Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehyde-3-phosphate dehydrogenase

Effects of Oxidative and Nitrosative Stress on Tetrahymena pyriformis Glyceraldehyde-3-Phosphate Dehydrogenase

LATIFA FOURRAT,^aABDELGHANI IDDAR,^bFEDERICO VALVERDE,^cAURELIO SERRANO^cand ABDELAZIZ SOUKRI^a

aLaboratoire de Physiologie et Ge´ne´tique mole´culaire (PGM), De´partement de Biologie, Faculte´ des Sciences Aı¨n-Chock, Universite´ Hassan-II, Km 8 route d’El Jadida, B.P. 5366 Maˆarif, Casablanca, Morocco, and

bUnite´ Radio-Immuno-Analyse, De´partement des Sciences du Vivant, Centre National de l’Energie, des Sciences et des Techniques Nucle´aires (CNESTEN) Centre d’Etudes Nucle´aires de la Maaˆmora (CENM), B.P. 1382 R.P. Rabat 10001, Morocco, and

cInstituto de Bioquı´mica Vegetal y Fotosı´ntesis (CSIC-Universidad de Sevilla), Centro de Investigaciones Cientı´ficas Isla de la Cartuja, Ame´rico Vespucio 49, 41092 Sevilla, Spain

ABSTRACT. Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformiswas chosen. Both reagents inhibited growth ofT. pyriformiscultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO- treated purified enzyme, but no effect was detected by the in vivo H2O2treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H2O2-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO.

Nevertheless, a decrease of GAPDH activity by H2O2and NO, mainly due to a decrease in itsVmaxwithout apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H2O2-dependent effects on GAPDH ofT. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.

Key Words.Central carbon metabolism, glycolysis, oxidative reagents, protein modification, regulatory network.

O XIDATIVE stress is an important physiological condition associated with the toxicity of many chemicals and with pathogenesis in many diseases (Halliwell and Gutteridge 1998).

This stress is linked to the presence of unusually high concentra- tions of toxic reactive oxygen species (ROS). Most of these spe- cies are highly oxidizing, and readily modify redox-sensitive proteins and enzymes, as well as attacking membranes and DNA (Beckman and Ames 1997; Berlett and Stadtman 1997; Vaughan 1997). Reactive oxygen species react frequently with cellular thiols to form disulfides, which are only mildly oxidizing under physiological conditions. Cells actively respond to oxidative stress by setting up many different reactions that increase cell defense or lead to adaptation to oxidant conditions (Wiese, Pacifici, and Davies 1995). One of the consequences of oxidative stress is the block of glycolysis (Spragg et al. 1985); this can be the conse- quence of the depletion of intracellular NAD

¹

pools [via the poly(ADP-ribosyl)polymerase-catalyzed NAD

¹

breakdown] and/

or of the inactivation of the glycolytic enzyme glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Colussi et al. 2000). This latter phenomenon is caused mainly by the interaction of radicals with the cysteine residue present in the active site, which has been reported to be S-thiolated by hydrogen peroxide (Schuppe-Kois- tinen et al. 1994) and S-nitrosilated by nitric oxide (Stamler et al.

1992). Nitric oxide radicals can lead also to inactivation of GAP- DH by favoring the modification of the active site cysteine residue by mono-ADP-ribosylation (Kots et al. 1992; Soukri et al. 1996).

This can be achieved by auto-ADP-ribosylation or by reversible post-translational modifications catalyzed by enzymes that add the ADP-ribose moiety of NAD

¹

onto acceptor proteins, which may finally result in the inactivation of the target protein (Shall 1995).

Besides its inhibitory effect on enzymatic activity, NO has been

reported to affect the GAPDH protein in mammalian cells in which the enzyme is a mediator of apoptotic cell death triggered by NO (Hara, Cascio, and Sawa 2006; Tao et al. 1997).

The phosphorylating NAD

¹

-dependent GAPDH (EC.1.2.1.12) is a key enzyme of the glycolytic pathway. It catalyzes the reversible interconversion of

D

-glyceraldehyde-3-phosphate (

D

-G3P) and 3-phosphoglycerate (Forthergill-Gilmore and Michels 1993). All glycolytic GAPDHs so far studied are homotetrameric proteins with 34–38 kDa subunits that have been remarkably con- served during evolution (Cerff 1995). Recently, GAPDHs from different sources have been shown to be involved in numerous cellular roles reflecting a much broader functional diversity than just the glycolytic function (Kim and Dang 2005; Sirover 1999).

In this work,

Tetrahymena pyriformis

was used as a eukaryotic model organism to assess the effects of hydrogen peroxide and nitric oxide on GAPDH activity and protein levels, both in vivo and in vitro. Its ability to grow in defined axenic media and the possibility to induce synchronic cultures (Zeuthen 1971) make it an appropriate system for a number of studies including cell morphogenesis, conjugation, cell division, and cytotoxicity (Darcy et al. 2002; Dias, Mortara, and Lima 2003; Wheatley, Rasmussen, and Tiedtke 1994) and, as demonstrated in this work for protein regulation studies. On the other hand, previous studies of our group had shown that GAPDH of

T. pyriformis

was coded by a single gene (Hafid et al. 1998) and hinted the possibility of a post-translational regulatory process that had already been de- scribed for the mammalian enzyme (Soukri et al. 1996). There- fore, to demonstrate the likelihood of a widespread regulatory process of glycolysis at the level of the GAPDH, the protozoan model appeared as a suitable choice.

MATERIALS AND METHODS

Organisms and growth conditions.

The amicronucleate strain E, ATCC 30005, of the ciliate

T. pyriformis

used in this work was grown axenically at 28

1

C for 72 h in proteose peptone yeast extract (PPY) medium (Rodrigues-Pousada, Cyrne, and Hayes 1979). To study the effect of hydrogen peroxide and

Corresponding Author: A. Soukri, Laboratoire de Physiologie et

Ge´ne´tique mole´culaire (PGM), De´partement de Biologie, Faculte´ des Sciences Aı¨n-Chock, Universite´ Hassan-II, Km 8 route d’El Jadida, B.P.

5366 Maˆarif, Casablanca, Morocco—Telephone number:1212 22 23 06 80/84; FAX number:1212 22 23 06 74; e-mail: a_soukri@hotmail.

com

338

Journal compilationr2007 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2007.00275.x

sodium nitroprusside (SNP, used as a NO-generating reagent), the PPY medium was supplemented with various concentrations of H

2

O

2

or SNP from stock solutions and inoculated with 1% (v/v) precultures of cell suspensions. H

2

O

2

was added from 0 to 600

mM

in 50-mM steps, while SNP was added from 0 to 1,400

mM in 100- mM steps. Growth curves were performed by following absorb-

ance at 600 nm in a spectrophotometer for 80 h at 6-h intervals.

Crude extract preparation.

Protozoan cells were harvested by centrifugation at 6,000

g

for 10 min and suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 2 mM ethylenediamine tetraacetic acid (EDTA), 10 mM 2-mercaptoethanol, 2 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, and 1% (v/v) glycerol. The cells were then disrupted in the cold with a Branson model B12 Sonifier (80 W, 60 s). The supernatant (soluble protein fraction) obtained after centrifugation at 15,000

g

for 45 min at 4

1

C was considered as the crude cell-free extract.

Enzyme purification.

Glyceraldehyde-3-phosphate dehydro- genase enzyme was purified from cultures grown on PPY medium (control) and PPY medium supplemented with 200

mM H2

O

2

or 500

mM SNP, using a procedure involving conventional anion-

exchange column chromatography (Iddar et al. 2002). Operations were performed at 4

1

C.

The crude extract was subjected to protein precipitation in the 66%–88% (w/v) saturation range of ammonium sulfate. The final precipitate was dissolved in a minimal vol. of 20 mM Tris-HCl (pH 7.5) containing 2 mM EDTA and 10 mM 2-mercaptoethanol (standard buffer). The protein preparation was dialyzed twice for 8 h against 2 L of the same buffer at 4

1

C.

The dialyzed extract was applied at a 10 ml/h flow rate to a DEAE-cellulose DE-52 (Whatman, Maidstone, U.K.) column (1.5 12 cm) previously equilibrated with standard buffer. Elu- tion was performed with a linear gradient of sodium chloride (0–400 mM; 250-ml total vol.) in standard buffer. Fractions of 1 ml were collected and the two fractions that showed the highest GAPDH enzymatic activity were pooled.

Enzyme assay.

The enzymatic reaction was started by adding enzyme to the assay mixture containing 50 mM Tricine buffer, pH 8.5, 10 mM sodium arsenate, 1 mM NAD

¹

, and 1 mM

D

-G3P at 25

1

C. The change in absorbance at 340 nm was followed in a spectrophotometer (Serrano, Maetos, and Losada 1993). Kinetic constants were calculated from initial rates. For the determination of kinetic parameters, the concentrations of the respective fixed substrates for the oxidative phosphorylation reaction were 1 mM NAD

¹

and 1 mM

D

-G3P. The

Km

and

Vmax

values of GAPDH for each of the substrates were determined at the optimal temperature of 35

1

C, using Lineweaver–Burk plots; means values SE of three independent determinations were calculated. One unit of enzyme is defined as the amount that catalyzes the production of 1

mmol NADH/min under the specified conditions.

The in vitro effect of NO and hydrogen peroxide was deter- mined by incubating the purified, dialyzed GAPDH in the pres- ence of various concentrations of the oxidative reagents SNP and H

₂

O

₂

, respectively, in 1-ml incubation mixtures (25 mM Tris- HCl, pH 7.0, 1 mM EDTA buffer). After different time intervals of incubation at 35

1

C, samples were removed and assayed for GAPDH activity as described above.

Protein techniques.

Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Sodium dodecyl sulfate-polyacrylamide gel electropho- resis (SDS-PAGE) was performed as described by Laemmli (1970) on one-dimensional 12% (w/v) polyacrylamide slab gels containing 0.1% (w/v) SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, CA).

After electrophoresis, gels were stained with Coomassie brilliant blue R-250 at 0.2% (w/v) in a mixture of methanol/acetic acid/

water 4:1:5 (v/v/v) for 30 min at room temperature. The apparent

subunit molecular mass was determined by measuring relative mobilities and comparing with pre-stained SDS-PAGE molecular mass protein standards (Precision Plus Protein TM; Bio-Rad, Her- cules, CA). Non-denaturing electrophoresis was carried out in 9%

(w/v) acrylamide gels in the absence of SDS employing the same electrophoretic unit described above.

For pI determination, isoelectric focusing was performed in the same electrophoretic unit described above employing 5% (w/v) polyacrylamide slab gels holding ampholite-generated pH gradi- ents (pH range 3.5–10.0; Pharmalyte 3.5–10.0; Amersham Biosciences, Freiburg, Germany). Cathode and anode solutions were 25 mM NaOH and 20 mM CH

3

COOH, respectively. A set of proteins of different known pI was used as standard (Amersham Biosciences). Analytical column chromatofocusing in the pH range 9.0–5.5 was performed on a column (1 18 cm) of Poly- buffer Exchanger PBE-94 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM Tris-HCl (pH 9.8) containing 1 mM EDTA and 5 mM 2-mercaptoethanol (starting buffer). Glyceralde- hyde-3-phosphate dehydrogenase preparations were dialyzed against starting buffer. After application of the GAPDH prepara- tion, the column was washed with 5 ml of starting buffer. Elution of the enzyme was performed at a flow rate of 12 ml/h by washing the column with 10 bed vol. of a 10-fold diluted mixture of Poly- buffer 96/Polybuffer 74 (30/70, v/v) adjusted to pH 5.5 with acetic acid. The fractions corresponding to the activity peaks were con- centrated and resuspended in standard buffer as described above for eventual catalytic and physico-chemical analyses.

Immunochemical techniques.

A rabbit was injected with 800

mg of purified T. pyriformis

GAPDH protein in aqueous solution 1:1 with incomplete Freund’s coadjuvant. After 21 d, a blood sample was collected, and a second dose of 500

mg of the

protein was injected. After 1 wk, 50-ml rabbit blood were collect- ed and serum was purified by letting it coagulate overnight at 4

1

C followed by centrifugation.

The serum, containing monospecific anti-GAPDH polyclonal antibodies, was brought to 25% (w/v) saturation with solid am- monium sulfate [(NH4)

2

SO

4

], stirred for 1 h, and then centrifuged at 20,000

g

for 45 min. The pellet was then dissolved in a minimal vol. of phosphate-buffered saline (PBS), pH 7.4. The antibody solution was dialyzed twice against 5 L of the same buffer over- night. The dialyzed antibody preparation was applied at a flow rate of 6 ml/h to a DEAE-cellulose (Whatman) column (1.5 12 cm) previously equilibrated with PBS. The column was extensively washed at the same flow rate with equilibrating buffer solution. Fractions of 2 ml were collected and those con- taining the anti-GAPDH specificity were pooled. All the fractions containing anti-GAPDH activity were pooled and applied to CNBr-activated Sepharose 4B coupled to the purified GAPDH.

After incubation at 4

1

C overnight, the column was washed with PBS. Elution was performed with 0.1 M of glycine (pH 2.5). Frac- tions of 1 ml were collected and neutralized with Tris-HCl, pH 9.0, at a final concentration of 50 mM. Those containing the anti- GAPDH activity were dialyzed against PBS buffer, pooled, and conserved at 20

1

C.

Western blot.

Immunoblot assays (Western blot) of protein samples were carried out after SDS-PAGE on 12% (w/v) polya- crylamide slab gels. Proteins were electroblotted onto a nitrocell- ulose membrane (Bio-Rad) employing a Biometra Fast-Blot semi-dry system and incubated with 1:800-fold diluted antiserum in Tris-buffered saline (TBS) containing 5% (w/v) skimmed milk.

The membrane was then washed 4 times (15 min each) in TBS

plus 0.05% (v/v) Tween 20 (TBSt) and incubated for 45 min with

a goat anti-rabbit immunoglobulin G antibody–peroxidase conju-

gate (1:1,000; Boehringer Mannheim, Indianapolis, IN). After

four 15-min rounds of washing with TBSt, the nitrocellulose filter

was developed under 2 mM H

2

O

2

and 10 mM 4-chloro-3-naphtol

in TBS. Filters were processed and when necessary quantified with an analytical imaging instrument (BioImage; Millipore, Bedford, MA).

RESULTS

In vitro effect of NO and H2O2onTetrahymena pyriformis GAPDH.

The

T. pyriformis

GAPDH protein was purified ca. 37- fold after the DEAE-cellulose chromatography (Table 1). Values of ca. 26 U/mg protein were obtained for the purified enzyme, with final yields of ca. 50%. The ammonium sulfate precipitation represented by far the largest contribution to the overall purifica- tion (the purification factor was 25.4). The enzyme activity was eluted from the ionic-exchange chromatography column as a sin- gle symmetrical peak.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the different fractions obtained during the purification procedure showed a progressive enrichment of a 36-kDa protein (Fig. 1A). Only this protein band, which corresponds to the sub- unit of the phosphorylating NAD

¹

-dependent GAPDH, was ob- served in the electrophoretically homogeneous final enzyme preparation (Fig. 1A, lane d). The polyclonal antibody raised against this purified GAPDH clearly recognized the same single 36-kDa protein band, corresponding to the GAPDH subunit, in Western blot analyses of both

T. pyriformis

crude extracts and the purified preparations (cf. Fig. 1B and 7B).

In order to determine the effects of the oxidating reagents on

T.

pyriformis

GAPDH activity, the native purified enzyme was dia- lyzed against 25 mM Tris-HCl (pH 7.0) buffer plus 1 mM EDTA to eliminate any trace of endogenous GAPDH substrates, and was incubated in the presence of different concentrations of either SNP or H

2

O

2

, which produced a time- and concentration-dependent inhibition of the enzyme that was detectable at 10

mM SNP and

H

2

O

2

(Fig. 2). After 30-min incubation, the inhibition reached approximately 50% at 50- and 75

mM SNP and H₂

O

2

, respectively.

Maximum inhibition was observed with concentrations of 200

mM

SNP and H

2

O

2

although even at this concentration the inhibition was not complete (Fig. 2).

To investigate the basis of the NO- and H

2

O

2

-mediated inhibi- tion of

T. pyriformis

GAPDH activity, we examined the effects of SNP and H

₂

O

₂

on the kinetic parameters of the enzyme. The

K_m

values of glyceraldehyde-3-phosphate and NAD

¹

(0.147 0.011 and 0.064 0.006 mM, respectively) did not change when the enzyme was incubated with SNP or H

₂

O

₂

(Fig. 3). On the other hand, the

Vmax

of the GAPDH without incubation with SNP or H

2

O

2

was 32.9 2.8 U/mg of protein, and this value exhibited a reagent concentration-dependent decrease that was more impor- tant with H

2

O

2

. Hydrogen peroxide should be, therefore, a strong- er inhibitor of GAPDH activity than SNP.

To gain further insights into NO and H

2

O

2

inhibition of GAP- DH activity, we examined their effects on the electrophoretic mo- bility of the native enzyme. No change in GAPDH migration was observed on non-denaturing gels after treatment with H

2

O

2

or SNP (data not shown). The data above indicated that the purified enzyme remained mostly unmodified or without any sign of de- gradation after exposure to NO or H

2

O

2

.

On the other hand, we investigated the capacity of NO or H

2

O

2

to mediate the inhibition of the GAPDH substrate-binding activ- ity. NAD

¹

is a redox cofactor for the enzymatic activity and a substrate for GAPDH and as such it binds to the active site. The result showed that the incubation of the enzyme with SNP (50

mM) or H₂

O

₂

(75

mM) for 30 min resulted in a 50% inhibition

of GAPDH activity. However, the addition of 10

mM NAD¹ Table1. Purification of the glyceraldehyde-3-phosphate dehydrogenase

fromTetrahymena pyriformis.

Fraction Total

protein (mg)

Total activity

(U)

Specific activity (U/mg)

Factor (fold)

Yield (%)

Cell-free extract 73 52.6 0.72 1 100

66%–88% ammonium sulfate 2 33.5 16.8 23.3 63.7 DEAE-cellulose eluate 0.9 23.8 26.4 36.7 45.3

kDa

36 kDa

75 50 37 25

20

a b c d e f b c d e f

A B

Fig.1. Purification of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) from control and oxidative stress-induced cultures ofTetrahymena pyriformisstrain E, ATCC 30,005. (A) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing fractions from different purification steps ofT. pyriformisGAPDH. (B) Immunoblot analysis of (A) using a monospecific antibody against theT. pyriformisGAPDH.

Lane a, protein markers; lane b, cell-free protein extract fromT. pyriformiscultured in peptone yeast extract (PPY) control medium; lane c, ammonium sulfate precipitate fromT. pyriformiscultured in control medium; lane d, purified GAPDH fromT. pyriformiscultured in control medium; lane e, purified GAPDH fromT. pyriformisgrown in PPY medium supplemented with 200mM H2O2. Lane f, purified GAPDH fromT. pyriformisgrown in PPY medium supplemented with 500mM sodium nitroprusside. About 40mg of total protein was loaded per lane. The 36-kDa protein band corresponding to the GAPDH subunit is indicated by arrows.

clearly conferred a protective effect; these treatments produced only 12% and 8% inhibition of GAPDH activity, respectively.

In vivo effect of NO and H2O2 onTetrahymena pyriformis GAPDH.

Both H

₂

O

₂

and SNP completely inhibited growth of

T. pyriformis

cultures at concentrations of 400

mM and 1 mM,

respectively (data not shown). Therefore, we employed non-lethal concentrations of H

₂

O

₂

(200

mM) and SNP (500mM) to culture T. pyriformis

and obtained growth curves that allowed us to evaluate the effect of these oxidative reagents on a normally growing pop- ulation (Fig. 4A). The typical growth curve (short lag phase, steep exponential phase, and long stationary phase) was drastically modified by the addition of either oxidative reagents. Neverthe- less, while H

2

O

2

slowed the growth of

T. pyriformis

and never allowed it to reach the same cell number in stationary phase than the control (Fig. 4B), SNP-treated cultures presented a longer lag phase that eventually developed into a naturally growing culture and reached higher cell densities than the control (Fig. 4C). The growth was performed in three separates experiments and the results were repeatable.

In an effort to elucidate the nature of the effects of these ox- idative reagents on

T. pyriformis

native GAPDH, we determined GAPDH activity levels in cell-free extracts obtained from cultures grown in the presence of 50 and 200

mM of H2

O

2

, or 250 and 500

mM of SNP. Although H2

O

2

did not significantly alter GAPDH-specific activity levels, NO diminished GAPDH specific activity by 10%–20% (data not shown).

On the other hand, a specific increase of a major 36-kDa protein band, the expected molecular mass of the GAPDH subunit, was observed in SDS-PAGE analyses of the soluble protein fraction of

T. pyriformis

cells grown on PPY medium supplemented with 250- and 500

mM SNP (Fig. 5A, lanes b and c) or 50- and 200-mM H₂

O

2

(Fig. 5A, lanes d and e), compared with control cells grown on PPY medium (Fig. 5A, lane a). A polyclonal antibody raised against the purified GAPDH was used for Western blot analysis of these crude protein preparations. The antibody clearly recognized a single 36-kDa protein band corresponding to the

T. pyriformis

GAPDH subunit in crude protein extracts from the control culture (see Fig. 5B, lane a). Western blot analysis showed an 5-fold increase of the single immunostained 36-kDa GAPDH subunit in crude extracts of

T. pyriformis

cells grown in the presence of 250- and 500-mM SNP (Fig. 5B, lanes b and c) and an 2-fold increase of the single immunostained GAPDH subunit in crude extracts of cells grown in the presence of 50- and 200-mM H

2

O

2

(Fig. 5B, lanes d and e), consistent with the SDS-PAGE protein analysis (cf. Fig. 5A). On the other hand, as also shown in SDS-PAGE gels, the other protein bands remained substantially unchanged in these crude extracts (Fig. 5A, but see also Fig. 1A).

Further information was obtained after GAPDH purification from

T. pyriformis

cells grown on PPY media supplemented with the oxidative reagents (500

mM SNP or 200mM H₂

O

2

). Although the H

₂

O

₂

-treated enzyme was eluted from the ionic-exchange chromatography column as a single symmetrical peak, resem- bling the behavior of the native enzyme, the NO-treated GAPDH presented a delayed elution that resulted in a broader peak (data not shown). In all cases the two fractions presenting most GAPDH activity were chosen for further analysis. The different binding capacity of the NO-treated enzyme to the DEAE-cellulose column could be due to an alteration in its physico-chemical condition caused by the NO-induced stress. Nevertheless, no significant al- teration in electrophoretic mobility was observed in the purified GAPDHs from

T. pyriformis

cultured in PPY supplemented with NO and H

2

O

2

when compared with the control (Fig. 1A, lanes d and e, respectively). This was confirmed by immunoblot analysis employing the specific antibodies raised previously (Fig. 1B, lanes d and e).

The purified GAPDHs from the control and the oxidative stress treatments allowed us to do a comparative study of their physico- chemical properties. All these dehydrogenases exhibited similar molecular masses under denaturing (see Fig. 1) and non-denatur- ing PAGE, a homotetrameric structure with an estimated 145-kDa molecular mass being obtained for native proteins, as determined by the Hedrick and Smith method (1968). Besides, no significant differences were found for optimal pH and temperature (8.5

1

C and 35

1

C, respectively). Nevertheless, an important change oc- curred in the specific activity of the purified GAPDH from

T.

pyriformis

grown in the presence of NO (19 U/mg) but not H

2

O

2

(25 U/mg) when compared with the controls (26 U/mg).

Furthermore, a difference in the isoelectric point was observed by isoelectric focusing for the GAPDH purified from NO-treated

T. pyriformis

cultures when compared with that obtained for the protein from cells of control or H

2

O

2

-supplemented PPY media (Fig. 6). A slightly more acidic isoelectric point value was observed for the GAPDH purified from

T. pyriformis

cultures supplemented with NO. The isoelectric point of the control GAPDH (pI 8.8) corresponded to that previously obtained by

100

75

50

25

0

% of activity

100

75

50

25

0

% of activity

0 10 20 30 40 50 60 70 80 90

0 10 20 30 40 50 60 70 80 90 Time (min)

Time (min)

Control

10 µM

100 µM 200 µM 25 µM 50 µM 75 µM Control

10 µM

100 µM 200 µM 25 µM 50 µM

A

B

Fig.2. Inactivation of purified glyceraldehyde-3-phosphate dehydro- genase (GAPDH) ofTetrahymena pyriformisstrain E, ATCC 30005 by oxidative reagents in vitro. (A) Glyceraldehyde-3-phosphate dehydrogen- ase (1 U in 1-ml incubation mixtures) was incubated with 10, 25, 50, 100, and 200mM of sodium nitroprusside (SNP). (B) Glyceraldehyde-3-phos- phate dehydrogenase was incubated with 10, 25, 50, 75, 100, and 200mM of H2O2. Values are given as means of three independent experiments. The data are presented as the percentage of initial GAPDH activity. Data are meansSE of three independent experiments.

chromatofocusing for the GAPDH from

T. pyriformis

(Hafid et al.

1998) while for the GAPDH purified preparation from NO-treated cultures a some periodically repetitive more acidic signals could be detected in the isoelectric focusing gel (marked by asterisks in Fig. 6). The subsequent use of column chromatofocusing, a high- resolution technique of protein separation according to pI, allowed us to separate a single basic GAPDH isoform (pI 8.8) in the purified preparations from control (not shown, cf. Hafid et al.

1998) and H

₂

O

₂

-treated cells (Fig. 7A), and three more acidic isoforms of pIs 8.6 (major isoform), 8.0 and 7.3 from SNP-treated cells (Fig. 7B). No co-purified protein peak was found by this chromatographic process, confirming the presence of a single 0.25

0.2 0.15

0.05 0.1

100 µM 50 µM 25 µM Control

100 µM 50 µM 25 µM Control

100 µM

50 µM 75 µM

Control 100 µM

50 µM 75 µM

Control

1/ V (U/mg of protein)

−1

1/ V (U/mg of protein)

−1

1/ V (U/mg of protein)

−1

1/ V (U/mg of protein)

−1

1 / [G

₃

P] mM

⁻¹

1 / [G

₃

P] mM

⁻¹

1 / [NAD] mM

⁻¹

1 / [NAD] mM

⁻¹

− 0.015

− 0.06 − 0.01

−0.1

− 0.005 0.005 −0.005

0.04 − 0.02

− 0.2 0.09

0.005

0.02 0.04 0.01 0.015 0.015 0.025

−0.05

0.5 0.4 0.3 0.2

0.5 0.4 0.3 0.2

0.2 0.4 0.6 0.8

0.1

− 0.1

A1 B1

A2 B2

Fig.3. Lineweaver–Burk plots of oxidative reagent-induced inhibition of purified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Tetrahymena pyriformisstrain E, ATCC 30005. (A) Glyceraldehyde-3-phosphate dehydrogenase preincubated 30 min with 25, 50, and 100mM sodium nitroprusside (SNP). (B) Glyceraldehyde-3-phosphate dehydrogenase preincubated 30 min with 50, 75, and 100mM H2O2. Controls were incubated without SNP and H2O2. Kinetic constants were determined for G3P (A1,B1) and NAD¹(A2,B2). TheKmof glyceraldehyde-3-phosphate (G3P) and NAD¹remained unchanged (0.1470.011 and 0.0640.006 mM, respectively). TheVmaxof the GAPDH not incubated with SNP or H2O2was 32.92.8 U/mg of protein.

4

3

2

1

0

0 20 40 60 80

Time (h)

Absorbance (600 nm)

Control H₂O₂ 200 µm SNP 500 µm

A

B C

Fig.4. Growth inhibition ofTetrahymena pyriformisstrain E, ATCC 30005 in the presence of sodium nitroprusside (SNP) and H2O2. (A) Stan- dard growth curve of culture in control peptone yeast extract (PPY) me- dium ( ); (B) growth curve of culture in PPY medium supplemented with 200mM H2O2( ); (C) growth curve of culture in PPY medium supplemented with 500mM SNP ( ). These experiments were per- formed at least 3 times with consistent and repeatable results. A typical result was represented.

GAPDH protein in all conditions tested. These results agree with those obtained in the isoelectric focusing and strongly suggest an NO-dependent post-translational modification of the enzyme without apparent changes in subunit molecular masses (see SDS-PAGE analysis in Fig. 7).

The GAPDH isoforms from SNP-treated cells exhibited clearly lower specific activities than the single isoform from control cul- tures, in particular the two minor more acidic ones (5%–10% of untreated enzyme-specific activity level) (Table 2).

DISCUSSION

The effect of hydrogen peroxide and the NO-generating reagent SNP on enzymatic activity and protein levels of GAPDH has been examined in

T. pyriformis

strain E, ATCC 30005 cells.

The effects of SNP and H

2

O

2

on the kinetic parameters of the purified enzyme from

T. pyriformis

grown on PPY medium can be explained in two ways: either NO and H

₂

O

₂

reduce the cata- lytic capacity of the enzyme population without altering the bind- ing of its substrates or, alternatively, the decrease in

V_max

may reflect an inactivation of a fraction of the enzyme population, and the

K_m

values estimated are those of the unaltered population.

Therefore, the two reagents did not inhibit GAPDH activity by causing the enzyme to dissociate into its subunits, albeit it had been previously demonstrated for the mammalian enzyme that dissociation of the holoenzyme to either dimer of monomers by oxidative reagents resulted in loss of activity (Harris and Waters 1976).

Our results suggest that GAPDH was probably inhibited by interactions of reactive oxygen radicals with the well-known cysteine residue present at the enzyme active site. A previous study showed that cysteine (as a thiol protecting reagent) con- ferred also protection against SNP inhibition of GAPDH activity

a b c d e a b c d e

A B

36 kDa

Fig.5. Accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inTetrahymenacells caused by oxidative stress. (A) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing the protein patterns corresponding to crude-extract preparations from Tetrahymena pyriformisstrain E, ATCC 30005 grown on peptone yeast extract (PPY) medium (lane a), PPY supplemented with 250mM sodium nit- roprusside (SNP) (lane b), 500mM SNP (lane c), 50mM H2O2(lane d), 200mM H2O2(lane e). (B) Western-blot analysis using a mono-specific antibody against theT. pyriformisGAPDH in crude extracts from cells grown on PPY medium (lane a), PPY supplemented with 250mM of SNP (lane b), 500mM of SNP (lane c), 50mM of H2O2(lane d), 200mM of H2O2(lane e). About 30–50mg of protein was used per lane. The 36-kDa protein band corresponding to the GAPDH subunit is indicated by an arrow.

9.30 8.65 8.45 8.15 7.35 6.85 6.55 5.85 5.20

pI 8.8

a b c

*

*

*

*

*

Fig.6. Modification of the isoelectric point of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) fromTetrahymena pyriformisstrain E, ATCC 30005 caused by in vivo treatment with the reagents sodium nitroprusside (SNP) and H2O2. Isoelectric focusing analyses of the T.

pyriformisGAPDH purified from cells grown on different peptone yeast extract (PPY) media: control (lane a), supplemented with 200mM H2O2

(lane b) and supplemented with 500mM SNP (lane c), were carried out in 5% (w/v) polyacrylamide slab gels holding an ampholite-generated pH gradient (pH range, 3.5–10.0). Aliquots of ca 20mg of purified protein were applied per lane. A more acidic isoelectric point value was observed by isoelectric focusing in the GAPDH purified fromT. pyriformiscultures supplemented with SNP (lane c) than that obtained for the control or the H2O2supplemented PPY medium (lanes a, b). In the GAPDH purified from NO-treated cultures some periodically repetitive more acidic signals were detected (asterisks, lane c).

(Bode, Blumenstein, and Raftery 1975). The GAPDH protection by NAD

¹

or cysteine shows that NO and H

2

O

2

probably reduce the catalytic capacity of the GAPDH by altering the binding of its substrates to the active site, where there is a cysteine residue essential for its activity. This cysteine has been reported to be S-thiolated by hydrogen peroxide (Schuppe-Koistinen et al. 1994) and S-nitrosilated by nitric oxide (Stamler et al. 1992). NO radicals can also lead to GAPDH inactivation by favoring the modification of the active site cysteine residue by mono-ADP- ribosylation (Kots et al. 1992).

The growth curve of

T. pyriformis

was modified by the addition of either oxidative reagents. Something similar has also been re- cently described for

Yarrowia lipolytica

cultures grown in the presence of different oxidative reagents, where a higher toxicity was observed in exponentially growing cells than those in sta- tionary phase (Biryukova et al. 2006). A slight effect of NO but not H

₂

O

₂

on the enzymatic activity was observed in vivo, while a significant increase of the GAPDH protein level could be detected by immunoblots in both cases. This is in agreement with previous reports showing that NO-generating reagents inhib- ited the specific activity of GAPDH in vivo in the amoeba sp.

Dictyostelium discoideum

(Tao et al. 1997) and in mammalian cells (Dimmeler and Bru¨ne 1992; Dimmeler, Lottspeich, and Bru¨ne 1992; Zhang and Snyder 1992). In some cases, a decrease of GAPDH has been also observed after cell injury caused by H

2

O

2

(Hyslop et al. 1988). The increase GAPDH protein level suggests a regulation at the level of GAPDH expression in order to compensate for the inhibitory effect observed both in vitro and in vivo.

A possible explanation for the higher GAPDH accumulation in the in vivo treatment with SNP may be due to the probable pro- tective effect of catalase, known to be very active in

T. pyriformis

cells (Roth and Buccino 1965).

Tetrahymena thermophila

has at least one catalase-encoding gene, TTHERM_01146030 (http://

db.ciliate.org/cgi-bin/locus.pl), so

T. pyriformis

may likely be able to cope more easily with H

₂

O

₂

stress than with NO stress.

A higher enzyme inactivation by SNP could be compensated for by a higher GAPDH protein level, as it was found under this treatment by the SDS-PAGE and immunoblot analyses presented in this paper.

It is interesting to note that GAPDH and cytosolic fatty acid synthase (a subunit) have been reported to be the major targets in response to stress conditions in

Saccharomyces cerevisiae

(Cab- iscol et al. 2000). A 38-kDa protein overexpressed in cultured cerebellar granule cells before age-dependent apoptosis was iden- tified as GAPDH, implying that this enzyme might acquire a gain- of-toxic function during apoptosis (Ishitani et al. 1996). Similar observations were obtained from cerebral cortical culture and cer- ebellar granule cells with a variety of stressors (Ishitani and Chuang 1996; Ishitani et al. 1999). In this regard, in addition to the various functions of GAPDH (Sirover 1999), recent studies have clarified a role for this molecule during cell death, frequently

3

2.5 2

1.5

1

0

0 10 20 30 40 50 60 70 80 90

0.5

3 3.5

2.5 2 1.5 1

0 0.5

FRACTION NUMBER

0 10 20 30 40 50 60 70 80 90

FRACTION NUMBER ENZYMATIC ACTIVITY (U/ml)ENZYMATIC ACTIVITY (U/ml)

Fraction: 51

36 kDa

Fractions: 54 36 kDa

64 72

pH 8.6 pH 8.8

pH 8.0 pH 7.3

10

8

6

4

pH ( )

10

8

6

4

pH ( )

0 0.5 1 1.5

0 0.5 1 1.5 2

ABSORBANCE AT 280 nmABSORBANCE AT 280 nm

A

B

Fig.7. Resolution by column chromatofocusing of the glyceralde- hyde-3-phosphate dehydrogenase (GAPDH) purified fromTetrahymena pyriformisstrain E, ATCC 30005. A sample of 100mg of protein was ap- plied to a Polybuffer Exchanger PBE94 column (118 cm) and the en- zyme was eluted using a pH gradient (o) generated by 10 bed volumes of a 10-fold diluted mixture of Polybuffer 96:Polybuffer 74 (30/70, v/v) ad- justed with acetic acid to pH 5.5. Fractions of 1 ml were collected. Ab- sorbance at 280 nm and enzyme activity were measured. (A)Tetrahymena pyriformisgrown on peptone yeast extract (PPY) medium supplemented with 200mM H2O2. Control culture exhibits the same chromatographic pattern in agreement with previous report (Hafid et al. 1998). Only one peak of enzymatic activity (fraction 51) was found, indicating a single basic isoform (pI 8.8). The inset shows Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the activity peak fraction exhibiting a single 36-kDa protein band corresponding to the GAPDH subunit. (B) Tetrahymena pyriformis grown on PPY medium supplemented with 500mM sodium nitroprusside. The inset shows Coo- massie-stained SDS-PAGE electrophoretogram of the three activity peak fractions (54, 64, 72). The arrow indicates the 36 kDa protein band cor- responding to the GAPDH subunit. Note that the main isoform is slightly acid (pI 8.6) compared with the single peak of the control and H2O2-treat- ed cultures and that further more acidic minor isoforms (pIs 8.0 and 7.3) are found suggesting post-transcriptional protein modification.

Table2. Specific activity of the glyceraldehydes-3-phosphate dehydrogenases (GAPDHs) isoforms in the eluted fractions from column chromato- focusing.

Source culture Fraction GAPDH specific

activity (U/mg)

Tetrahymena pyriformisgrown on PPY medium Fraction 51 (pI 8.8) 26.8

Tetrahymena pyriformisgrown on PPY medium supplemented with 200mM H2O2 Fraction 51 (pI 8.8) 25.9 Isoforms fromT. pyriformisgrown on PPY medium supplemented with 500mM SNP Fraction 54 (major isoform, pI 8.6) 13.1

Fraction 64 (pI 8.0) 1.9

Fraction 72 (pI 7.3) 2.3

PPY, peptone yeast extract; SNP, sodium nitroprusside.

associated with oxidative stress (Chuang, Hough, and Senatorov 2005; Hara et al. 2006). Overexpression of GAPDH as a target for oxidative stress may give cells an advantage when dealing with this stressing condition. We are currently investigating the mo- lecular mechanisms involved in the NO- and H

2

O

2

-dependent in- duction of the NAD

¹

-dependent GAPDH in

T. pyriformis. This

enzyme could be a main target of these effectors and may have a function in the response to nitrosative and oxidative stress in pro- tists and other organisms.

ACKNOWLEDGMENTS

This work was supported by grants from CNRST, project PARS (Morocco), by AECI (Spain), and Collaboratives Grants from the Andalusian Government, Spain (Consejerı´a de Presiden- cia, Proyectos de Cooperacio´n al Desarrollo en el Ambito Univ- ersitario de la Agencia Andaluza de Cooperacio´n Internacional no. 52/02 and 54/04, and Consejerı´a de Innovacio´n, Ciencia y Empresa, PAIDI CVI-261 group).

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Received: 02/14/07, 03/18/07; accepted: 03/23/07