/ . Biochem., 78, 1215-1224 (1975)
Hajime NAWATA2 and Tomoya KAMIYA3 Division of Chemistry, Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812 Received for publication, March 7, 1975
Two forms (Peak A and Peak B) of thymidine kinase [EC 2.7.1. 75] from regenerating rat liver cytosol were resolved and partially purified by DEAE-cellulose chromatography. Both fractions were identical with respect to their substrate requirement, pH optima, metal requirements, and molecular weight, as judged by their sedimentation in sucrose density gradient centrifugation. Peak B differed from Peak A in heat sensitivity, inhibition by dCTP and Km for thymidine and ATP. Peak B enzyme was the only enzyme found in normal adult liver and Peak A enzyme was the form increasing predominantly in regenerating liver.
Thymidine kinase [EC 2.7.1.75], a salvage enzyme in the pathway of DNA synthesis, is of particular interest because its activity in a variety of cells has been correlated with the occurrence of cell division. There is an increase of thymidine kinase activity in regenerating mammalian liver (1, 2), mammalian tumor (5, 6), embryonic liver (7, 8), regenerating kidney after uninephrectomy (10), tumor virus-infected mammalian cells (11, 12) and adipose tissue (13) and adrenal gland (14, 15) in response to hormonal influences. Several distinct molecular forms of thymidine ki1
This study was supported in part by a research grant for cancer research from the Ministry of Education, Japan. 2 Present address: Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan. 3 Present address: Department of Biochemistry, Nara Medical University, Kashihara, Nara, Japan. Abbreviation: PEI, polyethyleneimine.
Vol. 78, No. 6, 1975
nase probably exist in mammalian systems. Thymidine kinase from both normal and rapidly metabolizing cells, such as regenerating rat liver, is subject to feedback inhibition by dTTP ( 7 ) . However comparison of the properties of thymidine kinase in various tissues, such as inhibition by nucleotides ( 7 ) and patterns of electrophoresis (8, 9, 16), provides evidence that some forms of thymidine kinase differ from the adult form. The present work showed that DEAEcellulose chromatography of partially purified thymidine kinase from regenerating rat liver results in the isolation of two forms of this enzyme. Each form has been characterized with respect to certain biochemical and catalytic properties. In addition, our data indicate that one of the enzyme forms appearing in regenerating liver is only synthesized after the start of liver regeneration and is the major component of thymidine kinase in regenerating liver, while the other form is the same as the
1215
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
Two Molecular Forms of Thymidine Kinase in the Cytosol of Regenerating Rat Liver1
1216
H. NAWATA and T. KAMIYA.
EXPERIMENTAL PROCEDURE Animals—Donryu male albino rats weighing 160 to 200 g were obtained from the Inbred Animal Supply Center of Kyushu University and were maintained on Oriental solid chow and water ad libitum. Reagents — [uC]thymidine (45 mCi per mmole) and [UC]ATP (550 mCi per mmole) were products of C.E.N. Dep. des Radioisotopes, Belgium. Crystalline ATP, thymidine, dTTP, and other ribo- and deoxyribonucleotides were obtained from Boehringer Mannheim, Germany. DEAE-cellulose was a product of Brown Co. DEAE-cellulose paper was from Toyo Roshi, Tokyo, Japan. Preformed PEIcellulose plates were from Merck Sharp and Dohme. Ammonium sulfate and ethylene glycol were biochemical analysis grade from Nakarai, Osaka, Japan. Preparation of Enzyme Extract—Partial hepatectomy was done by the method of Higgins and Anderson, removing approximately two-thirds of the liver under ether anesthesia (17). Partially hepatectomized rats were sacrificed by neck dislocation 40 hr after the operation, when the induction of thymidine kinase reached the maximum level (inset, Fig. 5). Regenerating livers were collected, chilled quickly, minced with scissors, added to three volumes of 50 mM Tris-HCl (pH 7.2), 0.25 M sucrose, 2.2 mM CaCl2, and 20 fiM dTTP, then homogenized in a Potter-Elvehjem glass homogenizer with a teflon pestle by means of 10 strokes in crushed ice (homogenates). Subcellular Fractionation of Liver Cells— The liver was minced and suspended in three volumes of 0.25 M sucrose, 50 mM Tris-HCl buffer (pH 7.3), and 3 mM CaCl2, then homogenized with a teflon pestle homogenizer and filtered through four layers of gauze. The nuclear fraction was obtained by centrifugation at 700 X g for -15 min, then the precipitates were homogenized, layered on 2.2 M sucrose and 50 mM Tris-HCl buffer (pH 7.3) and cen-
trifuged at 40,000 xg for 60 min, sedimenting^ the nuclei. The 700xg supernatant was centrifuged at 8,000xg for 15 min, sedimenting^ the mitochondria. The 8,000Xg supernatant was the centrifuged at 105,000xg for 30 min, sedimenting microsomes. The mitochondriaL fraction was resuspended in the homogenizing: buffer. A part of all the fractions was treated with 1% Triton X-100. Assay of Thymidine Kinase—Thymidine kinase assays were perfomed by the DEAEcellulose paper disk method as modified by Bresnick (18). The reaction mixture included 0.2 ml of the enzyme, 2.5 /imoles of ATP, 2.5 /^mols of MgCl2, and 40 /imoles of Tris-HCl buffer (pH 7.3) at 37° in a total volume of 0.50 ml. The reaction was started by adding 25 ^1 of 200 /*M [ u C]thymidine. The incubation was conducted at 37° for 30 min. The reaction was stopped by immersing the tubes in a boilingwater bath for 2 min. The precipitates were removed by mild centrifugation and 0.2 ml aliquots from the supernatants were applied to DEAE-cellulose paper squares (3x3 cm) and dried at room temperature. The dried squares were immersed for 10 min in a tray containing 1 mM ammonium formate, then the papers were washed in distilled water for further 10 min. .. This procedure was repeated twice. Finally the papers were immersed in 95%ethanol, dried at 80° and counted by directly placing the dried paper in a counting vial with 10 ml of toluene-based scintillation fluid in a Beckman liquid scintillation counter. Whea the activity of enzyme preparations containing dTTP was assayed during the purification, the concentraton of dTTP in the reaction mixture was kept below 1 pM, where its inhibiting activity was negligible. Protein concentration was estimated by the method of Lowry et aL (19) with bovine serum albumin as a reference standard. Sucrose Density Centrifugation — Centrifugation in a sucrose density gradient was performed in linear gradients of 5% to 20%> (w/v) sucrose in 0.1 M Tris-HCl buffer (pH 7.3). Each tube for the SW39 rotor (Beckman Instruments) was filled with 4.5 ml of the gradient and 0.1 ml of the sample was layered on top. Centrifugation was carried out at / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
only enzyme found in normal adult liver and is the minor component in regenerating liver. The latter form resembles the thymidine kinase present in mitochondria.
TWO MOLECULAR FORMS OF THYMIDINE KINASE
RESULTS Initial Experiments — It was previously shown that thymidine kinase from various sources is quite unstable and the preparations require stabilizers such as thymidine (4, 5) and albumin (3). When the stabilizing effects of thymidine and various nucleotides were compared, dTTP was found to be best stabilizer. Some stabilizing effects were also observed with thymidine, ATP, dATP, CTP, dCTP, and ADP. Twenty % glycerol and 20% ethylene glycol were found to increase the activity by about 5 to 30% of the original activity upon incubation at 37°. Partial Purification of Thymidine Kinase Vol. 78, No. 6, 1975
—All purification steps were performed at 0 to4°. The homogenate from regenerating liver was centrifuged at 8,000 xg for 15 min. The supernatant fraction was further centrifuged at 105,000 Xfiffor 60 min (crude extract). Finely powdered solid ammonium sulfate was slowly added to the crude extract to 40% saturation. Next, the mixture was stirred for 101 min and centrifuged at 8,000xg for 15 min. The resulting pellets were dissolved in 3 ml of 200 mM Tris-HCl buffer (pH 7.2) containing 20 fiM dTTP. This solution was then applied to a Sephadex G-25 column (3x20 cm) previously equilibrated with 5 mM Tris-HCl (pH 7.2). The column was eluted with the same buffer; 4 ml fractions were collected and the fractions containing most of the enzyme activity were pooled. To this fraction, dTTP' and ethylene glycol were added to final concentrations of 20 [*M and 20% respectively (1st ammonium sulfate fraction). DEAE-cellulose (medium size, 0.9 meq per g) was processed as described elsewhere (25) except that adjustment of the pH of the absorbent was done by adding 50 mM Tris-HCl (pH 7.2). The DEAE-cellulose was then packed into a column (1X 20 cm) under gravity and equilibrated with 5 mM Tris-HCl (pH 7.2) containing 20% ethylene glycol. The enzyme solution (100' mg of protein) from the 1st ammonium sulfate fraction was applied to the column at a flow rate of 17 ml per hr. The column was washed with 50 ml of the same buffer, and a linear gradient (300 ml) of NaCl (0 to 0.25 M) in the same buffer was applied to the column. After gradient elution had been completed, the column was eluted with 50 ml of 0.4 M NaCl in the same buffer. Thymidine kinase activity was eluted at NaCl concentrations of 0 (breakthrough peak), 0.05M (Peak A), and 0.15 M (Peak B). Figure 1 shows a typical DEAEcellulose chromatogram of thymidine kjnase. It can be seen that the enzyme consists predominantly of two Peaks A and B. A further increase of the NaCl concentration in the elution buffer up to 0.4 M did not yield any appreciable activity.'
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
39,000 rpm for 18 hr using a Spinco L-2 ultracentrifuge at 4°. After centrifugation, the bottoms of the tubes were pierced and 20 fractions were collected. As enzyme markers, rat muscle aldolase [EC 4.1.2.13] and malate dehydrogenase [EC 1.1.1.37] from pig heart were used. Rat muscle aldolase was purified and crystallized after Ikehara et al. (20), and assayed according to Blostein and Rutter (21). Malate dehydrogenase was obtained from Sigma, and assayed according to Mehler et al. (22). Assay of ATP Phosphohydrolase and Thin Layer Chromatography of Nucleotides— The enzyme preparations were assayed for ATP phosphohydrolase (23) by incubating 50 l*\ samples at 37° for 30 min in the presence of 1.5 mM MgCl2, 50 mM Tris-HCl (pH 7.2) and [UC]ATP (5 nCi) in a total volume of 70 fi\. The reaction was stopped by adding 1 fil of 88% formic acid, and the resulting precipitates were spun down. The supernatant was put on PEI-impregnated cellulose plates (Merck). Resolution of ATP, ADP, and AMP was done by successively eluting the plates with water and 0.2 M ammonium formate in 4.0 M formic acid (24). Separated spots were detected by UV irradiation, scraped into vials and their radioactivities were determined with a Beckman liquid scintillation counter. The separation of thymidine, dTMP, dTDP, and dTTP was also carried out by the same thin layer chromatography procedure.
1217
In order to confirm the identity of these enzyme peaks, rechromatography was done in a stepwise fashion to avoid inactivation of the
1218
H. NAWATA and T. KAMIYA
240
280
320
Fig. 1. The elution profile of DEAE-cellulose chromatography of thymidine kinase. A 10-ml preparation (100 mg of protein) from the ammonium sulfate fraction containing 20 fiM dTTP and 20% ethylene glycol was applied to a column of DEAE-cellulose (1x20 cm) prepared as described in the text. Elution was carried as described in the text. Fractions of 4 ml were collected at a flow rate of 17 ml per hour. Each fraction was assayed for thymidine kinase activity (O) and the absorbance at 280 ( ) was determined. The arrow in the figure indicates the position of elution of free dTTP. This was confirmed by cochromatography with [3H]dTTP.
enzyme due to prolonged exposure on the column. Rechromatography of Peaks A and B did not change their elution positions. The addition of 20 ^M dTTP to the enzyme fraction before application to the second column gave the same results. The activity appearing in the breakthrough peak on DEAE-cellulose chromatography was not analyzed in this experiment. This peak fluctuated greatly in different experiments. The fractions from Peaks A and B (DEAE eluate) were concentrated by adding ammonium sulfate to 50% saturation and centrifuging at 50,000 Xg for 30 min. The resulting pellet was dissolved in 200 mM Tris-HCl (pH 7.2) and dialyzed against 50 mM Tris-HCl (pH 7.2) at 0° for 3 hr (2nd ammonium sulfate fraction). For comparison of the properties of the two forms of the enzyme, the presence of dTTP in these preparations should be avoided. dTTP added to the enzyme preparation during the preceding purification steps can be eluted by 0.1 M NaCl, which corresponds to the trough between Peaks A and B (arrow in Fig. 1).
Ammonium sulfate fractionation and dialysis after chromatography assure the elimination of dTTP from the enzyme preparations. Most of the experiments were performed with this preparation. A summary of the purification procedure is given in Table I. Normal adult rat liver enzyme was partially purified by the same method. The addition of dTTP during the various steps in the course of purification significantly augumented the recovery of the enzyme activity in various steps. When the purification was done without dTTP the overall recovery, as well as the recovery in the DEAE-cellulose step was markedly decreased. In addition no reliable pattern could be obtained in the DEAE-cellulose chromatography. Requirements for the Thymidine Kinase Reaction—Phosphorylation of thymidine with the two forms of the enzyme occurred only in the presence of ATP and was dependent on Mg2+. 5 mM AMP could not substitute for ATP, indicating that AMP-thymidine phosphotransferase {23) was not present in this enzyme preparation. Peaks A and B could uti-
/ . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
80 120 160 200 ELUTION VOLUME ( m l )
TWO MOLECULAR FORMS OF THYMIDINE KINASE TABLE I. Purification of thymidine kinase of regenerating rat liver. Twenty g of regenerating rat liver was used as starting material. Activity and protein were measured as described in " EXPERIMENTAL. PROCEDURE." One unit of thymidine kinase is defined as 1 nmole of TMP formed in 30 min at 37° and corresponds to 30 mU (international unit).
Homogenates Crude extract 1st Ammonium sulfate DEAE-eluate Peak A Peak B 2nd Ammonium sulfate Peak A Peak B
Total volume (ml)
Total activity (units)
Total protein (mg)
55
8,800 8,500 6,000
4,070 1,120
2.1
525
28.5
1,100
17
220
13
64.1 16.9
35 5 40 42
3 3
1,050 210
lize ATP and dATP as primary phosphate donors. CTP, GTP, dGTP, and dTTP were very poor donors for both enzymes. The reaction product formed by Peaks A and B was identified as dTMP by its behavior on a PEI-thin layer cellulose plate, as described in "EXPERIMENTAL PROCEDURE." Less than 2% of the label was found on spots corresponding to dTDP and dTTP. When ATPase activity was estimated in the two enzyme preparations, slight contamination was found in Peak B. The contamination was of the order of 5.9 nmoles of ATP degraded per mg of protein during 30 min incubation. To avoid errors in the kinetic plots due to the breakdown of ATP, 0.8 mM phosphoenolpyruvate and 1 Mg of pyruvate kinase [EC 2.7.1.40] were added to the reaction mixture in some kinetic studies. pH Optima—It was found that Peaks A and B both exhibit broad optima at pH 7.2 with a decline in activity in the range of pH 7.3 to 8.8. No significant difference was observed in the patterns of optimal pH of these two enzyme preparations. Metal Requirments—Both enzyme fractions required Mg2+ almost exclusively for maximum activity, as reported in other systems (3, 5). Mn2+ and Co2+ were slightly effective at comparable concentrations in Peaks A and B. When the reaction was performed in the presence of 5 mM MgCU, the order of inhibitory Vol. 78, No. 6, 1975
5 3
Specific activity (units/mg protein)
7.6
210.0 70.0
Yield (%) 100
96.5 68.0
14.3
activity of various divalent cations was Fe 2+ = Zn 2+ >Ni 2+ >Co 2+ >Mn 2+ >Ca 2+ and the extents of inhibition of Peaks A and B by these cations were quite similar. Effect of Temperature on Enzyme Stability—When aliquots of Peaks A and B were incubated at 37° for various times, the loss of enzyme activity followed quite different courses in Peaks A and B (Fig. 2). Peak B was clearly more stable than Peak A. The. mixing of Peaks A and B resulted in a simple additive effect, thus eliminating the possibility that stabilizing or labilizing factors were present in the preparations. Sucrose Density Gradient Studies — The molecular weights of Peaks A and B were estimated and compared accoding to the procedure of Martin and Ames (26). Rat muscle FDP-aldolase (1.5 xlO 5 mol. wt) and pig heart malate dehydrogenase (7.5x10* mol. wt) were used as internal markers. This method gave lxlO 5 mol. wt for both Peak A and B. The addition of 20 fiM dTTP to the buffered sucrose density gradient did not affect the molecular weight significantly. Kinetics for Thymidine—The kinetics for thymidine of Peaks A and B show that these two forms differ in apparent Km values at a level higher than 10 fiM thymidine; 38±10and 120±26 fiM for Peaks A and B, respectively. As shown in Fig. 3, the double
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
Step
H. NAWATA and T. KAMIYA
1220
Q3 5
10 TIME(min)
Fig. 2. Heat inactivation of Peaks A and B. Peak A (45 fig of protein) and Peak B (55 fig of protein) enzymes extracted from regenerating liver, and a mixture of Peak A and Peak B (100 fig of protein) were incubated in 50 mM Tris-HCl (pH 7.3) at 37° for the indicated times. After incubation, thymidine kinase activity was assayed as described in " EXPERIMENTAL PROCEDURE." O, Peak A; • , Peak B; A, Peak A+Peak B.
Fig. 3. Double reciprocal plot of thymidine kinase activity with respect to thymidine concentration in the presence of dTTP. Thymidine kinase assay was carried out as described in "EXPERIMENTAL PROCEDURE" except that various concentrations of [uC]thymidine were used. The reaction was started by adding 0.2 ml of Peak A (Fig. 3-a) or Peak B (Fig. 3-b) to the reaction mixture as described in " EXPERIMENTAL PROCEDURE." dTTP concentrations were as follows; A, none; • , 20/
Hajime NAWATA2 and Tomoya KAMIYA3 Division of Chemistry, Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812 Received for publication, March 7, 1975
Two forms (Peak A and Peak B) of thymidine kinase [EC 2.7.1. 75] from regenerating rat liver cytosol were resolved and partially purified by DEAE-cellulose chromatography. Both fractions were identical with respect to their substrate requirement, pH optima, metal requirements, and molecular weight, as judged by their sedimentation in sucrose density gradient centrifugation. Peak B differed from Peak A in heat sensitivity, inhibition by dCTP and Km for thymidine and ATP. Peak B enzyme was the only enzyme found in normal adult liver and Peak A enzyme was the form increasing predominantly in regenerating liver.
Thymidine kinase [EC 2.7.1.75], a salvage enzyme in the pathway of DNA synthesis, is of particular interest because its activity in a variety of cells has been correlated with the occurrence of cell division. There is an increase of thymidine kinase activity in regenerating mammalian liver (1, 2), mammalian tumor (5, 6), embryonic liver (7, 8), regenerating kidney after uninephrectomy (10), tumor virus-infected mammalian cells (11, 12) and adipose tissue (13) and adrenal gland (14, 15) in response to hormonal influences. Several distinct molecular forms of thymidine ki1
This study was supported in part by a research grant for cancer research from the Ministry of Education, Japan. 2 Present address: Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan. 3 Present address: Department of Biochemistry, Nara Medical University, Kashihara, Nara, Japan. Abbreviation: PEI, polyethyleneimine.
Vol. 78, No. 6, 1975
nase probably exist in mammalian systems. Thymidine kinase from both normal and rapidly metabolizing cells, such as regenerating rat liver, is subject to feedback inhibition by dTTP ( 7 ) . However comparison of the properties of thymidine kinase in various tissues, such as inhibition by nucleotides ( 7 ) and patterns of electrophoresis (8, 9, 16), provides evidence that some forms of thymidine kinase differ from the adult form. The present work showed that DEAEcellulose chromatography of partially purified thymidine kinase from regenerating rat liver results in the isolation of two forms of this enzyme. Each form has been characterized with respect to certain biochemical and catalytic properties. In addition, our data indicate that one of the enzyme forms appearing in regenerating liver is only synthesized after the start of liver regeneration and is the major component of thymidine kinase in regenerating liver, while the other form is the same as the
1215
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
Two Molecular Forms of Thymidine Kinase in the Cytosol of Regenerating Rat Liver1
1216
H. NAWATA and T. KAMIYA.
EXPERIMENTAL PROCEDURE Animals—Donryu male albino rats weighing 160 to 200 g were obtained from the Inbred Animal Supply Center of Kyushu University and were maintained on Oriental solid chow and water ad libitum. Reagents — [uC]thymidine (45 mCi per mmole) and [UC]ATP (550 mCi per mmole) were products of C.E.N. Dep. des Radioisotopes, Belgium. Crystalline ATP, thymidine, dTTP, and other ribo- and deoxyribonucleotides were obtained from Boehringer Mannheim, Germany. DEAE-cellulose was a product of Brown Co. DEAE-cellulose paper was from Toyo Roshi, Tokyo, Japan. Preformed PEIcellulose plates were from Merck Sharp and Dohme. Ammonium sulfate and ethylene glycol were biochemical analysis grade from Nakarai, Osaka, Japan. Preparation of Enzyme Extract—Partial hepatectomy was done by the method of Higgins and Anderson, removing approximately two-thirds of the liver under ether anesthesia (17). Partially hepatectomized rats were sacrificed by neck dislocation 40 hr after the operation, when the induction of thymidine kinase reached the maximum level (inset, Fig. 5). Regenerating livers were collected, chilled quickly, minced with scissors, added to three volumes of 50 mM Tris-HCl (pH 7.2), 0.25 M sucrose, 2.2 mM CaCl2, and 20 fiM dTTP, then homogenized in a Potter-Elvehjem glass homogenizer with a teflon pestle by means of 10 strokes in crushed ice (homogenates). Subcellular Fractionation of Liver Cells— The liver was minced and suspended in three volumes of 0.25 M sucrose, 50 mM Tris-HCl buffer (pH 7.3), and 3 mM CaCl2, then homogenized with a teflon pestle homogenizer and filtered through four layers of gauze. The nuclear fraction was obtained by centrifugation at 700 X g for -15 min, then the precipitates were homogenized, layered on 2.2 M sucrose and 50 mM Tris-HCl buffer (pH 7.3) and cen-
trifuged at 40,000 xg for 60 min, sedimenting^ the nuclei. The 700xg supernatant was centrifuged at 8,000xg for 15 min, sedimenting^ the mitochondria. The 8,000Xg supernatant was the centrifuged at 105,000xg for 30 min, sedimenting microsomes. The mitochondriaL fraction was resuspended in the homogenizing: buffer. A part of all the fractions was treated with 1% Triton X-100. Assay of Thymidine Kinase—Thymidine kinase assays were perfomed by the DEAEcellulose paper disk method as modified by Bresnick (18). The reaction mixture included 0.2 ml of the enzyme, 2.5 /imoles of ATP, 2.5 /^mols of MgCl2, and 40 /imoles of Tris-HCl buffer (pH 7.3) at 37° in a total volume of 0.50 ml. The reaction was started by adding 25 ^1 of 200 /*M [ u C]thymidine. The incubation was conducted at 37° for 30 min. The reaction was stopped by immersing the tubes in a boilingwater bath for 2 min. The precipitates were removed by mild centrifugation and 0.2 ml aliquots from the supernatants were applied to DEAE-cellulose paper squares (3x3 cm) and dried at room temperature. The dried squares were immersed for 10 min in a tray containing 1 mM ammonium formate, then the papers were washed in distilled water for further 10 min. .. This procedure was repeated twice. Finally the papers were immersed in 95%ethanol, dried at 80° and counted by directly placing the dried paper in a counting vial with 10 ml of toluene-based scintillation fluid in a Beckman liquid scintillation counter. Whea the activity of enzyme preparations containing dTTP was assayed during the purification, the concentraton of dTTP in the reaction mixture was kept below 1 pM, where its inhibiting activity was negligible. Protein concentration was estimated by the method of Lowry et aL (19) with bovine serum albumin as a reference standard. Sucrose Density Centrifugation — Centrifugation in a sucrose density gradient was performed in linear gradients of 5% to 20%> (w/v) sucrose in 0.1 M Tris-HCl buffer (pH 7.3). Each tube for the SW39 rotor (Beckman Instruments) was filled with 4.5 ml of the gradient and 0.1 ml of the sample was layered on top. Centrifugation was carried out at / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
only enzyme found in normal adult liver and is the minor component in regenerating liver. The latter form resembles the thymidine kinase present in mitochondria.
TWO MOLECULAR FORMS OF THYMIDINE KINASE
RESULTS Initial Experiments — It was previously shown that thymidine kinase from various sources is quite unstable and the preparations require stabilizers such as thymidine (4, 5) and albumin (3). When the stabilizing effects of thymidine and various nucleotides were compared, dTTP was found to be best stabilizer. Some stabilizing effects were also observed with thymidine, ATP, dATP, CTP, dCTP, and ADP. Twenty % glycerol and 20% ethylene glycol were found to increase the activity by about 5 to 30% of the original activity upon incubation at 37°. Partial Purification of Thymidine Kinase Vol. 78, No. 6, 1975
—All purification steps were performed at 0 to4°. The homogenate from regenerating liver was centrifuged at 8,000 xg for 15 min. The supernatant fraction was further centrifuged at 105,000 Xfiffor 60 min (crude extract). Finely powdered solid ammonium sulfate was slowly added to the crude extract to 40% saturation. Next, the mixture was stirred for 101 min and centrifuged at 8,000xg for 15 min. The resulting pellets were dissolved in 3 ml of 200 mM Tris-HCl buffer (pH 7.2) containing 20 fiM dTTP. This solution was then applied to a Sephadex G-25 column (3x20 cm) previously equilibrated with 5 mM Tris-HCl (pH 7.2). The column was eluted with the same buffer; 4 ml fractions were collected and the fractions containing most of the enzyme activity were pooled. To this fraction, dTTP' and ethylene glycol were added to final concentrations of 20 [*M and 20% respectively (1st ammonium sulfate fraction). DEAE-cellulose (medium size, 0.9 meq per g) was processed as described elsewhere (25) except that adjustment of the pH of the absorbent was done by adding 50 mM Tris-HCl (pH 7.2). The DEAE-cellulose was then packed into a column (1X 20 cm) under gravity and equilibrated with 5 mM Tris-HCl (pH 7.2) containing 20% ethylene glycol. The enzyme solution (100' mg of protein) from the 1st ammonium sulfate fraction was applied to the column at a flow rate of 17 ml per hr. The column was washed with 50 ml of the same buffer, and a linear gradient (300 ml) of NaCl (0 to 0.25 M) in the same buffer was applied to the column. After gradient elution had been completed, the column was eluted with 50 ml of 0.4 M NaCl in the same buffer. Thymidine kinase activity was eluted at NaCl concentrations of 0 (breakthrough peak), 0.05M (Peak A), and 0.15 M (Peak B). Figure 1 shows a typical DEAEcellulose chromatogram of thymidine kjnase. It can be seen that the enzyme consists predominantly of two Peaks A and B. A further increase of the NaCl concentration in the elution buffer up to 0.4 M did not yield any appreciable activity.'
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
39,000 rpm for 18 hr using a Spinco L-2 ultracentrifuge at 4°. After centrifugation, the bottoms of the tubes were pierced and 20 fractions were collected. As enzyme markers, rat muscle aldolase [EC 4.1.2.13] and malate dehydrogenase [EC 1.1.1.37] from pig heart were used. Rat muscle aldolase was purified and crystallized after Ikehara et al. (20), and assayed according to Blostein and Rutter (21). Malate dehydrogenase was obtained from Sigma, and assayed according to Mehler et al. (22). Assay of ATP Phosphohydrolase and Thin Layer Chromatography of Nucleotides— The enzyme preparations were assayed for ATP phosphohydrolase (23) by incubating 50 l*\ samples at 37° for 30 min in the presence of 1.5 mM MgCl2, 50 mM Tris-HCl (pH 7.2) and [UC]ATP (5 nCi) in a total volume of 70 fi\. The reaction was stopped by adding 1 fil of 88% formic acid, and the resulting precipitates were spun down. The supernatant was put on PEI-impregnated cellulose plates (Merck). Resolution of ATP, ADP, and AMP was done by successively eluting the plates with water and 0.2 M ammonium formate in 4.0 M formic acid (24). Separated spots were detected by UV irradiation, scraped into vials and their radioactivities were determined with a Beckman liquid scintillation counter. The separation of thymidine, dTMP, dTDP, and dTTP was also carried out by the same thin layer chromatography procedure.
1217
In order to confirm the identity of these enzyme peaks, rechromatography was done in a stepwise fashion to avoid inactivation of the
1218
H. NAWATA and T. KAMIYA
240
280
320
Fig. 1. The elution profile of DEAE-cellulose chromatography of thymidine kinase. A 10-ml preparation (100 mg of protein) from the ammonium sulfate fraction containing 20 fiM dTTP and 20% ethylene glycol was applied to a column of DEAE-cellulose (1x20 cm) prepared as described in the text. Elution was carried as described in the text. Fractions of 4 ml were collected at a flow rate of 17 ml per hour. Each fraction was assayed for thymidine kinase activity (O) and the absorbance at 280 ( ) was determined. The arrow in the figure indicates the position of elution of free dTTP. This was confirmed by cochromatography with [3H]dTTP.
enzyme due to prolonged exposure on the column. Rechromatography of Peaks A and B did not change their elution positions. The addition of 20 ^M dTTP to the enzyme fraction before application to the second column gave the same results. The activity appearing in the breakthrough peak on DEAE-cellulose chromatography was not analyzed in this experiment. This peak fluctuated greatly in different experiments. The fractions from Peaks A and B (DEAE eluate) were concentrated by adding ammonium sulfate to 50% saturation and centrifuging at 50,000 Xg for 30 min. The resulting pellet was dissolved in 200 mM Tris-HCl (pH 7.2) and dialyzed against 50 mM Tris-HCl (pH 7.2) at 0° for 3 hr (2nd ammonium sulfate fraction). For comparison of the properties of the two forms of the enzyme, the presence of dTTP in these preparations should be avoided. dTTP added to the enzyme preparation during the preceding purification steps can be eluted by 0.1 M NaCl, which corresponds to the trough between Peaks A and B (arrow in Fig. 1).
Ammonium sulfate fractionation and dialysis after chromatography assure the elimination of dTTP from the enzyme preparations. Most of the experiments were performed with this preparation. A summary of the purification procedure is given in Table I. Normal adult rat liver enzyme was partially purified by the same method. The addition of dTTP during the various steps in the course of purification significantly augumented the recovery of the enzyme activity in various steps. When the purification was done without dTTP the overall recovery, as well as the recovery in the DEAE-cellulose step was markedly decreased. In addition no reliable pattern could be obtained in the DEAE-cellulose chromatography. Requirements for the Thymidine Kinase Reaction—Phosphorylation of thymidine with the two forms of the enzyme occurred only in the presence of ATP and was dependent on Mg2+. 5 mM AMP could not substitute for ATP, indicating that AMP-thymidine phosphotransferase {23) was not present in this enzyme preparation. Peaks A and B could uti-
/ . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
80 120 160 200 ELUTION VOLUME ( m l )
TWO MOLECULAR FORMS OF THYMIDINE KINASE TABLE I. Purification of thymidine kinase of regenerating rat liver. Twenty g of regenerating rat liver was used as starting material. Activity and protein were measured as described in " EXPERIMENTAL. PROCEDURE." One unit of thymidine kinase is defined as 1 nmole of TMP formed in 30 min at 37° and corresponds to 30 mU (international unit).
Homogenates Crude extract 1st Ammonium sulfate DEAE-eluate Peak A Peak B 2nd Ammonium sulfate Peak A Peak B
Total volume (ml)
Total activity (units)
Total protein (mg)
55
8,800 8,500 6,000
4,070 1,120
2.1
525
28.5
1,100
17
220
13
64.1 16.9
35 5 40 42
3 3
1,050 210
lize ATP and dATP as primary phosphate donors. CTP, GTP, dGTP, and dTTP were very poor donors for both enzymes. The reaction product formed by Peaks A and B was identified as dTMP by its behavior on a PEI-thin layer cellulose plate, as described in "EXPERIMENTAL PROCEDURE." Less than 2% of the label was found on spots corresponding to dTDP and dTTP. When ATPase activity was estimated in the two enzyme preparations, slight contamination was found in Peak B. The contamination was of the order of 5.9 nmoles of ATP degraded per mg of protein during 30 min incubation. To avoid errors in the kinetic plots due to the breakdown of ATP, 0.8 mM phosphoenolpyruvate and 1 Mg of pyruvate kinase [EC 2.7.1.40] were added to the reaction mixture in some kinetic studies. pH Optima—It was found that Peaks A and B both exhibit broad optima at pH 7.2 with a decline in activity in the range of pH 7.3 to 8.8. No significant difference was observed in the patterns of optimal pH of these two enzyme preparations. Metal Requirments—Both enzyme fractions required Mg2+ almost exclusively for maximum activity, as reported in other systems (3, 5). Mn2+ and Co2+ were slightly effective at comparable concentrations in Peaks A and B. When the reaction was performed in the presence of 5 mM MgCU, the order of inhibitory Vol. 78, No. 6, 1975
5 3
Specific activity (units/mg protein)
7.6
210.0 70.0
Yield (%) 100
96.5 68.0
14.3
activity of various divalent cations was Fe 2+ = Zn 2+ >Ni 2+ >Co 2+ >Mn 2+ >Ca 2+ and the extents of inhibition of Peaks A and B by these cations were quite similar. Effect of Temperature on Enzyme Stability—When aliquots of Peaks A and B were incubated at 37° for various times, the loss of enzyme activity followed quite different courses in Peaks A and B (Fig. 2). Peak B was clearly more stable than Peak A. The. mixing of Peaks A and B resulted in a simple additive effect, thus eliminating the possibility that stabilizing or labilizing factors were present in the preparations. Sucrose Density Gradient Studies — The molecular weights of Peaks A and B were estimated and compared accoding to the procedure of Martin and Ames (26). Rat muscle FDP-aldolase (1.5 xlO 5 mol. wt) and pig heart malate dehydrogenase (7.5x10* mol. wt) were used as internal markers. This method gave lxlO 5 mol. wt for both Peak A and B. The addition of 20 fiM dTTP to the buffered sucrose density gradient did not affect the molecular weight significantly. Kinetics for Thymidine—The kinetics for thymidine of Peaks A and B show that these two forms differ in apparent Km values at a level higher than 10 fiM thymidine; 38±10and 120±26 fiM for Peaks A and B, respectively. As shown in Fig. 3, the double
Downloaded from https://academic.oup.com/jb/article-abstract/78/6/1215/829919 by Leiden University / LUMC user on 16 January 2019
Step
H. NAWATA and T. KAMIYA
1220
Q3 5
10 TIME(min)
Fig. 2. Heat inactivation of Peaks A and B. Peak A (45 fig of protein) and Peak B (55 fig of protein) enzymes extracted from regenerating liver, and a mixture of Peak A and Peak B (100 fig of protein) were incubated in 50 mM Tris-HCl (pH 7.3) at 37° for the indicated times. After incubation, thymidine kinase activity was assayed as described in " EXPERIMENTAL PROCEDURE." O, Peak A; • , Peak B; A, Peak A+Peak B.
Fig. 3. Double reciprocal plot of thymidine kinase activity with respect to thymidine concentration in the presence of dTTP. Thymidine kinase assay was carried out as described in "EXPERIMENTAL PROCEDURE" except that various concentrations of [uC]thymidine were used. The reaction was started by adding 0.2 ml of Peak A (Fig. 3-a) or Peak B (Fig. 3-b) to the reaction mixture as described in " EXPERIMENTAL PROCEDURE." dTTP concentrations were as follows; A, none; • , 20/
