Mammalian Deoxynucleoside Kinases

Deoxyadenosine kinase was partially purified 140-fold from calf thymus by fractionation with streptomycin, protamine, and ammonium sulfate and by column chromatography on Sephadex G-150 and DEAE-cellulose. The molecular weight was estimated to be about 63,000 by gel filtration chromatography. The enzyme appears to catalyze the phosphorylation of deoxyadenosine, deoxyguanosine, and cytidine to their corresponding nucleoside 5’-monophosphates in the presence of a divalent cation and a nucleoside 5’-triphosphate. All common riboand deoxfnucleoside S’-triphosphates, with the exception of deoxycytidine 5’-triphosphate, could act as phosphate donors.

with streptomycin, protamine, and ammonium sulfate and by column chromatography on Sephadex G-150 and DEAE-cellulose.
The molecular weight was estimated to be about 63,000 by gel filtration chromatography.
The enzyme appears to catalyze the phosphorylation of deoxyadenosine, deoxyguanosine, and cytidine to their corresponding nucleoside 5'-monophosphates in the presence of a divalent cation and a nucleoside 5'-triphosphate.
All common ribo-and deoxfnucleoside S'-triphosphates, with the exception of deoxycytidine 5'-triphosphate, could act as phosphate donors.
Deoxyadenosine kinase has been found to catalyze the transfer of a phosphate group from specific nucleoside 5'-triphosphate donors to the 5' position of dAdol (1). Interest in this enzyme was aroused by Klenow's observation (2) that the addition of 2 mM dAdo to Ehrlich ascites tumor cells in vitro almost completely inhibited DNA synthesis. This inhibition could be alleviated if dCyd and dGro were added simultaneously to the incubation mixture (3). Similar results were reported by Maley and Maley (4) who demonstrated that dAdo exerted a marked inhibitory effect on the incorporation of Cyd and Urd into chick embryo DNA cytosine and thymine.
When tumor cells were exposed to dAdo, dATP was shown to accumulate in these cells (5). Since dATP is known to act as a potent allost.eric inhibitor of ribonucleotide reductase in mammalian cells (6), it has been suggested that the inhibition of DNA synthesis caused by dAdo could be mediated through its end product, dATP.
It would therefore be of value to study the mechanism and control of the phosphorylation of dAdo so as to understand the role of dAdo kinase in deoxynucleotide metabolism. Previous work indicates that the activity of the enzyme, dAdo * This study was supported by Grant MA-2843 from the Medical Research Council of Canada and by the National Cancer Institute.
The previous paper in this series is Reference 16. A preliminary report of this work has been published (1 kinase, is regulated by the inhibition produced by the deoxynucleotides of adenine, guanine, and cytosine as well as by its relatively high apparent K, value for dAdo (1). This paper describes more extensively the purification and some of the properties, and the accompanying paper (7), the kinetics of dAdo kinase.

EXPERIMENTAL PROCEDURE
MaterialsThe 14C-or tritium-labeled purine and pyrimidine nucleosides were obtained from Schwarz BioResearch.
The labeled nucleosides were purified by descending chromatography for 24 hours on Whatman No. 3M paper in 86% l-butanol-concentrated ammonium hydroxide (94.5 : 5.5, v/v). The chromatograms were air-dried at room temperature, and the radioactive spots corresponding to the material to be purified were eluted with 50% ethanol and stored at -20".
Before use the ethanol was evaporated and the concentration of the nucleoside was adjusted.
Assay of Enzyme-dAdo kinase activity was assayed by measuring the incorporation of '4C-dildo or 3H-dAdo into deoxyadenosine 5'-monophosphate by the binding of this latter compound to DEAE-cellulose discs (8). The incubation mixture (0.1 ml) contained 10.0 pmoles of Tris-HCI (pH 8.0), 1.0 pmole of MgC&, 0.1 pmole of ATP, 0.1 pmole of dithiothreitol (or 1.0 pmole of 2-mercaptoethanol), 100 nmoles of 14C-dAdo (9 x lo4 cpm) or %dAdo (1.9 x lo5 cpm), and 0.1 to 2.0 units of enzyme. Following the addition of the enzyme to the previously heated (37") reaction mixture, the mixture was incubated for 5 min at 37" and then immediately diluted to 5 ml with water. The solution was permitted to flow by gravity through 2.5-cm diameter DEAE-cellulose discs that had been previously washed with 1 ml of 0.01 N HCl and 20 ml of water.
The discs were then washed with 20 ml of water, dried, and placed in scintillation vials containing 10 ml of scintillation solution (4 g of 2,5diphenyloxazole and 100 r.lg of p-bis[2-(5-phenyloxazolyl)]benzene in 1 liter of toluene).
The vials were counted in a Packard Tri-Carb liquid scintillation counter with a counting efficiency for 14C of 30% and for tritium of 2y0 under these conditions. One unit of enzyme was defined as the amount catalyzing the conversion of 1 nmole of dhdo to dAMP in 1 min under the described assay conditions. Protein was determined by the method of Lowry et al (9) with crystalline albumin as a standard.

Ammonium
Sulfate Treatment-To 102 ml of Fraction III, 21.3 g of ammonium sulfate (0 to 35% saturation) were added with st.irring over a 15-min period.
After 20 min of additional stirring, the suspension was centrifuged for 30 min at 23,000 x g. The precipitate was discarded whereas the supernatant was retained.
To 105 ml of the supernatant from the previous step 17.2 g of ammonium sulfate (35 to 60% saturation) were added with stirring over a lo-min period.
After an additional stirring of 20 min, the precipitate was obtained by centrifugation for 30 min at 23,000 x g and dissolved in 15 ml of 50 mM Tris-HCl, pH 8.0, and 20 InM 2-mercaptoethanol (Fraction IV). Pur$ication of Enzyme Extract-Frozen (-60") calf thymus (300 g) was homogenized Chromatography on Xephadex G-15&A column of Sephadex in a Phillips blender in 600 ml of 20 mM cold Tris-HCl, pH 8.0, G-150 (10 to 40 p, 19.4 cm2 x 50 cm) was prepared and equilifor 3 min and centrifuged at 23,000 x g for 30 min. The super-brated with 1200 ml of 50 mM Tris-HCl (pH 8.0) and 20 mM natant (565 ml) was passed through three layers of gauze. To 2-mercaptoethanol (Buffer A). Through the column were passed the supernatant 0.81 ml of 14 M 2-mercaptoethanol was then by upward flow 14.5 ml of Fraction IV (445 mg of protein). added, yielding Fraction I (Table I). Throughout the purifica-The protein was eluted from the column with Buffer A at a flow tion procedure the temperature was maintained at about 4". rate of 12.5 ml per hour. Streptomycin Treatment-To 565 ml of Fraction I, 28.2 ml of 10% streptomycin sulfate, pH 7.0, were added slowly with stirring.
After mixing for 30 min the suspension was centrifuged as above and the supernatant was collected.
Since the pH was found to have fallen to 6.5 it was raised to 8.0 with NH,OH (Fraction II).
Protamine Sulfate Treatment-To 565 ml of Fraction II, 114 ml of 2yo protamine sulfate, pH 7.0, were added with stirring over a lo-min period.
After the mixture was stirred for 30 min, it was centrifuged at 23,000 X g for 30 min and the precipitate was retained.
To the protamine sulfate precipitate 100 ml of 2% ammonium sulfate in 50 mM Tris-HCl, pH 8.0, and 20 InM 2-mercaptoethanol were added. An ultrasonic probe was used with continuous stirring for 17 min to disperse the precipitate. After the suspension was centrifuged at 23,000 x g for 30 min, the supernatant was collected (Fraction III). 90-min intervals, and protein concentration and enzyme activity were determined (Fig. 1). The two fractions (Nos. 15 and 16) of maximal specific activity and containing 45% of the activity applied to the column were pooled and concentrated 4.1-fold by dialyzing them (in a-inch diameter tubing) against 500 ml of 70% glycerol in Buffer A for 4 hours. Then in preparation for the next column they were dialyzed against 500 ml of 20% glycerol in 20 mM Tris-HCl, pH 8.0, and 20 rnM 2-mercaptoethanol (Buffer B) to yield Fraction V.
Chromatography on DEAE-cellulose-A column of DEAE- cellulose (0.63 cm2 x 16 cm) was prepared and equilibrated with Buffer B. About 9 ml of Fraction V (44 mg of protein) were placed on the column and eluted with a linear KC1 gradient (Fig. 2). The mixing chamber contained 43 ml of Buffer B and the reservoir contained 43 ml of 0.3 M KC1 in Buffer B. Fractions of 3 ml were collected every 8 min and assayed for protein and enzyme activity. The fractions containing the highest specific activities, 16,17,18,19, and 20, were combined and concentrated by dialysis against 250 ml of three different concentrations of glycerol in Buffer A: an 1%hour dialysis against 70% glycerol, followed by a 5% glycerol dialysis for 4 hours, and a final dialysis against 60% glycerol for another 4 hours.
A 140-fold purification was achieved with 13% recovery. The enzyme can be stored in 60% glycerol at -20" for at least 3 months with slight loss of activity.
A summary of the purification is given in Table I.

Properties of Partially Purijied Enzyme
Properties of Reaction-The presence of a divalent cation and ATP in the reaction mixture was essential for activity (Table II). Of the three cations tried, Mg ++ (10 mM) produced the highest activity, with Mn++ and Ca++ producing a lower rate of reaction. ADP was not able to replace ATP as the phosphate donor. The sensitivity of the enzyme to mercuric ions in the absence of the sulfhydryl compound, dithiothreitol, was shown by the presence of 0.01 and 0.1 mM heavy metal, causing 25% and almost complete inhibition, respectively.
The rate of formation of dAMP from deoxyadenosine with ATP as the phosphate donor was linear with respect to time up to 5 min and enzyme concentration up to 7 ~1 or 33 units per ml (Fig. 3) Effect of pH on Rate of Reaction-Under conditions of the routine assay, the partially purified enzyme (Fraction VIII) had a broad pH optimum range with the Tris-maleate-Bicine buffer (Fig. 4). There was hardly any difference (10%) in activity within the pH range of 6.5 to 8.5. Below pH 6.5 a sharp drop was evident.

Molecular Weight
Determination-The molecular weight of  dAdo kinase was determined by gel filtration according to the method of Andrews (10). The molecular weights of the proteins used to calibrate the column were: myoglobin, 17,800; ovalbumin, 45,000; bovine serum albumin, 67,000; and -y-globulin, 160,000 (10). Fig. 5 shows that a linear relationship between the logarithm of the molecular weight of a protein and the ratio of its elution volume to the void volume from a Sephadex G-150 (10 to 40 p) column exists up to a molecular weight of at least 70,000. By applying this relationship to dAdo kinase, a molecular weight of about 63,000 can be obtained. Stoichiometry-The stoichiometry of the reaction was determined as shown in Table  III  (and ULMP) and dAMP.
In the absence of dAdo from the reaction medium there was a slight increase in the amount of UMP produced and a decrease in UTP concentration. During the incubation, with both substrates and enzyme present, a somewhat larger amount of UMP than UDP was formed.
These results suggest the presence of trace amounts of UTP and UDP phosphatases in Fraction VIII.
Incubating ATP (the usual phosphate donor) alone with Fraction VIII resulted in no ADP or AMP production, indicating the absence of an ATPase.2 In another experiment the absence of nucleoside monophosphokinase or nucleoside diphosphokinase was demonstrated as no formation of %J-dADP or 14C-dATP was detected upon routine incubation with IJC-dAdo, ATP, and enzyme Fraction VIII.2 2 V. Krygier and R. L. Momparler, unpublished data. were tested for their ability to phosphorylate dAdo and dGuo (Table IV).
ATP and GTP were the most effective phosphate donors, followed by UTP and dTTP which were 90 and 75%, respectively, as active.
When either dAdo or dGuo was used as the substrate the order of activity of the donors was the same. Deoxyguanosine, cytidine, and deoxyadenosine 5'4riphosphates were much less capable of serving as phosphate donors. Deoxycytidine 5'-triphosphate was inactive as a phosphate donor.
Spec$city for Phosphate Acceptors-Fraction VIII was able to catalyze the phosphorylation, to varying extents, of dAdo, dGuo, dCyd, and Cyd (Table V). Neither Ado, Guo, Urd, nor dThd was active as substrates, nor were their corresponding kinases present in Fraction VIII.
Stability to Dialysis-Fraction IV was subjected to dialysis in buffer with and without 2-mercaptoethanol (Table VI). The dialyzed fract,ion was tested for kinase activity by using different nucleosides as phosphate acceptors.
When dCyd was tested as the substrate the amount of product formed was not affected by dialysis under these conditions. However, when either dAdo, dGuo, or Cyd was used as the substrate, there was about a 50% decrease in product formation with the fraction dialyzed in the absence of 2-mercaptoethanol.

Mammalian
Deoxynucleoside Kineses. II Vol. 246,No. 9 E$ect of Nucleosides and Nucleotides on Phosphrylation of Deoxycytidine and Cytidine-Ia Table VII it can be seen that there was no effect on the amount of WdCMP formed from 3H-dCyd upon the addition of either cold dAdo or dGuo in 200-fold excess with respect to dCyd. In contrast to this last experiment the addition of either cold dAdo, dGuo, or dCyd but not Urd decreased the conversion of WCyd to aH-CMP (Table VIII).
The monophosphorylated derivatives of these deoxynucleosides were also tested for their effect on aH-CMP formation.
The addition of dAMP resulted in a slight inhibition of the phosphorylation of 3H-Cyd, dGMP had no effect, and dCMP was completely inhibitory.

DISCUSSION
It has been shown that the addition of dAdo to mammalian cells results in an inhibition of DNA synthesis (2), possibly due to the accumulation of dATP (5), a potent allosteric inhibitor of ribonucleotide reductase (6). These findings stimulated an interest in the enzyme responsible for the phosphorylation of dAdo.
dAdo kinase was purified about 140-fold from calf thymus. The requirements of the enzyme for a divalent cation and a phosphate donor are similar to the other deoxynucleoside kinases (11,12). Throughout the purification procedure the enzyme was fairly unstable, necessitating the presence of a reducing agent, 2-mercapt,oethanol, and glycerol. The molecular weight of the enzyme, estimated to be about 63,000 by gel filtration, is similar to that of dCyd kinase (ll), whereas the molecular weight of mammalian dThd kinase from tumor cells is about 700,000 (12). Certain enzymes involved in deoxynucleotide metabolism such as dThd kinase (13, 14) and ribonucleotide reductase (15) have been shown to form aggregates under various conditions.
There was no evidence of aggregation of dAdo kinase in the presence of the inhibitor, dCTP.2 As with the other deoxynucleoside kinases there is no absolute specificity for phosphate donors (11,16). ATP and GTP serve almost equally well as phosphate donors, whereas UTP and dTTP are somewhat less effective. CTP, dATP, and dGTP are nearly inactive and dCTP is inactive as a phosphate donor.
The enzyme fraction appeared to catalyze the phosphorylation of dAdo, dGuo, Cyd, and dCyd. There are three possible explanations for this observation.
These nucleosides could be phosphorylated by (a) the same enzyme, (5) different enzymes, or (c) the same enzyme but at different catalytic sites. This latter possibility cannot be excluded at the present time.
In a previous report data were presented that suggested that the phosphorylation of dCyd is catalyzed by an enzyme distinct from dAdo kmase (16). This was confirmed here by the inability of either dAdo or dGuo, added in 200-fold excess, to interfere with the phosphorylation of dCyd (Table VII). Further proof was shown by the distinct difference in the ability of the enzyme fraction to catalyze the phosphorylation of dAdo and dCyd after dialysis. dCyd kinase remained stable during dialysis in the absence of 2-mercaptoethanol whereas dAdo kinase lost about half of its activity under the same conditions (Table VI).
Cellular studies of Bernard and Brent? offer additional evidence to support that dCyd kinase and dAdo kinase are distinct en-3 0. Bernard and T. P. Brent, unpublished data. zymes in mammalian cells. Kinase activity toward four different deoxynucleosides was measured during the cell cycle of 3T3 cells derived from mouse fibroblasts.
Both dCyd and dThd kinases increased about 4-and IO-fold, respectively, during S phase, i.e. during the period of DNA synthesis, whereas the activity toward dAdo and dGuo remained constant at a relatively high level throughout the cell cycle. The same enzyme, dAdo kinase, however, appears to catalyze the phosphorylation of dGuo as well as Cyd. In the case of dGuo this hypothesis is substantiated by the following observations: (a) the addition of cold dGuo resulted in a decrease in the rate of conversion of W-dAdo to W-dAMP (l), (b) a similar decrease in kinase activity toward dAdo and dGuo was observed in dialysis study (Table VI), (c) the same pattern of activity with inhibitors2 and phosphate donors was shown for dAdo and dGuo (Table IV), and (d) kinetic data presented in the following paper (7).
The finding that dAdo kinase could possibly catalyze the phosphorylation of Cyd was unexpected since Skiild (17) and Orengo (18) have reported that Urd kinase can catalyze the phosphorylation of Urd and Cyd but not dAdo or dGuo. That Cyd could be a substrate for dAdo kinase was supported by several experiments.
It was observed that the addition of either cold dAdo or dGuo decreased the conversion of i4C-Cyd to 14C-CMP (Table VIII).
It could be argued that the phosphorylated products of these deoxynucleosides, dAMP and dGMP, inhibit the formation of CMP.
However, it was shown that the addition of either dAMP or dGMP at sufficiently high concentration (0.3 mM) had no effect on the phosphorylation of Cyd (Table VIII).
The complete inhibition of CMP formation in the presence of dCyd is most likely caused by dCMP as dCyd kinase has been shown to be present in enzyme Fraction VIII. If dAdo kinase catalyzes the phosphorylation of Cyd, as it appears to do, it is understandable that dCMP would interfere with the reaction as it is a potent inhibitor of this enzyme (1). Since the addition of an excess of Urd had no effect on CMP formation it is most likely that the enzyme which catalyzes the phosphorylation of Cyd is not the same as the one found by Skold (17) and Orengo (18). Furthermore, the same pattern of inhibition with deoxynucleoside triphosphates as observed with dAdo (1) and dGuo emerged when Cyd was the substrate for the enzyme.2 Kinetic evidence is presented in the following paper which also suggests that the phosphorylation of Cyd is catalyzed by dAdo kinase (7).