[CANCER RESEARCH 37, 1598-1601, June 1977]
A New Deoxyuridine-5'-triphosphatase in Yoshida Sarcoma Cells
Involved in Deoxyuridine 5'-Triphosphate Metabolism1
Teruo Arima, Hiroto Aklyoshi, and Setsuro Fujli2
Departmentof EnzymePhysiology,Institute for EnzymeResearch,School of Medicine,TokushimaUniversity,Kuramoto-Cho,Tokushima770,Japan
SUMMARY
MATERIALS AND METHODS
A magnesium-independent deoxyuridine-5'-triphospha
tase was found in Yoshida sarcoma cells but not in normal
rat liver. The phosphatase is specific for deoxyuridine 5'diphosphate and deoxyuridine triphosphate, and its Kmfor
deoxyuridine triphosphate is 2.7 x 10@ M. The enzyme was
not inhibited by fluoride and required no divalent cations.
Thus it differs from known nucleotide phosphatases.
Deoxyuridine monophosphokinase , which is detectable
Materials. Nucleotides were purchased from Sigma
Chemical Co. , St. Louis, Mo. , and from Boehringer-Mann
heim Japan Co., Ltd., Japan. All radioactive nucleotides
were obtained from Japan Radioisotope Association, To
kyo, Japan. ECTEOLA-3 and DEAE-cellulose were commer
cial products. Hydroxylapatite was prepared by the method
of Tiselius et a!. (10). Male Donryu rats weighing 80 to 100 g
were maintained on laboratory chow with water given ad
!ibitum and were used for i.p. transplantation of Yoshida
sarcoma cells. For experiments on normal rat liver, male
albino Wistar-King rats were used.
Assay of Mononucleotide Kinases. The reaction mixture
(0.25 ml) for dUMP kinase containing 10 @moles
of Tris-HCI
(pH 8.0), 2.5 @moles
of MgCI2, 2 @moles
of ATP, 0.25 @mole
of [5-3H]dUMP (0.25 MCi), and enzyme was incubated at 37°
for 30 mm and heated for 3 mm in a boiling water bath to
stop the reaction. For assay of dTMP, CMP, and dCMP
kinases, 0.25 @moIe
of [5-3H]dTMP (0.25 pCi), 0.12 @moIe
(0.12 pCi) of [2-'4C]CMP, and 0.12 @moIeof [2-14CIdCMP
(0.12 MCi), respectively, were used as substrates in place of
dUMP. The products of dTMP kinase and dUMP kinase were
analyzed on an ECTEOLA-cellulose column as described
elsewhere (8, 12). The products of CMP and dCMP kinase
were analyzed by paper chromatography with isobutyric
acid:concentrated NH4OH:water (66:1 :33, v/v/v) as solvent.
The RF values of dCMP, dCDP, and dCTP were 0.5, 0.32,
and 0.19, respectively. Radioactivity of the fractionated nu
cleotides was counted in 10 ml of scintillator (4 g of PPO,
100 mg of POPOP, 1 liter of toluene, and 500 ml of Triton X
100). Kinase activity was expressed as nmoles of mononu
cleotide converted to di- or trinucleotide per 30 mm.
Determination of Masking of dUMP Kinase. A mixture of
dUMP kinase (105,000 x g supernatant of normal rat liver
homogenate in 0.25 M sucrose:10 mM Tris buffer) and the
fraction containing the masking factor was incubated for 15
in a crude extract of normal rat liver, could not be detected
in an extract of Yoshida sarcoma cells. However, with hy
droxylapatite
column chromatography
deoxyuridine
5'-monophosphate
of the extract, a
kinase activity as high as
that in normal rat liver was found in fractions separated
from the phosphatase activity. Thus the absence of detecta
ble deoxyuridine
5'-monophosphate
kinase activity
in the
crude extract of Yoshida sarcoma cells is due to the pres
ence of this nucleotide phosphatase.
INTRODUCTION
Mammalian DNA has no uracil residues, although purified
DNA polymerase can incorporate dUTP into DNA. This ab
sence of uracil residues could be due to inhibition of uridine
phosphorylation or increased dUTP degradation. Previ
ously, we reported (1) that the cytopiasmic fraction of Yosh
ida sarcoma cells inhibited the dUMP-phosphoryiating ac
tivity of normal rat liver. No inhibitor could be detected in
normal rat liver cytoplasm,
even when it was fractionated.
It
has also been reported (9) that no dUMP kinase is detecta
ble in a crude calf thymus extract, but that it appears with
fractionation of the extract. The mechanism of its appear
ance is unknown. These studies, along with our results on
the variability of dUMP kinase activity in different cell spe
cies, suggested that the apparent activity must be due to the
level of an inhibitor in cell extracts. In this work, we purified
this inhibitor
and found that it is a new nucleotide
phospha
tase that is specific for dUDP and dUTP, and that it has a
very low Kmfor dUTP. Thus the apparent absence of dUMP
kinase was shown to be due to product degradation.
mm. Then 10
@molesof Tris-HCI
(pH 8), 2.5
@moIesof
MgCI2, 2 j.@moIesof ATP, and 0.25 @moleof [5-3H]dUMP
(0.25 MCi) were added, and the mixture in a final volume of
0.25 ml was incubated for 30 mm at 37°.The products were
analyzed as described before. Masking is expressed as the
percentage reduction in activity of the dUMP kinase added.
Fractionation of dUMP Kinase on a Hydroxylapatite Col
umn. A 20% homogenate of normal rat liver or Yoshida
sarcoma cells in 10 mr@ipotassium phosphate buffer (pH
IThis
work
was
supported
inpart
by
agrant-in-aid
for
cancer
research
2Present
address:
Institute
for
Protein
Research,
Osaka
University,
Suita,
3The
abbreviation
used
is:
ECTEOLA,
epichlorohydrin
triethanolam
from the Ministry of Health and Welfare, and by a grant-in-aid for scientific
research from the Ministry of Education, Science, and Culture, Japan.
Osaka 565, Japan.
ReceivedOctober13, 1975;acceptedJanuary18, 1977.
1598
CANCER
RESEARCH
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VOL. 37
A New dUTPase
7.5)was centrifuged at 105,000 x g for60 mm. The resulting
supernatant was mixed with 10 volumes of cold acetone at
minus 10°.The precipitate was collected by centrifugation
at 105,000 x g for 30 mm and air dried. One g of acetone
powder was extracted with 20 ml of ice-cold 10 mM potas
sium phosphate buffer with the use of a Teflon homoge
nizer, and the mixture was centrifuged for 30 mm at 10,000
x g. Theresultingsupernatantwasappliedto a hydroxyla
patite column previously equilibrated with 10 mM potassium
phosphate buffer. In this way, dUMP kinase was purified
about 10-fold from the extract of the acetone powder of
normal rat liver. The preparation was not contaminated with
dUDP kinase.
Nucleotide Phosphatases with High KmValues. A reac
tion mixture (0.25 ml) containing 2.5 @moles
of Tris-HCI (pH
8.0), 1.25 @moles
of MgCI2, 0.25 @moIe
of various kinds of
unlabeled nucleotide, and enzyme was incubated at 37°for
30 mm. The reaction was stopped by the addition of 1 ml of
cold 5% trichloroacetic acid, and the P1formed was esti
mated colorimetrically
by the method of Fiske and
SubbaRow (3).
Nucleotide Phosphatase with a Low Km. The reaction
mixture was the same for nucleotide phosphatase with high
Km'S, except that 0.025
@moIeof labeled nucleotides
used instead of unlabeled substrates.
analyzed by paper chromatography or
lose column chromatography. Protein
estimated by the method of Lowry et
serum albumin used as a standard.
was
The products were
by ECTEOLA-ceilu
concentration was
a!. (7) with bovine
1.5
Fraction [email protected]'
Chart2. DEAE-cellulose
columnchromatographyof Yoshidasarcomacell
extract. Fraction 2 (10 ml) described in the text was charged on a DEAE
cellulose column that had been equilibrated with 10 [email protected] buffer (pH 8.0).
The column was eluted with 600 ml of a linear gradient of zero to 0.4 M NaCI
in 10 m@iTris buffer. dUMP kinase, masking factor, and nucleotide phospha
tases were assayed as described in “Materials
and Methods―and the legend
to Chart 1. 0, masking activity for dUMP kinase; U, low-K,,, dUTPase; 0,
high-K,,, dUTPase; •,high-K,@dUDPase; A, high-K,,, dUMPase; . . ‘
. , pro
tein.
RESULTS
1.0
EvIdence
for Latent
dUMP Kinase
Activity
in Yoshida
Sarcoma Cell Extracts. We reported previously (1) that
dUMP kinase activity was rather low in regenerating rat liver
:@52 and very low in rapidly growing tissues such as Yoshida
sarcoma, Ehrlich ascites tumor cells, rat bone marrow, and
embryonic rat liver. This could be due to masking of the
0 @‘enzyme. To test this possibility, extracts of Yoshida sar
@
i.5
@:coma
cells
and
normal
ratliver
were
fractionated
byhydrox
1.0
)5
0
Fraction Number
Chart 1. Hydroxylapatite column chromatography of crude extracts of
Yoshida sarcoma cells and normal rat liver. The acetone powder extract of
Yoshida sarcoma cells (A) or normal rat liver (B) was charged on a hydroxyl
apatite column and eluted with 800 ml of a linear gradient of 0.01 to 0.2 N
phosphate buffer. Shown are dUMP kinase (C) and the activity of the mask
ing factor (0) on normal rat liver dUMP kinase (20 @.tmoles
of input activity
taken as 100%); ‘
‘
‘
‘
, protein.
ylapatite column, as described in “Materials
and Methods.―
Chart 1A shows the appearance of dUMP kinase activity
with hydroxylapatite column chromatography of the Yosh
ida sarcoma cell extract. A comparison ofA and B in Chart 1
shows that, in fact, Yoshida sarcoma cells have as high a
dUMP kinase activity as normal rat liver. Thus, the activity
must be masked in these cells. The factor that masks the
activity was detected in an adsorbed fraction of the Yoshida
sarcoma cell extract but not in a normal rat liver extract.
involvement of a Nucleotide Phosphatase In Masking of
dUMP Kinase. The factor causing masking of dUMP kinase
activity was partially purified as follows: a homogenate of
Yoshida sarcoma cells in 200 ml of 0.25 M sucrose:10 mM
Tris-HCI buffer (pH 8.0) was centrifuged at 105,000 x g for
60 mm and the supernatant fluid was obtained (Fraction 1).
Ammonium sulfate was added to 30% saturation, and the
resulting precipitate was removed by centrifugation. Fur
JUNE 1977
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1599
T. Arima et a!.
@
ther solid ammonium sulfate was added to the supernatant
to 60% saturation. The precipitate formed was collected and
dissolved in 10 ml of 0.01 M Tris buffer (Fraction 2). This
fraction was analyzed by DEAE-cellulose column chroma
tography. As shown in Chart 2A, the factor was eluted as a
single peak from the column in a position
@
@
@
coinciding
with a
dUTPase with a low Km.The addition of this fraction to an
assay mixture of dUMP kinase that had been incubated for
30 mm decreased the amount of newly synthesized dUDP or
dUTP. Thus, the dUMP kinase activity in the Yoshida sar
coma cell extract forming dUDP or dUTP was masked by the
dUTPase. Chart 28 shows that other nucleotide phospha
tases with high Km'Sdid not participate in masking of dUMP
kinase activity. The fractions of eluate containing the
dUTPase were combined , concentrated , and dialyzed
against 0.01 M potassium phosphate buffer (Fraction 3).
Then 4 ml of Fraction 3 were further fractionated on a
hydroxylapatite column. The elution profile in Chart 3
shows that the dUTPase activity in the adsorbed fraction
coincided completely with the dUMP kinase-masking activ
ity. Some masking activity was observed in the unadsorbed
fraction, but we could not detect the dUTPase activity in this
fraction. The masking activity in the unadsorbed fraction
was also detected in the same fraction of normal rat liver.
Therefore, this masking activity is different from that in the
adsorbed fraction, but its properties are unknown.
Phosphatases for dUMP, dUDP, and dUTP with high Km'S
were notdetectable
intheadsorbedfraction
(Fraction
4).In
this way, the dUTPase with a low Kmwas purified about 11fold from the crude extract. This fraction was used for
further
I
A
2
3
‘
2
3
mooo
E
0
5
R@C@
i a
3
@‘
@‘
C
2
4
3
S
I,
-
I
E
C
I
II
2
II
cm
cm
3
I
S
studies.
Substrate Specificity of the dUTPase. Chart 4 shows
paper chromatograms obtained after treatment of deoxyri
bonucleoside triphosphates with Fraction 4 enzyme. dATP,
dGTP, dCTP, and dTTP were not hydrolyzed significantly,
but dUTP was hydrolyzed to dUDP and dUMP. To determine
whether production of dUMP from dUTP was due to release
of PP@or to secondary hydrolysis of dUDP, hydrolysis of
tritium-labeled dUDP by Fraction 4 was examined. As
shown in Table 1, dUDP was significantly hydrolyzed to
dUMP. The Kmof Fraction 4 enzyme was calculated to be 2.7
x 10-i M from a Lineweaver-Burk plot.
>‘
.@
0.8
0.62
@
cm
Chart 4. Paper chromatograms of deoxyribonucleoside triphosphates
after incubation with Fraction 4 enzyme; 0.1 [email protected] triphos
phates (1 pCi) were incubated with Fraction 4 enzyme for 30 mm as de
scribed in “Materials
and Methods,―and 0.1-mi samples of the hydrolysates
were spotted on Tokyo filter paper No. 50 and developed with isobutyric
acld:water:ammonia:O.1 N EDTA (100:56:4.2:1 .6 by volume) for 20 hr at room
temperature. The paper was cut into 5-mm wide strips and radioactivity was
counted in a toluene scintillator. A, dUTP; B, dCTP; C, dTTP; D, dGTP; E,
dATP. Arrows 1, 2, and 3 Indicate the positions of deoxyribonucleoside
triphosphate, diphosphate, and monophosphate, respectively. The abscissa
Indicates the distance from the origin without enzyme ( ‘
‘
. . ) and with Frac
tion 4 enzyme (—).
Table 1
Comparisonof hydro!yzing activity of Fraction 4 for dUDPand
dUTP
0.4
Concentration
(nM)Activity―
(dUMP
3
,@
formed)InitialResidualdUDP230119111dUTP217107110
0.2
0
@
0
0
20
FroctionNL(nber
Chart 3. Hydroxylapatite column chromatography of Fraction 3. Four ml
of Fraction 3 were charged on a hydroxylapatite column (2 x 6 cm) equil
ibrated with 0.01 U potassium phosphate buffer (pH 7.5). The column was
developed with a linear gradient of 0.01 to 0.2 N phosphate buffer. The
maskingactivity and Iow-K,,@
dUTPaseactivitywereassayedas describedin
the legend to Chart 2.
kinase.
1600
@,
low-K,..,dUTPase; 0, masking activity for dUMP
a The hydroiyzing activity of Fraction 4 was assayed with the
indicated
concentration
of tritium-labeled
dUDP or dUTP as sub
strates.
Effects of Deoxyribonucleoside Di- and Triphosphates
on dUTP Hydrolysis by the Fraction 4 Enzyme. As shown in
Table 2, of the deoxyribonucleoside di- and triphosphates
tested, only dUDP and dUTP had dilution effects on dUTP
CANCER RESEARCHVOL. 37
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A New dUTPase
Table 2
Effects
of variousnucleotides on dUTPhydrolysisby Fraction
4AdditionConcentration
(mM)Activity'
(%)None
dUDP
36
dUTP
dCDP
0.4
0.4
0.2
20
15
90
0.4
97
dCTP
0.4
dTDP
95
0.2
105
0.4
97
dTTP
0.4
dADP
0.2
89
94
0.4
100
dATP
0.4
91
dGDP
0.2
105
100
0.4
0.4100
dGTP0.2
97
a One hundred % enzyme activity indicates 8.2 nmoles dUMP
produced per 30 mm incubation. The activity was assayedwith 0.1
mM dUTP as substrate and the products were analyzed
by EC
TEOLA-cellulosecolumn chromatography.
ISO
(A)
100
normal rat liver. Thus its function seems to be to regulate
the size of the dUTP pool, probably by its controlled synthe
sis in various phases of cell growth, and its specific action
on dUDP and dUTP seems functionally significant. We did
not examine the hydrolyses of ribonucleoside triphosphates
directly, but it seems unlikely that these compounds are
susceptible to the dUTPase, because Yoshida sarcoma cells
have as high mono- and diphosphokinase activities for
AMP, CMP, and UMP as normal rat liver, so that only dUMP
kinase activity was masked. Moreover, we found that the
addition of Fraction 4 did not affect the assays of AMP,
CMP, and UMP kinases, so that the products of these en
zymes, ribonucleoside di- or triphosphates, were not hydro
lyzed by the dUTPase. This nucleotide phosphatase differs
from microbial dUTPases, such as T2, T4 phage-induced
dUTPases (5, 11, 13) and Escherichia coli dUTPase (2, 6),
which have 10 to 40 times higher Kmvalues. It also differs
from the dUTPase in leukemia
L1210 cells (4), which hydro
lyzes dUTP to dUMP and PP1,but does not hydrolyze dUDP
and is inhibited by fluoride. Magnesium-dependent nucieo
tide phosphatases seem to have no influence on dUTP
formation because normal rat liver has a higher dUMP ki
nase activity than Yoshida sarcoma cells, although it also
contains higher magnesium-dependent nucleotide phos
phatase activity than the latter.
.@
ACKNOWLEDGMENTS
:150
The authors express their gratitude to Kazuko Fujiwara for expert techni
cal assistance.
)
5
I:
@
0
IC
IS
M9CI2 ( mM)
20
REFERENCES
1. Arima, T., and Fujii, S. Inhibitor of Pyrimidine Metabolism from Tumor
Tissues. Biochem. Biophys. Res. Commun., 55: 410-416, 1973.
2. Bertani, L. E., Haggmark, A., and Reichard, P. Enzymatic Synthesis of
Deoxyribonucleotldes II. Formation and Interconversion of Deoxyuridine
Phosphates. J. Biol. Chem., 238: 3407-3413, 1963.
(B)
3. Fiske, C. H., and SubbaRow,Y. The Colorimetric Determinationof
10
20
Phosphorus. J. Biol. Chem., 66: 375—400,
1925.
4. Grindey, B. G., and Nichol, C. A. Mammalian Deoxyuridlne 5'-Triphos
phate Pyrophosphatase. Biochim. Biophys. Acts, 240: 180-183, 1971.
40
5. Greenberg,G. R. NewdUTPaseanddUDPaseActivitiesafterInfectionof
NoF ( mM)
Chart 5. Effects of MgCI, and NaF on the dUTP phosphatase activity.
dUTPase in Fraction 4 was assayed as described in “Materials
and Methods―
with various concentrations of MgCI, (A) and NaF (B).
hydrolysis. This indicates that the enzyme hydrolyzed dUDP
but not dADP, dCDP, dGDP, and dTDP, confirming the
result shown in Chart 4. Thus the nucleotide phosphatase is
specific for dUDP and dUTP.
Effects of MgCl and Sodium Fluoride. As shown in Chart
5, various concentrations of MgCI2 had no stimulatory ef
fect, and the addition of NaF was not inhibitory.
7. Lowry,0. H., Rosebrough,N. J., Farr,A. L., and Randall,R. J. Protein
Measurement with the Folin Phenol Reagent. J. BioI. Chem. , 193: 265275, 1951.
8. Shlosaka,T., Omura,Y., Okuda,H., andFujii, 5. ActIvationof Thymidine
Kinase of Adult Rat Liver by the Culture Filtrate of Clostridlum perfrin
gens. Biochim. Biophys. Acta, 240: 352-358, 1970.
9. Sugino,Y., Tanaka,F., and Miyoshi,Y. LatentDeoxyuridineMonophos
phokinasein CalfThymus.Biochem.Biophys.Res.Commun.,16:362367, 1964.
10. Tlsellus, A., Hjerten, S., and Levin, 0. Protein Chromatography on
Calcium Phosphate Column. Arch. Blochem. Biophys., 65: 132-155,
1956.
11. Warner, H. A., and Barnes, J. E. Evidencefor a Dual Role for the
DISCUSSION
This work showed the presence of a magnesium-inde
pendent nucleotide phosphatase specific for dUDP and
dUTP in Yoshida sarcoma cells. This enzyme hydrolyzed
di- and triphosphates
Escherichia coil by T, Bacteriophage. Proc. NatI. Acad. Sd. U. 5., 56:
1226—1232,
1966.
6. Greenberg, G. R., and Somerville, R. L. Deoxyuridylate Kinase Activity
and Deoxyuridine Triphosphatase in Escherichia coli. Proc. NatI. Acad.
Sd. U. 5., 48: 247-257, 1962.
of deoxyuridine
formed
by dUMP ki
nase and so masked its activity. It could not be detected in
Bacteriophage Tclnduced Deoxycytidine Triphosphate Nucleotidohy
drolase. Proc. NatI. Aced. Sci. U. S., 56: 1233-1240, 1966.
12. Weissmann, S. M., Smellie, R. M. S., and Paul, J. Studies on the
Biosynthesis of Deoxyribonucleic Acid by Extracts of Mammalian Cells.
lv. The Phosphorylatlon of Thymidine. Blochim. Biophys. Acts, 45: 101-
110,1960.
13. Zimmerman,S. B., andKornberg.A. DeoxycytidineDi-andTriphosphate
Cleavage by an Enzyme Formed in Bacteriphage-infected Escherichia
coil. J. BioI. Chem., 236: 1480-1486, 1961.
JUNE 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
1601
A New Deoxyuridine-5′-triphosphatase in Yoshida Sarcoma
Cells Involved in Deoxyuridine 5 ′-Triphosphate Metabolism
Teruo Arima, Hiroto Akiyoshi and Setsuro Fujii
Cancer Res 1977;37:1598-1601.
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