Oxidative Stress Promotes Specific Protein Damage inSaccharomyces cerevisiae (2000)

We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are exposed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aerobically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (α subunit) and glyceraldehyde-3-phosphate dehydrogenase were the major targets. In addition we also report the in vivomodification of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main protein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide generation as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features. We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are exposed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aerobically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (α subunit) and glyceraldehyde-3-phosphate dehydrogenase were the major targets. In addition we also report the in vivomodification of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main protein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide generation as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features. reactive oxygen species 2′,7′-dichlorofluorescein diacetate yeast extract-peptone-dextrose medium yeast extract-peptone-glycerol medium α-ketoglutarate dehydrogenase pyruvate dehydrogenase thiobarbituric acid-reactive substances thiamine pyrophosphate-dependent 2-oxo-acid dehydrogenase dihydrolipoamide acyltransferase FAD-containing dihydrolipoamide dehydrogenase 2,4-dinitrophenylhydrazine Cells growing in an aerobic environment need to cope with prooxidant conditions. Superoxide anion radical (O⨪2), hydrogen peroxide (H2O2), and hydroxyl radical (OH⋅) are normal byproducts of aerobic respiration (1Cadenas E. Annu. Rev. Biochem. 1989; 58: 79-110Crossref PubMed Scopus (899) Google Scholar). These reactive oxygen species (ROS)1 also derive from external environmental factors such as redox active drugs, radiation, and heavy metals (1Cadenas E. Annu. Rev. Biochem. 1989; 58: 79-110Crossref PubMed Scopus (899) Google Scholar). As a result, ROS cause damage to proteins, lipids, and nucleic acids and thereby compromise cell viability. A common property of prokaryotic and eukaryotic cells is their ability to develop defenses against ROS. In Escherichia coli, the adaptive response to oxidative stress has been well characterized. OxyR and SoxR/SoxS are transcriptional regulators that control the expression of several proteins under H2O2 or O⨪2 stress, respectively (2Farr S.B. Kogoma T. Microbiol. Rev. 1991; 55: 561-585Crossref PubMed Google Scholar, 3Hidalgo E. Demple B. Lin E.C.C. Lynch S. Regulation of Gene Expression in Escherichia coli. R. G. Landes Co., Austin, TX1996: 433-450Google Scholar, 4Demple B. Amábile-Cuevas C.F. Cell. 1991; 67: 837-839Abstract Full Text PDF PubMed Scopus (211) Google Scholar). In addition, therpoS-encoded ςs subunit of RNA polymerase is involved in controlling the expression of several antioxidant defense genes. Our previous observations in vivo (5Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 1998; 273: 3027-3032Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) indicated that in E. coli selective protein oxidative damage occurs when the cells were exposed to H2O2 or O⨪2stress. Proteins such as alcohol dehydrogenase, elongation factor G, the heat-shock protein DNA K, and the β-subunit of F0F1-ATPase were identified as major targets. Those results gave a better comprehension of how such stressing conditions affects specific cellular processes. In the yeast Saccharomyces cerevisiae, this adaptive response involves several transcription factors (Yap1, Yap2, Ace1, Mac1, and Hap1) that trigger the response to H2O2, O⨪2, and metal ions. The STRE element is present in the promoter of some of these inducible genes (6Kobayashi N. McEntee K. Mol. Cell. Biol. 1993; 13: 248-256Crossref PubMed Scopus (172) Google Scholar). The STRE-dependent induction of such genes also requires the binding of two zinc-finger proteins, Msn2 and Msn4. (7Estruch F. Carlson M. Mol. Cell. Biol. 1993; 13: 3872-3881Crossref PubMed Scopus (204) Google Scholar, 8Martı́nez-Pastor M.T. Marchler G. Schüller C. Marchler-Bauer A. Ruis H. Estruch F. EMBO J. 1996; 15: 2227-2235Crossref PubMed Scopus (875) Google Scholar, 9Moradas-Ferreira P. Costa V. Piper P. Mager W. Mol. Microbiol. 1996; 19: 651-658Crossref PubMed Scopus (236) Google Scholar). Variations in the amounts of proteins, which are induced or repressed in yeast adaptive responses to hydrogen peroxide stress, have been described (10Collinson L.P. Dawes I.W. J. Gen. Microbiol. 1992; 138: 329-335Crossref PubMed Scopus (176) Google Scholar, 11Jamieson D.J. Rivers S.L. Stephen D.W.S. Microbiology. 1994; 140: 3277-3283Crossref PubMed Scopus (120) Google Scholar) and analyzed (12Godon C. Lagniel G. Lee J. Buhler J.M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Despite adaptive responses, cells exhibit a background level of oxidative damage to their macromolecules, especially those from mitochondria. About 2% of the oxygen consumed by the mitochondrial respiratory chain generate O⨪2 (13Richter C. Schweizer M. Scandalios J.G. Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 169-200Google Scholar). There is a subsequent dismutation of O⨪2 to H2O2, and this molecule, in combination with Fe2+, produces the highly reactive hydroxyl radical (14Liochev S.I. Fridovich I. Free Radic. Biol. Med. 1994; 16: 29-33Crossref PubMed Scopus (370) Google Scholar, 15Stadtman E.R. Berlett B.S. J. Biol. Chem. 1991; 266: 17201-17211Abstract Full Text PDF PubMed Google Scholar). Increased production of these ROS is deleterious to mitochondria and thus to the metabolic and structural integrity of the cell. Oxidative damage to proteins can be evaluated by the titration of carbonyl groups generated in some amino acid side chains during stress conditions (16Levine R.L. Williams J.A. Stadtman E.R. Shacter E. Methods Enzymol. 1994; 233: 346-357Crossref PubMed Scopus (2338) Google Scholar, 17Shacter E. Williams J.A. Lim M. Levine R.L. Free Radic. Biol. Med. 1994; 17: 429-437Crossref PubMed Scopus (414) Google Scholar). The development of such methodologies has allowed the establishment of a correlation between protein oxidation and aging (18Stadtman E.R. Science. 1992; 257: 1220-1224Crossref PubMed Scopus (2417) Google Scholar) and also with several diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, respiratory distress syndrome, muscular dystrophy, Werner's syndrome, and progeria (19Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2847) Google Scholar). Given that identification of proteins affected by oxidative stress in vivo has only been done in prokaryotic cells (5Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 1998; 273: 3027-3032Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), we have usedS. cerevisiae as a general model for eukaryotic cell type to: (i) identify oxidatively damaged proteins under different oxidative stress conditions; (ii) compare its oxidation targets with those ofE. coli and evaluate the type of damage suffered by them when similar targets are shared; and (iii) test the physiological effects of the loss of function of some of these proteins. Hydrogen peroxide (30% solution), menadione sodium bisulfite, 2,4-dinitrophenylhydrazine (DNPH), and protease fromStaphylococcus aureus V8 were purchased from Sigma. Acrylamide/bisacrylamide solution was supplied by Bio-Rad. Polyvinylidene difluoride membranes (Immobilon Pseq) were from Millipore Corp. The chemiluminescent detection kit (Western Light) was from Tropix. 2′,7′-Dichorofluorescein diacetate (H2DCFDA) was from Molecular Probes (Ref. D-399). Polyclonal rabbit antibodies against lipoic acid were donated by Luke Szweda (Case Western University). The strain used in this work was S. cerevisiae CML 128 (20Gallego C. Garı́ E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (137) Google Scholar). Yeast cells were grown exponentially at 30 °C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or YPG medium (1% yeast extract, 2% peptone, 3% glycerol) by incubation in a rotary shaker at 200 rpm. To calculate cell viability, appropriate dilutions of the cultures were spread on plates with solid YPD medium; these were incubated at 30 °C and colony-forming units were determined after 3 days. The level of cell viability in stressed cultures was determined relative to untreated control cultures taken at the same optical density. Cultures of E. coli K12 (kindly provided by E. C. C. Lin) were grown at 37 °C in minimal mineral medium (34 mmNaH2PO4, 64 mmK2HPO4, 20 mm(NH4)2SO4, 1 μmFeSO4, 0.1 mm MgSO4, and 10 μm CaCl2) plus 0.2% glucose and aerated in a rotary shaker at 200 rpm. Exponentially growing cells at 1 × 107 cells/ml were treated with hydrogen peroxide or menadione under the conditions indicated in each experiment. In preadaptation experiments, cells growing either in YPG or YPD were treated with a low dose of hydrogen peroxide (0.25 mm) for 30 min followed by 5 mm hydrogen peroxide for 45 min. The same time protocol was used for menadione using 1 and 40 mm, respectively. When desired 1% succinate was added to cultures. Cultures of E. coli were challenged with 10 mm menadione for the indicated period of time. Samples of S. cerevisiae for analytical Western blot experiments were taken from exponentially growing cultures (A 600 = 0.6, 1 × 107 cells/ml) and prepared as described (21Rodrı́guez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (268) Google Scholar). For preparative purposes, cells from 1-liter cultures were broken with a French press (SLM Aminco) at a gauge pressure of 2200 p.s.i., using a FA-030 chamber. To obtain E. coliextracts, cells were disrupted with a French press at a gauge pressure of 900 p.s.i., using an FA-003 chamber. Crude extracts were clarified by centrifugation at 5000 rpm for 10 min. For Western blot experiments, samples were prepared as described previously (5Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 1998; 273: 3027-3032Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Protein concentration was determined by the Bio-Rad protein assay. Protein carbonyl content of crude extracts was measured according to the dinitrophenylhydrazine derivatization method described by Levineet al. (16Levine R.L. Williams J.A. Stadtman E.R. Shacter E. Methods Enzymol. 1994; 233: 346-357Crossref PubMed Scopus (2338) Google Scholar). Quantification was performed using a Zorbax GF 250 high pressure liquid chromatography gel filtration column at 1 ml/min flow rate and kept at 25 °C. Absorbance at 276 and 370 nm was monitored using a Waters 996 diode array detector. Values of carbonyl content for crude extracts are given in nmol/mg protein. Immunodetection of protein-bound 2,4-dinitrophenylhydrazones in crude extracts of S. cerevisiae and E. coli was conducted as described in Shacter et al. (17Shacter E. Williams J.A. Lim M. Levine R.L. Free Radic. Biol. Med. 1994; 17: 429-437Crossref PubMed Scopus (414) Google Scholar). The anti-2,4-dinitrophenol antibody (DAKO Ref. V0401) was used at a 1/4000 dilution. To detect lipoic acid bound to proteins, the antibody against lipoic acid was used at a 1/50,000 dilution. In both cases, the secondary antibody (goat anti-rabbit conjugated with alkaline phosphatase, Tropix) was used at a 1/25,000 dilution. Two approaches were used to isolate the proteins of interest, ion exchange chromatography and preparative electrophoresis. Crude extracts (typically 250 mg of protein) were introduced on a Waters DEAE 15HR column using a fast protein liquid chromatography system. After a 20-min wash with solvent A (50 mm Tris-HCl, pH 7.5), a linear gradient from 0 to 50% of solvent B (50 mm Tris-HCl, pH 7.5, plus 0.5 m NaCl) was developed over 40 min at a flow rate of 5 ml/min. Collected fractions were derivatized with DNPH and analyzed by Western blot as described above. Preparative electrophoresis, sample preparation for NH2-terminal sequencing, and limited proteolysis, used to further assess protein identification, were performed according to Tamarit et al. (5Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 1998; 273: 3027-3032Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). To detect cytosolic enzyme activities cell extracts were broken using glass beads and assayed as described in the respective references: enolase (24Maitra P.K. Lobo Z. J. Biol. Chem. 1971; 246: 475-488Abstract Full Text PDF PubMed Google Scholar), fatty acid synthase (25Lynen F. Methods Enzymol. 1969; 14: 17-33Crossref Scopus (153) Google Scholar), pyruvate decarboxylase (26Flikweert M.T. Van der Zanden L. Janssen W.M. Th. M. Steensma H.Y. Van Dijken J.P. Pronk J.T. Yeast. 1996; 12: 247-257Crossref PubMed Scopus (201) Google Scholar), and glyceraldehyde-3-phosphate dehydrogenase (27McAllister L. Holland M.J. J. Biol. Chem. 1985; 260: 15019-15027PubMed Google Scholar). To measure mitochondrial enzyme activities, yeast mitochondria were partially purified as described (28Rickwood D. Wilson M.T. Darley-Usmar V.M. Darley-Usmar V.M Rickwood D. Wilson M.T. Mitochondria: A Practical Approach. IRL Press, Oxford1987: 1-16Google Scholar). α-Ketoglutarate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH) activities were measured as previously reported (29Kresze G.B. Ronft H. Eur. J. Biochem. 1981; 119: 573-579Crossref PubMed Scopus (67) Google Scholar). Aconitase activity was determined by the cis-aconitate detection method (30Fansler B. Lowenstein J.M. Methods Enzymol. 1969; 13: 26-30Crossref Scopus (96) Google Scholar). The oxidant-sensitive probe H2DCFDA was used to measure the intracellular oxidation level in yeast (31Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5116-5121Crossref PubMed Scopus (379) Google Scholar, 32Inoue Y. Matsuda T. Sugiyama K. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Exponentially growing cells at A 600 of 0.5, either in YPG or YPD, were washed with 10 mm potassium phosphate buffer, pH 7.0, and incubated for 30 min in the same buffer with 10 μm H2DCFDA (dissolved in dimethyl sulfoxide). H2DCFDA-loaded cells were incubated with 20 mmmenadione as indicated. Cells were then washed, resuspended in distilled water, and disrupted using glass beads. Cell extracts (100 μl) were mixed in 1 ml of distilled water, and fluorescence was measured with λEX = 490 nm and λEM = 519 nm using a Shimadzu RF 5000 spectrofluorimeter. The value of λEM = 519 nm was normalized by protein concentration. Detection of thiobarbituric acid reactive substances (TBARS) was carried out by means of a fluorimetric assay. Values were referred to a standard curve using malondialdehyde bis(dimethyl acetal) ranging from 0 to 0.25 nmol (33Brown R.K. Kelly F.J. Punchard N.A. Kelly F.J. Free Radicals: A Practical Approach. IRL Press, Oxford1996: 119-131Google Scholar). Sequence analysis was carried out using the BLAST 2.0 program. Accession numbers were: for E2 subunit of yeast PDH, GI6324258; for E2 subunit of E. coli PDH, GI434011; for E2 subunit of yeast αKGDH, GI171783; for E2 subunit of αKGDH, GI43022; for yeast heat-shock protein 60, GI171719; and forE. coli DNA K, GI145773. Exponentially growing cultures ofS. cerevisiae under fermenting conditions with glucose as carbon source (YPD) or under respiring conditions with glycerol (YPG) were challenged with hydrogen peroxide for 45 min. They were subsequently analyzed for protein oxidative damage, cell viability, and lipid peroxidation. As shown in Table I, basal levels of protein carbonyl content in crude extracts of cells grown in YPG were 1.6 times higher than those in cells grown in YPD. By contrast, there was a marked increase in protein damage after hydrogen peroxide treatment in cells grown in YPD, whereas those growing in YPG only showed a moderate increase in protein carbonyls. These results are consistent with a higher loss of cellular viability after the stress on cells in fermentative metabolism (treatment with 5 mmhydrogen peroxide resulted in 18% survival on YPD compared with 48% survival on YPG grown cells).Table ICarbonyl content of crude extracts and cell viability after exposure to hydrogen peroxide stressCulture conditionsSurvivalCarbonyl contentTBARSmmYPD control1000.510.040 0.25720.78 2401.110.130 5181.58 5 (pretreated with 0.25 mm)680.85YPG control1000.800.045 2510.980.170 5481.12Cell viability is the percentage of the corresponding YPG and YPD cultures and the values are the mean of three independent experiments with a variation of ±5%. Carbonyl content, given in nmol of carbonyl/mg of protein, was determined after 45 min of treatment of the indicated H2O2 concentration. The values summarized here are mean values for three separate experiments with a variation of ±0.05. TBARS values are given in nmols/mg protein with a variation of ±0.01. Open table in a new Cell viability is the percentage of the corresponding YPG and YPD cultures and the values are the mean of three independent experiments with a variation of ±5%. Carbonyl content, given in nmol of carbonyl/mg of protein, was determined after 45 min of treatment of the indicated H2O2 concentration. The values summarized here are mean values for three separate experiments with a variation of ±0.05. TBARS values are given in nmols/mg protein with a variation of ±0.01. of cells with low of allowed them to higher of the P. Costa V. Piper P. Mager W. Mol. Microbiol. 1996; 19: 651-658Crossref PubMed Scopus (236) Google Scholar, L.P. Dawes I.W. J. Gen. Microbiol. 1992; 138: 329-335Crossref PubMed Scopus (176) Google Scholar). To protein carbonyl content in such preadaptation experiments, cells grown in YPD medium were treated for 30 min with 0.25 mm hydrogen peroxide were challenged with 5 mm hydrogen peroxide for 45 min. As shown in Table I, the carbonyl content was nmol of carbonyl/mg of protein, a value the for lower increase be of antioxidant defense induced under the preadaptation was consistent with a higher cell viability, which increased to compared with 18% of the 1 B the time for increased protein damage from exposure to mm hydrogen peroxide in cultures grown under respiratory (YPG) and fermentative (YPD) conditions. Protein is shown in 1 A. of the main proteins was carried out as described under can be that when hydrogen peroxide stress was performed in the mitochondrial proteins were detected as major targets for oxidative dihydrolipoamide subunit of dihydrolipoamide subunit of and aconitase, of them involved in and the heat-shock protein the cytosolic proteins the were glyceraldehyde-3-phosphate dehydrogenase, involved in glycerol elongation factor in the protein and the of fatty acid the enzyme activity values of fatty acid synthase, aconitase, PDH, and glyceraldehyde-3-phosphate dehydrogenase after hydrogen peroxide The of was different for each enzyme and from for glyceraldehyde-3-phosphate dehydrogenase to for and for activity after oxidative acid the of the yeast cells grown aerobically on YPG or YPD were challenged for 45 min with or 20 mm Cell extracts or mitochondrial were to measure enzyme activities as described under The table the percentage of activity with to control conditions as YPG growing control given in of protein, were: for fatty acid synthase, for aconitase, for pyruvate decarboxylase, for PDH, for and for glyceraldehyde-3-phosphate dehydrogenase. For YPD growing were for fatty acid synthase, for pyruvate decarboxylase, for enolase, and for glyceraldehyde-3-phosphate dehydrogenase. Values given are the mean of at three independent experiments with a variation of Open table in a new In the of the yeast cells grown aerobically on YPG or YPD were challenged for 45 min with or 20 mm Cell extracts or mitochondrial were to measure enzyme activities as described under The table the percentage of activity with to control conditions as YPG growing control given in of protein, were: for fatty acid synthase, for aconitase, for pyruvate decarboxylase, for PDH, for and for glyceraldehyde-3-phosphate dehydrogenase. For YPD growing were for fatty acid synthase, for pyruvate decarboxylase, for enolase, and for glyceraldehyde-3-phosphate dehydrogenase. Values given are the mean of at three independent experiments with a variation of Under fermentative conditions pyruvate decarboxylase was the main protein by the The of the proteins were the same as those in YPG the mitochondrial in amounts in these conditions, were acid synthase and pyruvate decarboxylase activity levels at as that of glyceraldehyde-3-phosphate dehydrogenase, at 50% values measured under this stress are shown in Table I. There was a increase of similar in in YPG and YPD, conditions. a can be in vivo to which in is to oxygen to anion the Fridovich I. Biochem. PubMed Scopus Google Scholar). To the of this different of menadione were added to yeast cultures growing either with glycerol or glucose as a carbon In both cases, the addition of the stressing at 0.5 mm for YPD and 1 mm for The results in with to basal menadione higher amounts of protein carbonyl content in cells grown on YPD than in cells grown on In of cell viability, these results were in with the higher stress of YPG grown especially when the dose of menadione was 5 mm or survival on YPD and on YPG after 40 The results in preadaptation experiments are summarized in Table Protein carbonyls showed a moderate increase nmol/mg which is the value of In with these viability of cells increased to a value higher than the for content of crude extracts and cell viability after exposure to menadione stress conditionsSurvivalCarbonyl contentTBARSmmYPD 40 (pretreated with 1 control1000.800.045 viability is the percentage of the corresponding YPG and YPD control cultures and is the mean value of three independent experiments with a variation of ±5%. Values of carbonyl content in nmol of carbonyl/mg of protein) were determined in exponentially YPD and YPG growing cells and after 45 min of treatment with the indicated menadione concentration. The values summarized here are mean values for three separate experiments with a variation of ±0.05. TBARS values are given in nmols/mg protein with a variation of ±0.01. Open table in a new Cell viability is the percentage of the corresponding YPG and YPD control cultures and is the mean value of three independent experiments with a variation of ±5%. Values of carbonyl content in nmol of carbonyl/mg of protein) were determined in exponentially YPD and YPG growing cells and after 45 min of treatment with the indicated menadione concentration. The values summarized here are mean values for three separate experiments with a variation of ±0.05. TBARS values are given in nmols/mg protein with a variation of ±0.01. of TBARS in cells stressed by incubation with 20 mm menadione a with to untreated both in YPD and YPG grown cells The in lipid after this stress on the metabolic conditions of the cell and were by on generated the cell measured with the probe H2DCFDA The of fluorescence menadione stress indicated that present in cells grown in YPD medium were the values in YPG grown The value in YPD grown cells was of that in YPG The of damaged proteins is in 1 C. Two proteins highly by menadione treatment in YPG grown The NH2-terminal identified them as the E2 subunits of and PDH, which are and similar both involved in the oxidative oxidation the low activity values for both enzymes a of was enzymes such as fatty acid synthase and also that a different from those with carbonyl is for the of these these is that both acid and to the respiratory chain be To of respiratory chain be deleterious to the cultures were treated with 40 mm menadione in the of 1% a to Under this a in cell viability was when compared with the values succinate The addition of succinate on cell viability In YPD grown the major after menadione stress were fatty acid synthase and three enzymes involved in glucose pyruvate decarboxylase, enolase, and glyceraldehyde-3-phosphate dehydrogenase 1 The of the oxidative of and in metabolism to their structural to better