Deoxyribonucleotide Synthesis Research Articles

Cloning and Sequencing of a Pea cDNA Fragment Coding for Thioredoxin m

Thioredoxins are low molecular mass proteins (about 12 kD) engaged in different redox processes (Holmgren, 1985; Buchanan, 1992). From a functional point of view two types of thioredoxins coexist in the plant kingdom. Chloroplast thioredoxins are concerned with activation of some organellar enzymes through a light-mediated reduction mechanism (Jacquot, 1984; Cseke and Buchanan, 1986), whereas cytosolic thioredoxins appear to be engaged in other redox functions, such as the synthesis of deoxyribonucleotides from the corresponding ribonucleotides. Two types of thioredoxins have been found in the chloroplast: thioredoxin f is especially competent in Fru-1,6-bisphosphatase activation, whereas thioredoxin m is more effective in the activation of NADP+malate dehydrogenase (Cseke and Buchanan, 1986; Buchanan, 1992). However, in spite of the same cellular location, both chloroplast thioredoxins show a clear phylogenetic divergence. The m form is structurally close to prokaryotic thioredoxins, and the f type is more similar to those from mammals and yeasts (Hartman et al., 1990). AI1 the thioredoxins so far sequenced show the active center Cys-X-ProCys (X = Gly or Ala), which fulfills the redox function through the establishment of a Cys-Cys bridge (Holmgren, 1985). The only chloroplast thioredoxins from higher plants so far sequenced are the f types from spinach (Kamo et al., 1989) and pea (Lepiniec et al., 1992) and the m type from spinach (Wedel et al., 1992). We now describe the isolation of a cDNA clone coding for pea thioredoxin m, as well as the nucleotide sequence and the deduced primary structure of this chloroplast thioredoxin (Table I).

Enhancement of tissue glutathione for antioxidant and immune functions in malnutrition

Glutathione (t-y-glutamyl-L-cysteinyl-glycine, GSHT), a cysteine-containing tripeptide and the most abundant non-protein thiol in mammalian cells, is receiving considerable research attention (over 1200 citations in the Medline Database for 1992). The structural uniqueness of GSH, conferred by the y-glutamyl bond, contributes to its intracellular stability (resistance to intracellular peptidases) and determines tissue specificity for uptake of extracellular GSH via y-glutamyl transpeptidase. Tissue concentrations of GSH, like many other metabolically important compounds, are highly regulated. For example, it is difficult to deplete hepatic GSH to less than 30% of control values even with xenobiotic challenge or prolonged starvation [l-6]. Also, it is difficult to exceed the physiological ma~mum concentration for hepatic GSH with supplementation of GSH precursors unless hepatic GSH stores have been depleted previously with xenobiotics or by fasting [4,6]. GSH, a substrate for GSH-S-transferase (EC 2.5.1.18) and GSH peroxidase (EC 1.11.1.9), was initially studied for its role in detoxification of xenobiotics and antioxidation of reactive oxygen species and free radicals, This also led to an appreciation of GSH for transport and storage of cysteine, and for the effects of nutritional status (e.g. sulfur amino acid deficiency) and physiological state on tissue GSH concentrations. With increasing knowledge, the recognized functions of GSH have been expanded to include many aspects of cell biology, such as regulation of cellular redox balance, leukotriene and prostaglandin metabolism, deoxyribonucleotide synthesis, immune function and cell proliferation [for recent GSH reviews, see Refs. 7-111. At the same time, there has been increased interest in the potential therapeutic use of GSH or GSH precursors for treatment of toxicity or diseases, especially those conditions that are believed to be free radical-mediated and that have depleted stores of tissue GSH. The concept of using GSH or GSH precursors

Null thioredoxin and glutaredoxin Escherichia coli K-12 mutants have no enhanced sensitivity to mutagens due to a new GSH-dependent hydrogen donor and high increases in ribonucleotide reductase activity

This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase. To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance. Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin. A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells. Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold. The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.

Ribonucleotide reductases and their occurrence in microorganisms: A link to the RNA/DNA transition

The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.

Ribonucleotide reductases and their occurrence in microorganisms: A link to the RNA/DNA transition

The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.

Serum transferrin receptor level is not altered in invasive adenocarcinoma of the breast.

The transferrin receptor is expressed on the surface of rapidly dividing cells that require iron as a co-factor for essential redox reactions and deoxyribonucleotide synthesis. Transferrin receptors are expressed on the surface of breast carcinoma cells but not on benign breast tumor cells. In this study, the authors investigated whether transferrin receptor concentrations in the serum were elevated in patients with invasive adenocarcinoma of the breast. The transferrin receptor was isolated and purified from human placenta by affinity chromatography. The serum transferrin receptor concentration was determined using an enzyme-linked immunosorbent assay in 19 patients with invasive breast adenocarcinoma, 12 of whom had involvement of axillary lymph nodes. These results were compared with those from 16 normal age-matched female controls. In the invasive breast cancer group, the range of transferrin receptor concentrations was 2.60-7.34 mg/L (mean, 4.44 mg/L) compared with 2.85-8.80 mg/L (mean, 5.49 mg/L) in the control group. Nine patients with in situ adenocarcinoma of the breast had transferrin receptor concentrations of 3.68-6.66 mg/L (mean, 4.94 mg/L). For both the invasive carcinoma group and the in situ group, the means were not significantly different from those of the control group (P = 0.06 and 0.32, respectively). It was concluded that the differential expression of transferrin receptor on the surface of malignant tumor cells in adenocarcinoma of the breast was not reflected by changes in circulating transferrin receptor concentrations.

Deoxyribonucleotide Synthesis in Phycovirus-Infected Green Algae. A New Virus-Induced Ribonucleotide Reductase

Infection of Chlorella-like green algae with freshwater phycoviruses is associated with a large and rapid demand for DNA precursors which cannot be met by the algal deoxyribonucleotide-synthesizing enzymes. We have demonstrated in these cells an up to ten-fold increase of the key enzyme, ribonucleotide reductase, 1-2 h post infection. The enzyme activity has been partially enriched from cell extracts. In vitro, it differs from that of uninfected algae in three characteristic parameters, viz. eight-fold higher resistance to millimolar hydroxyurea concentrations, much higher optimum concentration of an allosteric effector nucleotide, thymidine triphosphate, and an unusually low temperature optimum at 20 °C. We conclude that the large DNA phycoviruses, like Herpes and pox viruses, code for their own specific ribonucleotide reductase.

Cancer chemoprevention with the adrenocortical steroid dehydroepiandrosterone and structural analogs

Dehydroepiandrosterone (DHEA) is an adrenocortical steroid that produces broad-spectrum cancer chemopreventive action in mice and rats. In the mouse two-stage skin tumorigenesis model, DHEA treatment inhibits tumor initiation, as well as tumor promoter-induced epidermal hyperplasia and promotion of papillomas. There is considerable evidence that DHEA exerts its anti-proliferative and tumor-preventive action through the inhibition of glucose-6-phosphate dehydrogenase and the pentose phosphate pathway, which generate NADPH (required for mixed-function oxidase activation of chemical carcinogens, as well as for deoxyribonucleotide synthesis) and ribose 5-phosphate (also required for deoxyribonucleotide synthesis). Long-term DHEA treatment of mice also reduces weight gain (apparently by enhancing thermogenesis), and appears to produce many of the beneficial effects of food restriction, which have been shown to inhibit the development of many age-associated diseases, including cancer. Using the mouse two-stage skin tumorigenesis model, we found that adrenalectomy completely reverses the anti-hyperplastic and antitumor-promoting effects of food restriction. It is not unlikely that food restriction stimulates enhanced levels of adrenocortical steroids, such as the anti-inflammatory glucocorticoids and DHEA, which in turn mediate the tumor-inhibitory effect of underfeeding.

Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.

Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.

Mechanisms of collateral sensitivity to fluorouracil of a cis-diamminedichloroplatinum(II)-resistant human non-small lung cancer cell line.

A cisplatin(CDDP)-resistant subline of a human lung cancer cell line, PC-7/CDDP, was 4.7-fold more resistant to CDDP than the parent line in a colony-forming assay. The sensitivity of this cell line to anthracyclines, vinca-alkaloid, etoposide, mitomycin C, and bleomycin was similar to that of the parental line, PC-7. However, PC-7/CDDP exhibited 4-fold higher sensitivity to fluorouracil (FUra). Possible mechanisms associated with the collateral sensitivity to FUra were studied in PC-7/CDDP cells. The sensitivity of both cell lines to FUra did not correlate with the effect of FUra on RNA. On the other hand, FUra induced a greater reduction in dTTP pools and more single strand breaks in PC-7/CDDP than in PC-7 cells. These results suggest that the pathway for de novo deoxyribonucleotide synthesis may be a target for FUra in PC-7/CDDP cells. However, inhibition of thymidylate synthase after FUra treatment did not correlate with the DNA-directed activity of FUra. Based on the above findings, the decreased salvage synthesis of dTTP was considered a possible mechanism of the greater reduction of dTTP pools in PC-7/CDDP cells. However, the activity of dThd kinase was the same in both cell lines. In the presence of physiological concentrations of exogenous dThd in the serum, uptake of dThd was less in PC-7/CDDP cells than that in PC-7 cells. Our data suggest that FUra-induced cytotoxicity in PC-7/CDDP cells is associated with the inhibition of dTTP synthesis and that the decreased uptake of dThd is a possible mechanism of the collateral sensitivity to FUra in PC-7/CDDP cells.

Deoxyribonucleotide metabolism in hydroxyurea‐resistant V79 hamster cells

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.

Targeting iron-dependent DNA synthesis with gallium and transferrin-gallium.

While iron is essential for numerous intracellular processes, its critical role in DNA synthesis relates to the activity of the iron-containing M2 subunit of ribonucleotide reductase, the enzyme responsible for the synthesis of deoxyribonucleotides. Gallium, a metal which resembles iron with respect to transferrin (Tf) binding, cellular uptake by the Tf receptor and incorporation into ferritin, blocks the cellular uptake of iron and inhibits cell growth. Exposure of HL60 cells to Tf-gallium (Ga) results in decreased deoxyribonucleotide synthesis and a diminution in the electron spin resonance (ESR) spectroscopy signal of ribonucleotide reductase, findings consistent with inhibition of this enzyme. In the present study, Ga nitrate blocked the uptake of 59Fe by L1210 cells and inhibited their proliferation. The ribonucleotide reductase M2 subunit ESR signal in cell cytoplasmic extracts was markedly inhibited in Ga-treated cells; however, the signal was restored to normal within 10 min of exposure of these cytoplasmic extracts to ferrous ammonium sulfate. These results confirm that Ga inhibits DNA synthesis by specifically limiting the amount of intracellular iron needed for the activity of the M2 subunit of ribonucleotide reductase. Further studies utilizing HL60 cells made resistant to Ga showed that these cells were also more resistant to growth inhibition by an anti-Tf receptor monoclonal antibody and deferoxamine. Ga blocks cell growth through inhibition of iron-dependent DNA synthesis. Cells appear to overcome the effects of Ga through compensatory mechanisms involving cellular iron metabolism.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation in mice by 16α-fluoro-5-androsten-17-one and its reversal by deoxyribonucleosides

The work of ourselves and others has demonstrated that dehydroepiandrosterone (DHEA) dispalys a broad spectrum of cancer preventive action in laboratory rodents, with little toxicity. In the two-stage skin tumorigenesis model in mice, topical application of the synthetic DHEA analog 16 alpha-fluoro-5-androsten-17-one, a more potent preventive agent than DHEA without the sex-hormonal side-effects of the parent steroid, markedly inhibited promotion of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated tumor development by 12-O-tetradecanoylphorbol-13-acetate (TPA). DHEA is a powerful inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), suggesting that its inhibiting effect in carcinogenesis may be due to a lack of NADPH and ribose-5-phosphate production for deoxyribonucleotide synthesis and subsequent DNA replication. Further evidence of a reduced NADPH and ribose-5-phosphate pool on the lowering of intracellular deoxyribonucleotide levels has been demonstrated in this paper by completely reversing the 16 alpha-fluoro-5-androsten-17-one-induced inhibition of tumor promotion by the addition of the four deoxyribonucleosides-deoxyadenosine, deoxycytidine, deoxyguanosine and thymidine--to the drinking water during the promotion period of tumorigenesis.

Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis

We have shown previously that Escherichia coli cells constructed to lack both thioredoxin and glutaredoxin are not viable unless they also acquire an additional mutation, which we called X. Here we show that X is a cysA mutation. Our data suggest that the inviability of a trxA grx double mutant is due to the accumulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in the sulfate assimilation pathway. The presence of excess cystine at a concentration sufficient to repress the sulfate assimilation pathway obviates the need for an X mutation and prevents the lethality of a novel cys+ trxA grx double mutant designated strain A522. Mutations in genes required for PAPS synthesis (cysA or cysC) protect cells from the otherwise lethal effect of elimination of both thioredoxin and glutaredoxin even in the absence of excess cystine. Both thioredoxin and glutaredoxin have been shown to be hydrogen donors for PAPS reductase (cysH) in vitro (M. L.-S. Tsang, J. Bacteriol. 146:1059-1066, 1981), and one or the other of these compounds is presumably essential in vivo for growth on minimal medium containing sulfate as the sulfur source. The cells which lack both thioredoxin and glutaredoxin require cystine or glutathione for growth on minimal medium but maintain an active ribonucleotide reduction system. Thus, E. coli must contain a third hydrogen donor active with ribonucleotide reductase.

Effects of cytosine arabinoside and hydroxyurea on the synthesis of deoxyribonucleotides and DNA replication in L1210 cells

Experiments were carried out in L1210 cells to examine the importance of ‘substrate cycles’ in regulating the intracellular levels of deoxyribonucleoside 5′-triphosphate. L1210 cells were incubated with [ 14C]cytidine or [ 14C]adenosine in the presence and absence of hydroxyurea or cytosine arabinoside (araC). These incubations were carried out for either 30 or 120 min. Inhibition of ribonucleotide reductase by hydroxyurea resulted in the blockage of the flux of ribonucleotides to deoxyribonucleotides (>90%) as expected. When DNA synthesis was inhibited with araC, there was a marked decrease in the incorporation of [ 14C]cytidine or [ 14C]adenosine into DNA as deoxyribonucleotides. However, there was not a corresponding increase in the deoxyribonucleotide levels in the acid-soluble fraction or deoxyribonucleosides in the culture medium. AraC treatment decreased the total formation of deoxyribonucleotides. These data indicate that L1210 cells do not regulate the intracellular pools of dNTPs via ‘substrate cycles’ which involve activation of phosphatases when DNA synthesis is blocked or activation of kinases when ribonucleotide reductase is inhibited.

The metabolism of 3′-azido-2′,3′-dideoxyguanosine in CEM cells

When CEM cells were incubated with [ 3H]-labeled 3′-azido-2′,3′-dideoxyguanosine (AzddGuo), the isotope was largely recovered as ribo- and deoxyribonucleotides in the acid soluble fraction of CEM cells, due to extensive catabolism of AzddGuo and recycling of the guanine formed by purine nucleoside phosphorylase. Only 10% was found as the AzddGuo nucleotides, with the diphosphate of AzddGuo as the dominating nucleotide at all time points. Thus nucleoside diphosphate kinase was rate limiting for the formation of AzddGuo triphosphate, responsible for the toxic and antiviral activity of the nucleoside. Inhibition of de novo deoxyribonucleotide synthesis with hydroxyurea increased the phosphorylation of AzddGuo twofold.

Hydroxyurea increases the phosphorylation of 3'-fluorothymidine and 3'-azidothymidine in CEM cells.

The triphosphates of the nucleoside analogues 3'-azidothymidine and 3'-fluorothymidine inhibit reverse transcriptase and are of therapeutic interest for the treatment of retrovirus infections. At equimolar concentrations 3'-fluorothymidine was more effectively transformed to the triphosphate by human CEM cells than azidothymidine which mainly accumulates as the monophosphate. Hydroxyurea, a drug that inhibits de novo synthesis of deoxyribonucleotides, considerably increased the ability of cells to phosphorylate both analogues. Addition of as little as 50 microM hydroxyurea decreased the amount of dideoxynucleoside required to attain a given intracellular concentration of its triphosphate by an order of magnitude. Hydroxyurea is known to shift the balance of substrate cycles between natural deoxynucleosides and their 5'-phosphates in the direction of synthesis and thereby to increase the import and intracellular phosphorylation of the nucleoside. The present results demonstrate a similar effect for the two analogues and raise the possibility of using this effect in therapy.

Essential role of T4 phage deoxycytidylate hydroxymethylase in a multienzyme complex for deoxyribonucleotide synthesis

Several enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis interact in T4 phage-infected Escherichia coli to form a multienzyme aggregate, the T4 dNTP synthetase complex. To test the specificity of enzyme interactions seen in vitro with this complex, we analyzed bacteria infected with four T4 gene 42 amber mutants, which specify truncated forms of dCMP hydroxymethylase, one of the constituent enzymes in the complex. Mutants that specify a nearly full length gene 42 product can form an intact complex, as revealed by two criteria: kinetic coupling among constituent enzymes in crude extracts of infected bacteria, and co-elution of enzyme activities from a gel filtration column. By these criteria, mutations that specify truncated proteins less than half the size of the full length protein cause disruption of the complex. These findings suggest that an enzymatically inactive form of dCMP hydroxymethylase can contribute toward assembly of an intact complex, so long as the incomplete protein is of sufficient size to fold normally, allowing interaction with other proteins in the complex.

Deoxyribonucleotide synthesis in an Escherichia coli mutant (H 1491) which lacks ribonucleotide reductase subunit B2.

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase-catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."