Eur J Biochem 270, 1578–1589 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03529.x H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae An experimental and theoretical approach ´ Hugo Osorio1,2, Elisabete Carvalho1, Mercedes del Valle1, Mara A Gunther Sillero1, ă Pedro Moradas-Ferreira2 and Antonio Sillero1 Departamento de Bioquı´mica, Instituto de Investigaciones Biome´dicas Alberto Sols UAM/CSIC, Facultad de Medicina, Madrid, Spain; 2Instituto de Biologı´a Molecular e Celular, Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade Porto, Portugal When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mM hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP-ribose levels was estimated Concomitantly a net increase in the inosine levels was observed Treatment with 83 mM menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine Application of a mathematical model of differential equations previously developed in this laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mM (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2 Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium The strong decrease in the ATP level upon H2O2 treatment is an important factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress Our laboratory has been engaged for several years in the study of the metabolism and function of dinucleoside polyphosphates [1,2] and purine nucleotides [3] Initially, the aim of the work presented here was to investigate potential changes in the level of diadenosine tetraphosphate (Ap4A) in Saccharomyces cerevisiae subjected to oxidative stress, based on previous work by others describing the increase of Ap4A in yeast and in other microorganisms, when subjected to heat shock or oxidative stress [4,5] However, whereas we did not observe significant changes in the level of Ap4A, important variations in the concentration of other nucleotides were noticed; as shown below, this finding prompted us to investigate in more detail the influence of oxidative stress in yeast nucleotide metabolism Oxygen is both the support to maintain the aerobic metabolism of organisms and a source of damaging reactive free radicals [6] Molecular oxygen (O2) contains two unpaired electrons, both with the same spin, and its reactivity as a free radical is rather limited Upon accepting one electron, molecular oxygen generates a very reactive superoxide radical (ỈO2–), with one unpaired electron Further additions of electrons and combination with protons generate a variety of oxygen derivatives of biological interest [6,7] The reduced NADH and FADH2 are reoxidized by molecular oxygen with formation of H2O [8,9] Although this process is very efficient, the electron flow throughout the respiratory chain may produce reactive oxygen species (ROS) as byproducts, such as superoxide anion radical, hydroxyl radical and hydrogen peroxide Some of these reactive species can also be formed during the oxidation of arachidonic acid, and in different reactions catalyzed by nitric oxide synthase, xanthine oxidase, glucose oxidase, monoamine oxidase, and P450 enzymes [6,10] Although H2O2 itself is not a free radical, it can be decomposed through the Fenton reaction to generate hydroxyl radical (Fe2+ + H2O2 À Fe3+ + ỈOH + ! OH–) Moreover, H2O2, superoxide and hydroxyl radical (ỈOH) can be interconverted via the Haber–Weiss reaction Correspondence to A Sillero, Departamento de Bioquı´ mica, ´ Facultad de Medicicina, Universidad Autonoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain Tel.: + 34 91 3975413; Fax: + 34 91 5854401; E-mail: antonio.sillero@uam.es Abbreviations: ROS, reactive oxygen species; Ino, inosine Enzymes: adenosine deaminase (EC 3.5.4.4); adenosine kinase (EC 2.7.1.20); AMP deaminase (EC 3.5.4.6); AMP 5¢ nucleotidase (EC 3.1.3.5); IMP 5¢ nucleotidase (EC 3.1.3.5); nucleoside phosphorylase (EC 2.4.2.1); adenylate kinase (EC 2.7.4.3) (Received 19 December 2002, revised 13 February 2003, accepted 20 February 2003) Keywords: Saccharomyces cerevisiae; hydrogen peroxide; menadione; glycolysis; oxidative stress Ó FEBS 2003 Effect of H2O2 and menadione on S cerevisiae (Eur J Biochem 270) 1579 Fe3ỵ ỵ ặ O $ Fe2ỵ ỵ O2 Fe2ỵ ỵ H2 O2 ! Fe3ỵ þ Ỉ OH þ OHÀ Ỉ Ồ þ H2 O2 !ặ OH ỵ OH ỵ O2 Menadione is a cytotoxic quinone acting through a cycling reaction, implying its one-electron reduction to a semiquinone radical and subsequent reaction with molecular oxygen with the formation of the quinone and superoxide [11] The oxygen reactive species may oxidatively damage nucleic acids (producing double-strand breaks, apurinic and apyrimidic bases), lipids (formation of lipid peroxides), and proteins (oxidation of the amino acids side chains) [7,12–15] The yeast S cerevisiae has been used as model system to explore the mechanisms underlying the oxidative stress response, such as exposure to H2O2 or menadione [16–19] In this study we have assessed the effect of H2O2 and menadione on the metabolism of purine nucleotides Whereas menadione did not alter significantly the levels of these nucleotides, H2O2 promoted a drastic decrease in the level of adenine nucleotides and a concomitant increase in the level of inosine A plausible explanation of the effect of H2O2 as inhibitor of glycolysis is presented Materials and methods Materials Hydrogen peroxide (30%) solution, menadione sodium bisulfite, auxiliary enzymes, cofactors and substrates were purchased from Sigma or Roche Molecular Biochemicals Yeast nitrogen base was from Difco (catalogue no 233520) Hypersil ODS column (4.6 · 100 mm) was from HewlettPackard Strain and growth conditions The strain used in this work was the wild-type W303 1A from S cerevisiae, genotype: MATa leu2-3, 112 his3-11, 15 trp1-1, can1-100, ade2-1, ura3-1 [20] Cells were grown aerobically at 30 °C in a gyratory shaker (at 180 r.p.m), in a minimal medium containing (per litre): yeast nitrogen base without amino acids and ammonium sulfate 1.7 g; ammonium sulfate g; galactose, glucose or mannose 20 g; leucine 0.08 g; tryptophan, adenine, histidine and uracil 0.04 g each For growth on nonfermentable carbon sources the minimal medium contained 3% (v/v) glycerol or 2% (v/v) ethanol Cell growth was followed by optical absorbance readings at 600 nm (D600 ¼ corresponds to a concentration of 1.5 · 107 cellsỈmL)1) To determine the wet weight, portions of cell cultures grown to different cell densities were rapidly filtered and the filter plus the cells weighed out One gram of wet yeast has been found to contain an average of 24 mg of protein H2O2 and menadione treatment: control of cell viability Exponentially growing yeast cells, with a density of about 1.5 · 107 cellsỈmL)1, were treated with H2O2 or menadione as indicated in each experiment The extraction of nucleotides and the determination of enzyme activities were performed as indicated below When required, the number of viable cells after H2O2 or menadione exposure was determined by spreading appropriate dilutions of cells onto YEPD plates containing 1.5% agar, and counting the colonies formed after incubation at 30 °C for 2–3 days Extraction of nucleosides and nucleotides The sampling method was essentially as described in [21] 100-mL portions of the cell culture grown to a density of around 1.5 · 107 cellsỈmL)1 (1.2 mg wet weightỈmL)1), were rapidly collected by filtration on a nitrocellulose membrane filter (Millipore, pore size 1.2 lm, 47 mm diameter) and washed once with mL of a mixture of methanol/water (1 : 1, v/v) at )40 °C The yeast pellicle was immediately gathered with the help of a spatula and immersed in liquid nitrogen The samples were kept at )70 °C until extraction To prepare the acidic extracts 1.2 M HClO4 was added to the frozen yeast (0.4 mL per 100 mg wet weight) and the suspension was frozen and thawed three times to extract metabolites [22] Cell debris was removed by centrifugation and the pellet re-extracted once with 0.2 M HClO4 (0.1 mL per 100 mg wet weight) The supernatants were combined, neutralized with KOH/K2CO3 and analyzed by HPLC as described previously [23] The amount of the nucleosides/nucleotides was determined from the areas of the corresponding peaks, using the absorption coefficients obtained from standard curves; their intracellular concentration was calculated assuming that g of yeast (wet weight) contains 0.6 mL of intracellular volume [24] NADH did not interfere with NAD+ measurements, because it was destroyed by the acid extraction procedure Inosine (Ino) was identified by its retention time and its nature confirmed by treating the sample, before analysis by HPLC, with commercial E coli purine nucleoside phosphorylase In our assay conditions the detection limit was nmoles per gram of yeast cell dry weight Energy charge Energy charge is defined in terms of actual concentrations as ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]) [25] Preparation of cell extracts All the procedures were carried out at 0–4 °C Yeast (200 mL) grown to a cell density of around 1.5 · 107 cellsỈ mL)1 was harvested by centrifugation, and washed twice with 10 mL of extraction buffer (20 mM sodium phosphate pH 7.0, 0.1 M KCl; 0.1 mM dithiothreitol) The cells (1 g wet weight) were disrupted in the presence of mL of buffer plus g of glass beads (500 lm diameter) by vortexing at top speed on a tabletop mixer for periods of separated by 1-min periods of cooling on ice The homogenate was centrifuged for at 750 g and the supernatant centrifuged further at 550 000 g for 30 The final supernatant was dialyzed for h against 200 volumes of 20 mM sodium phosphate buffer, pH 7.0; 50 mM KCl; 0.1 mM dithiothreitol, followed by a second dialysis of 12 h against the same buffer All enzyme determinations were performed with freshly prepared supernatants Protein content was determined by the method of Bradford [26] 1580 H Osorio et al (Eur J Biochem 270) Ó FEBS 2003 Enzymatic assays Except when indicated, the reaction mixtures (0.15 mL) contained: 50 mM imidazole/HCl buffer pH 7.0, 0.1 M KCl; 0.1 mM dithiothreitol and mM MgCl2; the appropriate nucleoside and/or nucleotide, and inorganic phosphate or ribose-1-phosphate, when required The reaction, initiated by the addition of yeast cytosol (around 0.07 mg protein) was incubated at 30 °C and analyzed by HPLC as follows Aliquots of 20 lL were withdrawn from the reaction mixture at different times of incubation, transferred into 180 lL of water and kept in a boiling water bath for 1.5 After chilling, the mixture was filtered and 50 lL injected into a Hypersil ODS column Elution was performed as described previously [3] The nature and the concentration of the products formed in the course of the reaction were established by comparison with standards Quantification was made from data obtained under linear conditions of substrate consumption One unit is defined as lmol of substrate transformed per The following enzyme activities were estimated in the presence of the indicated substrates or cofactors: adenosine deaminase (EC 3.5.4.4) (0.5 mM adenosine); adenosine kinase (EC 2.7.1.20) (0.2 mM adenosine and mM ATP); AMP deaminase (EC 3.5.4.6) (5 mM AMP and mM ATP); AMP 5¢ nucleotidase (EC 3.1.3.5) (1 mM AMP); IMP 5¢ nucleotidase (EC 3.1.3.5) (1 mM IMP, mM MgCl2 and mM ATP); nucleoside phosphorylase (EC 2.4.2.1) (0.5 mM inosine and mM inorganic phosphate) or (1 mM hypoxanthine and mM ribose-1-phosphate) Adenylate kinase (EC 2.7.4.3) was determined spectrophotometrically in the presence of mM ADP Glucose and ethanol were determined in the medium, after the yeast cells had been removed by centrifugation, by the hexokinase/glucose-6-P dehydrogenase [27] and the alcohol dehydrogenase/acetaldehyde dehydrogenase coupled assays [28], respectively Results Effect of H2O2 on the nucleotide content of yeast cells, grown in the presence of galactose, glucose or mannose Exponentially growing yeast cells, with galactose as carbon source, were challenged with mM H2O2 for 0, 7, 11, 20 and 30 incubation (Fig 1A), and the nucleotide content analyzed by HPLC as described in Material and methods After 11 incubation in the presence of mM H2O2, the total amount of adenine nucleotides (AMP, ADP and ATP) decreased by around 50%, with concomitant appearance of inosine (Fig 1A) Incubation times longer than 30 in the presence of H2O2 did not greatly change the ratio SATP + ADP + AMP/Ino Similar changes in ATP and inosine concentrations were observed when yeast cultures were treated for 30 with different concentrations (0, 0.3, 0.6, 1.0 and 1.5 mM) of H2O2 (Fig 1B) The results presented in Fig were confirmed by growing several batches of yeast cells in galactose as carbon source, in the absence (6 batches) or presence (7 batches) of 1.5 mM H2O2 for h (Table 1) In addition Fig Effect of H2O2 on (AMP + ADP + ATP) and inosine pools of S cerevisiae grown in the presence of galactose as carbon source Yeast cells were challenged with mM H2O2 for the indicated times of incubation (A) or with different concentrations of H2O2 for 30 (B) AMP, ADP, ATP and inosine contents were determined as described in Materials and methods to AMP, ADP, ATP and Ino, the following compounds were also quantified: CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, UDP, UTP, UDP-sugars, NAD+, NADP+, ADP-ribose and hypoxanthine IMP was not detected Representative HPLC nucleotide profiles obtained from yeast cultures grown in galactose and in the absence (A) or presence (B) of 1.5 mM H2O2 are shown in Fig Treatment with 1.5 mM H2O2 for h gave rise to a 10-fold increase in the amount of inosine, a 17-fold decrease in the ATP level, and a five- to sixfold decrease in the levels of ADP, CTP, GTP, UTP and ADP-ribose (Table 1) Changes in the concentration of the other nucleotides analyzed were less relevant The nucleoside mono-, di- and triphosphate pools of adenosine, cytidine, guanosine and uridine in the untreated vs the H2O2treated cells decreased around seven-, two-, two- and 1.5-fold, respectively Although the interpretation of these results is currently not possible, it seems that, in the Ó FEBS 2003 Effect of H2O2 and menadione on S cerevisiae (Eur J Biochem 270) 1581 Table Nucleoside and nucleotide content of S cerevisiae (strain W303) grown in galactose and treated for h in either 1.5 mM H2O2 or 83 mM menadione Exponentially growing yeast cells were challenged with either H2O2 or menadione Analysis of the nucleotide content was performed as described in Materials and methods The data represent mean values ± SE of 6, and experiments for the control, H2O2-treated and menadionetreated cells, respectively The concentrations of the indicated compounds are expressed in mM Parameters Starting wet weight (mg) Total protein (mg) Adenylic charge AMP ADP ATP S (ATP + ADP + AMP) CMP CDP CTP S (CTP + CDP + CMP) GMP GDP GTP S (GTP + GDP + GMP) UMP UDP UTP S (UTP + UDP + UMP) ADP-Rib NAD+ NADP+ UDP-sugars Hypoxanthine Inosine Unknown a 147 3.7 0.78 0.21 0.52 1.51 2.24 0.18 0.07 0.21 0.46 0.06 0.18 0.30 0.54 0.15 0.22 0.33 0.70 0.24 0.97 0.06 1.30 0.11 0.20 – H2O2 Control a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 70 1.5 0.03 0.04 0.18 0.32 0.49 0.03 0.01 0.03 0.08 0.01 0.03 0.02 0.06 0.06 0.04 0.08 0.16 0.11 0.39 0.01 0.16 0.05 0.04 185 3.0 0.41 0.14 0.10 0.09 0.33 0.14 0.04 0.04 0.22 0.08 0.11 0.07 0.26 0.31 0.14 0.05 0.50 0.04 0.95 0.05 1.18 0.16 2.07 – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Menadione 34 1.2 0.15 0.09 0.02 0.05 0.12 0.09 0.02 0.02 0.03 0.02 0.07 0.03 0.08 0.08 0.08 0.01 0.09 0.03 0.34 0.02 0.16 0.06 0.69 H2O2/Control MD/Control 173 2.5 0.86 0.08 0.26 1.15 1.49 0.18 0.03 0.13 0.34 0.03 0.10 0.19 0.32 0.27 0.12 0.07 0.46 0.05 0.88 0.05 1.58 0.11 0.94 0.33 – – 0.53 0.67 0.19 0.06 0.15 0.78 0.57 0.19 0.48 1.33 0.61 0.23 0.48 2.07 0.64 0.15 0.71 0.17 0.98 0.83 0.91 1.45 10.3 – – – 1.10 0.38 0.50 0.76 0.67 1.00 0.43 0.62 0.74 0.50 0.55 0.63 0.59 1.80 0.54 0.21 0.66 0.21 0.91 0.83 1.21 1.00 4.70 – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9.0 0.5 0.01 0.01 0.01 0.08 0.08 0.08 0.01 0.01 0.07 0.00 0.03 0.02 0.05 0.03 0.04 0.02 0.05 0.03 0.04 0.02 0.41 0.05 0.57 0.03 The concentration (mM) of this compound has been calculated assuming the extinction coefficient of adenosine Fig HPLC nucleotide profile obtained from yeast cells grown in galactose or glucose, as carbon source and in the absence or presence of H2O2 Yeast cells grown in the presence of galactose or glucose were challenged, when indicated, with 1.5 mM H2O2 for h Thereafter nucleotides were extracted and analyzed by HPLC as described in Materials and methods The chromatographic peaks, identified by its UV spectra and time of elution, correspond to (1) Hyp, (2) Ino, (3) NAD+, (4) (unknown compound whose spectrum has a maximum at 280 nm), (5) UDP-sugars, (6) AMP, (7) ADP-rib, (8) NADP+, (9) ADP, (10) GTP, (11) UTP and (12) ATP Ó FEBS 2003 1582 H Osorio et al (Eur J Biochem 270) presence of H2O2, the adenine nucleotide content is diverted towards inosine, and that the adenylic charge value decreases, from a standard value of around 0.8 to a value of around 0.4 Similar results to those obtained with galactose, concerning variations in the levels of ATP and Ino (Fig 1) were obtained when yeast cells grown in glucose were treated with different concentrations (0, 0.5, 1.0 and 1.5 mM) of H2O2 (results not shown) As was the case for galactose, the experiments were performed using five different batches of yeast cells, in the absence or presence of 1.5 mM H2O2 for h (Table 2) In the presence of H2O2 there was a decrease of about threefold in the content of adenosine, cytidine and uridine, and twofold for guanosine nucleotides The nucleoside triphosphates were the most affected by the H2O2 treatment The decrease in ATP (5.6-fold) was almost coincident with the increase in Ino (5.7-fold) By contrast, the concentration of NAD+ remained almost constant after H2O2-treatment of yeast cells growing either in galactose or glucose A representative chromatographic profile of a batch of yeast cells growing in glucose, in the absence or presence of 1.5 mM H2O2 is also depicted in Fig 2C,D When mannose was used as a carbon source, similar results to those described for glucose were obtained (results not shown) Effect of menadione on the nucleotide content of yeast cells Here we tried to compare the effect of H2O2 on yeast cells with that of menadione, a different oxidative agent As for H2O2, we started by assaying the effect of different concentrations of menadione (10, 30, 83, 90 and 110 mM) on cell viability (results not shown) and noticed that 83 mM menadione produced a viability similar to that evoked by 1.5 mM H2O2 (around 40% after 60 treatment.) Based on these experiments, three batches of yeast cells growing exponentially in a medium containing galactose (Table 1) or glucose (Table 2), were treated for h with 83 mM menadione In general, the variations in the concentration of the nucleoside triphosphates induced by menadione are lower than those promoted by H2O2 treatment In all the chromatograms corresponding to menadione-treated yeast cells, a new peak with a retention time of around 4.0 was observed (Tables and 2, and results not shown) Although its UV spectrum coincides with that of adenosine, both compounds are different because (a) they elute in a slightly different chromatographic position (not shown) and (b) they behave differently as substrates of adenosine deaminase: the new chromatographic peak is insensitive to the enzyme, in the same experimental conditions that adenosine is transformed to inosine (results not shown) Table Nucleoside and nucleotide content of S cerevisiae (strain W303) grown in glucose and treated for h with either 1.5 mM H2O2 or 83 mM menadione Exponentially growing yeast cells were challenged with either H2O2 or menadione Analysis of the nucleotide content was performed as described in Materials and methods The data represent mean values ± SE of 5, and experiments for the control, H2O2-treated and menadionetreated cells, respectively The concentrations of the indicated compounds are expressed in mM Parameters Starting wet weight (mg) Total protein (mg) Adenylic charge AMP ADP ATP S (ATP + ADP + AMP) CMP CDP CTP S (CTP + CDP + CMP) GMP GDP GTP S (GTP + GDP + GMP) UMP UDP UTP S (UTP + UDP + UMP) ADP-Rib NAD+ NADP+ UDP-sugars Hypoxanthine Inosine Unknowna 123 2.7 0.89 0.05 0.18 1.07 1.30 0.04 0.04 0.22 0.30 0.02 0.05 0.16 0.23 0.06 0.06 0.53 0.65 0.11 0.62 0.04 0.49 0.10 0.20 – H2O2 Control a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 42 0.8 0.02 0.01 0.02 0.08 0.09 0.01 0.02 0.03 0.04 0.01 0.01 0.02 0.14 0.03 0.01 0.07 0.07 0.03 0.12 0.02 0.09 0.01 0.06 124 3.2 0.53 0.16 0.12 0.19 0.47 0.03 0.02 0.05 0.10 0.03 0.05 0.06 0.14 0.07 0.06 0.10 0.23 0.02 0.97 0.02 0.59 0.15 1.16 – ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Menadione 1.4 0.10 0.04 0.03 0.10 0.12 0.02 0.01 0.03 0.03 0.02 0.01 0.03 0.04 0.06 0.01 0.04 0.05 0.00 0.12 0.01 0.19 0.05 0.28 H2O2/ Control MD/ Control 147 4.7 0.85 0.08 0.18 0.90 1.16 – 0.05 0.28 – – 0.05 0.18 – – 0.06 0.41 – 0.05 0.64 0.04 0.40 0.04 0.22 0.44 – – 0.60 3.20 0.67 0.18 0.36 0.75 0.50 0.23 0.33 1.50 1.00 0.37 0.61 1.17 1.00 0.19 0.35 0.18 1.59 0.50 1.20 1.50 5.80 – – – 0.96 1.60 1.00 0.84 0.89 – 1.25 1.27 – – 1.00 1.12 – – 1.00 0.77 – 0.45 1.05 1.00 0.82 0.40 1.10 – ± ± ± ± ± ± ± 17 1.6 0.05 0.04 0.05 0.14 0.06 ± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.01 ± 0.08 ± ± ± ± ± ± ± 0.01 0.12 0.00 0.12 0.01 0.06 0.09 The concentration (mM) of this compound has been calculated assuming the extinction coefficient of adenosine Ó FEBS 2003 Effect of H2O2 and menadione on S cerevisiae (Eur J Biochem 270) 1583 This new unknown chromatographic peak, not present in the preparation of menadione used, may correspond to a derivative of adenosine Effect of H2O2 on yeast cells growing in the presence of glycerol or ethanol To obtain further insight into the oxidative effect of H2O2 (see below), yeast cells were grown in the presence of 3% glycerol as carbon source, and challenged with 1, 2, and mM H2O2 A concentration of H2O2 as high as mM did not change the HPLC nucleotide profile obtained with untreated cells (results not shown) In a different experiment, yeast cells were grown in the presence of 2% ethanol as a carbon source, and challenged with 1.5 mM H2O2 for h; again, no significant changes in the nucleotide content were observed in relation to the control cells (results not shown) It seems that, with respect to nucleotide metabolism, yeast cells grown in ethanol or glycerol as carbon sources are more resistant to H2O2 than those grown in the presence of galactose or glucose Search for a plausible mechanism A mechanism to explain the different effects of H2O2 on the nucleotide content of yeast grown in the presence of hexoses (galactose, glucose or mannose), glycerol or ethanol was sought To explore the reasons for the decrease in ATP and the increase in Ino promoted by H2O2 in yeast cells growing in the presence of galactose, glucose or mannose, we followed an approach partially based on a previous study from our laboratory [3] In that work, the metabolic pathways of AMP, GMP, IMP and XMP catalyzed by rat brain cytosol were explored using two complementary (experimental and theoretical) approaches Experimental approach – determination of enzyme activities related to adenine metabolism Enzyme activities related to adenine metabolism were determined in the cytosol of yeast cells, grown in glucose and in the absence or presence of 1.5 mM H2O2 The pathways considered here to approach the metabolism of adenine nucleotides in yeast cells subjected (or not) to oxidative stress, together with the differential equation describing these pathways are represented in Figs and 4, respectively The enzymes considered in the pathway from ATP to Ino were E1 (AMP 5¢-nucleotidase), E2 (IMP-GMP specific 5¢–nucleotidase), E3 (AMP deaminase), E4 (adenosine deaminase), E5 (purine nucleoside phosphorylase), E6 (adenylate kinase), E7 (adenosine kinase), E8 (a hypothetical enzyme catalyzing two general and reversible reactions), E8d (synthesis of ATP through the glycolytic pathway) and V8r (degradation of ATP through general anabolic processes) Enzyme activities were determined as described in Materials and methods To avoid enzyme inactivation, only fresh (not frozen) cytosol was used Reaction mixtures were set up containing the yeast cytosol, and the concentration of substrate(s) and buffering conditions that we considered pertinent (based on the literature) to render linear formation of products The results obtained and the Fig Adenine and hypoxanthine nucleotide metabolism in yeast cytosol The pathways considered are those shown in the Figure The enzymes involved are E1, 5¢-nucleotidase acting on AMP and IMP; E2, IMP-GMP specific 5¢-nucleotidase; E3, AMP deaminase; E4, adenosine deaminase; E5, purine nucleoside phosphorylase; E6, adenylate kinase; E7, adenosine kinase; E8, hypothetical enzyme recycling ATP Fig Differential equations describing the fluxes operating in the pathways from ATP to hypoxanthine, as described in Fig Vn, maximum velocity of the reaction catalyzed by En, on the substrate indicated The velocity equations considered for E1, E3, E4, E5, E6 and E7 were as in Torrecilla et al [3]; those for E2 and E8 are indicated in the text and Table kinetic constants taken from the literature are compiled in Table As a representative example, mixtures containing 1.8 mM ATP, 0.8 mM ADP and 0.34 mM AMP were incubated with cytosol from yeast cells grown in glucose in Ó FEBS 2003 1584 H Osorio et al (Eur J Biochem 270) Table Vmax, Km and Ki values of enzymes involved in the adenine and hypoxanthine metabolism in yeast cytosol Vmax values represent the average of a minimum of three determinations obtained from different batches of yeast cells grown in glucose, with H2O2 results determined in the yeast cytosol, and grown for 30 in the presence of 1.5 mM H2O2 Ki values of the products were assumed equal to the Km values of the substrate in the cases of enzymes E2, E5, E6, E7 and E8 En represents the enzymes as specified in Fig Vmax (mmg)1) Enzyme Substrate Control H2O2 Km (lM) Ki (lM) E1 5¢-AMP nucleotidase AMP IMP 10.4 ± 1.4 12.7 ± 0.9 11.6 ± 2.2 11.8 ± 2.2 200 [29] 540 [29] E2 5¢-nucleotidase IMP-GMP specific IMP 640 (Ado) [29] 8600 (Ino) [29] 27.2 ± 5.4 (ATP) 37.5 ± 8.9 (ADP) 400 (Ino) 300 (Ino) 2000 (Ino) E3 AMP deaminase E4 Adenosine deaminase E5 Nucleoside phosphorylase AMP 29.2 ± 2.4 Ado 0.8 ± 0.2 0.6 ± 0.2 10.9 ± 1.7 11.6 ± 1.0 E6 Adenylate kinase E7 Adenosine kinase E8 a c 5.7 ± 0.8a a 3.1 ± 0.7 24.4 ± 7.5 Ino Pi Hyp Rib-1P AMP ADP ATP Ado 29.4 ± 1.4 33.4 ± 0.6 4000 c 2414 ± 621 4000 c 203 ± 97 4297 c 2593 ± 557 4297 c 216 ± 61 ATP X ADP X-P 200 c 200 c 4000 c 4000 c a a 400 [30] 300 [30] 2000 b 1800 a,b a 2670 [31] 500 [31] a 40.7 [32] 166 [34] 1600 22 [35] 320 [35] 34 [36] 23 [36] 63 [36] 2.8 [35] 220 (ATP) [35] 33 100 33 100 5000 (Pi) [30] 4700 (IMP) [31] 28 (Ino) [32,33] 166 1600 22 320 34 23 63 2.8 (Ado) 220 (ATP) 200 (AMP) [35] 1200 (ADP) [35] 33 100 33 100 Values obtained in the presence of ATP b Km values determined in yeast cytosol, and used in the theoretical simulation depicted in Fig Values calculated using MATHEMATICA-3.0 program the absence (Fig 5A) or presence of 1.5 mM H2O2 (not shown) The rate of adenine nucleotide degradation and the appearance of intermediate products were essentially the same in both cases and, above all, no appreciable differences in the rates of disappearance of ATP or appearance of Ino were observed Theoretical approach – mathematical simulation of some metabolic pathways related to adenine nucleotide metabolism This was a theoretical approach The simulation was started by stating the metabolic pathways from ATP to Ino (Fig 3), writing the opportune differential equations (Fig 4) and solving them with the help of the MATHEMATICA-3.0 program [37] The equation velocities considered for the enzymes involved were essentially as described in [3], with the following main modifications The kinetic properties of E2 (5¢-nucleotidase for IMP) from yeast [30] are different to those described for the enzyme from rat brain [38] The sigmoidal kinetic toward IMP reported for the yeast enzyme changed to near-hyperbolic in the presence of ATP [30], i.e a behavior similar to that previously described for the AMP deaminase Accordingly [3] the velocity equation used for 5Â-IMP nucleotidase was settled as: m2 ẳ V2IMP ẵIMPn ẵIMPn ỵ ẵS0:5 n where n ẳ 1.71.2[ATP]/(Ka2ATP + [ATP]) and S0.5 ¼ Km2IMP – (F2K [ATP]/(Ka2ATP + [ATP])) From both the enzyme properties reported by Itoh [30], and experiments from this laboratory (not shown), the following values were used: Ka2ATP ¼ 1250; F2K ¼ 200 (Fig 5) or 100 (Fig 6) (see [3], and Table 3, for further explanations on the significance of these parameters) The equation described as V8d and V8r, and the corresponding substrates and products were not considered at this stage (i.e V8d ¼ V8r ¼ 0, see below) Taking into account the above values, application of the MATHEMATICA-3.0 program [37] to the case of a reaction mixture containing ATP, ADP and AMP (at the same concentrations as those present in the experimental approach, Fig 5A) produced Ó FEBS 2003 Effect of H2O2 and menadione on S cerevisiae (Eur J Biochem 270) 1585 speculated that changes in the relative rates of both processes could affect the actual concentration of ATP and hence the rate of synthesis of inosine It is here assumed that the complex processes of syntheses and degradation of ATP in vivo (involving many enzymes) is carried out by a hypothetical unique enzyme (E8) catalyzing both the synthesis of ATP in the direct reaction (E8d) and the phosphorylation/transformation of substrates with participation of ATP in the reverse direction (E8r): ADP ỵ X-P $ ATP ỵ X where X and X-P represent a pool of unphosphorylated and phosphorylated unspecified substrates, respectively This hypothetical enzyme has been used to test, with the help of the mathematical model described in [3], whether different rates of synthesis of ATP would modify the intracellular pool of inosine The reaction catalyzed by E8 is here supposed to be similar to that catalyzed by adenylate kinase, i.e randombireactant [41], and the corresponding velocity equation is: m8 ¼ [ADP][SÀP]V8d [ATP][S]V8r À Km8ADP Km8SÀP Km8ATP Ki8S [ADP] [SP] [ADP][SP] [ATP] [S] [S][ATP] 1ỵ ỵ þ þ þ þ Ki8ADP Ki8sÀp Km8ADP Ki8SÀP Ki8ATP Ki8S Km8S Ki8ATP The following kinetic constants were established to solve the equation: Km8ADP ¼ Ki8ADP ¼ Km8ATP ¼ Ki8ATP ¼ 0:033 mm Km8SÀP ¼ Ki8SÀP ¼ Km8S ¼ Ki8S ¼ 0:1 mm Fig Metabolism of ATP, ADP, AMP in the presence of cytosol from yeast growing in glucose, in the absence or presence of H2O2 – theoretical simulation The reaction mixtures contained: 50 mM imidazole/HCl buffer, pH 7.0; 0.1 M KCl; 0.1 mM dithiothreitol; mM MgCl2; 1.8 mM ATP; 0.8 mM ADP; 0.34 mM AMP and cytosol from yeast cells grown in the absence (A) or presence (result not shown) of 1.5 mM H2O2, for 60 Aliquots were taken at the indicated times and analyzed by HPLC In (B) application of the theoretical model was performed with the MATHEMATICA-3.0 program, as described in the text and in [3] The V-values, from control cells, and the kinetic parameters described in Table were used similar rates of disappearance of substrates and appearance of products (Fig 5B) Inhibition of glycolysis as a plausible theoretical explanation for increased inosine Yeast cells treated with H2O2 and grown in galactose, glucose or mannose showed an increase in inosine level, for which the inhibition of glycolysis was proposed as a possible theoretical explanation The results from Fig and Table 1, suggested that (a) the main if not unique source of inosine is the intracellular pool of adenine nucleotides, (b) the decrease in ATP and the increase in inosine, promoted by H2O2, cannot be explained solely by a change in the level of the enzymes more directly involved in the nucleotide pathway from ATP to inosine (Fig 3), so that (c) other factors should account for those changes When yeast cells are grown in glucose, galactose or mannose, ATP is generated mainly through the glycolytic pathway, and used in diverse anabolic pathways [39,40] We ẵX ẳ ẵX-P ẳ 0:1 mm: As this hypothetical activity represents the activity of many enzymes, we have chosen representative mean values for the kinetic constants of the enzyme E8, in the order of mM, while the concentrations of ATP and ADP were considered as variables With these characteristics, the maximum velocities in the direct (V8d, synthesis of ATP) and in the reverse (V8r, synthesis of ADP) directions were mathematically adjusted, using the MATHEMATICA-3.0 program (to 4000 and 200, respectively) to keep the level of ATP during the application of the mathematical procedure nearly constant (Fig 6B) Metabolic situations conveying diminution in the rate of formation of ATP from ADP (i.e inhibition of glycolysis) were simulated by decreasing V8d from 4000 (Fig 6B) step by step to 500, and leaving constant V8r at 200 (Fig 6C–F) The graph in Fig 6A represents an extreme situation in which V8d ¼ V8r ¼ Together, the graphs depicted in Fig 6B–F show that the inhibition in the rate of synthesis of ATP from ADP is accompanied by an increase in the rate of synthesis of Ino, without any need to modify the kinetic parameters or activities of the enzymes involved in the pathway from ATP to Ino Being aware of the simplifications involved in these calculations (where so many more enzymes participate in vivo), these results would indicate that H2O2 diminishes the rate of synthesis of ATP, probably through inhibition of glycolysis It is worth noting that H2O2 has no appreciable effect on the level of ATP on yeast cells grown in ethanol or glycerol, that are metabolized through an oxidative pathway Ó FEBS 2003 1586 H Osorio et al (Eur J Biochem 270) Fig Influence of the hypothetical enzyme (E8) recycling ATP, on the rate of synthesis of inosine Application of the theoretical model was performed with the MATHEMATICA-3.0 program, as described in the text and in [3] Simulation was made considering the kinetic values for the enzymes E1–E7 determined in the cytosol of control cells, grown in glucose In the case of enzyme E2, the Km values described in [30] were used (Table 3) Graphs A–F were computer made using the following additional values, respectively, for V8d and V8r: A (0,0); B (4000, 200); C (3500, 200); D (3000, 200); E (2500, 200); F (2000, 200) Effect of H2O2 on glycolysis From the above, it seemed obvious to verify in our experimental conditions the effect of H2O2 on either the rate of glucose consumption or on the rate of synthesis of ethanol At the usual concentrations of both glucose (2%) and yeast cells (around 1.2 D600 units per mL), at which the effect of H2O2 was previously tested, the consumption of glucose (in control and treated cells) was so low that its disappearance from the culture medium could not be detected However, at higher yeast cells (91 D600 units per mL) and H2O2 (15 mM) concentrations, a decrease in the consumption of glucose was clearly observed a few minutes after the onset of the H2O2 treatment (results not shown) The rate of ethanol production by yeast cells growing in glucose was also determined as a parameter to measure potential inhibition of glycolysis by H2O2 As shown in Fig 7, treatment of yeast cells with 0, 0.05, 0.1, 0.3, 0.5 and 1.5 mM H2O2 promoted a dose-dependent decrease in the rate of synthesis of ethanol Recovery of yeast cells after the oxidative stress caused by H2O2 A yeast culture grown in glucose was challenged with 1.5 mM H2O2 for 30 After this treatment, cells were separated by centrifugation, resuspended in fresh medium without H2O2, and aliquots taken at 0, 30, 60, 90 and 120 incubation As expected, the ATP content was very low after the H2O2 treatment (time zero) and the inosine concentration very high (Fig 8) After 30 incubation in Fig Effect of H2O2 on the synthesis of ethanol by S cerevisiae Yeast cells, grown in glucose as carbon source, were challenged with 0; 0.05; 0.1; 0.3; 0.5 and 1.5 mM H2O2 Ethanol was determined in the medium, at the indicated times, as described in Materials and methods the absence of H2O2, the recovery of ATP was almost complete, while the return of inosine to normal values was much slower Discussion The results presented above are clear, concerning the effect of H2O2 on the yeast strain W303 of Saccharomyces cerevisiae In the presence of glucose, galactose or mannose, Ó FEBS 2003 Effect of H2O2 and menadione on S cerevisiae (Eur J Biochem 270) 1587 Fig Recovery of ATP after treatment of yeast cells with H2O2 Yeast cells grown in glucose, were treated with 1.5 mM H2O2 for 30 min, collected by centrifugation, resuspended in fresh medium (without H2O2) and incubated further for 120 At the times indicated, the adenylic charge, ATP and inosine were determined as described in Materials and methods H2O2 evokes a decrease and an increase in the intracellular concentration of ATP and inosine, respectively Searching for the rationale for these phenomena, possible changes in the specific activities of enzymes directly involved in the pathway from ATP to Ino were explored in extracts from normal and oxidatively stressed cells (Table 3) At first glance, the changes in the activities of those enzymes did not account for the changes in the ATP or inosine levels This impression was quantified with the help of a mathematical model of differential equations describing the changes in substrate and product concentration in a metabolic pathway as a function of the kinetic constants of the enzymes involved in that pathway [3] Application of this method pointed to the inhibition of the rate of synthesis of ATP by the glycolytic route as a potential reason for the changes in ATP and inosine levels, provoked by H2O2 This assumption was experimentally tested by measuring the consumption of glucose and the synthesis of ethanol in yeast cells treated with H2O2, which produced a decrease in both the consumption of glucose and synthesis of ethanol The apparent effect of H2O2 on glycolysis was further confirmed by the lack of effect of H2O2 when yeast cell were grown in glycerol or ethanol, two oxidative substrates The possibility that the resistance to H2O2 in these last two cases may be due to a stronger expression of antioxidant enzymes has not been explored The effect of H2O2 on yeast cells had been previously analyzed from several perspectives Cabiscol et al [42] observed the formation of carbonyl groups in several amino acid side chains of proteins after treatment of yeast with H2O2 and menadione Here, mitochondrial proteins (E2 subunits of both pyruvate kinase and a-ketoglutarate dehydrogenase, aconitase and heat shock protein 60) and the cytosolic fatty acid synthetase and glyceraldehyde 3-phosphate dehydrogenase were the enzymes mainly affected by the H2O2 treatment [42] In line with the results reported in this study, the activity of glyceraldehyde 3-phosphate dehydrogenase (one of the two enzymes in glycolysis responsible for the synthesis of ATP through substrate level phosphorylation) was 85 and 53% (in relation to an untreated control) in yeast cells subjected to H2O2 treatment and grown in glycerol or glucose, respectively [42] A similar observation concerning carbonylation of key metabolic enzymes by H2O2 has been described recently by Costa et al [43] These authors observed an 80% reduction of glyceraldehyde 3-phosphate dehydrogenase upon incubation of yeast cells with 1.5 mM H2O2 [43] In this regard, preliminary results from our laboratory showed a fivefold increase in the level of fructose 1,6-bisphosphate concentration in H2O2 treated cells (unpublished results) Moreover, it seems to us important to emphasize that the effect of H2O2 on glycolysis is likely to be reversible, as ATP and inosine levels are restored upon washing H2O2 from the cells and resuspending them in fresh medium The recovery appears quite fast, which probably suggests covalent modification of protein(s) (i.e glyceraldehyde 3-phosphate dehydrogenase) and precludes any in vivo protein synthesis Considering that ATP is the center of a very important metabolic crossroads [44], other possibilities could be contemplated to explain the decrease of ATP promoted by H2O2, such as the inhibition of the transport of hexoses (what could be considered as an inhibition of glycolysis) or an increase in ATPase activity This latter possibility does not seem to be operative in this case, as the ATPase activities found by us in the cytosol from untreated or H2O2-treated cells were 5.2 ± 2.3 and 5.5 ± 1.9 mmg)1 protein, respectively Moreover, application of the theoretical method, taking into accounts these values, did not alter significantly the rate of ATP degradation The decrease in ATP promoted by H2O2 could be also compared with the decrease of this nucleotide promoted by the mutation in the gene responsible for the synthesis of trehalose 6-phosphate (TPS-1), which is accompanied also by an increase in glucose 6-phosphate In the case of tps-1 mutants, the decrease in ATP and the increase in glucose 6-phosphate in yeast grown in glucose could be explained by an enhanced activity of hexokinase produced by both the release of its inhibition by trehalose 6-phosphate and/or by the proper effect of the TPS-1 gene product [45–49], two conditions most probably not prevalent in the H2O2-treated yeast cells, where the decrease of ATP is accompanied by a decrease of about twofold in the glucose 6-phosphate level (unpublished results from this laboratory) Godon et al [50] approached the effect of H2O2 on S cerevisiae in a different way Yeast grown in minimal medium containing 2% glucose were treated with 0.4 mM H2O2 for 15 and subsequently pulse-labeled with [35S]methionine from 15 to 30 Total proteins were then extracted and subjected to two-dimensional gel electrophoresis They observed that at least 115 proteins were repressed and 52 induced by this treatment Two isozymes of glyceraldehyde 3-phosphate dehydrogenase were repressed by this treatment Godon et al [50] did not perform the same experiment growing yeast in the presence of glycerol or ethanol as carbon sources The response of S cerevisiae to stress is also dependent on its redox state However, as shown in [51] the metabolic basis for this behavior is still not clear Deficiency in glutathione reductase promotes a higher imbalance in the ratio of reduced glutathione to total glutathione than that produced by glucose 6-phosphate dehydrogenase deficiency However, in contrast to what would be expected, cells 1588 H Osorio et al (Eur J Biochem 270) deficient in this enzyme are comparatively more sensitive to H2O2 stress than those deficient in glutathione reductase Izawa et al [51] concluded that glucose 6-phosphate dehydrogenase appears to play other important roles in the adaptive response to H2O2 stress besides supplying NADPH for the recycling of glutathione Our work is also in line with previous reports indicating that H2O2 and menadione have different effects on yeast [52–54] S cerevisiae cells subjected to treatment with H2O2 (0.2 mM for 60 min) were more resistant to mM menadione However, pretreatment with menadione did not induce resistance to H2O2 and different polypeptides were synthesized as response to treatment with menadione or H2O2 [53] Partially different results were reported later [54] using Schizosaccharomyces pombe Cells pretreated with a low dose of menadione became resistant to a lethal dose of H2O2, whereas cells pretreated with H2O2 became only partially resistant to a lethal dose of menadione The pattern of induction of several oxidative defence enzymes promoted by H2O2 or menadione was also slightly different [54] The study of oxidative response of S cerevisiae, and of other cell types, to stress can be focused under different aspects: the oxidative defence systems of the cell, inducible adaptive responses and their genetic regulation, signal transduction, etc One of the main conclusions that can be derived from this report is that the steady state of the nucleotide level is an important factor to be considered in relation to the general response of S cerevisiae to oxidative stress, as illustrated by the different response to H2O2, depending on whether the yeast uses glucose or glycerol as carbon source It seems to us evident that the intracellular concentration of nucleotides is a key factor to be considered in 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Fig and Table 1, suggested that (a) the main if not unique source of inosine is the intracellular pool of adenine nucleotides, (b) the decrease in ATP and the increase in inosine, promoted by H2O2,. .. degradation and the appearance of intermediate products were essentially the same in both cases and, above all, no appreciable differences in the rates of disappearance of ATP or appearance of Ino