Purification and Some Properties of a Deoxyribonucleoside Kinase from L1210 Cells1

Home , Coomassie Brilliant Blue, Deoxyguanosine

[CANCER RESEARCH 42, 3033-3039, August1982] 0008-5472/82/0042-0000302.00 Purification and Some Properties of a Deoxyribonucleoside Kinase from L1210 Cells1

Chi-Hsiung Chang,2 R. Wallace Brockman, and L. Lee Bennett, Jr.

Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255

ABSTRACT phosphorylation of each nucleoside analog is essential for a full understanding of its mode of action. Such identification is A nucleoside kinase has been purified about 1680-fold from difficult because of the uncertainty about the number and L1210 leukemia cells with deoxyadenosine as the phosphate substrate spÃ©cificitÃ©sof kinases acting on deoxyribonucleo acceptor and with adenosine triphosphate as phosphate donor. sides. The literature on the deoxyribonucleoside kinases has The molecular weight of the enzyme, determined by Sephadex been reviewed by Anderson (1) and more recently by Hender G-200 column chromatography, was found to be about 100,- son et al. (12). Several studies of partially purified enzymes 000. Purified enzyme, after the final step of purification and obtained from calf thymus (8, 17, 19) showed that deoxyaden also after disc gel electrophoresis (Rf 0.66), catalyzed the osine, deoxyguanosine, deoxycytidine, and cytidine were phosphorylation of 2'-deoxyadenosine, 2'-deoxyguanosine, 2'- phosphorylated by the same enzyme; however, results of other deoxycytidine, 9-/S-D-arabinofuranosyladenine, 9-/S-D-arabino- studies with partially purified enzymes from calf thymus (9, 24) furanosyl-2-fluoroadenine, 1-/ÃŸ-o-arabinofuranosylcytosine, indicated that cytidine was not an active phosphate acceptor and cytidine. for this enzyme. Gower ef al. (10) reported the isolation of both The enzyme exhibited a broad pH optimum ranging from 7.8 a mitochondrial and a cytoplasmic enzyme from calf thymus to 8.7. Magnesium ion and nucleoside triphosphates were that catalyzed the phosphorylation of deoxyguanosine. The essential for activity. The addition of Ni2+, Zn2+, or Co2+ to the cytoplasmic enzyme apparently is the nonspecific enzyme cat reaction mixture containing 2 HIM MgCI2 produced 90 to 100% alyzing the conversion of deoxyguanosine, deoxyadenosine, inhibition of the enzyme activity. The kinase had broad speci deoxycytidine, and cytidine to their corresponding nucleotides. ficity for phosphate donors; adenosine triphosphate, guanosine The mitochondrial enzyme catalyzes the phosphorylation of triphosphate, uridine triphosphate, inosine triphosphate, deox- deoxyguanosine, guanosine, and deoxyinosine but not deox yuridine triphosphate, and deoxythymidine triphosphate ac ycytidine and cytidine. Kinases specific for deoxyguanosine tively donated phosphate to deoxyadenosine as did several have been purified from pig skin and neonatal mouse skin (2, nucleoside triphosphate analogs. The Michaelis constant for 11). Meyers and Kreis (22) reported that a purified enzyme deoxyadenosine varied with the phosphate donor; the apparent from mouse murine neoplasm P815 catalyzed the phosphoryl Kmvalues with adenosine triphosphate and uridine triphosphate ation of deoxyguanosine and deoxyadenosine; this enzyme as donor were 1.25 and 0.13 mM, respectively. Apparent Km was distinct from deoxycytidine kinase. Schrecker (26) sug values (0.25 mw) with deoxyuridine triphosphate and deoxy gested that the phosphorylation of deoxycytidine and deoxy thymidine triphosphate also were lower than those obtained guanosine was mediated by the same enzyme in cell-free with adenosine triphosphate as donor, but guanosine triphos extracts of L1210 cells. Adenosine kinase must also be con phate and inosine triphosphate gave the same constant as sidered as being potentially responsible for the phosphorylation adenosine triphosphate. The apparent Vmaxfor phosphorylation of deoxyadenosine analogs, for it is the most abundant purine of deoxyadenosine was severalfold higher with adenosine tri nucleoside kinase in mammalian cells and homogenous prep phosphate than with uridine triphosphate. The apparent Mi arations from various mammalian sources have been found to chaelis constants for deoxyguanosine, deoxycytidine, and cy catalyze the phosphorylation of deoxyadenosine and ara-A tidine with adenosine triphosphate as phosphate donor were (23, 29, 30). There is evidence that certain deoxyribonucleo 1.37, 8.0, and 33 mw, respectively. Since deoxyadenosine sides or analogs may be substrates for more than one kinase. and deoxyguanosine were the best substrates, this enzyme Thus, Ullman et al. (27) found deoxyadenosine to be a sub may be regarded as a purine deoxyribonucleoside kinase. strate for both adenosine kinase and deoxycytidine kinase from cultured human lymphoblasts; however, in intact cells, adeno INTRODUCTION sine kinase apparently was primarily responsible for the phos phorylation of deoxyadenosine. The phosphorylation of 2-F- Analogs of deoxyribonucleosides, such as ara-C,3 ara-A, and ara-A is mediated primarily by deoxycytidine kinase (4, 7) and 2-F-ara-A, require the action of a nucleoside kinase as the first that of ara-A by both adenosine kinase and deoxycytidine step in their conversion to triphosphates, which apparently are kinase (28). the active forms. Identification of the kinase responsible for the It is evident that there is a need for extensive purification and characterization of these enzymes as well as for more infor ' This investigation was supported by USPHS Grant R01 -CA-23155 awarded mation on structure-activity relationships among substrates. by the National Cancer Institute and Grant SO7-RR-05676, Division of Research Resources, Department of Health and Human Services. Since leukemia L1210 is widely used for evaluation of antitumor 2 To whom requests for reprints should be addressed. agents and study of their metabolism and metabolic effects, we 3 The abbreviations used are: ara-C, 1-/8-D-arabinofuranosylcytosine; ara-A, have undertaken to isolate and characterize the nucleoside 9-/8-D-arabinofuranosyladenine; 2-F-ara-A, 9-/S-r>arabinofuranosyl-2-fluoroadenine; CM-cellulose, carboxymethyl cellulose. kinases present in L1210 cells. We have reported results of a Received December 3, 1981 ; accepted May 6, 1982. study with adenosine kinase from L1210 cells (6); we report

AUGUST 1982 3033

here the results of a study of a deoxyribonucleoside kinase described. Gels from other tubes were stained for protein by means of isolated with deoxyadenosine as the phosphate acceptor. 0.04% (v/w) Coomassie Blue G and 3.5% (w/v) perchloric acid for 16 hr and then destained in 7% acetic acid (14). Determination of Molecular Weight by Sephadex G-200 Column MATERIALS AND METHODS Chromatography. The molecular weight of the enzyme was determined by Sephadex G-200 column Chromatography. The column (1 x 110 Materials. Nucleoides, nucleosides, lactate dehydrogenase, he moglobin, ovalbumin, a-chymotrypsinogen A, myoglobin, DEAE-cellu- cm) was equilibrated and eluted with Buffer B (see below) containing 20% sucrose. Lactate dehydrogenase, hemoglobin, ovalbumin, a-chy lose, and CM-cellulose were purchased from Sigma Chemical Co., St. Louis, Mo. 2-F-ara-A was synthesized at Southern Research Institute motrypsinogen A, and myoglobin served as reference proteins of known molecular weight. (25). The compound was tritiated by New England Nuclear, Boston, Enzyme Purification. All steps indicated in Table 1 were performed Mass., and then purified chromatographically in this laboratory by Dr. at 0-4Â° within a period of no more than 6 days. The final preparation R. F. Struck. All other 14C-or 3H-labeled nucleosides were supplied by of enzyme was stored at -20Â°. The enzyme in the presence of 40% Moravek Biochemicals, City of Industry, Calif. All other chemicals were of reagent grade. dGTP-Sepharose was generously provided by Drs. sucrose was stable at least 1 month. Buffers used for the purification steps are as follows: Buffer A, 50 mM Tris-HCI (pH 8.0), 50 JUMEDTA, P. J. Hoffman and R. L. Blakley (13). Blue-Sepharose and octyl- and 1 mM dithiothreitol; and Buffer B, 50 mM Tris-HCI (pH 8.0), 50 Sepharose were purchased from Pharmacia Fine Chemicals, Inc., UM EDTA, 1 mM dithiothreitol, and 1 mM MgCI2. Details of the purifi Piscataway, N. J. cation procedure are described under "Results." Enzyme Assay. The total volume of the standard enzyme assay mixture was 0.1 ml and included 50 mM Tris-HCI (pH 8.0; determined at 4Â°),0.7 mM ATP, 12 ÃŸC\[2-3H]deoxyadenosine (1.5 mM), 12 ,uCi 3H- RESULTS nucleoside (1.5 mw), or 0.1 /xCi ""C-nucleoside (1.5 mM), and MgCI2 at a concentration 0.4 mM in excess of the sum of nucleoside triphos- Preparation of Cytosol Fractions. L1210 cells free of eryth- phates. It is specified in the text, the tables, or chart legends where rocytes were prepared as described previously (6). Freshly DTP was substituted for ATP as phosphate donor. The assay mixture prepared cells (about 8.3 g) were allowed to swell for 10 min was incubated at 37Â°for 1 hr. The enzyme reaction was found to be in 13.5 ml of 25 mw 4-(2-hydroxyethyl)-1-piperazineeth- linear with respect to time and enzyme concentration during this anesulfonic acid, pH 7.5, containing 0.5 mM CaCI?, 1.0 mM incubation period. The reaction was stopped by immersion in a boiling- MgCI2, 2.0 mM ethyleneglycol[bis(/?-aminoethyl ether)]- water bath for 2 min; 50 jil of the reaction mixture were then spotted on A/,A/,A/',A/'-tetraacetic acid, and 1.0 mM dithiothreitol. Cells Whatman DE81 paper discs. The DE81 discs were washed with sol vents that differed for the various substrates as follows. For deoxycy- were then disrupted with 30 strokes in a Dounce homogenizer. tidine, discs were washed 3 times with H2O and once with alcohol; for To the suspension were added 1.5 ml of 200 mM 4-(2-hydrox- thymidine and cytidine, discs were washed 3 times with 1 mM ammo yethyO-1 -piperazineethanesulfonic acid, pH 7.5, containing 2.5 nium formate, once with H2O, and once with alcohol; for adenosine, HIM sucrose and 0.5 M KCI; it was then centrifuged first at deoxyadenosine, uridine, deoxyuridine, and ara-C, discs were washed 10,000 x g for 20 min and then at 100,000 x g for 60 min. 3 times with 2 mM ammonium formate, once with H2O, and once with The supernatant (22 ml; cytosol fraction) was stored at -20Â° alcohol; for deoxyguanosine, inosine, and ara-A, discs were washed 3 until needed. times with 3 mM ammonium formate, once with H2O, and once with Streptomycin Sulfate Fractionation. A solution of strepto alcohol; for guanosine and 2-F-ara-A, discs were washed 3 times with mycin sulfate (40%, w/v) was added dropwise to the cytosol 5 mM ammonium formate, once with H2O, and once with alcohol. Discs containing 14C-labeled compounds were then dried and inserted into a fraction (22 ml) until a final concentration of 2% was obtained. The solution was stirred for 25 min at 4Â°,and the precipitate vial containing 5 ml of scintillation solution (Scintiverse; Fisher Scien tific Co.). Discs containing 3H-labeled compounds were dried and was removed by centrifugation at 10,000 x g for 20 min. The inserted into a vial containing 1.5 ml of aqueous HCI/KCI (0.1 M/0.2 supernatant solution (22 ml) was used in the following steps. M) (15). Vials were shaken gently for 30 min, and then 10 ml of Ammonium Sulfate Fractionation. Ammonium sulfate (114 Scintiverse were added. Quantitative determination of radioactivity was g/liter) was added to the supernatant obtained from the pre accomplished in a Packard Tri-Carb Model 314E liquid scintillation vious step. After being stirred at 4Â°for 30 min, the suspension spectrometer. was centrifuged at 10,000 x g for 20 min; the precipitate was Protein Determination. Protein concentration was determined by discarded. More ammonium sulfate (300 g/liter) was added to the method of Bradford (3). Bovine serum albumin was used as the the supernatant, and after being stirred for another 30 min, the standard. Disc Gel Electrophoresis. Electrophoresis was performed with a precipitate was collected by centrifugation and was dissolved Model 155 Bio-Rad apparatus. The lower gel was polymerized from in 6.1 ml of Buffer A containing 20% sucrose. The enzyme acrylamide (10%), bis-acrylamide (0.3%), ammonium persulfate solution (6.1 ml), after filtration through Sephadex G-25, was (0.03%), and W,W,A/',N'-tetramethylethylenediamine (0.05%) contain used in the following step. ing 1 mM MgCI2 and 1 mM dithiothreitol in 0.4 M Tris-HCI (pH 8.0) DEAE-Cellulose Column Chromatography. The enzyme so buffer adjusted to a final concentration of 10% polyacrylamide. The lution was applied to a DEAE-cellulose column (1.5 x 10 cm) upper gel was the same as the lower gel except that the buffer was equilibrated previously with Buffer A containing 20% sucrose. 0.125 M Tris-HCI (pH 8.0) containing a final concentration of 2% After the column was loaded with the enzyme solution, it was polyacrylamide. Samples (150 /il) were layered on the top of the gel washed with the same buffer. Fractions were collected and with bromophenol blue (0.0001 %) added as a marker. Electrophoresis was performed at 4Â° with a constant current of 3 ma/tube applied analyzed for protein concentration and enzyme activity. The across the gel for approximately 5 hr. The running buffer contained enzyme was not retained on the column under these conditions 0.1 M Tris-glycine (pH 8.4), 50 Â¡J.MEDTA,and 1 mM dithiothreitol. At and was eluted with Buffer A containing 20% sucrose; fractions the end of the run, gels were removed from the tubes and sliced in 3- containing enzyme activity were pooled for further purification. mm segments. Each segment was immersed in 150 Â¡i\ofthe nucleoside dGTP-Sepharose Column Chromatography. The pooled kinase reaction mixture, incubated overnight at 37Â°, and assayed as fractions (10.7 ml) containing enzyme activity obtained from

3034 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Deoxyribonucleoside Kinase from L1210 Cells the DEAE-cellulose column were adjusted to 10 HIM with re ml of 0.12 M KCI and 16 ml of 0.6 M KCI in Buffer B containing spect to NaF and loaded on a dGTP-Sepharose column (1.5 5% sucrose. After dialysis against Buffer B containing 45% x 10 cm) equilibrated previously with Buffer A containing 10 sucrose, fractions were analyzed for enzyme activity and pro rriM NaF and 20% sucrose. The column was washed with the tein concentration; enzyme activity was retained on the column same buffer until the absorbance at 280 nm was less than and was eluted with Buffer B containing 0.6 M KCI and 5% 0.05. After dialysis against Buffer B containing 40% sucrose, sucrose. The fractions containing enzyme activity were pooled the fractions were analyzed for protein concentration and en for further purification. zyme activity. The enzyme was eluted from the column in the CM-Cellulose Column Chromatography. The pooled en unadsorbed fractions. zyme fractions (12.4 ml), after DEAE-cellulose column Chro A second enzyme catalyzing phosphorylation of deoxyaden- matography and dialysis against Buffer B containing 45% osine was eluted from the dGTP-Sepharose column with 0.9 sucrose, were loaded on a CM-cellulose column (1.5 x 10 cm) M KCI in buffer containing 10 mw NaF and 5% sucrose. This equilibrated previously with Buffer B containing 5% sucrose. enzyme had the same molecular weight, pH optimum, and The column was then washed with 16 ml of the same buffer. electrophoretic mobility as the enzyme that was not retained After dialysis against Buffer B containing 45% sucrose, frac on the column and was similar in other properties. We suspect tions were analyzed for protein concentration and enzyme that this second enzyme activity may be an isozyme or an activity. Enzyme activity was eluted in the unadsorbed fraction. artifactual variant of the principal (unretained) enzyme. Be Phenyl-Sepharose Column Chromatography. The enzyme cause it affords a separation of this other enzyme activity, the obtained from CM-cellulose column Chromatography was ad dGTP-Sepharose column was retained as a step in the purifi justed to 1 M with respect to ammonium sulfate and loaded on cation of the principal enzyme activity. a phenyl-Sepharose column (1.5 x 10 cm) equilibrated previ Blue-Sepharose Column Chromatography. The combined ously with Buffer B containing 20% sucrose and 1 M ammonium fractions (22 ml) obtained from dGTP-Sepharose were adjusted sulfate. The column was washed with 8 ml of the same buffer to 1 rriM with respect to MgCI2 and loaded on a blue-Sepharose and then eluted with 18 ml of Buffer B containing 5% ethylene column (1.5 x 8 cm) equilibrated previously with Buffer B glycol; this was followed by 25 ml of Buffer B containing 35% containing 20% sucrose. The column was then washed with 8 ethylene glycol. Fractions were collected and dialyzed against ml of 35 mM Tris-HCI at pH 8.0 containing 20% sucrose. Each 50 volumes of Buffer B for 3 to 4 hr. Each fraction was fraction was analyzed for protein concentration and enzyme concentrated by further dialysis overnight against 50 volumes activity. The enzyme was not retained on the column. The of Buffer B containing 45% sucrose. The enzyme activity was unadsorbed fractions containing enzyme activity were pooled present in the fractions that contained 35% ethylene glycol. for further purification. This was the final step of purification of the enzyme. Ammonium Sulfate Fractionation. Ammonium sulfate (243 The purification procedures are summarized in Table 1. The g/liter) was added to the pooled (22-ml) fractions obtained enzyme was purified to about 1680-fold and was free of aden- from the previous step. After 30 min of stirring at 4Â°, the osine kinase, dAMP kinase, adenosine deaminase, and ATP- suspension was centrifuged at 10,000 x g for 20 min; the ase. The overall recovery of the purified enzyme was 70%. The precipitate was discarded. More ammonium sulfate (132 g/ enzyme preparations used for all subsequent studies were liter) was added to the supernatant, and after 30 min of stirring, carried through the final steps of purification (Table 1). The the precipitate was collected by centrifugation and dissolved in enzyme preparations in the presence of 45% sucrose were 5.3 ml of Buffer B containing 20% sucrose. The enzyme stable for more than 1 month at â€”20Â°,provided they were not solution (5.3 ml) was used in the following step. subjected to frequent freezing and thawing. Octyl-Sepharose Column Chromatography. The enzyme obtained from ammonium sulfate fractionation was dissolved in Buffer B containing 20% sucrose and 1 M ammonium sulfate and loaded on an octyl-Sepharose column (1.5 x 10 cm) equilibrated previously with Buffer B containing 20% sucrose and 1 M ammonium sulfate. The column was washed with 8 ml of the same buffer and then eluted with 18 ml of Buffer B containing 8% ethylene glycol; this was followed by 25 ml of Buffer B containing 40% ethylene glycol. Fractions were col lected and dialyzed against 50 volumes of Buffer B for 3 to 4 hr. Each fraction was concentrated by further dialysis overnight against 50 volumes of Buffer B containing 45% sucrose. The volume of the concentrated fraction was adjusted to 1 ml and analyzed for protein concentration and enzyme activity, as 8 10 12 14 16 18 shown in Chart 1. The enzyme activity was present in the Fraction (3.5 mO fractions that contained 40% ethylene glycol. Chart 1. Octyl-Sepharose column Chromatography. Protein (16.43 mg) from DEAE-Cellulose Column Chromatography. The pooled frac ammonium sulfate fractionation (40 to 60%) was dissolved in Buffer B containing tions (9.6 ml), after octyl-Sepharose column Chromatography 20% sucrose and 1 M ammonium sulfate. This solution was loaded on an octyl- Sepharose column (1.5 x 10 cm) equilibrated previously with Buffer B containing and dialysis against Buffer B containing 45% sucrose, were 20% sucrose and 1 M ammonium sulfate, and the column was eluted with 8 and loaded on a DEAE-cellulose column (1.5 x 10 cm) equilibrated 40% ethylene glycol in Buffer B as indicated. Fractions, after overnight dialysis against Buffer B containing 45% sucrose, were analyzed for protein concentra previously with Buffer B containing 5% sucrose. The column tion and enzyme activity as described in the text. ATP was used as a phosphate was then washed with 15 ml of the same buffer followed by 15 donor. Fractions 11 to 15 were pooled for further purification.

AUGUST 1982 3035

Table 1 Purification of a deoxyribonucleoside kinase from L1210 cells with deoxyadenosine as substrate activity(pmol productformed/min/ml sam (mg/ml)7.95.410.05.22.21.53.10.20.040.0240.008Specific(pmol/min/mgactivity stepCrudePurification ple)'300 protein)38 extract0Streptomycin (514)c295 (65)54 (2%)Ammoniumsulfate (491)750 (91)75 sulfate"DEAE-cellulosedGTP-SepharoseBlue-SepharoseAmmonium(1,160)785 (116)151 (1,264)546 (243)248 (1,472)519 (669)346 (1,215)1,082 0)349 (81 sulfate"Octyl-SepharoseDEAE-celluloseCM-cellulosePhenyl-SepharoseVolume(ml)22.022.06.110.722.022.05.39.68.012.49.0Enzyme(2,517)1,308 (812)6,540 (1,208)376 (6,340)9,400 (334)461 (8,350)19,208(18,917)63,875 (454)51 1 (403)Protein (50,375) a Assayed at pH 8.0, using 1.5 mM [2-3H]deoxyadenosine(12fiCi/ml), 1.1 mw MgCU, and 0.7 mw ATP. b The crude extract is the supernatant after centrifugation of whole-cell homogenate at 10,000 x g. c Numbers in parentheses, amount obtained in assays at pH 8.0, using 1.5 mw [2-3H]deoxyadenosine (12/igCi/ml), 0.7 mw UTP, and 1.1 mM MgCI2. Ammonium sulfate fractionation is specified under "Results."

Molecular Weight and Electrophoretic Mobility. The molec ular weight was determined by means of Sephadex G-200 column chromatography, as described under "Materials and BPB Methods." A single peak of the enzyme activity was found with a molecular weight of about 100,000. When electrophoresis Â«*e was performed under the conditions described in "Materials and Methods," activity was found at R( 0.66 with deoxyaden 4 osine as substrate (Chart 2). Each slice of gel was cut in half; one half of the gel was assayed for capacity to phosphorylate deoxyadenosine and the other half was assayed for capacity to phosphorylate deoxyguanosine, deoxycytidine, cytidine, a o a ara-C, ara-A, or 2-F-ara-A. Enzyme activity for all substrates 0 0.2 0.4 0.6 0.8 1.0 was found in the same gel fraction (Rf 0.66). Rf Effects of pH, ATP, and Magnesium Concentration on Chart 2. Polyacrylamide disc gel electrophoresis. Methods are described Enzyme Activity. Enzyme activity was measured in the pH under "Materials and Methods." Ri values were calculated with bromophenol range of 7 to 9.5 at 37Â° in the presence of 1.1 M MgCI2 and blue (BPB) as the standard. Gel sections were assayed overnight (16 hr) at 37Â°. 0.7 mM ATP (or UTP); results are shown in Chart 3. The enzyme The amount of enzyme used was 3 jig, which gave a rate of 50 nmol/min/mg for phosphorylation of deoxyadenosine when assayed at pH 8.0, using 1.5 mw showed optimal enzyme activity from pH 7.8 to 8.7. The effects [2-3H]deoxyadenosine (12 fiCi/ml), 1.1 mM MgCI2, and 0.7 mM ATP(O O)or of MgCI2 on the enzyme activity were also studied. When the 0.7 mM UTP (O O). Ri values were calculated with bromophenol blue as the standard. R. is defined as the ratio of the displacement of the protein band from concentration of ATP or UTP was 0.7 mM, the addition of MgCI2 the origin to that of bromophenol blue. increased the enzyme activity; maximum activity was obtained at a MgCI2 concentration of 1 mM (results not shown). Effects of Divalent Cation Chlorides and Polyamines on enzyme. Phosphorylations of ara-A, ara-C, and 2-F-ara-A also Enzyme Activity. The effects of these agents were examined were accomplished by the purified enzyme. The enzyme did in the absence and in the presence of MgCI2. In the absence of not catalyze the phosphorylation of adenosine. added metal salts, the reaction catalyzed by the enzyme pro Specificity for Phosphate Donor. Various nucleoside tri- ceeded at less than 2% of the rate observed in the presence of phosphates were tested as phosphate donors. Results indicate 2 mM MgCI2. Chloride salts of Mn2+, Ca2+, Ni2H~,and Co2+ that for this purified enzyme UTP, GTP, ITP, dUTP, and dTTP could partially replace MgCI2. Polyamines were ineffective in substituted for ATP as a phosphate donor when deoxyadeno replacing MgCI2. In the presence of 2 mM MgCI2 and 0.7 mM sine was the acceptor (Table 3). Several nucleoside triphos- ATP (or UTP), Ni2+, Mn2+, Ca2+, and Co2+ produced 50% or phate analogs, such as 8-bromoadenosine S'-triphosphate, greater inhibition of enzyme activity; Zn2+ was completely 1,A/6-etheno-2'-deoxyadenosine S'-triphosphate, 5-bromo-2'- inhibitory to both enzymes. Among those polyamines tested, deoxyuridine 5'-triphosphate, 5-bromouridine 5'-triphosphate, putrescine, spermidine, and spermine produced 50% or 5-iodo-2'-deoxyuridine S'-triphosphate, and 3,W4-etheno cy- greater inhibition of enzyme activity. tosine S'-triphosphate, also replaced ATP as a phosphate Nucleoside Specificity of Oeoxyadenosine Kinase. Purified donor for deoxyadenosine, deoxyguanosine, and deoxycyti enzyme was assayed for capacity to catalyze the phosphoryl- dine (Table 4). 9-/3-D-Arabinofuranosyladenine S'-triphosphate ation of nucleosides with UTP or ATP as the phosphate donor; and 1-ÃŸ-D-arabinofuranosylcytosine S'-triphosphate served as results are presented in Table 2. Among the natural nucleo phosphate donors for deoxycytidine. sides tested, deoxyadenosine, deoxyguanosine, deoxycyti Initial Velocity Studies with Respect to Substrates. Initial dine, and cytidine were active phosphate acceptors for the velocity measurements were performed using 1.1 mM MgCI2

3036 CANCER RESEARCH VOL. 42

of ATP is greater than the value obtained in the presence of UTP. Kâ€ž,and Vmaxvalues for deoxyadenosine with various phosphate donors are summarized in Table 5. The apparent Km values for the substrates other than deoxyadenosine were

Table 4 Phosphorylation of deoxyadenosine, deoxyguanosine, and deoxycytidine with nucleoside triphosphate analogs as phosphate donor Kinase activity was assayed at pH 8.0, using 1.5 mM 3H-nucleosides (12 /iCi / ml), 1.1 mM MgClz, and 0.7 mM nucleoside triphosphate. protein)Phosphate Nucleotide formed (nmol/min/mg

10 donorNoneATPIdUTP"ara-ATPara-CTPBrATP1

PH Chart 3. Effects of pH on deoxyadenosine kinase activity. Enzyme activity was determined by the radiochemical assay using 50 HIM Tris-HCl buffer, 1.5 mu [2-3H]deoxyadenosine (12 /iCi/ml), 1.1 DM MgCI2, and 0.7 mw ATP (O- O) or 0.7 mW UTP (O--- O). ,N6-etheno-2'-dATPBrdUTPBrUTP3,N"-etheno-CTPDeoxyadenosine0.1655344131807344199Deoxyguanosine0.136691936658617070Oeoxycytidine0.1115347546356604186 Table 2 Phosphate acceptor specificity of a deoxyribonucleoside kinase from L1210 cells Kinase activity was measured at pH 8.0, using 1.5 mM "C-nucleoside (1 /iCi/ â€¢IdUTP,5-iodc-2'-deoxyuridine 5'-triphosphate; ara-ATP, 9-/8-o-arabinofur- ml) or 3H-nucleoside (1 2 fiCi/ml), 1.1 mM MgCI2, and 0.7 mw UTP or ATP. anosyladenme S'-triphosphate; ara-CTP, 1-/3-o-arabinofuranosylcytosi.ne 5'-triphosphate; BrATP, 8-bromoadenosine 5'-triphosphate; 1, Ne-etheno-2'-dATP, Specific activity 1,N6-etheno-2'-deoxyadenosine 5'-triphosphate; BrdUTP, 5-bromo-2'-deoxyuri- (nmol/min/mg protein) dine 5'-triphosphate; BrUTP, 5-bromo-uridine 5'-triphosphate; 3,N4-etheno-CTP, 3,N4-etheno-cytosine 5'-triphosphate. Phosphate acceptor UTP ATP

AdenosineUndineGuanosineCytidineInosineDeoxyadenosineDeoxyu (A) o.os 0.04 0.03 ridineDeoxyguanosineDeoxycytidineThymidineara-A2-F-ara-Aara-C<0.1<0.1<0.120.0<0.150.0<0.181.011.0<0.129.038.040.0<0.1<0.1<0.113.4<0.165.5<0.135.711.0<0.113.422.023.0 0.0

-10 -8 -6 -4 -2 6 10 14 18 1/DeoÂ«yadenoÂ»lne(mM)

Table 3 Specificity for natural phosphate donors with 2'-deoxyadenosine as acceptor Kinase activity was determined at pH 8.0, using 1.5 mM (2- 'H Jdeoxyadenosine (12 /iCi/mi). 1.1 mM MgClz, and 0.7 mM nucleoside triphosphate. Specific activity Phosphate donor (nmol/min/mg protein) NoneATPUTPGTPCTPITPdATPdUTPdGTPdCTPTTP0.1655065983107717677

-1.0 -0.6 -0.2 0.2 0.8 1.0 1.4 1.8 1/Deoxyadenosine (mM) Chart 4. Double-reciprocal plots of initial deoxyadenosine concentration with respect to the reaction velocity. Enzyme activity was determined at pH 8.0 using various concentrations of [2-3H]deoxyadenosine, 1.1 mM MgCI2, and 0.7 mM dUTP, dTTP, or UTP (A) or 0.7 mM ATP, GTP. or ITP (8). Velocity of reaction, v, is expressed as nmol/min/mg enzyme.

Table 5 and 0.7 mM nucleoside triphosphate at pH 8.0 with various Kinetic constants for phosphorylation of deoxyadenosine with various concentrations of deoxyadenosine. The results are shown in phosphate donors Km and vm,. values were obtained by the double-reciprocal plots of initial Chart 4. The apparent Km values for deoxyadenosine in the deoxyadenosine concentration with respect to the reaction velocity as shown in presence of UTP and ATP were 0.13 and 1.25 rnw (Chart 4). Chart 4. The apparent Km value for deoxyadenosine calculated from Phosphate Chart 4A for the reaction in the presence of 0.7 mM dUTP was donorATP (nmol/min/mgprotein)122 0.25 mM. The apparent Km value for the reaction in the pres ence of dTTP is the same as that in the presence of dUTP. The GTP 1.25 122 ITP 1.25 160 apparent Km values calculated from Chart 46 for the reaction UTP 0.13 55 in the presence of ATP, GTP, or ITP were all 1.25 mM. The dUTP 0.25 83 0.25Vâ„¢, 83 Vmaxfor the reaction catalyzed by the enzyme in the presence dTTPKm(mM)1.25

AUGUST 1982 3037

Table 6 significance of this property of the enzyme cannot be assessed, Kinetic constants for the phosphorylation of nucleosides but since pools of UTP in L1210 cells are about 10 to 15% of Enzyme activity was determined at pH 8.0, using various concentrations of 3H-nucleosides, 1.1 rriM MgCI2, and 0.7 mM ATP. Km and Vmaxvalues were those of ATP, UTP may play a role in the normal functioning of obtained by the double-reciprocal plots of initial nucleoside concentration with the kinase. The lack of specificity of this enzyme for phosphate respect to the reaction velocity. donors is indicated further by the fact that several triphosphates Km Vmax of nucleoside analogs were equal or superior to ATP (Table 4). Substrate (mM) (nmol/min/mg protein) It is unlikely that the analog triphosphates could reach a con Deoxyadenosine 1.25 122 centration at which they might function as phosphate donors in Deoxyguanosine 1.37 33 Deoxycytidine 8.0 61 cells exposed to these nucleoside analogs. Cytidine 33.0 805 Because of the different Km values obtained with ATP and UTP, both phosphate donors were used in the determination of also determined for the reaction in the presence of 0.7 mM the properties of the enzyme (Tables 1 and 2; Charts 2 and 3). ATP; results are summarized in Table 6. For the 3 substrates, A standard concentration of deoxyadenosine of 1.5 mw was deoxyguanosine, deoxycytidine, and cytidine, the apparent Km used and the rates of reaction were calculated from initial values (mw) were 1.37, 8.0, and 33.0, respectively, and the velocities. This concentration of deoxyadenosine is close to apparent Vmaxvalues (nmol/min/mg enzyme) were 33, 61, and the Kmwith ATP as phosphate donor but more than 10-fold the 805, respectively. Kmwith UTP as donor. Because of these differences, any small The apparent Km values for DTP and ATP in the presence of difference in results with the 2 phosphate donors may not be deoxyadenosine were 45 and 100 /Â¿M,respectively (data not significant. However, it is clear that substitution of UTP for ATP presented). did not result in a different pH optimum (Chart 3), did not alter substrate specificities (Table 2), and did not reveal enzyme activity in different fractions (Table 1; Chart 2). DISCUSSION Michaelis constants in the range of 0.33 to 5 mM have been The kinase isolated here from L1210 cells catalyzed the reported for the phosphorylation of deoxyadenosine by various phosphorylation of several natural nucleosides: deoxyadeno nucleoside kinases (5, 17, 18, 20, 21, 23, 29, 30), and the sine, deoxyguanosine, deoxycytidine, and cytidine. Since the value for our enzyme falls in this range. Deoxyadenosine is Kmvalues for deoxyadenosine and deoxyguanosine were lower thus a poor substrate for all of the kinases that have been than those for deoxycytidine and cytidine, the enzyme perhaps reported. Henderson et al. (12) have suggested that, despite should be regarded as a purine deoxyribonucleoside kinase. the high Michaelis constants, deoxyadenosine can be an effec With regard to substrate specificity, this enzyme is similar to tive substrate in intact cells because of the amounts of enzymes the nonspecific deoxycytidine kinase from calf thymus that has present and the duration of their action on the substrate. been studied in several laboratories (9, 17, 24). However, the Another consideration is that the L1210 purine deoxyribonu calf thymus enzyme preparations differed from our L1210 cleoside kinase can catalyze phosphorylation of deoxyadeno enzyme in that they had much lower Km values for deoxycyti sine with a variety of phosphate donors and, as mentioned dine (17, 24). Since the enzymes from calf thymus were only above, with a lower Km value when pyrimidine nucleoside partially purified, contamination with uridine-cytidine kinase, a triphosphates are the donors. Therefore, because of the com well-characterized enzyme (1 ), could not be ruled out as being bined effects of possible phosphate donors, the true Michaelis responsible for the phosphorylation of cytidine. However, for constants in the intact cells may differ from that estimated with the L1210 enzyme, the retention of cytidine kinase activity a single phosphate donor. after extensive purification, the low level of uridine kinase ara-A and 2-F-ara-A, 2 deoxyribonucleoside analogs that activity, and the association of cytidine kinase activity with have antitumor activity, were substrates for the purine deox activities for deoxyadenosine, deoxyguanosine, and deoxycy yribonucleoside kinase from L1210 cells. The question re tidine after gel electrophoresis indicate that phosphorylation of mains, however, as to the number of the kinases that may act cytidine is a property of this kinase. The Kmfor phosphorylation on these analogs. We have reported that a partially purified of cytidine was high, and it remains to be determined whether deoxycytidine kinase from L1210 cells catalyzed phosphoryl this enzyme plays a role in the metabolism of cytidine and its ation of 2-F-ara-A but not deoxyadenosine (4). Thus, there analogs. A deoxycytidine kinase also has been partially purified appear to be at least 2 kinases for 2-F-ara-A; yet cells that had from L1210 cells by Kessel (16). This enzyme apparently is lost deoxycytidine kinase activity were resistant to this analog different from our enzyme because it has a low Km (11 Â¡IM)for with a halogen substituent (4). In intact cells, the phosphoryl deoxycytidine and a lower molecular weight (60,000); its activ ation of ara-A itself apparently is catalyzed by adenosine kinase ity with other nucleosides was not reported. Thus, it appears and deoxycytidine kinase (28); if our purine deoxyribonucleo that in L1210 cells there are at least 2 kinases that catalyze side kinase is, in fact, different from deoxycytidine kinase, then phosphorylation of deoxycytidine. Since our enzyme was iso there appear to be at least 3 enzymes that can catalyze lated from the cytosol, the possibility remains that there may phosphorylation of ara-A. The number and identity of the be still other deoxyribonucleoside kinases in the mitochondria kinases responsible for the phosphorylation of these and other or nuclei. analogs are under investigation in this laboratory. An interesting property of the L1210 enzyme is that the Michaelis constants for the phosphorylation of deoxyadenosine ACKNOWLEDGMENTS were lower with pyrimidine nucleoside triphosphates as phos We wish to thank Mary Trader and Maxie Witt, Chemotherapy Department, for phate donors (Table 5). For example, the apparent Kmwith ATP passage of L1210 ascites tumor cells and Nancy DuBois, Biochemistry Depart as donor was 10-fold that with DTP as donor. The metabolic ment, for technical assistance in harvesting and preparing L1210 leukemia cells.

3038 CANCER RESEARCH VOL. 42

REFERENCES 15. IvÃ©s,D. H., Durham, J. P., and Tucker, V. S. Rapid determination of nucleoside kinase and nucleotidase activities with tritium-labeled substrates. 1. Anderson, E. P. Nucleoside and nucleotide kinases. In: P. D. Boyer (ed.). Anal. Biochem., 28. 192-205, 1969. The Enzymes, Vol. 9, pp. 49-96. New York: Academic Press, Inc., 1973. 16. Kessel, D. Properties of deoxycytidine kinase partially purified from L1210 2. Barker, J., and Lewis, R. A. Deoxyguanosine kinase of neonatal mouse skin cells. J. Biol. Chem., 243: 4739-4744, 1968. tissue. Biochim. Biophys. Acta, 658: 111 -123, 1981. 17. Krenitsky, T. A.. Tuttle, J. V., Koszalka, G. W., Chen, l. S., Beacham, L. M., 3. Bradford, M. M. A rapid and sensitive method for the quantitation of micro- Ill, Rideout, J. L., and Elion, G. B. Deoxycytidine kinase from calf thymus. gram quantities of protein utilizing the principle of protein-dye binding. Anal. Substrate and inhibitor specificity. J. Biol. Chem., 25Ã•: 4055-4061, 1976. 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AUGUST 1982 3039

Chi-Hsiung Chang, R. Wallace Brockman and L. Lee Bennett, Jr.

Cancer Res 1982;42:3033-3039.

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