CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is
the enzyme responsible for the biosynthesis of the nucleotide
CTP(1, 2) . CTP is a precursor for the synthesis of
RNA, DNA, and membrane phospholipids(3, 4) . Thus, CTP
synthetase activity plays a major role in the growth and metabolism of
all organisms. Regulation of CTP synthetase activity is critical to
normal cell growth in mammalian cells. Mutant mammalian cell lines
lacking normal regulation of CTP synthetase activity exhibit abnormally
high intracellular levels of CTP and dCTP(5, 6) ,
resistance to nucleotide analog drugs used in cancer
chemotherapy(7, 8, 9, 10) , and an
increased rate of spontaneous mutations (8, 10, 11) . Moreover, elevated levels of
CTP synthetase activity are characteristic of rapidly growing tumors of
liver(12) , colon(13) , and lung(14) .
We
are using the yeast Saccharomyces cerevisiae as a model
eucaryote to study the regulation of CTP synthetase activity. CTP
synthetase is encoded by the URA7 and URA8 genes in S. cerevisiae(15, 16) . Comparison of the
nucleotide and deduced amino acid sequences of the open reading frames
of the URA7 and URA8 genes show 70 and 78% identity,
respectively(15, 16) . Although there is a high degree
of identity between the two CTP synthetases, they are not functionally
identical. The CTP concentration in cells possessing only a functional URA7 gene is 78% of that found in wild-type cells whereas the
CTP concentration in cells with only a functional URA8 gene is
36% of that found in wild-type cells(16) . Moreover, the
reduced CTP concentration found in cells possessing only one of the CTP
synthetase genes correlates with a reduced rate of cell
growth(16) . Simultaneous presence of null alleles for both CTP
synthetase genes is lethal(16) . Thus, the CTP synthetases
encoded by the URA7 and URA8 genes are functionally
overlapping and play an essential role in the growth of yeast cells.
The URA7-encoded CTP synthetase has been purified to
homogeneity and characterized with respect to its physicochemical and
kinetic properties(17) . URA7-encoded CTP synthetase
activity exhibits complex cooperative kinetics characteristic of
allosteric enzymes and is allosterically regulated by CTP product
inhibition(17) . In this study, we questioned whether the
activities of the CTP synthetases encoded by the URA7 and URA8 genes were regulated differentially through their
biochemical properties. The URA8-encoded CTP synthetase was
purified to homogeneity so its biochemical properties could be studied
under well-defined conditions. URA8-encoded CTP synthetase
differed from URA7-encoded CTP synthetase with respect to
several enzymological properties and sensitivity to inhibition by CTP.
Thus, these CTP synthetase isoforms were regulated differentially on a
biochemical level. In addition, we showed that the URA7-encoded CTP synthetase mRNA was 2-fold greater than the URA8-encoded CTP synthetase mRNA. The differential regulation
of the two CTP synthetases may account for the relative contribution of
each enzyme for CTP synthesis.
EXPERIMENTAL PROCEDURES
Materials
All chemicals were reagent grade. Growth medium supplies were
purchased from Difco Laboratories. Restriction endonucleases, random
primer NEBlot kit, and the protein fusion kit used to make
maltose-binding protein-CTP synthetase fusion protein were purchased
from New England Biolabs. Radiochemicals and GeneScreen were purchased
from DuPont NEN. Nucleotides, L-glutamine,
phenylmethanesulfonyl fluoride, benzamide, aprotinin, leupeptin,
pepstatin, molecular mass standards for gel filtration chromatography,
and bovine serum albumin were purchased from Sigma. Centricon-10
concentration filters were purchased from Amicon. Protein assay
reagent, Affi-Gel Blue, DEAE-Affi Gel Blue, molecular mass standards
for SDS-polyacrylamide gel electrophoresis, electrophoresis reagents,
and immunochemical reagents were purchased from Bio-Rad. Q-Sepharose,
Mono Q, and Superose 6 were purchased from Pharmacia Biotech Inc.
Methods
Strains and Growth Conditions
Wild-type strain FL100 (MATa, American Type
Culture Collection 28383) was used to examine CTP synthetase mRNA
abundance. CTP synthetase was purified from strain OK8 (MAT
leu2 trp1 ura3 ura7
::TRP1 ura8) bearing the URA8 gene on the multicopy plasmid pOK10.1(16) . Strain OK8 has
mutations in both the URA7 and URA8 genes (15, 16) . Cells were grown in complete synthetic
medium (18) without uracil to the exponential phase of growth
(1-2
10
cells/ml) at 30 °C. Cell numbers
were determined by microscopic examination with a hemacytometer.
Purification of URA8-encoded CTP Synthetase
All steps were performed at 5 °C.
Step 1: Preparation of Cytosol
Cells were
disrupted with glass beads with a Bead-Beater (Biospec Products) in
Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM
Na
EDTA, 20 mML-glutamine, 0.3 M sucrose, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, 1 mM benzamide, 5
µg/ml aprotinin, 5 µg/ml leupeptin, and 6 µg/ml pepstatin)
as described previously(19) . Unbroken cells and glass beads
were removed by centrifugation at 1,500
g for 5 min.
The cytosolic fraction was obtained by centrifugation at 100,000
g for 1.5 h.
Step 2: Ammonium Sulfate Fractionation
The
cytosolic fraction was diluted to a protein concentration of 5 mg/ml
with Buffer A. Enzyme grade ammonium sulfate was added to the cytosol
to 45% saturation with slow stirring. After stirring for 2 h,
precipitated protein was removed by centrifugation at 12,000
g for 20 min and dissolved in a minimum volume of Buffer B (50
mM Tris-HCl, pH 7.5, 4 mML-glutamine, 1
mM Na
EDTA, 10 mM 2-mercaptoethanol, and
10% glycerol). The enzyme preparation was then desalted by dialysis
against Buffer B.
Step 3: Q-Sepharose Chromatography
A Q-Sepharose
column (1.5
12 cm) was equilibrated with Buffer B. The ammonium
sulfate fraction was applied to the column at a flow rate of 30 ml/h.
The column was washed with Buffer B until all of the unbound protein
had been removed from the column. CTP synthetase was then eluted from
the column in 3-ml fractions with 10 column volumes of a linear NaCl
gradient (0-1.0 M NaCl) in Buffer B. The peak of CTP
synthetase activity eluted from the column at a NaCl concentration of
0.16 M. The most active fractions were pooled and diluted with
Buffer B to a NaCl concentration of 0.1 M.
Step 4: Affi-Gel Blue Chromatography
An Affi-Gel
Blue column (1.5
12 cm) was equilibrated with Buffer B
containing 0.1 M NaCl. Q-Sepharose-purified enzyme was applied
to the column at a flow rate of 30 ml/h. The column was washed with
Buffer B containing 0.1 M NaCl until all of the unbound
protein eluted from the column. CTP synthetase was eluted from the
column in 2-ml fractions with 10 column volumes of a linear NaCl
gradient (0.1-0.7 M NaCl) in Buffer B. The peak of CTP
synthetase eluted from the column at a NaCl concentration of 0.17 M NaCl. The most active fractions were pooled and diluted with
Buffer B to a NaCl concentration of 0.1 M NaCl.
Step 5: Mono Q Chromatography
A Mono Q column (0.5
5 cm) was equilibrated with Buffer B containing 0.1 M
NaCl. Affi-Gel Blue-purified enzyme was applied to the column at a flow
rate of 30 ml/h. The column was washed with 3 column volumes of Buffer
B containing 0.2 M NaCl. CTP synthetase was eluted from the
Mono Q column in 1-ml fractions with 30 column volumes of a linear NaCl
gradient (0.2-0.9 M) in Buffer B. The peak of CTP
synthetase activity eluted from the column at a NaCl concentration of
0.32 M. The most active fractions were pooled and concentrated
using an Amicon Centricon-10 filter.
Step 6: Superose 6 Chromatography
A Superose 6
column (1
24 cm) was equilibrated with Buffer B and calibrated
with blue dextran 2000 (for the void volume), thyroglobulin (669 kDa),
apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), and carbonic anhydrase (29 kDa). The Mono Q-purified
enzyme was applied to the column at a flow rate of 15 ml/h. CTP
synthetase activity was then eluted from the column in 1-ml fractions
using buffer B. CTP synthetase activity and protein eluted from the
column as a single peak. Fractions containing activity were pooled and
the glycerol concentration in the enzyme preparation was increased to
30%. Purified CTP synthetase was stable at -20 °C for at
least 3 months.
Preparation of Anti-URA8-encoded CTP Synthetase
Antibodies
An inducible pMAL-c-URA8 plasmid was constructed for
the production of a maltose-binding protein-CTP synthetase fusion
protein. The fusion protein was used for the generation of
anti-URA8-encoded CTP synthetase antibodies. A 0.7 kb (
)fragment was isolated from the plasmid pOK10.1 (16) by HpaI and HindIII digestion by
standard recombinant DNA techniques(20) . This fragment
contained the URA8 open reading frame from codon 245 to 477 (16) . The pMal-c plasmid was digested with StuI and HindIII. The 0.7-kb URA8 fragment was ligated into
the pMal-c plasmid at these sites, placing URA8 in frame with
the malE gene, to create the inducible pMal-c-URA8 plasmid.Maltose-binding protein-URA8-encoded CTP
synthetase fusion protein was produced in Escherichia coli strain DH5
by induction of its expression from the
pMAL-c-URA8 plasmid and purified as described
previously(17) . Antibodies to the maltose-binding
protein-URA8-encoded CTP synthetase fusion protein were raised
in New Zealand White rabbits by standard procedures (21) at
the Pocono Rabbit Farm (Canadensis, PA). IgG antibodies were isolated
as described previously(17) .
Electrophoresis and Immunoblotting
SDS-polyacrylamide gel electrophoresis (22) was
performed with 10% slab gels. Proteins on polyacrylamide gels were
visualized with Coomassie Blue. Immunoblot analyses of cytosol and pure
CTP synthetase were performed as described
previously(23, 24) . Immunoblot signals were optimized
by analyzing a number of antigen and antibody concentrations and were
in the linear range of detectability. The density of the CTP synthetase
bands on immunoblots was quantified by scanning densitometry.
Enzyme Assay and Protein Determination
CTP synthetase activity was determined by measuring the
conversion of UTP to CTP (molar extinction coefficients of 182 and 1520 M
cm
, respectively) by
following the increase in absorbance at 291 nm(2) . The
standard reaction mixture contained 50 mM Tris-HCl, pH 7.5, 10
mM MgCl
, 10 mM 2-mercaptoethanol, 2
mML-glutamine, 0.1 mM GTP, 2 mM ATP, 2 mM UTP, and an appropriate dilution of enzyme
protein in a total volume of 0.2 ml. Enzyme assays were performed in
triplicate with an average standard deviation of ±3%. All assays
were linear with time and protein concentration. A unit of enzyme
activity was defined as the amount of enzyme that catalyzed the
formation of 1 µmol of CTP/min under the assay conditions described
above. Specific activity was defined as units/milligram of protein.
Protein was determined by the method of Bradford (25) using
bovine serum albumin as the standard. Protein was monitored from column
chromatography fractions by measuring absorbance at 280 nm.
Analysis of Kinetic Data
Kinetic data were analyzed according to the Michaelis-Menten
and Hill equations using the EZ-FIT Enzyme Kinetic Model Fitting
Program (26) . EZ-FIT uses the Nelder-Mead Simplex and
Marquardt/Nash nonlinear regression algorithms sequentially and test
for the best fit of the data among different kinetic models.
Preparation of RNA and Northern Blot Analysis
Total RNA was extracted from cells using hot phenol as
described by Schmitt et al.(27) . The RNA was
separated by electrophoresis under denaturing conditions using 1%
agarose formaldehyde gels(28) . Following electrophoresis, RNA
was transferred to GeneScreen and probed with a radiolabeled fragment
of the URA7 and URA8 genes. URA7 probe was a
1.6-kb fragment isolated from YEp352-URA7(17) by EcoRI and HindIII digestion. The URA8 probe
was a 1-kb fragment generated by the polymerase chain reaction (29) using plasmid pOK10.1 (16) as a template and the
primers 5`-GGATCCCGATATGATTGCCTG-3` and 5`-GGATCCAGCACCTTCGATGTA-3`.
The probes were labeled with [
-
P]dCTP by
the random priming reaction using a NEBlot kit. Prehybridization and
hybridization of blots were carried out at 60 °C in modified Church
buffer (30) as recommended by U. S. Biochemical Corp. Ribosomal
subunit L32 mRNA (31) was used as a constitutive
standard and loading control. The density of the CTP synthetase mRNA
bands on Northern blots was quantified by scanning densitometry.
RESULTS AND DISCUSSION
Purification of URA8-encoded CTP Synthetase
The
purification of the URA8-encoded CTP synthetase was
facilitated by the overexpression of the URA8 gene on a
multicopy plasmid in a strain which lacked the URA7-encoded
CTP synthetase. A summary of the purification of the URA8-encoded CTP synthetase is presented in Table 1. The
purification scheme included ammonium sulfate fractionation of the
cytosolic fraction followed by chromatography with Q-Sepharose,
Affi-Gel Blue, Mono Q, and Superose 6 (Fig. 1). The ammonium
sulfate and Mono Q steps resulted in yields of activity over 100%. The
reason for this was unclear. However, the removal of an inhibitor or
protease could account for the increased yields of activity in these
preparations. Overall, CTP synthetase was purified 1263-fold over the
cytosolic fraction with an activity yield of 43.7% to a final specific
activity of 4.8 µmol/min/mg. This purification scheme resulted in
the isolation of an essentially homogeneous enzyme preparation as
judged by SDS-polyacrylamide gel electrophoresis (Fig. 2). The
purification scheme for the URA8-encoded CTP synthetase was
similar, but not identical to, the scheme used for the purification of
the URA7-encoded enzyme(17) .
Figure 1:
Elution profiles of URA8-encoded CTP synthetase activity after chromatography with
Q-Sepharose, Affi-Gel Blue, Mono Q, and Superose 6. CTP synthetase was
subjected to chromatography with Q-Sepharose (panel A),
Affi-Gel Blue (panel B), Mono Q (panel C), and
Superose 6 (panel D) as described under ``Experimental
Procedures.'' Fractions were collected and assayed for CTP
synthetase activity (
) and protein (-). NaCl gradient
profiles are indicated by a dashed
line.
Figure 2:
SDS-polyacrylamide gel electrophoresis and
immunoblot analysis of URA8-encoded CTP synthetase. Purified CTP
synthetase was subjected to SDS-polyacrylamide gel electrophoresis (lane 1) and immunoblot analysis (lane 3) as
described in the text. Molecular mass standards (lane 2) were
phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin
inhibitor (21.5 kDa).
The minimum subunit
molecular mass of URA8-encoded CTP synthetase was 67 kDa (Fig. 2). This value was in close agreement with the size (64.5
kDa) of the deduced protein sequence of the open reading frame of the URA8 gene(16) . In addition, antibodies raised against
the maltose-binding protein-URA8-encoded CTP synthetase fusion
protein constructed from the coding sequences of the URA8 gene
and expressed in Escherichia coli reacted with purified CTP
synthetase (Fig. 2). These results supported the conclusion that
the product of the URA8 gene was indeed a CTP synthetase.
Enzymological Properties of URA8-encoded CTP
Synthetase
CTP synthetase activity was measured with a
Tris-maleate-glycine buffer at pH values ranging from 5.5 to 9.5 (Fig. 3A). Optimum CTP synthetase activity was obtained
at pH 7.5. The dependence of CTP synthetase activity on the
concentration of magnesium ions is shown in Fig. 3B.
Maximum CTP synthetase activity was dependent on 6 mM magnesium ions. In these experiments, activity was measured at
saturating concentrations of ATP (2 mM), UTP (2 mM),
and GTP (0.1 mM) (see below). At 6 mM magnesium ions,
all of the nucleotides present in the enzyme assay would be present as
magnesium-nucleotide complexes (32) . The dependence of
activity on magnesium ions was cooperative. Analysis of the data
according to the Hill equation yielded a K
value
for magnesium ions of 2.4 mM and a Hill number of 3. The
cooperative dependence of activity on magnesium ions was likely due to
the formation of magnesium-nucleotide complexes and subsequent
cooperative binding to CTP synthetase(17) . Manganese ions
could not substitute for the magnesium ion requirement. CTP synthetase
activity was totally inhibited by 0.8 mMp-chloromercuriphenylsulfonic acid and 0.8 mMN-ethylmaleimide. The addition of 10 mM 2-mercaptoethanol to the assay system prevented the inhibition of
activity by these thioreactive compounds. When 10 mM 2-mercaptoethanol was added to the assay system by itself, it
stimulated CTP synthetase activity by 52%.
Figure 3:
Dependence of URA8-encoded CTP
synthetase activity on pH and magnesium ions. CTP synthetase activity
was assayed at the indicated pH values with 50 mM Tris-maleate-glycine buffer (panel A) and at the
indicated concentrations of MgCl
(panel
B).
CTP synthetase activity
eluted from the Superose 6 chromatography column at a position
consistent with a molecular mass of 150 kDa (Fig. 1D).
This indicated that native URA8-encoded CTP synthetase was a
dimer. A characteristic of the URA7-encoded CTP synthetase is
nucleotide-dependent oligomerization (17) . This property was
examined for URA8-encoded CTP synthetase by subjecting the
enzyme to Superose 6 chromatography in the presence of 2 mM UTP and 2 mM ATP as described previously(17) .
Indeed, the enzyme eluted from the column at a position consistent with
a molecular mass of 300 kDa (data not shown). Thus, under optimum assay
conditions URA8-encoded CTP synthetase existed as a tetramer.
A summary of these properties and those for the URA7-encoded
CTP synthetase is presented in Table 2.
Kinetic Properties of URA8-encoded CTP
Synthetase
CTP synthetase catalyzes an ATP-dependent transfer of
the amide nitrogen from glutamine to the C-4 position of UTP to form
CTP(2, 33) . GTP activates the reaction by
accelerating the formation of a covalent glutaminyl enzyme catalytic
intermediate(2, 33) . We first examined the dependence
of CTP synthetase activity on UTP and ATP using saturating
concentrations of glutamine, GTP, and magnesium ions. Under these
conditions all three nucleotides in the reaction mixtures existed as
magnesium-nucleotide complexes(32) . The kinetics of the enzyme
with respect to the UTP concentration at various set concentrations of
ATP was complex (Fig. 4A). CTP synthetase exhibited
saturation kinetics when the UTP concentration was varied (K
= 74 µM) and ATP was held
constant at a saturating concentration (0.8 mM). On the other
hand, when ATP was held constant at subsaturating concentrations, the
enzyme showed positive cooperative kinetics with respect to UTP. For
example, the Hill number with respect to UTP at 0.1 mM ATP was
2.2. This complex kinetic behavior was also observed when CTP
synthetase activity was measured with respect to ATP at various fixed
concentrations of UTP (Fig. 4B). At a saturating
concentration of UTP (0.6 mM), CTP synthetase showed
saturation kinetics toward ATP (K
= 22
µM). However, the dependence of CTP synthetase activity on
ATP at subsaturating concentrations of UTP was cooperative. For
example, the Hill number with respect to ATP at 75 µM UTP
was 1.6.
Figure 4:
Dependence of URA8-encoded CTP
synthetase activity on UTP and ATP. Panel A, CTP synthetase
activity was measured as a function of the concentration of UTP at the
indicated set concentrations of ATP. Panel B, CTP synthetase
activity was measured as a function of the concentration of ATP at the
indicated set concentrations of UTP. The concentrations of glutamine,
GTP, and MgCl
were maintained at 2, 0.1, and 10
mM, respectively.
The dependence of CTP synthetase activity on glutamine and
GTP was examined using saturating concentrations of UTP, ATP, and
magnesium ions. CTP synthetase showed saturation kinetics with respect
to glutamine at saturating and subsaturating concentrations of GTP (Fig. 5A). The enzyme also showed saturation kinetics
with respect to GTP at saturating and subsaturating concentrations of
glutamine (Fig. 5B). GTP did not affect the
enzyme's affinity for glutamine (K
=
0.14 mM) but did cause an increase in maximum velocity (Fig. 5A). GTP was not an absolute requirement for CTP
synthetase activity (Fig. 5B). However, at a saturating
concentration of glutamine, GTP stimulated (K
= 26 µM) CTP synthetase activity 12-fold. The
activation constant for GTP was not affected by glutamine.
Figure 5:
Dependence of URA8-encoded CTP
synthetase activity on glutamine and GTP. Panel A, CTP
synthetase activity was measured as a function of the concentration of
glutamine at the indicated set concentrations of GTP. Panel B,
CTP synthetase activity was measured as a function of the concentration
of GTP at the indicated set concentrations of glutamine. The
concentrations of UTP, ATP, and MgCl
were maintained at 2,
2, and 10 mM, respectively.
The URA7- and URA8-encoded CTP synthetases exhibited
significant differences in their kinetic properties. In contrast to the URA8-encoded CTP synthetase, the URA7-encoded enzyme
shows positive cooperative kinetics toward UTP and ATP and negative
cooperative kinetics toward glutamine and GTP even when kinetic
experiments are performed with saturating substrate
concentrations(17) . Differences in the kinetic properties of
the URA7- and URA8-encoded enzymes were also
reflected in the kinetic constants for ATP, glutamine, and GTP (Table 2). The cellular concentrations of UTP (0.75 mM)
and ATP (2.3 mM) are saturating for both the URA7-
and URA8-encoded CTP synthetase activities (15, 17) . Thus, the URA8-encoded CTP
synthetase would be expected to exhibit saturation kinetics toward UTP
and ATP in vivo. On the other hand, the URA7-encoded
enzyme would be expected to exhibit positive cooperative kinetics
toward its substrates in vivo. Thus, the URA7- and URA8-encoded CTP synthetase activities may be regulated
differentially in vivo through the cellular concentrations of
UTP and ATP.
Effect of CTP on URA8-encoded CTP Synthetase
Activity
CTP inhibited the URA8-encoded CTP synthetase
activity in a dose-dependent manner with an IC
value of 85
µM (Fig. 6A). The CTP-mediated inhibition
of CTP synthetase activity followed a cooperative (n =
3.2) kinetic pattern (Fig. 6A, inset). The
effect of CTP on the kinetics of CTP synthetase activity with respect
to UTP was examined using saturating concentrations of ATP, glutamine,
GTP, and magnesium ions. As indicated above, CTP synthetase activity
exhibited saturation kinetics toward UTP when ATP was set at a
saturating concentration. However, in the presence of 0.1 mM
CTP, the enzyme exhibited positive cooperative kinetics (n = 2.8) with respect to UTP (Fig. 6B). CTP
also caused a decrease in the apparent V
and an
increase in the apparent K
values with respect to
UTP (Fig. 6B). An important characteristic of the URA7-encoded CTP synthetase is product inhibition of its
activity by CTP(17) . The inhibition of CTP synthetase activity
by CTP regulates the cellular concentration of CTP in growing
cells(17) . Strikingly, the inhibitor constant for CTP (85
µM) for URA8-encoded CTP synthetase was 3.5-fold
lower when compared with the inhibitor constant (0.3 mM) for
the URA7-encoded enzyme (Table 2). Furthermore, the
inhibitor constant for the URA8-encoded enzyme was 8-fold
lower than the cellular concentration (0.77 mM) of
CTP(34) . These results indicated that in vivo, the URA8-encoded enzyme would be more sensitive to product
inhibition by CTP when compared with the URA7-encoded enzyme.
Figure 6:
Effect of CTP on URA8-encoded CTP
synthetase activity. Panel A, CTP synthetase activity was
measured under standard assay conditions with 0.1 mM UTP in
the absence and presence of the indicated concentrations of CTP. The inset in panel A is a replot of the CTP-mediated
inhibition of CTP synthetase activity. Panel B, CTP synthetase
activity was measured as a function of the concentration of UTP in the
absence and presence of 0.1 mM CTP as
indicated.
Abundance of the URA7- and URA8-encoded CTP Synthetase
mRNAs
The abundance of the URA7- and URA8-encoded CTP synthetase mRNAs in wild-type cells was
examined by Northern blot analysis. Under the high stringency
hybridization conditions used in these studies, the URA7 probe
did not hybridize to URA8-encoded CTP synthetase mRNA, and the URA8 probe did not hybridize to the URA7-encoded CTP
synthetase mRNA. The level of the URA7-encoded CTP synthetase
mRNA was 2-fold more abundant than the level of the URA8-encoded CTP synthetase mRNA (Fig. 7). Ribosomal
subunit L32 mRNA was used as an internal standard in these
experiments. Thus, the difference in the abundance of these mRNA levels
was attributed to differential expression of these genes as opposed to
RNA degradation or to differences in the amount of RNA loaded on the
gel.
Figure 7:
Abundance of the URA7- and URA8-encoded CTP synthetase mRNAs. Wild-type cells were grown
in complete synthetic medium to the exponential phase of growth. CTP
synthetase mRNA encoded by the URA7, and URA8 genes
were determined by Northern blot analysis as described in the text. The
amount of the L32 mRNA loading control was set at 1 in the
figure.
Based on codon bias values, the URA7 gene product
would be expected to be expressed more than the URA8 gene
product (16) . This notion was supported by the fact that the URA8-encoded enzyme required a greater fold purification to
obtain pure enzyme when compared with the URA7-encoded enzyme (Table 2). We were unable to determine differential expression of
the URA7 and URA8 gene products by immunoblot
analysis. Anti-URA8-encoded CTP synthetase antibodies reacted
with pure URA7-encoded CTP synthetase, and
anti-URA7-encoded CTP synthetase antibodies (17) reacted with pure URA8-encoded enzyme (data not
shown). In addition, the subunit molecular masses of the two CTP
synthetases were too similar to differentiate on immunoblots.
Effect of Growth Phase on CTP
Synthetases
Anti-URA7-encoded CTP synthetase antibodies
were used to examine the expression of the CTP synthetases during the
exponential and stationary phases of growth in wild-type cells. Maximum
expression of the CTP synthetases was found in the early exponential
phase of growth (Fig. 8). As cells entered the stationary phase
of growth, the expression of the CTP synthetases decreased 5-fold (Fig. 8). We also examined the activity of CTP synthetase during
the exponential and stationary phases of growth. The specific activity
of the enzyme was 4.3-fold higher in early exponential phase cells when
compared with stationary phase cells. The regulation of CTP synthetase
expression with growth phase was consistent with the essential role CTP
plays in the synthesis of nucleic acids and membrane phospholipids
during active cell growth (16) .
Figure 8:
Effect of growth phase on CTP synthetases.
Wild-type cells were grown in complete synthetic medium. Cells were
harvested in the early exponential (9
10
cells/ml),
late exponential (1.3
10
cells/ml), and stationary
(4.2
10
cells/ml) phases of growth. Cytosolic
fractions were prepared, and 25 µg of each sample were subjected to
immunoblot analysis using a 1:500 dilution of IgG
anti-URA7-encoded CTP synthetase antibodies. CTP synthetase
bands on immunoblots were quantified by scanning densitometry. The
amount of CTP synthetase found in the early exponential phase of growth
was set at 1 in the figure. The inset shows a growth curve of
wild-type cells. Cell numbers were determined by counting under a
microscope on a hemacytometer.
Concluding Discussion
The URA7- and URA8-encoded CTP synthetases are functionally overlapping
enzymes responsible for the synthesis of CTP in S.
cerevisiae(16) . Studies performed with mutants defective
in the URA7 or URA8 genes indicated that the URA7-encoded CTP synthetase is responsible for the majority
(78%) of the CTP synthesized in vivo(16) . We
questioned whether the relative contribution of the URA7- and URA8-encoded CTP synthetases for the synthesis of CTP was
influenced by differential biochemical regulation of these enzymes. To
address this question under well-defined conditions, we previously
purified and characterized the URA7-encoded CTP synthetase (17) and in this work, we purified and characterized the URA8-encoded enzyme. The URA7- and URA8-encoded CTP synthetases had striking differences with
respect to several enzymological and kinetic properties including
turnover number, pH optimum, substrate dependences, and sensitivity to
inhibition by CTP. Importantly, the differential kinetic properties of
the enzymes with respect to UTP and ATP and inhibition by CTP were seen in vitro at concentrations within the the range of
concentrations which occur in vivo. In addition, the products
of the URA7 and URA8 genes were differentially
expressed. The increased sensitivity of the URA8-encoded
enzyme activity to product inhibition by CTP coupled to the lower
expression of the URA8-encoded CTP synthetase was consistent
with the URA7-encoded CTP synthetase being responsible for the
majority of the CTP synthesized in vivo.