Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. We have produced and characterized rat polyclonal and monoclonal antibodies directed against protein M2 of mouse ribonucleotide reductase. Using these antibodies for immunocytochemical studies, an exclusively cytoplasmic localization of protein M2 was demonstrated both in cultured parent and hydroxyurea-resistant, M2-over-producing mouse TA3 cells, and in cells from various mouse tissues. These data, together with the previously demonstrated cytoplasmic localization of the M1 subunit, clearly show that ribonucleotide reductase is a cytoplasmic enzyme. Combining the anti-M2 antibodies with a monoclonal anti-M1 antibody allowed for double-labelling immunofluorescence studies of the two subunits in individual cells. Only approximately 50% of the cells in a logarithmically growing culture contained immunodetectable protein M2, while the M1-specific staining was present in all cells. The M2 staining correlates well with the proportion of cells in the S-phase of the cell cycle. In tissues, only actively dividing cells stained with either antibody and there were always fewer cells stained with the M2-antibodies than with the M1-antibody. Our data therefore present independent evidence for the earlier proposed model of a differential regulation during the cell cycle of the M1 and M2 subunits of ribonucleotide reductase.     

Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents: an EPR study

The inhibition of ribonucleotide reductase (RR) of intact Ehrlich ascites tumor cells by different antitumor agents was compared using EPR spectroscopy. The inactivation of M2 subunit was measured via quenching of the functionally essential tyrosine radical. Inhibitors of different classes, for example, hydroxyurea, pyrogallol, and thiosemicarbazones, differ in their efficiency by three orders of magnitude. Most effective inhibition was found for isoquinoline-1-aldehyde-thiosemicarbazone (IQ-1) with an IC50 value of 0.18 microM. Inhibition of RR inside tumor cells is comparable with that reported for isolated enzymes.     

Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Improvement of a simple method to purify ribonucleotide reductase

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in yoshida ascites hepatoma AH 130

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP⁺ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP⁺ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.     

Improvement of a Simple Method to Purify Ribonucleotide Reductase

Abstract

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported.¹ The column selectively retains < 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KC1 for elution. The enzyme could also be recovered Intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KC1 prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12 degrees C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state. The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]...Tyr+. was suggested as the structure of this two-electron oxidized state of the enzyme. A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C-13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.     

Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.     

A mixed valence form of the iron cluster in the B2 protein of ribonucleotide reductase from Escherichia coli.

A mixed valent form of the iron cluster (Fe(II)Fe(III) in the B2 protein of ribonucleotide reductase has been isolated and characterized. The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum. This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleotide reductase.     

Mechanism of Lactobacillus leichmannii ribonucleotide reductase studied with Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (Pseudocoenzyme B12) as coenzyme.

Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (pseudocoenzyme B12) purified from Clostridium tetanomorphum has been reacted with ribonucleotide reductase purified from Lactobacillus leichmannii under various conditions, and the properties of the products obtained have been compared by electron paramagnetic resonance (EPR) with those previously reported for products formed from the normal coenzyme (adenosylcobalamin). The rapidly formed intermediate and the slowly formed "doublet" species from the pseudocoenzyme have EPR spectra identical with those formed from the normal coenzyme. This and other considerations make it less likely that the unusual magnetic properties of the rapidly formed intermediate are due to strongly distorted octahedral symmetry about Co(II) as previously postulated. Instead it is probable that the EPR spectrum is due to interaction of the radical pair by both exchange coupling and magnetic dipole--dipole coupling. Although Coalpha-[alpha-(aden-9-YL)]cob(II)amide in solution does not show superhyperfine splitting in the EPR spectrum because of its base-off configuration, the cob(II)amide formed by degradation of the pseudocoenzyme within the catalytic site of the enzyme did show triplets due to a nitrogen axially coordinated to cobalt. This suggests that binding of the cob(II)amide to the reductase catalytic site causes a shift to the base-on form.     

Inhibition of iron uptake in HL60 cells by 2-formylpyridine monothiosemicarbazonato Cu(II).

The electron paramagnetic resonance (EPR) signal of the tyrosyl radical attributed to ribonucleoside diphosphate reductase decreases after treatment of promyelocytic leukemic HL60 cells with 2-formylpyridine thiosemicarbazonato copper(II) (CuL). According to EPR studies, CuL forms adducts with both histidine and cysteine-like Lewis bases associated with isolated membranes from HL60 cells. After the addition of CuL, the EPR signal for the cysteine-like adduct decreases substantially over a 15-min period. The reduction of signal is consistent with oxidation of thiols as shown by an analysis of sulfhydryl content. It is hypothesized that receptor-mediated transferrin internalization is inhibited by oxidation of critical thiols. Since the uptake of 59Fe-transferrin is greatly inhibited by the treatment of HL60 cells with CuL, the reduced uptake of iron by cells, in the presence of CuL, may lead to decreased iron availability for the activity of the M2 subunit of ribonucleotide reductase and a subsequent decrease in the tyrosyl radical signal of the enzyme. Moreover, the intact subunit M2 is no longer detected by EPR, even after the addition of excess iron.     

Detection of a Stable Free Radical in the B2 Subunit of the Manganese Ribonucleotide Reductase (MN-RRase) of Corynebacterium ammoniagenes

Ribonucleotide reductases catalyze the irreversible reductive formation of 2′-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Coynebacterium ammoniagenes ATCC 6872. Enrichment by 2′5′-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with Superose 12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hy-droxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa 82 subunit of the Mn-RRase of Corynebacteriurn ammoniagenes.