From the Departments of Pediatrics and
§ Clinical Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, ¶ Zentrum Biochemie und
Molekuläre Zellbiologie, Biochemie II, Universität
Göttingen, 37073 Göttingen, Germany, and
Department of Medical Chemistry, Institute of Biomedicine,
University of Helsinki, 00016 Helsinki, Finland
Received for publication, February 28, 2001, and in revised form, March 15, 2001
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ABSTRACT |
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Previous studies suggest that the steps of
the CDP- choline pathway of phosphatidylcholine synthesis are tightly
linked in a so-called metabolon. Evidence has been presented that only
choline that enters cells through the choline transporter, and not
phosphocholine administered to cells by membrane
permeabilization, is incorporated into phosphatidylcholine. Here, we
show that [14C]phosphocholine derived from the
lysosomal degradation of [14C]choline-labeled
sphingomyelin is incorporated as such into phosphatidylcholine in human
and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct
[14C]sphingomyelin to the lysosomal degradation pathway.
Free labeled choline was not found either intracellularly or in the
medium, not even when the cells were energy-depleted. Deficiency of
lysosomal acid phosphatases in mouse or alkaline phosphatase in human
fibroblasts did not affect the incorporation of lysosomal
[14C]sphingomyelin-derived
[14C]phosphocholine into phosphatidylcholine, supporting
our finding that phosphocholine is not degraded to choline prior to its
incorporation into phosphatidylcholine. Inhibition studies and analysis
of molecular species showed that exogenous [3H]choline
and sphingomyelin-derived [14C]phosphocholine are
incorporated into phosphatidylcholine via a common pathway of
synthesis. Our findings provide evidence that, in fibroblasts,
phosphocholine derived from sphingomyelin is transported out of the
lysosome and subsequently incorporated into phosphatidylcholine without
prior hydrolysis of phosphocholine to choline. The findings do not
support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.
De novo synthesis of phosphatidylcholine
(PC)1 predominantly occurs
via the CDP-choline pathway (1). Choline can enter the cell via the
ATP-dependent choline transporter present in the cell
membrane and is subsequently phosphorylated to phosphocholine (PCho) by the cytosolic enzyme choline kinase (EC 2.7.1.32). PC is
then synthesized in two steps, catalyzed by the rate-limiting cytosolic
and membrane-bound enzyme CTP:phosphocholine cytidylyltransferase (CT;
EC 2.7.7.15) and the membrane-bound enzyme
CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase
(EC 2.7.8.2). For some cell types, including fibroblasts, it has been
reported that the steps of PC synthesis are tightly linked in a
so-called metabolon (2-4). In these cell types only choline that
entered the cells through the choline transporter was incorporated into
PC. In contrast, PCho administered to cells by partial membrane
permeabilization was not incorporated into PC (2, 4).
A possible endogenous source of PCho and choline is provided by the
lysosomal degradation of PC and sphingomyelin (SM) by acid
sphingomyelinase (5). We hypothesized that if only choline, and not
PCho, can enter the PC metabolon and be incorporated into PC in human
fibroblasts, then PCho that is formed during the degradation of SM by
acid sphingomyelinase in the lysosome must be degraded to choline to be
incorporated into PC. Candidate enzymes for intralysosomal hydrolysis
of PCho to choline are lysosomal acid phosphatase (LAP) and
tartrate-resistant acid phosphatase (TRAP) (6-8). Their in vivo substrates and functional roles are unclear. LAP is
ubiquitously expressed, and TRAP is predominantly expressed in alveolar
macrophages and osteoclasts (9), suggesting a specific function of the latter enzyme in these cell types. Another possible route for lysosomal
SM-derived PCho is transport across the lysosomal membrane (by an
unknown transporter), followed by hydrolysis to choline and subsequent
incorporation of choline into the PC synthesis metabolon. A candidate
enzyme for the hydrolysis of cytosolic PCho is alkaline phosphatase. In
human skin fibroblasts, alkaline phosphatase is predominantly located
at the plasma membrane (10, 11). Phosphoethanolamine and
pyridoxal-5'-phosphate have been described as physiological
substrates for this enzyme (10). We have investigated the possible
role of LAP, TRAP, and alkaline phosphatase in the hydrolysis of PCho
derived from the late endosomal/lysosomal degradation of SM.
In this study we compared exogenous choline and late
endosome/lysosome-derived PCho as substrates for PC synthesis.
Hexadecylphosphocholine (HePC) was used as an inhibitor of PC synthesis
(12-16). The results indicate that PCho derived from lysosomal SM is
not degraded to choline but is incorporated into PC as such. We
conclude that the PC synthesis metabolon either does not exist in
fibroblasts or is not as inaccessible to PCho as indicated by previous studies.
Materials--
Culture flasks, Ham's F-10, Dulbecco's modified
Eagle's medium with Glutamax, fetal bovine serum (FBS),
penicillin, and streptomycin were obtained from Life Technologies,
Inc. Culture plates (6 wells) were obtained from Greiner,
Nurtingen, Germany. Recombinant human apolipoprotein E3 (apoE) was
obtained from PanVera Corporation (Madison, WI). HePC,
2-deoxyglucose, digitonin, and bovine serum albumin were obtained from
Sigma. HPTLC plates (number 5633) and NaN3 were from Merck.
[Choline-methyl-14C]sphingomyelin (specific
activity, 54.5 Ci/mol; >99% purity) was obtained from ICN
Biomedicals, Costa Mesa, CA, and
[methyl-3H]choline chloride (specific
activity, 81 Ci/mmol) was obtained from PerkinElmer Life Sciences.
Cell Culture--
Human fibroblasts from three healthy subjects
and from hypophosphatasia (alkaline phosphatase deficiency), familial
hypercholesterolemia (low density lipoprotein (LDL) receptor
deficiency), and Niemann-Pick A (NP-A) (acid sphingomyelinase
deficiency) patients were cultured as described previously (5). Primary
embryonic (embryonic day 12.5) fibroblasts from LAP-deficient
(17), TRAP-deficient (18), LAP/TRAP double-deficient, and control mice
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% FBS, penicillin (10 IU/ml), and streptomycin (10 IU/ml).
LAP/TRAP double-deficient mice are viable but more severely affected
than single-deficient mice.2
[14C]SM Incubations--
Cells were plated in
6-well plates (1.5-2.0 × 105 cells/well) and
cultured for 4-5 days until confluence. They were then starved on
Ham's F-10 or Dulbecco's modified Eagle's medium with 10%
lipoprotein-deficient serum for 24 h to enhance LDL receptor
expression. Subsequently, the medium was replaced with medium
containing 0.8 nmol of [14C]SM (dissolved in ethanol;
final concentration, 0.2%, v/v) in complex with apoE (5 µg/ml)
([14C]SM·apoE). Cells were incubated for 30 min
at 37 °C, then the medium was replaced by medium (10% FBS) without
[14C]SM·apoE, and cells were kept at 18 °C for 30 min to enable internalization of the lipid·apoE complex. Medium was
replaced again with fresh medium (10% FBS) for chase studies at
37 °C for various periods of time. In some experiments 50 µM HePC was present during the chase. For energy
depletion, 10 mM NaN3 and 10 mM
2-deoxyglucose were dissolved in the chase medium. After incubations
cells were trypsinized and pelleted.
[3H]Choline Labeling--
Fibroblasts were plated
as described above and incubated with 2 µCi of
[3H]choline per well (dissolved in ethanol) in medium
(10% FBS) for 2 h. In some experiments 50 µM HePC
or 10 mM NaN3 + 10 mM 2-deoxyglucose was present during the incubation.
Digitonin Permeabilization--
Digitonin permeabilization was
done according to Wanders et al. (19), with slight
modifications. Briefly, cells were incubated for 30 min with
[14C]SM·apoE, washed, trypsinized and taken up in
buffer (final pH 7.4) containing 150 mM KCl, 25 mM Tris-HCl, 2 mM EDTA, 10 mM
KH2PO4, 0.1% (w/v) bovine serum albumin, and
digitonin at different concentrations. After a 5-min incubation at
37 °C, the cells were pelleted by centrifugation. Hexosaminidase
activity was measured in the pellets and the supernatant to determine
the intactness of the lysosomal membrane (20). The protein
concentration was determined according to Lowry et al.
(21).
Determination of Degradation Products--
The lipids in the
pellets, supernatant, or medium were separated from aqueous metabolites
with Folch extraction (22). The radiolabeled lipids were separated by
HPTLC (5), and aqueous metabolites were separated by ion-exchange
chromatography (23). Quantification of each radiolabeled product was
achieved by liquid scintillation counting in 10 ml of Ultima Gold
(Packard Instrument Co.).
Analysis of the Molecular Species of PC, Synthesized from
Exogenous [3H]Choline and from
[14C]SM-derived [14C]PCho--
Fibroblasts
were incubated with [3H]choline or
[14C]SM·apoE as described above. The fibroblast pellets
were homogenized, and lipids were extracted with chloroform:methanol
(2:1; v/v) in the presence of 0.01 mM butylated
hydroxytoluene and washed according to Folch et al.
(22). The lower phase was dried under N2, dissolved in 100 µl of chloroform:methanol (2:1; v/v), and PC was purified by
preparative TLC as described (24). The PC molecular species were then
separated by reverse-phase HPLC (25), and their radioactivity was
determined by liquid scintillation counting. The identity of the major
species was determined based on the retention time of standards, as
well as by electrospray mass spectrometry, as described elsewhere
(26).
Validation of the Specific Uptake of [14C]SM·apoE
by the LDL Receptor and Its Lysosomal Degradation--
To study the
fate of lysosomal SM-derived PCho, fibroblasts were incubated with
[14C]SM·apoE. The lipid·apoE complex was specifically
taken up by the LDL receptor. This was verified by measurement of the
uptake of [14C]SM·apoE by LDL receptor-deficient
fibroblasts from a familial hypercholesterolemia patient. In these
cells the uptake of [14C]SM was less than 8% compared
with normal fibroblasts. When normal fibroblasts were incubated with
[14C]SM, not in complex with apoE but in the presence of
bovine serum albumin, the uptake of [14C]SM was
negligible. Furthermore, LDL receptor-mediated uptake of
[14C]SM·apoE ensured exclusive delivery of the labeled
lipid to the lysosomal compartment. This was verified by incubating
NP-A fibroblasts, which are deficient in the lysosomal enzyme acid
sphingomyelinase, with [14C]SM·apoE. In these cells,
[14C]SM·apoE was taken up in similar amounts as in
normal cells, but [14C]SM was not degraded. Even after
prolonged incubation, no labeled PCho or PC was formed (Fig.
1). These experiments show that
[14C]SM·apoE uptake was mediated by the LDL receptor.
[14C]SM was subsequently transported to the late
endosomal/lysosomal compartment and degraded by acid sphingomyelinase
in normal fibroblasts. Thus, incubation of fibroblasts with
[14C]SM·apoE as described results in the hydrolysis of
[14C]SM by acid sphingomyelinase in the lysosomal
compartment, producing lysosomal SM-derived (endogenous)
[14C]PCho.
Lysosomal Degradation of [14C]SM--
The
degradation of [14C]SM was determined after a 30-min
incubation with [14C]SM·apoE (pulse) and during a 3-h
chase in normal medium (Fig. 1). During the pulse period 36% of total
intracellular SM was degraded to PCho. During the initial phase of the
chase, production of labeled PCho was prominent. Thereafter, the amount
of radiolabeled PCho gradually decreased followed by the appearance of
labeled PC. At all time points the amount of radioactivity in the
medium was negligible. The recovery of total radioactivity for all cell lines at each time point was 98.7 ± 3.2% as compared with
t = 0. Free choline was not detected during pulse or
chase, neither intracellularly nor in the medium.
Role of Acid Phosphatases in Incorporation of SM-derived PCho into
PC--
To determine the role of LAP and TRAP in the incorporation of
lysosomal [14C]SM-derived PCho into PC, primary embryonic
fibroblasts from normal, LAP-deficient, TRAP-deficient, or LAP/TRAP
double-deficient mice were pulsed with [14C]SM·apoE and
subsequently chased. In each deficient cell type, [14C]SM
was similarly degraded compared with cells from normal mice (Fig.
2A). The degradation of
[14C]SM produced [14C]PCho in all cell
types (Fig. 2B). No labeled free choline was detected.
Furthermore, labeled PCho was incorporated into PC in the deficient
cells at rates comparable with or even faster than in normal cells
(Fig. 2C). Thus, in mouse fibroblasts, acid phosphatase activity was not required for the incorporation of lysosomal
[14C]SM-derived PCho into PC.
Role of Alkaline Phosphatase in Incorporation of SM-derived PCho
into PC--
The role of alkaline phosphatase in the incorporation of
lysosomal [14C]SM-derived PCho into PC was determined
with alkaline phosphatase-deficient fibroblasts. These cells were
pulsed with [14C]SM·apoE and subsequently chased.
[14C]SM was degraded to PCho, which was then incorporated
into PC (Fig. 3). Labeled free choline
was found neither in the cellular fraction nor in the medium. These
results were similar to the degradation of [14C]SM in
normal fibroblasts (Fig. 1). This indicated that alkaline phosphatase
did not play a role in the incorporation of lysosomal [14C]SM-derived PCho into PC.
Effect of HePC and Energy Depletion on the Incorporation of
Exogenous Choline and SM-derived PCho into PC--
Administration of
radiolabeled choline to normal fibroblasts via the culture medium
resulted in its incorporation in intracellular PCho and PC (Fig.
4). The presence of the PC synthesis
inhibitor HePC caused accumulation of PCho and decreased PC synthesis,
in agreement with the reported specific inhibition by HePC of CT, the
rate-limiting enzyme of PC synthesis (12-14, 16). Energy depletion,
accomplished by the addition of NaN3 and 2-deoxyglucose, caused accumulation of intracellular choline and decreased the amounts
of labeled PCho and PC (Fig. 4). The total choline uptake in the
presence of NaN3 and 2-deoxyglucose was ~25% of the
uptake in the absence of these additives (with or without HePC),
showing that choline uptake is energy-dependent.
Addition of HePC after the incubation with [14C]SM·apoE
had no effect on the degradation of SM, but it inhibited PC synthesis from the endogenous PCho (Fig. 5). Energy
depletion by the addition of NaN3 and 2-deoxyglucose during
the chase resulted in slower degradation of SM and slower synthesis of
labeled PC (Fig. 6). Thus, the
incorporation of SM-derived PCho into PC is
energy-dependent. Under conditions of energy depletion
labeled free choline was detected neither intracellularly nor in the
medium.
Subcellular Localization of [14C]SM-derived
PCho--
To study the subcellular localization of
[14C]SM-derived PCho, normal human fibroblasts,
pre-labeled with [14C]SM·apoE, were treated with
increasing concentrations of digitonin to selectively permeabilize the
plasma membrane. Leakage of hexosaminidase activity to the
permeabilization buffer was used as an indicator of the integrity of
the lysosomal membrane. At low digitonin concentrations (5-10
µg/ml), ~88% of [14C]PCho, but only a small percent
of the cellular hexosaminidase, was found in the permeabilization
buffer (Fig. 7). Increasing the digitonin
concentration above 10 µg/ml markedly enhanced the release of
hexosaminidase, and it was complete at a concentration of 160 µg/ml
digitonin. These data indicate that most of the
[14C]SM-derived PCho was present in the cytoplasm.
Notably, the addition of 5 µg/ml digitonin or more caused leakage of
cytoplasmic proteins to the permeabilization buffer (40-60% of total
cell-associated protein). This indicates that the absence of
hexosaminidase activity in the permeabilization buffer of cells treated
with digitonin concentrations of 10 µg/ml or less was indeed because
of intactness of the lysosomal membrane, rather than inability of the
hexosaminidase molecules to pass through the plasma membrane.
Analysis of the Molecular Species of PC, Synthesized from
[3H]Choline and [14C]SM--
To
investigate further whether SM-derived PCho could enter the PC
synthesis metabolon, the molecular species of PC synthesized from
[3H]choline or [14C]SM were analyzed. As
can be seen in Fig. 8, the distribution of the radioactivity among PC species synthesized from both precursors is nearly identical, suggesting a common biosynthetic pathway.
In the present study we describe the incorporation of labeled PCho
derived from the late endosomal/lysosomal degradation of SM into PC via
the CDP-choline pathway of PC synthesis (1). We have shown that the
uptake of [14C]SM·apoE was LDL receptor-mediated and
that [14C]SM was exclusively metabolized in the late
endosomal/lysosomal compartment, because NP-A fibroblasts, which are
deficient in acid sphingomyelinase, did not metabolize
[14C]SM. Also, in this cell line no evidence was found
for the incorporation of SM-derived [14C]choline into PC,
even after prolonged incubation. This finding excludes the involvement
of the reverse reaction catalyzed by SM synthase (27) in our
experimental setup. In addition we have clearly shown that during the
initial degradation of [14C]SM to
[14C]PCho, [14C]PC synthesis was
negligible, indicating a lag phase in the process of PC synthesis from
SM-derived PCho.
In these in situ experiments, we did not observe production
of radiolabeled free choline from SM, indicating that SM-derived PCho
was not hydrolyzed to choline prior to its incorporation into PC. Even
under conditions of energy depletion, when resynthesis of PCho from
choline would be inhibited, no radiolabeled free choline was detected.
These results corroborate our previous in vitro studies with
fibroblast homogenates at acidic pH, where we found PCho, and not
choline, as an end product of PC degradation. PCho, which was
predominantly generated by acid sphingomyelinase activity, was
apparently not further degraded by acid phosphatases (5). Furthermore,
in another in vitro study the incubation of purified rat
liver lysosomes with PC or PCho at acidic pH levels produced virtually
no free choline (28).
The possible role of acid phosphatases in the degradation of PCho in
the present in situ study was investigated with embryonic fibroblasts from LAP and/or TRAP (double)-deficient mice. These mice
have been developed to study the physiological role of the acid
phosphatases (17, 18).2 The natural substrates for
these enzymes have not been elucidated. However, an early study
reported that PCho was a poor substrate for a purified rat liver acid
phosphatase in vitro (29). Our study shows that PCho is not
an in situ substrate for acid phosphatases present in
mouse fibroblasts.
Because we have not found any indication that SM-derived
PCho was hydrolyzed to free choline, it is likely that PCho is an end
product of lysosomal degradation. However, most of the intracellular SM-derived PCho was present in the cytoplasm, not in the lysosomes, as
was shown by digitonin-induced membrane permeabilization. This suggests
that PCho must be transported out of the lysosomes by an as yet unknown
transporter. Subsequently, it can serve as a substrate for PC
synthesis. The experiments with alkaline phosphatase-deficient human
fibroblasts indicate that PCho is not hydrolyzed in the cytosol by
alkaline phosphatase. In conclusion, all results indicate that PCho is
not hydrolyzed prior to its incorporation into PC.
Our data point to a direct incorporation of cytosolic PCho,
endogenously produced by the late endosomal/lysosomal breakdown of SM,
into PC. This is not compatible with previous reports that concluded
that in C6 rat glioma cells,
C3H10T1/2 fibroblasts, and rat
hepatocytes the intermediates of PC synthesis are channeled to the
enzymes involved, defining a so-called PC synthesis metabolon (2, 4).
This conclusion was based on experiments in which PCho, which was
delivered to the cytosol via partial membrane permeabilization, was not
used for PC synthesis. However, our results show that PCho, which is
derived from the lysosomal degradation of SM, is directly incorporated
into PC via the CDP-choline pathway of PC synthesis. The latter is
supported by the fact that HePC, a specific inhibitor of this pathway,
inhibited the incorporation of both exogenous choline and endogenous
PCho into PC. HePC has been shown by several groups to inhibit PC
synthesis in different cell types (12-16), including human breast
fibroblasts (30). HePC inhibits CT, the rate-limiting enzyme of PC
synthesis, which results in the accumulation of PCho. In those previous
studies with HePC, the incorporation of exogenous choline into PC was investigated. In the present study with human skin fibroblasts, the
inhibition of PC synthesis from exogenous radiolabeled choline by HePC
and the subsequent accumulation of PCho were confirmed. HePC also
inhibited PC synthesis from lysosomal SM-derived PCho in these cells,
suggesting that this PCho, like exogenous choline, entered the
CDP-choline pathway of PC synthesis that is regulated by CT. The fact
that the molecular species of PC synthesized from exogenous
[3H]choline and of PC synthesized from late
endosomal/lysosomal [14C]PCho were nearly identical
indicates a common diacylglycerol precursor pool. This supports the
idea that [14C]SM-derived PCho enters the normal PC
biosynthetic pathway, rather than incorporating to PC via another route.
In conclusion, our results clearly indicate that in human skin
fibroblasts, PCho that is produced by acid sphingomyelinase activity is
transported intact from the late endosomal/lysosomal compartment to the
cytoplasm and then enters the CDP-choline pathway of PC synthesis. This
means that if the proposed PC synthesis metabolon exists, it is not as
impermeable to intermediates of PC synthesis as indicated by previous
studies (2, 4).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Metabolism of [14C]SM,
specifically directed to the late endosomal/lysosomal compartment.
Control fibroblasts (n = 4; closed symbols)
and Niemann-Pick A (acid sphingomyelinase-deficient) fibroblasts
(n = 1; open symbols) were incubated with
[14C]SM and apoE for 30 min as described under
"Experimental Procedures." At indicated times during chase,
radioactivity in intracellular SM (circles), PCho
(triangles), and PC (squares) was determined.
Data are expressed as percentages of total cell-associated
radioactivity at t = 0. The mean total cell-associated
radioactivities were 32.9 × 103 dpm/mg cellular
protein (control) and 39.8 × 103 dpm/mg cellular
protein (NP-A). Values are given as means ± S.D.
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Fig. 2.
Role of acid phosphatases in the
incorporation of SM-derived PCho into PC. Primary embryonic (E
12.5) fibroblasts from LAP-deficient (open squares),
TRAP-deficient (open triangles), LAP/TRAP double-deficient
(open circles), and control (closed circles) mice
were incubated with [14C]SM and apoE for 30 min as
described under "Experimental Procedures." At indicated times
during chase, radioactivity in intracellular SM (A), PCho
(B), and PC (C) was determined. Data are
expressed as percentages of total cell-associated radioactivity at
t = 0. The mean total cell-associated radioactivities
were 26.0 × 103 dpm/mg cellular protein
(LAP-deficient), 22.4 × 103 dpm/mg cellular protein
(TRAP-deficient), 23.4 × 103 dpm/mg cellular protein
(LAP/TRAP double-deficient), and 26.7 × 103 dpm/mg
cellular protein (control). Values are given as means ± S.D. from
three independent experiments.
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Fig. 3.
Role of alkaline phosphatase in the
incorporation of SM-derived PCho into PC. Fibroblasts from an
alkaline phosphatase-deficient patient were incubated with
[14C]SM and apoE for 30 min as described under
"Experimental Procedures." At indicated times during chase,
radioactivity in intracellular SM (circles), PCho
(triangles), and PC (squares) was determined.
Data are expressed as percentages of total cell-associated
radioactivity at t = 0. The mean total cell-associated
radioactivity was 32.3 × 103 dpm/mg cellular protein.
Values are given as means from two independent experiments.
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Fig. 4.
Effect of HePC and energy depletion on the
incorporation of exogenous [3H]choline into PCho and
PC. Control fibroblasts were incubated with
[3H]choline for 2 h without additives
(control) or in the presence of 50 µM HePC or
10 mM NaN3 + 10 mM 2-deoxyglucose
(NaN3+2-DG). The percentage of total cell-associated
radioactivity in PC (black bars), PCho (gray
bars), and choline (white bars) is depicted. The mean
total cell-associated radioactivities were 42.7 × 104 ± 14.0 × 104 dpm/mg cellular protein (control),
39.3 × 104 ± 12.8 × 104 dpm/mg
cellular protein (HePC), and 11.9 × 104 ± 2.1 × 104 dpm/mg cellular protein (NaN3+2-DG).
Results are depicted as means ± S.D. from three independent
experiments.
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Fig. 5.
Effect of HePC on the incorporation of
SM-derived PCho into PC. Control fibroblasts were incubated with
[14C]SM and apoE for 30 min as described under
"Experimental Procedures." At indicated times during the chase, in
the absence (closed symbols) or presence (open
symbols) of 50 µM HePC, radioactivity in
intracellular SM (circles), PCho (triangles), and
PC (squares) was determined. Data are expressed as percentages of total
cell-associated radioactivity at t = 0. The mean total
cell-associated radioactivity was 19.5 × 103 dpm/mg
cellular protein. Results are means from a representative experiment
performed in duplicate. The experiment was repeated with two different
control cell lines giving similar results.
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Fig. 6.
Effect of energy depletion on the
incorporation of SM-derived PCho into PC. Control fibroblasts
(n = 4) were incubated with [14C]SM and
apoE for 30 min as described under "Experimental Procedures." At
indicated times during the chase, in the absence (closed
symbols) or presence (open symbols) of energy poison
(10 mM NaN3 + 10 mM
2-deoxyglucose), radioactivity in intracellular SM
(circles), PCho (triangles), and PC
(squares) was determined. Data are expressed as percentages
of total cell-associated radioactivity at t = 0. The
mean total cell-associated radioactivity was 32.9 × 103 dpm/mg cellular protein. Values are given as means ± S.D.
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Fig. 7.
Subcellular localization of radiolabeled
PCho. Control fibroblasts were incubated for 30 min with
[14C]SM and apoE and subsequently permeabilized for 5 min
with different concentrations of digitonin (mg/l). The percentage of
total radioactivity in PCho (squares) and the percentage of
total hexosaminidase activity (circles) in the
permeabilization buffer were determined. Results are means of two
independent experiments.
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Fig. 8.
Analysis of molecular species of PC
synthesized from [3H]choline and
[14C]SM·apoE. Normal human fibroblasts were
incubated with (A) [3H]choline or
(B) [14C]SM·apoE, PC was isolated by TLC,
and the PC molecular species were separated by reverse phase HPLC. The
elution positions of the most abundant molecular species,
i.e. 16:0/20:4, 16:0/18:1, and 18:0/18:1
(identified by electrospray mass spectrometry; see "Experimental
Procedures") are indicated in panel A. CPM, counts per
minute.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. L. M. G. van Golde and M. Houweling (Laboratory of Veterinary Biochemistry, Utrecht University, Utrecht, The Netherlands) for helpful discussions and critically reading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Pediatrics, Leiden University Medical Center, Bldg. 1, P3-P, P. O. Box 9600, 2300 RC Leiden, The Netherlands. Tel.: 31715262904; Fax: 31715266876; E-mail: B.J.H.M.Poorthuis@kgc.azl.nl.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M101817200
2 A. Suter, V. Everts, R. Lüllmann-Rauch, D. Hartmann, A. R. Hayman, T. M. Cox, M. J. Evans, S. J. Jones, A. Boyde, T. Meister, K. von Figura, and P. Saftig, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PC, phosphatidylcholine; PCho, phosphocholine; CT, CTP:phosphocholine cytidylyltransferase; SM, sphingomyelin; LAP, lysosomal acid phosphatase; TRAP, tartrate resistant acid phosphatase; HePC, hexadecylphosphocholine; FBS, fetal bovine serum; apoE, apolipoprotein E; LDL, low density lipoprotein; NP-A, Niemann-Pick A; LPC, lysophosphatidylcholine; HPTLC, high-performance thin-layer chromatography..
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REFERENCES |
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