ORIGINAL PAPER

Glutathione peroxidase3 of Saccharomyces cerevisiaeprotects phospholipids during cadmium-inducedoxidative stress

Kannan Muthukumar • Selvaraj Rajakumar •

Mary Nirmala Sarkar • Vasanthi Nachiappan

Received: 22 October 2010 / Accepted: 4 January 2011 / Published online: 13 January 2011

� Springer Science+Business Media B.V. 2011

Abstract The present study was undertaken to

determine the role of glutathione peroxidase3

(gpx3) in phospholipid protection in cells. Wild-type

(WT) cells showed an overall increase in phospho-

lipids upon 50 lM cadmium (Cd)-treatment, whereas

an untreated gpx3D strain showed a drastic reduction

in overall phospholipids which was further reduced

with 50 lM Cd. In WT cells, Cd-exposure increased

the short chain fatty acids and decreased the unsat-

urated fatty acids and the magnitude was high in Cd-

treated gpx3D cells. Purified recombinant gpx3p

showed higher activity with phospholipid hydroper-

oxides than shorter hydroperoxides. An increase in

gpx activity was observed in Cd-treated WT cells and

no such alteration was observed in gpx3D. WT cells

treated with Cd showed an increase in MDA over

untreated, while untreated gpx3D cells themselves

showed a higher level of MDA which was further

enhanced with Cd-treatment. Iron, zinc and calcium

levels were significantly altered in WT and gpx3Dcells during Cd-treatment.

Keywords Glutathione peroxidase � Phospholipids �Fatty acids � Antioxidants � Cadmium �Saccharomyces cerevisiae and oxidative stress

Abbreviations

Cd Cadmium

CL Cardiolipin

Gpx Glutathione peroxidase

IPTG Isopropyl b-D-1-thiogalactopyranoside

LPL Lysophospholipids

MDA Malondialdehyde

Ni2?NTA nickel nitrilo acetic acid-agarose

PC Phosphatidylcholine

PE Phosphatidylethanolamine

PHGpx Phospholipid hydroperoxide glutathione

peroxidase

PI Phosphatidylinositol

PLOOH Phospholipid hydroperoxide

PS Phosphatidylserine

ROS Reactive oxygen species

Introduction

In cells phospholipids play diverse roles that are

essential for growth and metabolism. The major

phospholipids found in the cell membranes of Saccha-

romyces cerevisiae include phosphatidylcholine (PC),

phosphatidylethanolamine (PE), phosphatidylinositol

K. Muthukumar � S. Rajakumar � V. Nachiappan (&)

Department of Biochemistry, Bharathidasan University,

Tiruchirappalli 620 024, Tamilnadu, India

e-mail: vasanthibch@gmail.com

M. N. Sarkar

Department of Biochemistry, Indian Institute of Science,

Bangalore 560012, India

123

Antonie van Leeuwenhoek (2011) 99:761–771

DOI 10.1007/s10482-011-9550-9

(PI), and phosphatidylserine (PS), and the mitochon-

drial membranes also contain a significant amount of

phosphatidylglycerol (PG) and cardiolipin (CL) (Car-

man and Han 2009). Phospholipid biosynthetic

enzymes are regulated to cope with a variety of stress

conditions (Carman and Han 2009; Iwanyshyn et al.

2004). Changes in the phospholipid content were

shown to be an important factor in providing tolerance

against organic solvents (Ghosh et al. 2008). Elegant

work on mechanisms of solvent tolerance in Pseudo-

monas putida strain showed that the strain was able to

repair the damaged phospholipids through increased

biosynthesis and this was necessary to stabilize the cell

membrane structure (Pinkart and White 1997). Cell

membranes contain a carefully balanced mixture of

different phospholipid classes, each with a certain fatty

acyl chain composition. These compositions are

important for the physical properties of the membrane

and are altered with variations in the temperature and

other environmental conditions (Aguilar and de Men-

doza 2006; Martin et al. 2007; Falcone et al. 2004). The

most common fatty acids esterified to the glycero-

phosphate backbone of the phospholipids include

palmitic acid (C16:0), palmitoleic acid (C16:1), stearic

acid (C18:0), and oleic acid (C18:1) (Rattray et al.

1975). Unsaturated acyl chains of phospholipids in cell

membranes are prominent targets of oxidant attack.

This can result in lipid peroxidation, a degenerative

process that perturbs structure and function of the

target system, often with cytopathological conse-

quences (Girotti 1985).

A variety of environmental toxicants cause oxida-

tive stress through peroxidation of membrane lipids,

(Steffensen et al. 1994) and heavy metals are

considered to be one of the potent causative agents.

Cadmium (Cd) is one of the most toxic heavy metal

pollutants, inducing a broad spectrum of toxicolog-

ical and biochemical dysfunctions constituting seri-

ous hazard to health. Cd is a multi-target toxicant for

most organisms, causing severe damage to lung,

liver, kidney, testis and placenta (Patrick 2003). The

main routes of poisoning are either through occupa-

tional exposure or through ingestion of contaminated

food and water (Thorsen et al. 2009). Cd has an ionic

radius close to calcium (Ca2?) while its electro

negativity is similar to zinc (Zn2?). These properties

allow Cd to enter the cells and interfere with the

physiological functions of both Ca and Zn (Suwalsky

et al. 2004). It has a high affinity for protein thiol

groups and also induces membrane damage by

interacting with lipids (Girault et al. 1998). Cd

induces the production of a variety of reactive oxygen

species (ROS) including O2•- and •OH. Cytoprotec-

tion against lipid peroxidation and other types of

oxidative damage is accomplished by diverse enzy-

matic and non-enzymatic antioxidants. Saccharomy-

ces cerevisiae has evolved with many antioxidants

that include glutathione (GSH), catalase (CAT),

superoxide dismutase (SOD) (Temple et al. 2005)

and glutathione peroxidase (gpx) (Arthur 2000).

Gpx is an important participant in antioxidant

protection, which can reduce and detoxify various

organic hydroperoxides at the expense of GSH.

Saccharomyces cerevisiae expresses three different

gpx namely gpx1, gpx2 and gpx3 (Inoue et al. 1999)

with phospholipid hydroperoxidase activity (Avery

and Avery 2001). Among them, gpx3 is major

cellular enzyme that prevents the peroxidation of

membrane lipids and it also plays a major role in

enzymatic defence against oxidative damage (Ursini

et al. 1995). It has been reported that gpx3D is

hypersensitive to peroxides, whereas gpx1D or gpx2Dare resistant to oxidative stress (Ursini et al. 1995).

Gpx3 has multiple functions, by adapting to oxidative

stress response, acting as a ROS scavenger and also

as an efficient linker in repairing proteins during

oxidative stress damage (Temple et al. 2005). Two

major forms of gpx have been characterized in cells:

classical gpx and phospholipid hydroperoxide gluta-

thione peroxidase (PHGPx) (Arthur 2000). The

inorganic and organic hydroperoxides are substrates

for both enzymes and PHGPx alone reduces lipid

hydroperoxides esterified to bio membranes. Thus,

PHGPx is often considered the principal cellular

enzyme capable of repairing membrane lipid perox-

idation, the highly damaging process that has been

linked to pathological conditions such as ischemic

injury, atherosclerosis and carcinogenesis (Halliwell

and Gutteridge 1999).

Hence we determined the differences in phospho-

lipid and its fatty acid composition of wild-type (WT)

and gpx3D strains during Cd-treatment. The substrate

preference assay of gpx3 was studied with recombi-

nant purified gpx3p. To assess lipid peroxidation, the

levels of malondialdehyde (MDA) was quantified and

the activity of gpx was also assayed. The levels of

intracellular contents of divalent iron (Fe), Ca, Zn, Cd

and copper (I) (Cu) were analysed. In this study we

762 Antonie van Leeuwenhoek (2011) 99:761–771

123

investigated the possible relationship of gpx3 and

phospholipids in Saccharomyces cerevisiae during

oxidative stress.

Materials and methods

Materials

WT strain of Saccharomyces cerevisiae BY4741

[MATa his3D1 leu2D0 met15D0 ura3D0] was pro-

cured from Open Biosystems, USA and its isogenic

gpx3D strain was a gift from Prof. William A. Prinz,

NIH. The pET14b-gpx3 plasmid was kindly provided

by Prof. Simon V. Avery, University of Nottingham,

UK. Yeast extract and peptone were obtained from

Difco. The nickel-nitrilotriacetic acid (Ni2?NTA)

matrix was from Qiagen India Pvt. Ltd. All chemicals

and solvents were purchased from Sigma unless

specifically mentioned.

Growth conditions

Yeast cells were grown in YPD (1% yeast extract, 2%

peptone, and 2% dextrose) medium (pH 7.0) with

aeration at 30�C. For growth in Cd-containing medium,

the yeast cells were grown in 5 ml of YPD medium for

12 h. The cells were harvested and transferred to 25 ml

of YPD media, so that A600 of 0.1 OD was reached and

cells were grown at 30�C (with or without Cd) till the

final A600 of 1.00 OD was achieved.

Lipid extraction and analysis

Lipids from yeast cells were extracted by the method

of Bligh and Dyer (1959). The individual phospho-

lipids were separated by two-dimensional TLC on

silica plates (Wagner and Paltauf 1994). Individual

phospholipids were identified by comparing the Rf

values of the unknown with the Rf values of the

standard. Spots corresponding to individual phospho-

lipids were scraped off and quantified by estimating

phospholipid phosphorus after perchloric acid diges-

tion by the method of Siakotos et al. (1966).

Fatty acids analysis

Fatty acids of phospholipids were analyzed by gas

chromatography. Phospholipids were separated by

TLC and individual phospholipids were extracted

from the silica gel with chloroform/methanol (2:1

v/v), and subjected to methanolysis using BF3-

methanol for conversion to methyl esters (Morrison

and Smith 1964). Fatty acid methyl esters were

separated by gas chromatography and quantification

was referred to heptadecanoic methyl ester, as an

internal standard.

Determination of MDA

Lipid peroxidation was measured by the content of

MDA as described by Steels et al. (1994).

Preparation of cell-free extract

Cells were centrifuged (6,000 rpm for 5 min),

washed and sonicated in buffer (100 mM Tris–HCl

pH 7.4, 5 mM MgCl2, 1% glycerol, 1 mM phenyl

methyl sulfonyl fluoride, and 0.03 mM leupeptin).

The homogenate was then centrifuged at 10,000 rpm

for 15 min at 4�C and the supernatant was used to

determine enzyme activity. Total protein was deter-

mined by the method of Bradford (1976).

Expression and purification of recombinant gpx3p

The pET14b-gpx3 construct was transformed into E.

coli BL21 (DE3) cells and the transformed cells were

induced with 0.5 mM IPTG for 2 h. Briefly, IPTG

induced cells were centrifuged and the cell pellet was

resuspended in lysis buffer containing 50 mM Tris–

HCl (pH 8.0) and 300 mM NaCl (buffer A). Cells

were disrupted by sonication. The supernatant was

allowed to bind to the Ni2?-NTA matrix. The column

was washed with buffer A containing 50 mM imid-

azole. The bound protein was eluted with 250 mM

imidazole in buffer A. One-millilitre fractions were

collected and checked for activity. The fractions

showing activity were analyzed for the presence of

protein in 10% SDS-PAGE (Laemmli 1970) followed

by Coomassie Blue staining.

Synthesis and quantification of phospholipid

hydroperoxide

Phospholipid hydroperoxide (PLOOH) was synthesised

using soybean type IV lipoxygenase (Maiorino et al.

1990). The separation of peroxidized phospholipids was

Antonie van Leeuwenhoek (2011) 99:761–771 763

123

done by TLC and identified using tetramethyl-P-

phenylenediamine detection (Kriska and Girotti

2004). The concentration of PLOOH was estimated

by a colorimetric reaction using the ferrous oxidation of

xylenol orange reagent (FOX 2) with reference to a

calibration curve produced with known H2O2 concen-

trations (Wolff 1994).

Assay of gpx

Enzymatic activities of gpx in crude cell extracts or

purified gpx3p were assayed at 25�C with PLOOH or

other substrates according to standard protocols

(Inoue et al. 1999).

Determination of intracellular metals

Cells were harvested by centrifugation at 7,000 rpm

for 10 min. Crude extracts were prepared by glass

bead lysis and then resuspended in nitric acid and

heated at 200�C for 4 h. After complete digestion,

1 ml was diluted to 10 ml in metal-free water and

subjected to analysis using an inductively coupled

plasma atomic emission spectrometer (ICP-OES)

(Perkin Elmer 3300). This analysis was done at

Central Institute of Medicinal and Aromatic Plants

(CIMAP), Lucknow, India following the method of

Doner and Ege (2004).

Statistics

Data were analyzed using the programs of SPSS 10.0

(Statistical Package for the Social Science for Win-

dows 10.0). All the values reported in the paper were

the mean of three replicates. Statistical analysis was

carried out by analysis of variance (ANOVA) test. A

significant difference among means was determined

at P \ 0.05.

Results

Exposure of Cd alter phospholipid composition

in WT and gpx3D cells

To analyze the alteration of phospholipids under Cd-

stress, WT and gpx3D cells were grown in the

presence or absence of 50 lM Cd up to mid log

phase. It was observed that on exposure of WT cells

to Cd significantly (P \ 0.05) increases the levels of

total and individual phospholipids (PC, PE, PS and

LPL) when compared to WT control cells (Fig. 1).

Fig. 1 Exposure to Cd alters the yeast phospholipid compo-

sition. WT and gpx3D cells were grown up to mid log phase on

YPD medium in the absence or presence of 50 lM Cd. Equal

number of cells was taken in each group, lipids were extracted

and resolved in two-dimensional silica TLC. Phospholipid was

quantified and presented as lg of phosphorus/A600 cells 9 103.

PC phosphatidylcholine, PE phosphatidylethanolamine, PIphosphatidylinositol, PS phosphatidylserine, CL cardiolipin,

LPL lysophospholipids. The data shown are mean from three

independent experiments. *P \ 0.05, compared to WT control

cells, �P \ 0.05 compared to gpx3D untreated cells

764 Antonie van Leeuwenhoek (2011) 99:761–771

123

On the contrary to WT cells, Cd-treated gpx3D cells

showed a significant (P \ 0.05) reduction in both

total and individual phospholipids when compared to

gpx3D untreated cells (Fig. 1). Control gpx3D cells

themselves showed a significant (P \ 0.05) decrease

in PC, PE, PI and CL when compared to WT control

cells (Fig. 1).

Fatty acid profile of Cd-treated WT and gpx3Dcells

To elucidate the changes in the acyl composition of

individual phospholipids, fractions were analyzed by

GC–MS. Table 1 shows the changes in acyl compo-

sition of phospholipids in WT and gpx3D cells. In

contrast to phospholipid results not much difference

was observed in fatty acid content of gpx3D control

cells when compared to WT control cells. Exposure

of yeast cells to Cd, showed an overall increase in

short chain fatty acids (C 10:0) and decrease in

unsaturated fatty acids (16:1 and 18:1) in all phos-

pholipids (Table 1). In WT cells Cd-treatment sig-

nificantly increases the level of short chain and

medium chain (C12:0 and C14:0) fatty acids in PC,

PE, PS and CL. The levels of saturated fatty acids

(C16:0 and 18:0) were also increased in PC, PE, PS,

PI and CL when compared to WT control cells.

Similar results was found with Cd-treated gpx3D cells

and the magnitude was high when compared to WT

cells (Table 1).

Determination of MDA in Cd-treated WT

and gpx3D cells

The role of gpx3 in elimination of lipid peroxides was

studied in WT and gpx3D cells. The level of MDA in

Cd-treated and untreated conditions are shown in

Fig. 2. In response to Cd, WT and gpx3D cells

accumulated (P \ 0.05) more MDA than its control

cells. The untreated gpx3D cells themselves showed

an increase in MDA levels and the levels were closer

to Cd-treated WT cells. The gpx3D cells showed a

further increase in MDA upon Cd- treatment (Fig. 2).

Expression and purification of gpx3p

The recombinant gpx3p was expressed in E. coli and

this protein was purified by Ni2?NTA affinity

chromatography. Protein bands corresponding to the

recombinant product were evident from 10% SDS-

PAGE as 18.6 kDa, respectively (Fig. 3).

Substrate preference of the gpx3p

To determine the substrate specificity, the gpx activ-

ities were measured in WT, gpx3D cell extracts and

with purified recombinant gpx3 using different sub-

strates (Fig. 4). Functionally active purified recombi-

nant gpx3 protein showed activity against PCOOH,

PEOOH, PSOOH, LPCOOH, t-BHP and H2O2

(Fig. 4). However higher activity was noted in

PCOOH, PEOOH followed by PSOOH and a

decreased activity was observed against t-BHP, H2O2

and LPCOOH. A similar trend was also observed with

WT cell extract. A significant (P \ 0.05) decrease in

the gpx activity was observed in gpx3D cells against

hydroperoxides when compared to WT cells (Fig. 4).

Activity of gpx with H2O2 and PCOOH

The activity of gpx in WT and gpx3D cells exposed to

Cd was studied. Increased activity was observed in

Cd-treated WT cells and PCOOH was a preferred

substrate over H2O2 (Fig. 5). A decrease in activity

was observed in gpx3D as compared to WT control

cells. No significant changes were observed between

Cd-treated or untreated gpx3D cells (Fig. 5).

Determination of intracellular metals

Figure 6 shows the levels of biologically important

metal ions in WT and gpx3D cells. Cd-treated WT

and gpx3D cells showed significant (P \ 0.05)

increase in Fe and Zn. Exposure of WT and gpx3Dcells to Cd showed a decrease in Cu and Ca content

(Fig. 6). WT and gpx3D cells absorb Cd equally.

Discussion

Yeast cells respond to environmental toxicant by

altering the composition of cellular phospholipids

(Vijayaraj et al. 2010). The main aim of this study is to

elucidate the role of gpx3 in protecting phospholipids

against Cd-induced oxidative stress in Saccharomy-

ces cerevisiae. Previously the PHGPx activity was

reported in gpx1, gpx2, and gpx3 of Saccharomy-

ces cerevisiae (Avery and Avery 2001). Gpx3 has

Antonie van Leeuwenhoek (2011) 99:761–771 765

123

Ta

ble

1E

ffec

to

fC

do

nal

tera

tio

no

ffa

tty

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com

po

siti

on

iny

east

ph

osp

ho

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id

Fat

tyac

ids

(%)

C1

0:0

C1

2:0

C1

4:0

C1

6:0

C1

6:1

C1

8:0

C1

8:1

18

:16

UF

A/S

FA

PC W

Tco

ntr

ol

3.6

8±

0.1

96

.78

±0

.24

13

.64

±0

.86

17

.61

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.71

29

.62

±1

.44

17

.83

±1

.56

12

.46

±1

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1.0

71

.22

WT

Cd

9.5

3±

0.5

7*

10

.10

±0

.61

*1

8.3

0±

1.1

2*

19

.43

±1

.16

*1

4.6

4±

0.9

3*

22

.31

±1

.24

*6

.51

±0

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*1

.14

0.5

0

Gp

x3

con

tro

l4

.21

±0

.36

*7

.13

±0

.42

12

.27

±0

.61

17

.26

±0

.59

28

.13

±1

.12

19

.28

±1

.17

11

.22

±0

.54

*1

.09

1.0

1

Gp

x3

Cd

8.6

2±

0.4

1*

,�

13

.76

±0

.94

*,

�2

1.3

1±

0.5

8*

,�

21

.26

±1

.21

*,

�6

.52

±0

.08

*,

�2

5.7

6±

1.3

1*

,�

4.1

9±

0.1

5*

,�

1.2

10

.22

PE W

Tco

ntr

ol

2.6

0±

0.1

14

.15

±0

.23

8.2

3±

0.4

93

4.6

9±

2.7

21

4.4

2±

2.8

72

1.5

7±

1.6

41

2.1

4±

1.5

40

.62

0.4

7

WT

Cd

4.8

4±

0.2

9*

7.2

1±

0.3

8*

8.7

4±

0.5

64

0.7

1±

3.4

3*

5.6

2±

0.4

6*

26

.61

±1

.45

*5

.36

±0

.31

*0

.65

0.1

6

Gp

x3

con

tro

l3

.11

±0

.16

*5

.42

±0

.36

9.6

8±

0.3

23

5.5

8±

2.1

21

5.7

9±

0.7

12

0.4

3±

1.2

01

1.7

1±

1.2

60

.57

0.4

9

Gp

x3

Cd

6.8

7±

0.3

2*

,�

8.7

4±

0.4

2*

,�

9.2

1±

0.4

14

3.8

3±

2.8

1*

,�

4.4

0±

0.2

5*

,�

25

.79

±1

.71

*,

�4

.36

±0

.23

*,

�0

.58

0.1

2

PS W

Tco

ntr

ol

4.2

1±

0.1

2–

9.1

3±

0.5

13

9.1

5±

2.2

69

.74

±0

.34

17

.25

±1

.35

19

.69

±1

.31

0.4

40

.52

WT

Cd

6.3

4±

0.3

1*

–1

3.6

2±

1.1

2*

43

.91

±3

.42

*5

.31

±0

.12

*2

6.9

3±

2.1

2*

6.2

6±

0.3

4*

0.6

10

.16

Gp

x3

con

tro

l4

.82

±0

.17

–9

.87

±0

.36

38

.72

±2

.34

10

.52

±0

.61

*1

6.6

2±

1.2

11

8.4

8±

1.8

00

.42

0.6

2

Gp

x3

Cd

7.9

6±

0.3

1*

,�

–1

5.3

2±

1.1

5*

,�

44

.73

±2

.69

*,

�3

.84

±1

.22

*,

�2

1.8

4±

1.6

8*

,�

6.7

3±

0.3

5*

,�

0.4

80

.15

PI W

Tco

ntr

ol

–5

.75

±0

.35

18

.33

±1

.24

37

.67

±2

.08

4.2

9±

0.6

92

4.9

6±

1.3

29

.21

±0

.43

0.6

80

.22

WT

Cd

–8

.21

±0

.42

*1

9.2

7±

1.6

73

8.7

6±

2.4

13

.19

±0

.17

*2

6.4

7±

1.5

65

.11

±0

.21

*0

.68

0.1

2

Gp

x3

con

tro

l–

6.2

2±

0.2

81

7.4

5±

1.2

83

6.8

4±

2.3

63

.71

±0

.13

23

.84

±1

.94

10

.79

±0

.62

*0

.59

0.2

6

Gp

x3

Cd

–9

.87

±0

.39

*,

�1

7.8

1±

1.1

64

1.9

6±

2.1

2*

,�

1.5

9±

0.1

1�

22

.74

±2

.11

7.7

3±

0.3

50

.54

0.1

4

CL W

Tco

ntr

ol

1.3

8±

0.1

12

.38

±0

.14

8.2

1±

0.3

42

2.8

7±

1.3

11

7.0

8±

1.1

92

8.1

4±

1.2

52

0.3

4±

1.9

11

.27

0.7

1

WT

Cd

4.6

1±

0.3

6*

4.0

2±

0.2

1*

10

.24

±0

.66

*2

9.6

4±

1.2

8*

5.3

5±

0.2

4*

34

.73

±2

.32

*1

3.8

9±

0.8

2*

1.1

70

.29

Gp

x3

con

tro

l1

.74

±0

.18

2.6

7±

0.1

67

.32

±0

.43

23

.31

±1

.47

*1

6.1

1±

0.7

32

7.2

9±

1.6

41

8.1

3±

1.5

11

.07

0.6

5

Gp

x3

Cd

6.1

2±

0.3

3*

,�

6.9

3±

0.4

3*

,�

12

.73

±0

.82

*,

�3

1.6

7±

1.2

9*

,�

5.2

9±

0.3

1*

,�

32

.53

±1

.42

*,

�5

.72

±0

.33

*,

�1

.27

0.1

7

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766 Antonie van Leeuwenhoek (2011) 99:761–771

123

been shown to play a crucial role in resistance to

oxidative stress by efficiently reducing the PLOOH

(Avery et al. 2004), however the relationship of gpx3

with phospholipids under oxidative stress conditions is

yet to be studied. Hence we studied here the alteration

of phospholipid levels during gpx3 deficiency and Cd-

induced oxidative stress conditions.

Increased major phospholipid levels were

observed in WT cells when treated with Cd

(Fig. 1). Inversely reduction was observed in gpx3Dcells and further decreased with Cd, This suggests the

role of gpx3 in the maintenance of phospholipids

under oxidative stress. Previously it was reported that

gpx3D was hypersensitive to Cd (Avery et al. 2004).

We observed that gpx3D cells showed a decrease in

growth rate when exposed to Cd (data not shown).

Reported evidence suggested that increase in the

phospholipid content was shown to be an important

factor in providing tolerance against organic solvents

(Ghosh et al. 2008). Our results suggest that increased

phospholipid levels in WT cells (Fig. 1) may be

required to resist the Cd-induced oxidative stress.

Stress tolerance mechanism is known to be associated

with increased phospholipid biosynthesis in Saccha-

romyces cerevisiae (Ghosh et al. 2008). Phospholip-

ids were substantially decreased in gpx3D control

cells, thus indicating the partial loss or damage of

phospholipids in the absence of gpx3 and this was

further reduced on Cd-treatment thereby, leading to

major compositional changes (Fig. 1). Previously it

was reported that the deficiency of antioxidants, leads

to marked decrease in the levels of PC and also

leading to mitochondrial damage (Jain et al. 1992).

When treated with Cd the levels of CL were

decreased in both WT and gpx3D cells. Mitochondria

is an important cellular source of O2•- and H2O2

(Lambert and Brand 2009) and are intracellular

targets for different stressors including Cd (Sanni

et al. 2008). Mechanisms of metal-induced mito-

chondrial damage are not fully understood. From our

observation we can suggest that, in the absence of

gpx3, cells were unable to protect phospholipids in

Cd-treated and untreated conditions, as a result

phospholipid content was decreased.

Our phospholipid data suggested that gpx3 is linked

with maintenance of cellular phospholipids during Cd-

induced oxidative stress. The functions of phospholip-

ids mainly depend on its fatty acyl composition.

Previously it was reported that the fatty acid

Fig. 2 Effect of Cd on formation of MDA. WT and gpx3Dcells were grown up to midlog phase on YPD medium in the

absence or presence of 50 lM Cd. The level of MDA was

determined in WT and gpx3D cells as described in ‘‘Materials

and methods’’ section. The data shown are mean from three

independent experiments. *P \ 0.05, compared to WT control

cells, �P \ 0.05 compared to gpx3D untreated cells

Fig. 3 Purification of recombinant gpx3p. The gpx3p from E.coli BL21 (DE3) was purified using nickel-nitrilotriacetic acid

affinity column. Induced gpx3p was visualized by coomassie

blue-staining of 10% SDS-polyacrylamide gel. M molecular

weight marker, Lane 1 isopropyl b-D-1-thiogalactopyranoside

(IPTG) uninduced, Lane 2 induced with 0.4 mM IPTG, Lane3 Ni2?–NTA purified gpx3p

Antonie van Leeuwenhoek (2011) 99:761–771 767

123

composition of CL plays an important role in the

function of the lipid, as aberrant CL remodelling causes

Barth syndrome (Schlame et al. 2005). To understand

molecular species alterations in the absence of gpx3,

we studied the alterations of fatty acyl composition of

phospholipids. Exposure of WT and gpx3D cells to Cd

significantly increases short chain (C10:0), and satu-

rated fatty acids (C16:0, C18:0) in all phospholipids

(Table 1). Oxidative degradation of unsaturated fatty

acids was supported by the lower cellular fatty acid

unsaturation indices evident during exponential

growth in the presence of Cd; depletion of unsaturated

fatty acids is a useful marker of lipid peroxidation (De

Vos et al. 1993). We also found higher reduction in

unsaturated fatty acids (C16:1 and C18:1) (Table 1).

Previous studies with higher organisms have indicated

that susceptibility to heavy-metal toxicity may be

partly dependent on the lipid composition and physical

properties of cellular membranes (e.g. Vossen et al.

1995; De Vos et al. 1993). Cd is suspected to induce the

production of ROS which can attack and damage

biological molecules, including DNA, proteins and

lipids (Esterbauer et al. 1991). Polyunsaturated fatty

acids are esterified in membrane or storage lipids are

subjected to ROS-induced peroxidation and may

yield cytotoxic aldehydes, like 4-hydroxy-2-nonenal

(HNE), MDA and acrolein (Esterbauer et al. 1991).

Fig. 4 Substrate preference of the gpx3p The activities of gpx

from WT, gpx3D cells extract and purified gpx3p are expressed

as U/mg protein. One unit of activity is defined as the amount

of protein required to oxidize 1 lMol of NADPH/min at 25�C.

PCOOH oxidized phosphatidylcholine; PEOOH oxidized

phosphatidylethanolamine, PSOOH oxidized phosphatidylser-

ine, LPCOOH oxidized lysophosphotidylcholine, t-BHP tert

butyl hydro peroxide, H2O2 hydrogen peroxide. The data

shown are mean from three independent experiments.

*P \ 0.05, compared to WT control cells

Fig. 5 Gpx3 activities in Saccharomyces cerevisiae. The

enzyme activity was assayed with PCOOH or H2O2 in WT

and gpx3D cells that were grown on YPD medium in the

absence or presence of 50 lM Cd. Activity of gpx was

expressed as U/mg protein. One unit of activity was defined as

the amount of protein required to oxidize 1 lMol of NADPH/

min at 25�C. The data shown are mean from three independent

experiments. *P \ 0.05, compared to WT control cells,�P \ 0.05 compared to gpx3D untreated cells

768 Antonie van Leeuwenhoek (2011) 99:761–771

123

Heavy metals generally exert their cytotoxic effect

by damaging cell membranes, by initiating the

formation of lipid hydroperoxides and the level of

MDA was measured. Exposure with Cd led to an

increase in the lipid peroxidation in gpx3D cells

(Fig. 2). Avery et al. 2004 observed that gpx3 plays a

major role in heavy metal resistance through its

phospholipid hydroperoxidase activity.

A relationship between oxidative damage of Cd

and the inhibiting effects of antioxidants were known

in Saccharomyces cerevisiae (Muthukumar and Na-

chiappan 2010). Reported evidence suggested that

marked activity against PLOOH is unique to PHGPx

among the cellular enzymes. Thus PHGPx is capable

of directly reversing the process of membrane lipid

peroxidation (Avery and Avery 2001). We studied

the substrate preference assay for gpx3 with PLOOH,

t-BHP and H2O2. From our study, we found that the

WT cell extract and purified recombinant gpx3p

preferred PLOOH particularly PCOOH followed by

PEOOH and PSOOH however lower activities were

also found with LPCOOH, t-BHP and H2O2 (Fig. 4).

Interestingly, no significant increase in activity was

observed in gpx3D with Cd and also not much change

in activity was found between PLOOH and other

hydroperoxides (Fig. 5). From our results we can

suggest that oxidized phospholipids are preferred

by gpx3 (Fig. 5) and gpx3 function is important for

the protection of phospholipids against oxidative

damage. Exposure of Saccharomyces cerevisiae to

Cd increases the activity of gpx in WT cells and

higher activity was observed with PCOOH than H2O2

(Fig. 5). These results suggest that, gpx3 of Saccha-

romyces cerevisiae reduces the level of oxidized

phospholipids during Cd-induced oxidative stress and

the function of gpx3 was predominant for the

maintenance of phospholipids under such conditions.

We analysed the interference of on Fe(II), Cu(I),

Ca(II) and Zn(II) in both WT and gpx3D (Fig. 6). The

level of Ca was decreased and Fe was increased in

Cd-treated WT and gpx3D cells. The decreased Ca

may due to Cd competition (Suwalsky et al. 2004). It

was already reported that, Cd can enter the cells and

potentially interfere with the physiological functions

of Zn and Cu (Girault et al. 1998). Cd perturbs

intracellular Fe metabolism, both Fe and Cu have the

ability to gain or transfer the electrons from molec-

ular oxygen to form ROS, which makes these metals

potentially toxic (Lesuisse and Labbe 1995). Free Cu

and Fe can rapidly catalyse superoxide radical

production which is the precursor for H2O2. This

H2O2 reacts with free Fe(II) and Cu (I) by the Fenton

reaction to generate the potent oxidant hydroxyl

radical. This may be a main route for cellular toxicity,

caused by heavy metals.

In conclusion, Cd treatment in WT cells signifi-

cantly increases the phospholipids except CL, whereas

gpx3D shows a decrease in phospholipid content. WT

Fig. 6 Determination of

intracellular metals. WT

and gpx3D cells were grown

on YPD medium in the

absence or presence of

50 lM Cd. The intracellular

level of divalent Fe, Zn, Ca,

Cd and Cu (I) were

determined as described in

‘‘Materials and methods’’

section. The data shown are

mean from three

independent experiments.

*P \ 0.05, compared to

WT control cells, �P \ 0.05

compared to gpx3Duntreated cells

Antonie van Leeuwenhoek (2011) 99:761–771 769

123

and gpx3D cells showed an increase in short chain fatty

acids, at the same time the levels of unsaturated fatty

acids were decreased. Gpx3 might be required for the

protection of phospholipids during oxidative stress.

Cd-treatment increases the gpx activity in WT cells

whereas no such change was observed in gpx3D cells.

The results obtained from the study indicate that gpx3

might protect the phospholipids from Cd-induced

oxidative damage in Saccharomyces cerevisiae. Fur-

ther investigations must be carried out to understand

the relationship between phospholipid biosynthetic/

degradative enzymes and gpx3 during Cd-induced

oxidative stress.

Acknowledgement The financial support from Bharathidasan

University, Tiruchirappalli, Tamilnadu, India is gratefully

acknowledged.

Conflict of interest None.

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