono Q 5/5 column (Waters
HPLC system) equilibrated with buffer B containing 0.05% Triton X-100,
10% sucrose, and 15 mM potassium phosphate. The column was
washed for 10 min with equilibration buffer at 1 ml/min, and then a
linear gradient of 0.015-0.5 M potassium phosphate was
applied. Fractions of 1 ml were collected in tubes containing 250 µl
of 5 M NaCl. Sphingosine kinase activity was eluted as two
broad peaks. Each peak was pooled and reapplied to a small
calmodulin-Sepharose column (1 ml) to concentrate the sample since
concentration by ultrafiltration usually resulted in a marked loss of
activity. Furthermore, this second calmodulin-Sepharose column
decreased the Triton X-100 concentration, which would have resulted
from ultrafiltration of the Mono Q fractions and which would interfere
with further purification.
Gel Filtration Chromatography on Superdex 75--
The eluate
from the calmodulin affinity column was concentrated to 200 µl using
Microcon-10 concentrators and then injected onto a Superdex 75 gel
filtration column (Waters HPLC system) pre-equilibrated with buffer B
containing 0.05% Triton X-100, 10% sucrose, and 1 M NaCl.
Proteins were eluted at a flow rate of 0.4 ml/min, and 0.4-ml fractions
were collected. The column was calibrated using bovine serum albumin,
ovalbumin, chymotrypsinogen, and lysozyme as standard proteins to
determine the apparent molecular mass of native sphingosine kinase.
SDS-Polyacrylamide Gel Electrophoresis--
SDS-polyacrylamide
gel electrophoresis was performed essentially as described by Laemmli
(33). Protein samples were boiled for 5 min in sample buffer with or
without a reducing agent and loaded onto 12% gels. Molecular masses of
the various protein bands were estimated with the low molecular mass
range prestained standard proteins from Bio-Rad. Proteins were
visualized by silver staining. Sample buffer containing
-mercaptoethanol often produced artificial bands using the sensitive
silver stain procedure, and thus, most gels were run under nonreducing
conditions.
Protein Determination--
Proteins were determined with either
the Coomassie dye binding method (Pierce) or the Lowry procedure after
precipitation with 7% trichloroacetic acid in the presence of 0.015%
deoxycholate (Peterson variation (34)). After Superdex 75 gel
filtration, the protein concentration was too low for determination by
these methods and was estimated from optical densities obtained by
scanning silver-stained gels using bovine serum albumin as a
standard.
Characterization--
Purified sphingosine kinase obtained after
the gel filtration step was used for characterization studies. To
determine pH dependence, the following buffers were used: pH 4-5, 200 mM sodium acetate; pH 6.0-6.6, 200 mM MES; pH
7.0-7.5, 200 mM HEPES; and pH 7.4-9.0, 200 mM
Tris-HCl. For inhibition studies, sphingosine kinase was incubated with
sphingosine and the indicated concentrations of
N,N-dimethylsphingosine or
DL-threo-dihydrosphingosine dissolved in
dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in
assays was <1%.
 |
RESULTS AND DISCUSSION |
Sphingosine Kinase Activity in Various Tissues--
It has been
reported that sphingosine kinase is a ubiquitous enzyme (12, 22-27,
35, 36). Using our recently developed quantitative sphingosine kinase
assay (12, 35), we measured sphingosine kinase activity in various rat
tissues to determine which source would be most appropriate for
purification. In agreement with a previous qualitative study (24), we
found that spleen and kidney have higher specific activities than
liver, which has about twice the activity of brain (Table
I). More than 50% of the sphingosine
kinase activity in kidney, liver, and brain was in the cytosolic
fraction, independently of ionic strength of the extraction buffer. SPP
levels have also been measured in these tissues (37). However, there
does not appear to be a good correlation between sphingosine kinase
activity and SPP levels, as the highest levels of SPP were found in
brain and spleen. Furthermore, kidney has more SPP than liver, which
contains only very low levels (37, 38). Although we found that spleen
has high sphingosine kinase activity and SPP levels, we selected
kidneys as the source for the purification of sphingosine kinase since
initial experiments demonstrated that the majority of the sphingosine
kinase activity in spleen was associated with membranes (Table I) and
unstable. Bovine kidneys were also examined as a potential source for
purification of sphingosine kinase since they are much larger. However,
the specific activity in the cytosolic fraction from bovine kidneys was
lower than that in rat kidney cytosol (Table I), and very poor
recoveries were found after preliminary ammonium sulfate fractionations
(data not shown). Interestingly, we have found that most of the
sphingosine kinase in Saccharomyces cerevisiae is in the
microsomal fraction and not in the cytosol (Table I). These results
suggest that various forms of sphingosine kinase may exist depending on
the tissue and species.
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Table I
Comparison of sphingosine kinase activity from various sources
Cytosolic fractions of various tissues and S. cerevisiae
were obtained after ultracentrifugation at 100,000 × g
essentially as described for rat kidneys under "Experimental
Procedures." The specific activity of sphingosine kinase from each
source is expressed as pmol of SPP formed per min/mg relative to rat
kidneys. Percent cytosolic = (activity in 100,000 × g supernatant/total activity in homogenate) × 100.
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Purification of Rat Kidney Sphingosine Kinase--
Table
II summarizes the purification of
sphingosine kinase from 2500 rat kidneys. Sphingosine kinase was
purified ~6 × 105-fold to near homogeneity, with a
total activity recovery of 0.6%. After an initial 20,000 × g centrifugation of the homogenate, it was important to
carry out two subsequent centrifugations at 100,000 × g since after ammonium sulfate precipitation of the 20,000 × g supernatant and consequent dialysis, 60%
of the sphingosine kinase activity became insoluble and could not be
solubilized by detergents or in different buffers with pH values
ranging from 6.0 to 8.0. However, when the second 100,000 × g supernatant was fractionated with ammonium sulfate, most
of the sphingosine kinase activity could be resolubilized, and very
little was irreversibly associated with insoluble material.
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Table II
Purification of sphingosine kinase from rat kidney
Sphingosine kinase was purified from 2.5 kg of rat kidneys
(~2500 kidneys) as described under "Experimental Procedures." One
unit of sphingosine kinase activity is 1 pmol of SPP formed from
sphingosine per min. Up to the ammonium sulfate fractionation step,
sphingosine kinase activity was determined by incubation for 5 min at
37 °C to ensure linearity of reactions. With more purified
fractions, assays were carried out for 15 min. After the blue
A-Sepharose step, the activity was frozen and stored at 70 °C.
This fraction was usually thawed within several weeks and used for the
next purification steps. It should be noted that concentration and
dialysis of the blue A-Sepharose eluate resulted in loss of 30-50% of
the sphingosine kinase activity.
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In contrast to bovine brain sphingosine kinase (26), rat kidney
sphingosine kinase binds to DEAE-cellulose at pH 7.5 in Tris buffer,
but not in phosphate buffer. The activity was eluted with 0.2 M NaCl from DEAE-cellulose as a single broad peak (Fig. 1). As expected for a nucleotide-binding
protein, sphingosine kinase binds tightly to both green A and blue A
dye-matrix columns (Fig. 2, A
and B), even in the presence of relatively high salt concentrations (0.2 M for the green A column and 0.5 M for the blue A column). This makes it possible to
directly apply the pooled DEAE fractions containing sphingosine kinase
activity to these dye-matrix columns in a sequential manner. This was
advantageous since either concentration or dialysis of sphingosine
kinase activity resulted in considerable loss of activity. Sphingosine
kinase was purified 270-400-fold after the dye-ligand chromatography steps, with a yield of 30-50%. Sphingosine kinase activity eluted from the blue A column in 2 M NaCl could be stored for
several weeks at
70 °C after quick freezing in liquid nitrogen. It
should also be noted that concentration and dialysis of the blue A
column eluate resulted in a loss of ~30-50% of the activity.
Subsequent purification steps were then repeated two times with half of
this fraction since poor recoveries were found when larger columns were
used for the chromatography separations described below. Addition of
0.05% Triton X-100, but not Nonidet P-40 or
-octyl glucopyranoside,
markedly improved both the recovery and the resolution of proteins and
was thus included in subsequent steps. Triton X-100 has previously been
successfully used to stabilize a number of other lipid enzymes, such as
phospholipase A2, ceramidase, acid sphingomyelinase, and
phosphoinositide 4-kinase, since it prevents aggregation and
nonspecific adsorption to surfaces (39-42).

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Fig. 1.
Anion exchange chromatography of sphingosine
kinase on DEAE-cellulose. The dialyzed 25-45% ammonium sulfate
precipitate was applied to a DEAE-cellulose column pre-equilibrated
with buffer B, and the proteins were eluted with a linear gradient
(0-0.5 M) of NaCl, collecting 20-ml fractions. Sphingosine
(Sph) kinase activity (units/fraction; ) and protein
(mg/fraction; ------) were measured as described under "Experimental
Procedures." Similar results were obtained in at least six
experiments.
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Fig. 2.
Purification of sphingosine kinase on green
A- and blue A-Sepharose columns. A, sphingosine
(Sph) kinase activity eluted from the DEAE-cellulose column
was applied to a green A-Sepharose column equilibrated with buffer B
containing 0.2 M NaCl and eluted stepwise with the
indicated concentrations of NaCl as described under "Experimental
Procedures." B, the sphingosine kinase-containing fraction
from the green A column was diluted 1:1 with buffer B, applied to a
blue A-Sepharose column equilibrated with buffer B containing 0.5 M NaCl, and eluted stepwise with increasing NaCl
concentrations as described under "Experimental Procedures."
Protein concentration and sphingosine kinase activity were
measured in each fraction. Results are expressed as percentage of total
protein or activity applied to each of the columns. Similar results
were obtained in at least six experiments. RT,
run-through.
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Several attempts were made to purify sphingosine kinase by affinity
chromatography on immobilized sphingosine (43) or ATP and by
hydrophobic chromatography, but they were unsuccessful probably due to
the necessity of maintaining the enzyme in solutions containing high
salt concentrations and detergent. We found that sphingosine kinase
binds tightly to 1,6-diaminohexane covalently linked to Sepharose 4B
(EAH-Sepharose), but could not be eluted from this matrix by substrates
containing primary amino groups, such as sphingosine or choline, in a
similar manner, as was previously found for purification of choline
kinase (44). In contrast, at least 70% of the applied sphingosine
kinase activity was eluted from EAH-Sepharose with 0.15 M
NaCl, whereas only 14-18% of the applied proteins were eluted in this
fraction, resulting in 3-5-fold purification (Fig.
3A).

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Fig. 3.
Separation of sphingosine kinase by
chromatography on EAH-Sepharose and calmodulin-Sepharose columns.
A, a portion of the blue A-Sepharose 2 M NaCl
eluate was concentrated and dialyzed. Triton X-100 was added to a final
concentration of 0.05% before application to EAH-Sepharose. The column
was eluted stepwise with increasing NaCl concentrations. B,
the sphingosine (Sph) kinase-containing fractions from the
EAH column were applied to a calmodulin-Sepharose column after addition
of CaCl2, and the proteins were then eluted stepwise with
buffer B containing 10% sucrose, 0.05% Triton X-100, and 1 mM EGTA without and then with 1 M NaCl added.
Results are expressed as percentage of the total protein or sphingosine
kinase activity applied to each of the columns. Similar results were
obtained in at least six experiments. RT, run-through.
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This EAH fraction was immediately applied to a calmodulin-Sepharose
column after addition of CaCl2. Most of the protein applied (96-98%) did not bind and was eluted with the wash buffer. In contrast, most of the sphingosine kinase activity was tightly retained,
and only a small fraction of the activity could be eluted with EGTA in
the absence of CaCl2 (Fig. 3B). However, >95%
of the activity was eluted when the calmodulin column was eluted with 2 mM EGTA solution containing 1 M NaCl (Fig.
3B), resulting in at least 20-fold purification. Similarly,
another calmodulin-binding lipid kinase, inositol-1,4,5-trisphosphate
3-kinase, from either human platelets (45) or rat brain (46) could not
be eluted from a calmodulin-Sepharose column unless the elution buffer
contained EGTA as well as 0.5% Triton X-100 or 0.2% SDS,
respectively. Despite purification of sphingosine kinase by >9000-fold
at this stage, several protein bands were still evident on
silver-stained SDS-polyacrylamide gels. Detectable amounts of
calmodulin also coeluted with the sphingosine kinase activity (Fig.
4B). One of the difficulties we encountered in later steps of the purification of sphingosine kinase
was that the kinase rapidly lost activity. However, we found that
addition of 10% sucrose, which has been shown to stabilize other lipid
enzymes, including squalene synthetase (47), to buffers containing
0.05% Triton X-100 and high salt concentrations further increased
stability at these stages of purification.

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Fig. 4.
Purification of sphingosine kinase by
hydroxylapatite chromatography. A, sphingosine
(Sph) kinase activity eluted from the calmodulin-Sepharose
column was loaded onto a hydroxylapatite column and then eluted with a
gradient of potassium phosphate buffer (pH 7.4). Sphingosine kinase
activity in the fractions was immediately measured as described under
"Experimental Procedures." B, aliquots of fractions were
analyzed by SDS-polyacrylamide gel electrophoresis and visualized by
silver staining. The molecular masses of standard proteins are
indicated. The second lane contains the calmodulin-Sepharose
eluate.
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Sphingosine kinase was then further purified by hydroxylapatite
chromatography followed by HPLC on a Mono Q column. As shown in Fig. 4,
sphingosine kinase activity was eluted from a hydroxylapatite column as
a single peak with a gradient of increasing concentrations of potassium
phosphate at ~0.06 M, resulting in 6-fold additional purification. Although sphingosine kinase is tightly bound to the
strong anion exchanger Mono Q and can be eluted at an ionic strength of
0.15 M with a salt gradient (data not shown), it is not as
tightly bound when applied in the presence of 15 mM
potassium phosphate. We found that most of the activity could be eluted with the wash buffer alone in this case, greatly improving separation from other more tightly bound proteins (Fig.
5). Sphingosine kinase was resolved into
two activity peaks by chromatography on Mono Q. For further
purification, only the first activity peak was utilized since the major
protein band in this fraction was a 49-kDa polypeptide (Fig.
5B), and preliminary experiments utilizing several different
sequences of purification suggested that this 49-kDa polypeptide
correlated with the sphingosine kinase activity.

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Fig. 5.
Separation of sphingosine kinase by anion
exchange chomatography on Mono Q. Sphingosine (Sph)
kinase activity eluted from the hydroxylapatite-Sepharose column was
concentrated, desalted, injected onto a Mono Q column (Waters HPLC
system), and eluted as described under "Experimental Procedures."
A, elution profile of sphingosine kinase on Mono Q;
B, silver-stained SDS-polyacrylamide gel of the Mono Q
fractions. The molecular masses of standard proteins are indicated. The
first lane contains the hydroxylapatite eluate.
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Because SDS-polyacrylamide gel electrophoresis analysis suggested that
sphingosine kinase was highly purified at this stage, we decided to do
a final purification by gel filtration chromatography. However, it was
necessary to concentrate the Mono Q eluate to a small volume before
injection onto a Superdex 75 gel filtration HPLC column.
Ultrafiltration at this stage resulted in large losses of activity and
high concentrations of Triton X-100, which interfere with the gel
filtration. Thus, we used a 1-ml calmodulin-Sepharose column to
concentrate the sample 9-fold, eluted exactly as above described for
the large-scale calmodulin-Sepharose column. Further concentration of
the calmodulin eluate by ultrafiltration did not result in major losses
of activity, and the resulting concentration of Triton X-100 did not
interfere with the resolution of the gel filtration column.
As shown in Fig. 6, the sphingosine
activity was eluted from a Superdex 75 gel filtration column at a
volume corresponding to an apparent native molecular mass of ~59 kDa
when compared with standard proteins (Fig. 6B).
Silver-stained SDS-polyacrylamide gels revealed that the fractions with
the highest sphingosine kinase activity (fractions 23-25) contained a
single 49-kDa polypeptide under both reducing and nonreducing
conditions. Thus, sphingosine kinase isolated from rat kidney is likely
active as a monomer since the apparent native molecular mass of
sphingosine kinase was similar to its molecular mass on
SDS-polyacrylamide gel. However, the possibility that sphingosine
kinase purified to homogeneity from rat kidney cytosol may be an active
fragment of membrane-bound sphingosine kinase cannot be excluded. It
should be noted that the native molecular mass of Swiss 3T3 fibroblast
sphingosine kinase determined by gel filtration was identical to that
of highly purified rat kidney sphingosine kinase (data not shown). The
specific activity of highly purified rat kidney sphingosine kinase (100 µmol/min/mg) is ~10-100-fold higher than that of several other highly purified lipid kinases, including phosphoinositide 3-kinase (48,
49), phosphoinositide 4-kinase (40), phospholipase D (50), and
phospholipase A2 (41), but it is the same order of
magnitude as that reported for acid sphingomyelinase from human urine
(51).

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Fig. 6.
Gel filtration chromatography of sphingosine
kinase on Superdex 75. Sphingosine (Sph) kinase
activity from the Mono Q column was concentrated on a small
calmodulin-Sepharose column and applied to a Superdex 75 gel filtration
column as described under "Experimental Procedures." To calibrate
the column, standard molecular mass markers were loaded onto the
column, and the elution volumes were noted. A, elution
profile of sphingosine kinase on Superdex 75; B,
silver-stained SDS-polyacrylamide gel of the Superdex 75 fractions. The
molecular masses of standard proteins are indicated. The first
lane contains the Mono Q eluate. BSA, bovine serum
albumin.
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Characterization of Sphingosine Kinase--
To characterize
purified sphingosine kinase, a number of experiments were carried out
to examine the pH dependence, substrate specificity, and enzyme
kinetics. Purified sphingosine kinase was active from pH 6 to >8, with
a broad pH optimum between pH 6.6 and 7.5 (Fig.
7A). At pH 7.4, activity
increased linearly with the incubation time for the first 60 min of the
reaction and then gradually decreased. Sphingosine kinase activity was maximal at a concentration of 5-10 mM MgCl2
(Fig. 7B), whereas physiological concentrations of calcium
(1-100 µM) had no effect on its activity. Activity with
D-erythro-sphingosine showed typical Michaelis-Menten kinetics, with Km = 5.1 ± 1.7 µM and Vmax = 101 ± 22 µmol/mg/min (Fig. 7C). The Km for ATP was 93 µM (Fig. 7D), similar to
Km values for other lipid kinases (40, 48). Next, we
examined the substrate specificity for sphingosine kinase. The
naturally occurring
D(+)-erythro-trans-isomer was the
best substrate for purified rat kidney sphingosine kinase. DL-erythro-Dihydrosphingosine was also
phosphorylated, but to a lesser extent (30% compared with
D(+)-erythro-sphingosine), whereas
DL-threo-dihydrosphingosine,
L-threo-dihydrosphingosine, ceramide,
diacylglycerol, and phosphatidylinositol were not phosphorylated (Fig.
8A).
N,N-Dimethylsphingosine and
DL-threo-dihydrosphingosine have previously been
used to decrease SPP levels stimulated by various physiological stimuli
(7, 8, 18). We have now found that both
N,N-dimethylsphingosine and
DL-threo-dihydrosphingosine are potent
competitive inhibitors of purified sphingosine kinase, with
Ki values of 9.9 ± 1.0 and 5.2 ± 0.5 µM, respectively (Fig. 8, B and C).
These results further substantiate the usefulness of these compounds as
tools to inhibit sphingosine kinase activity in vivo and to
examine the role of SPP in diverse cellular responses.

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Fig. 7.
Characterization of sphingosine kinase.
A, pH dependence of sphingosine (Sph) kinase
activity. The activity of purified sphingosine kinase was measured as
described under "Experimental Procedures." The pH was adjusted by
addition of the following buffers: 200 mM sodium acetate
(pH 4-5), 200 mM MES (pH 6.0-6.6), 200 mM
HEPES (pH 7.0-7.5), and 200 mM Tris-HCl (pH 7.4-9.0).
B, concentration-dependent activation of
sphingosine kinase by MgCl2. Sphingosine kinase activity
was measured in the presence of increasing concentrations of
MgCl2. C, Michaelis-Menten and Lineweaver-Burk
(inset) plots for sphingosine kinase with
D-erythro-sphingosine. The activity of purified
sphingosine kinase was determined in the presence of increasing
concentrations of sphingosine. Km and
Vmax values were 3.7 µM and 78.4 µmol/mg/min, respectively. Data are the average of triplicate
determinations in a representative experiment. Similar results were
found in at least three experiments. D, Michaelis-Menten and
Lineweaver-Burk (inset) plots for sphingosine kinase with
ATP. Sphingosine kinase activity was measured in the presence of 50 µM D-erythro-sphingosine and
increasing concentrations of ATP. The Km for ATP was
93 µM.
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Fig. 8.
Substrate specificity (A) and
competitive inhibition of sphingosine kinase by
N,N-dimethylsphingosine
(B) and DL-threo-dihydrosphingosine
(C). A, phosphorylation of various sphingosine
(Sph) analogs or other lipids (50 µM) by
purified sphingosine kinase was measured as described under
"Experimental Procedures." Data are expressed as percentage of
activity with D-erythro-sphingosine.
B, N,N-dimethylsphingosine is a
competitive inhibitor of sphingosine kinase. Sphingosine kinase
activity was determined with varying concentrations of
D-erythro-sphingosine in the absence ( ) or
presence of 20 µM ( ) or 40 µM ( )
N,N-dimethylsphingosine. Inset,
Lineweaver-Burk plot. C,
DL-threo-dihydrosphingosine is a more potent
competitive inhibitor of sphingosine kinase. Sphingosine kinase
activity was determined with varying concentrations of
D-erythro-sphingosine in the absence ( ) or
presence of 10 µM ( ) or 20 µM ( )
DL-threo-dihydrosphingosine. Inset,
Lineweaver-Burk plot.
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In summary, rat kidney sphingosine kinase has been purified to
homogeneity by >6 × 105-fold, indicating that
sphingosine kinase is a low abundance protein in rat kidney and
probably in other tissues as well. Similarly, another kinase, choline
kinase, has been partially purified from rat kidney by >200,000-fold
(44). This study provides the basis for molecular characterization of
sphingosine kinase and will aid in elucidation of its role in various
physiological processes.
We thank Drs. Elliot Crooke and Anton
Wellstein for helpful suggestions and discussions during this
study.