Demonstration of Two Components and Association of Adenosine Diphosphate-Cytidine Diphosphate Reductase from Cultured Human Lymphoblast Cells (Molt-4F)
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[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 ±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. 436 CANCER RESEARCH VOL. 39 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 MgCl2•6H20,8g StreptomycinSulfate Fractionation.A solutionof strep NaCI, and 2.16 g Na2HPO4•7H20).Afterthis treatment, the tomycin sulfate (20%, w/v) was added dropwise to the cells were stored at —70°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 4°,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, [‘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 4°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, [‘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 37°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.