Demonstration of Two Components and Association of Adenosine Diphosphate-Cytidine Diphosphate Reductase from Cultured Human Lymphoblast Cells (Molt-4F)

Home , Adenosine diphosphate

[CANCER RESEARCH 39, 436-442, February 1979] 0008-5472/79/0039-0000$02.00 Demonstrationof Two Components and Association of Adenosine Diphosphate-CytidineDiphosphate Reductase from Cultured Human LymphoblastCells (Molt-4F)1

Chi-Hsiung Chang and Yung-chi Cheng2

Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263

ABSTRACT Under the influence of different activators, the same en zyme molecule is capable of catalyzing the reduction of all Ribonucleotide meductase was isolated from a human 4 natural mibonucleotides at the diphosphate level (20). lymphoblast line (Molt-4F). Most of the meductase activity The enzyme obtained from mammalian cells has not been was present in the cytosol fraction. Two components (A and completely described due to difficulties in purification. B) were found which were readily separable by deoxy Studies of some properties of the partially purified enzyme guanosine triphosphate Sepharose column chromatogma derived from matNovikoff hepatoma (22, 23), Ehnlich ascites phy. Only Component B was retained on this column and cells (13), rabbit bone marrow (18) and regenerating rat could be eluted by high concentrations of KCI. Components liven (19) have been reported. More than one subunit has A and B were purified further by blue Sepharose, diethyl been shown for the enzyme derived from rabbit bone aminoethyl cellulose and phenyl-Sephamose column chro marrow (18), matNovikoff hepatoma (23), and Ehnlich ascites matography, as well as by sucrose gradient sedimentation. cells (12). The possible existence of different enzymes The apparent molecular weights estimated by sucrose gra responsible for the reductions of ADP and COP has been dient sedimentation were 100,000 for both Components A proposed in the case of Chinese hamster cells (24), rat and B, and 210,000 for the nondissociated mibonucleotide regenerating liven (10), and Ehmlich ascites cells (13). No reductase. The cytidine diphosphate (COP) and adenosine detailed study of the isolation and properties of nibonucIe diphosphate (AOP) reductase activities cochnomatogmaphed otide reductase from human origin has been reported. throughout the purification procedure with a constant ratio Because this enzyme has the potential of being a target for of 1.73 Â±0.19 (5.0.) Variation of the ratio of purified cancer chemotherapy, we have undertaken the study of the Component A to B led to subsequent variation in overall properties of the enzyme derived from a cultured human activity. However, the ratio of COP to AOP enzyme activity lymphoblast cell line (Molt-4F). In this communication, we remained constant. The enzyme activities of reconstituted demonstrate that the enzyme consists of at least 2 compo purified A and B components were further characterized nents and that the ADP and COP meductaseactivities were with reference to cation requirements. Of those divalent associated throughout the purification procedure. We also cations tested, magnesium ion was found to be essential describe some of the properties of the 2 components. A for maximal enzyme activity, while calcium ion gave only preliminary report of this work has appeared previously (5). partial activation. Addition of zinc or manganese ion, at concentrations higher than 0.4 mM, to the reaction mixture MATERIALSAND METHODS containing 6 mM MgCl2 caused a marked inhibition of the enzyme activity for both ADP and CDP reduction. Spermi The sodium salts of COP, ADP, ATP, and dGTP; DTT,3 dine and spemmine can partially replace the MgCI2 require HEPES, pymuvatekinase, lactic dehydrogenase, and hemo ment for COP and ADP reduction. The optimal concentra globin were all purchased from Sigma Chemical Co. , St. tions of MgCl2 and dithiothreitol were 6 and 3 mM, mespec Louis, Mo. Ammonium salts of all 14C-labeled nucleotides tively. were supplied by Amersham/Seamle Corp. , Arlington Heights, Ill. Oowex 1-Cl was obtained from Bio-Rad Labo natory, Richmond, Va. All materials required for cell cul INTRODUCTION tunes were from Grand Island Biological Co. , Grand Island, Ribonucleotide reductase is the key enzyme responsible N. Y. All other chemicals were of reagent grade. dGTP for the synthesis of deoxynibonucleotides via the direct Sepharose was generously provided by Hoffmann and Blak reduction of mibonucleotides. The enzyme from Escherichia ley (16). Blue Sepharose, DEAE-celiulose, and phenyl coli has been purified and well characterized (2, 17, 27). It Sephanose were purchased from Pharmacia Fine Chemi is made of 2 nonidentical subunits, B1 and B2, both of cals, Piscataway, N. J. which are required to form the enzymatically active complex Culture Conditions. Molt-4F cells, which were isolated in the presence of magnesium ion (4). The enzyme contains from peripheral blood of acute lymphocytic leukemia pa nonheme iron which is essential for enzyme activity (3). tients, were cultured in 1-liter spinner flasks with Roswell Park Memorial Institute Medium 1640 containing 5% heat inactivated fetal calf serum, penicillin (100 units/mI), and 1 This work was supported by USPHS Project Grant CA-18499 and Core stneptomycin sulfate (100 @g/ml).The cells were maintained Grant CA-13038 from the National Cancer Institute. 2 An American Leukemia Society Scholar. To whom requests for reprints should be addressed. 3 The abbreviations used are: DTT, dithiothreitol; HEPES, 4-(2-hydroxy Received June 5, 1978: accepted November 3, 1978. ethyl)-1-piperazmneethanesultOnic acid.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. Two Ribonuc!eotide Reductase Components from Lymphob!ast Ce!!s in the log phase of growth by feeding the cultures with an with 25 strokes in a Dounce homogenizer. The homogenate equal volume of the fresh medium every 24 hr. The cultured was centrifuged at 100,000 x g for 60 mm, and the super cells were harvested by centnifugation and washed 6 times natant (16 ml) was used immediately in the next purification with phosphate-buffered saline (pH 7.2, 1 liter containing step. 0.1 g CaCI2, 0.2 g KCI, 0.2 g KH2PO4,0.1 g MgCl2â€¢6H20,8g StreptomycinSulfate Fractionation.A solutionof strep NaCI, and 2.16 g Na2HPO4â€¢7H20).Afterthis treatment, the tomycin sulfate (20%, w/v) was added dropwise to the cells were stored at â€”70Â°untilneeded. crude extract (16 ml) to yield a final concentration of 1%. Enzyme Assay. COP reductase was assayed by the The solution was stirred for 20 mm at 4Â°,andthe precipitate method of Steepen and Steuart (25) with the use of Dowex was removed by centnifugation at 10,000 x g for 20 mm. 1-borate ion-exchange chromatography. The assay mixture The supemnatant (16 ml) was used in the following step. contained, in a final volume of 0.2 ml, [â€˜4CJCOP(0.2pCi; Ammonium Sulfate Fractionation. Ammonium sulfate 0.15 mM), OTT (3 mM), MgCI2 (6 mM), ATP (5 mM), and a was added to the supemnatant obtained from the previous specified amount of the enzyme. AOP neductase activity step to 35% saturation. After a stirring at 4Â°for 30 mm, the was determined by the method of Conyet a!. (14). The assay suspension was centrifuged at 10,000 x g for 20 mm, and mixture contained, in a final volume of 0.2 ml, [â€˜4CJADP the precipitate was discarded. More ammonium sulfate was (0.22 pCi; 0.15 mM), OTT (3 mM), MgCI2 (6 mM), and dGTP added to the supennatant to give 50% saturation. After (5 mM), and a specified amount of the enzyme. An enzyme being stirred for another 30 mm, the precipitate was col sample heated for 2 mm in a boiling water bath prior to the lected by centnifugation and was dissolved in 4 ml of Buffer addition of the labeled substrate served as the reaction B. The enzyme solution was dialyzed overnight against the blank. The incubation was at 37Â°for 60 mm, and the same buffer. reaction was linear with respect to time and enzyme con Separation of Components A and B on dGTP-Sepharose centration during this incubation period. The inclusion of Chromatography.Thedialysate(4 ml) was madeto 10 mM ATP in the COP meductase assay and dGTP in the ADP with respect to NaF and loaded on a dGTP-Sephamose meductase assay was essential for COP and AOP meductase column (1.5 x 10 cm) previously equilibrated with Buffer B activities, respectively. The specificity of the activators for containing 10 mM NaF. The column was washed with the COP and ADP reductase activity will be reported in a same buffer until the absorbance at 280 nm was less than subsequent communication. A preliminary report of the 0.05. Four consecutive-step elutions were then performed kinetic behaviors of this enzyme has appeared previously with 0.5 mM dGTP, 50 mM KCI, 1 M KCI, and 2 M KCI in (6). The activities of Components A and B as shown in Buffer B as indicated in Chart 1. After dialysis against Buffer Charts 1 to 5 and Table 1 were determined by adding an C, the fractions were analyzed for protein concentration excess amount of B on A, respectively. The amount of and enzyme activity. No activity was detected in any of the Component A on B used to saturate the respective compo fractions collected. Fractions 3 to 12, 13 to 19, 20 to 26, 27 nent under investigation was sufficient to give a minimum to 31, and 32 to 38 were pooled and dialyzed overnight activity of 90 pmol COP reduced per mm penml and 50 pmol against Buffer C containing 30% sucrose. Various combi ADP reduced per mm per ml of Component A on B. These nations of each pooled fraction were assayed for both AOP components were obtained from the dGTP-Sephamose col and COP reductase activities. Only the combination of the umn. pooled fractions from 3 to 12 (Component A) and 27 to 31 Cellular Fractionation.The proceduresusedfor obtain (Component B) gave both AOP and COP meductaseactivity. ing various subcellulam fractions of the MoIt-4F cells have The other pooled fractions could neither enhance non been previously described (8). inhibit the activity observed with this combination of Corn Protein Determination. Proteinwas determinedby the fluorometnic method of BOhlen and Stein (1). Bovine serum Ad@@O@'@TP SO,, â€˜@â€˜@ 2M@ albumin was used as the standard. Enzyme Purification. All steps were performed at 0-4Â°as indicated in Table 1 within a period of no more than 4 days. The final preparation of the enzyme and aliquots of partially purified components were stoned at â€”70Â°.Under these conditions, no significant loss of the enzyme activity occurs

during 1 week. Buffers used for the purification steps are as & 40'- follows. Buffer A contains 100 mM HEPES (pH 7.5), 1 mM MgCl2, and 2 mM OTT. Buffer B includes 50 mM HEPES, (pH 7.5), and 2 mM OTT. Buffer C contains 50 mM HEPES V Frachons 1S5inVfroct,on) (pH 7.5), 1 mM MgCI2, and 2 mM OTT, and 0.05 mM EOTA. Details of the purification procedure are described under Chart 1. dGTP-Sepharase column chromatography at ribonucleotide re ductase derived tram MoIt-4F cells. Protein (17.3 mg) tram the ammanium â€œResults.â€• sulfate fractionation step (35 to 50%) was loaded an a dGTP-Sepharase column (1.5 x 10.0 cm), and the column was eluted with Buffer B containing RESULTS various additives as shown. Fractions after dialysis against Buffer C were analyzed far ribanucleatide reductase activities for CDP and ADP reductions Purification of Ribonucleotide Reductase as describedin the text. A, protein profile. B, the enzymeactivity of each fraction assayed with an excess amount of Component B. Fractions 3 to 8 were pooled as Component A. C, the enzymeactivity at each fraction Preparationof Crude Extract. Molt-4Fcells (about9 x assayed with an excess amount at Component A. Fractions 27 to 31 were 10@cells)were suspended in 14 ml of Buffer A and disrupted pooled as Component B.

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(A) @ ponents A and B (data not shown). Each fraction from the Addd,m (IC) 60 025M dGTP column chromatography was dialyzed overnight against Buffer C and was assayed for both ADP and COP meductase activity in the presence of excess amount of @ either Component A on Component B. The results are JO.6 @ shown in Chart 1, B and C. Component A was eluted from .@o4 the column in the unabsorbed fractions, while Component B could only be eluted with Buffer B containing 1 M KCI. Fractions having the respective activity of each component were combined. Component A was further purified as described below. Component B was dialyzed against 2 changes of 40 volumes of Buffer C containing 30% sucrose 4 over a 6-hr period. The dialysate was further purified as described below. Chromatographyof ComponentA on Blue Sepharose Column.The pooledComponentA(10 ml) fromthe dGTP Sepharose column was loaded on a blue Sephamosecolumn (1.5 x 7.5 cm) previously equilibrated with Buffer C. The Fraction (I8mVfroction) column was then washed with the same buffer until the Chart 3. Chromatograph of Components A and B on DEAE-cellulose columns. A , Component A (3.1 mg) from blue Sepharose column chromatog absorbance of the eluant at 280 nm was less than 0.05, at raphy was loaded on a DEAE-cellulose column (1.5 x 9.0 cm), and the which point Buffer C containing 2 M KCI was applied. Each column was eluted with Buffer C containing various additives as indicated. Fractions after dialysis against Buffer C were assayed for CDP and ADP fraction was dialyzed against Buffer C and then analyzed reductase activities as described in â€˜â€˜MaterialsandMethods.â€•Fractions 20 for protein concentration and enzyme activity. More than to 23 were pooled for further purification. In B, Component B (3.6 mg) from 96% of the activity of Component A was not retained on the dGTP-Sepharose column chromatography was loaded on a DEAE-cellulose column (1.5 x 9.0 cm), and the column was eluted with solutions as column, as shown in Chart 2. The reason for retention of indicated. After dialysis against Buffer C, fractions were assayed for CDP the small amount of activity on the column (less than 4%) is and ADP reductase activities as described in â€˜â€˜Materialsand Methods,â€• unknown at this time. However, it may be due to nonspecific Fractions 3 to 5 were pooled for further purification. adsorption. Chromatography of Component A on DEAE-Cellulose. Chromatography of Component A on a Phenyl-Sepha The combined fractions (17 ml) obtained from blue Sepha rose Column. Component A (7 ml), obtained from DEAE nose chromatography were applied to a OEAE-cellulose cellulose chromatography, was made 1 M with respect to column (1.5 x 9 cm) previously equilibrated with Buffer C. ammonium sulfate and loaded onto a phenyl-Sepharose After loading Component A, the column was washed with 5 column (1.5 x 12.5 cm) previously equilibrated with Buffer ml of the same buffer and was then eluted with Buffer C C containing 1 M ammonium sulfate. The column was containing 0.08 M, 0.25 M, and 2 M KCI, respectively. After washed with 5 ml of the same buffer and then eluted with dialysis against Buffer C, fractions were analyzed for pro 25 ml of Buffer C containing 25% ethylene glycol, followed tein concentration and Component A activity. The results by 25 ml of Buffer C containing 50% ethylene glycol. are shown in Chart 3A. Component A was retained on the Fractions were collected and dialyzed against 40 volumes column and could be eluted with Buffer C containing 0.25 of Buffer C for 3 to 4 hr. Each fraction was concentrated by M KCI. The fractions containing Component A activity were further dialysis overnight against 40 volumes of Buffer C pooled for further purification. containing 45% sucrose. The volume of the concentrated fraction was adjusted to 1 ml and analyzed for protein Add'h@s concentration and enzyme activity as shown in Chart 4. The activity of Component A was present in the fractions eluted with 50% ethylene glycol. 20 2.0 Chromatography of Component B on DEAE-cellulose Column. The pooled Component B (8 ml), after dGTP 6 .6 Sephamose chromatography and dialysis against Buffer C .2 @ 12 1.2 containing 30% sucrose, was loaded on a DEAE-cellulose column (1.5 x 9 cm) previously equilibrated with Buffer C 8 0.8 containing 10% sucrose. The column was then washed with 12 ml of the same buffer followed by 15 ml of Buffer C 4 o_4 I containing 2 M KCI. After dialysis against Buffer C, fractions were analyzed for protein concentration and enzyme activ 4 8 2 6 20 ity. Component B activity was found in the void volume of Froction ( 85 ml I fraction) the column (Chart 3B). Chart 2. Chromatography at Component A on a blue sepharase column. Component A (8.2 mg) from the dGTP-Sepharose column chromatography Sedimentationof Component B by Sucrose Density was loadedon blue Sepharosecolumn(1.5 x 7.5 cm), and the columnwas Gradient Centrifugation. The pooled Component B (4 ml) eluted with the solutions as indicated. After dialysis against Buffer C, obtained from the previous step was dialzyed overnight and fractions were assayed for CDP and ADP reductase activities as described in the text. Fractions 2 to 10 were pooled for further purification. concentrated against 40 volumes of Buffer C containing

438 CANCERRESEARCHVOL. 39

EthyleneGlycol 25 50 The pooled Component B obtained from dGTP-Sepha @ )%( rose chromatography following dialysis with 2 changes of @ 30 40 volumes of Buffer C was loaded on a blue Sephamose E column. The activity was retained on the column and could Â£ be eluted with 1 M KCI (data not shown). However, the @ 20 specific activity of Component B after this chromatography I was not increased, due to the poor recovery of the activity of Component B. Therefore, blue Sepharose chnomatogna

0.4 3 phy was omitted for purification of Component B. : In contrast to the behavior of Component A on a phenyl Sepharose column, Component B came through the col umn with unabsorbed proteins and with a recovery of less @ 4 2 6 20 than 10%. Therefore, this step also was not used for the Frocton (tOrn)Ifraction) purification of Component B. Chart 4. Chromatography of Component A an a phenyl-Sepharose cal umn. Component A (1.4 mg) from DEAE-cellulose column chromatography MolecularWeightDetermination after addition of 1 M ammanium sulfate was loaded an a phenyl-Sepharose column (1.5 x 12.5 cm) previously equilibrated with Buffer C containing 1 M The molecular weight of nondissociated nibonucleotide ammonium sulfate, and the column was eluted with 25 and 50% of ethylene glycol in Buffer C as indicated. The volume at each traction after overnight reductase (after the ammonium sulfate fractionation step), dialysis against Buffer C containing 45% sucrose was adjusted to 1 ml, and as well as that of the purified A and B components, was each fraction was assayed tar ADP and CDP reductase activities as described in â€œMaterialsandMethods.â€•Fractions 13 to 15 were pooled. estimated by the method of sucrose density gradient. Sam pIes (1 ml) were layered onto 11-ml linear sucrose gradients, 5 to 20% (w/v), prepared in Buffer C, and centrifuged at 30% sucrose. After removal of sucrose by Sephadex G-25 100@000x g for 20 hr at 2Â°in a SW 41 rotor. Fractions (0.9 column chromatography, the dialysate (1 ml) was layered ml) were collected by puncturing the bottom of the tube onto an 11-mi linear sucrose gradient 5 to 20% (w/v) and were assayed for the enzyme activity. The results are prepared in Buffer C, and centrifuged at 100,000 x g for 20 hr at 2Â°in a SW 41 rotor. Fractions (0.9 ml) were collected by puncturing the bottom of the tube and assayed for PK LDH Hb @ â€˜(A)@. Component B activity. The results are shown in Chart SC 80 and will be discussed under â€˜â€˜MolecularWeight Oetermina 60 E tion.â€• 40 C Commentsonthe PurificationofComponentsAand B E 20 . /@, ...... (â€˜I(B) I The scheme used to purify both Components A and B of 0 24- â€˜@ 8 12 16 20 E mibonucleotide neductase derived from Molt-4F cells is sum @20

mamized in Table 1. The specific activity (pmol/min/mg C protein) of the final preparation of Component A was 384 0 16 U @ for CDP reduction and 243 for ADP reduction. However, the 12 specific activity (pmol/mmn/mg protein) for the final prepa a, ration of Component B was 669 for COP and 372 for ADP @ reduction. The ratio of COP to ADP meductase activity is I â€¢...... S.. I4 I 1.73 Â±0.19 throughout the purification procedure. Com a. 8 12 16 20 24 ponent A and Component B are both required to give the @ ,@ 20 â€˜(C)@. enzyme activity; neither of them, by itself, has any detecta 0 ble catalytic activity. The addition of either component A or Component B to the enzyme preparation obtained from crude extract, streptomycin sulfate fractionation, or am monium sulfate fractionation did not alter the enzyme activity. The final preparations of Component A and Corn . ponent B are not homogeneous as judged by electropho â€¢..S....SSS..1@4 I 8 12 16 20 24 metictechniques. However, they were purified to such an Bottom extent that nucleotide phosphatases and nucleoside di Fraction (O.45 mI/fraction) phosphate kinases which would interfere with kinetic stud Chart 5. Ribanucleatide reductase activity profile after sucrose density ies of the enzyme were not present in the purified Compo gradient centrifugatian. The sucrose density gradient centrifugatian condi nents A and B (data not shown). tions were performed as described in the text. Hemoglobin (Hb), lactate dehydragenase (LDH), and pyruvate kinase (Pk) were used as markers. The When Component B was applied to a OEAE-ceilulose volume at each sample layered on the gradient was 1 ml. Fractions were column, its activity was not retained on the column. Under collected and assayed tar ADP and CDP reductase activities. A , the ribanu the same condition, Component A was absorbed to the cleatide reductase preparation obtained from the ammonium sulfate frac tionatian (35 to 50%); B, Component A obtained from blue Sepharose column (Chart 3). This observation suggested that Compo column chromatography; C, Component B obtained tram DEAE-cellulose nent B is relatively cationic as compared to Component A. column chromatography.

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Table 1 Purification of ribonucleotide reductasederivedfrom human Mo!t-4Fcells This representsthe purification of enzymesfrom 9 x 10@cells. (pmol/ activity0 mm) (pmol/min/mg)

ADPCDP/ADPbUndissociatedStepProtein (mg)ActivityCDPC ADPdSpecificCDP enzyme@'Crudeextract114348 2.31.35Streptomycin 2663.1 291.@9tionationAmmoniumsulfate frac 57.72657 169446

691.71tionationComponentsulfate frac 17.32054 1197118

A1dGTP-Sepharose 91.67tographyBlue chroma 8.2126 7615

351.69tographyDEAE-CelluloseSepharose chroma 3.1183 10959 521.77tographyPhenyl-Sepharosechroma 1.4128 7392

2431.@8matographyComponentchro 0.1247 30384

BÂ°dGTP-Sepharose 362.03tographyDEAE-cellulasechroma 3.6266 13073

492.08tographySucrosechrama 1.84 -1 87 901 02

3721.80centrifugationdensity gradient0.16103 60669

a pmal of substratereduced per mm per mg of protein in the component being studied. An excessamount of one componentwas usedto determinethe specific activity of the other component. This is to ensure that the latter component produces the maximumactivity. b The ratio of COP to ADP specific activity. C COP was used as the substrate. The detailed procedure of the assay is described in the text. d ADP was used as the substrate. The detailed procedure of the assay is described in the text. t, The addition of either Component A or Component B to the enzyme preparation obtained from crude extract, streptomycin sulfate fractionation, and ammoniumsulfate fractionation did not alter the enzymeactivity. I Assays were performed with an excess of Component B as described in â€˜â€˜Materials and Methods.â€• 0 Assays were @rformed with an excess of Component A as described in â€˜â€˜Materials and Methods.â€•

depicted in Chart 5. The apparent molecular weight was Effectsof DivalentCationsand Polyamines estimated to be 210,000 for the nondissociated nibonucleo The requirements for divalent cations and polyamines for tide neductase and 100,000 for both Component A and either AOP or COP meductase activity were examined by Component B. The activities for both ADP and COP reduc using a mixture of purified A and B components. The tase cosedimented in all studies. results are presented in Table 3. No reaction took place in Requirements for the Enzyme Activity the absence of divalent cations. Among the divalent cations and polyamines tested at a concentration of 6 mM, MgCI2 The requirements for the reductions of COP and AOP are gave the highest velocity for both AOP and COP reduction. shown in Table 2. Like the nibonucleotide neductases de MgSO4 gave the same velocity as MgCl2 for COP reduction nived from other sources (2, 13, 17-20, 22), the enzyme but not for AOP reduction. This result was due to the obtained from MoIt-4F cells has a requirement for a specific substitution of the Cl anion by the SO@= ion. Ca2@could activator. ATP was required as an activator for COP reduc replace Mg2@and maintain full activity for CDP reduction tion, as was dGTP for ADP reduction. Magnesium ions and but resulted in lower activity for AOP reduction. Mn2@could OTT were also essential for COP and ADP reduction. OTT partially substitute for Mg2@for COP reduction, but not for was used in this study to substitute for thioredoxin reduc AOP reduction. Zn2@and Fe2@could not substitute for Mg2@ tase which is a natural reducing protein (21). The optimal for either AOP or COP reduction. Spenmidine and spermine concentration of OTT and MgCI2 for COP and ADP neduc at the same concentration as MgCl2 gave 70% activity for tase activity was 3 and 6 mM, respectively, in the presence COP reduction and 30 to 40% activity for AOP reduction as of activator at a concentration of 5 mM (data not shown). compared to the rate seen when MgCl2 was used. Further

440 CANCERRESEARCHVOL. 39

Table 2 Table 4 reductionTheRequirementsfor COPand AOP Reduction of COP and ADP by ribonucleotide reductase when reductionwascomplete incubation mixture for COP and ADP ComponentA wastitrated by ComponentB theseassays,the sameas described in â€˜â€˜MaterialsandMethods.'â€˜For The amount of Component A used was fixed at 6 pg/assay. thepurificationComponentsA and B from the respectivelast steps of ComponentsA and B usedfor theseassayswereobtained from the (w/w),respectively.procedurewere combined in the ratio of 1.4:1.0 last respectivestepsofprocedure.CDP the purification The maximalactivity with the reconstituted nibonucle neductase AOPreductase otideand12 reductasewas equal to 20 pmol of COPreduced per hr Component B activity (pmol/ activity (pmol/ pmol of ADPreduced per hr.% addedCDP/ADPâ€•0 (/.Lg) hr) hr) activityCOPTotal 0 0 0 6 4.6 2.7 1.70 reduc- ADPreduc 11 8.5 5.1 1.67 tionNoneComponentomitted tion 16 9.5 5.7 1.67 100Activator 100 21 10.8 6.4 1.69 4MgCI2 (ATP or dGTP) 4 a The ratio of CDP to ADP reductase activity. 4DTT 5 1Enzyme 2 0 0 DISCUSSION When streptomycin sulfate was added to the crude ho Table 3 mogenate to remove nucleic acid contaminants, the total Effectsof the substitution of MgCI2byvariouschloride and sulfate forms of divalent cations and polyamineson ADPand COP enzyme activity for AOP and COP reduction increased about reduction catalyzed by ribonucleotide reductase derived from 7-fold (Table I ). In view of the report by Cory et a!. (11) that Molt-4Fcells RNA and oligomibonucleotides markedly inhibit nibonucleo Purified reconstituted ribonucleotide reductasewas usedwhich tide reductase activity, it seems plausible that this observa gave an activity of 60 pmol/hr reduction of COP in the standard tion was due to the precipitation of these inhibitors by the assayconditions. SeeTable 2 for a description of the reconstituted streptomycin sulfate. In addition, as suggested by Cohen et ribonucleotide reductase. a!. (9), there may be some competitive endogenous precun activityâ€•COP sons, formed from breakdown of DNA and ANA, which are Additionreduction@'None00MgCI,100100MgSO49563MnCl,350CaCI29058FeSO400ZnSO400Putrescine3320Spermidine6739Spemmine7130 (6 mM)%of reductionb ADP removed during the subsequent dialysis of the streptomycin sulfate pellet. The observation that the nibonucleotide reductase from Molt-4F cells was a cytoplasmic enzyme is in agreement with other published work on the mammalian enzyme (15, 19). Efforts to purify nibonucleotide reductase from Molt-4F cells have resulted in the separation of 2 components (A and B). After the 2 components of nibonucleotide reductase were dissociated by dGTP-Sepharose chromatography, a a Percentage of activity was calculated by comparing the activity substantial loss of activities of Components A and B was under different conditions with that found with 6 mMMgCI2. observed when these activities were assayed using an b The assay for COP reduction was the same as described in â€œMaterialsandMethods.â€• excess amount of Components B and A, respectively (Table C The assay for ADP reduction was the same as described in 1). This might be due to the fact that, when Components A â€œMaterialsandMethods.â€• and B were reconstituted under the assay conditions used, they did not assume their native conformation. This may be more, in the presence of 6 mM MgCl,, Mn2@and Zn2@had supported by the observation that, when purified Compo strong differential inhibitory effects on both COP and AOP nents A and B were mixed and centrifuged in sucrose reduction (data not shown). density gradient, no activity was observed with the same sedimentation rate as that of nondissociated nibonucleotide CellularLocalizationofthe Enzyme meductase(data not shown). Molt-4F cells in the log phase of growth were fractionated Some properties of the Components A and B were re into subcellulam fractions according to the procedure de vealed by their behavior during the process of purification. scnibed previously (8). More than 95% of the activity for The 2 components have similar apparent molecular weights both ADP and COP reduction was present in the cytosol which are comparable to those for the 2 active components fraction, only 1 to 3% of the activity was associated with the from Ehrlich tumor cells (12). This is in contrast to 2 nuclear fraction, and no detectable activity was found in subunits form Escherichia co!i which have molecular either the mitochondnial on the endoplasmic reticulum frac weights of 160,000 (protein B1) and 78,000 (protein B2) (2, tions. 26). Component A is relatively anionic and hydrophobic Titrationof ComponentA by ComponentB when compared with Component B. The binding sites for tniphosphate nucleotides, which could serve as either acti Table 4 shows the titration of Component A by Compo vators or inhibitors, appear to be present on Component B, nent B. The ratio of COP to AOP reductase activity was based on the observation that only Component B has constant at any ratio of Component B to A tested. binding affinity for dGTP-Sepharose and blue Sepharose.

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Mg2@may enhance the association of Component A and Assay of Proteins in the Nanogram Range. Arch, Biochem. Biophys., 155: 213â€”220,1973. Component B, because no clear resolution of the 2 corn 2. Brown, N. C., Canellakis, Z. N., Lundin, B. , Reichard, P., and Thelander, ponents was obtained when the undissociated enzyme was L. Ribanucleaside Diphosphate Reductase. Purification of the Two Subunits, Protein B1 and B2. European J. Biachem., 9: 561-573, 1969. applied to a dGTP-Sephanose column in the presence of 3. Brawn, N. C. , Eliassan, R., Reichard, P., and Thelander, L. Spectrum [email protected] for Mg2@inthe binding of Subunits B1 and Iran Content at Protein B2 from Ribonucleaside Diphasphate to B2 of nibonucleotide neductase derived from E. coli has Reductase. European J. Biochem., 9: 512-518, 1969. 4. Brown, N. C., Larssan, A., and Reichard, P. On the Subunit Structure of been reported previously (4). Ribanucleaside Diphosphate Reductase. J. BioI. Chem. , 242: 4272- Mg2@isthe most effective divalent cation among all those 4273, 1967. tested to fulfill both ADP and COP meductase activity. 5, Chang, C-H., and Cheng, V-C. Purification and Characterization of Human Ribonucleotide Reductase. Pharmacologist, 19: 135, 1977. Replacement of MgCI2 in the reaction mixture by various 6. Chang, C-H., and Cheng, V-C. Properties of Ribonucleatide Reductase cations, or addition of various divalent cations to the Isolated from a Human Lymphoblast Line (Malt 4F). Proc. Am. Assoc. Cancer Res., 19: 68, 1978. reaction mixture containing 6 mM MgCl,, has different 7. Cheng, V-C., Chang, C-H., Williams, M. V. , Cass, C. E. , and Paterson, effects on the ADP and COP reduction catalyzed by the A. R. P. Fluctuations at Ribonucleatide Reductase Activity and Deoxyri reconstituted enzyme. The different effect of MgCl, and bonucleatide Pools in Synchronized HeLa Cells. Proc. Am. Assoc. Cancer Res., 18: 185, 1977. MgSO4 on COP and ADP enzyme activity must relate to the 8. Cheng, Y-C., and Ostrander, M. Deaxythymidine Kinase Induced in HeLa difference in the anions (Cl versus SO4) . Unlike the TK Cells by Herpes Simplex Virus Type I and Type II. J. Biol. Chem., enzymes for other mammalian systems, which require low 251: 2605-2610, 1975. 9. Cohen, S. S., and Barner, H. D. Spermidine in the Extraction of the concentration of exogenous ferrous or femnicion for optimal Deaxyribasyl-synthesizing System tram T6r'-intected Escherichia coli. activity (18, 19, 22), addition of Fe2@will not alter COP and J. Biol. Chem., 237: 1376â€”1378,1962. 10. Collins, T., David, F., and Van Lancker, J. L. CDP and ADP Reductase in AOP meductaseactivity in Molt-4F cells. In the presence of 6 Rat Regenerating Liver. Federation Proc., 31: 641, 1972. mM MgCI,, Mn2@ and Zn2@ have inhibitory effects that are 1 1 . Cory, J. G. Inhibition of Ribanucleotide Reductase from Ehrlich Tumor more pronounced on AOP reduction than on COP reduction Cells by RNA. Cancer Res., 33: 993-998, 1973. 12. Gary, J. G., Fleischer, A. E., and Munro, J. B., III. Reconstitution of the (data not shown). Cohen and Banner (9) reported that, in Ribonucleatide Reductase Enzyme tram Ehrlich Tumor Cells. J. Bial. the absence of MgCI2, the enzyme reduction system from Chem., 253: 2898-2901 , 1978. T6n@-infected E. coli could be stabilized or activated by 13. Cory, J. G., Mansell, M. M., and Whitfard, T. W., Jr. Control of Ribonucleatide Reductase in Mammalian Cells. Advan. Enzyme Regula polyamines. It has been observed in this laboratory that tion, 14: pp. 45â€”62,1975. polyamines could partially replace Mg2@in fulfilling the 14. Cory, J. G., Russell, F. A., and Mansell, M. M. A Convenient Assay for ADP Reductase Activity Using Dawex-1-Borate Columns. Anal. Bio metal ion requirements. On the contrary, in the presence of chem., 55: 449-456, 1973. MgCl, at 6 mM, none of the polyamines at concentrations 15. Elford, H. L. Subcellular Localization of Ribanucleatide Reductase in higher than 0.4 mM tested were demonstrated to stimulate Navikaff Hepatoma and Regenerating Rat Liver. Arch. Biochem. Bia phys., 155: 213-220, 1973. either COP or ADP meductaseactivity (data not shown). 16. Hoffmann, P. J., and Blakley, R. L. An Affinity Adsorbent Containing The differences observed in the sensitivity of AOP and Large Scale Preparation of Ribanucleatide Reductase of Lactobacillus COP reduction to various agents may be explained in 2 Ieichmannii. Biochemistry, 14: 4804â€”4812,1975. 17. Halmgren, A., Reichard, P., and Thelander, L. Enzymatic Synthesis of ways. AOP and COP neductase activities might reside in 2 Deaxyribanucleatides, VIII. The Effects of ATP and dATP in the CDP separate enzyme entities, or they might exist in the same Reductase System from E. coli. Proc. NatI. Acad. Sci. U. S., 54: 830- 836, 1965. enzyme but have different active sites. The following obser 18. Hopper, S. Ribanucleotide Reductase at Rabbit Bane Marrow. 1. Purifi vations tend to support the concept that the 2 neductase cation, Properties, and Separation into Two Protein Fractions. J. Biol. activities are associated with the same molecule: (a) both Chem., 247: 3336-3340, 1972. 19. Larssan, A. Ribanucleotide Reductase from Regenerating Rat Liver. COP and ADP reductase activities remain associated EurapeanJ.Biochem.,11: 113-121,1969. throughout the purification with a constant ratio; (b) the 20. Larsson, A., and Reichard, P. Enzymatic Synthesis of Deaxyribanuclea rate of reduction of COP and AOP flucttiates similarly tides. IX. Allosteric Effects in the Reduction of Pyrimidine Ribonuclea tides by the Ribanucleaside Diphosphate Reductase System of Esche throughout the HeLa cell cycle (7) [this observation is richia coIl. J. Biol. Chem., 241: 2533-2539, 1966. different from the results reported by Peterson and Moore 21. Laurent, T. C., Moore, E. C., and Reichard, P. Enzymatic Synthesis at Deaxyribanucleatides. IV. Isolation and Characterization of Thioredoxin, using Chinese hamster fibnoblast cells (24)]; and (C) the the Hydrogen Donor from Escherichia coli, B. J. Bial. Chem., 239: 3436- ratio of COP to ADP reductase activity was the same at any 3444, 1964. tested ratio of Component B to A. 22. Moore, E. C. [21] Mammalian Ribanucleaside Diphosphate Reductase Methods Enzymal., 12: 155-164, 1967. 23. Moore, E. C. Components and Control at Ribonucleatide Reductase System of the Rat, Advan. Enzyme Regulation, 15: 101-114, 1976. ACKNOWLEDGMENTS 24. Peterson, M. D., and Moore, E. C., Independent Fluctuations of Cytidine and Adenasine Diphasphate Reductase Activities in Cultured Chinese We wish to thank Joanne Cobler, Linda Roberts, and Susan Grill far their Hamster Fibroblasts. Biachim. Biophys. Acta, 432: 80-91 , 1976. excellent technical assistance and to express our appreciation to Dr. Dennis 25. Steeper, J. R., and 5teuart, C. D. A Rapid Assay for CDP Reductase R. Conrad far his valuable advice and comments. Activity in Mammalian Cell Extracts. Anal. Biachem., 34: 123-130, 1970. 26. Thelander, L. Physicachemical Characterization of Ribanucleaside Di phosphate Reductase from Escherichia coIl. J. Bial. Chem., 248: 4591- REFERENCES 4601, 1973. 27. Thelander, L. Reaction Mechanism of Ribanucleaside Diphasphate 1 . BOhIen, P., Stein, S., Dairman, W., and Udenfriend, 5. Fluarametric Reductase tram Escherichia coli. J. Bial. Chem., 249: 4858-4862, 1974.

442 CANCERRESEARCHVOL. 39

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. Demonstration of Two Components and Association of Adenosine Diphosphate-Cytidine Diphosphate Reductase from Cultured Human Lymphoblast Cells (Molt-4F)

Chi-Hsiung Chang and Yung-chi Cheng

Cancer Res 1979;39:436-442.

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