Nucleoside kinase activities of Chinese hamster ovary cells

Biochimica et Biophysica Acta, 761 (1983) 135-'141

135

Elsevier BBA 21615

N U C L E O S I D E KINASE ACTIVITIES OF C H I N E S E H A M S T E R OVARY CELLS PRISCILLA P. SAUNDERS and MILDRED M. LAI Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute Houston, TX 77030 (U.S.A.)

(Received June 13th, 1983) (Revised manuscript received September2nd, 1983)

Key words: Nucleoside kinase; Nucleoside metabolism; (CHO cell)

Chinese hamster ovary (CHO) cells and appropriate drug-resistant mutants derived from them have been analyzed for nucleoside kinase activities relevant to the phosphorylation of adenosine, deoxyadenosine, deoxyguanosine and deoxycytidine and for resistance to a variety of nucleoside analogs. Fractionation of extracts by DEAE-cellulose chromatography revealed three major peaks of activity. Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20), the first to elute from the column is responsible for the majority of the deoxyadenosine phosphorylation in cell extracts and, according to resistance data, appears to phosphorylate most adenosine analogs tested, including 9-/~-D-arabinosyladenine (ara-A). A deoxyguanosine kinase, the second enzyme to elute from the column, was responsible for the majority of deoxyguanosine and deoxyinosine phosphorylation in cell extracts. The function of this enzyme in cell metabolism is unclear. 2-Chlorodeoxyadenosine, on the other hand, appeared from resistance data to be pbosphorylated, at least in part, by deoxycytidine kinase (ATP: deoxycytidine 5'-phosphotransferase, EC 2.7.1.74), which in cell extracts could also phosphorylate deoxyguanosine and deoxyadenosine, though much less efficiently than deoxycytidine.

Introduction The action of nucleoside analogs is generally dependent upon either phosphorylation of the analog by an appropriate kinase or degradation via a nucleoside phosphorylase to the free base and subsequent activation to the monophosphate form by a phosphoribosyltransferase. While many of tfiese enzymes have been purified from a variety of Abbreviations: CHO, Chinese hamster ovary; ara-C, 1-fl-Darabinofuranosylcytosine; ribavirin, 1-fl-o-ribofuranosyl-l,2,4triazole-3-carboxamide;ara-A, 1-fl-D-arabinofuranosyladenine; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine;HPLC, high pressure liquid chromatography;tubercidin, 7-deazaadenosine; cordycepin, Y-deoxyadenosine;formycin A, 7-amino-3-(fl-Dribofuranosyl)-pyrazolo[4,3-d]pyrimidine; formycin B, 7-hydroxy-3-(fl-ribofuranosyl)-pyrazolo[4,3-d]pyrimidine. 0304-4165/83,/$03.00 © 1983 ElsevierSciencePublishers B.V.

sources, considerable confusion remains regarding their roles in natural cell metabolism and in the phosphorylation of analogs. The relevant literature which is too copious to be discussed here, has been carefully reviewed by He.nderson [1]. Chinese hamster ovary (CHO) cells have been widely used in studies of purine and pyrirhidine analog metabolism and mechanism of action. This report describes several nucleosideTphosphorylating activities of CHO cells and relates them, using drug-resistant cell lines, to the activation of a variety of nucleoside analogs. Experimental Procedure Chemicals. [8-14C]Adenosine (59 m C i / m m o l ) [5-H]deoxycytidine (27 C i / m m o l ) , [8-3H]de -

136

oxyguanosine (7 Ci/mmol), [8-3H]deoxyadenosine (11 C i / m m o l ) were purchased from ICN Pharmaceuticals, Inc. (Irvine, CA). The specific activities of the deonynucleosides were adjusted to 4 C i / m m o l prior to use. Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purchased from Sigma Chemical Co. (St. Louis, MO) as a suspension in (NH4)2SO 4 (235 units/ mg). Fetal bovine serum was purchased from Irvine Scientific (Santa Ana, CA) and McCoy's 5A medium was obtained from Grand Island Biological Co (Grand Island, NY). Erythro-9-(2-hydroxy3-nonyl)adenine (EHNA) was obtained through W. Plunkett from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, M D . Ribavirin and 3-deazaguanosine were provided by R.K. Robins. All other chemicals were reagent grade and obtained from appropriate commercial sources. Cell lines and medium. CHO cells were carried in monolayer culture using McCoy's 5A growth medium supplemented with 10% fetal bovine serum as previously described [2]. The mutant RbR-1 [3] is deficient in adenosine kinase and line aCg-7 [4] is deficient in deoxycytidine kinase. Both mutants were derived from the CHO line. Determination of minimum inhibitory drug concentration. The minimum inhibitory drug concentration was determined as described elsewhere [4]. Briefly, cells were plated in varying concentrations of drug, allowed to form clones for 7-8 days and stained. The minimum inhibitory drug concentration was the lowest tested concentration that inhibited clone formation (fewer than 100 cells per clone). Preparation of extracts. Ceils were grown in 670 cm 2 roller bottles, washed, removed by scraping into phosphate-buffered saline, centrifuged, and stored at - 2 0 ° C until used. To prepare extracts, cells were resuspended in 2 vol. of buffer A (50 mM potassium phosphate, pH 7.5, 10 mM 2mercaptoethanol) and disrupted by sonication. The suspension was centrifuged at 14500 x g for 20 min in a Sorvall RC-5 refrigerated centrifuge, and the supernatant employed as the crude extract after dialysis against 100 vol. of buffer A. Enzyme assays. Adenosine kinase reaction mixtures contained 0.1 M Tris-maleate buffer, pH 5.5, 4 mM ATP, 1.5 mM MgC12, 5 /~M EHNA

(adenosine deaminase inhibitor), 100 /~M [814C]adenosine (59 mCi/mmol), and extract in a final volume of 100/~1 [5]. Incubation was at 37°C. At appropriate intervals, 20-/~1 samples were removed and applied to DE-81 filter paper discs, which were then washed three times for 10-15 min in 1 mM ammonium formate, twice with deionized H20, and once with 95% ethanol, dried, and counted in 10 ml of aquasol (New England Nuclear, Boston, MA). Deoxynucleoside kinase activities were determined in reaction mixtures containing 50 mM Tris-HC1, pH 7.8, 5 mM MgCI z, 8 mM ATP (adjusted to pH 7.0 25 /~M [3H]deoxynucleoside (4 Ci/mmol), and extract in a final volume of 100/~1. At 10-min intervals 20-~tl samples were removed and processed as described for the adenosine kinase assay. Purine nucleoside phosphorylase was assayed by the method described by Milman [6]. In this assay, the conversion of nucleoside to purine base and ribose 1-phosphate was measured by the formation of ribose-l-[14C]phosphate from [U-14C]-labeled guanosine (350 m C i / m m o l ) catalyzed by 60/~1 of eluate. The radioactive assay described by Nygaard [7] was used to determine adenosine deaminase activity. Assay mixtures contained 12.5 /~i of column fractions and incubation was for 1 h. In this assay, the formation of [14C]inosine from [14C]adenosine (58 mCi/mmol) was measured after thin-layer chromatographic separation of the two. Specific activities are expressed as nmol product formed per min per mg protein. Protein was determined with the Bio-Rad protein reagent using bovine serum albumin as standard. DEAE-cellulose chromatography. 5 ml of crude extract were applied to a 1.8 x 13 cm column of DEAE-cellulose (Whatman DE-52) that had been previously equilibrated with buffer A. Elution was carried out with a linear gradient (500 ml) of KC1 from 0 to 0.3 M in 50 mM Tris-HC1, pH 7.5, containing 10 mM 2-mercaptoethanol. Fractions of approx. 5.3 ml were collected and every third fraction was assayed for kinase activity. For ease in assaying numerous fractions, reaction mixtures were placed in multi-well plates for incubation. A reaction mixture for each deoxynucleoside substrate containing 8 mM ATP, 0.1 M Tris-HC1 pH 7.8, 10 mM MgC1 z and 0.017 mM [3H]nucleoside (4 C i / m m o l ) was dispensed into each of 35 wells

137

(25 #l each) in a 96-well microtiter plate. 25 /~l samples of the desired column fractions were then added, the plate covered with parafilm, and incubation carried out by floating the plate in a 37°C water bath for 60 rain. Adenosine kinase was assayed similarly using the incubation mixture described earlier. 40/~l samples of each incubation mixture were then applied to DE-81 paper discs, which were washed and counted as described. Identification of reaction products. The products of the nucleoside kinase reactions were characterized by HPLC techniques. Using the column fraction containing the peak of each enzyme activity (Fig. 1, bottom) 0.2-ml reaction mixtures were set up with the composition described for analysis of column fractions and incubated for 2 h at 37°C. Duplicate 20-/~1 samples were removed from each, applied to DE-81 discs, washed and counted as described for enzyme assays to determine the amount of product formed. A third sample of each (50 #1) was also applied to a DE-81 disc, which was washed by the same procedure. The phosphorylated product was then removed from the disc by soaking in 0.4 ml of 0.5 M ammonium acetate, pH 6.4. 200 #l of this eluate were fractionated using a Waters Associates (Milford, MA) ALC 204 high-pressure liquid chromatograph equipped with a Model 6000A pump and 3.9 mm × 30 cm reversed phase column of /xBondapak Cls (#C18) from Waters Associates (Milford, MA). Samples were injected by means of the U6K-LC injection system, and isocratic elution carried out with 0.5 M ammonium acetate. pH 6.4. Eluted compounds were detected at 254 nm with the model 440 detector and quantitated

with a Waters Data Module. Retention times in minutes of standard compounds were: dAMP, 27.9; dGMP, 14.1; dIMP, 11.0; dUMP, 14.5; GMP, 5.1; and dCMP, 4.4. Fractions (1.0 ml) were collected directly into scintillation vials and counted. This experiment was carried out twice with similar results. Results

The apparent specific activities of adenosine kinase, deoxyadenosine kinase, deoxyguanosine kinase, and deoxycytidine kinase in crude extracts of three cell lines are shown in Table I. The ribavirin-resistant cell line, RbR-1, though clearly deficient in adenosine kinase, shows an incomplete deficiency of deoxyadenosine-phosphorylating activity. Line aCR-7, however, which is resistant to ara-C and deficient in deoxycytidine kinase activity, appears to have parental levels of both deoxyadenosine kinase and deoxyguanosine kinase activities. These observations suggested that in CHO cells there might be an enzyme (or enzymes) other than adenosine kinase or deoxycytidine kinase that could phosphorylate deoxyadenosine and deoxyguanosine. Alternatively, metabolic alteration of the nucleoside (i.e., deamination) could render it susceptible to phosphorylation by one of the other kinases. The roles of these enzymes in nucleoside analog activation can be estimated in part by determining the cross-resistance properties of the enzyme-deficient cell lines (Table II). The dramatic resistance of line RbR-1 to ribavirin, tubercidin, and cordycepin indicates that in CHO cells phosphory-

TABLE I SPECIFIC ACTIVITIES O F N U C L E O S I D E KINASES IN C H O CELL C R U D E E X T R A C T Reaction mixtures for adenosine kinase and deoxyadenosine kinase contained 10/~M E H N A to inhibit adenosine deaminase activities. The values shown are representative of several determinations. The values for adenosine kinase m a y be underestimates since assays were carried out with relatively high (100 ~M) concentrations of adenosine. Cell line

CHO RbR-1 acR-7

Selective agent

-ribavirin ara-C

Enzyme specific activity adenosine kinase

deoxyadenosine kinase

deoxyguanosine kinase

deoxycytidine kinase

1.12 0.01 1.19

0.018 0.007 0.025

0.011 0.011 0.012

0.023 0.029 0.005

138 t i a t e d to i d e n t i f y those enzymes c o n t a i n e d in these cells a n d to clarify their roles in nucleoside a n a l o g activation. Fig. 1 ( b o t t o m ) shows a typical elution Each of these determinations has been carried out several times profile of kinases from a C H O cell crude extract with the same result. fractionated by DEAE-cellulose chromatography Approximate minimum inhibitory concen- using a linear g r a d i e n t of KC1 from 0 to 0.3 M. Nucleoside analog centration of drug (/LM) toward cell line A d e n o s i n e kinase elutes first from the column. CHO RbR-1 aCR-7 D e o x y a d e n o s i n e kinase activity is resolved into Ribavirin 50 500 50 two peaks, one that elutes c o i n c i d e n t with a d e n o Ara-C 0.5 0.5 100 sine kinase a n d a n o t h e r that is n e a r l y c o i n c i d e n t Ara-A a 5.0 50 5.0 with d e o x y g u a n o s i n e kinase activity. The latter Tubercidin 0.005 0.5 0.01 activity is distinct a n d s e p a r a b l e from deoxycytiFormycin A ~ 5.0 50 5.0 d i n e kinase, the last p e a k to elute f r o m the colCordycepin a 0.1 10 0.1 umn. W i t h the exception of s o m e variation in the 2-Cl-deoxyadenosine 5.0 1 50 Forymycin B 10 5 10 size of the d e o x y a d e n o s i n e kinase second peak, 3-Deazaguanosine 5 5 5 this profile is highly r e p r o d u c i b l e even t h o u g h 2'-Deoxyguanosine 100 50 200 m o s t of the enzymes are quite unstable. Since a Determined in the presence of 5/~M EHNA to inhibit adenosine p u r i n e nucleoside p h o s p h o r y l a s e or a d e n o s i n e d e a m i n a s e could c o n c e i v a b l y interfere with the deaminase. a s s a y of certain kinase activities, their l o c a t i o n was d e t e r m i n e d in the D E A E - c h r o m a t o g r a p h i c profile l a t i o n b y a d e n o s i n e kinase is a p r i m a r y require(Fig. 1, top). m e n t for activity b y these agents. Line RbR-1 W e have e m p l o y e d this m e t h o d to analyze the d e m o n s t r a t e s o n l y p a r t i a l resistance to a r a - A a n d k i n a s e c o m p o s i t i o n of the nucleoside analog-ref o r m y c i n A, suggesting either that these nucleosistant cell lines shown in T a b l e I. The ribavirinsides are p h o s p h o r y l a t e d b y a n o t h e r k i n a s e o r that resistant cell line, RbR-1, is deficient in a d e n o s i n e t h e y are active b y a n o t h e r m e c h a n i s m as well. T h e k i n a s e a n d shows a c o r r e s p o n d i n g deficiency of d e o x y c y t i d i n e k i n a s e deficient line, aCR-7, while the first p e a k of d e o x y a d e n o s i n e , kinase activity (Fig. 2), thus c o n f i r m i n g the a s s u m p t i o n that b o t h h i g h l y resistant to ara-C, is o n l y slightly resistant to d e o x y g u a n o s i n e a n d is a p p a r e n t l y slightly reactivities are c a t a l y z e d b y the same enzyme. The sistant to tubercidin. N e i t h e r cell line is resistant s e c o n d p e a k of d e o x y a d e n o s i n e kinase activity was u n a f f e c t e d b y this m u t a t i o n . T h e a r a - C - r e s i s t a n t to 3-deazaguanosine. A n a l y s i s of the various cell line aCR-7, d e m o n s t r a t e d an absence of den u c l e o s i d e kinases p r e s e n t in C H O cells was iniTABLE II

SUMMARY OF CROSS-RESISTANCE

TABLE III IDENTIFICATION OF KINASE REACTION PRODUCTS A CHO cell extract was fractionated by DEAE-cellulose chromatography as described for Fig. 1 and the peak fractions for each enzyme activity used in this determination: adenosine kinase/deoxyadenosine kinase (fraction 10); deoxyadenosine kinase/deoxyguanosine kinase (fraction 40); deoxycytidine kinase (fraction 76). Reaction mixtures were as described for assay of deoxynucleoside kinases. Calculations are based on 50/~1 aliquots of the reaction mixtures. This experiment was carried out twice with similar results. Substrate [ 3H]dAdo [3H]dAdo [3H]dAdo [ 3H]dGuo [3H]dCR

Enzyme fraction adenosine kinase/deoxyadenosine kinase deoxyadenosine kinase/deoxyguanosine kinase deoxyadenosine kinase/deoxyguanosine kinase deoxyadenosine kinase/deoxyguanosine kinase deoxycytidine kinase

EHNA (20/~M)

Total product formed (cpm)

+ -

79 023 13610 64635 34 310 24777

% of product clam in dCMP dGMP dlMP 0

0

0

0 0 0 > 95

0 0 94 0

73.3 96.3 1.7 0

dAMP 82

20 2.4 0 0

139 5O

2.0

L* ,~

0.08

,0% o.0e

il dAK 1

!!oo

!ii

J'

'

10

i / !

J

20

30

40

50

eo

70

80

90

Fig. 3. DEAE-cellulose chromatography of aC R-7 cell kinases. A dialyzed extract of aCR-7 cells containing 23 mg of protein was fractionated and analyzed as described for Fig. 1.

o.o.i 2

0.02

Friction Number

°"°!

~" 11o

<~

"'L.L." }

!

0.04.

- Friction Numblw

Fig. 1. DEAE-cellulose chromatography of CHO cell kinases (bottom) and adenosine deaminase (ADA) and purine nucleoside phosphorylate (PNPase) (top). A dialyzed extract of CHO cells containing 31 mg of protein was applied to a 1.8 × 13 cm column of Whatman DEAE-cellulose (DE-52). Elution was with a 500 ml linear gradient of KCI, 0 to 0.3 M and fractions of approx. 5.13 ml were collected. Each third fraction was assayed for the following enzyme activities: adenosine kinase (AK); deoxyadenosine kinase (dAK); deoxyguanosine kinase (dGK); and deoxycytidine kinase (dCK).

oxycytidine kinase, while the other kinase activities were comparable to those of the parent CHO line (Fig. 3). While the identities of the peaks corresponding to adenosine kinase (fraction 10) and deoxycyti-

dine kinase (fraction 80) were established by the mutant cell lines, the remaining peaks were more uncertain. The source of the second deoxyadenosine-phosphorylating activity became apparent during characterization of the products of the enzyme reactions (Table III). The product of the adenosine kinase reaction with deoxyadenosine was clearly dAMP and that of-the deoxyguanosine kinase (fraction 43) reaction with deoxyguanosine was dGMP. The apparent phosphorylation of deoxyadenosine by fraction 43, however, resulted in the formation of dIMP rather than dAMP. This reaction could be considerably reduced by preincubation with EHNA, an adenosine deaminase inhibitor. This strongly suggests that the deoxyadenosine and deoxyguanosine phosphorylating activities of fraction 43 both reflect the same enzyme, which appears to be a deoxyinosine, deoxyguanosine kinase.

dGK

5 dGK

0.15 "~

dAK 2

0.10

,<

dCK

""'2

i! Q

0

0,08 E 1

~d

20

~Fraction Number

0

10

20

30 40 50 60 Fraction Number

70

80

90

Fig. 2. DEAE-cellulose chromatography of RbR-1 cell kinases. A dialyzed extract of RbR-1 cells containing 26 mg of protein was fractionated and analyzed as described for Fig. 1.

Fig. 4. DEAE-cellulose chromatography of kinases from cells deficient in hypoxanthine guanine phosphoribosyltransferase and adenine phosphoribosyltransferase. A dialyzed extract of AAR-6/AGR-7 cells containing 30 mg protein was analyzed as described for Fig. 1.

140 The apparent deoxyadenosine-phosphorylating activity appears to be the result of deamination of the substrate by the tailing portion of the adenosine deaminase peak (Fig. 1) and subsequent phosphorylation of the product, deoxyinosine. The observation that the deoxyadenosine and deoxyguanosine phosphorylating peaks were not exactly coincident reflects decreasing amounts of adenosine deaminase in the fractions as the deoxyguanosine kinase peak eluted from the column, slightly shifting the apparent deoxyadenosine phosphorylating peak. This was supported by the observation that these extracts contain a very active deoxyinosine phosphorylating activity, which elutes coincident with the deoxyguanosine kinase activity (not shown). The possibility was considered that the apparent deoxyinosine and deoxyguanosine phosphorylating activities could result from phosphorolysis and subsequent phosphoribosylation by hypoxanthine guanine phosphoribosyltransferase. This possibility was eliminated by the observation that a cell line that is deficient in both hypoxanthine guanine phosphoribosyltransferase and adenine phosphoribosyltransferase, line AAR-6/AGR-7 [4], contains a deoxyguanosine phosphorylating activity that ap-

TABLE IV NUCLEOSIDE SPECIFICITYOF KINASES Assays for all substrates (including adenosine)were carried out in the reaction mixtures described for deoxynucleosidekinases with the addition of 50/~M EHNA. In each case the substrate concentration was 0.017 mM. A CHO extract was fractionated by DEAE-cellulosechromatographyas described for Fig. 1 and the peak fractions for each enzymeused in this determination: adenosine kinase (fraction 10), deoxyguanosinekinase (fraction 43), deoxycytidinekinase (fraction 80). Incubation was for 60 win. This experimentwas carried out twicewith similar results. Product formed (pmol)/reaction mix adenosine deoxyguanosine deoxycytidine kinase kinase kinase Adenosine 249 0.7 0.4 Deoxyadenosine 8.7 2.3 a 2.0 Deoxyguanosine 0.8 15.2 2.8 Deoxyinosine 0 41.6 1.5 Deoxycytidine 0 0 18 Nucleoside substrate

a Most of the product is probably dlMP as indicated by Table III.

pears, after chromatography, to be comparable to that of the parent CHO line (eluting in fractions 43-52 in the DEAE-cellulose profile, Fig. 4). The specificities of the three kinase activities were determined using the same reaction conditions for all (Table IV). Although adenosine kinase was relatively specific for adenosine, it phosphorylated significant amounts of deoxyadenosine because the enzyme appears to be present in large quantity. Deoxyguanosine kinase phosphorylated deoxyinosine more efficiently than deoxyadenosine (100:37) while the samll amount of phosphorylation of deoxyadenosine may, in fact, reflect a small amount of adenosine deaminase activity which would produce deoxyinosine. Deoxycytidine kinase p h o s p h o r y l a t e d d e o x y c y t i d i n e , deoxyguanosine and deoxyadenosine in the ratios 100 : 16 : 11 under these conditions. Since unusual enzyme activities in cultured mammalian cells can reflect the presence of mycoplasma contamination, this possibility was considered. Mycoplasma-free CHO-K1 cells, obtained from the American Type Culture Collection, were grown and assayed as quickly as possible to determine their kinase composition. The profile obtained was highly similar to that of our parent CHO line (Fig. 1) suggesting that none of these enzymes are the result of mycoplasma contamination. Discussion While it initially appeared that there were two peaks of enzyme activity relevant to the metabolism of deoxyadenosine, it soon became clear that one of these resulted from deamination of the substrate and subsequent phosphorylation of the product, deoxyinosine, by deoxyguanosine kinase. Although the adenosine deaminase inhibitor E H N A is relatively effective at high concentration in controlling this activity, some deoxyinosine was consistently formed, which can be misleading, particularly in crude extracts. Adenosine kinase is primarily responsible for the phosphorylation of adenosine and deoxyadenosine in CHO cell extracts. It should be pointed out that adenosine kinase from some sources is inhibited by adenosine above 2-5 #M. Since most of the experiments reported here (except Table IV) were carried out at

141 100 # M adenosine, the values shown for adenosine kinase may be underestimates. The data from mutants indicate that adenosine kinase also phosphorylates all of the adenosine analogs that were tested, with the exception of 2-chlorodeoxyadenosine, which appears to be activated at least in part by deoxycytidine kinase. Ara-A has been shown to be phosphorylated in human lymphoblastoid cells by both adenosine kinase and deoxycytidine kinase [8]. This appears not to be the case in CHO cells, since adenosine kinase deficiency renders the cells resistant to ara-A but deoxycytidine kinase deficiency does not. This observation is consistent with those of Chan and Juranka [9] who found ara-A resistant mutants of baby hamster kidney cells that were deficient in adenosine kinase, but none that demonstrated a deficiency in deoxycytidine kinase. Ullman et al. [10] found, in studying deoxyadenosine metabolism in cultured human cells, that although the physiologically important activity was associated with adenosine kinase, the majority of the activity in the extracts was associated with deoxycytidine kinase. Deoxycytidine kinase from some sources such as calf thymus [11,12] has been found to be relatively nonspecific towards deoxynucleosides, phosphorylating deoxyadenosine as well as deoxyguanosine and deoxycytidine. The C H O cell enzyme is similar in this respect, with deoxycytidine being the best substrate of the three and deoxyadenosine the poorest. As reviewed by Henderson [1] in a variety of cell types, this is frequently but not always the case. In addition to deoxycytidine kinase, C H O extracts contain a very active deoxyguanosine kinase that efficiently phosphorylates deoxyinosine as well. The intracellular location of this enzyme as well as its function in cell metabolism remain unclear. C H O cells have been reported [13] to contain two deoxycytidine kinases, one of which is of mitochondrial origin and does not phosphorylate ara-C. The possibility that the C H O cell deoxyguanosine kinase activity could reflect this mitochondrial enzyme was tested by locating another deoxycytidine kinase activity using high specific activity [3H]deoxycytidine as s u b s t r a t e (not shown). This p r e s u m a b l y mitochondrial kinase eluted from the column in fractions 30-35 and was quite distinct from deoxyguanosine kinase. A deoxyguanosine, deoxya-

denosine kinase from calf thymus has been described by Gower et al. [14] that does not phosphorylate deoxycytidine and is localized in the mitochondria. The deoxycytidine kinase is located in the cytosol and does phosphorylate deoxyguanosine. Deoxyguanosine kinases, distinct from deoxycytidine kinase, have also been observed in extracts of mouse thymocytes [15], neonatal mouse skin [16] and human placenta [17].

Acknowledgments The initial experiments in this study were supported by grant P01 CA 14528 from the National Cancer Institute. The later stages of the investigation were supported by grant CA 35788, also from the National Cancer Institute.

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