THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1959 by The American Society for Biochemistryand Molecular Biology, Inc.
Vol. 264, No. 18, Issue of June 25, pp. 10371-10377,1959
Printed in U.S.A .
Free Sphingosine Formation from EndogenousSubstrates by a Liver
Plasma Membrane System witha Divalent Cation Dependence and
a
Neutral pH Optimum*
(Received for publication, July 26, 1988)
Charles W. Slife$, Elaine Wang$, Rosemary Hunter$, SijianWang$, Carol Burgess$,
Dennis C. Liottas, and Alfred H. Merrill, Jr.$ll
From the Departments of IBiochrnistry and §Chemistry, Emory University, Atlanta, Georgia 30322
Long-chain (sphingoid) bases may serve as another nant species (1, 2).’ Sphingolipids have been associated with
category of “lipid second messenger” because they in- diverse cellular functions, such as maintenance of membrane
hibit protein kinase
C and affect multiple cellular func-structure, modulation of cell surface receptors, cell-cell comtions. Free sphingosine has been found in
rat liver munication, differentiation (and neoplastic transformation),
(Merrill, A. H., Jr., Wang, E.,Mullins, R. E.,Jamison, among others (3, 4); however, the mechanism of action of
W.C. L., Nimkar, S., and Liotta, D.C. (1988)Anal. these complex compounds has largely remained unclear.
Biochem. 171,373-381); hence, this study determined The recent finding that sphingosine and other long-chain
bases are potent inhibitorsof protein kinase C in vitro and in
if liverplasmamembranescontainfreelong-chain
bases and have the ability to form them from
endoge- intact cells (5-8) has presented a possible link between sphinnous enzymes and substrates. Isolated plasma mem- golipids and cell communication. Since these initial studies,
branes contained 0.45 nmol of sphingosine/mg of pro- long-chain bases have been found to modulate many other
tein which, based on the recovery of the membranes, protein kinase C-dependent cell functions (see Ref. 9 for a
was equivalent to
3.6 f 1.2 nmol/g of liver and at least recent review) as well as a number of other systems (10-12).
half of the total free sphingosine in liver. When the Long-chain bases are of additional interest because they are
membranes were incubated at 3 7 “C, the amount in- natural constituents of cells and might serve as endogenous
creased at an initial rate
of 5-25 pmol/min/mg, result- modulators of cell functions. Free sphingosine has been obing in a 2-3-fold increase over an hour. Sphingosine served in HL-60 cells (8), neutrophils (13), liver (14),and
other tissues (15). The amount of sphingosine in neutrophils
formation required divalent cations, was optimal at
neutral to alkaline pH, and was temperature-depend- is affected by phorbols and other agonists (13), which may
indicate that themodulation of endogenous sphingosine levels
ent. Activities with these characteristics were not identified in microsomesor lysosomes (lysosomalactivities serves a physiological function.
with acidic pH optima
were detected, however); hence, Nonetheless, the source and location of this free sphingosine is unknown. Lysosomes and plasma membranes contain
they appear to reflect a separate plasma membrane
system. Sphingosine formation was stimulated by cer-many sphingolipid hydrolases (3), but theformation of sphingosine by their action on endogenous sphingolipids has not
amides either added exogenously or formed endogenously by treating the membranes with
sphingomyeli- been evaluated. In this report, a recently developed method
for measuring free sphingosine (14) was used to show that
nase(butnotendoglycoceramidase).Sphingomyelin
isolated plasma membranes contain this compound, and that
hydrolysis to ceramide was also observed during incubation of the plasma membranesalone. Some of the they also possess an enzymatic system that acts on endogeproperties of this system resembledthe neutral sphin- nous substrates to form free sphingosine.
gomyelinase and ceramidase activitiesof liver. While
the physiologicalsignificance
of this endogenous
EXPERIMENTALPROCEDURES
sphingosine is not known, this system has the approMaterials-Phorbol 12-myristate 13-acetate was purchased from
priate subcellular location to provide sphingosineas a LC Services Corp. (Woburn, MA); erythro-dihydrosphingosine
participant in signal transduction.
(sphinganine), sphingosine, N-palmitoylsphingosine, N-palmitoyl-
Sphingolipids (sphingomyelin, gangliosides, cerebrosides,
etc.) are elaborations of several long-chain (sphingoid) bases,
of which sphingosine (4-trans-sphingenine) is the predomi* This work was supported by National Science Foundation Grant
DCB-871083and National Institutes of Health Grants GM33369 and
RR05364. Parts of this work were presented at the Seventy-ninth
Annual Meeting of the American Society for Biochemistry and Molecular Biology (41). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “aduertisement” in accordance with 18
U.S.C. Section 1734 solelyto indicate this fact.
ll To whom reprint requests should be addressed Dept. of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
sphinganine, sphingomyelin and ceramides (from bovine brain), dioctanoylglycerol, 1-oleoyl,2-acetylglycerol,and dioleoylglycerol were
obtained from Sigma. Sphingomyelinase (from Bacillus cereus) was
purchased from Boehringer Mannheim, and endoglycoceramidase
(from Rhodococcus) wasobtained from Genzyme Corp. (Boston, MA).
The chloroform (Chempure) and HPLC-grade methanol (Omnisolv
glass-distilled) were from Curtin Matheson Scientific (Houston, TX).
The other chemicals and biochemicals were of high quality.
Synthesis of the Long-chain Base Compounds-The internal standards used for analyses of sphingosine by high performance liquid
chromatography (HPLC)’ were synthesized by a recently developed
Throughout this manuscript, the generic term sphingosine refers
to thelong-chain base containing the 4-transdouble bond (which has
also been called 4-trans-sphingenine). Sphinganine is used for the
dihydro species, which is also called dihydrosphingosine.
The abbreviations used are: HPLC, high performance liquid chromatography; TLC, thin layer chromatography.
10371
10372
Sphingosine
Formation
procedure (16). The (3-3H]sphinganine was synthesized as described
previously (17,18) and the
[3H]sphingomyelinand [3H]ceramidewere
prepared by reduction of the 4-trans-double bond of the sphingosine
backbone with NaB3H4(19). Each of the compounds were pure based
on thin layer chromatography (TLC) on Silica Gel H plates developed
with the solvent systems described below for each lipid type, and the
long-chain bases were >95% pure by HPLC (14).
Membrane Preparations-Male Sasco/King (SD)BR rats (125-150
g) were obtained from Sasco, Inc. (Omaha, NE). They were housed
in group cages in a room with a 12-h light/dark cycle (beginning at
8 0 0 a.m.) and provided water and rat chow ad libitum. Prior to use,
the animals were fasted overnight, then killed between 8 and 10 a.m.
Plasma membranes were prepared from rat livers essentially as
described by Hubbard et ai. (20) as modified by Tyrrell and coworkers (21). Lysosomes were prepared by the procedure of Sawant
et el. (22) and microsomes as described by Williams et al. (23). The
final membrane pellets were resuspended in 0.25 M STM buffer (0.25
M sucrose, 5 mM Tris-HC1,0.5 mMMgC12, pH 7.5) or in other buffers
as indicated.
Membrane Marker Assays--6'-Nucleotidase, acid phosphatase,
and glucose-6-phosphatase activities were determined by measuring
the rate of release of inorganic phosphate from 5'-AMP (24), pglycerol phosphate (25), and glucose 6-phosphate (26), respectively.
The microsomal NADH-cytochrome c reductase was measured by the
method of Kreibich et al. (27), and lysosomal 0-glucuronidase was
measured as described in Fishman (28).
Sphingosine-generating Actiuity-For most experiments, membranes were resuspended to approximately 0.5-2 mg/ml in 0.25 M
STM buffer or in this buffer minus magnesium (for experiments on
the divalent cation dependence) or with 100 mM Tris-acetate buffer
(for experiments at different pH values). The membranes were incubated, and atvarious times duplicate or triplicate10O-pl aliquots were
removed and placed into 1.5 ml ofCHCkCH30H (1:2) for sphingosine
analysis.
Sphingosine Analysis-The samples weremixed with 1.5mlof
CHC13:methanol(1:2) and 50-250 pmol of eicosasphingosine or eicosasphinganine, which serve as an internal standards, then extracted
and analyzed by HPLC as the o-phthalaldehyde derivatives as described by Merrill et al. (14).
Treatment of Plasma Membranes with Sphingomyelinaseor Endoglycoceramidase-Plasma membranes were resuspended at approximately 2 mg/ml in 0.25 M STM and incubated at 3 'C with 0.3 unit/
mlof sphingomyelinase or0.2 milliunit/ml endoglycoceramidase.
Aliquots were removed after different times for analysis of sphingosine, sphingomyelin, or gangliosides, as described below.
Analysis of Other Plasma Membrane Sphingolipids-Plasma membranes were incubated with or without other additives (i.e. sphingomyelinase or endoglycoceramidase) as described above, then 1.5 ml
of CHC13:methanol (1:2) was added. For the analysis of sphingomyelin, 1 ml of CHCL was added, and the samples were stirred a t 37 "C
for 1h. The samples were centrifuged and thesupernatants collected.
(2:1),
The pellets were reextracted with 1 mlofCHC1s:methanol
centrifuged, and the supernatants werepooled. The solvents were
evaporated under reduced pressure and the residue was extracted
twice with CHCb using sonication. The extracts were filtered, and
the volumewasreduced
under vacuum before the samples were
applied to Silica Gel H (Brinkman) TLC plates and developed with
CHC13:methanol:acetic acidwater (50:30:6:4, v/v/v/v). The lipids
were visualized with iodine vapor, and theregions co-migrating with
the sphingomyelin and phosphatidylcholine standards were marked.
After the iodine had faded, the lipids were quantitated by phosphate
assays (26).
For glycolipids, the extract was prepared as described above but
the solvent system wasCHCl,,
methanol, 0.02%CaClz.2H20
(6040:9), and the gangliosides were visualized using resorcinol reagent. The loss of gangliosides was quantitated by measuring the
total lipid sialic acid in the extracts (29).
For ceramides, the lipids were extracted by the same procedure
used for sphingosine analyses; however, the CHC13 phase was evaporated, applied to a Silica Gel H
plate,
and
developed with
ether:methanol (99:l). The ceramides werevisualized with iodine,
and the region co-migrating with a ceramide standard was scraped
from the plate, eluted from the silica with CHCl,, and acid-hydrolyzed
to release the sphingosine, which was analyzed by HPLC. The samples were spiked with 0, 1, 3, and 5 nmol of ceramide standard to
allow correction for recoveries.
Incubation of Erogenous Sphingolipids with Isolated Plasma Membranes-To incubate sphingomyelin and ceramides with the mem-
in
Membranes
Plasma
branes, the lipids were solubilized with sodium deoxycholate then
added to the standard incubation mixture to yield a final detergent
concentration of 1%.
The I3H]sphinganine (47,000 cpm/nmol) was prepared as a 1:l
molar complex with fatty acid-free bovine serum albumin in phosphate-buffered saline, then 5 p1of a 2 mM solution was added to 100
~1 of plasma membranes and incubated in 0.25 M STM.
After different time intervals at 37 "C, the incubations with the
different lipids were terminated by adding 1.5 ml of CHCl3:methanol
(1:2) andextracting the lipids as described above. For unlabeled
substrates, the long-chain bases were analyzed by HPLC, the lipid
extracts of radiolabeled sphingolipids were applied to Silica Gel H
TLC plates and developedwith CHCI,, methanol, 2 N NH,OH
(40101). The radioactive regions of the chromatoplate were visualized by fluorography and quantitated by cutting the plate into portions and measuring the radioactivity by liquid scintillation counting
as described previously (18).
In an additional experiment to estimate losses of long-chain bases
during plasma membrane isolation, approximately 1 mg of isolated
plasma membranes were incubated for 5 min with 1 pCiof [3H]
sphinganine. The labeled membranes were recovered by centrifugation and mixed with a freshly prepared rat liver homogenate and
carried through the entire procedure for isolating plasma membranes.
The amount of radiolabel in the final fraction and the percentage
recovery of 5'-nucleotidase were compared.
Measurement of Sphingosine Synthesis de Nouo-Plasma membranes were incubated with [I4C]serine under the optimal assay
conditions for serine palmitoyltransferase (30). Since this detected
some activity, assays were also conducted using the buffer (i.e. STM)
and other conditions (i.e. palmitoyl-Cob was not added) that were
used elsewhere in this study to measure sphingosine formation. Under
these conditions, no activity was detected.
RESULTS
Free Sphingosine in Plasma Membranes-When lipid extracts from isolated ratliver plasma membranes were examined by HPLC (Fig. I), they were found to containfree
sphingosine (Fig. l), the identity of which was confirmed by
the other analyses (not shown)
described in Merrillet al. (14).
The sphingosine content of the plasma membranes was approximately 0.45 k 0.15 nmol/mg protein. When corrected
for the recovery of theplasmamembranes
based onthe
recovery of 5'-nucleotidase(which we find to be 10-18%,
which also agrees with that originally described by Hubbard
et al., (20)), the totalsphingosine was 3.5 -+ 1.2 nmol/g liver.
This estimateis somewhat greater thanhalf of the total free
sphingosine found in liver (14). Estimates of the free sphingosine in other subcellular fractions were not conducted in
detail; however, an analysis of microsomes and lysosomes
4
8
12
4
8 1 2
4
8 1 2
Retention time(mid
FIG. 1. HPLC elutionprofile for endogenous sphingosine
from rat liver plasma membranes. Panel A, elution of intrinsically
fluorescent compounds extracted from plasma membranes; panel B,
elution of o-phthalaldehyde-sphingosineextracted from plasma membranes;panel C, HPLC profile for sphingosine with eicosasphingosine
as an internal standard.
Sphingosine Formation in Plasma Membranes
found 0.08 f 0.1 and 0.13 f 0.1 nmol, respectively, of sphingosine/mg of protein.
Thisestimate of the amount of sphingosine in plasma
membranes assumes that it remains with the membranes
during the isolation procedure. To testthis assumption,
plasma membranes were spiked with [3H]sphinganine and,
after recovering the plasma membranes by centrifugation,
they were added to a freshly prepared rat liver homogenate
and the plasma membranes were reisolated. The recovery of
the radiolabel in the reisolated plasma membranes was 44%;
hence, the estimate may, in fact,be somewhat lower than the
amount of sphingosine that is actually in plasma membranes
in vivo.
Free Sphingosine Formation-When the isolated plasma
membranes were incubated at 37 "C, the amount of sphingosine increased 2-3-fold over an hour (Fig. 2), with an initial
rate of 5-25 pmol/min/mg protein, depending on the membrane preparation. Thus, the preparation contains both an
enzyme system and endogenous substrates for the formation
of free sphingosine. Subsequent experiments were conducted
to better define the optimal conditions for sphingosine formation from endogenous substrates.
The rate of sphingosine formation was temperature-dependent. No increase occurred upon incubation of the plasma
membranes at 4 "C (Fig. 2), or when they were boiledprior to
incubation at 37"C. The rate increased with temperature
between 4 and 45 "C, with an inflection point at about 30 "C
(Fig. 3).
Divalent cations were required for sphingosine formation
30
90
150
Time(min)
FIG. 2. Time course of sphingosine formation. Plasma membranes were incubated at 4 or 37 "C in 0.25 M STM, and at various
times duplicate 100-pl aliquots wereremoved, placed into chloroform:methanol, and the sphingosine content of the membranes was
measured.
10373
because EDTA completely inhibited the activity and it was
fully restored by magnesium (Fig. 4)and partiallyby calcium.
The activity in the plasma membranes alone (i.e. inthe
absence of EDTA or added divalent cations) is probably due
to residual magnesium from the buffer used to isolate the
membranes. Preliminary studies (not shown) indicated that
BaC12,CoC12, and MnC12 could also stimulate sphingosine
formation somewhat; whereas, zinc and monovalent cations
(sodium, potassium, and lithium)did not increase the amount
of sphingosine over that obtained with EDTA alone.
Comparison of Sphingosine Formation in Various Subcellular Fractions-Since lysosomes contain a catabolic system
for degrading complex sphingolipids (3), it was possible that
the sphingosine formation observed in theplasma membrane
fraction was the result of lysosomal contamination. This was
examined by iosolating lysosomes and comparing the activity
of the sphingosine generating system in these membranes to
the plasma membrane activity (Table I). The rate of sphingosine formation was similar for both fractions even though
the lysosomal fraction contained a 14.5-32-fold relative enrichment of the lysosomal markers acid phosphatase and @glucuronidase, respectively, in comparison tothe plasma
membranes. Also, the plasma membrane fraction containeda
7-8-fold relative enrichment of the plasma membrane markers alkaline phosphodiesterase and 5'-nucleotidase, respectively, in comparison to thelysosomal fraction. These results
indicate that sphingosine formation occurs in both plasma
membranes and in lysosomes.
It should be emphasized that theactivity of the sphingosineforming system was not determined with saturating concentrations of the substrates (as for the marker enzymes); but
rather, it is based on the interaction of the pertinent enzyme(s) of sphingosine formation with endogenous substrates.
This leads to the possibility that the activity reflects the
interaction of an enzyme in one compartment (for example,
the lysosomes) with substrates in another compartment (for
example, the plasma membranes) or vice versa. To test this,
lysosomes were mixedwith varying amounts of plasma membranes, andthe incubations were conducted as described
above. Under these conditions, the amount of sphingosine
formed was the sum of the individual fractions (data not
shown). This suggests that mixing of enzymes and substrates
in different membrane compartmentscontributeslittle
to
sphingosine formation under the assay conditions used here.
By examining the effects of pH on sphingosine formation,
the existence of both plasma membrane and lysosomal systems was confirmed. The activity in the lysosomal fraction
was maximal at acid pH, whereas the greatest activity was
I
None EDTA Ca++ Ms++
Temperature (OC)
FIG. 3. Temperature dependence of sphingosine formation.
Plasma membranes were incubated at different temperatures in 0.25
M STM. At various times, duplicate 100-p1 aliquots were removed,
placed into CHCls:methanol, andthe sphingosine content of the
membranes was measured.
FIG. 4. Ion requirements of Sphingosine formation. Plasma
membranes were washed one time with buffer containing 5mM TrisHCl, pH 7.5, and 0.25 M sucrose, then resuspended in this buffer
(None) or in buffer containing 1 mM EDTA, 2 mM calcium chloride,
or 2 mM magnesium chloride. After incubation at 37 "C for 60 min,
the sphingosine content of the membranes was measured.
Sphingosine Formation in Plasma Membranes
10374
TABLE
I
Sphingosine formation by different membrane fractions
Plasma membranes, lysosomes, and microsomes were isolated and assayed for sphingosine-generating activity
and various marker enzyme activities as described under “Experimental Procedures.”
Fraction
Marker enzyme
Plasma membranes
Lysosomes
Microsomes
gmollminlmg
0.2
0.07
2.4
0.13
Preparation A
5”Nucleotidase0.3
0.07
Alkaline phosphodiesterase
Acid phosphatase
48.0
@-Glucuronidase
1.5
Glucose-6-phosphatase
NADH-Cytochrome c reductase
Sphingosine formation
Preparation B
5’-Nucleotidase
Acid phosphatase1.5
Glucose-6-phosphatase
Sphingosine
20formation
0.11
0.79
pH
FIG.5. Sphingosine formation at different pH. Aliquots of
or lysosomal ( L y s ) fractions in 0.25 M
the plasma membrane (PM)
STM were diluted in an equal volume of 100 mM Tris-acetate buffer
of different pH values. The membranes were incubated at 37 “C, at
various times duplicate aliquots were removed, and the sphingosine
content was measured.
seen at neutral to alkaline pH for the plasma membranes
(Fig. 5). Hence, although plasma membranes and lysosomes
contain about equal activities at pH7.5 (Table I), theplasma
membrane activity decreased when the pHwas shifted to 4.5
and the lysosomal activity increased (Fig. 5).
Although the plasma membrane preparation used in these
studies is only minimally contaminated with most of the other
membranes, it is known to have significant amounts of microsomes (20). Microsomes were therefore isolated and sphingosine formation measured in this fraction (Table I). Although
the microsomes were enriched 6-79-fold in the microsomal
enzyme markers glucose-6-phosphatase and NADH-cytochrome c reductase, it contained about one-tenth the sphingosine forming activity of plasma membranes. Sphingosine
formation in the plasma membrane fraction is clearly not a
consequence of the microsomal contamination.
To determine if the activity in the plasma membrane fraction represents an appreciable portion of the neutral sphingosine forming activity in liver, the activities were compared
to thatof a liver homogenate. The initial rate for the homogenate incubated under identical conditions was 0.11 k 0.01
nmol/min/g liver. Since the rate for plasma membranes from
this same preparation was about 0.16 nmol/min/g liver, the
activity was comparable to the apparent total. It should be
emphasized that this comparison is only for the action of
endogenous enzymes on endogenous substrates at neutral pH
and clearly does not apply to total cellular sphingolipid hy-
2.6
0.49
0.06
1.5
0.4
0.01
5.3
0.88
1.1
1.6
0.12
0.4
20
0.2
7.1
0.6
0.2
0.01
0.9
1.1
drolases, which include the very active acidic lysosomal enzymes.
Effect of Various Reagents on Sphingosine Formation-A
likely pathway for sphingosine formation in plasma membranes, if the system is analogous to the lysosomal hydrolases,
is the removal of the headgroup of complex sphingolipids to
yield ceramide followed by removal of the amide-linked fatty
acid. To determine if plasma membrane ceramides can be
hydrolyzed to sphingosine, ceramide was generated by treating
the membranes with sphingomyelinase. This resulted in an
increase in the rate and extentof sphingosine formation (Fig.
6). To confirm that the exogenous sphingomyelinase was
active, sphingomyelin was quantitated in the starting preparation (16 ? 3.7 nmol) and after 1 h of sphingomyelinase
treatment (<0.2 nmol was detected). This establishes that at
least some of the ceramides generated from endogenous sphingomyelin can be hydrolyzed to free sphingosine. It also indicates that the ceramidase is not saturated with endogenous
substrates, otherwise increasing the amount of ceramide
would not have affected sphingosine formation.
Ceramide could also be derived from other sphingolipids.
Therefore, in an analogous experiment, plasma membranes
were treated with endoglycoceramidase, which cleaves the
oligosaccharide moieties of gangliosides. This reduced the
I
I
1
40
80
Time (mid
,
120
FIG.6. Effect of sphingomyelinase on sphingosine formation. Sphingomyelinase (SMase) was added to the plasma membranes in STM, and the sample was incubated a t 37 “C. At various
times, 100-rl aliquots were removed, placed in CHCla:methanol, and
assayed for free sphingosine. Control plasma membranes were incubated in the absence of added sphingomyelinase.
Sphingosine
Formation
sialic acid content of the lipid extracts from approximately 30
nmol at time zero to 9 nmol after 90 min of endoglycoceramidase treatment (theloss of gangliosides was also apparent by
TLC); hence, approximately 20 nmol of ceramide was released. Nonetheless, no increase in sphingosine formation
occurred (Fig. 7), which indicates that the ceramides generated from these gangliosides are less readily hydrolyzed to
sphingosine.
Disappearance of Endogenous Sphingomyelin-Since ceramides from sphingomyelin can be hydrolyzed to free sphingosine, the possibility that sphingomyelin might be the endogenous precursor of sphingosine was considered. The
freshly isolated plasma membranes contained approximately
46 2 nmol of sphingomyelin and 184 32 nmol of phosphatidylcholine/mg of protein. This ratio of sphingomyelin to
phosphatidylcholine (1:4.0) was similar to previous findings
(ie. L5.3 in Ref. 31).
Upon incubation at 37 “C,there was an initial rapid disappearance of sphingomyelin that slowed to reflect a loss of
about 14 nmol by 1 h(Table 11). This complemented the
appearance of sphingosine (cf. Fig. 2 and Table 11); however,
the amounts differed substantially because only 1-2 nmol of
free sphingosine was formed. The major product of the hydrolysis of endogenous sphingomyelin was ceramide, which
increased by about 9 nmol in an hour. No loss of phosphatidylcholine nor of sialic acid containing glycolipids was observed over this time course (not shown). This establishes
that hydrolysis of endogenous sphingomyelin occurs during
incubation of these membranes, but that ceramides accumulate. The accumulation of ceramides could be due to their
inaccessibility to the ceramidase(s) or because the activity of
this enzyme is rate-limiting.
Stimulation of Sphingosine Formation by Exogenous Pre-
Time (rnin)
FIG.7. Effect of endoglycoceramidase on sphingosine formation. Endoglycoceramidase was added to plasma membranes in
STM and the sample was incubated at 37 T . At various times,
aliquots were removed, placed in CHCb:methanol, and assayed for
in Plasma Membranes
10375
cursors-To determine if a portion of the ceramide might be
inaccessible to the ceramidase, the plasma membranes were
solubilized with deoxycholate and sphingosine formation was
measured. The rateandtotalamount
of sphingosine was
increased dramatically (Fig. 8). Sphingosine formation was
further stimulated by addition of exogenous ceramide and, to
a much lesser extent, sphingomyelin. While several mechanisms may account for this behavior (for example, direct
activation of the enzyme by deoxycholate), it is consistent
with detergent facilitation of the access of the ceramidase to
endogenous and exogenous ceramides.
Long-chin Base Release from Exogenous Sphingomyelin
and Ceramide-To this point, the losses of sphingomyelin
and the appearance of cerarnide (a potential source of sphingosine) correlated with increased sphingosine formation, but
the actual hydrolysis of either of these to sphingosine has not
been established directly. To do so, plasma membranes were
incubated with sphingomyelin and ceramide labeled in the
backbone moiety (by reduction of the 4-trans-double bond),
and the release of [3H]sphinganine was measured. In 1 h, 18
k 7 pmol of [3H]sphinganine was released from 0.5 nmol of
[3H]ceramide and 4.5 f 0.4 pmol was formed from the same
amount of [3H]sphingomyelin (450 f 130 pmol of the sphingomyelin wasconverted to [3H]ceramide).Free [3H]sphinganine was also released from these substrates when added in
Triton X-100 (data not shown).
These results arequalitatively, but notquantitatively, similar to the findings with hydrolysis of endogenous sphingomyelin (cf. Table 11) andthe stimulation of sphingosine
formation by exogenous ceramides and sphingomyelin (Fig.
8). However, the amount of sphinganine formed from [3H]
ceramide was much smaller than the stimulation of sphingosine formation from unlabeled ceramide (i.e. 18 uersus 625
pmol) and only about 1%of the ceramide released from [3H]
sphingomyelin appeared as [3H]~phinganine (the amount of
sphingosine formed from endogenous substrates was about
10% of the amount of endogenous ceramide, Table 11). To
some extent, this probably reflects the dilution of the radiolabel into a large pool of unlabeled ceramides; however, an
additional factor was also discovered.
The radiolabeled substrates were synthesized by reduction
of the 4-trans-double bond, so that the backbone of the
ceramide moiety is sphinganine rather than sphingosine. To
see if this might affect the results, the hydrolysis of N palmitoylsphingosine and N-palmitoylsphinganine were compared (Table 111). Since hydrolysis of endogenous substrates
formed sphingosine and sphinganine, theamounts formed
from the exogenous substrates was estimated by subtraction
free sphingosine. Control plasma membranes were incubated in the
absence of added endoglycoceramidase.
TABLE
I1
Sphingomyelin hydrolysis over time inisolated plasma membranes
Isolated plasma membranes were incubated at 37 “C for the times
shown, then the lipids were extracted, separated by thin layer chromatography, and the amounts of sphingomyelin and ceramide determined as described under “Experimental Procedures.”
Sphingomyelin
Incubation
Amount
time
min
36
formed
nrnollmg protein
0
20
46
40
34
32
29
60
90
Ceramide
Amount
hydrolyzed
10
12
14
17
3.7
9.4
12.2
12.3
5.7
8.5
8.6
Not determined
Buffer DOC
SM
Cer
FIG.8. Stimulation of sphingosine formation by deoxycholate ( D O C ) ,sphingomyelin ( S M ) ,and ceramide (Cer).Plasma
membranes were incubated in STM buffer or buffer with 1% (w/v)
sodium deoxycholate alone or with 1 mg/ml of sphingomyelin in
ceramide, and the amount of sphingosine formed was determined by
HPLC asdescribed under “Experimental Procedures.”
in
Membranes
Plasma
Sphingosine
Formation
10376
TABLEI11
Hydrolysis of N-palmitoylsphingosineand -sphinganine
by plasma membranes
The membranes were incubated with the indicated sphingolipids
in 1% deoxycholate and the amount of free sphingosine and sphinganine (given as themean f S.D.) was determined by HPLC. For the
control, the net increase is the change uersus time zero; for the other
groups, the control andthe time zero for that group have been
subtracted.
Time of incubation
Condition
0 min
30 min
60 min
90 min
Pml
Control (deoxycholate alone)
Sphingosine
32 f 1
180f5
423 f 18
952 f 17
Net increase
148
391
920
Sphinganine
3.9 f 1.0
4.5
f 0.8
7.7
f 0.1
11.9
f 1.3
Net increase
0.6
3.8
8.0
+N-palmitoylsphingosine
Sphingosine
32 f 4
241 f 5
527 f 37 1137 + 4
Net increase
61
104
185
+N-palmitoylsphinganine
Sphingosine
29 f 5
199 f 33 452 f 50 1048 f 490
Net increase
22
32
99
Sphinganine
7 . 8 2 0.1 18.8 f 3.4 30.1 f 4.1
43 f 2.0
Net increase
27.2
18.5
10.4
formation, [3H]sphinganine was incubated with the plasma
membranes, and the radiolabeled products were examined
(see “Experimental Procedures”). No radiolabel was visible in
the ceramide (nor dihydroceramide) region of the TLC plates;
hence, there appears to
be little metabolism of the sphingosine
in isolated plasma membranes under the conditions used in
this study. This does not preclude the existence of an enzyme
system that requires other co-factors (e.g. fatty acyl-CoAs, for
example) for activity in vivo.
Other Analyses-It was recently shown that 1,2-diacylglycerols, but not phorbol esters, activate the acid (lysosomal)
sphingomyelinase in GH3 pituitary cell homogenates, but not
the neutral sphingomyelinase (32). We observed no effect on
sphingosine formation when the plasma membranes were
treated with either 1,L”diacylglycerolsor phorbol esters (data
not shown).
DISCUSSION
These investigations show that ratliver plasma membranes
contain free sphingosine and that half or more of the sphingosine in liver appears to be associated with this subcellular
fraction. In addition, the amount increases during incubation
of the membranes at 37 “C,indicating that plasma membranes
containboththe
endogenous substratesand enzymes for
sphingosine
formation.
The characteristics of this system
of the data for the group incubated with deoxycholate alone.
were
that
it
has
a
neutral
pH optimum, is divalent cationIt is evident from these results that thesphinganine-containing ceramide is hydrolyzed less well (by about 6-f0ld), and dependent, is highly temperature-dependent, and is stimuthis could account for the noted differences between the lated by adding exogenous ceramide and by treatment of the
membranes with sphingomyelinase. Ceramides were also rehydrolysis of labeled and unlabeled substrates.
Sphingosine Formation from Lysosphingomyelin-An alter- leased from endogenous sphingomyelin during incubation of
native mechanism for sphingosine formation from sphingo- the plasma membranes alone, and a portion of this may have
myelin could be removal of the amide-linked fatty acid fol- contributed to sphingosine formation.
The stimulation of sphingosine formation by ceramides
lowedbycleavage of the headgroup. Although we did not
indicates that the endogenous ceramides are not present at
observe sphingosine 1-phosphorylcholine during the HPLC
analyses, this pathway cannot be excluded onthis basis alone; “saturating” levels for this system. This indicates that either
and, hence, the fate of lysosphingomyelin was also examined. the enzyme activity per se, or the availability of the ceramide
When 500 pmol of sphingosine 1-phosphorylcholine was in- to the enzyme, is rate-limiting for sphingosine formation. It
cubated for 1h with 0.1 mg of plasma membranes the amount was evident that all ceramides were not utilized equally because treatment of the membranes with endoglycoceramidase
of sphingosine increased to 198 f 13 pmol, compared to 34 k
caused no increase in sphingosine formation. These observa4 pmol at zero time. The elution of o-phthalaldehyde-sphintions, plus the fact that sphingosine formation was stimulated
gosine 1-phosphorylcholine (at 7.1 min) did not interfere with
the analysis of o-phthalaldehyde-sphingosine(at 8.5 min). by deoxycholate, suggest that some of the ceramides are not
accessible.
Hence, plasma membranes also containapotent
activity
The system also did not appear to utilize ceramides concapable of releasing the phosphorylcholine group from lysotaining
a sphinganine backbone as effectively as those with
sphingomyelin.
Sphingosine Formation de Novo-Long-chain base synthe- sphingosine. This may be due to several causes (such as
differences in the physical properties of these compounds)
sis de novo involves the condensation of serine and palmitoylthat were beyond the scope of this investigation. Interestingly,
CoA and is thought to proceed via formation of N-acylsphinceramides with the sphinganine backbone are intermediates
ganine, which is converted to N-acylsphingosine (ceramide)
in the de novo pathway of sphingolipid biosynthesis (18);thus,
(18).Nonetheless, the possibility that sphingosine was formed
directly in plasma membranes was tested by assaying for the
incorporation of [14C]serine into long-chain bases (30). Although activity (approximately 20 pmol/min/mg protein) was
detected when the membranes were supplemented with palmitoyl-CoA (the optimal conditions for assaying the microsomal serine palmitoyltransferase), none was detected under
the assay conditions used to measure sphingosine formation
Probln Klnue C
Other Klnaws
(i.e. in STM buffer without addition of palmitoyl-CoA).
Hence, the sphingosine appearing in isolated plasma memFIG. 9. Schematic representation of complex sphingolipid
branes does not reflect de novo synthesis, even though serine hydrolysis to yield free sphingosine. Based on this study, it
palmitoyltransferase is present due to microsomal contami- appears that the headgroup of complex sphingolipids (SL),such as
nation and can synthesize a long-chain base (presumably 3- sphingomyelin, are cleaved to yield ceramides (Cer), which can be
hydrolyzed to free sphingosine. Evidence for modulation of sphingoketosphinganine) when palmitoyl-CoA is added.
sine formation has been seen in other systems, as has inhibition of
Metabolism of Exogenously AddedLong-chain Bases-Since
protein kinase C by exogenous sphingosine and the existence of
removal of the plasma membrane-associated sphingosine by kinases that appear to be activated by this compound (see “Discusmetabolism could result in underestimation of the rate of sion”).
sph
(Y
\“’
Sphingosine
Formation
Membranes
in Plasma
10377
REFERENCES
the lesser utilization of these compounds may help delineate
pools designated for sphingolipid biosynthesis versus turn1. Karlsson, K.-A. (1970) Chem. Phys. Lipids 5 , 6-43
2. Hakomori, S.I. (1981) Annu. Reu. Biochem. 5 0 , 733-764
over.
3. Kanfer, J. N., and Hakomori, %-I. (eds) (1983) Sphingolipid
Many of the characteristics of this system were similar t o
Biochemistry, Plenum Publishing Co., New York
thesphingomyelinaseandceramidaseactivitiesthat
have
4. Feizi, T., and Childs, R. A. (1985) Trends Biochem. Sci. 1 0 , 24been observed previously when plasma membranes were in29
cubated with exogenous radiolabeled substrates (33-36). For
5. Hannun, H., Loomis, C. R., Merrill, A. H., Jr., and Bell, R.M.
example, the plasma membrane sphingomyelinase is M$+(1986) J. Biol. Chem. 2 6 1 , 12604-12609
dependent and hasa neutral to alkaline pH optimum, and the6. Hannun, Y., and Bell, R. M. (1987) Science 235,670-674
7. Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and
ceramidase also has optimal activity above pH 7. However,
Lambeth, J. D. (1986) J. Biol. Chem. 2 6 1 , 12616-12623
earlier investigationshave not linked these activities to sphin8. Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A,,
gosine formation by the action of these enzymes on endogeBell, R. M., and Kinkade, J. M., Jr. (1986) J. Biol. Chem. 2 6 1 ,
nous substrates. More detailed comparisons
between our find12610-12615
ings and the earlier studies are complicated by the fact that 9. Merrill, A. H., Jr., and Stevens, V. L. (1989) Biochim. Biophys.
Acta 1 0 1 0 , 131-139
sphingosine formation as assayed in this study (from
endog10. Faucher, M., Giron&s,N., Hannun, Y. A., Bell, R. M., and Davis,
enous substrates)probably involves at least two activities.
R. J. (1988) J. Biol. Chem. 2 6 3 , 5319-5327
It is notknown if this sphingosine formationreflects mem11. Northwood, I. C., and Davis, R. J. (1988) J . Biol. Chem. 2 6 3 ,
brane degradation after disruptionof the liver cells, a normal,
7450-7453
ongoing process of plasma membrane remodeling, or the re- 12. Jefferson, A.B., and Schulman, H. (1988) J. Biol. Chem. 2 6 3 ,
lease of sphingosine for a functional purpose. In fact, very
15241-15244
little is known about the in viuo turnover of the sphingosine 13. Wilson, E., Wang, E., Mullins, R. E., Uhlinger, D. J., Liotta, D.
C., Lambeth, J. D., and Merrill, A. H., Jr. (1988) J. Biol. Chem.
backbone of cellular sphingolipids(37). Release of sphingosine
263,9304-9309
might haveeffects on cellular functions since sphingosine and
14. Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L.,
lysosphingolipids are potent inhibitorsof protein kinaseC ( 5 ,
Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171,3736) and affect numerous
cellular responses to agonists that act
381
via this enzyme (9) and other factors (10-12). Kolesnick and 15. Kobayashi, T., Mitsuo, K., and Goto, I. (1988) Bur. J . Biochem.
171,747-752
Clegg (38) have shown that treatment of GHs pituitary cells
16. Nimkar, S., Menaldino, D., Merrill, A. H., and Liotta, D.C.
with sphingomyelinase alters the phorbol ester-induced trans(1988) Tetrahedron Lett. 2 9 , 3037-3040
location of protein kinase C to membranes, which could be
17. Gaver, R., and Sweeley, C. C. (1966) J. Am. Chem. SOC.
88,3643due t o sphingosine release in their preparation. This implies
3647
that endogenously generated sphingosine might affect the 18. Merrill, A.H., Jr., and Wang, E. (1986) J. Biol.Chem. 2 6 1 ,
behavior of protein kinase C and/or other systems thathave
3764-3769
been shown t o be altered by sphingosine added exogenously 19. Schwarzmann, G. (1978) Biochim. Biophys. Acta 529, 106-114
20. Hubbard, A. L., Wall, D. A., and Ma, A. (1983) J . Cell Biol. 9 6 ,
(9-11).
21
"
. 7-229
-__
A model consistent with thesefindings, and illustrating this
21. Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1986) J . Biol. Chem.
possibility, is shown in Fig. 9. It suggests that sphingosine
261,14833-14836
could arise from sphingolipid hydrolysis via ceramides (and a 22. Sawant, P. L., Shibko, S., Kumta, U. S., and Tappel, A. L. (1964)
neutral ceramidase); however, the finding of an activity that
Biochim. Biophys. Acta 8 5 , 82-92
hydrolyzes sphingosine 1-phosphorylcholine prevents exclu- 23. Williams, R. D., Wang, E., and Merrill, A. H., Jr. (1984) Arch.
Biochem. Biophys. 2 2 8 , 282-291
sion of other routesvia lysosphingolipids. The model includes
the speculation that sphingosine formation can be activated 24. Aronson, N. N., and Touster, 0. (1974) Methods Enzymol. 3 1 ,
90-102
by appropriate stimuli. Little is known about this aspect of 25. Trouet, A. (1974) Methods Enzymol. 3 1 , 323-329
sphingolipid metabolism; however, the modulation of sphin- 26. Ames, B. N. (1966) Methods Enzymol. 8 , 115-118
gosine levels by various agonists has been seen in neutrophils27. Kreibich, G., Debey, P., and Sabatini, D. D. (1973) J . Cell Biol.
58,436-462
(13). If theneutral sphingomyelinase is importantinthe
control of sphingosinegeneration,thesefindings
may be 28. Fishman, W. H. (1974) in Methods ofEnzymaticAna1ysi.s (Bergmeyer, H.-U., ed) Vol. 2, pp. 929-943, Academic Press, New
pertinent to the action of dexamethasone, which has been
York
shown to alter sphingomyelin metabolism (39). It may also 29. Suzuki, K. (1964) Life Sci. 3 , 1227-1233
be relevant to the action (and/or toxicity) of volatile anes- 30. Merrill, A. H., Jr. (1983) Biochim. Biophys. Acta 7 5 4 , 284-291
thetics since Mooibroeck et al. (36) have shown that com- 31. Wood, R., Upreti, G. C., and deAntueno, R. J. (1986) Lipids 2 1 ,
292-300
pounds such as methoxyfurane and halothane stimulate
a
plasma membrane sphingomyelinase and the
loss of much of 32. Kolesnick, R. N. (1987) J. Biol. Chem. 262,16759-16762
the plasma membranesphingomyelin. In addition, Deshmukh 33. Rao, B. G., and Spence, M. W. (1976) J. Lipid Res. 17,506-515
K. Y., and Yazaki, P. J. (1979) J. Lipid Res. 2 0 , 456and Radin (40) have noted significantlability of sphingomye- 34. Hostetler,
463
lin in intact brain and brain
homogenates, which is suggestive 35. Spence, M. W., Beed, S., and Cook, H. W. (1986) Biochem. Cell
of asphingomyelinase that can be "triggered" byvarious
Biol. 6 4 , 400-404
stimuli. Since the plasma membranes
used in our studies were 36. Mooibroek, M. J., Cook, H. W., Clarke, J. T. R., and Spence, M.
W. (1985) J . Neurochem. 4 4 , 1551-1558
necessarily isolated from sedated animals, we do not know if
the anesthetics or the procedures involved in liver perfusion 37. Medlock, K. A., and Merrill, A. H., Jr. (1988) Biochem. Biophys.
Res. Commun. 157,232-237
andmembrane isolationhave alreadystimulated,orsup38. Kolesnick, R. N., and Clegg, S.(1988) J . Biol. Chem. 263,6534pressed, the systems that cause
sphingosine formation. These
6537
possibilities are under investigation.
39. Nelson, D. H., and Murray, D. K. (1986) Biochem. Biophys. Res.
Acknowledgments-We thank Dr. Sanjay Nimkar forhelp in preparingthe C2O-sphingosine andsphinganine used asaninternal
standard and Drs. Victoria L. Stevens and J. David Lambeth for
helpful discussions concerning this project.
Commun. 1 3 8 , 463-467
40. Deshmukh, G. D., and Radin, N. S.(1985) J. Neurochem. 4 4 ,
1152-1155
41. Slife,C. W., Wang,E.,Wang,
S., and Merrill, A. H. (1988)
FASEB J. 2 , A1416