Results Effect of H2O2on the nucleotide content of yeast cells, grown in the presence of galactose, glucose or mannose Exponentially growing yeast cells, with galactose as carbon source,
Trang 1H2O2, but not menadione, provokes a decrease in the ATP
An experimental and theoretical approach
Hugo Osorio1,2, Elisabete Carvalho1, Mercedes del Valle1, Marı´a A Gu¨nther Sillero1,
Pedro Moradas-Ferreira2and Antonio Sillero1
1 Departamento de Bioquı´mica, Instituto de Investigaciones Biome´dicas Alberto Sols UAM/CSIC, Facultad de Medicina, Madrid, Spain; 2 Instituto de Biologı´a Molecular e Celular, Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do 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
83mMmenadione 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 H2O2on 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) H2O2on 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 H2O2treatment is an import-ant factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress Keywords: Saccharomyces cerevisiae; hydrogen peroxide; menadione; glycolysis; 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
nucleo-tides 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 (ÆO2), with one unpaired electron Further additions of electrons and combination with protons generate a variety of oxygen derivatives of biolo-gical 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! Fe3++ÆOH +
OH–) Moreover, H2O2, superoxide and hydroxyl radical (ÆOH) can be interconverted via the Haber–Weiss reaction
Correspondence to A Sillero, Departamento de Bioquı´mica,
Facultad de Medicicina, Universidad Auto´noma 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 13February 2003,
accepted 20 February 2003)
Trang 2Fe3þ þÆO2 $ Fe2þþ O2
Fe2þþ H2O2! Fe3þ þÆOHþ OH
ÆO2 þ H2O2!ÆOHþ OHþ 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 H2O2or 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, H2O2promoted 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
H2O2as 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
Hewlett-Packard
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 30C 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;
ammo-nium sulfate 5 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
absorb-ance readings at 600 nm (D600¼ 1 corresponds to a
concentration of 1.5· 107cellsÆmL)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
H2O2and menadione treatment: control of cell viability
Exponentially growing yeast cells, with a density of about
1.5· 107cellsÆmL)1, were treated with H2O2or menadione
as indicated in each experiment The extraction of
nucleo-tides 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 30C for 2–3days 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· 107cellsÆmL)1(1.2 mg wet weightÆmL)1), were rapidly collected by filtration on a nitrocellulose membrane filter (Millipore, pore size 1.2 lm, 47 mm diameter) and washed once with 5 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 1.2M
HClO4was 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 0.2M
HClO4(0.1 mL per 100 mg wet weight) The supernatants were combined, neutralized with KOH/K2CO3 and ana-lyzed by HPLC as described previously [23] The amount of the nucleosides/nucleotides was determined from the areas
of the corresponding peaks, using the absorption coeffi-cients obtained from standard curves; their intracellular concentration was calculated assuming that 1 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 phos-phorylase In our assay conditions the detection limit was
5 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 0–4C Yeast (200 mL) grown to a cell density of around 1.5· 107cellsÆ
mL)1was harvested by centrifugation, and washed twice with 10 mL of extraction buffer (20 mMsodium phosphate
pH 7.0, 0.1M KCl; 0.1 mM dithiothreitol) The cells (1 g wet weight) were disrupted in the presence of 2 mL of buffer plus 4 g of glass beads (500 lm diameter) by vortexing at top speed on a tabletop mixer for 6 periods of 1 min separated by 1-min periods of cooling on ice The homo-genate was centrifuged for 5 min at 750 g and the super-natant centrifuged further at 550 000 g for 30 min The final supernatant was dialyzed for 2 h against 200 volumes of
20 mM sodium phosphate buffer, pH 7.0; 50 mM KCl; 0.1 mMdithiothreitol, 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]
Trang 3Enzymatic assays
Except when indicated, the reaction mixtures (0.15 mL)
contained: 50 mMimidazole/HCl buffer pH 7.0, 0.1MKCl;
0.1 mM dithiothreitol and 4 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 30C 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 min After chilling, the mixture was filtered and 50 lL
injected into a Hypersil ODS column Elution was
per-formed 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
1 lmol of substrate transformed per min The following
enzyme activities were estimated in the presence of the
indicated substrates or cofactors: adenosine deaminase (EC
3.5.4.4) (0.5 mMadenosine); adenosine kinase (EC 2.7.1.20)
(0.2 mMadenosine and 1 mMATP); AMP deaminase (EC
3.5.4.6) (5 mMAMP and 1 mMATP); AMP 5¢ nucleotidase
(EC 3.1.3.5) (1 mMAMP); IMP 5¢ nucleotidase (EC 3.1.3.5)
(1 mM IMP, 4 mM MgCl2 and 2 mM ATP); nucleoside
phosphorylase (EC 2.4.2.1) (0.5 mM inosine and 2 mM
inorganic phosphate) or (1 mM hypoxanthine and 2 mM
ribose-1-phosphate) Adenylate kinase (EC 2.7.4.3) was
determined spectrophotometrically in the presence of 2 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 H2O2on 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 1 mMH2O2for 0, 7, 11, 20
and 30 min incubation (Fig 1A), and the nucleotide
content analyzed by HPLC as described in Material and
methods After 11 min incubation in the presence of 1 mM
H2O2, the total amount of adenine nucleotides (AMP,
ADP and ATP) decreased by around 50%, with
concom-itant appearance of inosine (Fig 1A) Incubation times
longer than 30 min in the presence of H2O2did 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 min with different
concentrations (0, 0.3, 0.6, 1.0 and 1.5 mM) of H2O2
(Fig 1B)
The results presented in Fig 1 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 mMHO for 1 h (Table 1) In addition
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 mMH2O2are shown
in Fig 2 Treatment with 1.5 mMH2O2for 1 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 H2O2 -treated 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
Fig 1 Effect of H 2 O 2 on (AMP + ADP + ATP) and inosine pools of
S cerevisiae grown in the presence of galactose as carbon source Yeast cells were challenged with 1 m M H 2 O 2 for the indicated times of incubation (A) or with different concentrations of H 2 O 2 for 30 min (B) AMP, ADP, ATP and inosine contents were determined as des-cribed in Materials and methods.
Trang 4Table 1 Nucleoside and nucleotide content of S cerevisiae (strain W303) grown in galactose and treated for 1 h in either 1.5 m M H 2 O 2 or83 m M
menadione Exponentially growing yeast cells were challenged with either H 2 O 2 or menadione Analysis of the nucleotide content was performed as described in Materials and methods The data represent mean values ± SE of 6, 7 and 3experiments for the control, H 2 O 2 -treated and menadione-treated cells, respectively The concentrations of the indicated compounds are expressed in m M
Parameters Control H 2 O 2 Menadione H 2 O 2 /Control MD/Control Starting wet weight (mg) 147 ± 70 185 ± 34 173 ± 9.0 – –
Total protein (mg) 3.7 ± 1.5 3.0 ± 1.2 2.5 ± 0.5 – –
Adenylic charge 0.78 ± 0.030.41 ± 0.15 0.86 ± 0.01 0.53 1.10
S (ATP + ADP + AMP) 2.24 ± 0.49 0.33 ± 0.12 1.49 ± 0.08 0.15 0.67
S (CTP + CDP + CMP) 0.46 ± 0.08 0.22 ± 0.030.3 4 ± 0.07 0.48 0.74
S (GTP + GDP + GMP) 0.54 ± 0.06 0.26 ± 0.08 0.32 ± 0.05 0.48 0.59
S (UTP + UDP + UMP) 0.70 ± 0.16 0.50 ± 0.09 0.46 ± 0.05 0.71 0.66
ADP-Rib 0.24 ± 0.11 0.04 ± 0.030.05 ± 0.03 0.17 0.21
UDP-sugars 1.30 ± 0.16 1.18 ± 0.16 1.58 ± 0.41 0.91 1.21
Hypoxanthine 0.11 ± 0.05 0.16 ± 0.06 0.11 ± 0.05 1.45 1.00
Inosine 0.20 ± 0.04 2.07 ± 0.69 0.94 ± 0.57 10.34.70
a
The concentration (m M ) of this compound has been calculated assuming the extinction coefficient of adenosine.
Fig 2 HPLC nucleotide profile obtained from yeast cells grown in galactose or glucose, as carbon source and in the absence or presence of H 2 O 2 Yeast cells grown in the presence of galactose or glucose were challenged, when indicated, with 1.5 m M H 2 O 2 for 1 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.
Trang 5presence 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 1 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 H2O2is 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 H2O2on 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 83mM
menadione produced a viability similar to that evoked by 1.5 mMH2O2(around 40% after 60 min 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 1 h with 83mM mena-dione 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 min was observed (Tables 1 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 2 Nucleoside and nucleotide content of S cerevisiae (strain W303) grown in glucose and treated for 1 h with either 1.5 m M H 2 O 2 or83 m M
menadione Exponentially growing yeast cells were challenged with either H 2 O 2 or menadione Analysis of the nucleotide content was performed as described in Materials and methods The data represent mean values ± SE of 5, 5 and 3experiments for the control, H 2 O 2 -treated and menadione-treated cells, respectively The concentrations of the indicated compounds are expressed in m M
Parameters Control H 2 O 2 Menadione H 2 O 2 / Control MD/ Control Starting wet weight (mg) 123± 42 124 ± 6 147 ± 17 – –
Total protein (mg) 2.7 ± 0.8 3.2 ± 1.4 4.7 ± 1.6 – –
Adenylic charge 0.89 ± 0.02 0.53± 0.10 0.85 ± 0.05 0.60 0.96
S (ATP + ADP + AMP) 1.30 ± 0.09 0.47 ± 0.12 1.16 ± 0.06 0.36 0.89
S (CTP + CDP + CMP) 0.30 ± 0.04 0.10 ± 0.03 – 0.33 –
S (GTP + GDP + GMP) 0.23± 0.14 0.14 ± 0.04 – 0.61 –
S (UTP + UDP + UMP) 0.65 ± 0.07 0.23± 0.05 – 0.35 –
ADP-Rib 0.11 ± 0.030.02 ± 0.00 0.05 ± 0.01 0.18 0.45
NADP + 0.04 ± 0.02 0.02 ± 0.01 0.04 ± 0.00 0.50 1.00 UDP-sugars 0.49 ± 0.09 0.59 ± 0.19 0.40 ± 0.12 1.20 0.82 Hypoxanthine 0.10 ± 0.01 0.15 ± 0.05 0.04 ± 0.01 1.50 0.40 Inosine 0.20 ± 0.06 1.16 ± 0.28 0.22 ± 0.06 5.80 1.10
a
The concentration (m ) of this compound has been calculated assuming the extinction coefficient of adenosine.
Trang 6This new unknown chromatographic peak, not present in
the preparation of menadione used, may correspond to a
derivative of adenosine
Effect of H2O2on 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, 3and
4 mMH2O2 A concentration of H2O2as high as 4 mMdid
not change the HPLC nucleotide profile obtained with
untreated cells (results not shown) In a different
experi-ment, yeast cells were grown in the presence of 2% ethanol
as a carbon source, and challenged with 1.5 mMH2O2for
1 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
metabo-lism, yeast cells grown in ethanol or glycerol as carbon
sources are more resistant to H2O2than those grown in the
presence of galactose or glucose
Search for a plausible mechanism
A mechanism to explain the different effects of H2O2on 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 H2O2in 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
acti-vities 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 mMH2O2
The pathways considered here to approach the
meta-bolism of adenine nucleotides in yeast cells subjected (or
not) to oxidative stress, together with the differential
equation describing these pathways are represented in
Figs 3and 4, respectively The enzymes considered in the
pathway from ATP to Ino were E1 (AMP
5¢-nucleoti-dase), E2 (IMP-GMP specific 5¢–nucleotidase), E3 (AMP
deaminase), E4(adenosine deaminase), E5(purine
nucleo-side 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
concen-tration 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
kinetic constants taken from the literature are compiled in Table 3 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
Fig 3 Adenine and hypoxanthine nucleotide metabolism in yeast cyto-sol The pathways considered are those shown in the Figure The enzymes involved are E 1 , 5¢-nucleotidase acting on AMP and IMP; E 2 , IMP-GMP specific 5¢-nucleotidase; E 3 , AMP deaminase; E 4 , adeno-sine deaminase; E 5 , purine nucleoside phosphorylase; E 6 , adenylate kinase; E 7 , adenosine kinase; E 8 , hypothetical enzyme recycling ATP.
Fig 4 Differential equations describing the fluxes operating in the pathways from ATP to hypoxanthine, as described in Fig 3 V n , maximum velocity of the reaction catalyzed by E n , on the substrate indicated The velocity equations considered for E 1 , E 3 , E 4 , E 5 , E 6 and
E 7 were as in Torrecilla et al [3]; those for E 2 and E 8 are indicated in the text and Table 3.
Trang 7the 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
meta-bolism 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
MATHEMAT-ICA-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
proper-ties 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 5¢-IMP nucleotidase was settled as:
m2¼ V2IMP½IMP
n
½IMPnþ ½S0:5n where n¼ 1.7–1.2[ATP]/(Ka2ATP+ [ATP]) and S0.5¼
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:
Ka2ATP¼ 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 V8dand V8r, and the corres-ponding substrates and products were not considered at this stage (i.e V8d¼ V8r¼ 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
Table 3 V max , K m and K i values of enzymes involved in the adenine and hypoxanthine metabolism in yeast cytosol V max values represent the average
of a minimum of three determinations obtained from different batches of yeast cells grown in glucose, with H 2 O 2 results determined in the yeast cytosol, and grown for 30 min in the presence of 1.5 m M H 2 O 2 K i values of the products were assumed equal to the K m values of the substrate in the cases of enzymes E 2 , E 5 , E 6 , E 7 and E 8 E n represents the enzymes as specified in Fig 3.
V max (mUÆmg)1)
K m (l M ) K i (l M ) Control H 2 O 2
5¢-AMP nucleotidase IMP 12.7 ± 0.9 11.8 ± 2.2 540 [29] 8600 (Ino) [29]
27.2 ± 5.4 (ATP) 37.5 ± 8.9 (ADP)
1800a,b
2000 (Ino)
5000 (P i ) [30]
E 3 AMP 29.2 ± 2.4 a 24.4 ± 7.5 a 2670 [31] 4700 (IMP) [31]
Adenosine deaminase
Hyp 29.4 ± 1.4 33.4 ± 0.6 22 [35] 22
Adenylate kinase ADP 2414 ± 621 2593± 557 23[36] 23
200 (AMP) [35]
1200 (ADP) [35]
a Values obtained in the presence of ATP b K m values determined in yeast cytosol, and used in the theoretical simulation depicted in Fig 5.
c
Values calculated using MATHEMATICA -3.0 program.
Trang 8similar rates of disappearance of substrates and appearance
of products (Fig 5B)
Inhibition of glycolysis as a plausible theoretical
explan-ation 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 1 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
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 partici-pation of ATP in the reverse direction (E8r):
ADPþ X-P $ ATP þ 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 random-bireactant [41], and the corresponding velocity equation is:
m 8
¼
[ADP][S P] V 8d
K m8ATP K i8S
1 þ[ADP]
K i8ADP þ[SP]
K i8S þ [S][ATP]
K m8S K i8ATP
The following kinetic constants were established to solve the equation:
Km8ADP¼ Ki8ADP¼ Km8ATP¼ Ki8ATP¼ 0:033 mm
Km8SP¼ Ki8SP¼ Km8S¼ Ki8S¼ 0:1 mm
½X ¼ ½X-P ¼ 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 V8dfrom 4000 (Fig 6B) step
by step to 500, and leaving constant V8rat 200 (Fig 6C–F) The graph in Fig 6A represents an extreme situation in which V8d¼ V8r¼ 0 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 H2O2diminishes the rate of synthesis of ATP, probably through inhibition of glycolysis It is worth noting that H2O2has no appreciable effect on the level of ATP on yeast cells grown in ethanol or glycerol, that are metabolized through an oxidative pathway
Fig 5 Metabolism of ATP, ADP, AMP in the presence of cytosol from
yeast growing in glucose, in the absence or presence of H 2 O 2 – theoretical
simulation The reaction mixtures contained: 50 m M imidazole/HCl
buffer, pH 7.0; 0.1 M KCl; 0.1 m M dithiothreitol; 4 m M MgCl 2 ;
1.8 m M ATP; 0.8 m M ADP; 0.34 m M AMP and cytosol from yeast
cells grown in the absence (A) or presence (result not shown) of 1.5 m M
H 2 O 2 , for 60 min 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 3were used.
Trang 9Effect of H2O2on 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 D600units per mL), at which
the effect of H2O2was 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 D600units 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 mMH2O2promoted 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 min After this treatment, cells were
separated by centrifugation, resuspended in fresh medium
without H2O2, and aliquots taken at 0, 30, 60, 90 and
120 min incubation As expected, the ATP content was very
low after the H2O2treatment (time zero) and the inosine
concentration very high (Fig 8) After 30 min incubation in
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,
Fig 6 Influence of the hypothetical enzyme (E 8 ) 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 E 1 –E 7 determined in the cytosol of control cells, grown in glucose.
In the case of enzyme E2, the K m values described in [30] were used (Table 3) Graphs A–F were computer made using the following additional values, respectively, for V 8d and
V 8r : A (0,0); B (4000, 200); C (3500, 200);
D (3000, 200); E (2500, 200); F (2000, 200).
Fig 7 Effect of H 2 O 2 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 m M H 2 O 2 Ethanol was determined in the medium,
at the indicated times, as described in Materials and methods.
Trang 10H2O2evokes 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
path-way 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
assump-tion was experimentally tested by measuring the
consump-tion 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 H2O2on glycolysis was further confirmed
by the lack of effect of H2O2when yeast cell were grown in
glycerol or ethanol, two oxidative substrates The possibility
that the resistance to H2O2in 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
affec-ted by the H2O2 treatment [42] In line with the results
reported in this study, the activity of glyceraldehyde
3-phos-phate 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 H2O2treatment 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 incuba-tion 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 concentra-tion in H2O2treated 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 H2O2from 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 ± 2.3and 5.5 ± 1.9 mUÆmg)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 H2O2could 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 min and subsequently pulse-labeled with [35S]methionine from 15 to 30 min Total proteins were then extracted and subjected to two-dimensional gel elec-trophoresis 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
Fig 8 Recovery of ATP after treatment of yeast cells with H 2 O 2 Yeast
cells grown in glucose, were treated with 1.5 m M H 2 O 2 for 30 min,
collected by centrifugation, resuspended in fresh medium (without
H 2 O 2 ) and incubated further for 120 min At the times indicated, the
adenylic charge, ATP and inosine were determined as described in
Materials and methods.