[CANCER RESEARCH 40. 3555-3558. October 1980] 0008-5472/80/0040-OOOOS02.00 Effects of Deoxyadenosine Triphosphate and 9-/?-D-Arabinofuranosyl- adenine S'-Triphosphate on Human Ribonucleotide Reducíasefrom Molt-4F Cells and the Concept of "Self-Potentiation"1 Chi-Hsiung Chang2 and Yung-Chi Cheng3 Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill. North Carolina 27514 ABSTRACT conceivable that the intracellular activity of this enzyme is influenced or even regulated by the balance of the steady-state Deoxyadenosine triphosphate (dATP) acted as a noncom level of various nucleotides. That is, changes in pool sizes of petitive inhibitor with respect to the specific nucleoside tri nucleotides could influence the reduction of different ribonu- phosphate activator for the reduction of all four common ribo- cleoside diphosphates and result in changes in deoxyribonu- nucleoside diphosphates catalyzed by the reductase derived cleotide levels. Among the nucleotides examined, ATP acted from human Molt-4F (T-type lymphoblast) cells. The inhibition either as an activator for pyrimidine ribonucleoside diphos constant of dATP for different ribonucleotide reduction reac phate reduction or as an accessory activator for purine ribo tions was different, indicating that the binding of the nucleoside nucleoside diphosphate reduction. dATP is the only nucleotide triphosphate activator or substrate could modify the binding which inhibits reduction of all 4 natural ribonucleotides. The affinity of dATP to the enzyme. dATP also acted as a noncom Ki's (obtained by the replots of intercepts and slopes versus petitive inhibitor with respect to cytidine diphosphate (CDP) for inhibitors) were reported previously (4), but the detailed exper reductase-catalyzed CDP reduction. 9-/8-D-Arabinofuranosyl- adenine S'-triphosphate acted as a competitive inhibitor with imental results were not reported; these are included in this paper. It should be noted that the regulation of the enzyme respect to either adenosine triphosphate or guanosine triphos derived from Molt-4F cells is similar to that of the enzymes from phate for CDP or for adenosine diphosphate reduction, respec various sources such as Escherichia coli (9, 10) or other tively. The inhibition constant was 15 /¿MforCDP reduction and mammalian cells (12-14). However, they do differ in their 4 ¡IMfor adenosine diphosphate reduction. 1-/8-o-Arabinofu- ranosyladenine S'-triphosphate could not substitute for aden kinetic properties. Since 9-/?-D-arabinofuranosyladenine exerts cytotoxic ef osine triphosphate or guanosine triphosphate as the activator fects after its conversion to a nucleotide, the effect of ara-ATP4 for CDP or adenosine diphosphate reduction, respectively. The effects of 9-ß-D-arabinofuranosylcytosine 5'-triphosphate and on this enzyme is important for a better understanding of its action. The effects of ara-ATP and ara-CTP on a crude prepa 5-iodo-2'-deoxyuridine 5'-triphosphate on ribonucleotide re ductase were also included for comparison. The "self-potentia- ration of rat tumor ribonucleotide reductase were examined by Moore and Cohen (11 ). It was concluded that ara-ATP inhibited tion" mechanism of the action of 9-ß-D-arabinofuranosylade- nine and 5-iodo-2'-deoxyuridine is discussed. the reduction of all 4 common diphosphate ribonucleotides to about the same extent; inhibition appeared to be similar to that produced by dATP at a lower concentration. ara-CTP was a INTRODUCTION weak inhibitor of this enzyme activity (11 ). Since the purity and source of the enzyme that we studied are different, the effects Ribonucleotide reductase is one of the key enzymes respon of ara-ATP on the partially purified ribonucleotide reductase sible for the synthesis of deoxyribonucleotides which are re from human Molt-4F cells were examined in detail, and the quired for DMA synthesis. Therefore, this enzyme could be effects of ara-CTP and IdUTP on this enzyme are included for considered a target for anticancer agents. The activity of this comparison. enzyme in cells could also influence the cytotoxic effects of certain antitumor agents such as 1-/?-D-arabmofuranosylcyto- sine, 1-/?-D-arabinofuranosyladenine, 5-iodo-2'-deoxyuridine, MATERIALS AND METHODS and 5-fluoro-2'-deoxyuridine. Thus, it is of importance to un Materials. Nucleoside di- and triphosphates (sodium salts), derstand the kinetic behavior of this enzyme. DTT, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid We have partially purified ribonucleotide reductase from a were purchased from Sigma Chemical Company, St. Louis, human T-type lymphoblast tumor line (Molt-4F) (2). The kinetic Mo. ara-ATP, IdUTP, and ara-CTP were purchased from P-L behavior of this enzyme in utilizing ADP, GDP, CDP, and UDP Biochemicals, Inc., Milwaukee, Wis. Ammonium salts of all '"C- as substrates indicated that the enzyme can be regulated by labeled nucleotides were supplied by Amersham/Searle Corp., natural nucleoside triphosphates or diphosphates (3, 4). It is Arlington Heights, III. Dowex-1 CI was obtained from Bio-Rad Laboratories, Richmond, Calif. Materials required for cell cul ' This work was supported by USPHS Project Grant CA-27449 from the tures were purchased from Grand Island Biological Co., Grand National Cancer Institute. NIH. This is Paper 5 in a series on human ribonucleotide reductase from Molt-4F cells. Island, N. Y. All other chemicals were of reagent grade. 2 Present address: Biochemistry Department. Southern Research Institute. 2000 Ninth Avenue South, Birmingham, Ala. 35205. 3 An American Leukemia Society Scholar. To whom requests for reprint should " The abbreviations used are: ara-ATP. 9-/i-D-arabinofuranosyladenine 5'- be addressed. triphosphate; ara-CTP. 9-/i-D-arabinofuranosylcytosine 5'-triphosphate; IdUTP. Received February 4. 1980; accepted July 9. 1980. 5-iodo-2'-deoxyuridine S'-triphosphate; DTT. dithiothreitol. OCTOBER 1980 3555 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1980 American Association for Cancer Research. C-H. Chang and Y-C. Cheng Preparation of the Enzyme Components. The methods for (A) culturing Molt-4F cells and the purification of Components A \fu 50 ntercept and B of this ribonucleotide redactase have been described (x-x) 30 previously (2). The final preparations of Components A and B are not homogeneous as judged by electrophoretic techniques. However, they were purified to such an extent that nucleotide phosphatase and nucleoside diphosphate kinases, which would interfere with kinetic studies of the enzyme, were not present in the purified Components A and B. All of the studies reported here were performed using the reconstituted enzyme from highly purified components. 05 LO I5 2.0 Enzyme Assays. CDP reducíasewas assayed by the method ATPJ , mMJ of Steeper and Steuart (15) with the use of Dowex 1-borate ion-exchange chromatography. Under "standard conditions," l/v I Slope Intercept 5O the assay mixture contained, in a final volume of 0.2 ml, (x-x) [14C]CDP (0.2 /iCi; 0.15 ITIM),DTT (3 mvi), MgCI2 (6 rriM), ATP (5 rriM), and a specific amount of the enzyme. ADP reductase dATP.uM -50 -30 -IO IO 30 50 activity was determined by the method of Cory et al. (6). Under 50 dATP, uM standard conditions, the assay mixture contained, in a final volume of 0.2 ml, [14C]ADP (0.4 jnCi; 0.3 rriM), DTT (3 ITIM), MgCI2 (6 ITIM), dGTP (5 HIM), and a specific amount of the 20 40 60 enzyme. UDP and GDP reductase activities were determined CDPJ,mM-1 by the method reported previously (3). Under standard condi tions, the assay mixture contained, in a final volume of 0.2 ml, [14C]UDP (0.2 /iCi; 0.15 rriM) or [14C]GDP (0.2 ¿iCi;0.15 mw), Chart 1. Effects of dATP on the Lineweaver-Burk plots of the reciprocal of DTT (3 rriM), MgCI2 (6 rriM), ATP (5 ITIM)for UDP reduction or activator concentration (A) or substrate concentration (B) with respect to the dTTP (5 rriM) for GDP reduction, and a specific amount of the reciprocal of reaction velocity (nmol of dCDP produced in 1 hr). Standard incubation conditions were used except for addition of activator (A) or substrate enzyme. An enzyme sample heated for 2 min in a boiling water (8) at the indicated concentration. Each assay contained 14 /xg of purified bath prior to the addition of the labeled substrate served as the Component A and 12 fig of purified Component B of ribonucleotide reductase reaction blank. The incubation was at 37° for 60 min; the obtained from Molt-4F cells. reaction was linear with respect to time and enzyme concen tration during the incubation period. Protein Determination. Protein concentrations were deter mined by the method of Bradford (1 ). Bovine albumin was used as the standard. -IO -6 -2 246 8 RESULTS dATP, Inhibition by dATP of the Enzyme Activity for CDP, ADP, UDP, and GDP Reduction. In the preceding report (4), the inhibition constants of dATP on various ribonucleotide reduc tions were summarized without describing the kinetic data. 0.4 I.2 2.0 Detailed kinetic data are described here in Charts 1 and 2 for dGTP-l, mM-l purposes of comparison with the effects of ara-ATP. dATP behaves as a noncompetitive inhibitor of CDP reduction with respect to substrate (CDP) or activator (ATP) according to the definition of Cleland (5). The double reciprocal plot of initial velocity versus substrate or activator is shown in Chart 1. The -D -6-2 24 68 values of K¡intercept and K, slope for dATP with respect to ATP for CDP reduction were calculated based on the replot dATP,/iM and gave the same value of 40 JUM(Chart 1/4). The values of Ki intercept and K¡slope for dATP with respect to CDP for CDP reduction were 60 and 21 fiM, respectively (Chart IB). -I.2 -0.4 Q4 I.2 2.0 Since both GTP and dGTP act equally well as the activator GTP-1, mM-1 for ADP reduction, the effect of ATP was examined with respect to GTP or dGTP on the reduction of ADP.