Possible role of superoxide dismutases in the yeast Saccharomyces cerevisiae under respiratory conditions

Archives of Biochemistry and Biophysics 441 (2005) 35–40 www.elsevier.com/locate/yabbi

Possible role of superoxide dismutases in the yeast Saccharomyces cerevisiae under respiratory conditions Volodymyr Lushchak ¤, Halyna Semchyshyn, Serhij Mandryk, Oleh Lushchak Department of Biochemistry, Vassyl Stefanyk Precarpathian National University, 57 Shevchenko Str., Ivano-Frankivsk 76025, Ukraine Received 10 June 2005, and in revised form 18 June 2005 Available online 11 July 2005

Abstract We have analyzed the activity of antioxidant and tricarboxylic acid cycle enzymes as well as protein carbonyl content in budding yeast Saccharomyces cerevisiae cells grown in medium with glycerol using wild-strain cells and defective mutants in superoxide dismutases (SODs). The present report demonstrates that the activity of catalase, glucose-6-phosphate dehydrogenase, glutathione reductase, isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase, on average, was lower in the strains lacking SODs than that in the parental strain. On the other hand, under conditions used in this study, the content of carbonyl groups in proteins was relatively higher in the wild type as compared with SOD-defective strains. It may be suggested that in vivo SOD can demonstrate protective as well as pro-oxidant properties, and the Wnal result depends on particular conditions.  2005 Elsevier Inc. Allrights reserved. Keywords: Saccharomyces cerevisiae; Superoxide dismutases; Antioxidant enzymes; Tricarboxylic acid cycle enzymes; Carbonyl proteins

Superoxide anion (O2¡) is known as a potentially dangerous byproduct of oxygen metabolism in all aerobic organisms. Bakers’ yeast Saccharomyces cerevisiae, like most other eukaryotes, possesses two superoxide dismutases that catalyze the disproportionation of superoxide to hydrogen peroxide. A manganese-containing enzyme (Mn-SOD) encoded by the SOD2 gene accounts for 5–15% of the total superoxide dismutase activity and is the main superoxide scavenger in mitochondria [1,2]. The activity of Mn-SOD is rather low in fermentative cells and can be induced by starvation or respiration as well as exposure to ethanol [3–5]. A copper- and zinc-containing superoxide dismutase (Cu,ZnSOD) encoded by the SOD1 gene represents up to 90% of the total SOD 1 activity [6] and about 1% of soluble *

Corresponding author. Fax: +38 03422 31574. E-mail address: [email protected] (V. Lushchak). 1 Abbreviations used: G6PDH, glucose-6-phosphate dehydrogenase; GR, glutathione reductase; ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; SDH, succinate dehydrogenase; SOD, superoxide dismutase; TCA cycle, tricarboxylic acid cycle. 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.06.010

protein in the cell during fermentation and respiration [4,7]. Cu,Zn-SOD is believed to be located primarily in cytosol, but some of its part has been found in the mitochondrial intermembrane space of diVerent yeast species [6,8] as well as higher eukaryotic cells [9]. Cu, Zn-SOD protects mitochondrial and cytosolic constituents from oxidation and provides long term survival of the respiring yeast [6]. Defect in cytosolic SOD leads to increase of intracellular oxidation after heat shock [5] and quick death in the stationary phase [10]. The importance of SODs is demonstrated by hypersensitivity to oxygen of S. cerevisiae strains carrying mutations in SOD1 and SOD2. The lack of either SOD causes slow aerobic growth with a great number of mutations and a particular sensitivity to redox-cycling drugs [10,11]. Whereas SOD is an essential antioxidant enzyme, some reports have shown that elevated doses of SOD increase lipid peroxidation, generate hydroxyl radical, and induces cell killing [12–14]. Thus, SOD may act as antioxidant and/or pro-oxidant, depending on particular conditions.

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We report here the results of an investigation of the possible role of SODs in S. cerevisiae stationary culture grown on glycerol under respiratory conditions. The Wndings revealed that SODs protect antioxidant and associated enzymes under used conditions. Surprisingly, we have found that the level of oxidized proteins evaluated as the level of carbonyl proteins was signiWcantly lower in the defective strains than that in the wild type. Obtained data make possible speculation that in vivo SODs may behave directly or indirectly as a pro-oxidant.

Materials and methods Materials Phenylmethylsulphonyl Xuoride (PMSF), isocitrate, malate, succinate, dichlorphenolindophenol, glucoso6-phosphate were obtained from “Sigma” (USA); NADPH, NADP, NAD, oxidized glutathione, 2,4-dinitrophenylhydrazine (DNPH), N,N,N⬘,N⬘-tetramethyl ethylenediamine (TEMED) and quercetin were from “Reanal” (Hungary); guanidine–HCl was from “Fluka” (Germany). Inorganic chemicals were obtained from “Reachim” (Russia). All the other chemicals were of analytical grade. Yeast extract was from “Biogene” (Great Britain). Strains and growth condition Yeast strains used in this study were as follows: parental strain EG103 (MAT
leu2-3,112 his3
l trp1289a ura3-52) and its derivative strains containing coding region insertions URA3 in EG118 (sod1
A::URA1), TRP1 in EG110 (sod2
::TRP1) and URA3 and TRP1 in EG133 (sod1
A::URA1 sod2
::TRP1). The strains were kindly provided by Dr. Edith B. Gralla (University of California, Los-Angeles). Cells were grown to stationary phase (72 h) at 28 °C under aeration in a liquid medium containing 1% yeast extract, 2% bacto-peptone, 0.1% glucose, and 2% glycerol. For experiment, cells were prepared from overnight culture grown in YPD medium (1% yeast extract, 2% bacto-peptone, and 2% glucose). The resulting yeast cultures were counted for cell concentration of about 3 £ 106 cells/ml inoculated for the main culture. Preparation of cell extracts Cells from the experimental cultures were harvested by centrifugation (5 min, 7000g) and washed with 50 mM potassium phosphate (K-phosphate) buVer (pH 7.5). The yeast pellets were resuspended in lyses buVer (50 mM Kphosphate (pH 7.5), 1 mM PMSF, and 0.5 mM EDTA). The cell suspensions were vortexed for 15 cycles of 1 min of vortexing with 1 volume of glass beads (0.5 mm) fol-

lowed by 1 min of cooling on ice. Cell debris were removed by centrifugation for 10 min at 15,000g. The cell extract was kept on ice for immediate use. Enzyme activities assay The activity of SOD was assayed at 406 nm as the inhibition of quercetin oxidation by superoxide anion [15] in a medium containing (Wnal concentrations): 30 mM Tris–HCl buVer (pH 9.0), 0.3 mM EDTA, 0.8 mM (TEMED), 50 M quercetin, and 1–30 l of cell extract in a Wnal volume of 2.0 ml. One unit of SOD activity was deWned as the amount of soluble protein of supernatant which inhibited the maximal rate of quercetin oxidation by 50%. The activity of glutathione reductase (GR) was measured by following the consumption of NADPH in a reaction medium containing 50 mM potassium–phosphate buVer (pH 7.5), 0.5 mM EDTA, 1.0 mM oxidized glutathione, 0.25 mM NADPH, and 20 l of supernatant in a Wnal volume of 1.5 ml. Two blanks were run, without glutathione or cell extract. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was measured by monitoring NADP reduction in a reaction medium containing 50 mM K-phosphate buVer (pH 7.5), 5.0 mM MgCl2, 0.2 mM NADP, 1.0 mM glucose-6-phosphate, and 40 l of supernatant in a Wnal volume of 1.5 ml. The activity of isocitrate dehydrogenase (ICDH) was assayed in a medium containing 50 mM K-phosphate (pH 7.5), 2 mM MgCl2, 1 mM NAD, 0.5 mM isocitric acid, and 50 l of cell extract in a Wnal volume of 1.5 ml. The activity of malate dehydrogenase (MDH) was measured in a medium containing 50 mM K-phosphate (pH 7.5), 7.5 mM MgCl2, 0.15 mM NAD, and 1 mM L-malic acid and 40 l of cell extract in a Wnal volume of 1.5 ml. NAD or NADP reduction and NADH or NADPH oxidation by respective enzymes were registered at 340 nm and an extinction coeYcient for these coenzymes of 6.22 mM¡1 cm¡1 was used [16]. Dismutation of hydrogen peroxide by catalase was assayed in 2 ml of medium containing 50 mM K-phosphate buVer (pH 7.0), 0.5 mM EDTA, 10 mM hydrogen peroxide, and 20 l of cell extract in a Wnal volume of 2 ml. Blanks were run in the absence of hydrogen peroxide. Hydrogen peroxide consumption was measured at 240 nm using an extinction coeYcient for hydrogen peroxide of 39.4 M¡1 cm¡1 [16]. The reactions were started by addition of cell free extract. One unit of GR, G6PDH, MDH, ICDH and catalase activity is deWned as the amount of supernatant protein that utilizes or produces 1 mol of substrate or product per minute. The activity of succinate dehydrogenase (SDH) was assayed by modiWed method of T. Singer [17] in a medium containing 50 mM K-phosphate (pH 7.5), 0.5 mM EDTA, 1.5 mM succinate, 40 M of 2,6-dichlorophenolindophenol and 50 l of cell extract in a Wnal volume of 1.5 ml. Two blanks were run, without succinate or cell extract. One

V. Lushchak et al. / Archives of Biochemistry and Biophysics 441 (2005) 35–40

37

Results and discussion

to the YPG medium for easier cell adaptation during lag-phase and growth stimulation. The enzyme activity and carbonyl protein content of each mutant were measured and compared with its isogenic parental strain after growth to the post diauxic shift phase (72 h). Fig. 1 demonstrates the SOD activity in four investigated strains. Under conditions used in this study, the activity of Cu,Zn-SOD (EG110) and Mn-SOD (EG118) accounted for 71 and 2%, respectively, as compared with the wild type (EG103). In the double mutant (EG133) we have found about one unit of SOD activity per milligram of protein, which formed about 0.5% of activity in the parental strain. It should be noted that this activity could be removed by dialysis. A comparison of the relative part of the Cu,Zn-SOD and Mn-SOD enzymes in the mutants indicates that SOD activity of the parental strain is not a simple sum of the SODs activities in its derivatives. Based on the same growth characteristics of the wild-strain and sod2 mutant (data not shown), it may be supposed that SOD1 expression in the defective strain is suYcient to maintain cellular superoxide concentration at an appropriate level providing normal aerobic growth. On the other hand, despite the fact that Cu,ZnSOD is mainly known to be a cytoplasmic enzyme, its activity was detected in mitochondria fractions in amounts ranging from 5 to 29% depending on growth conditions [6,8]. Thus, it seems Cu,Zn-SOD to some extent can compensate for lack of the mitochondrial Mn-SOD, despite the diVerence in their cellular location, and could be more important than other SOD for growth on glycerol. This suggestion is conWrmed by slower growth of strain with mutation in SOD1 than wild type and strain lacking in SOD2 in YPG medium (data not shown). It may be assumed that the intracellular concentration of superoxide would be increased in the mutants

Exponential culture of S. cerevisiae growing on glucose as fermentable carbon source is a commonly used model system. Under these conditions, most of the antioxidant enzymes are repressed by glucose, and aerobically growing cells display higher levels of these enzymes, particularly SOD, catalase, and peroxidase [2,20,21]. On the other hand, the conditions usually used for yeast growth are rare in nature. Most microorganisms, including yeast, have evolved to survive glucose starvation in their environment. Stationary phase yeast depend on mitochondrial respiration for energy and survival [10]. Thus, the role of the antioxidant and TCA cycle enzymes of yeast should be enhanced in natural external surroundings. In this study, yeast were grown in YPG medium with glycerol using yeast strains EG103 (wild type), EG110 (
sod2), EG118 (
sod1), and EG133 (
sod1
sod2). Because yeast demonstrated slower growth on glycerol compared with one on glucose, 0.1% glucose was added

Fig. 1. SOD activity in the wild and SOD-deWcient Saccharomyces cerevisiae strains. Results are shown as means § SEM (n D 5–7). SigniWcantly diVerent from values for the wild type (EG103) with *P < 0.05 and ***P < 0.001.

unit of SDH activity was deWned as the amount of supernatant protein producing a change of 0.01 optical density units at 436 nm per minute. All activities were measured at 25 °C and expressed per milligram of soluble protein in supernatant. Measurement of carbonyl proteins The content of carbonyl groups in proteins was measured by determining the amount of 2,4-dinitrophenylhydrazone formed upon reaction with DNPH. After cells disruption and centrifugation, samples (>1.5 mg protein) were treated with 10 mM DNPH in 2 M HCl at room temperature for 60 min. Blanks contained 2 M HCl without DNPH. Proteins were precipitated by addition of trichloroacetic acid up to Wnal concentration 10%, centrifuged at 4000g for 10 min at 4 °C, and washed three times with 1 ml ethanol–ethyl acetate (1:1). The Wnal pellets were dissolved in 6 M guanidine hydrochloride in 5% (v/v) phosphoric acid. Carbonyl content was calculated from the absorbance maximum of 2,4-dinitrophenylhydrazone measured at 370 nm using an extinction coeYcient of 22 mM¡1cm¡1 [18]. The results are expressed in nanomoles per milligram of protein. Protein concentration and statistical analysis Protein concentration was determined by the Coomassie brilliant blue G-250 dye-binding method [19] with bovine serum albumin as the standard. Experimental data are expressed as means § SEM, and statistical testing used the Student’s t test.

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V. Lushchak et al. / Archives of Biochemistry and Biophysics 441 (2005) 35–40

with decreased SOD activity. Therefore, it was expected that diminished activity of SOD in the deWcient strains should contribute to oxidation of intracellular components. Surprisingly, when total carbonyl proteins data were plotted against SOD activity, the dependence demonstrated a complicated pattern (Fig. 2). On average, the strains lacking the SOD2, SOD1 genes and double mutant showed 1.5-fold lower protein carbonyl levels than that in the wild type. One may suggest that some additional defensive mechanisms are switched on in the defective strains. Besides that, reduced SOD activity may result in lower levels of H2O2 and «OH which are widely believed to be responsible for protein oxidation [22,23]. Some support for this concept was received earlier in our laboratory where a strong inverse correlation between catalase activity and protein carbonyl content was found [24]. On the other hand, present results can also be explained by the pro-oxidant properties of SOD in vivo under certain conditions. Earlier, a similar pattern was found for native Cu,Zn- and Mn-SOD and synthetic SOD mimics in a model system [12,13,25]. Investigation of the relationship between SOD activity and lipid peroxidation of isolated rabbit hearts indicated that the low SOD doses inhibited lipid peroxidation, but increased SOD doses dramatically increased it [12]. The authors concluded that some optimal SOD levels exist and any concentration of SOD other than the optimal leads to increased lipid peroxidation and therefore to oxidative stress. In another model experiment, it was found that Cu,Zn-SOD (but not Mn-SOD), under certain conditions, can catalyze the generation of highly reactive hydroxyl radicals from hydrogen peroxide [14]. Thus, our Wndings can indicate that in native cells, SOD may

also play a pro-oxidant role. However, this conclusion is in contradiction with most reports and generally accepted point of view on the protective role of SOD [26]. To resolve this discrepancy, we have investigated the activity of certain enzymes which are known to be sensitive to oxidation. They are antioxidant (catalase) and associated with antioxidant enzymes (GR and G6PDH), as well as some dehydrogenases of the TCA cycle (ICDH, SDH, and MDH). Because it is known that the superoxide anion can inactivate catalase [27,28], lower activity of this enzyme in the lacking strains could be expected. Fig. 3 shows a strong positive correlation (r2 D 0.89) between SOD and catalase activities. Comparison of parental and its derivative strains indicated 81% (
sod2), 63% (
sod1) and 45% (
sod1
sod2) of catalase activity. Similar relationships between SOD and MDH activities (r2 D 0.96) was also found (Fig. 4). The data show that wild type yeast demonstrated the highest MDH activity. The mutants demonstrated 81% (
sod2), 58% (
sod1), and 45% (
sod1
sod2) of the activity in the parental strain. This can also be explained by protective role of SOD in this case. The activities of other checked enzymes SDH, ICDH, GR, and G6PDH in the four strains used are given in Table 1. In all cases, the parental strain demonstrated signiWcantly higher activity of listed enzymes. However, the relative involvement of each SOD cannot be clariWed. For example, if SDH activity was similar in both single mutants, the activity of ICDH was 2.5-fold higher in the sod2 mutant, than in the sod1 strain. The last Wnding clearly shows the importance of localization in mitochondria of Mn-SOD for ICDH protection, but not for SDH.

Fig. 2. Correlation analysis of data obtained with the wild type and SOD-deWcient Saccharomyces cerevisiae strains: correlating between SOD activity and carbonyl protein levels. Results are shown as the mean § SEM (n D 3–7). SigniWcantly diVerent from respective values for the wild type (EG103) with* P < 0.05, **P < 0.0025 and ***P < 0.001.

Fig. 3. Correlation analysis of data obtained with the wild type and SOD-deWcient Saccharomyces cerevisiae strains: correlating between SOD and catalase activities. Results are shown as the mean § SEM (n D 3–7). SigniWcantly diVerent from respective values for the wild type (EG103) with *P < 0.05, **P < 0.025 and ***P < 0.001.

V. Lushchak et al. / Archives of Biochemistry and Biophysics 441 (2005) 35–40

Fig. 4. Correlation analysis of data obtained with the wild type and SOD-deWcient Saccharomyces cerevisiae strains: correlating between SOD and MDH activities. Results are shown as the mean § SEM (n D 3–7). *SigniWcantly diVerent from respective values for the wild type (EG103) with P < 0.05, ¤¤P < 0.025 and ***P < 0.001.

The data presented in Table 1 indicate that in the defective strains GR and G6PDH activities are diminished as compared with the parental strain. Despite the fact that O2¡ is mainly generated by the mitochondrial respiratory chain, the typical cytosolic enzymes GR and G6PDH can also be targets for ROS in respiring yeast. Because O2¡ carries a negative charge, it cannot easily pass through the mitochondrial inner membrane. However, it can be transported into the cytosol through an anion channel [4,29]. On the other hand, oxidative damage may be caused by secondary ROS which can be formed spontaneously from O2¡ and penetrate the membrane by simple diVusion. For example, spontaneous transformation of O2¡ into HO2¡, or H2O2 can occur near the mitochondrial membrane at low pH [30]. It is also known that O2¡ is generated bidirectionally, both into the cytosol and mitochondrial matrix [10,29]. Besides that, oxidative modiWcation of proteins by aldehydes resulting from lipid peroxidation by ROS is believed to play an important role in inactivation of

39

some enzymes [31,32]. Thus, the mechanisms whereby various enzymes may be inactivated under oxidative stress conditions are multiple and varied, depending on the speciWc enzyme. Comparison of the measured values for the activity of investigated enzymes gives one more interesting fact. Cytosolic enzymes GR and G6PDH as well as mitochondrial SDH were approximately equally protected by both SODs (Table 1), indicating that, although MnSOD and Cu,Zn-SOD account for diVerent levels of activity (Fig. 1) and have various cellular locations, their protective role could, in fact, be similar. This apparent disagreement may to some extent be explained by the complicated organization of eukaryotic cells. It is well known that above 90% of ROS are generated in mitochondria [26]. Colocalization of places of ROS production and Mn-SOD in mitochondria should make this SOD responsible for the detoxiWcation of most ROS in cell, because the last cannot escape mitochondria due to disproportion in primary production places. The situation is complicated when we look at the activities of ICDH, SDH, GR, and G6PDH in the double mutant strain. In this case, the activities of ICDH, SDH, and G6PDH occupied intermediate positions between the wild type cells and each of the single mutant strains. It appears that extremely low SOD activity also has beneWcial eVect for these three enzymes. It can be seen that the dependency of activities of ICDH, SDH, and G6PDH plotted against SOD activity demonstrates a bell-shaped curve similar to one found for the relationship between carbonyl protein levels and SOD activity (Fig. 2). Comparison of the data on ICDH, SDH, G6PDH activities and SOD activity clearly shows that the relationships between these parameters is quite complicated and needs more detailed investigation. It seems that SOD in vivo can play protective as well as pro-oxidant roles. The Wnal result should depend on compartmentalization in a cell of ROS sources, superoxide dismutases and possible targets for ROS attack. However, it should be added that transformation of diVerent ROS species has to be accounted for as well. Thus, present results provide additional support of the

Table 1 The activity of certain enzymes in the parental and SOD-deWcient strains of S. cerevisiae Enzyme

SDH (U/mg protein) ICDH (mU/mg protein) G6PDH (mU/mg protein) GR (mU/mg protein)

Activity EG103 (wild)

EG110 (
MnSOD)

EG118 (
Cu,ZnSOD)

EG133 (
MnSOD,
Cu,ZnSOD)

0.570 § 0.063 85.4 § 5.0 122 § 4 20.1 § 0.9

0.273 § 0.046¤¤ 37.2 § 5.1¤¤¤ 70.0 § 17.7¤¤ 10.7 § 1.9¤¤¤

0.333 § 0.083¤ 14.0 § 3.3¤¤¤ 67.9 § 5.6¤¤¤ 13.5 § 3.0¤

0.407 § 0.029¤ 52.9 § 3.8¤¤¤ 81.9 § 10.4¤¤ 12.8 § 0.9¤¤¤

Results are shown as the means § SEM (n D 4–7). ¤ SigniWcantly diVerent from respective values for the wild type (EG103) with P < 0.05. ¤¤ P < 0.005. ¤¤¤ P < 0.001.

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point of view that it is risky to transfer results obtained by in vitro experiments to native cells.

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