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Effects of Nucleoside Triphosphates on Human Ribonucleotide Reductase from Molt-4F Cell&

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Effects of Nucleoside Triphosphates on Human Ribonucleotide Reductase from Molt-4F Cell& (CANCERRESEARCH39,5087-5092, December19791 0008-5472/79/0039-0000$02.00 Effects of Nucleoside Triphosphates on Human Ribonucleotide Reductase from Molt-4F Cell& ChlHsiung chang2 and Yung-chi Cheng3 Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 ABSTRACT INTRODUCTION The effects of nucleoside triphosphates on various nucleo The specificity of nibonucleotide reductase obtained from side diphosphate reductions catalyzed by a highly purified bacterial sources has been reported to be strongly influenced nbonucleotide reductase from MoIt-4F cultured human cells by different nucleoside tniphosphates (1, 10, 11, 16). Reduc were examined. It was found that deoxyadenosine 5'-tmiphos tion of pymimidine nibonucleotides catalyzed by the enzyme phate strongly inhibitedall four reductions.The reduction of system from Escherichia co!i B was stimulated by ATP and pyrimidine nucleoside diphosphate in the presence of an acti dTTP. GOP reduction was stimulated by dTTP, and AOP reduc vaton [adenosine 5'-tniphosphate (ATP)] was inhibited in a tion was stimulated by dGTP (1 0, 11). dATP strongly inhibited noncompetitive manner with respect to ATP by deoxyguano all 4 reductions. Moreover, COP and UDP inhibited the meduc sine 5'-trlphosphate (dGTP) and deoxythymidine 5'-tniphos tion of each other in the enzyme system from E. co!i B (10). phate (dTTP). For cytidine 5'-diphosphate reduction, the value The regulation of the reduction of nibonucleotides to deoxyni of the K, intercept for dGTP was 47 @tMandfor dTTP, it was bonucleotides has also been described for enzyme obtained 270 @u@i;theK slope was 25 @MfordGTP, 100 g@MfordTTP. from mammalian systems (1 2—14).The reduction of pynimidine Similarly, for unidine diphosphate reduction, the K intercept nucleoside diphosphates required ATP as activator for the was 4.3 @LMfordGTP, 25 @sMfordTTP. The K slope was 1.5 enzyme system from Novikoft tumor (12). Reduction of ADP @zMfor dGTP and 9 @LMfordTTP. The reduction of ADP in the and GOP required, respectively, dGTP and dTTP as activators. presence of its activator (dGTP) was inhibited noncompetitively Reduction of pynimidine nibonucleoside diphosphates was in by dTTP. The values of K intercept and slope of dTTP for hibited by dATP, dGTP, dTTP, and dUTP, reduction of GOP adenosine 5'-diphosphate (ADP) reduction were 1.8 and 0.9 was inhibited by dATP and dGTP, and reduction of ADP was mM, respectively. Although guanosine 5'-tniphosphate(GTP) inhibited by dATP. Similar results have been obtained with the and dGTP were found to serve equally well as activators for enzyme of rat embryos (13) and chick embryo (14), and calf ADP reductions with the same apparent K,, and Vmax,the thymus (9). inhibition pattern of GTP and dGTP on the enzyme activity for The regulation of the reduction of mibonucleotidesto deoxy cytidine 5'-diphosphate reduction was different. ATP was found ribonucleotides by the enzyme obtained from human Molt-4F to be an accessory activator for ADP reduction due to the fact cells has now been studied (3). In this communication, we have that ATP at 1.0 or 0.3 mM concentration decreased the appar further studied the effects of nucleoside tniphosphates on the ent K@ofGTP for ADP reduction from 1.1 to 0.14 or 0.08 m@i, enzyme from this cell line. respectively. In the absence of ATP, the V@ for ADP reduction was increased 2-fold. ATP at a concentration of 1.0 or 0.3 m@ MATERIALS AND METHODS also changed the apparent Ka of dTTP for guanosine 5'-di phosphate reduction from 1.25 to 0.9 or 0.6 @M,respectively, Materials. All the materials used were same as those de but V,,,@for guanosine 5'-diphosphate reduction was in scmibedin the preceding paper (5). creased. Preparation of the Enzyme Components. Molt-4F mibonu GTP at a concentration of 0.3 or 0.5 m@idecreased the cleotide reductase was prepared as described previously (4). apparent Ka'S of ATP for pynimidine nucleoside reduction. At All of the studies reported here were performed using the these concentrations of GTP, the Vm@for cytidine 5'-diphos reconstituted enzyme from the components after the final step phate reduction was decreased 7 and 12%, and the Vmaxfor of purification (phenyl Sephamose for Component A and sucrose uridine 5'-diphosphate reduction was decreased 33 and 45% density gradient centnifugation for Component B). when they were compared with those in the absence of GTP. Enzyme Assays. COPreductasewas assayedbythe method Therefore, a change in any of these nucleotideconcentrations of Steeper and Steuart (15), and ADP reductase activity was could lead to changes in nibonucleotide reductase activity. ATP determined by the method of Cory et a!. (8). The details for emerges as a most important factor in controlling the reduction assaying ADP, COP, UOP, and GOP were described in the of all four nbonucleoside diphosphates. preceding paper (5). Each experiment was repeated at least 3 times, and assays were done in duplicate. Protein Determination. Protein concentrationswere deter 1 This work was supported by USPHS Project Grant CA-i 8499 and Core mined by the method of Bradford (2). Bovine serum albumin GrantCA-i3038fromtheDivisionofCancerTreatment,NationalCancerInstitute, NIH.DepartmentofHealth,Education,andWelfare. was used as the standard. 2 Present address: Biochemistry Department, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Ala. 35205. RESULTS 3 An American Leukemia Society Scholar. To whom requests for reprints shouldbeaddressed.Presentaddress:DepartmentofPharmacology,University of North Carolina, Chapel Hill, N. C. 27514. Effects of Nucleoside Tniphosphateson CDP, UDP, ADP, ReceivedJune22, 1979;acceptedSeptember17, 1979. and GOP Reductions in the Presence of the Best Activators. DECEMBER 1979 5087 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. C-H. Chang and V-c. Cheng The effects of various nucleoside triphosphates at a concentra 20 tion of 2.5 mM on the enzyme activities for COP, UOP, ADP, and GDP reductions in the presence of the best activators are I00 shown in Table 1. In the absence of nucleoside tniphosphates, >@ except for the best activators, the enzyme activities for all 4 > .@ 80 reductions were determined and set as 100%. dATP was the 0 most potent inhibitor among those nucleoside tniphosphates 0 tested. It inhibited the enzyme activities for all 4 reductions at a concentration of 2.5 [email protected] the concentration of dATP was at 0.25 mM,as shown in Table 1 (numbers in parentheses), I:: dATP was still the most potent inhibitor. dTTP inhibited the reduction of COP, UOP, and AOP; dGTP inhibited the reduction 20 of pynimidine nucleoside diphosphates. However, ATP at a concentration of 2.5 m@islightly stimulated the reduction of GOP in the presence of the best activator, dTTP. Although GTP and dGTP served equally well as activators for AOP reduction, dGTP (X—X),and GTP (0-0); mM dGTP was a more potent nucleoside triphosphate than was Chart 1. Effects of GTP and dGTP on the enzyme activity for COP reduction. Standard incubation conditions for CDP reduction were used. Each reaction GTP as an inhibitor of COP meductase activity. mixture contained 14 @gofpurified Component A and 12 @zgofpurified Compo Different Effects of GTP and dGTP on the Enzyme Activity nentB of ribonucleotidereductaseobtainedfromMolt-4Fcells. for CDP Reduction. The effects of GTP and dGTP on the enzyme activity for COPreduction were different. The inhibition dGT@M pattern of these nucleoside triphosphates is shown in Chart 1. I00 Al 2r I 30 When the concentration of GTP and dGTP was varied, and the io[@ @( 25 enzyme activity for COP reduction in the presence of activator I/I' 81. 1 20 Intercept6 I 5 Slope (ATP) was measured, it was observed that dGTP at a concen (x—xI4 I 0 tration higher than 0.1 mt@igavemore than 50% inhibition of 2 0.5 lIz' the enzyme activity. However, GTP at a concentration of be @@L( III 25 tween 0.1 and 0.3 mM slightly increased the enzyme activity. -50 50 50 dGTF@j@M Inhibition was seen at GTP concentrations higher than 1.0 mt@i. Inhibition Constants for dGTP on the Enzyme Activity for 0 CDP and UDP Reductions. The inhibitionconstantsfor dGTP on the enzyme activity for pymimidinenucleoside diphosphate reduction are shown in Chart 2. The pattern of inhibition and kinetic constants are determined according to the definition of ATP1, mM' Cleland (7). The concentration of ATP was varied for COP or UOP reduction at several fixed concentrations of dGTP. By IB) means of double reciprocal plots, it was demonstrated that 50 05 dGTP behaved as a noncompetitive inhibitor with respect to I/v 40@@@J04 Intercept I Slope @ ATP for COP (Chart 2A) and UOP reduction (Chart 28). The )X—X) 103 0@ 20 ..@02 @-@-@;?0 .101 @ Table 1 .4.3-2-I ; @ diphosphatereductionsEffects of various nucleoside triphosphates on ribonucleotide dGTP,/.LM dGTt@/.LM catalyzed byinthe ribonucleotide reductase derived from Molt-4F cells activatorThe presence of the best 50 activatorforconcentrations used for the activator are all at 2.5 mM.The best lIz' GDPreduction,COP and UDP reduction is ATP; for ADP reduction, it is GTP; for reductionwasit is dTTP. The enzyme activity for CDP, UDP,ADP, and GDP 85, 110, 110 and 92 pmol/hr, respectively.% ofactivityUDP -10 -05 0.5 10 GDPNTP reduc- ADP reduc- ATP1, mM1 duction8None(2.5 mM) CDP reductiona tiona tionC Chart 2. Effects of dGTP on the Lineweaver-Burk plots of the reciprocal of 100 100 100 100 activator concentration with respect to the reciprocal of reaction velocity (nmol ATP130UTP 100 100 NDb of deoxyribonucleotides produced in 1 hr). Standard incubation conditions were 119GTP 68 ND 100 used except for addition of activator at the indicated concentration.
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