From the Division of Genetic Information, Institute
for Genome Research and the ¶ Otsuka Department of Clinical and
Molecular Nutrition, School of Medicine, The University of Tokushima,
Tokushima 770-8503, Japan
Received for publication, December 11, 2000, and in revised form, March 7, 2001
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ABSTRACT |
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To clarify the contributions of
amidophosphoribosyltransferase (ATase) and its feedback regulation to
the rates of purine de novo synthesis, DNA synthesis,
protein synthesis, and cell growth, mutated human ATase (mhATase)
resistant to feedback inhibition by purine ribonucleotides was
engineered by site-directed mutagenesis and expressed in CHO ade
Purine nucleotides are synthesized both via the de novo
pathway and via the salvage pathway and are vital for cell functions and cell proliferation through DNA and RNA syntheses and ATP energy supply. Amidophosphoribosyltransferase
(ATase)1 is the rate-limiting
enzyme in the de novo pathway of purine ribonucleotide
synthesis (1) and is regulated by feedback inhibition by AMP and GMP.
Hypoxanthine (Hx) and hypoxanthine guanine phosphoribosyltransferase (HPRT) are the most important substrate and enzyme, respectively, of
the salvage pathway (1). The Lesch-Nyhan syndrome is caused by a
complete deficiency of HPRT. Although patients with this syndrome show
hyperuricemia with accelerated de novo purine synthesis, the
mechanism of activation of the de novo pathway is not fully understood.
In a previous study, we demonstrated that the expression level of ATase
limits the growth rate of cultured fibroblasts, and purine salvage
strongly inhibits purine de novo synthesis (1). Two
mechanisms by which the salvage pathway inhibits the de novo pathway were presumed. 1) Consumption of 5-phosphoribosyl
1-pyrophosphate (PRPP) by the salvage pathway decreases the activity of
the de novo pathway because PRPP is the common source of
both pathways. 2) Purine nucleotides synthesized via the salvage
pathway inhibit ATase by feedback regulation. However, it is unclear
which mechanism plays the more important role.
To resolve these questions, feedback-resistant human ATase (hATase) was
engineered by site-directed mutagenesis and expressed in CHO ade
Furthermore, we generated transgenic mice expressing mhATase under the
control of the CAG promoter (mhATase-Tg mice). Because this promoter
functions in most cells, overexpression of mhATase was expected in all
tissues of the transgenic mice. In mhATase-Tg mice, the following data
were examined: ATase activity in the liver and spleen, the metabolic
rate of the de novo pathway in the liver, urinary excretion
of allantoin and uric acid (UA), serum UA concentration, and T
lymphocyte proliferation by phytohemagglutinin (PHA).
Site-directed Mutagenesis--
In Escherichia coli
ATase, amino acid replacements of K326Q and P410W result in decreased
binding affinity for AMP and GMP and lead to corresponding reductions
in feedback inhibition (2). Because the amino acid sequences of these
two regions are conserved in E. coli ATase and hATase, these
mutations were expected to make hATase, as well as E. coli
ATase, feedback-resistant. Using a polymerase chain
reaction-based method, a K338Q/P422W double mutation, which corresponds
to K326Q/P410W of E. coli ATase, was generated in hATase
cDNA, resulting in mhATase cDNA.
Cell Culture--
CHO K1 and CHO ade Transfection of CHO ade Measurement of Cell Growth Rate--
Cultured cells during the
logarithmic growth phase were counted by an improved Neubauer
hemocytometer (1). The doubling time (h) was determined from cell
counts, and its reciprocal was defined as the cell growth rate.
Determination of Synthesis Rates of Purine Nucleotides and
Proteins--
The metabolic rates of the de novo and
salvage pathways were determined, respectively, by the incorporation of
[14C]glycine (Amersham Pharmacia Biotech) or
[3H]Hx (Amersham Pharmacia Biotech) in acid-soluble
purines (1, 6). In a 90-mm culture dish, 2 × 106
cells were plated. After the recovery of cell function from plating by
18 h of culture, [14C]glycine or
[3H]Hx was added to the medium at the final concentration
of 150 µM (0.3 MBq/ml) and 40 µM (0.15 MBq/ml), respectively. The cells were cultured in radioactive medium
for 30 min, washed three times with 10 ml of ice-cold
phosphate-buffered saline, and harvested with a rubber policeman.
Purine nucleotides were extracted from the cells in 1 ml of 0.4 N perchloric acid at 100 °C for 60 min. After
centrifugation at 12,000 × g at 4 °C for 5 min, the
supernatant was applied to a column (0.5 × 3 cm) of AG-50W-X8
(Bio-Rad) equilibrated with 0.1 N HCl. After washing with 5 ml of 1 N HCl, the acid-soluble purines were eluted with 5 ml of 6 N HCl and counted with Aquasol-2 (Packard
Instrument Co.) in a scintillation counter. The amount of purines was
determined by the absorbance of the eluate at 260 nm. The rates of both
purine de novo synthesis and protein synthesis were
simultaneously determined by the incorporation of
[14C]glycine (6). To measure the incorporation of
[14C]glycine in protein, the acid-insoluble precipitate
from the last centrifugation was washed three times in 10%
trichloroacetic acid and dissolved in 200 µl of 0.3 N
NaOH, and the radioactivity was counted with Aquasol-2 in a
scintillation counter. The protein concentrations were assayed by
Bradford's method (7).
Determination of Rate of DNA Synthesis--
The rate of DNA
synthesis was determined by bromodeoxyuridine (BrdUrd) incorporation.
In a 90-mm culture dish, 2 × 106 cells were plated.
After the recovery of cell function from plating by 18 h of
culture, the cells were cultured with 10 µM BrdUrd for 30 min. Incorporated BrdUrd was quantified with an enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) according to the
manufacturer's protocol.
Assay of ATase Activity--
Cell lysate for enzyme assay was
prepared by sonication and centrifugation (1). To assay ATase activity,
the cell lysate was incubated in 50 mM potassium phosphate
buffer (pH 7.4) containing 5 mM PRPP (Sigma), 5 mM MgCl2, 1 mM dithiothreitol, and
5 mM [14C]glutamine (Amersham Pharmacia
Biotech; 5.55 kBq/mmol) at 37 °C for 1 h. Formed
[14C]glutamate was separated from
[14C]glutamine by high voltage paper electrophoresis at
800 W for 15 min and counted with toluene scintillation mixture in a
scintillation counter. The PRPP-dependent hydrolysis of
glutamine to glutamate was regarded as representing ATase activity (8).
In the assay solution, ATase was activated by PRPP with the
conformational change from tetramer to dimer. Therefore, ATase activity
in this study indicates the amount of ATase protein, but not real
enzyme activity, in cells.
Subunit Structure of hATase--
The small active form of hATase
is a dimer of subunits, whereas the large inactive form of hATase is a
tetramer (9). These two molecular forms of hATase were identified by
the retention time of gel filtration by HPLC. The cell lysate prepared
for enzyme assay was separated through a TSK-G3000SWXL column (Tosoh,
Tokyo, Japan) in 50 mM potassium phosphate buffer (pH 7.4)
containing 0.2 M NaCl, 1 mM dithiothreitol, and
10 mM AMP at a flow rate of 0.4 ml/min. The retention time
of hATase was determined by ATase assay of eluate fractions.
Generation of mhATase Transgenic Mice--
An expression vector
containing the CAG promoter (chicken Measurement of Purine Derivatives--
Serum UA concentrations
of mice were determined by a uricase method with a UA-L kit (Serotec,
Sapporo, Japan). Urinary excretions of allantoin, UA, Hx + xanthine,
and creatinine were measured by HPLC through two µBondapak C18
columns (Waters Corp., Milford, MA) in 20 mM potassium
phosphate buffer (pH 4.0) at a flow rate of 0.5 ml/min. The effluent
was monitored at 205 nm, and peak areas of purine derivatives were
compared with those of the corresponding standard solutions.
T Lymphocyte Proliferation by PHA--
Splenocytes (5 × 105 cells/well) obtained from mice of 15-20 weeks of
age were cultured in RPMI 1640 medium containing 10% FCS and 6 µg/ml
PHA-L (Roche Molecular Biochemicals) in a 96-well plate. After 24, 48, and 72 h, BrdUrd incorporation for 6 h was measured with an
enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals)
according to the manufacturer's protocol.
Statistical Analysis--
The data are presented as means ± S.E. For a comparison of two means, Student's paired or unpaired
t test was used. A probability value (p) of less
than 0.05 was considered statistically significant.
Characterization of mhATase--
Using cell lysates of
hATase has two molecular forms: a homodimer and a homotetramer of
subunits. hATase activated by PRPP forms the homodimer, whereas hATase
inactivated by purine ribonucleotides forms the homotetramer (9). In
the presence of 10 mM AMP, the molecular form of ATase was
analyzed by gel filtration. The retention time of ATase showed that all
hATase forms were inactive tetramers, whereas most mhATase forms were
active dimers even in the presence of 10 mM AMP (Fig.
2).
Long Term Culture of Transfectants--
Several clones of
After a 2-week culture, the ATase activities of
Mutual Regulation of de Novo and Salvage Pathways--
The
metabolic rate of the de novo pathway in CHO K1 cells under
conditions where only the de novo pathway was functioning in
HamF + XO was 5 times higher than that under conditions where both
purine biosynthetic pathways were functioning in HamF, i.e. the salvage pathway strongly suppresses the de novo pathway
down to 20% of the initial value (Table
I).
The metabolic rate of the salvage pathway in CHO K1 cells under the
conditions where both pathways were functioning in HamF was only 13%
less than that under the conditions where only the salvage pathway in
CHO ade Contributions of ATase and Its Feedback Regulation to
Biosyntheses--
The contributions of ATase to various biosyntheses
are represented as the rates of biosyntheses relative to the ATase
level (Table II). The contributions of
ATase to biosyntheses in
The contribution of hATase to purine de novo synthesis in
HamF was smaller than that in Ham F + XO (37.1 versus 163.1)
because the salvage pathway inhibits the de novo pathway in
HamF (Table II). Therefore, the contribution of the feedback regulation
to purine de novo synthesis in HamF was greater than that in
HamF + XO (13.5 versus 3.7).
In hamster ATase, hATase, and mhATase, the contributions of ATase to
protein and DNA syntheses in HamF were modestly larger than those in
HamF + XO, although the differences are not statistically significant
(Table II). These findings indicate that the de novo pathway
alone in HamF + XO can supply almost all purine nucleotides necessary
for protein and DNA syntheses, and the additional effect of the salvage
pathway in HamF is trivial. The contributions of mhATase to protein and
DNA syntheses were, respectively, 1.6-1.8 and 3.5-3.9 times higher
than those of hATase, suggesting that the feedback inhibition of ATase
also regulates protein and DNA syntheses in cultured fibroblasts.
Contributions of ATase and Its Feedback Regulation to Cell Growth
Rate--
The growth rate of CHO K1 cells in HamF + XO was higher (6.1 versus 4.2) than that of CHO ade Generation of mhATase Transgenic Mice--
We obtained nine
founders of mhATase-Tg mice. The number of transgene copies in these
transgenic mice ranged from 5 to 100. In expectation of high level
mhATase expression, two founder mice with high copy numbers of more
than 50 were bred, and their F1 mice were used in this study. Body
weights measured weekly were similar in both lines of transgenic mice
and wild-type mice at least up to 25 weeks of age.
Purine Nucleotide Metabolism in mhATase-Tg Mice--
In mhATase-Tg
mice, liver ATase activity (1.8-fold),
spleen ATase activity (1.6-fold), the metabolic rate of the de
novo pathway in the liver (1.8-fold), urinary excretion of
allantoin + UA normalized to creatinine excretion (1.5-fold), and serum
UA concentration (1.5-fold) were significantly higher than these values
in wild-type mice (Figs. 4 and 5).
T Lymphocyte Proliferation by PHA--
T lymphocytes in mouse
splenocytes were stimulated by PHA. After 48 and 72 h, BrdUrd
incorporation by T lymphocytes in mhATase-Tg mice was significantly
greater than that in wild-type mice (Fig. 6).
Subunit Structure of ATase--
In the presence of 10 mM AMP, hATase formed inactive tetramers, whereas most
mhATase remained in the active dimeric form, i.e. mhATase
was resistant to a conformational change induced by AMP. A minor part
of the mhATase was in tetrameric form. This tetramer may be a complex
of mhATase subunits and hamster ATase subunits inactivated by a
mutation in CHO ade Mutual Regulation of de Novo and Salvage Pathways--
Purine
nucleotides are synthesized preferentially by the salvage pathway as
long as hypoxanthine is available, with concomitant inhibition of the
de novo pathway for sparing the energy expenditure required
for de novo synthesis (1, 11). In
In addition to the activation of the de novo
pathway, mhATase also strongly suppressed the salvage pathway. The
suppression of the salvage pathway by the de novo pathway is
probably due to consumption of PRPP by the de novo pathway
because the activity of the de novo pathway has no effect on
HPRT expression (1) and HPRT is not regulated by the feedback
inhibition. Indeed, a large consumption of PRPP by de novo
synthesis is suggested by the 16-fold increase in PRPP concentration in
human leukemia cells treated with 6-methylmercaptopurine riboside to
inhibit de novo synthesis (13), whereas the primary cultured
astroglia from HPRT-deficient mice showed only a 27.8% increase in
intracellular PRPP level (12).
Contributions of ATase and Its Feedback Regulation to Various
Biosyntheses and Cell Growth Rate--
In addition to the acceleration
of purine de novo synthesis, mhATase also increased the
rates of DNA and protein syntheses and of cell growth, suggesting that
ATase and its feedback inhibition regulate the growth rate via the
rates of DNA and protein syntheses. We previously reported that purine
de novo synthesis is a determinant of intracellular ATP
concentration and promotes G1 to S phase transition in the
cell cycle, i.e. the initiation of DNA synthesis (11).
Therefore, ATase and its feedback inhibition probably regulate DNA and
protein syntheses through ATP production.
Surprisingly, the minimum doubling time of Purine Nucleotide Metabolism in mhATase-Tg
Mice--
In transgenic mice expressing normal hATase, the
acceleration of purine de novo synthesis cannot be expected
because of the feedback inhibition of hATase. Indeed, T Lymphocyte Proliferation by PHA--
Resting human T lymphocytes
are reported to meet their metabolic demands via the salvage pathway,
whereas intact de novo synthesis is essential for the
proliferation of PHA-stimulated T lymphocytes (16). Even in patients
with Lesch-Nyhan syndrome, the proliferation of T lymphocytes in
response to mitogenic and antigenic stimulation was normal (17).
Moreover, in leukocytes from two gouty patients affected with a partial
deficiency of HPRT, de novo synthesis was accelerated to
more than 13 times that of normal controls (18). These reports suggest
that the large capacity of purine de novo synthesis can
compensate for the defect in the salvage pathway. In mhATase-Tg mice, T
lymphocytes stimulated by PHA proliferated more rapidly than those in
wild-type mice. The rate of purine de novo synthesis may
determine the growth rate of T lymphocytes under mitogen stimulation.
Indeed, severe immunological abnormalities are induced by deficiencies
in enzymes of purine nucleotide metabolism such as adenosine deaminase
and purine nucleoside phosphorylase. Recently, methotrexate, an
immunosuppressive agent, has been viewed as the drug of choice in
treating rheumatoid arthritis. Low dose methotrexate inhibits ATase and
mitogen-induced expansion of ATP and GTP pools in human T lymphocytes
and induces cell arrest at the G1 phase, thus exerting
immunosuppressive effects (19, 20).
In conclusion, the following concepts are supported by this study using
CHO transfectants with mhATase and mhATase-Tg mice. First, ATase and
its feedback inhibition regulate not only the rate of purine de
novo synthesis but also DNA and protein synthesis rates and the
growth rate in cultured fibroblasts. Second, the suppression of the
de novo pathway by the salvage pathway is primarily due to
the feedback inhibition of ATase by purine ribonucleotides produced
through the salvage pathway, whereas the suppression of the salvage
pathway by the de novo pathway is due to the consumption of
PRPP by the de novo pathway. Third, the feedback inhibition of ATase is more important for the regulation of the de novo
pathway than that of PRPP synthetase, and PRPP synthetase indirectly
regulates the de novo pathway via ATase activation by
producing PRPP. Finally, ATase superactivity leads to hyperuricemia and
increases BrdUrd incorporation in T lymphocytes stimulated by PHA.
Understanding the regulation of purine synthetic pathways and their
relationship to DNA and protein syntheses and cell growth is useful for
various medical applications such as developing therapies for malignant neoplasms and autoimmune diseases.
A cells (an ATase-deficient cell line of Chinese hamster
ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were
examined: ATase activity and its subunit structure, the metabolic rates
of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity
in the liver and spleen, the metabolic rate of the de novo
pathway in the liver, serum uric acid concentration, urinary excretion
of purine derivatives, and T lymphocyte proliferation by
phytohemagglutinin were examined. We concluded the following. 1) ATase
and its feedback inhibition regulate not only the rate of purine
de novo synthesis but also DNA and protein synthesis rates
and the rate of cell growth in cultured fibroblasts. 2) Suppression of
the de novo pathway by the salvage pathway is mainly due to
the feedback inhibition of ATase by purine ribonucleotides produced via
the salvage pathway, whereas the suppression of the salvage pathway by
the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3)
The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl
1-pyrophosphate synthetase. 4) ATase superactivity leads to
hyperuricemia and an increased bromodeoxyuridine incorporation in T
lymphocytes stimulated by phytohemagglutinin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A fibroblasts, an auxotrophic cell line deficient in
ATase. In CHO ade
A cells transfected with mutated hATase
cDNA (
A + mhATase cells), the following parameters
were examined and compared with those in CHO ade
A cells
transfected with normal hATase cDNA (
A + hATase
cells): ATase activity, the subunit structure of ATase, the metabolic
rates of de novo and salvage pathways, the rates of protein
and DNA syntheses, and the rate of cell growth. For a comprehensive
study, we used, in total, four cell lines: 1) CHO K1, a wild type of
CHO fibroblasts, 2) CHO ade
A cells, 3)
A + hATase cells, and 4)
A + mhATase cells.
We also used two culture media: 1) a medium rich in purine bases and 2)
a medium free of purine bases. In the purine-rich medium, both the
de novo and salvage pathways function in CHO K1,
A + hATase, and
A + mhATase cells, but only
the salvage pathway functions in CHO ade
A cells. In the
purine-free medium, only the de novo pathway
functions in CHO K1,
A + hATase, and
A + mhATase cells, and neither of the two pathways functions in CHO ade
A cells. Under various conditions, we examined the
effects of feedback regulation of ATase on the cell growth rate and on
the biosyntheses of purine nucleotides, DNA, and proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A cells (3)
were kind gifts from Dr. David Patterson (Eleanor Roosevelt Institute
for Cancer Research, Denver, CO). The CHO fibroblasts were cultured at
37 °C in an atmosphere of humidified air:CO2 (95:5) in
Ham's F-12 (HamF) purine-rich medium containing 30 µM Hx + 10% fetal calf serum (FCS) or HamF with 10% FCS treated with 2.5 mg
(1.8 unit)/liter xanthine oxidase (XO) from buttermilk (Sigma) at
37 °C overnight, serving as a Hx-free medium (HamF + XO). The
complete removal of Hx in HamF + XO was confirmed first by the
disappearance of the Hx peak in the reversed-phase high performance
liquid chromatography (HPLC) analysis through a C18 column (1) and
second by the absence of growth of CHO ade
A cells in
HamF + XO. Although RPMI 1640 + 10% dialyzed FCS was also used as a
completely purine-free medium, the results obtained from CHO cell
culture in this medium were essentially identical to those in HamF + XO
containing 10% nondialyzed FCS (data not shown). Therefore, adenine
and guanine carry-over from nondialyzed FCS was considered to be
negligible, and HamF + XO containing 10% nondialyzed FCS was used as
an essentially purine-free medium in this study. In HamF, both the
de novo and salvage pathways function in CHO K1,
A + hATase, and
A + mhATase cells, but only
the salvage pathway functions in CHO ade
A cells. In HamF + XO, only the de novo pathway functions in CHO K1,
A + hATase, and
A + mhATase cells, and
neither of the two pathways functions in CHO ade
A cells.
A Cells--
hATase or
mhATase cDNA (2.2 kilobase pairs) was inserted into the cloning
site downstream of the cytomegalovirus promoter in pBCMGSNeo (14.5 kilobase pairs), which includes the replication origin of bovine
papillomavirus leading to 10-500 copies/cell in mammalian cells
(4, 5). Each plasmid vector (5 µg) was mixed with Lipofectin reagent
(Life Technologies, Inc.) according to the manufacturer's protocol and
overlaid onto CHO ade
A cells (2 × 106
cells/90-mm tissue-culture plate) in 5 ml of Opti-MEM (Life
Technologies, Inc.). After incubation at 37 °C for 6 h in a
CO2 incubator, the DNA-containing medium was replaced with
10 ml of HamF containing 1 mg/ml G418 (Life Technologies, Inc.).
Several clones of
A + hATase and
A + mhATase cells were isolated after G418 selection for 7 days. To select
subclones with high ATase activity, these transfectants were further
cultured in HamF + XO for several weeks. Long term culture in this
purine-free medium positively selected the subclones with high ATase
activity associated with a high rate of cell growth.
-actin promoter with
cytomegalovirus enhancer) was a kind gift from Dr. Jun-ich Miyazaki
(Department of Nutrition and Physiological Chemistry, Osaka University,
Japan). mhATase cDNA was inserted into the cloning site of the
expression vector, and a SalI fragment excluding
plasmid-derived sequences was microinjected into the male pronuclei of
fertilized eggs obtained from superovulated BDF1 (C57BL/6 × DBA/2
F1) female mice crossed with males of the same strain. Injected embryos
were implanted into the oviducts of pseudopregnant female mice and
allowed to develop (10). DNA was extracted from tail snips of live
offspring by the proteinase K/SDS method. The integration of the
transgene into the mouse genome was detected by polymerase chain
reaction and Southern blot analysis. The copy numbers of integrated
transgenes were determined from the intensity of each radioactive band
in Southern blot analysis compared with indicator bands of 1, 10, and
100 copies of the transgene. F1 transgenic progeny were bred by
crossing transgenic founder mice with BDF1 mice and used for
experiments with sex-matched nontransgenic littermates (wild-type mice)
at the age of 15-25 weeks. Body weight was determined every week.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A + hATase and
A + mhATase cells, the
effects of AMP and GMP on ATase activity were examined. hATase was
allosterically inhibited by AMP and GMP, whereas mhATase was resistant
to inhibition by AMP and GMP (Fig.
1).
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Fig. 1.
Feedback inhibition of ATase by AMP and
GMP. Cell lysates of A + hATase (
;
n = 4) and
A + mhATase (
;
n = 4) cells were used for the ATase assay.
mhATase is resistant to inhibition by AMP and GMP.
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Fig. 2.
Subunit structure of ATase. Cell lysates
of A + hATase (
) and
A + mhATase
(
) cells were separated by HPLC through a TSK-G3000SWXL
column in 50 mM potassium phosphate buffer (pH 7.4)
containing 0.2 M NaCl, 1 mM dithiothreitol, and
10 mM AMP at a flow rate of 0.4 ml/min. ATase activity with
a peak retention time of 22.5 min represents the inactive tetrameric
form, whereas the enzyme activity with a peak retention time of 28.0 min represents the active dimeric form. In the presence of 10 mM AMP, all hATase formed tetramers, and most mhATase
formed dimers. Molecular markers: ferritin, 440 kDa; catalase, 232 kDa;
albumin, 67 kDa.
A + hATase and
A + mhATase cells were
further cultured in purine-free medium, HamF + XO. Because subclones with high ATase activity proliferate more rapidly than those with low
ATase activity, subclones with high ATase activity were gradually selected in the time course of cell culture. As the ATase activity of
A + hATase cells increased, the metabolic rate of the
de novo pathway, the synthesis rates of DNA and proteins,
and the growth rate also increased in parallel (Fig.
3).
A + mhATase cells
cultured in HamF + XO also showed similar results (data not shown).
These results showed strong links among ATase activity, the metabolic
rate of the de novo pathway, the synthesis rates of DNA and
proteins, and the cell growth rate.
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Fig. 3.
Long term culture of A + hATase
cells.
A + hATase cells were cultured in HamF + XO.
Because subclones with high ATase activity proliferate more rapidly
than those with low ATase activity, subclones with high ATase activity
were positively selected. As intracellular ATase activity gradually
increased, the metabolic rate of the de novo pathway, the
rates of DNA and protein syntheses, and the cell growth rate also
increased in parallel.
A + hATase and
A + mhATase cells increased
to the same level as ATase in CHO K1 cells (450-550
cpm/106 cells). When the ATase activity is measured
in vitro, ATase is activated by PRPP at the maximal level.
Therefore, the ATase activity indicates the amount of ATase protein in
cells but not the actual ATase activity in vivo. Using
A + hATase,
A + mhATase, and CHO K1 cells
with the same ATase levels, the mutual regulation of the de
novo and salvage pathways was analyzed.
A + hATase cells
also showed a similar result. In contrast, in
A + mhATase
cells, the suppression of the de novo pathway by the salvage
pathway was only 16%, and the metabolic rate of the de novo
pathway in HamF was 16 times higher than that in CHO K1 cells. These
findings indicate that the suppression of the de novo
pathway by the salvage pathway was primarily due to feedback
inhibition of ATase by purine ribonucleotides produced through the
salvage pathway. Moreover, even in HamF + XO, the metabolic rate of the de novo pathway in
A + mhATase cells was
3.8 times higher than that in CHO K1 cells, indicating that AMP and GMP
produced through the de novo pathway also inhibit ATase
in a negative feedback loop in vivo.
Metabolic rates of de novo and salvage pathways of four cell lines
A cells was functioning in HamF (Table I). A
similar result was obtained in
A + hATase cells. In
A + mhATase cells, however, the metabolic rate of the
salvage pathway was strongly suppressed by the de novo
pathway in HamF.
A + hATase and
A + mhATase cells were compared, and the contributions of feedback regulation of ATase to these biosyntheses are represented as the ratio
of the contributions of hATase and mhATase to the biosyntheses (mhATase/hATase ratio). The contributions of hamster ATase in CHO K1
cells to the biosyntheses were similar to those of hATase in
A + hATase cells (Table II).
Contributions of ATase and its feedback regulation to various
biosyntheses
A + hATase and
A + mhATase cells
were cultured in HamF + XO for several weeks. When their ATase levels
reached maximum plateau values (about 3 times the ATase level of CHO K1
cells), their maximal growth rates were determined in HamF and compared
with the growth rates of CHO K1 and CHO ade
A cells
(Table III). The maximal growth rates of
A + hATase and
A + mhATase cells were
higher than those of CHO K1 cells. In particular, the doubling time of
A + mhATase cells was shortened to 6.0 h by long
term culture for more than 8 weeks. To our knowledge,
A + mhATase cells grow at the highest rate of all cultured mammalian cells
investigated. These results show that ATase and its feedback inhibition
regulate the growth rate of cultured fibroblasts.
Growth of CHO fibroblasts
A cells in
HamF, i.e. the contribution of the de novo
pathway to the growth rate was greater than that of the salvage pathway (Table III). The additional contribution of the salvage pathway to the
growth rate was also small (from 6.1 to 6.6).
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Fig. 4.
Purine nucleotide metabolism in mhATase-Tg
mice. Liver and spleen homogenates in 50 mM potassium
phosphate buffer (pH 7.4) containing 5 mM dithiothreitol,
100 µg/ml phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin were
centrifuged at 12,000 × g at 4 °C for 20 min. The
supernatants were used for the ATase assay. For determination of the
metabolic rate of the de novo pathway, 200 µl of saline
containing 50 mM glycine with [14C]glycine
(1.85 MBq) were administered intraperitoneally. After 60 min, the mouse
liver was removed, and a liver homogenate in 0.4 N
perchloric acid was prepared for the extraction of radiolabeled
purines. The numbers of samples ranged from seven to nine for each bar.
White bars, wild-type mice; black bars,
mhATase-Tg mice. *, p < 0.05; **, p < 0.01.
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Fig. 5.
Urinary excretion of purine derivatives.
a, standard solution; b, a wild-type mouse; and
c, an mhATase-Tg mouse. Under the chromatographic conditions
in this study, Hx and xanthine were not separated, and the peak of Hx + xanthine was not detected in mouse urine. Therefore, the urinary
excretion of allantoin + UA was regarded as the urinary excretion of
total purine derivatives and was normalized to creatinine excretion
(Fig. 4).
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Fig. 6.
T lymphocyte proliferation by PHA.
Splenocytes obtained from mice of 15-20 weeks of age were cultured in
RPMI 1640 medium containing 10% FCS and 6 µg/ml PHA in a 96-well
plate, and BrdUrd incorporation was determined every 24 h. , mhATase-Tg mice (n = 7);
,
wild-type mice (n = 7). *, p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A cells because hamster ATase was
always found in the form of tetramers, even in the presence of PRPP,
and because the less common tetrameric forms of hATase and
mhATase were detected along with the major dimeric forms in the
presence of PRPP (data not shown).2
A + mhATase
cells, the suppression of the de novo pathway by the salvage
pathway was strongly inhibited even in the presence of sufficient Hx in
HamF. Therefore, the mechanism by which the salvage pathway suppresses
the de novo pathway was shown to be mainly due to the
feedback inhibition of ATase rather than to the consumption of PRPP,
the common substrate of both pathways. From these results, the
mechanism of the accelerated de novo pathway in patients
with Lesch-Nyhan syndrome can also be explained as follows: AMP and GMP
produced through the salvage pathway decrease because of complete HPRT
deficiency; decreased AMP and GMP reduce the feedback inhibition of
ATase, and activated ATase increases the de novo synthesis of purine nucleotides. Indeed, primary cultured astroglia from HPRT-deficient mice showed reduced levels of intracellular purine ribonucleotides with a 9.4-fold acceleration of de novo
purine synthesis (12). Even under conditions where only the de
novo pathway functions without the suppression by the salvage
pathway, mhATase activated the de novo pathway by a factor
of 3.7-3.8. Therefore, it was first demonstrated in cultured cells
that purine ribonucleotides produced through the de novo
pathway inhibit ATase in a negative feedback loop.
A + mhATase
cells was as short as only 6.0 h, thus representing one of the
most rapidly growing cell types. This result also indicates the great contribution of ATase and its feedback inhibition to regulating the
growth rate of cultured fibroblasts.
A + hATase cells with an ATase level twice that of CHO K1 cells showed only
a 1.2-fold increase in the rate of purine de novo synthesis
(1). Therefore, mhATase was used to generate transgenic mice in this
study. In mhATase-Tg mice, increases in ATase levels in the liver
(1.8-fold) and spleen (1.6-fold) were comparable to the increase in the
metabolic rate of the de novo pathway (1.8-fold). Furthermore, ATase activities in the liver and spleen of mhATase-Tg mice were resistant to inhibition by AMP (data not shown). These results suggest that mhATase is primarily expressed in mhATase-Tg mice,
and the endogenous expression of mouse ATase is suppressed. Normal
ATase protein may have a short half-life in the presence of excessive
mhATase, whereas mhATase may also be resistant to protein degradation.
mhATase-Tg mice also showed increases in serum UA concentration and
urinary excretion of allantoin + UA. These findings suggest that the
superactivity of ATase leads to hyperuricemia in humans, although the
germline mutation of the human ATase gene has not been reported. Purine
de novo synthesis is also regulated by the feedback
inhibition of PRPP synthetase, and the superactivity of this enzyme by
point mutations results in hyperuricemia and gout (14). Both in
A + mhATase cells and in mhATase-Tg mice, the feedback
regulation of PRPP synthetase by purine ribonucleotides was intact, but
the acceleration of the de novo pathway was not suppressed
by the feedback regulation of PRPP synthetase. This indicates that the feedback inhibition of ATase is more important for the regulation of
the de novo pathway than that of PRPP synthetase and that
PRPP synthetase indirectly regulates the de novo pathway via
ATase activation by producing PRPP. Indeed, the studies using
fibroblasts from patients with Lesch-Nyhan syndrome and with PRPP
synthetase superactivity supported the following concepts. 1) The rate
of purine de novo synthesis is regulated at both PRPP
synthetase and ATase reactions, and the latter reaction is more
sensitive to small changes in purine ribonucleotide concentrations. 2)
PRPP is a major regulator of the purine synthesis rate because it is a
determinant of ATase activity. 3) Activation of ATase by PRPP is nearly
maximal at baseline in fibroblasts with PRPP synthetase superactivity
(15).
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FOOTNOTES |
---|
* This study was supported in part by the Gout Research Foundation of Japan and the Otsuka Pharmaceutical Factory, Inc.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. Tel.: 81-88-633-9483; Fax: 81-88-633-9484; E-mail: yamaoka@genome.tokushima-u.ac.jp.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M011103200
2 T. Yamaoka and M. Itakura, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ATase, amidophosphoribosyltransferase;
hATase, human ATase;
mhATase, mutated
human ATase;
A + hATase, CHO ade
A cells
transfected with normal human ATase cDNA;
A + mhATase, CHO ade
A cells transfected with mutated human
ATase cDNA;
BrdUrd, bromodeoxyuridine;
CHO, Chinese hamster ovary;
HamF, Ham's F-12;
HPLC, high performance liquid chromatography;
HPRT, hypoxanthine guanine phosphoribosyltransferase;
Hx, hypoxanthine;
mhATase-Tg mice, transgenic mice expressing mhATase under the control
of the CAG promoter;
PHA, phytohemagglutinin;
PRPP, 5-phosphoribosyl
1-pyrophosphate;
UA, uric acid;
XO, xanthine oxidase;
FCS, fetal calf
serum.
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