[CANCER RESEARCH 39, 3102-3106, August 1979] 0008-5472/79/0039-0000$02.O0 Some Properties of Uridine-Cytidine Kinase from a Human Malignant Lymphoma1

Nahed K. Ahmed2and Arnold D. Welch

Division of Biochemical and Clinical Pharmacology, St. Jude Children ‘sResearchHospital, Memphis, Tennessee38101

ABSTRACT partially purified from rat liver (4, 17), muninetumor cells (5, 6, 13), calf thymus (12), human neoplastic lymphoid cells (3), Unidine-cytidine kinase has been partially purified from a Ehrlich ascites tumor (21), Novikoff ascites rat tumor (17), and malignant human lymphoma, and some of its properties have also from Tetrahymena pyriformis (18). Urd-Cyd kinase cata been described. The apparent Michaelis constants for unidine lyzes the following reaction: and adenosine 5'-triphosphate are 7 x 1O@and 4 x i O@M, .. .. (Urd-Cyd)-kinase respectively. Maximal enzyme activity is observed between pH Urldine or cytldlne + NTP 6.5 and 8.5, and temperatures of incubation are observed uridine5'-monophosphateorcytidine5'-monophosphate+ NDP between 37 and 42°.The enzyme requires Mg2@for full enzy matic activity and exists in two isozymic forms, as indicated by where NTP is nucleoside tniphosphate, and NDP is nucleoside isoelectric focusing and column chromatography on Sepharose diphosphate. Previous partial purifications and the proper 6B. The roles of these two isozymes, i.e., the adult (I) and the ties of the enzyme have been reviewed (1, 2). The enzyme embryonic (II) forms, are not yet clear; conceivably, however, appears to require the nibofuranosyl moiety because 2'-deoxy they may have relevance to the problem of the development of nibonucleosides and certain arabinofuranosyl- and xylofura resistance to chemotherapeutic analogs that require unidine nosyl-containing nucleosides tested have not been phospho @ cytidine kinase for their ‘activation.― rylated (i 3, 18, 21). Urd-Cyd kinase has a requirement for Potent inhibitors (pyrazofunin and N-phosphonacetyl-L-as Mg2@that was only partially fulfilled by Mn2―(13, 16) and Fe2@ partic acid) of the biosynthesis de novo of unidine 5'-mono (i 3, 18). Various pH optima, mostly between 6.5 and 8.5, have phosphate have not inhibited significantly the growth of other been reported for different preparations of the enzyme (2, 13, than a relatively few experimental tumors. Most neoplastic cells 17, 21). The apparent Km'5of the enzyme have been described; also can derive their essential supplies of unidine 5'-mono these approximate 1O@ M for the nucleoside substrate and phosphate by one on another route, especially through the i03 to i0@4 M for ATP (10, 18, i9, 21). Also, patterns salvage of unidineand cytidine, as catalyzed by uridine-cytidine obtained with Urd-Cyd kinase purified 250-fold from a murine kinase. No effective inhibitor of this key enzyme in the salvage mast cell neoplasm, P815, showed a Kmof 1.5 x 1O'@M for pathway has been reported. Among a variety of compounds uridine and 4.5 x 1O@ M for cytidine (14). Although the (20 substances) so far examined by us for their possible ability molecular weight of the enzyme has been reported to be quite to interfere with the phosphorylation of [5-3H]unidine by aden high, namely, in the region of 150,000 to 200,000 as judged osine 5'-triphosphate, as catalyzed by unidine-cytidine kinase, by gel filtration (13, 18), those kinases obtained from rat liver none is a powerful inhibitor of this enzyme, although leads for (1 1) and from Ehrlich ascites cells (10), respectively, were future development have been obtained. In the meantime, described as existing in 2 isozymic species: one of molecular information concerning the uridine-cytidine kinase activity of a weight of about 120,000, and the other with a molecular weight variety of munine and human neoplasms (particularly of cob of about 30,000. rectal origin), as well as in normal tissues, is being expanded; The reaction that this enzyme catalyzes is part of the ana these findings may contribute to the design and prospective bolic pathway by which preformed unidine and cytidine can be syntheses of potential chemotherapeutic agents, functioning salvaged for nucleic acid biosynthesis. Because our primary as inhibitors of the salvage of uridine and cytidine. interest is in delineating the roles of salvage, and in discovering effective inhibitors of these processes, especially that step INTRODUCTION catalyzed by Urd-Cyd kinase, the present investigation was undertaken. The principal objective of the investigation has Urd-Cyd3-kinase (ATP:unidine 5'-phosphotransferase, EC been to provide more information concerning the properties of 2.7.1 .48) catalyzes the phosphorylation, particularly by ATP, a partially purified Urd-Cyd kinase isolated from a human of unidine, cytidine, and some nucleoside analogs (6-azauni malignant lymphoma and its activity relative to different unidine dine, 5-fluorounidine, 5-fluorocytidine, 5-azacytidine, 3-dea and cytidine derivatives (potential inhibitors), which could affect zauridine, etc.). This enzyme has been previously isolated and the activity of this enzyme and its role in the salvage pathway.

I This investigation was supported by the Whitehall Foundation, Grant MATERIALSAND METHODS CA21677 from the National Cancer Institute, Department of Health, Education, and Welfare, and American Lebanese Syrian Associated Charities. [5-3H]Uridine, DE81 discs, and ATP were purchased from 2 To whom requests for reprints should be addressed, at St. Jude Children's Research Hospital, P. 0. Box 318, Memphis, Tenn. 38101. Monavek Biochemicals (City of Industry, Calif.), Whatman, Inc. 3The abbreviations used are: Urd-Cyd kinase, uridine-cytidine kinase; DE81, (Clifton, N. J.) and Sigma Chemical Co. (St. Louis, Mo.), re DEAE-cellulese paper; BME, fl-mercapteethanol; PALA, N-phosphenacetyl-L-as partic acid. spectively. RiaSolve II was obtained from Research Products Received January 24, 1979; accepted May 8, 1979. International Corp. (Elk Grove Village, Ill.); all other reagents

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and solvents were of the purest grade available. an LKB 8101 ebectrofocusing column (1 10-mI capacity) with 1% carrier ampholyte (pH range, 3 to 10) and a stabilizing sucrose-gradient containing 0.5 mM dithiothreitol. Dialyzed METHODS ammonium sulfate fractions were introduced into the middle of Enzyme Preparation. A human malignant lymphoma was the sucrose gradient of the column. Electrofocusing lasted obtained at autopsy (within 6 hr of death), and the neoplastic about 24 hr at 4°,with an initial power of 5.0 watts. After tissue was separated, insofar as possible, from fibrous and electrofocusing was completed, the column contents were necrotic materials. The neoplastic mass was then divided into collected in 1-ml fractions at a flow rate of 60 ml/hr; pH small portions, wrapped, and frozen for subsequent prepara measurements and assays of Urd-Cyd kinase activities were tions of the enzyme; all steps concerning the preparation and performed immediately. uses of the enzyme were performed at 0—4°. Determination of pH Profile. The pH profile of the kinase The tissues were homogenized in ice-cold Tris-HCI buffer was determined in the indicated buffers at concentrations (200 mM) (pH 7.5), containing 10 mM BME. The supernatant giving conductivity readings of 4700 to 5200 m@2;otherwise, fraction of the homogenate, obtained after a preliminary cen assays were identical to those described under . Materials and trifugation at 15,000 x g, was then subjected to high-speed Methods.― centrifugation (100,000 x g) for 90 mm at 4°.Thesupernatant Chromatography with Sepharose 6B. The dialyzed 30 to fluid was treated with solid ammonium sulfate to yield a level of 50% ammonium sulfate fraction, equivalent to 10.6 mg of saturation of 30%; the supernatant material obtained after protein, was applied to a 2.3- x 27-cm Sepharose 6B column. centnfugation was retained for retreatment with solid ammo The Sepharose column was equilibrated, and 1-ml fractions nium sulfate to yield a level of 50% saturation. The recovered were eluted with 200 mt@iTris—HCIbuffer(pH7.5), 20 [email protected], precipitate was dialyzed against 2 changes (each of i OOx and 20% glycerol; 8O-@tlaliquots of the eluted fraction were volume at 4°)of the homogenizing buffer; this fraction served assayed for Urd-Cyd kinase activity. as a source of the enzyme activity used in all the experiments described in this study. RESULTS Protein Determinations. Protein determinationswere per formed by the method of Lowry et a!. (15). The enzymatic formation of uridine nucbeotides, as functions Uridine Kinase Assay. Uridine kinase activitywas assayed of enzyme concentration and time, was studied. A linear reba by a recently modified procedure4 utilizing DE81 discs. The tionship was obtained up to 200 j@gofenzyme protein; nucleo following mixture ordinarily was used in a final volume of 0.2 tide formation also was linear with respect to time for periods ml: Tris-HCI buffer (pH 7.5 to 7.7), 200 mM; BME, 10.0 mM; of up to 10 mm. Accordingly, most of the assays were carried MgCl2, 7.5 mM;ATP, 3.9 mM;[5-3H)uridine(0.1 MCi),0.5 mM; out with 100 to 150 ,sgof enzyme protein during an incubation and 100 to 200 ,@gofthe dialyzed ammonium sulfate fraction. timeof 5 mm. Incubation was carried out in tubes for 5 to 10 mm (37—40°), Dependence on Substrate Concentration. The Lineweaver and the reaction was stopped by immersion of the tubes in Burk plots in Charts 1 and 2 demonstrate the dependence of water at 100°for 3 mm; denatured protein was removed by the reaction rate on the concentrations of uridine and ATP. centrifugation. The content of [5-3Hjuridine 5'-phosphate (as Slight inhibition was observed at a concentration of ATP of well as any higher phosphate esters of the radioactive uridine) about 18 x Km.The apparent Km'5for ATP and uridine were 4 was measured by spotting the supernatant fluid on DE8i discs. x 1o-@and 7 x 10-@M, respectively, at saturation concentra The discs were washed carefully with water (3 times), dried, tions of the second substrate. Accordingly, the concentrations and eluted with 1 ml of 0.1 M HCI:0.5 M NaCI in scintillation of both ATP and undine used in all assays were 7 to 10 x Km. vials, which were shaken for 30 mm to permit the labeled Dependence of Urd-Cyd Kinase Activity on pH and Tem nucleotide(s) to be eluted from the discs. Subsequently, 9 ml of RiaSolve II were added to the vials, which were shaken vigorously until the discs disintegrated; the vials were then placed in a liquid scintillation counter for determinations of the amounts of radioactivity present. To correct for any irreversible adsorption on the DE81 discs, blanks containing the reaction mixture and aliquots of boiled enzyme were processed identi cabby,and the apparent radioactivity, in dpm, was subtracted IS) —I> from all experimental values. 0.75 The kinase activity5 is expressed as nmoi of unidine nucleo tides/mm/mg protein and calculated as follows: 0.50

— dpm in sample dpm in blank 0.25 dpm in control 008 0.04 004 0.08 0(2 Isoelectric Focusing. Isoelectnic focusing was performed in (•k) Chart 1. Dependence of Urd-Cyd klnase activity on the concentration of 4 N. K. Ahmed and A. D. Welch. Determination of uridine-cytidine kinase uridine. Standard conditions were used, as described under ‘‘Materialsand actMty by an assay using DEAE-paper disks, submitted for publication to Ana Methods.“Thereaction, run at 37°,was inItiated by the addItion of 100 @gof lytical Chemistry, 1979. enzyme protein and incubated for 5 mm. Ordinate, enzyme activity (nmol/mln/ 5 An aliquot of the reaction mixture is counted directly. mg protein); abscissa, amounts of uridine (nmol present in the reaction mixture).

AUGUST 1979 3i03

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. N. K. Ahmed and A. D. Welch 9.6 Kinase from a Human Malignant Lymphoma. Amonga variety of compounds (for sources, see Table 1) so far examined by @ ; 6.4 us for their possible ability to interfere with the phosphorylation of [5-3H]uridine by ATP as catalyzed by the kinase (derived @ 32 U from the human malignant lymphoma) (Table 1), most exerted little or no inhibition when tested at a level 7-fold higher than 200 400 600 800 )OOO'i@O0 S) that of the Km of uridine (7 x 1O@ M). Among the several —I> compounds so far examined, some of particular interest have been the 2',3'-isopropylidene derivative of undine; the so 0.75 called dialdehyde of cytidine (formed by opening the ribofura nose ring between carbons 2' and 3'); and the 1-methoxy-2,3- 0.50 dialdehyde derived from 1-methoxyribofuranose, 6-aminocyti dine, 5-bromo-, and 5-iodo-unidine, as well as xanthosine 0.25 (which may be regarded as a derivative of uridine). A possible hint of activity has been observed with 5-cyanouridine, but not 0.0) 0.0) 0.02 0.03 004 with either 6-cyanocytidmneor 6-aminocytidine. 5'-Nitrouridine (Uk) possesses definite (but still moderate) activity, i.e. , it caused a Chart 2. Dependence of Urd-Cyd kinase activity on ATP concentration (using reduction of about 40% in the formation of UMP; studies of its [5-3H]urldine as substrate). The reaction, run at 37°,wasinitiated by the addition possible effect on the conversion of UMP to UDP are to be of 100 M9of enzyme protein and incubated for 5 mm. Standard conditions were pursued. used, as described under “MaterialsandMethods.―Ordinate,enzyme activity (nmol/min/mg protein); abscissa, amounts of ATP (nmol present in the reaction mixture). DISCUSSION The utilization of uracil, uridine, and cytidine for the synthesis perature. A dialyzed ammonium sulfate fraction was used to examine the activity profile relative to pH of the human malig nant lymphoma kinase. Optimal activity occurred at pH levels between 6.5 and 8.9 and at incubation temperatures between 37 and 45°; accordingly, the enzyme activity ordinarily was assayed at 40°and pH 7.5. Stimulation by Cations. Mg2@wasrequiredfor full enzyme activity. The dependence of the reaction rate on the concen 0 tration of Mg2'@was studied at a fixed concentration of ATP (3.9 mM). The maximal concentration of Mg2―was between 2.5 0. and 8 mM; higher concentration than 8 m@iresulted in a slight inhibition of enzyme activity, while concentrations of about 30 mMreduced enzyme activity by about 50%.6 The concentration of Mg2@usedin all our assays was 7.5 [email protected] was also tested 20 40 60 80 for its effect on the activity of Urd-Cyd kinase (concentrations TUBENO. between 1.25 and 35 mM were tested in the presence of 7.5 Chart 3. Iseelectric focusing of Urd-Cyd kinase of malignant humanIymphoma mM MgCl2); because no effect on the activity of the enzyme over the pH range of 3 to 10. lsoelectnc focusing and kinase assays were was observed, KCI was not included in the standard enzyme performed as described under “MaterialsandMethods.―Fractionsof 1 ml each were collected and read at 280 nm; 100 p1were used for Urd-Cyd kinase assays, assay. Incubated at 37°for 10 mm, and processed as described under “Materialsand Isolation of Urd-Cyd Kinase Isozymes by Isoelectric Fo Methods.― cusing and Sepharose 68 Chromatography. Evidencefor the existence of 2 forms of uridine kinase in the human malignant lymphoma are presented. Isoelectnic focusing of the dialyzed 0 ammonium sulfate fraction over a pH gradient from 3 to 10 @@i02 A 0.8 resolved the enzyme activity into 2 separate activity peaks, one 0.6 with a p1of 4.6 to 4.8 and a second with a p1of 6.2 to 6.5 F') 0.) (Chart 3). Minor activities were detected occasionally, but 04 these 2 major peaks of activity were evident even from one ! 0.) jl'\j'9/@\@q@ enzyme preparation to others. The existence of 2 major iso @. @4I @°@‘ 02 @ zymes also was confirmed by column chromatography on _o,@J! ‘I @\,°“q,,, Sepharose 6B. A typical elution profile of Urd-Cyd kinase 0 20 30 40 50 60 70 activity in the 30 to 50% ammonium sulfate fraction is shown FractionNumber in Chart 4. Again, 2 activity peaks were resolved, a finding that Chart 4. Sepharese 6B chromatography of the 30 to 50% ammonium sulfate confirmed the existence of 2 isozymes in this human malignant fraction from a malignant human lymphoma. An allquot of the ammonium sulfate fraction (10.6 mg enzyme protein) was applied to Sepharese 6B column (2.3 x lymphoma. 27 cm), equilibrated, and eluted with 200 [email protected] (pH 7.5) 20 m@ Effect of Certain Compounds on the Activity of Urd-Cyd $-mercapteethanol, and 20% glycerol. Fractions of 1 ml were collected and read at 280 nm. Eighty f41wereused for the kinase assays, and these were incubated 6 N. K. Ahmed and A. D. Welch, unpublished observations. at 37°for 15 mm and processed as described under “MaterialsandMethods.―

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. Properties of a Human Lymphoma Uridine Kinase of RNA pynimidines represents an alternative pathway to the Table1 biosynthesis de novo of UMP via orotic acid and orotidine 5'- Effectof certain compounds @ptheactivity of Urd a%kinase from monophosphate. During recent years, particular interest has .humanmalignantlymphomaCyd been developed in connection with work on growth-inhibiting InhlbltienCCompoundbExperi- pynimidine analogs or related antimetabobites, most of which Experi are active only after transformation to nucleotides (8, 20). In 212',3'-lsopropylidene ment 1d ment mammalian tissues, this often involves the participation of ‘—@21 uridine1 2 enzymes of either the uracil or the uridine-cytidine pathways. —35-Bromo-6-azauridine1-Methexy ribese dlaldehyde1 2 —4Cytidine 2 Urd-Cyd kinase is one of the important enzymes in these —5Pyrazefurin14dialdehyde1 7 alternative pathways, and it was therefore of much interest to —6xanthesine1 study the properties of this enzyme, when derived from a —75-lodouridine16 3 —85-Bromouridine1 human neoplasm. —95-Chlorouridine1 6 The enzyme has a requirement for Mg2@,and the apparent —1 6 pH optimum lies between 6.5 and 8.9. The Kmfor unidine is 7 16105-Cyaneuridine1 9 —116-Aminocytidine— x 1o-@M, while that for ATP is 4 x 1O@ M. Most of these 1126-Cyanocytldine1 1 1 properties were in agreement with properties cited for other 213Cytidine-6-methylcarbeximidate— Urd-Cyd kinases, partially purified from other sources. There 614Cytidine-6-carboxylic acid— 9156-Tetrazolouridine— have been suggestions from other studies that multiple forms 9boxamide161-f3-o-Ribefuranesyl-2-exo-4-imidazoline-4-car — of Urd-Cyd kinase may exist (10, 11). In the present study, the —175-Diazouridine1 5 heterogeneity of the enzyme derived from the human lymphoma —185-(3,3-Dimethyl-1 -triazeno)undine1 3 was evidenced by both isoelectnic focusing and column chro 9205'-Nitrouridine4591 ,3-Dihydroxy-4-fl-ribofuranesylbenzene— matography on Sepharose 6B, which led to the separation of 36 2 major peaks of activity. a Compounds 1 and 6 were purchased from Sigma Chemical Co. Compounds 2 and 4 were kindly supplied by Dr. Richard Cysyk of the National Cancer It has been reported that potent inhibitors (pynazofurin and Institute. Compounds 3 and 20 were synthesized by Dr. D. J. Smith and Dr. T. L. PALA) of steps in the de novo pathway for the biosynthesis of Chwang, St. Jude Children's Research Hospital. Compound 5 was kindly supplied UMP have failed to affect very significantly the growth of other by Dr. Irvin9 Johnson of the Lilly Laboratories, Indianapolis, Ind. Compounds 7 to 16 were gifts from Dr. T. Ueda, Hekkaido University, Sapporo, Japan. Com than relatively few experimental tumors. It also has been re pounds 17 and 18 were gifts from Dr. L. B. Townsend, Department of Medicinal ported that pyrazofunin (7) and PALA (9) are without effect on Chemistry, University of Utah, Salt Lake City, Utah. Compound 19 was provided the growth of Li 210 cells in vitro. Evidence that the biosyn by the Drug Synthesis and Chemistry Branch, National Cancer Institute, Be the@Ja, Md. thesis de novo of UMP by Li 210 cells is blocked by pyrazofurin All compounds were tested at 5 x 1O@ N, and solutions were freshly has been obtained both by Brockman [discussion of the paper prepared within 1 hr prior to each experiment. by Gutowski et a!. (7)] and by Handschumacher,7 who have C Inhibition of control values. d Experiments 1 and 2 represent 2 different enzyme preparations. The cem demonstrated that pyrazofunin causes the accumulation of both pounds were tested at least 3 times (each time in duplicate or triplicate). orotic acid and orotidine in Li 210 cells; thus, the possibility e nodifferencefromcontrolvalues. that Li 210 cells can salvage uridine and cytidine was afforded. Indeed, we have found that the Urd-Cyd kinase activity of neoplasms studied). Also, the Urd-Cyd kinase isozymes de Li 2i 0 cells is unusually high as compared to that of other nivedfrom Li 210 cells are being studied because these provide neoplastic cells studied.8 a readily available source of the enzyme, as compared to the Although several inhibitors of the biosynthesis of UMP de limited amounts of tissue provided by samples obtained by novo exist in addition to pyrazofunin 5'-phosphate, 6-azaunidine biopsy or at autopsy with most human, as well as rodent, 5'-phosphate, and PALA, none of practical significance for neoplasms. either Und-Cyd kinase activity or other pynimidine-salvaging The basic information obtained in this study should prove enzymes has been developed. It is possible, however, that a useful in further characterizations of this enzyme, for compar useful inhibitor of Urd-Cyd kinase could reduce salvage signif isons with those of other neoplastic, as well as normal, tissues, icantly; hence, preliminary studies of potential antagonists of and for correlating the activity of this enzyme in vivo to that the utilization of either uracil, uridine, cytidine, or their corre obtained subsequent to the use of various chemotherapeutic sponding derivatives, eventually may disclose compounds of agents. chemotherapeutic utility, particularly when used judiciously in combination with short-acting inhibitors of the de novo biosyn REFERENCES thesis of UMP. As shown in Table 1, none of 20 compounds so far tested has proved to be a really good inhibitor of Urd-Cyd 1. Anderson, E. P. Nucleoside and nucleetide kinases. In: P. D. Beyer (ed), The Enzymes, Vol. 9, pp. 49-96. New York, Academic Press, Inc.. 1973. kinase. 2. Cihák,A., and Rada, B. Uridine kinase: properties, blolegical significance Extensions of the information so far obtained with the Urd and chemotherapeutic aspects (a review). Neoplasma (Bratisl.), 23: 233— Cyd kinase isozymes derived from the human malignant lym 257, 1976. 3. Drake, J. C., Stoller, R. G., and Chabner, B. A. Characteristics of the enzyme phoma are now being made; these will involve a wide variety of uridine-cytidine kinase isolated from a cultured human cell line. Biochem. murine and human neoplasms (especially those of coborectal Pharmacol., 26: 64—66,1977. origin, preliminary studies of which suggest that their Urd-Cyd 4, Fulchigneni-Lataud, M. C., Tuilie, M., and Roux, J. M. Uridine-cytidine kinase from feetal and adult rat liver. Purification and Study of Some kinase activities are exceptionally low, relative to those of other Properties. Eur. J. Biochem., 69: 217-222, 1976. 5. Furner, R. L., and Mellet, L. B. Kinase and deaminase activity in a variety of subcutaneous mouse tumors. Cancer Res., 35: 1799—1803,1975.

7 R. E. Handschumacher, personal communication. 6. Greenberg, N., Schumm, D. E., Hurtubise, P. E., and Webb, T. E. Uridine 8 N. K. Ahmed and A. D. Welch, unpublished data. kinase activities in normal and neoplastic lymphoid cells. Cancer Res., 37:

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1028-1034, 1977. Biophys., 168: 66-73, 1975. 7. Gutowski, G. E., Sweeney, M. J. , DeLong, D. C. . Hamill, R. L., and Gerzon, 14. Liacouras, A. S., and Anderson, E. P. Uridine-cytidine kinase. Kinetic studies K. Biochemistry and biological effects of the pyrazofurins (pyrazomycins): and reaction mechanism. Arch. Biochem. Biephys., 168: 74—80,1975. Initial clinical trial. Ann. N. Y. Acad. Sci., 255: 544—551,1975. 15. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein 8. Handschumacher, R. E., and Pastemak, C. A. Inhibition of orotodylic acid measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265—275, decarbexylase, a primary site of carcinostasis by 6-azauracil. Biochim. 1951. @ Biophys. Acta, 30: 451 —452,1958. 16. Orengo, A. Feedback regulation in the ribopyrimidine salvage'‘pathway. 9. Johnson, R. K., lnouye, T., Goldin, A., and Stark, G. R. Antitumor activity of Exp. Cell Res., 41: 338-349, 1966. N-(phosphonacetyl)-L-aspartic acid, a transition-state inhibitor of aspartate 17. Orange, A. Regulation of enzymic activity by metabolites. I. Uridine-cytidine transcarbamylase. Cancer Res., 36: 2720—2725,1976. kinase of Novikoff ascites rat tumor. J. Biol. Chem., 244: 2204—2209,1969. 10. Krystal, G., and Scholefield, P. G. The partial purification and properties of 18. Plunkett, W., and Mener, J. C. The role of uridine-cytidine kinase in the uridine kinase from Ehrlich ascites tumor cells. Can. J. Biochem., 5 1: 379— regulation of pyrimidine ribenucleotide synthesis in Tetrahymenapyriformis 389, 1973. GL. Biochim. Biophys. Acta, 250: 92—102,1971. 11. Krystal, G., and Webb, T. E. Multiple forms of uridine kinase in normal and 19. Reichard, P., and Sköld,0., Pyrimidine synthesis and breakdown. Methods neoplastic rat liver. Biochem. J., 124: 943—947,1971. Enzymol., 6: 194-1 97, 1963. 12. Lee, T., Karen, M., and Momparler, R. L. Kinetic studies en phosphorylation 20. Reichard, P., Skbld, 0. , and Klein, G. Possible enzymic mechanism for the of 5-azacytidine with the purified uridine-cytidine kinase from calf thymus. development of resistance against 5-fluerouracil in ascites tumors. Nature Cancer Res., 34: 2482—2488,1974. (Lond.),183:939-941, 1959. 13. Liacouras, A. S., and Anderson, E. P. Uridine-cytidine kinase. Purification 21. Sköld,0. Uridine kinase from Ehrlich ascites tumor: Purification and prop from a murine neoplasm and characterization of the enzyme. Arch. Biochem. erties. J. Biol. Chem., 235: 3273-3279, 1960.

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Nahed K. Ahmed and Arnold D. Welch

Cancer Res 1979;39:3102-3106.

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