From the Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606
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ABSTRACT |
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To probe the mechanism of lipid activation of
CTP:phosphocholine cytidylyltransferase (CCT CTP:phosphocholine cytidylyltransferase
(CCT)1 is a critical
participant in the CDP-choline pathway, catalyzing the synthesis of
CDP-choline for the biosynthesis of phosphatidylcholine (PC), a major
component of eukaryotic cell membranes (1, 2). CCT is rate-limiting for
the CDP-choline pathway and is extensively regulated at the enzymatic
level. Mammalian CCT is present as both soluble and membrane-associated
forms. Activation of CCT often occurs simultaneously with the
translocation of the enzyme from a soluble form to membrane-associated
form, resulting in an increase in the rate of PC synthesis. Consistent
with in vivo membrane activation, the soluble form of CCT is
activated by the addition of certain lipids in vitro. There
are two isoforms of mammalian CCT; the most extensively studied,
CCT Mammalian CCT These studies have contributed to the general understanding that the
M/L region is truly responsible for membrane binding in the cell and
lipid activation in vitro. On the other hand, two important
questions regarding the M/L segment remain. First, what is the
mechanism by which the M/L segment controls the activation of the
enzyme by lipids? Second, what is the importance of the M/L segment
in vivo? The answer to the first question depends on an
understanding of the level of activity remaining in the truncated
enzyme lacking the M/L region, which has been a matter of some debate.
The truncation expressed by Wang and Kent (5), CCT), we have characterized
a catalytic fragment of the enzyme that lacks the membrane-binding
segment. The kinetic properties of the purified fragment, CCT
236,
were characterized, as well as the effects of expressing the fragment in cultured cells. CCT
236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The
activity of purified CCT
236 was independent of lipids and about
50-fold higher than the activity of wild-type CCT
assayed in the
absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. The kcat/Km values for
CCT
236 were only slightly lower than those for lipid-activated
CCT
. The importance of the membrane-binding segment in
vivo was tested by expression of CCT
236 in CHO58 cells, a cell
line that is temperature-sensitive for growth and CCT
activity.
Expression of wild-type CCT
in these cells complemented the
defective growth phenotype when the cells were cultured in complete or
delipidated fetal bovine serum. Expression of CCT
236, however, did
not complement the growth phenotype in the absence of serum lipids.
These cells were capable of making phosphatidylcholine in the
delipidated medium, so the inability of the cells to grow was not due
to defective phosphatidylcholine synthesis. Supplementation of the
delipidated medium with an unsaturated fatty acid allowed growth of
CHO58 cells expressing CCT
236. These results indicate that the
membrane-binding segment of CCT
has an important role in cellular
lipid metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, is nuclear and apparently ubiquitously expressed (2). The
recently discovered CCT
is cytoplasmic and exhibits tissue-specific
expression (3).
contains several functional regions: an N-terminal
nuclear localization signal, a central catalytic domain, a
membrane/lipid (M/L) activation segment, and a C-terminal
phosphorylation region (see Fig. 1). There is a high degree of sequence
similarity within the catalytic domain of all known forms of CCT, with
the yeast catalytic domain being 56% identical to that of mammalian CCT
, and the catalytic domains of CCT
and CCT
being 90%
identical. By contrast, there is much less conservation of primary
structure in the N-terminal sequence or the C-terminal M/L and
phosphorylation regions. Truncated forms of rat CCT
have been
constructed to determine the regions responsible for lipid activation.
Both full-length CCT
and truncated forms lacking the phosphorylation
segment require lipids for full activity (4, 5). Truncations lacking
the M/L region, however, are not lipid-activated (4-6) and do not translate to membranes in the cell (5). It is notable that CHO58 cells
that are temperature-sensitive for growth and CCT
activity can be
complemented by exogenous expression of the truncated CCT
lacking
the M/L region, which suggested that this segment is not important for
growth under normal cell culture conditions (5).
236 (Fig.
1), is constitutively active as assessed
by measuring enzyme activity in crude extracts and enzyme levels by
quantitative immunoblotting. Slightly different forms expressed by Yang
et al. (6) or Cornell et al. (4) are less than
10% as active as full-length, lipid-activated CCT
(7). Thus the
precise role of the M/L segment in enzyme activation is not clear. Wang and Kent (5) proposed that the M/L segment interacts with the catalytic
domain to cause inhibition and that removal of the M/L segment by lipid
binding results in activation of catalysis. Jackowski and co-workers
(6), however, proposed that lipids and the M/L domain are co-activators
of the catalytic domain.
View larger version (3K):
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Fig. 1.
Functional regions of CCT
and CCT
236. Full-length wild-type
rat CCT
contains a nuclear localization sequence (NLS),
catalytic domain, membrane and lipid binding segment (M/L),
and phosphorylation region (P). CCT
236 is truncated after
residue 236, so it lacks the M/L segment and phosphorylation
region.
In this paper we report the kinetic properties of purified CCT236,
showing that it is nearly as active as full-length CCT
and is indeed
lipid-independent, supporting the model that the M/L segment is
inhibitory. In addition, we report conditions under which the presence
of the M/L segment in CCT
is necessary for cell growth.
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EXPERIMENTAL PROCEDURES |
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Materials
CTP, phosphocholine, protease inhibitors, lipids, CM-Sepharose, DEAE-Sepharose, and Sephacryl S-200-HR were obtained from Sigma. The cross-linkers, BS3 and Sulfo-MBS, were from Pierce. [3H]choline and [14C]phosphocholine were from Amersham Pharmacia Biotech. The Bac-to-Bac baculovirus expression system, Sf9 cells adapted for serum-free growth, Sf900II serum-free medium, and Ham's F-10 and F-12 media were from Life Technologies, Inc. Restriction endonucleases were from New England Biolabs. Fetal bovine serum was from BioWhittaker. Blue-Sepharose was from Amersham Pharmacia Biotech.
Baculovirus Expression of CCT and CCT
236
A cDNA encoding rat liver CCT (8) was placed in the
baculovirus donor plasmid pFASTBAC1 using the BamHI and
SphI sites of the multiple cloning site. To generate a
cDNA encoding truncation mutant CCT
236 with appropriate
restriction sites for the vector, oligonucleotide-directed polymerase
chain reaction mutagenesis was used to introduce a stop codon following
amino acid 236. The following oligonucleotides were utilized:
5'-oligo, 5'-CGCGGATCCAGATCTATGGATGCACAGAGTTCA-3', and 3'-oligo,
5'-ACATGCATGCGGTACCTTAGTTGATAAAGCTGACATT-3'.
Stocks of virus encoding wild-type CCT and CCT
236 were prepared
as described in the Bac-to-Bac Baculovirus expression system instruction manual. For protein production, 1-liter cultures of Sf9 cells at 1 × 106 cells/ml were infected at
a multiplicity of infection of 2. Cells were harvested at 48 h
post-infection for protein purification.
Protein Purification
Wild-type CCT--
Sf9 cells expressing wild-type
CCT
were collected, lysed, and centrifuged as described (8). The
100,000 × g Sf9 cell supernatant was loaded
directly onto a DEAE-Sepharose column equilibrated with buffer A (10 mM Tris-Cl, pH 7.5, 2 mM dithiothreitol, 1 mM EDTA) containing 150 mM NaCl. The column was
washed with 10 column volumes of buffer A containing 150 mM
NaCl and 1% Nonidet P-40 followed by 10 column volumes of buffer A
containing 150 mM NaCl. The enzyme was eluted from the
column with a 150-400 mM NaCl gradient in buffer A. The
fractions constituting the peak of CCT
activity were pooled, diluted
5-fold in buffer A, and loaded onto a CM-Sepharose column equilibrated
with buffer A containing 30 mM NaCl. The enzyme was eluted
from the column with a gradient of NaCl from 30 to 150 mM,
and the fractions containing the peak of the CCT
activity were pooled.
CCT236--
The high speed Sf9 cell supernatant was
loaded onto a CM-Sepharose column equilibrated with buffer A. The
column was washed sequentially with 10 column volumes each of buffer A
and buffer A containing 0.1 M NaCl. CCT
236 was eluted
from the CM-Sepharose column with buffer A containing 0.2 M
NaCl. The CM-Sepharose elution fraction was loaded onto a
Blue-Sepharose 6 fast flow column. The column was washed with 10 column
volumes of buffer A containing 0.2 M NaCl. CCT
236 was
eluted from Blue-Sepharose with buffer A containing 0.5 M NaCl.
Kinetic Characterization
Activity of purified enzymes was determined with a charcoal binding assay as described previously (9). The kinetic parameters kcat and Km with respect to substrates CTP and phosphocholine were determined from secondary plots. Kinetic parameters were determined in the presence and absence of PC:oleate vesicles (10); the final concentration of each lipid was 100 µM.
Cell Growth and Maintenance
CHO58 cells expressing full-length CCT or CCT
236 were
obtained previously (5). Cell stocks were maintained at 34 °C in 5%
CO2 in Ham's F-12 medium supplemented with 25 mM HEPES and10% fetal bovine serum (FBS). The stably
transfected cell lines were maintained in the presence of 0.8 mg/ml
G418 during one-third of the passages. For growth curves, cells were
plated at 34 °C in Ham's F-12, 10% FBS at 50,000 cells/35-mm dish
or 50,000 cells/well of a 6-well plate. After 24 h the medium was
removed, cells were washed twice in Ca2+- and
Mg2+-free phosphate-buffered saline, and then F-10 medium
supplemented with 25 mM HEPES, 50 µg/ml gentamycin, and
either 10% FBS or 10% delipidated serum was added. The cells were
then maintained at 37 °C, and viable cell density was determined
after trypsinization by counting in trypan blue with a hemocytometer.
Lipids were diluted into the medium from a 1000× stock in ethanol. For
the choline uptake experiment, cells were plated at 250,000 cells/60-mm
dish in Ham's F-10, 10% FBS, and the medium changed as for growth curves.
Delipidation of Serum
Fetal bovine serum was delipidated by extraction with isopropyl ether and n-butanol as described (11) and then sterilized by filtration.
Incorporation of [3H]Choline
The protocol used is a modification of that described previously (5). 24 h after plating, cells were washed twice with Ca2+- and Mg2+-free phosphate-buffered saline and shifted into Ham's F-10, 25 mM HEPES, 10% delipidated serum, and 50 µg/ml gentamycin. Cells were then grown for another 24 h at 37 °C. Cells were labeled by incubating at 37 °C in 1.5 ml of medium supplemented with 2 µCi/ml [3H]choline for the indicated times. Cells were harvested by washing three times in Ca2+- and Mg2+-free phosphate-buffered saline and scraping into 1.0 ml of water at 0 °C. Total lipids were extracted from 0.8 ml of the extract as described previously (12).
Protein Assay
Protein concentration was determined by the assay of Bradford
(13) or Lowry et al. (14).
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RESULTS |
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Protein Purification--
Because we were interested in lipid
activation of CCT, we devised a new purification procedure for
CCT
that does not include treatment with lipids, as did the
classical procedure (7). The new procedure involves ion exchange
chromatography on both DEAE-Sepharose and CM-Sepharose at pH 7.5. Apparently the distribution of charged residues in the enzyme is
suitable to allow it to bind to both resins at pH 7.5, unlike most
other proteins. The baculovirus-expressed enzyme that was purified by
this procedure appeared homogeneous by Coomassie Blue staining of SDS
gels (Fig. 2A). The specific activity of the purified enzyme was similar to the activity of the
enzyme purified by the classical procedure (7, 8) (Table I).
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Purification of the truncation mutant CCT236 to homogeneity involved
cation exchange chromatography on CM-Sepharose as well as
Blue-Sepharose chromatography (Table I). The enzyme was homogeneous as
assessed by SDS gels (Fig. 2B). CCT
236 did not bind to
DEAE-Sepharose at pH 7.5, suggesting that the C-terminal regulatory
segments in CCT
are negatively charged at that pH and mediate
interaction with the anion exchange support.
CCT236 Is Constitutively Active--
The ability of the
purified forms of CCT
to be activated by lipids was determined by
assaying in the presence and absence of vesicles of PC:oleate, 1:1
(10). The activity of wild-type CCT
, purified in the absence of
lipids, is very responsive to the addition of lipid (Fig.
3). Over the range of 0-20
µM lipid, CCT
is activated approximately 50-fold. In
contrast, the activity of CCT
236 was not affected by the addition of
lipid; the specific activity of CCT
236 with and without lipid was
comparable with lipid-activated wild-type CCT
(Fig. 3). The kinetic
parameters of purified wild-type CCT
and CCT
236 were measured in
the presence and absence of 100 µM PC:oleate (1:1), a
concentration at which CCT
is maximally active. The
kcat value for wild-type CCT
in the absence
of lipid was 86-fold less than that for wild-type CCT
with 100 µM lipid (Table II). The
kcat values for CCT
236 were independent of
the presence of lipid and were only about 30% lower than that for
wild-type CCT
in the presence of lipid. The Km
values for phosphocholine were similar for both enzyme forms in the
presence and absence of lipids. The Km values for
CTP were somewhat higher for CCT
236 than for wild-type CCT
. The
catalytic efficiency
(kcat/Km) values, therefore, for CCT
236 in the absence of lipid were 30-50% as high as those for wild-type CCT
in the presence of lipids. In contrast, the kcat/Km values for CCT
236
were 30-70-fold higher than those for wild-type CCT
in the absence
of lipids (Table II). The fact that the activity of CCT
236 is so
much greater than the activity of wild-type CCT
in the absence of
lipids supports the model in which the M/L region is inhibitory to
catalysis.
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CCT236 Is a Homodimer--
Wild-type CCT
is known to be a
homodimer (15, 16), but the residues interacting at the dimer interface
have not been determined. If the M/L region were important in
determining the oligomerization state of CCT
, then CCT
236 might
be expected to be monomeric. The quaternary structure of CCT
236 was
therefore assessed by intermolecular cross-linking using the
bifunctional cross-linkers BS3 and sulfo-MBS. BS3 is specific for
cross-linking two amine-containing functional groups, whereas sulfo-MBS
targets an amine and sulfhydryl. Both wild-type CCT
and CCT
236
were cross-linked by BS3 and sulfo-MBS (Fig.
4). The major cross-linked form for both
species appeared to be dimeric. Wild-type CCT
also appeared to form
high molecular weight species, whereas CCT
236 formed only the
cross-linked dimer.
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The quaternary structure of CCT236 was also investigated by gel
filtration on Sephacryl S-200-HR. The truncated enzyme eluted between
the protein standards bovine serum albumin and ovalbumin. The
calculated molecular weight was 50,300, and the expected molecular weight of a dimer of CCT
236 is 53,500. Thus, both cross-linking and
gel filtration indicated that CCT
236 is a homodimer and support the
concept that the dimerization interface of CCT
involves residues in
the catalytic domain and not the C-terminal regulatory domains.
Expression of CCT236 in CHO58 Cells in Delipidated
Medium--
Although it is clear that the M/L region of CCT
has a
profound effect on enzyme activity, the function of this domain in the
cell remains open to question. Expression of CCT
236 in the CHO58
cell line, which is temperature-sensitive for growth and CCT
activity (17), fully complements the temperature-sensitive growth
defect (5). This suggests that the M/L region is not required for
routine lipid metabolism and cell cycling. However, we have more
recently discovered conditions under which expression of CCT
236 is
not sufficient to complement the CHO58 defect. When CHO58 cells stably
transfected with CCT
236 were transferred to medium containing
delipidated serum, the cells could not continue to grow at 40 °C
(data not shown). Furthermore, these cells could not grow in
delipidated serum at 37 °C, a temperature that is permissive for the
growth of untransfected CHO58 in either complete serum or delipidated
serum (Fig. 5). Thus the inability of
cells expressing CCT
236 to grow in delipidated medium is a dominant negative effect. CHO58 cells expressing wild-type CCT
were capable of growth at 37 °C in either complete or delipidated serum (Fig. 5).
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To determine whether a single specific lipid would restore the ability
of CHO58 cells expressing CCT236 to grow, a number of different
lipids were added to the delipidated medium. The unsaturated fatty acid
oleate was capable of supporting growth at the same rate as in complete
serum (Fig. 6). Optimal growth was seen
at oleate concentrations from 40 to 100 µM oleate. Other unsaturated fatty acids, linoleate and palmitoleate, could also fully
support growth at 40 µM (not shown). The saturated fatty acids myristate, palmitate, and stearate at 40 µM could
not support growth. The combination of 20 µM cholesterol
plus 200 µM mevalonate could not support growth, and the
addition of cholesterol and mevalonate to the oleate-containing medium
did not enhance growth further than oleate alone. These results
indicate that expression of CCT
236 had transformed CHO58 cells into
unsaturated fatty acid auxotrophs.
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Synthesis of Phosphatidylcholine in CCT236/58 Cells--
We had
observed previously that CHO58 cells expressing CCT
236 in complete
serum were considerably more active at making PC than cells expressing
wild-type CCT
(5), which is consistent with the CCT
236 being
constitutively active. To discover whether the ability of these cells
to make PC was defective in delipidated serum, rates of choline
incorporation into PC were measured 24 h after transfer to
delipidated medium, a time at which the cells were still viable as
determined by trypan blue exclusion. As indicated in Fig.
7, CCT
236/58 cells were fully capable
of making PC in delipidated serum. The rate of PC synthesis was higher
by a factor of 1.7 ± 0.6 (n = 5) in cells
expressing CCT
236 than in cells expressing wild-type CCT
, as had
been observed when these cells were grown in complete serum. The higher
rate of PC synthesis in cells expressing CCT
236 is consistent with
the M/L domain being inhibitory in the cell.
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DISCUSSION |
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Role of M/L Region--
Two distinct models can be envisioned for
the role of lipid binding in regulating catalysis by CCT (Fig.
8). In model A, the M/L region interacts
with the catalytic domain in the absence of lipids, and this
interaction inhibits enzyme activity. Binding of lipids to the M/L
segment causes it to dissociate from the catalytic domain, and the
inhibition is relieved. In model B, the catalytic domain in the
inactive enzyme does not associate with the M/L region. When the M/L
region becomes complexed with lipids it then associates with the
catalytic domain to activate catalysis. Both models predict that
CCT
236 would have lipid-independent activity. Model A, however,
predicts that CCT
236 would be as active as the wild-type enzyme in
the presence of lipids, whereas model B predicts that CCT
236 would
be as inactive as the wild-type enzyme in the absence of lipids.
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The results in this manuscript show that the activity of CCT236 is
indeed independent of lipids. Moreover, CCT
236 is nearly as active
as full-length CCT
in the presence of lipids and much more active
than full-length CCT
in the absence of lipids. The presence of the
regulatory regions in the absence of lipid lowers the maximal velocity
attainable by CCT
about 50-fold. These data clearly support model A,
in which the M/L region acts as an inhibitor in the absence of lipids.
Other truncated forms of rat CCT have been expressed with
considerably lower activity than CCT
236. Two of these, CCT
228 (4)
and CCT
230 (6), were missing several residues that are highly
conserved in all CCTs from yeasts to mammals (2). It is possible that
these residues are important for catalysis or for enzyme structure. In
addition both these truncations contained additional amino acids at
their C termini, which may have adversely affected activity. CCT
256
also had low activity (6), which presents the interesting possibility
that residues 237-256 contain part of the inhibitory region of the M/L
region. We originally chose to end our truncation at 236 based on
sequence conservation (5), but later analysis of the mouse CCT
gene
indicated that a splice junction terminates the last exon of the
catalytic domain at this site (18). This suggests that the catalytic
domain does truly end at residue 236.
The role of lipid in the activation of CCT has been reported to be
due to both a lowering of the Km for CTP as well as
an increase in Vmax (6). Our analysis of kinetic
parameters of wild-type CCT
revealed only an increase in
kcat in the presence of lipids, with no
substantial decrease in Km for CTP (Table II). It is
not yet clear why there is a discrepancy in these Km
determinations. It should be remembered that the kinetic constant
Km is a complex term of rate constants that is
rarely a simple reflection of substrate binding affinity (19). It has
been argued that the effect of an activator or inhibitor on
Km is not as useful as a determination as the effect
on Vmax/Km or
kcat/Km (20). It is
interesting that the Vmax/Km
values for CCT
± lipids vary by a factor of 220 in the studies of
Yang et al. (6), and the
kcat/Km values for CCT
± lipids in the present study differed by a factor of 114. That these
values are quite similar supports the argument that the ratio of
catalytic constant to Km rather than
Km itself is the appropriate constant for comparing
the effects of lipids on activity.
Quaternary Structure of CCT236--
Results obtained by
cross-linking (Fig. 4) and gel filtration indicate that CCT
236 is a
dimer. This would indicate that the residues responsible for dimer
formation in wild-type CCT
are largely within the catalytic domain
or the N terminus. CTP:glycerol-3-phosphate cytidylyltransferase (GCT),
a small enzyme similar in primary sequence to the catalytic domain of
CCT, is also a homodimer (21), supporting the concept that residues in
the catalytic domain are sufficient for dimer formation. We have
recently completed the crystal structure of
GCT.2 The majority of
residues at the dimer interface of GCT are similar or identical to the
corresponding residues in CCT
, suggesting that the interface within
the catalytic domain is a conserved feature of this
cytidylyltransferase family. A role for the M/L or phosphorylation
regions in modulating a monomer-dimer equilibrium cannot be ruled out
but does not seem likely.
Cellular Expression of CCT236--
Determining the role of the
M/L region in vitro should aid in our understanding of the
function of this enzyme in the cell. It was disappointing, therefore,
that previous expression results had indicated that the regulatory
regions of CCT
are not needed by cells, at least under the usual
cell culture conditions (5). The present results indicate, however,
that the presence of the M/L region can be critical under certain
conditions. Expression of CCT
236 in CHO58 cells maintained in
delipidated serum was toxic unless the cells were supplied with an
unsaturated fatty acid. The toxicity was a dominant effect in that it
occurred even at a temperature permissive for growth of untransfected
CHO58 cells. The toxicity was not due to an inability of the cells to make PC under these conditions (Fig. 7). Although we do not yet know
the reason for the requirement for an unsaturated fatty acid, two
possibilities come to mind. One is simply that the cells are making so
much more PC that the desaturase responsible for making unsaturated
fatty acids cannot keep up with the demand, with the result that too
much saturated phospholipid is made and put into membranes. The other
possibility is that CCT
is somehow involved in the induction of the
desaturase in lipid-free medium, and the M/L region plays a role in
that induction. The latter possibility would mean that CCT
has a
role in coordinating lipid biosynthesis, which might explain its
puzzling nuclear location. The demonstration of a role for the M/L
region in lipid-free medium has afforded an experimental system in
which that role can be elucidated.
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ACKNOWLEDGEMENTS |
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We thank Patricia Gee for cell culture media, T. Y. Chang for assistance with serum delipidation, and Paul Weinhold, Douglas Feldman, Joel Clement, and Subramaniam Sanker for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health National Research Service Award Grants 1 F32 GM19286-01 (to J. A. F.) and RO1 CA64159 (to C. K.). The work was supported in part by core services funded by National Institutes of Health Grants 5P60DK-20572 and MO1-RR00042.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 Biological
Chemistry, 4417 Medical Science I, University of Michigan Medical
School, Ann Arbor, MI 48109-0606. Tel.: 734-647-3317; Fax:
734-764-3509; E-mail: ckent{at}umich.edu.
2 C. H. Weber, Y. S. Park, S. Sanker, C. Kent, and M. Ludwig, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CCT, CTP:phosphocholine cytidylyltransferase; PC, phosphatidylcholine; FBS, fetal bovine serum; M/L, membrane/lipid; GCT, CTP:glycerol-3-phosphate cytidylyltransferase.
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REFERENCES |
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