348
Biochimica et Biophysica Acta. 1079 (1991) 348-352 ,,~ 1991 Elsevier Science Publisl:ers B.V. All rights reserved 0167-4838/91/$03.50 A DONIS t) 16748389 I00294L
t~BAPR() 34trl~
Purification and characterization of thymidine kinase from regenerating rat liver I k u y o T s u k a m o t o ~, Y u m i k o T a n i g u c h i J, M a s a m i t s u M i y o s h i ~ a n d S h o s u k e Kojo z I Department of Food Science and Nutrition. Nara Women ~" Unit'ersit),, Nara (JapatO and 2 Department ~(Life and Health Sciences, Hyogo Unit'ersity of Teacher Education. Yashiro, t/yogo (Japan) (Received 28 March 1991)
Key words: Thymidine kinase: Regenerating liver: (Rat)
Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54 000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 pM. ATP acts as a sigmoidal substrate wittt a 'K a' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.
Introduction
Thymidine kinase (EC 2.7.1.21) is an enzyme of the pyrimidine salvage pathway which catalyzes the phosphorylation of thymidine to thymidine 5'-monophosphate. The activity of thymidine kinase is very low in resting eukaryotic cells, but increases dramatically in a proliferative phase [1-6]. We have demonstrated that thymidine kinase is a rate-limiting enzyme of DNA synthesis in regenerating rat liver and its activity is closely linked to the grow,~h rate of the cells [7-20]. Little is known about the molecular mechanisms of the cell cycle-dependent regulation of the enzyme. Recently, thymidine kinase genes have been cloned from human [21-24], hamster [25,26], mouse [27], rat [28] and chicken [39,30] cells. These cloned genes will be useful for identifying genetic elements which are important in the regulation of thimidine kinase. However, a complete understanding of the regulatory mechanism will also require detailed biochemical studies. I"o this end, we have purified and characterized thymidine kinase from rat regenerating liver. Some attempts have been made to purify the enzyme from regenerating rat liver [31-33], but hampered by the extremely low abundance of this enzyme in tissue and its instability. Recently, the development of an affinity column has been successfully employed to
Correspondence: i. Tsukamoto. Department of Food Science and Nutrition, N~ra Women's University, Nara 630, Japan.
isolate the enzyme from human normal adult liver [34], human placenta [35,36], Hela cells [37] and rat hepatoma [38]. In this report, we describe a purification procedure for cytosolic thymidine kinase from regenerating rat liver and some characteristics of the purified enzyme. Materials and Methods
Materials [Methyl-~H]dThd (70 Ci/mmol) were purchased from ICN Biomedicals. Nucleosides and nucleotides were obtained from Yamasa Japan. p-Nitrophenyl thymidine-3'-phosphate and phenylmethylsulfonyi fluoride (PMSF) were from Sigma. Activated CH-Sepharosc and Sephacryl S-200 were from Pharmacia Fine Chemical. DE81 ion exchange paper was from Whatman. All other reagents were of analytical grade.
Regenerath~g lit'er Male Wistar rats (200-220 g) given commercial laboratory chow were partially (70%) hepatectomized according to the procedure of Higgins and Anderson [39]. At 48 h after partial hepatectomy, the rats wzre killed under diethyl ether anesthesia. The liver was perfused in situ with saline and immediately excised.
Affinity co&mn preparation p-Aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity gel was prepared according to a similar method in the literature [40].
2oj!
Enzyme assay Thymidine kinase activity was assayed by the method previously described ~,ith a slight modification [7]. The standard reaction mixture (30/.tl) which contained 0.1 M Tris-HCl buffer (oH 8.0), I0 mM ATP, 10 mM MgCI 2, 0.1 M NaF, 1 mM [3H]dThd (5 m C i / m m o l ) and the enzyme preparation was incubated at 37 ° C for 15 rain. The reaction was stopped by heating in a boiling water bath for 2 min. 20/xl of the supernatant fraction resulting from centrifugation was spotted on W h a t m a n DES1 paper disk, which was washed three times with 1 m M ammonium formate and once with methanol. One unit of thymidine kinase activity represented the formation of 1 nmol of d T M P per min under the standard reaction conditions.
Protein assay Protein concentrations were determined according to Lowry et al. [41] with prior precipitation of proteins with T C A in the presence of deoxycholate [42] using bovine serum albumin as the ~;tandard. Eiectrophoresis of the purified preparation was performed on a slab gel made of 10% polyacrylamide containing SDS as described by Laemmli [43] and followed by silver staining
[~1. Results
Purification of thymidine kinase Crude extract fraction. The regenerating livers (60 g wet weight) were homogenized with 5 vols. of 50 m M Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 10 mM /3-mercaptoethanol, 2 mM E D T A and 1 m M PMSF at 4 ° C. All subsequent operations were carried out at 4 ° C . After centrifugation at 100000 × g for 60 rain, the supernatant was collected. Ammonium sulfate fractionation. To stirred crude extract solid ammonium sulfate was added to bring it to 40% saturation. The mixture was centrifuged at 36 000 x g for 30 min after 30 rain. The precipitate was suspended in a half volume of original supernatant of the homogenate buffer containing 1 mM thymidine. Calcium phosphate gel fractionation. Ca 3 (PO4) 2 gel was added to the suspension of ammonium sulfate fraction in the amount of 0.6 mg of g e l / r a g protein.
349
2
u
1
0 ~
40
60
BO
520
g
540
FroctJon
Fig. 1. Elution pattern of thymidine-3'-(p-aminophenylphosphate) affinity chromatography of thymidine kinase. After addition of ATP and MgC! z (2 raM), the pooled fractions from Ca3(PO4) 2 get fractionation were applied Io the columr and fractions (i ml) were collected. The a b ~ r b a n c e at 280 nm and TK activity were determined. Arrow indicates the start point of elution after the washing. - - - - , absorbance at 280 nm: e. TK activity.
The suspension was stirred for 15 rain and centrifuged at 36000 x g for 10 min. The supernatant was brought to 40% saturation with ammonium sulfate, stirred and centrifuged as described above. The precipitate was dissolved in a small amount of 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM /3-mercaptoethanol, 2 mM E D T A and 1 m M PMSF. The solution was applied to the column (3.5 x 24 cm) of Sephadex G-25 which has been equilibrated with 20 mM Tris-HCl buffer ~oH 7.5) containing 10 mM O-mercaptoethanol, and eluted with the equilibration buffer. Protein fractions were pooled.
Affinity chromatography on Thymidine-3'-(p-am#~ophenylphosphate)-CH-Sepharose. After addition of ATP and MgCI 2 in the final con'zentrations of 2 raM, the pooled fractions were applied slowly to a column of thymidine-3'(p-aminophenylphosphate)-CH-Sepharose (1.3 x 2.5 c m ) w h i c h had been equilibrated previously with 20 mM Tris-HC! buffer (pH 7.5) containing 5 mM dithiothreitol. The column was washed with 300 bed vols. of the 0.5 M Tris-HC1 buffer tpH 7.5) with 5 mM dithiothreitol and eluted with the above buffer containing 0.4 mM thymidine. Enzyme act:,, ty emerged as a single symmetrical peak as shown in Fig. 1. The results of the purification described above are summarized in Table I. The total purification achieved
TABLE I
Purification of thymidine kina.se from regenerating rat liter Step
Total protein (mg)
Total activity (units}
Specific activity (units/rag protein)
Purification (fold)
Recover)' (%)
1. 2. 3. 4.
3 200 990 4'00 t).02
640 580 540 280
0.20 0.60 1.10 14 000
1.0 3.0 5.5 70 000
100 91 84 44
Crude extract Ammonium sulfate Ca3 (PO4)2 gel and G-25 gel filtration ~ffinity chromatography
E
350 T i
J
b
3
lg
94 K >s
67K ,,,,,,g
43K
o.,
..,...,..,.,,,._.,_
0.2
0.3
0'.4
o~5
A'I'P (raM) Fig. 3. Thymidihe kinase activity as a function of ATP concentrati:~a. The activities of thymi~ine kinase were determined with various amounts of ATP at fixed concentration of thymidine (1 raM) as described in Materials and Methods.
25 K ,,,,,,,,
- -
",~F---- 26 K
=:
Fig. 2. SDS-polyacrylamide gel electrophoresis of purified thymidine kinase. Electrophoresis was performed on 10% polyacrylamide gel containing SDS and followed by silver staining as described in Materials and Methods. The left lane contained molecular weight standards; phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000) and chymotrypsinogen (25000). The right lane was the final purified thymidine kinase.
was approx. 70000-fold wi~a recovery of 44%. In a typical pttrification, 20 /.tg of enzyme were obtained from 60 ~ of 48 h-regenerating rat liver. The protein obtained ~as apparently homogeneous, as judged by the single band observed upon electrophoresis in polyacrylamide gel containing SDS (Fig. 2).
Properties of purified thyrnidine kinase Molecular weight. Thymidine kinase activity was eluted at a position corresponding to a molecular weight of approx. 54000 upon Sephacryl S-200 gel filtration. As estimated from its mobility with respect to protein standards, the single band observed upon electrophoresis in polyacrylamide gets containing SDS had a molecular weight of about 26000. This result suggests that native enzyme is a dimer composed of very similar or identical subunits. Substrate kinetics and reaction mechanism. For a kinetic study of the purified enzyme, dThd had to be completely removed from the final affinity gel fraction. The enzyme preparation was passed through a minicolumn (1 : ~) of Sephadex G-25 equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM dithiothreitol and bovine serum albumin (1 mg/ml). initial velocity studies were performed by keeping one substrate at a fixed concentration and varying the concentration of the other substrate. ATP-Mg ~+ acted
as a sigmoidal substrate as shown in Fig. 3. Linear plots were obteined when l/t, was plotted against 1 / ( A T P _ M g : ~ )2 at a fixed concentration. When the intercepts of these plots on ( l / v ) axis were plotted against 1/dThd, the K,,, of dThd was calculated to be 2.2/zM. When A T P - M g 2+ was used at a fixed concentration, Lineweaver-Burk plots of l / t , vs. 1 / d T h d were linear and the K m of A T P - M g 2+ was determined to be 0.2 mM by replotting the intercept of each line. All K m values obtained from these plots were consistent with values obtained at the saturation levels of the other substrate. The results of product inhibition studies were shown in Table II. The inhibition of the enzyme activity by dTMP was apparently competitive with thymidine as the variable substrate ( K i =0.65 mM). When A T P - M g 2+ was the variable substrate, the inhibition of dTMP appeared noncompetitive (K~ = 0.65 mM) and A D P - M g 2+ inhibition was competitive (K i = 0.4 mM). The inhibition of the enzyme activity by A D P - M g z+ measured with thymidine as the variable substrate was not observed at the highest cencentration of 5 mM. These results suggest that the enzyme follows a sequential mechanism.
TABLE I1
Product inhibition siudies of the reaction of thymidine kinase The enzyme was used after the get filtration of the final preparation as described in Results. The concentration of [3H]thymidine (2.5 mci//J.mol) was varied from 0.5 to 2 #M. The concentration of A T P - M g 2+ was varied from 0.1 to 0.25 mM. The fixed concentrations of dTMP (0.5-3.0 raM) and A D P (0.5-5.0 raM) were incubated with thymidine as the variable substrate (10 mM A T P - M g 2+ ) and with A T P - M g 2÷ as the variable substrate (10 # M thymidine). Product
Variable substrate
Apparent K i (mM)
Type of inhibition
dTMP dTMP ADP ADP
thymidine ATP thymidine ATP
0.65 0.65 0.40
competitive noncompetitive no inhibition competitive
351
Enzyme stability. The purified enzyme preparation was stabilized by the presence of dithiothreitol and the substrate dThd. With 5 mM dithiothreitol and 400 # M dThd, the enzyme retained its activity for about 3 days when stored at 4 ° C. Without dithiothreitol and dThd, the purified thymidine kinase was inactivated totally in 1 day. Phosphate donor specificity. The efficiency as a phosphate donor of various nucleotides was determined at the equimolar concentration of MgCI 2 (10 mM). The maximal rate of reaction was obtained with ATP and dATP, whereas UTP, CTP, GTP and dGTP had about 20% activity of the maximum. The other nucleotides such as dCTP, d'lq'P, dTDP, dTMP, ADP, and CDP were poor phosphate donors in the catalysis of thymidine kinase. Discussion The cytosolic rat tia.~xnidine kinase is purified to apparent homogeneity from regenerating liver utilizing the thymidine affinity matrix originally developed by Kowal and Markus [40]. A number of investigators have reported the protocols for purification of thymidine kinase from human and rat ti~,sues [31-38,40]. Human thymidine kinase has been purified to homogeneity from normal adult liver [34], placenta [35,36] and Hela cells [37]. However, several attempts to purify the enzyme from rat tissues have been unsuccessful [31-33,40], whereas thymidine kinase from rat hepatoma was isolated to near homogeneity [38]. The result that about 70000-fold purification was required to obtain the pure protein indicated that even regenerating liver possezsed a very low level of this enzyme. The specific activity of the final electrophoreticaily homogenec, uz preparation of the rat liver enzyme, 14 / z m o l / m i n per mg of protein was higher than that of the enzyme isolated from Hela cell (5.4 ~ m o l / m i n per mg of protein) [37] or rat hepatoma (4 / z m o ! / m i n per mg of protein) [38]. The turnover number of the enzyme at 37 ° C was calculated as 757 min- ~. The molecular weight of the native rat liver enzyme was found to be 54000 and that of its monomer was 26000. The subunit molecular weights predicted from thymidine Kinase e D N A for the human [23], hamster [26], mouse [27] and chicken [30] enzymes are all quite similar (25 504, 25 625, 25 873 and 24 844, respectively). Thus, it seems likely that previous preparation of rat thyraidine kinase contained major contaminants [38]. The native molecular weight of rat liver cytosolic thymidine kinase was 54000 and distinct from the molecular weight of 96000 reported for the enzyme of Hela cells [37] or 92000 or 70900 for that of human placenta [35,36]. This result suggests that the structure of rat liver cytosolic thymidine kinase is different from that of the human enzyme.
References I Bresnick. U., Williams, S.S. and Mosse. i-1. (1967) Cancer Res. 27, 469 -475. 2 Brent, T.P. (1971) Cell Tissve Kinet. 4, 297-305. 3 Kit. S., Dubbs, D.R. and Frearson, P.M. (1965)J. Biol. (?hem. 240. 2565-2573. 4 Adler, R. and McAuslan. B.R. (1974) Cell. 2, 113-117. 5 Kit, S. ~1976) Mol. Cell. Biochem. 11, 161-182. 6 Munch-Petersen. B. and T}rsted, G. (1977) Biochem. Biophys. Acta 478, 364-375. 7 Nakata, R., Tsukamoto, I., Miyoshi, M. and Kojo, S. (t985) Biochem. Pharmacol. 34, 586-588. 8 Nakata, R., Tsukamoto, I., Nanme, M., Makino, S., Miyoshi, M. and Kojo, S. (1985) Eur. J. Pharmacol. 114, 355-360. 9 Nakata, R., Tsukamoto, I., Miyoshi, M. and Kojo, S. (1986) Biochem. Pharmacol. 35, 865-867. 10 Tsukamoto, I. and Kojo. S. (1987) Biochem, Pharmacol. 36, 2871-2872. 11 Nakata, R., Tsukamoto, I., Miyoshi, M. and Kojo, S. (1987) Ctin. Sci. 72, 455-461. 12 Tsukamoto, 1. and Kojo, S. (1987) Chem. Lett. 2313-2316. 13 Tsukamoto, !. and Kojo, S. (1987) Eor. J. Pharmacol. 144, 159162. 14 Tsukamoto, !. and Kojo, S. (1989) Gut 30, 387-390. 15 Tsukamoto, I. and Kojo, S. (1989) Biochim. Biophys. Acta, 1009, 191-193. 16 Tsukamoto, !. and Kojo, S. (1990) Biochim. Biophys. Acta, 1033, 287-290. 17 Ochi, Y., Yumori, Y., Morioka, A., Miura. K., Tsukamoto, I. and Kojo, S. (1990) Biochem. Pharmacol. 39, 2066-2069. t8 Tsukamoto, !. and Kojo, S. (1990) J. Nutr. Sci. Vitaminol. 36, 357-363. 19 Tsukamoto, 1. and Kojo, S. (1991) Biochim. Biophys. Acta 1074, 52 -55. 20 Tsukamoto, I. and Kojo, S. (1991) Biochem. Int. 23, 619-624. 21 Bradshaw, H.D., Jr. (1983) Proc, Natl. Acad. Sci. U.S.A. 80, 5588-5591. 22 Lin, P., Zhao, S. and Ruddle, F.H. (1983) Proc. Natl. Acad. Sci. USA 80, 6528-6532. 23 Bradshaw, H.D., Jr. and Deininger, P.L. (1984) Mol. Cell. Biol, 4, 2316- 2320. 24 Lau, Y.-F. and Kan, Y.W. (1984) Proc. Natl. Acad. Sci. USA 81, 414-418. 25 Lewis, J.A.. Shimizu, K. and Zipser, D. (1983) Mol. Cell. Biol. 3, 1815-1823, 26 Lewis, J.A. (1986) Mol. Cell. Biol. 6, 1998-2010. 27 Lin, P.-F., Lieberman, ll.B., Yeh, D.-B., Xu, T., Zhao, S.-Y. and Ruddle, F.H. (1985) Moi. Ceil. Biol. 5, 3149-3156. 28 Murphy, A.J.M. and Efstratiadis. A. (1987) Proc Natl. Acad. Sci. USA 84, 8277-8281. 29 Perucho, M., t- .~ahan, D., Lipsich, L. and Wigler, M. (1980) Nature, 285, 2~,,-210. 5(! K~voh, T.J. and Engler, J.A. (t984) Nucleic Acids Res. 12, 39593971. 31 Bresoick, E., Mainigi, K.D., Bucc!no, R and Burleson, S.S. (1970) Cancer Res. 30, 2502-2506. 32 Kizer, D.E. and Holman, L. (1974) Biochim. Biophys. Acta, 350, 193-200. 33 Nawata, H. and Kamiya, T. (1975)J. Biochem. 78, 1215-1224. 34 Etlims, P.H. and Vail Der Weyden, M.B. (1080) J, Biol. Chem. 255, 11290-11295. 35 Gan, T.E., Brumley, JL. and Van der Weyden. M.B. (1983) J. Biol. Chem. 258, 7(X~)-7004. 36 Tamiya, N., Yusa, T., Yamaguchi. Y., Tsukifuji, R., Kuroiwa, N., Moriyama, Y. and Fujimura, S. (1989) Bit~chim. Biophys. Acta, 995.28-35.
352 37 Shcrley, J.L. and Kelly. T.]. (1988) J, Biol. (,hem. 263, 375-382. 38 Lai, MALT. and Weber. G. (t983) Bichem. Biophy:,. Res. Commun. I I i, 280-287. 39 Higgins. G.M, and Anderson, R.M, (10317 Arch. Pathol, 12, 186-202. 40 Kowal, E.P. and Markus, G. (t976) Prep. Biochem. ft. 369-385. 41 Lx~wry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biot. Chem. 193. 265-275.
42 Bensadoun, A. and Weinslein, D. (1076) Anal. Biochem. 70, 241-250. 43 Laemmli, U.K. (1970) Nature 227, 680-685. 44 Merril, C.R., Goldman, D., Sedman, S.A. and Ebert, M.H. (198!) Science. 211, 1437-I438.
Biochimica et Biophysica Acta. 1079 (1991) 348-352 ,,~ 1991 Elsevier Science Publisl:ers B.V. All rights reserved 0167-4838/91/$03.50 A DONIS t) 16748389 I00294L
t~BAPR() 34trl~
Purification and characterization of thymidine kinase from regenerating rat liver I k u y o T s u k a m o t o ~, Y u m i k o T a n i g u c h i J, M a s a m i t s u M i y o s h i ~ a n d S h o s u k e Kojo z I Department of Food Science and Nutrition. Nara Women ~" Unit'ersit),, Nara (JapatO and 2 Department ~(Life and Health Sciences, Hyogo Unit'ersity of Teacher Education. Yashiro, t/yogo (Japan) (Received 28 March 1991)
Key words: Thymidine kinase: Regenerating liver: (Rat)
Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54 000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 pM. ATP acts as a sigmoidal substrate wittt a 'K a' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.
Introduction
Thymidine kinase (EC 2.7.1.21) is an enzyme of the pyrimidine salvage pathway which catalyzes the phosphorylation of thymidine to thymidine 5'-monophosphate. The activity of thymidine kinase is very low in resting eukaryotic cells, but increases dramatically in a proliferative phase [1-6]. We have demonstrated that thymidine kinase is a rate-limiting enzyme of DNA synthesis in regenerating rat liver and its activity is closely linked to the grow,~h rate of the cells [7-20]. Little is known about the molecular mechanisms of the cell cycle-dependent regulation of the enzyme. Recently, thymidine kinase genes have been cloned from human [21-24], hamster [25,26], mouse [27], rat [28] and chicken [39,30] cells. These cloned genes will be useful for identifying genetic elements which are important in the regulation of thimidine kinase. However, a complete understanding of the regulatory mechanism will also require detailed biochemical studies. I"o this end, we have purified and characterized thymidine kinase from rat regenerating liver. Some attempts have been made to purify the enzyme from regenerating rat liver [31-33], but hampered by the extremely low abundance of this enzyme in tissue and its instability. Recently, the development of an affinity column has been successfully employed to
Correspondence: i. Tsukamoto. Department of Food Science and Nutrition, N~ra Women's University, Nara 630, Japan.
isolate the enzyme from human normal adult liver [34], human placenta [35,36], Hela cells [37] and rat hepatoma [38]. In this report, we describe a purification procedure for cytosolic thymidine kinase from regenerating rat liver and some characteristics of the purified enzyme. Materials and Methods
Materials [Methyl-~H]dThd (70 Ci/mmol) were purchased from ICN Biomedicals. Nucleosides and nucleotides were obtained from Yamasa Japan. p-Nitrophenyl thymidine-3'-phosphate and phenylmethylsulfonyi fluoride (PMSF) were from Sigma. Activated CH-Sepharosc and Sephacryl S-200 were from Pharmacia Fine Chemical. DE81 ion exchange paper was from Whatman. All other reagents were of analytical grade.
Regenerath~g lit'er Male Wistar rats (200-220 g) given commercial laboratory chow were partially (70%) hepatectomized according to the procedure of Higgins and Anderson [39]. At 48 h after partial hepatectomy, the rats wzre killed under diethyl ether anesthesia. The liver was perfused in situ with saline and immediately excised.
Affinity co&mn preparation p-Aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity gel was prepared according to a similar method in the literature [40].
2oj!
Enzyme assay Thymidine kinase activity was assayed by the method previously described ~,ith a slight modification [7]. The standard reaction mixture (30/.tl) which contained 0.1 M Tris-HCl buffer (oH 8.0), I0 mM ATP, 10 mM MgCI 2, 0.1 M NaF, 1 mM [3H]dThd (5 m C i / m m o l ) and the enzyme preparation was incubated at 37 ° C for 15 rain. The reaction was stopped by heating in a boiling water bath for 2 min. 20/xl of the supernatant fraction resulting from centrifugation was spotted on W h a t m a n DES1 paper disk, which was washed three times with 1 m M ammonium formate and once with methanol. One unit of thymidine kinase activity represented the formation of 1 nmol of d T M P per min under the standard reaction conditions.
Protein assay Protein concentrations were determined according to Lowry et al. [41] with prior precipitation of proteins with T C A in the presence of deoxycholate [42] using bovine serum albumin as the ~;tandard. Eiectrophoresis of the purified preparation was performed on a slab gel made of 10% polyacrylamide containing SDS as described by Laemmli [43] and followed by silver staining
[~1. Results
Purification of thymidine kinase Crude extract fraction. The regenerating livers (60 g wet weight) were homogenized with 5 vols. of 50 m M Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 10 mM /3-mercaptoethanol, 2 mM E D T A and 1 m M PMSF at 4 ° C. All subsequent operations were carried out at 4 ° C . After centrifugation at 100000 × g for 60 rain, the supernatant was collected. Ammonium sulfate fractionation. To stirred crude extract solid ammonium sulfate was added to bring it to 40% saturation. The mixture was centrifuged at 36 000 x g for 30 min after 30 rain. The precipitate was suspended in a half volume of original supernatant of the homogenate buffer containing 1 mM thymidine. Calcium phosphate gel fractionation. Ca 3 (PO4) 2 gel was added to the suspension of ammonium sulfate fraction in the amount of 0.6 mg of g e l / r a g protein.
349
2
u
1
0 ~
40
60
BO
520
g
540
FroctJon
Fig. 1. Elution pattern of thymidine-3'-(p-aminophenylphosphate) affinity chromatography of thymidine kinase. After addition of ATP and MgC! z (2 raM), the pooled fractions from Ca3(PO4) 2 get fractionation were applied Io the columr and fractions (i ml) were collected. The a b ~ r b a n c e at 280 nm and TK activity were determined. Arrow indicates the start point of elution after the washing. - - - - , absorbance at 280 nm: e. TK activity.
The suspension was stirred for 15 rain and centrifuged at 36000 x g for 10 min. The supernatant was brought to 40% saturation with ammonium sulfate, stirred and centrifuged as described above. The precipitate was dissolved in a small amount of 20 mM Tris-HCl buffer (pH 7.5) containing 10 mM /3-mercaptoethanol, 2 mM E D T A and 1 m M PMSF. The solution was applied to the column (3.5 x 24 cm) of Sephadex G-25 which has been equilibrated with 20 mM Tris-HCl buffer ~oH 7.5) containing 10 mM O-mercaptoethanol, and eluted with the equilibration buffer. Protein fractions were pooled.
Affinity chromatography on Thymidine-3'-(p-am#~ophenylphosphate)-CH-Sepharose. After addition of ATP and MgCI 2 in the final con'zentrations of 2 raM, the pooled fractions were applied slowly to a column of thymidine-3'(p-aminophenylphosphate)-CH-Sepharose (1.3 x 2.5 c m ) w h i c h had been equilibrated previously with 20 mM Tris-HC! buffer (pH 7.5) containing 5 mM dithiothreitol. The column was washed with 300 bed vols. of the 0.5 M Tris-HC1 buffer tpH 7.5) with 5 mM dithiothreitol and eluted with the above buffer containing 0.4 mM thymidine. Enzyme act:,, ty emerged as a single symmetrical peak as shown in Fig. 1. The results of the purification described above are summarized in Table I. The total purification achieved
TABLE I
Purification of thymidine kina.se from regenerating rat liter Step
Total protein (mg)
Total activity (units}
Specific activity (units/rag protein)
Purification (fold)
Recover)' (%)
1. 2. 3. 4.
3 200 990 4'00 t).02
640 580 540 280
0.20 0.60 1.10 14 000
1.0 3.0 5.5 70 000
100 91 84 44
Crude extract Ammonium sulfate Ca3 (PO4)2 gel and G-25 gel filtration ~ffinity chromatography
E
350 T i
J
b
3
lg
94 K >s
67K ,,,,,,g
43K
o.,
..,...,..,.,,,._.,_
0.2
0.3
0'.4
o~5
A'I'P (raM) Fig. 3. Thymidihe kinase activity as a function of ATP concentrati:~a. The activities of thymi~ine kinase were determined with various amounts of ATP at fixed concentration of thymidine (1 raM) as described in Materials and Methods.
25 K ,,,,,,,,
- -
",~F---- 26 K
=:
Fig. 2. SDS-polyacrylamide gel electrophoresis of purified thymidine kinase. Electrophoresis was performed on 10% polyacrylamide gel containing SDS and followed by silver staining as described in Materials and Methods. The left lane contained molecular weight standards; phosphorylase b (94000), bovine serum albumin (67000), ovalbumin (43000) and chymotrypsinogen (25000). The right lane was the final purified thymidine kinase.
was approx. 70000-fold wi~a recovery of 44%. In a typical pttrification, 20 /.tg of enzyme were obtained from 60 ~ of 48 h-regenerating rat liver. The protein obtained ~as apparently homogeneous, as judged by the single band observed upon electrophoresis in polyacrylamide gel containing SDS (Fig. 2).
Properties of purified thyrnidine kinase Molecular weight. Thymidine kinase activity was eluted at a position corresponding to a molecular weight of approx. 54000 upon Sephacryl S-200 gel filtration. As estimated from its mobility with respect to protein standards, the single band observed upon electrophoresis in polyacrylamide gets containing SDS had a molecular weight of about 26000. This result suggests that native enzyme is a dimer composed of very similar or identical subunits. Substrate kinetics and reaction mechanism. For a kinetic study of the purified enzyme, dThd had to be completely removed from the final affinity gel fraction. The enzyme preparation was passed through a minicolumn (1 : ~) of Sephadex G-25 equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM dithiothreitol and bovine serum albumin (1 mg/ml). initial velocity studies were performed by keeping one substrate at a fixed concentration and varying the concentration of the other substrate. ATP-Mg ~+ acted
as a sigmoidal substrate as shown in Fig. 3. Linear plots were obteined when l/t, was plotted against 1 / ( A T P _ M g : ~ )2 at a fixed concentration. When the intercepts of these plots on ( l / v ) axis were plotted against 1/dThd, the K,,, of dThd was calculated to be 2.2/zM. When A T P - M g 2+ was used at a fixed concentration, Lineweaver-Burk plots of l / t , vs. 1 / d T h d were linear and the K m of A T P - M g 2+ was determined to be 0.2 mM by replotting the intercept of each line. All K m values obtained from these plots were consistent with values obtained at the saturation levels of the other substrate. The results of product inhibition studies were shown in Table II. The inhibition of the enzyme activity by dTMP was apparently competitive with thymidine as the variable substrate ( K i =0.65 mM). When A T P - M g 2+ was the variable substrate, the inhibition of dTMP appeared noncompetitive (K~ = 0.65 mM) and A D P - M g 2+ inhibition was competitive (K i = 0.4 mM). The inhibition of the enzyme activity by A D P - M g z+ measured with thymidine as the variable substrate was not observed at the highest cencentration of 5 mM. These results suggest that the enzyme follows a sequential mechanism.
TABLE I1
Product inhibition siudies of the reaction of thymidine kinase The enzyme was used after the get filtration of the final preparation as described in Results. The concentration of [3H]thymidine (2.5 mci//J.mol) was varied from 0.5 to 2 #M. The concentration of A T P - M g 2+ was varied from 0.1 to 0.25 mM. The fixed concentrations of dTMP (0.5-3.0 raM) and A D P (0.5-5.0 raM) were incubated with thymidine as the variable substrate (10 mM A T P - M g 2+ ) and with A T P - M g 2÷ as the variable substrate (10 # M thymidine). Product
Variable substrate
Apparent K i (mM)
Type of inhibition
dTMP dTMP ADP ADP
thymidine ATP thymidine ATP
0.65 0.65 0.40
competitive noncompetitive no inhibition competitive
351
Enzyme stability. The purified enzyme preparation was stabilized by the presence of dithiothreitol and the substrate dThd. With 5 mM dithiothreitol and 400 # M dThd, the enzyme retained its activity for about 3 days when stored at 4 ° C. Without dithiothreitol and dThd, the purified thymidine kinase was inactivated totally in 1 day. Phosphate donor specificity. The efficiency as a phosphate donor of various nucleotides was determined at the equimolar concentration of MgCI 2 (10 mM). The maximal rate of reaction was obtained with ATP and dATP, whereas UTP, CTP, GTP and dGTP had about 20% activity of the maximum. The other nucleotides such as dCTP, d'lq'P, dTDP, dTMP, ADP, and CDP were poor phosphate donors in the catalysis of thymidine kinase. Discussion The cytosolic rat tia.~xnidine kinase is purified to apparent homogeneity from regenerating liver utilizing the thymidine affinity matrix originally developed by Kowal and Markus [40]. A number of investigators have reported the protocols for purification of thymidine kinase from human and rat ti~,sues [31-38,40]. Human thymidine kinase has been purified to homogeneity from normal adult liver [34], placenta [35,36] and Hela cells [37]. However, several attempts to purify the enzyme from rat tissues have been unsuccessful [31-33,40], whereas thymidine kinase from rat hepatoma was isolated to near homogeneity [38]. The result that about 70000-fold purification was required to obtain the pure protein indicated that even regenerating liver possezsed a very low level of this enzyme. The specific activity of the final electrophoreticaily homogenec, uz preparation of the rat liver enzyme, 14 / z m o l / m i n per mg of protein was higher than that of the enzyme isolated from Hela cell (5.4 ~ m o l / m i n per mg of protein) [37] or rat hepatoma (4 / z m o ! / m i n per mg of protein) [38]. The turnover number of the enzyme at 37 ° C was calculated as 757 min- ~. The molecular weight of the native rat liver enzyme was found to be 54000 and that of its monomer was 26000. The subunit molecular weights predicted from thymidine Kinase e D N A for the human [23], hamster [26], mouse [27] and chicken [30] enzymes are all quite similar (25 504, 25 625, 25 873 and 24 844, respectively). Thus, it seems likely that previous preparation of rat thyraidine kinase contained major contaminants [38]. The native molecular weight of rat liver cytosolic thymidine kinase was 54000 and distinct from the molecular weight of 96000 reported for the enzyme of Hela cells [37] or 92000 or 70900 for that of human placenta [35,36]. This result suggests that the structure of rat liver cytosolic thymidine kinase is different from that of the human enzyme.
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