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Deoxyuridine triphosphatase in human hepatoma
hr. J. Bidwn.
Pergamon
Vol. 26, No. 4, pp. 4X7--490. 1994 Copyright tc, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0020-711x/94 $7.00 + 0.00
0020-7llX(93)E~21-G
DEOXYURIDINE SHEERU HOKARI,‘*
TRIPHOSPHATASE HEPATOMA
IN HUMAN
IWAO KOYAMA,’ KEI SHIODA” and YO~HIKATSUSAKAGISHI’
‘Department of Biochemistry and *Department of Pathology, Saitama Medical School, 38 Morohongo, Moroyama-machi, Saitama 350-04, Japan [Received
20 September
f99.7
Abstract-l. Deoxyuridine triphosphatase (dUTPase) in human hepatoma was investigated. The apparent activity in a gram weight tissue was about 6 times that of the activity in rat livers
after partial hepatecomy. 2. Most catalytic properties of the hepatoma dUTPase were similar to the enzymes from other sources. 3. The hepatoma dUTPase reacted with although the molecular size of the enzyme 4. The immunochemicai studies indicated subunits, whose molecular mass was about
the antibodies against the rat spleen dUTPase, (4648 kDa) was different to the rat enzyme. that the enzyme was composed of two identical 22 kDa.
INTRODUCTION Deoxyribonucleotides, which must be supplied for cellular DNA replication, were produced by special synthetic pathways. In various types of growing cells, we have recognized that the DNA replication correlates with increase in the enzyme activities and participates in the deoxyribonucleotide synthesis (Reichard, 1988). Deoxyuridine triphosphatase(dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and PPi, and it may result in preventing the incorporation of uridylic acid to DNA. This enzyme also acts for supplement of dUMP, a unique precursor of thymidylic acid. We have reported on the structure and function of the dUTPases from some mammalian origin (Hokari et al., 1982, 1989; Hokari and Sakagishi, 1987), while the enzymic studies from various sources are also being accumulated (Shlomai and Kornberg, 1978; Williams and Cheng, 1979; Caradonna and Adamkiewicz, 1984; Williams, 1984; Giroir and Deutsch, 1987; Cedergren-Zeppezauer et al., 1992; Pri-Hadash et al., 1992). All the dUTPases have similar catalytic properties and they are homodimers or homotrime~ composed of about 20 kDa subunit. Although the structure of these subunit proteins just differed from one *To whom correspondence
should be addressed.
another, no isozyme of the dUTPase has been detected. In the present report, we investigated the enzyme molecule of the human hepatoma dUTPase using antibodies against rat spleen dUTPase, which partially reacted with the human hepatoma enzyme. MATERIALSAND METHODS Blue Sepharose CLdB, Phenyl-Sepharose CL4B and Sephacryl S-300 were purchased from Pharmacia (Uppsala, Sweden). Peroxidaseconjugated IgG preparations were obtained from Organon Teknika Corp.-Cappel Products. Polyvinylidene difluoride (PVDF) membrane filters were purchased from Millipore Corp. (Bedford, U.S.A.). Other chemicals used were the analytical grade products as described previously (Hokari and Sakagishi, 1987; Hokari et al., 1988, 1989). A small piece of human hepatoma tissue was homogenized with 10~01 of 0.25 M sucrose containing 20 mM Tris-HCl buffer (pH 7.6), 3 mM MgSO, and 0.2 mM dithiothreitol in the presence of a protease inhibitor mixture (100 pggiml each of chymostatin, leupeptin, antipain, elastatinal and pepstatin (Peptide Institute, Osaka), and the cytosol was collected by centrifugation at 150,OOOgfor 60 min. The dUTPase was partially purified from the cytosol 487
488
SHIGERU HOKARI et al
by the procedure as described in the previous study on the calf thymus dUTPase (Hokari et al., 1989), the sequential fractionation on the columns of Blue Sepharose, Phenyl-Sepharose and Sephacryl S-300. The dUTPase activity was assayed as described earlier (Hokari and Sakagishi, 1987) by the determination of Pi, which was produced by a coupled reaction with the inorganic pyrophosphatase. One unit of the enzyme activity was defined as the amount that produced 1 nmol PPi/min. Protein was determined by the method of Bradford (1976), using bovine serum albumin as standard. Antibody against the rat spleen dUTPase was raised in rabbit (Hokari et al., 1988), and it against the calf thymus dUTPase was obtained from rats as described previously (Hokari et al., 1989). Western blot analysis was carried out by sodium dodecyl sulfate(SDS)-polyacrylamide gel (12% mini-slab gels) electrophoresis, electrophoretical transfer of the proteins to PVDF membrane, and immunoblot assay with antibodies against dUTPase and with peroxidaseconjugated IgG preparations. RESULTS
AND DISCUSSION
In a couple of studies we have indicated that the increase of dUTPase activity closely related to the replication of DNA (Hokari et al., 1982, 1987). While, a considerable dUTPase activity has been detected in several types of cultured human cells (Williams and Cheng, 1979; Williams, 1984; Caradonna and Adamkiewicz, 1984), of course they have sufficient capacity on growing. So, it is easily supposed that the dUTPase gene(s) constantly expresses in most tumor tissues. In the present study, we fractionated the proteins of human hepatoma to investigate the dUTPase molecule. As mentioned earlier (Hokari et al., 1982), a nonspecific nucleotidohydrolase activity can be eliminated by the chromatography on Blue Sepharose so that, we detected the dUTPase activity of the human hepatoma after such treatment. It apparently showed 78 U/g weight tissue. This value was about 6 times of the regenerating rat liver, 12.5 U/g tissue (Hokari et al., 1988). Roughly comparing with the activities in the HeLa cells (Caradonna and Adamkiewicz,l984) and the human lymphocytic leukemia cells (Williams and Cheng, 1979), the human hepatoma dUTPase was about a quarter of them. However, the amount of dUTPase
Table
I. Purification
of human Total protein
Purification
step
Cytosol Blue Sepharose Phenyl-Sepharose Sephacryl S-300
(mg)
176.6 12.6 1.05 0.069
hepatoma dUTPase Total
units
(*390) 342 185 79.8
dUTPase activity Units/mg (j2.2) 27.1 176 1152
5 g of hepatoma tissue was fractionated by the purification procedure as described in “Materials and Methods”. An activity in the cytosol(*) was deduced from the activity which assayed the eluting fractions from Blue Sepharose column.
activity seemed to be significant in the hepatoma DNA replication. Then we tried to purify the hepatoma dUTPase (Table 1) for the purpose of investigation for the characteristics of the enzyme. But, it was so difficult that the partially purified enzyme preparation (Sephacryl S-300 fraction) was still in about 5% purity, which was inferred from a purification grade of the rat spleen dUTPase (Hokari and Sakagishi, 1987). Although the catalytic properties of the hepatoma dUTPase were similar to the rat enzyme, the two enzymes were different in their molecular properties; a KC1 concentration which, required for elution of the enzyme from Blue Sepharose column, was about 0.3 M (Fig. l), while the rat spleen dUTPase and the calf thymus enzyme required 0.1 and 0.15 M KC1 for elution, respectively. Apparent molecular mass of the hepatoma dUTPase estimated by gel filtration was 46-48 kDa (data was not shown), on the other hand, the rat spleen enzyme has been calculated to be 58,500 Da (Hokari and Sakagishi, 1987). The molecular mass of the dUTPase from human cells was reported to be 43 kDa (Williams and Cheng, 1979) or 45 kDa (Caradonna and Adamkiewicz, 1984). A more recent study on a sequencing of the human dUTPase cDNA (McIntosh et al., 1992) indicated that the subunit molecular mass of the enzyme was 16.6 kDa. We then tried to investigate the molecular nature of the dUTPase, but a sufficient amount of hepatoma tissue could not be obtained for the enzyme purification. So, we attempt to analyze them by immunochemical techniques. We have prepared the antibodies against the dUTPases of rat spleen and calf thymus. The immunoprecipitation reactions indicated that the anti-rat spleen dUTPase antibody reacted slightly with the human hepatoma enzyme, but the anti-
dUTPase in human hepatoma ‘0. s 6 7 8 Q10111213l41516
”
5 6 7 8 910111213141516 Fraction
number
Fig. 1. Analyses of the activity and the subunit protein of the human hepatoma dUTPase. 5ml of the hepatoma cytosol was applied to a column (1.0 x 8 cm) of Blue Sepharose. After the coiumn was washed thoroughly with TMD-buffer (20 mM Tris-HCl buffer, pH 8.0, 4mM MgSO, and 0.2mM dithiothreitol), the proteins retained were eluted stepwise with TMD-buffer containing 0.1, 0.3 and 3.0 M KC1 in 1ml-fractions. The dUTPase activity or a non-specific activity was assayed as described previously (Hokari et af., 1988) with dUTP (a) or UTP (El) as the substrate. 20~1 each of the fractions were analyzed by immunoblot assay (upper panel) using the antibodies against the rat spleen dUTPase and peroxidase-conjugated goat anti-rat IgG. Arrow indicates the migrating position of the subunit protein.
489
The subunit compositions of various dUTPases are reported to be homodimers in HeLa S3 cells (Caradonna and Adamkiewicz, 1984) and in calf thymus (Hokari et af., 1989) or homotrimers in rat spleen (Hokari and Sakagishi, 1987) and in E. coli (Cedergren-Zeppezauer et al., 1992). As described in this report, the human hepatoma dUTPase was strongly supposed to be a dimer. Also the molecular mass of the most dUTPase subunits was 16-23 kDa, except that the enzyme from herpes simplex virus type 1 was 35 kDa (Caradonna and Adamkiewicz, 1984) or 39 kDa (Bjornberg et al., 1993) of monomer enzyme. The results presented here indicate that the structure of the human hepatoma dUTPase is similar to the HeLa S3 dUTPase (Caradonna and Adamkiewicz, 1984), and it may be composed of two identical subunits whose molecular mass is about 22 kDa. The human hepatoma dUTPase and the rat spleen enzyme seem to share similar antigen epitopes on the subunit structure from their immuno-reactivity. While McIntosh et al. (1992) indicated in their study using cloned human dUTPase cDNA that the sequence homology was higher to the cow dUTPase gene than
-114 67
calf thymus dUTPase antibody did not. So, a subunit protein of the human enzyme was analyzed by Western blot assay using the anti-rat dUTPase antibody after the enzyme was fractionated on a Blue Sepharose column. As shown in Fig. 1, an immuno-reactive band was detected in the fractions with dUTPase activity, which was eluted from the Blue Sepharose column with 0.3 M KCl. The reactive band must be the subunit of the dUTPase, because no other band was detected in the blotting assay and the partially purified enzyme preparation showed the same result (Fig. 2). The subunit molecular mass of the human hepatoma dUTPase was about 22 kDa, and the enzyme protein seemed to be sensitive to proteases, so that an additional protein band (about I8 kDa) appeared in the absence of protease inhibitors. Such limited cleavage was also observed in the rat dUTPase preparations (Hokari et al., 1988), but endogenous proteolytic rate in human hepatoma seemed to be higher than in the rat tissues.
-
Fig. 2. Immunoblot analysis of the human hepatoma dUTPase. Partially purified preparations of the human hepatoma dUTPase were analyzed by immunoblot assay as described in Fig. 1. Fr.II: Blue Sepharose fraction, 50 and IOOpg of protein; Fr.lV: Sephacryl S-300 fraction, 0.7 and I .4 ng of protein. Numbers indicate the molecular mass of marker proteins in kDd. Arrow shows the migrating position of the rat spfeen dUTPase subunit.
SHIGERU
490
Ho Km
to the rat one. Our antibody against the ,calf thymus dUTPase did not react with the human dUTPase, however, sufficient data have not been accumuiated to discuss on the detailed structures of these enzyme proteins. We still have no observation as to whether the dUTPase activity increases by the progression of human tumor. However, a high level of the dUTPase activity was detected in the human hepatoma as described in this report. Increasing dUTPase activity in rats, as shown in regenerating liver (Hokari et al,, 1982) hematogenic spleen (Hokari ef al., 1987) and correlation between the activity and growing rate of rat hepatoma cells (Xiao el’ al., 1992), lead to the assumption that the activity and also the enzyme protein may increase in human hepatoma and in the other human tumors. REFERENCES Bjornberg O., Bergman A.-C., Rosengren A. M., Persson R., Lehman I. R. and Nyman P. 0. (1993) dUTPase from herpes simplex virus type 1: purification from infected green monkey kidney (Vero) cells and from an overproducing ~cherich~u co& strain. Prot. Express. Puri$$ 1499159. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 2488254. Caradonna S. J. and Adamkiewicz D. M. (1984) Purification and properties of the deoxyuridine triphosphate nucleotido-hydrolase enzyme derived from HeLa S3 cells. 1. biol. Chem. 259, 5459-5464.
Cedergren-Zeppezauer E. S., Larsson G., Nyman P. O., Dauter Z. and Wilson K. S. (1992) Crystal structure of a dUTPase. Nature 355, 746743. Giroir L. E. and Deutsch W. A. (1987) Drosophifo deoxyuridine triphosphatase. Purification and characterization. J. biol. Chem. 262, 130-134.
et al.
Hokari S. and Sakagishi Y. (1987) Purification and characterization of deoxyuridine triphosphate nucleotidohydrolase from anemic rat spleen: a trimer composition of the enzyme protein. Archs Biochem. Biophys. 253, 350-356. Hokari S., Sakagishi Y. and Tsukada K. (1982) Enhanced activity of deoxyuridine 5’-triphosphatase in regenerating rat liver. Biochem. b~o~hy~. Res. Commun. 108, 95-101. Hokari S., Hasegawa M., Sakagishi Y. and Kikuchi G. (1987) Deoxyuridine triphosphate nucleotidohydrolase activity and its correlation with multiplication of erythroid cells in rat spleen. Biochem. Int. 14, 851-857. Hokari S., Takizawa A., Tanaka M. and Sakagishi Y. (1989) Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition. Biochem. Int. 19, 453461.
Hokari S.. Hasegawa M., Tanaka M., Sakagishi Y. and Kikuchi G. (1988) Deoxyuridine triphosphate nucleotidohydrolase: Distribution of the enzyme in various rat tissues. J. 3joc~em. ~ok~Io~ 104, 21 l-214. McIntosh E. M., Ager D. D., Gadsden M. H. and Haynes R. H. (1992) Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme. Proc. nafn. Acad. Sci. U.S.A. 89, 8020-8024. Pri-Hadash A., Hareven D. and Lifschitz E. (1992) A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and Roral meristems. The Plant Ceil 4, 1499159. Reichard P. (1988) Interaction between deoxyribonucleotide and DNA synthesis. A. Rev. Biochem. 57, 349-374. Shlomai J. and Kornberg A. (1978) ~oxyuridine triphosphatase of Eseherich~o coli. Purification, properties, and use as a reagent to reduce uracil incorporation into DNA. J. bioi. Chem. 253, 3305-3312.
Williams M. V. (1984) Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type I. Purification and characterization of induced enzyme. J. biol. Chem. 259, 10080-10084. Williams M. V. and Cheng Y.-C. (1979) Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nu~leotidohydrolase from acute lympho~ytic leukemia. J. biol. Chem. 254, 2897-2901. Xiao Q., Luke A. and Lea M. A. (1992) dUTP pyrophosphatase and uracil-DNA glycosylase in rat liver and hepatomas. Int. J. Biochem. 24, 437445.
Pergamon
Vol. 26, No. 4, pp. 4X7--490. 1994 Copyright tc, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0020-711x/94 $7.00 + 0.00
0020-7llX(93)E~21-G
DEOXYURIDINE SHEERU HOKARI,‘*
TRIPHOSPHATASE HEPATOMA
IN HUMAN
IWAO KOYAMA,’ KEI SHIODA” and YO~HIKATSUSAKAGISHI’
‘Department of Biochemistry and *Department of Pathology, Saitama Medical School, 38 Morohongo, Moroyama-machi, Saitama 350-04, Japan [Received
20 September
f99.7
Abstract-l. Deoxyuridine triphosphatase (dUTPase) in human hepatoma was investigated. The apparent activity in a gram weight tissue was about 6 times that of the activity in rat livers
after partial hepatecomy. 2. Most catalytic properties of the hepatoma dUTPase were similar to the enzymes from other sources. 3. The hepatoma dUTPase reacted with although the molecular size of the enzyme 4. The immunochemicai studies indicated subunits, whose molecular mass was about
the antibodies against the rat spleen dUTPase, (4648 kDa) was different to the rat enzyme. that the enzyme was composed of two identical 22 kDa.
INTRODUCTION Deoxyribonucleotides, which must be supplied for cellular DNA replication, were produced by special synthetic pathways. In various types of growing cells, we have recognized that the DNA replication correlates with increase in the enzyme activities and participates in the deoxyribonucleotide synthesis (Reichard, 1988). Deoxyuridine triphosphatase(dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and PPi, and it may result in preventing the incorporation of uridylic acid to DNA. This enzyme also acts for supplement of dUMP, a unique precursor of thymidylic acid. We have reported on the structure and function of the dUTPases from some mammalian origin (Hokari et al., 1982, 1989; Hokari and Sakagishi, 1987), while the enzymic studies from various sources are also being accumulated (Shlomai and Kornberg, 1978; Williams and Cheng, 1979; Caradonna and Adamkiewicz, 1984; Williams, 1984; Giroir and Deutsch, 1987; Cedergren-Zeppezauer et al., 1992; Pri-Hadash et al., 1992). All the dUTPases have similar catalytic properties and they are homodimers or homotrime~ composed of about 20 kDa subunit. Although the structure of these subunit proteins just differed from one *To whom correspondence
should be addressed.
another, no isozyme of the dUTPase has been detected. In the present report, we investigated the enzyme molecule of the human hepatoma dUTPase using antibodies against rat spleen dUTPase, which partially reacted with the human hepatoma enzyme. MATERIALSAND METHODS Blue Sepharose CLdB, Phenyl-Sepharose CL4B and Sephacryl S-300 were purchased from Pharmacia (Uppsala, Sweden). Peroxidaseconjugated IgG preparations were obtained from Organon Teknika Corp.-Cappel Products. Polyvinylidene difluoride (PVDF) membrane filters were purchased from Millipore Corp. (Bedford, U.S.A.). Other chemicals used were the analytical grade products as described previously (Hokari and Sakagishi, 1987; Hokari et al., 1988, 1989). A small piece of human hepatoma tissue was homogenized with 10~01 of 0.25 M sucrose containing 20 mM Tris-HCl buffer (pH 7.6), 3 mM MgSO, and 0.2 mM dithiothreitol in the presence of a protease inhibitor mixture (100 pggiml each of chymostatin, leupeptin, antipain, elastatinal and pepstatin (Peptide Institute, Osaka), and the cytosol was collected by centrifugation at 150,OOOgfor 60 min. The dUTPase was partially purified from the cytosol 487
488
SHIGERU HOKARI et al
by the procedure as described in the previous study on the calf thymus dUTPase (Hokari et al., 1989), the sequential fractionation on the columns of Blue Sepharose, Phenyl-Sepharose and Sephacryl S-300. The dUTPase activity was assayed as described earlier (Hokari and Sakagishi, 1987) by the determination of Pi, which was produced by a coupled reaction with the inorganic pyrophosphatase. One unit of the enzyme activity was defined as the amount that produced 1 nmol PPi/min. Protein was determined by the method of Bradford (1976), using bovine serum albumin as standard. Antibody against the rat spleen dUTPase was raised in rabbit (Hokari et al., 1988), and it against the calf thymus dUTPase was obtained from rats as described previously (Hokari et al., 1989). Western blot analysis was carried out by sodium dodecyl sulfate(SDS)-polyacrylamide gel (12% mini-slab gels) electrophoresis, electrophoretical transfer of the proteins to PVDF membrane, and immunoblot assay with antibodies against dUTPase and with peroxidaseconjugated IgG preparations. RESULTS
AND DISCUSSION
In a couple of studies we have indicated that the increase of dUTPase activity closely related to the replication of DNA (Hokari et al., 1982, 1987). While, a considerable dUTPase activity has been detected in several types of cultured human cells (Williams and Cheng, 1979; Williams, 1984; Caradonna and Adamkiewicz, 1984), of course they have sufficient capacity on growing. So, it is easily supposed that the dUTPase gene(s) constantly expresses in most tumor tissues. In the present study, we fractionated the proteins of human hepatoma to investigate the dUTPase molecule. As mentioned earlier (Hokari et al., 1982), a nonspecific nucleotidohydrolase activity can be eliminated by the chromatography on Blue Sepharose so that, we detected the dUTPase activity of the human hepatoma after such treatment. It apparently showed 78 U/g weight tissue. This value was about 6 times of the regenerating rat liver, 12.5 U/g tissue (Hokari et al., 1988). Roughly comparing with the activities in the HeLa cells (Caradonna and Adamkiewicz,l984) and the human lymphocytic leukemia cells (Williams and Cheng, 1979), the human hepatoma dUTPase was about a quarter of them. However, the amount of dUTPase
Table
I. Purification
of human Total protein
Purification
step
Cytosol Blue Sepharose Phenyl-Sepharose Sephacryl S-300
(mg)
176.6 12.6 1.05 0.069
hepatoma dUTPase Total
units
(*390) 342 185 79.8
dUTPase activity Units/mg (j2.2) 27.1 176 1152
5 g of hepatoma tissue was fractionated by the purification procedure as described in “Materials and Methods”. An activity in the cytosol(*) was deduced from the activity which assayed the eluting fractions from Blue Sepharose column.
activity seemed to be significant in the hepatoma DNA replication. Then we tried to purify the hepatoma dUTPase (Table 1) for the purpose of investigation for the characteristics of the enzyme. But, it was so difficult that the partially purified enzyme preparation (Sephacryl S-300 fraction) was still in about 5% purity, which was inferred from a purification grade of the rat spleen dUTPase (Hokari and Sakagishi, 1987). Although the catalytic properties of the hepatoma dUTPase were similar to the rat enzyme, the two enzymes were different in their molecular properties; a KC1 concentration which, required for elution of the enzyme from Blue Sepharose column, was about 0.3 M (Fig. l), while the rat spleen dUTPase and the calf thymus enzyme required 0.1 and 0.15 M KC1 for elution, respectively. Apparent molecular mass of the hepatoma dUTPase estimated by gel filtration was 46-48 kDa (data was not shown), on the other hand, the rat spleen enzyme has been calculated to be 58,500 Da (Hokari and Sakagishi, 1987). The molecular mass of the dUTPase from human cells was reported to be 43 kDa (Williams and Cheng, 1979) or 45 kDa (Caradonna and Adamkiewicz, 1984). A more recent study on a sequencing of the human dUTPase cDNA (McIntosh et al., 1992) indicated that the subunit molecular mass of the enzyme was 16.6 kDa. We then tried to investigate the molecular nature of the dUTPase, but a sufficient amount of hepatoma tissue could not be obtained for the enzyme purification. So, we attempt to analyze them by immunochemical techniques. We have prepared the antibodies against the dUTPases of rat spleen and calf thymus. The immunoprecipitation reactions indicated that the anti-rat spleen dUTPase antibody reacted slightly with the human hepatoma enzyme, but the anti-
dUTPase in human hepatoma ‘0. s 6 7 8 Q10111213l41516
”
5 6 7 8 910111213141516 Fraction
number
Fig. 1. Analyses of the activity and the subunit protein of the human hepatoma dUTPase. 5ml of the hepatoma cytosol was applied to a column (1.0 x 8 cm) of Blue Sepharose. After the coiumn was washed thoroughly with TMD-buffer (20 mM Tris-HCl buffer, pH 8.0, 4mM MgSO, and 0.2mM dithiothreitol), the proteins retained were eluted stepwise with TMD-buffer containing 0.1, 0.3 and 3.0 M KC1 in 1ml-fractions. The dUTPase activity or a non-specific activity was assayed as described previously (Hokari et af., 1988) with dUTP (a) or UTP (El) as the substrate. 20~1 each of the fractions were analyzed by immunoblot assay (upper panel) using the antibodies against the rat spleen dUTPase and peroxidase-conjugated goat anti-rat IgG. Arrow indicates the migrating position of the subunit protein.
489
The subunit compositions of various dUTPases are reported to be homodimers in HeLa S3 cells (Caradonna and Adamkiewicz, 1984) and in calf thymus (Hokari et af., 1989) or homotrimers in rat spleen (Hokari and Sakagishi, 1987) and in E. coli (Cedergren-Zeppezauer et al., 1992). As described in this report, the human hepatoma dUTPase was strongly supposed to be a dimer. Also the molecular mass of the most dUTPase subunits was 16-23 kDa, except that the enzyme from herpes simplex virus type 1 was 35 kDa (Caradonna and Adamkiewicz, 1984) or 39 kDa (Bjornberg et al., 1993) of monomer enzyme. The results presented here indicate that the structure of the human hepatoma dUTPase is similar to the HeLa S3 dUTPase (Caradonna and Adamkiewicz, 1984), and it may be composed of two identical subunits whose molecular mass is about 22 kDa. The human hepatoma dUTPase and the rat spleen enzyme seem to share similar antigen epitopes on the subunit structure from their immuno-reactivity. While McIntosh et al. (1992) indicated in their study using cloned human dUTPase cDNA that the sequence homology was higher to the cow dUTPase gene than
-114 67
calf thymus dUTPase antibody did not. So, a subunit protein of the human enzyme was analyzed by Western blot assay using the anti-rat dUTPase antibody after the enzyme was fractionated on a Blue Sepharose column. As shown in Fig. 1, an immuno-reactive band was detected in the fractions with dUTPase activity, which was eluted from the Blue Sepharose column with 0.3 M KCl. The reactive band must be the subunit of the dUTPase, because no other band was detected in the blotting assay and the partially purified enzyme preparation showed the same result (Fig. 2). The subunit molecular mass of the human hepatoma dUTPase was about 22 kDa, and the enzyme protein seemed to be sensitive to proteases, so that an additional protein band (about I8 kDa) appeared in the absence of protease inhibitors. Such limited cleavage was also observed in the rat dUTPase preparations (Hokari et al., 1988), but endogenous proteolytic rate in human hepatoma seemed to be higher than in the rat tissues.
-
Fig. 2. Immunoblot analysis of the human hepatoma dUTPase. Partially purified preparations of the human hepatoma dUTPase were analyzed by immunoblot assay as described in Fig. 1. Fr.II: Blue Sepharose fraction, 50 and IOOpg of protein; Fr.lV: Sephacryl S-300 fraction, 0.7 and I .4 ng of protein. Numbers indicate the molecular mass of marker proteins in kDd. Arrow shows the migrating position of the rat spfeen dUTPase subunit.
SHIGERU
490
Ho Km
to the rat one. Our antibody against the ,calf thymus dUTPase did not react with the human dUTPase, however, sufficient data have not been accumuiated to discuss on the detailed structures of these enzyme proteins. We still have no observation as to whether the dUTPase activity increases by the progression of human tumor. However, a high level of the dUTPase activity was detected in the human hepatoma as described in this report. Increasing dUTPase activity in rats, as shown in regenerating liver (Hokari et al,, 1982) hematogenic spleen (Hokari ef al., 1987) and correlation between the activity and growing rate of rat hepatoma cells (Xiao el’ al., 1992), lead to the assumption that the activity and also the enzyme protein may increase in human hepatoma and in the other human tumors. REFERENCES Bjornberg O., Bergman A.-C., Rosengren A. M., Persson R., Lehman I. R. and Nyman P. 0. (1993) dUTPase from herpes simplex virus type 1: purification from infected green monkey kidney (Vero) cells and from an overproducing ~cherich~u co& strain. Prot. Express. Puri$$ 1499159. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 2488254. Caradonna S. J. and Adamkiewicz D. M. (1984) Purification and properties of the deoxyuridine triphosphate nucleotido-hydrolase enzyme derived from HeLa S3 cells. 1. biol. Chem. 259, 5459-5464.
Cedergren-Zeppezauer E. S., Larsson G., Nyman P. O., Dauter Z. and Wilson K. S. (1992) Crystal structure of a dUTPase. Nature 355, 746743. Giroir L. E. and Deutsch W. A. (1987) Drosophifo deoxyuridine triphosphatase. Purification and characterization. J. biol. Chem. 262, 130-134.
et al.
Hokari S. and Sakagishi Y. (1987) Purification and characterization of deoxyuridine triphosphate nucleotidohydrolase from anemic rat spleen: a trimer composition of the enzyme protein. Archs Biochem. Biophys. 253, 350-356. Hokari S., Sakagishi Y. and Tsukada K. (1982) Enhanced activity of deoxyuridine 5’-triphosphatase in regenerating rat liver. Biochem. b~o~hy~. Res. Commun. 108, 95-101. Hokari S., Hasegawa M., Sakagishi Y. and Kikuchi G. (1987) Deoxyuridine triphosphate nucleotidohydrolase activity and its correlation with multiplication of erythroid cells in rat spleen. Biochem. Int. 14, 851-857. Hokari S., Takizawa A., Tanaka M. and Sakagishi Y. (1989) Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition. Biochem. Int. 19, 453461.
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