Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene  

1096-4959/02/$- see front matter

2002 Elsevier Science Inc. All rights reserved.

PII: S 1 0 9 6 - 4 9 5 9Ž 0 1

0 0 5 1 8 - 8

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a

GapC gene

Khadija Mounaji , Nour-Eddine Erraiss , Abdelghani Iddar , Maurice Wegnez ,

Aurelio Serrano *, Abdelaziz Soukri

Laboratoire de Biologie et Physiologie de la Reproduction et du Developpement, Faculte des Sciences I, BP5366, Maarif,

a

´ ´

Casablanca, Morocco

Laboratoire de Biochimie, Biologie Cellulaire et Moleculaire, Faculte des Sciences I, BP5366, Maarif, Casablanca, Morocco

b

´ ´

Laboratoire d’Embryologie Moleculaire et Experimentale, UPRES-A 8080 du CNRS, Universite Paris XI, Batiment 445,

c

´ ´ ´ ˆ

91405 Orsay, France

Instituto de Bioquımica Vegetal y Fotosıntesis

CSIC-Universidad de Sevilla

,

d

´ ´

Centro de Investigaciones Cientıficas Isla de la Cartuja, Americo Vespucio syn, 41092 Seville, Spain ´ ´

Received 16 August 2001; received in revised form 12 November 2001; accepted 22 November 2001 Abstract

The NAD -dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase

(GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl ( Amphibia, Urodela ) . The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD . Only one GAPDH isoform

( pI 7.57 ) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose.

A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme — the first GAPDH from an amphibian of the Caudata group studied so far — with other GAPDHs of major vertebrate phyla.

2002 Elsevier Science Inc. All rights reserved.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; GapC; Amphibia; Caudata; Pleurodeles waltl; Protein purification; cDNA;

Molecular phylogeny

1. Introduction

One of the most studied enzymes in the glycol-

Abbreviations: bp, base pair

s

;

-G3P,

-glyceraldehyde- 3-phosphate; GapC, gene

DNA, RNA

encoding glycolytic GAPDH; GAPDH, glyceraldehyde-3-phosphate dehydrogen- ase; kDa, kiloDaltons; PCR, polymerase chain reaction; pI, isoelectric point; RT, reverse transcription; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

*Corresponding author. Tel:

34-5-448-9524; fax:

34-5- 446-0065.

E-mail address: aurelio@cica.es

A. Serrano

.

ytic pathway is glyceraldehyde-3-phosphate dehy-

drogenase ( GAPDH, EC 1.2.1.12 ) , which

reversibly catalyses the oxidative phosphorylation

of

-glyceraldehyde-3-phosphate to form 1,3-

diphosphoglycerate in the presence of the NAD

and inorganic phosphate ( Harris and Waters,

1976 ) . This enzyme is widely distributed in nature

in a variety of species ranging from bacteria to

humans ( Fothergill-Gilmore and Michels, 1993 ) .

It is found mainly in the cytosol, in the mitochon-

dria and chloroplasts. Organellar GAPDHs are

encoded by nuclear genes as precursor polypep-

tides and post-translationally imported into the organelles ( Cerff, 1995 ) . This enzyme has been well characterized not only because of its central role in the intermediary metabolism, but also because of its abundance and ease of preparation.

GAPDH is well conserved during evolution, being a protein with native molecular weight in the range of 140–150 kDa and composed of four identical subunits of approximately 35–37 kDa ( Fothergill- Gilmore and Michels, 1993 ) .

The ubiquity and evolutionary conservation of GAPDH indicate a highly important physiological function. In addition to its well characterized glycolytic activity, a housekeeping function essen- tial for the normal metabolism of all cells, there is now accumulating evidence that this protein is implicated in a large spectrum of cellular functions ( Sirover, 1999 ) . These included: a nuclear activity as DNA repair enzyme ( Baxi and Vishwanatha, 1995 ) , specific binding to 39 and 59 regions of mRNA ( Schultz et al., 1996 ) , a nuclear RNA export activity ( Singh and Green, 1993 ) , and possible roles in neuronal apoptosis ( Saunders et al., 1999 ) , a neurodegenerative disease ( Mazzola and Sirover, 2001 ) and in prostate cancer ( Rondi- nelli et al., 1997 ) .

In the present study we have asked whether some of the distinguishing characteristics of other GAPDHs are in any way evident in the features of amphibian GAPDH. Basis for choosing the newt Pleurodeles waltl for such study is manifold.

This species, which possess particularly organized lampbrush chromosomes, has been well studied and a considerable amount of work has been done on this amphibian providing a useful model to study GAPDH in the context of oogenesis and embryonic development ( Callan, 1986; Angelier et al., 1996 ) . So far, except for Xenopus GAPDH ( Nickells and Browder, 1988; Nickells et al., 1989 ) , no information was available about the structure of this dehydrogenase in amphibians and how they are related to other GAPDHs. In this paper we report the isolation and characterization of the skeletal muscle GAPDH from Pleurodeles waltl on the basis of its apparent native and subunit molecular weights, isoelectric point, and Western blot analyses using monospecific polyclonal anti- bodies against it. The GAPDH is recognized by an antiserum against the skeletal-muscle protein in the different tissues analyzed. A cDNA fragment of a GapC gene encoding a glycolytic GAPDH was amplified by polymerase chain reaction tech-

niques, sequenced and identified as the internal region of the gene corresponding to the catalytic site. The phylogenetic relationships of the amphib- ian GAPDHs with the homologous dehydrogenases of other vertebrate phyla are discussed.

2. Materials and methods

2.1. Animals

Iberian ribbed newts, Pleurodeles waltl ( Amphi- bia, Batrachia, Caudata w Urodela x , Salamandridae ) , are originated from Morocco. Animals used in this study were from our breeding stocks.

2.2. Enzyme purification

The enzyme was purified to electrophoretic homogeneity from crude cell extracts by the pro- cedure previously described ( Soukri et al., 1995, 1996 ) .

All steps were performed at 4 8C. Centrifuga- tions were carried out at 20 000 = g for 45 min.

2.2.1. Preparation of crude extracts

Adult newts were anesthetized by immersion in 0.1% MS 222 ( ethyl m-aminobenzoate methane sulfonate ) . Skeletal muscle tissue ( approx. 8 g, fresh weight ) was ground and homogenized using an Ultra-Turrax homogenizer in 25 mM Tris–HCl buffer ( pH 7.5 ) , containing 2 mM EDTA, 10 mM 2-mercaptoethanol and protease inhibitors ( 2 mM phenylmethylsulfonyl fluoride, 2 mM benzamidi- ne, and 5 mM ´ -amino-n-caproic acid ) at a ratio of 3 mlyg of fresh tissue. The supernatant ( soluble protein fraction ) obtained after centrifugation was considered as the crude extract.

2.2.2. Ammonium sulfate fractionation

The crude extract was subjected to protein precipitation in the 66–88% ( w y v ) saturation range of ammonium sulfate. The final pellet was dissolved in a minimal volume of 25 mM Tris–

HCl ( pH 7.5 ) , containing 2 mM EDTA and 10 mM 2-mercaptoethanol ( buffer A ) . The protein solution was dialyzed twice against 1 l of the same buffer.

2.2.3. Blue Sepharose CL-6B chromatography

The dialyzed enzyme preparation was applied

to a Blue Sepharose CL-6B column ( 1 = 6 cm )

equilibrated with two bed volumes of buffer A.

The column was washed with three bed volumes of buffer A and two bed volumes of the same buffer adjusted to pH 8.5 ( buffer B ) . The enzyme was eventually eluted with buffer B containing 10 mM NAD

at a flow rate of 6 ml yh. Active fractions were collected and concentrated by ultra- filtration on a Diaflo PM10-Amicon membrane.

2.3. Analytical procedures

2.3.1. GAPDH activity determination

GAPDH activity in the oxidative phosphoryla- tion was determined spectrophotometrically at 25 8C by monitoring NADH generation at 340 nm ( Serrano et al., 1993 ) . The reaction mixture of 1 ml contained 50 mM Tricine–NaOH buffer ( pH 8.5 ) , 10 mM sodium arsenate, 1 mM NAD

and 2 mM

-G3P. A coupled assay in which aldolase ( 1 unit yml ) produced the stoichiometric breakage of

-fructose 1-6 biphosphate ( 2 mM ) to

-G3P and dihydroxyacetone-phosphate, the first product being the actual substrate of the oxidative reaction ( Serrano et al., 1991 ) , was usually used during enzyme purification. For kinetic studies, however,

D

-G3P in aqueous solution was used as above described at a final concentration of 2 mM. Kinetic constants were calculated from initial rates esti- mated from initial absorbance changes. To deter- mine the kinetic parameters, the concentration of the respective fixed substrate for the reaction was 1 mM NAD

or 0.2 mM G3P in the presence of 10 mM PO

. K

and V

were determined from Lineweaver–Burk double reciprocal plots. To determine optimal pH, enzymatic activity was measured over a wide range of pH ( from 5 to 10 ) with different buffers ( acetate, imidazole, Tris and carbonatey bicarbonate ) adjusted to the same ionic strength than the standard reaction mixture. To determine apparent optimal temperature, reactions were carried out in the 5–70 8C temperature range using a thermostated cuvette holder connected with a refrigerated bath circulator. One unit of enzyme is defined as the amount which catalyses the formation of 1 mmol of NADH per min under the conditions used. Protein was estimated by the method of Bradford ( Bradford, 1976 ) using bovine serum albumin as a standard. Activity levels in cell-free extracts were expressed as specific activ- ity ( unitsy mg of protein ) .

2.3.2. Chromatofocusing

Concentrated GAPDH preparations were dia- lyzed against 25 mM Tris–HCl buffer ( pH 8.5 ) ,

containing 1 mM EDTA and 10 mM 2-mercaptoe- thanol ( starting buffer ) . Column chromatofocusing in the pH range 8.5–5.5 was performed on a Polybuffer Exchanger PBE-94 column ( 1 = 18 cm ) equilibrated with starting buffer, following the instructions of the manufacturer ( Pharmacia Bio- tech, Uppsala, Sweden ) . After application of the dialyzed enzyme preparation, the column was washed with 5 ml of starting buffer. The GAPDH was eventually eluted at a flow rate of 12 ml yh by washing the column with 10 bed volumes of a 10-fold diluted mixture of Polybuffer 96 y Polybuf- fer 74 ( 30 y70, vy v ) adjusted to pH 5.5 with acetic acid. The pooled fractions corresponding to the only activity peak were concentrated and equili- brated in standard buffer by ultrafiltration as described above.

2.3.3. Polyacrylamide gel electrophoresis

Determination of native molecular weight was carried out by electrophoresis on non-denaturing polyacrylamide slab gels ( BIO-RAD ) by using the following protein standards: ferritin ( 440 kDa ) , catalase ( 232 kDa ) , aldolase ( 154 kDa ) and oval- bumin ( 43 kDa ) . As described by the method of Hedrick ( Hedrick and Smith, 1968 ) , a calibration curve can be calculated from the relative mobilities of standard proteins on non-denaturing polyacryl- amide gels with different acrylamide concentra- tions ( 5%, 7%, 9% and 10%, wy v ) .

Isoelectric focusing ( Robertson et al., 1987 ) was done with the same electrophoresis system in 5% polyacrylamide slab gels holding ampholite- generated pH gradients in the range 3.5–10.0 ( Pharmalite 3.5-10; Pharmacia Biotech, Uppsala, Sweden ) , 25 mM NaOH and 20 mM acetic acid as cathode and anode solutions, respectively, and the standard proteins of the Sigma 3.6-9.3 IEF- Mix isoelectric focusing protein marker kit ( Sigma Chem. Co. ) .

SDS-polyacrylamide gel electrophoresis ( SDS- PAGE ) was performed as described by Laemmli ( 1970 ) on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit ( Hoefer Scientific Instruments ) . After electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 at 0.2% ( w y v ) in methanol y acetic acid y water ( 4:1:5, vy vy v ) for 30 min at room temperature.

The apparent subunit molecular weight was deter-

mined by measuring relative mobilities and com-

paring with the following pre-stained SDS-PAGE

molecular weight standards ( Low Range MW, BIO-RAD ) : phosphorylase b ( 104 kDa ) , bovine serum albumin ( 82 kDa ) , ovalbumin ( 48.3 kDa ) , carbonic anhydrase ( 33.4 kDa ) , soybean trypsin inhibitor ( 28.3 kDa ) and lysozyme ( 19.4 kDa ) . 2.3.4. Preparation of polyclonal antibodies

Polyclonal antibodies were raised in New Zea- land White rabbits to GAPDH that had been purified to electrophoretic homogeneity from Pleu- rodeles skeletal muscle. The enzyme ( approx. 0.3 mg ) was mixed with Freund’s complete adjuvant and injected subcutaneously to rabbits in multiple places as described by Vaitukaitis ( 1981 ) . Rabbits were boosted four times at 3-week intervals and bleeding was done 10 days after.

2.3.5. Western blot analysis

Proteins were separated by SDS-PAGE as described previously. Separated protein bands were electrophoretically transferred from the gel slab to a nitrocellulose filter ( Schleicher & Schuell ) using a BIO-RAD Trans-Blot system. Transferred pro- teins were then visualized by prestaining in 0.2%

( w y v ) Ponceau Red in trichloroacetic acid. The nitrocellulose paper was then incubated for 1 h in blocking solution containing 5% ( w y v ) non-fat dry milk, 50 mM Tris–HCl ( pH 7.5 ) , 150 mM NaCl, 0.01% ( w y v ) NaN

and 0.05% ( vy v ) Tween-20, followed by incubation with the anti- GAPDH antiserum ( 1:1000 dilution ) as the first antibody. Western blots were eventually visualized by coupled immunoreaction with peroxidase-con- jugated goat anti-rabbit IgG ( Sigma Chemical Co. ) as the second antibody using 3,39-diaminobenzidi- ne as the chromogenic substrate.

2.4. Nucleic acid techniques

2.4.1. RNA isolation and reverse transcriptase- polymerase chain reaction

Total RNA was isolated from skeletal muscle using the method of Chomczynski and Sacchi ( 1987 ) . First-strand cDNA was produced by reverse transcription ( RT ) using MMLV reverse transcriptase ( Promega ) in conjunction with 2 mg total RNA and the reverse primer named Gap2;

59- ACCATG ( A ) CTG ( A ) TTG ( A ) CTC ( T ) ACC ( G ) CC-39 during 1 h at 42 8C. An aliquot from this template ( 1y 10 of the reaction ) was used in a subsequent polymerase chain reaction ( PCR ) using Taq DNA polymerase ( Promega ) , Gap2 and

forward primer named Gap1; 59-GCC ( T ) T ( A ) C ( G ) C ( T ) TGC ( T ) ACG ( C ) ACG ( C ) AAC ( T ) TG-39. PCR conditions were 30 cycles of 92 8C for 1 min, 458C for 1 min and 72 8C for 1 min.

Gap 1 and Gap2 are degenerated oligonucleotides constructed from conserved regions ( ASCTTNC, WYDNEW ( C ) G ) present in all GAPDHs so far studied ( Fothergill-Gilmore and Michels, 1993 ) . Agarose gel electrophoresis of the PCR-amplified cDNA from four independent amplification exper- iments showed a single fragment of approximately 0.5 kb — the expected size for the internal GapC fragment to be amplified with the above described primers — comprising approximately half of the complete coding region.

2.4.2. Cloning and sequencing

Two PCR products from independent experi- ments were purified using the Genclean II Kit ( BIO 101, La Jolla, CA ) . These PCR products were subcloned into the pGEM-T vector system ( Promega ) and the nucleotide sequence was deter- mined on both strands using universal primers T7 and SP6 ( Eurogentec s.a. DNA sequencing depart- ment, Belgium ) .

2.5. Phylogenetic analyses

Multiple sequences alignment of GAPDH pro- tein regions corresponding to the cDNA fragment of Pleurodeles GapC was performed with the

CLUSTAL X

v.1.8 program ( Thompson et al., 1997 ) and was used to construct phylogenetic trees using the distance ( Neighbor-Joining, Kimura distance calculations ) , maximum likelihood and maximum parsimony methods with the programs

v.1.8,

-

v.5.0 ( Strimmer and von Hae- seler, 1996 ) and

v.3.573c (

pack- age v.3.5c w 1993 x Felsenstein, J., Dept. of Genetics, Univ. of Washington, Seattle, USA ) , respectively.

Bootstrap analyses ( values being presented on a

percentage basis ) were computed with 1000 rep-

licates for the distance and maximum parsimony

trees; for maximum likelihood analysis estimations

of support ( also expressed as percentages ) were

assigned to each internal branch by the algorithm

quarter puzzling ( Strimmer and von Haeseler,

1996 ) . Published amino acid sequences of animal

GAPDHs used for the alignment were: teleost fish

( Onchorhyncus mykiss, accession number

AAB82747 ) ; Amphibia ( Xenopus leavis, P51469 ) ;

Avian ( Gallus gallus, P00356; and Columba livia,

Table 1

Purification of GAPDH from skeletal muscle of Pleurodeles waltl

Fraction Total protein Total activity Specific activity Purification Yield

(

mg

U

U

fold

%

Crude extract 446 2054 4.6 1 100

Ammonium sulfate 41 1466 35.7 8 71

(

66–88%

Blue Sepharose 8.4 945 112.5 24 46

CL-6B

Fig. 1. Purification of Pleurodeles waltl skeletal muscle GAPDH. Analysis by SDS- PAGE and immunoblotting. Purification was carried out from caudal skeletal muscle. Proteins at each purification step were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue

a

or subjected to Western blot

b

using polyclonal antibodies specific to Pleurodeles GAPDH. Crude extract

40

was loaded in lane 2. Protein preparation from the ammonium sulfate fractionation

20

was in lane 3. Blue-Sepharose fractions pool

20

was loaded in lane 4. The positions and apparent molecular weights of pre-stained standard proteins

loaded in lane 1

a

have been indicated. Bound antibody was located by coupled immunoreaction with peroxidase conjugated goat anti-rabbit IgG. The arrow indicates the band corresponding to the 37-kDa GAPDH subunit.

AAB88869 ) ; Mammalia ( Mus musculus, P16858;

Jaculus orientalis, P80534; Oryctolagus cuniculus, P46406; Sus scrofa, P00355; Bos taurus, P10096;

and Homo sapiens, P00354 ) . A bacterial GAPDH encoded by the Escherichia coli gap1 gene ( acces- sion number P06977 ) was also included.

3. Results and discussion

3.1. Purification and physicochemical, catalytic and immunological properties of Pleurodeles GAPDH

GAPDH has been purified from a soluble pro- tein fraction from Pleurodeles waltl skeletal mus- cle to electrophoretic homogeneity by a straightforward procedure involving only one chro- matography step, namely dye-affinity chromatography.

Table 1 summarizes a representative purification protocol. Values of approximately 110–115 units y mg of protein were obtained for the purified enzyme with a yield of approximately 45% and a purification factor of approximately 24-fold. As for other NAD -dependent GAPDHs

( Thompson et al., 1975; Soukri et al., 1995; Hafid et al., 1998 ) dye-affinity chromatography on Blue Sepharose was very effective for this purification and no additional purification steps were required to obtain homogeneous samples. SDS-PAGE analysis of the different fractions obtained during the puri- fication procedure showed a progressive enrich- ment in a 37-kDa protein ( Fig. 1a ) . Only this protein band, having the same size than the GAPDH subunit, was seen in the electrophoreti- cally homogeneous final enzyme preparations ( Fig.

1a, lane 4 ) . Concerning the physicochemical par-

ameters of the purified GAPDH from Pleurodeles,

Fig. 3. Column chromatofocusing of GAPDH purified from Pleurodeles skeletal muscle. A sample containing approxi- mately 1.8 mg of protein was applied to a Polybuffer Exchang- er PBE 94 column

1

18 cm

and the enzyme was eluted by using a pH gradient generated by 10 bed volumes of a 10-fold diluted mixture of Polybuffer 96y Polybuffer 74

30y70, vyv

adjusted with acetic acid to pH 5.5. Fractions of 2 ml were collected. Absorbance at 280 nm and the enzyme activity were measured. Only one peak of activity eluted at approximately pH 7.57, the estimated pI of the protein.

Fig. 2. Determination of native Pleurodeles GAPDH molecular weight by non-denaturing polyacrylamide gel electrophoresis. Proteins were electrophoresed on various acrylamide concentration gels

concentration range 5, 7, 9 and 10%

under non-denaturing conditions.

Molecular weight marker proteins were ferritin

Fer, 440 kDa

, catalase

Cat, 232 kDa

, aldolase

ALD, 154 kDa

and ovalbumin

OVA, 43 kDa

. Relative mobilities of proteins plotted as log R

vs. acrylamide concentration are indicated on the inset. A plot of the obtained slopes vs. molecular weight was linear and used to determine native GAPDH molecular weight

Pw,

.

non-denaturing PAGE yielded a value of approxi- mately 146 kDa for the native molecular mass ( Fig. 2 ) . As stated above, SDS-PAGE of the purified enzyme showed a single band correspond- ing to a 37-kDa protein ( Fig. 1a, lane 4 ) , thus indicating that Pleurodeles GAPDH should have an homotetrameric structure like other GAPDHs ( Fothergill-Gilmore and Michels, 1993 ) . However, the Pleurodeles enzyme subunit displayed an esti- mated molecular weight of approximately 37 kDa that is somewhat higher than the one ( 35 kDa ) reported for the GAPDH subunit of Xenopus ( Nickells et al., 1989 ) .

Column chromatofocusing, a chromatographic technique of protein separation according to pI values, showed a single protein peak with a max- imum at pH 7.57 ( the estimated pI for the enzyme ) which perfectly overlapped with that of GAPDH activity ( Fig. 3 ) . The same value of pI ( approx.

7.6 ) was found with the isoelectric focusing tech- nique ( data not shown ) . Chromatofocusing has been proved as an analytical technique that effi- ciently resolves slightly different isoforms of diverse proteins ( Serrano, 1986; Soukri et al., 1996 ) . Therefore, the results presented here indi-

cate that only one slightly-basic isoform of the

enzyme occurs in skeletal muscle of the urodele

Pleurodeles waltl, and strongly suggest that a

single GapC gene is expressed in this tissue. A

Fig. 4. Immunodetection of GAPDH levels in various tissues from Pleurodeles. Crude extracts from liver

and adult ovaries

were separated by SDS-PAGE and stained for proteins with Coomassie Blue

or blotted and probed with the antibody raised against skeletal muscle GAPDH

GAPDH

20

from skeletal muscle was loaded in lane 2 as a reference. Pre-stained molecular weight markers described in Fig. 1 were loaded in lane 1. The arrow indicates the band corresponding to the 37-kDa GAPDH subunit.

single GAPDH isoform has been also found in some other animal tissues and microorganisms, both prokaryotes and eukaryotes, but it seems not to be a general rule since the presence of several GAPDH isoforms has been reported in organisms phylogenetically very different ( Cerff, 1995; Hafid et al., 1998; Mateos and Serrano 1992; Soukri et al., 1996 ) .

We have produced rabbit polyclonal antibodies using purified Pleurodeles waltl muscle GAPDH as immunogen. These antibodies selectively react- ed by the immunoblotting procedure with a single immunoreactive band in both crude extracts and purified preparations. Fig. 1b shows that the rela- tive molecular mass of the detected protein ( 37 kDa ) is the expected one for the GAPDH mono- mer. The same protein band was recognized by the antiGAPDH antiserum in all tissues analyzed ( Fig. 4 ) including skeletal muscle, liver and ova- ries. No protein bands were detected by non- immune rabbit serum confirming specificity ( data not shown ) .

GAPDH catalyzes a two-substrate reaction. The K

values for NAD

and G3P, which have been determined at saturating concentrations of the other substrate, were 0.06 " 0.01 mM and 0.027 " 0.011 mM, respectively. The value obtained for V

was 33.24 " 5.73 mmol min

. Whereas the K

( G3P ) is similar to those found for cytosolic GAPDHs from mammalian tissues ( Heinz and Freimuller, 1982; Soukri et al., 1996 ) , the K

( NAD

) of

Pleurodeles GAPDH is clearly higher, having therefore a lower affinity for the nucleotide co- enzyme. An optimal pH value of approximately 8.5 and an apparent optimal temperature of 40 8C have been determined for the enzyme activity.

From the Arrhenius plot a value of 5.60 kcal mol

was calculated for the apparent E

of the Pleurodeles GAPDH.

3.2. Cloning and sequencing of a cDNA fragment of a GapC gene from Pleurodeles

RT-PCR amplification using primers constructed

from two highly conserved GAPDH regions pro-

duced a single cDNA fragment of the expected

size ( approx. 0.5 kb ) comprising approximately

half of the coding region of a GapC gene. The

nucleotide sequence determined for the amplified

cDNA fragment ( 510 bp; GenBank yEMBL acces-

sion no. AF343978 ) is shown in Fig. 5. Two

clones from independent RT-PCR experiments

were sequenced and found to be identical. The

derived amino acid sequence corresponds to a

highly conserved region of the catalytic subunit

including many residues strictly conserved in

GAPDHs from very diverse organisms ( Fothergill-

Gilmore and Michels, 1993 ) . This sequence was

aligned and compared with other 11 GAPDHs,

selected to include representatives of the main

aquatic and terrestrial vertebrate phyla and one

bacterial species, by using the

( v. 1.8 )

Fig. 5. Nucleotide and deduced amino acid sequences of a partial RT-PCR amplified cDNA fragment of the GapC gene from Pleurodeles waltl. The nucleotide sequence of the cDNA clone

510 bp, accession number AF343978

generated by RT-PCR represents approxi- mately half of the predicted complete coding sequence; the deduced amino acid sequence

170 amino acids

is shown in the upper line using the single letter code. The two arrows indicate amino acid sequences corresponding to the synthetic primers Gap1 and Gap2 used for PCR amplification.

program ( EMBL-Align database accession no.

ALIGN 000209 ) . Highest homology was obtained when the partial sequence deduced from the Pleu- rodeles GapC gene was aligned with Xenopus laevis GAPDH, a match of 148 identical amino acid stretch over 170 amino acid between residues 148 and 317 being observed. The amphibian X.

laevis — African clawed frog, a member of the Anura group — is the only other amphibian GAPDH cDNA sequence available so far in GenBank yEMBL databases ( accession no.

U41753 ) . The above described multiple sequences alignment was used to construct phylogenetic trees or cladograms using the distance ( Neighbor-Join- ing ) , maximum likelihood and maximum parsi- mony methods ( Fig. 6 ) . These trees show analogous phylogenetic relationships of the GAPDHs of the main vertebrate groups and reveal

that Pleurodeles GAPDH is more closely related to the avian and mammalian enzymes than Xeno- pus GAPDH. Moreover, in spite of the fact that both species are members of the same class ( Amphibia ) a paraphyletic relationship is observed between their GAPDH sequences. However, it should be noted that these results depict the molec- ular phylogeny of the GAPDH protein solely and they do not necessarily represent phylogenetic relationships between species. In fact, anomalous phylogenetic relationships due to horizontal gene transfer or enzyme functional substitution were reported among GAPDH-based phylogenies ( Foth- ergill-Gilmore and Michels, 1993; Cerff, 1995;

Hafid et al., 1998 ) . However, phylogenetic analy-

ses of amphibian relationships using ribosomal

RNAs showed contradictory results, supporting

Amphibia either as a monophyletic ( 18S rRNA )

419 Mounaji et al. / Comparative Biochemistry and Physiology Part B 131 (2002) 411–421 Fig. 6. Distance

a

and maximum likelihood

b

phylogenetic trees of GAPDH proteins including the amino acid sequence deduced from the

partial

sequence.

The presented trees were obtained with the

v.1.8

a

and

-

v.5.0

b

programs from a multiple sequence alignment of the GAPDH regions corresponding to

the partial

sequence of

as described in Section 2.5. A maximum parsimony tree obtained with the

program of the

v.3.5c package was very

similar to the maximum likelihood tree presented here

data not shown

. Values in the nodes indicate on a percentage basis the statistical support

e.g. bootstrap values

of the

associated group. Scale bars indicate percent estimated differences in protein sequences. The two amphibian sequences are boxed. A bacterial GAPDH encoded by the

gene was also included and used as an outgroup. Accession numbers of used GAPDH sequences are listed in Section 2.5.

or a non-monophyletic ( 25S rRNA ) group ( Hedg- es et al., 1990 ) . Therefore, any definitive conclu- sion of extensive divergence within Amphibia remains to be demonstrated in further studies using other molecular markers.

Although GAPDH has been considered for a long time as a ‘house-keeping’ constitutive enzyme, recent evidence demonstrate that it should have a number of diverse functions not related with its enzymatic role in central carbon metabo- lism and that it exhibits both genetic and post- translational regulations ( Sirover, 1999 ) . In this work our attention has been focused on Pleuro- deles skeletal muscle GAPDH — a single protein isoform was found suggesting the expression of a single GapC gene — but immunological analyses of ovarian proteins also detected in this tissue a single protein band that appeared to possess the same characteristics that muscle GAPDH ( see Fig.

4, lane 4 ) . A possible GAPDH regulation ( at the genetic or protein levels ) and its connection with a specific role of this protein in embryogenesis and associated tissue differentiation are worth to be investigated using Pleurodeles as an animal model system. Moreover, by using biochemical and immunological techniques it will be of interest to explore whether ovarian GAPDH shows specific intracellular distribution changes linked to these processes. These experiments are currently under- way in our laboratories.

Acknowledgments

This work was supported by research grants from the Moroccan National Research Fund ‘Pro- gramme d’Appui a la Recherche Scientifique ` ( PARS-CNCPRST ) ’, ‘CNR Convention’ attributed to the unite de Biochimie, AECI ´ ( Spain ) and a collaborative grant of the Andalusian Regional Government ( Convenio Colaboracion Univ. Mar- ´ roquies, grupo PAI CVI-261 ) . The authors thank Professor Manuel Losada ( University of Seville, Spain ) for his generous encouragement and help ( PB 97-1135 ) and to all the members of Labora- toire d’Embryologie Moleculaire, Universite Paris- ´ ´ Sud, Orsay, for their able assistance, especially Dr Marie Laure Samson for her helpful in cDNA cloning techniques.

References

Angelier, N., Penrad-Mobayed, M., Billoud, B., Bonnanfant- Jaıs, M.L., Coumailleau, P., 1996. What role might lamp- ¨

brush chromosomes play in maternal gene expression? Int.

J. Dev. Biol. 40, 645–652.

Baxi, M.D., Vishwanatha, J.K., 1995. Uracil DNA- glycosylase

an Ap4A binding protein. Biochemistry 34, 9700–9707.

Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

Callan, H.G., 1986. Lampbrush Chromosomes, Springer, Ber- lin, pp. 1–254.

Cerff, R., 1995. The chimeric nature of nuclear genomes and the antiquity of introns as demonstrated by GAPDH gene system. In: Go, M., Schimmel, P.

Evolution in Protein and Gene Structures, Elsevier, Amster- dam, pp. 205–227.

Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol- chloroform extraction. Anal. Biochem. 162, 156–159.

Fothergill-Gilmore, L.A., Michels, P.A., 1993. Evolution of glycolysis. Prog. Biophys. Mol. Biol. 59, R105–235.

Hafid, N., Valverde, F., Villalobo, E., et al., 1998. Glyceral- dehyde-3-phosphate dehydrogenase from Tetrahymena pyr- iformis: enzyme purification and characterization of a gap C gene with primitive eukaryotic features. Comp. Biochem.

Physiol. B 119, 493–503.

Harris, J.I., Waters, M., 1976. Glyceraldehyde-3-phosphate dehydrogenase. In: Boyer, P.D.

Ed.

, The enzymes, third ed. Academic Press, New York, pp. 1–49.

Hedges, S.B., Moberg, K.D., Maxon, L.R., 1990. Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and review of the evidence for amniote relation- ships. Mol. Biol. Evol. 7, 607–633.

Hedrick, J.L., Smith, A.J., 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155–164.

Heinz, F., Freimuller, B., 1982. Glyceraldehyde-3-phosphate dehydrogenase from human tissues. Methods Enzymol. 89, 301–305.

Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.

Mateos, M.I., Serrano, A., 1992. Occurrence of phosphorylat- ing and non- phosphorylating NADP-dependent glyceralde- hyde-3-phosphate dehydrogenases in photosynthetic organisms. Plant. Sci. 84, 163–170.

Mazzola, J.L., Sirover, M.A., 2001. Reduction of glyceralde- hyde-3-phosphate dehydrogenase activity in Alzheimer’s disease and in Huntington’s disease fibroblasts. J. Neuro- chem. 76, 442–449.

Nickells, R.W., Browder, L.W., 1988. A role for glyceralde- hyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos. J. Cell. Biol.

107, 1901–1909.

Nickells, R.W., Browder, L.W., Wang, T.I., 1989. Factors influencing the heat shock response of Xenopus laevis embryos. Biochem. Cell. Biol. 67, 687–695.

Robertson, E.F., Dannelly, H.K., Malloy, P.J., Reeves, H.C.,

1987. Rapid isoelectric focusing in a vertical polyacrylamide

minigel system. Anal. Biochem. 167, 290–294.

Rondinelli, R.H., Epner, D.E., Tricoli, J.V., 1997. Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in late pathological state human prostate cancer. Prostate Cancer Prostatic Dis. 1, 66–72.

Saunders, P.A., Chen, R.W., Chuang, D.M., 1999. Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoforms during neuronal apoptosis. J. Neurochem. 72, 925–932.

Schultz, D.E., Hardin, C.C., Lemon, S.M., 1996. Specific interaction of glyceraldehyde-3-phosphate dehydrogenase with the 59-nontranslated RNA of hepatitis A virus. J. Biol.

Chem. 271, 14134–14142.

Serrano, A., 1986. Characterization of cyanobacterial ferredox- in-NADP

oxidoreductase molecular heterogeneity using chromatofocusing. Anal. Biochem. 154, 441–448.

Serrano, A., Mateos, M.I., Losada, M., 1991. Differential regulation by trophic conditions of phosphorylating and non phosphorylating NADP

-dependent glyceraldehyde-3- phosphate dehydrogenases in Chlorella fusca. Biochem.

Biophys. Res. Commun. 181, 1077–1083.

Serrano, A., Mateos, M.I., Losada, M., 1993. ATP-driven transhydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenase

phosphorylat- ing and non-phosphorylating

model system. Biochem. Bio- phys. Res. Comm. 197, 1348–1356.

Singh, R., Green, M.R., 1993. Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogen- ase. Science 259, 365–368.

Sirover, M.A., 1999. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phos- phate dehydrogenase. Biochim. Biophys. Acta 1432, 159–184.

Soukri, A., Valverde, F., Hafid, N., Elkebbaj, M.S., Serrano, A., 1995. Characterization of muscle glyceraldehyde-3- phosphate dehydrogenase isoforms from euthermic and induced hibernating Jaculus orientalis. Biochim. Biophys.

Acta 1243, 161–168.

Soukri, A., Hafid, N., Valverde, F., ElKebbaj, M.S., Serrano, A., 1996. Evidence for a posttranslational covalent modifi- cation of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa

phys. Acta 1292, 177–187.

Strimmer, K., von Haeseler, A., 1996. Quarter puzzling: a quarter maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13, 964–969.

Thompson, S.T., Cass, K.H., Stellwagen, E., 1975. Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc. Natl. Acad. Sci. USA 72, 669–672.

Thompson, J.D., Gibson, T.J., Plewnisk, F., Jeanmougin, F., Higgins, D.G., 1997. The

Windows interface:

flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 25, 4876–4882.

Vaitukaitis, J.L., 1981. Production of antisera with small doses

of immunogen: multiple intradermal injections. Methods

Enzymol. 73, 46–52.