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Purification and Some Properties of a Deoxyribonucleoside Kinase from L1210 Cells1

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Purification and Some Properties of a Deoxyribonucleoside Kinase from L1210 Cells1 [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 spécificitésof 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-/ß-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-fluoroad- enine; 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. C-H. Chang et al. 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 0-4° 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 -20°. 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 4°),0.7 mM ATP, 12 ßC\[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 37°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.
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