Different Thermostability of
Skeletal Muscle Glyceraldehyde-3-phosphate Dehydrogenase from Hibernating and
Euthermic Jerboa (Jaculus orientalis)

IDDAR
Abdelghani, CAMPOS Luis A.¹,
SANCHO Javier¹, SERRANO Aurelio², SOUKRI Abdelaziz*

( Laboratoire de Biochimie, Unite＇ de Ge＇nie Enzymatique et Biologie Mole＇culaire, Faculte＇ des Sciences-Ain Chock,
Casablanca, Morocco; 1Departamento de Bioquímica y Biología Molecular y
Celular, Universidad de Zaragoza, Zaragoza, Spain; 2Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de
Sevilla, Sevilla, Spain )

Abstract        In
previous study, we demonstrated that the specific activity of
D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in skeletal
muscle of induced hibernating jerboa (hibernating GAPDH) was 3－4 folds lower than that of the one
in the skeletal muscle of the euthermic jerboa (euthermic GAPDH). A significant
decrease in both GAPDH protein and GapC mRNA levels occurs when hibernating,
but the purified hibernating GAPDH is less active than the euthermic GAPDH. To
investigate the physico-chemical basis of this lower activity, the behaviour
during thermal inactivation of skeletal muscle GAPDH from hibernating and
euthermic tissues was examined by a variety of spectroscopic techniques,
including fluorescence emission, circular dichroism and ultraviolet absorption.
A clear resistance to thermal denaturation was observed in the hibernating
GAPDH compared with the euthermic GAPDH. The different temperature of denaturation
found in these proteins by both fluorimetry and circular dichroism indicates
that there might exist conformational changes of GAPDH upon hibernation that
could affect the stability of this enzyme.

Key
words     glyceraldehyde-3-phosphate
dehydrogenase; hibernation; thermal denaturation; circular dichroism
spectroscopy; protein stability

D-Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, EC 1.2.1.12) is a key enzyme of the glycolytic pathway
that is present in the cytosol of all organisms so far studied[1]. The
glycolytic GAPDH has been remarkably conserved during evolution, having a
homotetrameric structure with subunits of 35－37 kD[1]. GAPDH has been isolated from a variety of species[2],
including mesophilic, moderately thermophilic and hyperthermophilic
microorganisms[3]. These enzymes, which differ in thermal stability, have been
shown to be highly similar in amino acid sequence, subunit composition, and
enzymatic behaviour[2]. Comparison of these homologues may shed light on the
mechanism of adaptation to extreme environmental conditions at the molecular
level. The skeletal muscle enzyme, composed of four identical subunits and
believed to be a symmetric dimer of dimers[4], has been used as a model to
study the unfolding, refolding, dissociation and re-association of oligomeric
proteins[5]. Kinetic analysis of the reactivation of denatured GAPDH indicated
that dimer dimerization is the rate-limiting step[6].

Hibernation is a
seasonal adaptation by which some mammalian species survive low environmental
temperature and a scarcity of food by drastically lowering their body
temperature, metabolism and respiratory function, thereby dramatically reducing
their energy requirements[7]. The changes involved in hibernation are precisely
controlled and can only be reverted by internally-driven mechanisms, which
suggests a specific biochemical regulation[8]. The jerboa (Jaculus orientalis),
a small rodent from dessert areas of Moroccan Highlands, is a convenient
organism to study metabolic regulation, not only because of its remarkable
tolerance to heat and dry diet but also because it is one of the few small
mammals that can undergo hibernation[9]. In previous articles[10,11], we
demonstrated that the specific activity of GAPDH found in skeletal muscle and
in liver of induced hibernating jerboa was 3－4 folds lower than that in the euthermic animal. This decrease is
associated in the skeletal muscle with a decrease in both enzyme concentration
and GapC mRNA expression[12].

In this article,
the thermal stabilities of hibernating and euthermic GAPDH are studied and
compared. The significant differences found between these enzymes may be
related with their different catalytic efficiencies at low temperature.

1    Materials and Methods

1.1   Preparation of biological material

Jerboa (Jaculus
orientalis) was captured in the sub-desert Moroccan East Highlands and raised
in a pre-acclimated room (22 ± 2) ℃ with food
and free access to water. To induce hibernation, young adult jerboas of both
sexes, 4－6 months old,
100－150 g of body
weight, were transferred to the cold room and kept at (4±1) ℃ for 4－5 weeks of
adaptation with food and free access to water. Then, food was removed, which
soon induced hibernation. Animals of this group are defined as “hibernating jerboa”.

1.2   Enzyme purification

Unless specially
indicated, all the steps were performed at 4 ℃, and centrifugations were carried out at 20 000 g for 45 min. 100 g
of skeletal muscle, from both hibernating and euthermic groups, were ground and
homogenized using a Sorvall mixer in 25 mmol/L Tris-HCl buffer, pH 7.5,
containing 2 mmol/L EDTA, 10 mmol/L 2-mercaptoethanol and a protease inhibitor
mixture (2 mmol/L PMSF, 2 mmol/L benzamidine). Next, a 60%－90% saturated ammonium sulfate
fraction was carried out and the precipitate was dissolved in buffer A (25
mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA and 10 mmol/L 2-mercaptoethanol) and
applied onto a Blue Sepharose CL-6B column equilibrated with 2 bed volumes of
buffer A. The column was washed with buffer B (25 mmol/L Tris-HCl, pH 8.6, 2
mmol/L EDTA and 10 mmol/L 2-mercaptoethanol), and the enzyme was then eluted
with buffer B containing 10 mmol/L NAD at a flow rate of 20 mL/h. To remove NAD
from the enzyme, the active pool was dialyzed against 25 mmol/L Tris-HCl
buffer, pH 8.0, containing 1 mmol/L EDTA and 5 mmol/L 2-mercaptoethanol.

1.3   Chromatofocusing analysis

Column
chromatofocusing was carried out as described previously[10]. The active pool
was dialyzed against 25 mmol/L Tris-HCl buffer, pH 9.8, containing 1 mmol/L
EDTA and 5 mmol/L 2-mercaptoethanol (starting buffer). Chromatofocusing in the
pH range 9.0 to 5.5 was performed in a Polybuffer Exchanger PBE-94 (Pharmacia Biotech)
column (1 cm×18 cm)
equilibrated with starting buffer. After application of the concentrated enzyme
solution (about 10 mL), the column was washed with 5 mL of starting buffer. The
enzyme was eventually eluted at a flow rate of 12 mL/h by washing the column
with 10-bed volumes of a 10-fold diluted mixture of Polybuffer 96/Polybuffer 74
(30/74, V/V) (Pharmacia Biotech), adjusted to pH 5.5 with acetic acid. The
pooled active fractions were concentrated and equilibrated in standard buffer
supplemented with 0.1 mol/L NaCl by ultrafiltration on a Diaflo PM-10 (Amicon)
membrane.

1.4   Enzyme assays

Enzyme activity
was measured according to reference [13]. The reaction was started by adding
the enzyme to the assay mixture containing 45 mmol/L sodium pyrophosphate (pH
8.5), 3 mmol/L 2-mercaptoethanol, 10 mmol/L sodium arsenate, 1 mmol/L NAD, and
1 mmol/L D-glyceraldehyde-3-phosphate at 25 ℃. The change in absorbance at 340 nm was detected continuously.
Kinetic constants were calculated from initial rates. In the determination of
kinetic parameters, the concentration of fixed substrate for the oxidative
phosphorylation was 1 mmol/L NAD or 1 mmol/L GAP respectively. Km and Vmax were
determined from Eadie-Hofstee plots [14,15].

Concentration of the pure enzyme was determined
using extinction coefficients calculated from amino acid sequence[16]. The
spectral measurements were made using a Kontron UV-visible spectrophotometer
thermostated at 20 ℃. The absorbances of solutions of native protein were corrected for
light scattering by determining correction parameters over the 320－380 nm spectral range, and
extrapolating these parameters into the UV range.

1.5   Thermal denaturation experiments

Thermally
induced irreversible denaturation was monitored by activity measurements.
Enzyme preparations from euthermic and hibernating animals were incubated at
different temperatures for 15 min and then chilled on ice. Protein samples [500
mg/L (3.8 μmol/L)
protein in 50 mmol/L Tris-HCl (pH 7.5) containing 1 mmol/L EDTA and 1 mmol/L 2-mercaptoethanol]
were then diluted with incubation buffer pre-incubated at 25 ℃, and the enzyme activity was
immediately measured at 25 ℃.

The stability of the enzyme at 60 ℃ was determined by incubation of 30
μL-aliquots of
the stock enzyme solution in 3.0 mL 50 mmol/L Tris-HCl, pH 7.5, with 1 mmol/L
EDTA and 1 mmol/L 2-mercaptoethanol, preheated at 60 ℃. The incubation enzyme
concentration was about 0.1 mmol/L. 20 μL-aliquots of this mixture were withdrawn at different times and
assayed for activity. To avoid reactivation of the heat-treated enzyme upon
mixing, the assay mixture (3 mL containing 0.25 mmol/L G3P and NAD) was kept at
60 ℃.

1.6   Fluorescence emission

Fluorescence
emission spectra were recorded with an Aminco Bowman series 2
spectrofluorimeter. The excitation wavelength was 280 nm and the emission was
recorded from 300 nm to 400 nm. The influence of the temperature on the
intrinsic protein fluorescence was monitored at an emission wavelength of 320
nm (excitation at 280 nm). The experimental curves were fitted as described in
reference [17] to calculate the temperature of denaturation.

1.7   Circular dichroism (CD)
spectroscopy

CD spectra (near
and far UV) of the muscle euthermic and hibernating GAPDH were recorded out
using a Jasco CD spectropolarimeter, with a constant temperature cuvette
holder. The enzymes were dissolved in 5 mmol/L boric acid (pH 7.5) containing 1
mmol/L EDTA, placed in a 1 cm cuvette, and the CD spectrum was recorded at room
temperature. The temperature was increased stepwisely and the ellipticity was
recorded at 273 nm for euthermic GAPDH and at 270 nm for hibernating GAPDH. A
Shimadzu UV 250 spectrophotometer was used for simple light absorption
measurements.

2    Results and Discussion

In our previous
studies, the GAPDH specific activity in the soluble protein fraction from
skeletal muscle of euthermic jerboas was found to be 3 folds higher than the
one measured in preparations from the same tissue of induced hibernating
animals (1.26 U/mg of protein and 0.45 U/mg of protein for euthermic and
hibernating animals, respectively). A relevant marked reduction of GAPDH
protein level in the crude preparations of the hibernating tissue was also
found by using Western blot assay[10]. As previously reported for other
NAD+-dependent GAPDHs[18], dye-ligand chromatography on Blue Sepharose is a
very effective purification step. But purified GAPDHs from ethermic and
hibernating jerboas exhibited different specific activity with 21.5 U/mg and
6.7 U/mg respectively. What’s more, these two GAPDHs exhibited same subunit and
native molecular mass. A decrease (about 3-fold) in the amount of GapC mRNA, a
single 1.2 kb transcript, was also observed in muscle of hibernating jerboa
compared with the euthermic animal[12]. Analytical chromatofocusing of purified
GAPDH preparations from skeletal muscle of euthermic and hibernating jerboa
resolved two main enzyme isoforms (GAPDH Ⅰ and GAPDH Ⅱ) with pI values in 7.5－8.2, exhibiting 
different relative contributions to the total enzymatic activity: GAPDH
I accounted for more than 90% of the activity of euthermic muscle whereas the
activity due to GAPDH II reached up to 65% in the preparation from hibernating
jerboa[10]. The chromatofocusing analysis has been proved to efficiently
resolve closely related GAPDH isoforms in other organisms[19]. Determination of
kinetic parameters showed that euthermic isoform GAPDH Ⅰ (pI 8.1) exhibited significantly
higher affinities for both substrates, G3P and NAD, than hibernating isoform
GAPDH Ⅱ (pI 7.85)
(Table 1). The catalytic efficiency of the hibernating GAPDH Ⅱ isoform was clearly lower, as
indicated by the corresponding kcat/Km ratios (Table 1). It should be pointed
out that the N-terminal sequences of the two main GAPDH isoforms purified from
euthermic and hibernating tissues are identical[11]. These results suggested
that dramatic decrease of body temperature during hibernation might change the
structures of these two main GAPDH isoforms purified from euthermic and
hibernating tissues.

The influence of
hibernation on GAPDH stability was investigated as follows. The result of
incubating GAPDH from euthermic and hibernating jerboa at different
temperatures for 15 min is shown in Fig.1(A). It is clear that the GAPDH from
hibernating jerboa is more resistant to this treatment than the euthermic one,
as indicated by the residual activity. Fig.1(B) shows thermal inactivation
time-courses incubation at 60 ℃. After 40 min, the inactivation of the euthermic GAPDH is
significantly higher than that of the hibernating GAPDH (70% and 25%
respectively).

In a previous
study[10], we described a change in the slope of the Arrhenius plots of the
euthermic and hibernating GAPDH preparations over the temperature range
investigated. These thermal denaturation studies reveal a clear difference between
the two proteins that could reflect subtle differences at their catalytic sites
or rather a different stability of the two proteins at high temperatures. To
clarify this point, we have recorded and analysed several observable thermal
transitions of the two proteins using intrinsic protein fluorescence, far UV CD
and near UV CD spectroscopy. The emission spectra of euthermic and hibernating
GAPDHs are shown in Fig.2. The spectrum of hibernating GAPDH differs from that
of the euthermic GAPDH (maxima at 315.0 nm and 309.5 nm for the euthermic and
hibernating enzymes, respectively), which indicated a different exposure of the
tryptophan residues. Tryptophan residues are more prone to be buried into the
apolar interior of GAPDH when hibernating.

Table
1   Kinetic parameters of
skeletal muscle main GAPDH isoforms purified from euthermic and hibernating J.
orientalisa

Data correspond to the enzyme preparations used in this work and are
means of three determinations. The GAPDH isoforms were isolated by column chromatofocusing
as described in Materials and Methods.

Fig.1       The
effect of temperature on the activity of euthermic and hibernating GAPDH

(A) Thermal inactivation of GAPDH. 3.8 μmol/L enzyme from euthermic
and hibernating jerboas were incubated at different temperatures for 15 min in
50 mmol/L Tris-HCl, pH 7.5, containing 1 mmol/L EDTA and 1 mmol/L
2-mercaptoethanol. After rapid cooling, the enzyme activity was immediately
measured at 25 ℃. (B) The stability of the GAPDH at 60 ℃. Add of 3.8 μmol/L of the
enzyme to 3.0 mL of an incubation mixture, preheated to the required
temperature, containing 50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA and 1 mmol/L
2-mercaptoethanol. Aliquots were then withdrawn for the activity assay.

Fig.2       Fluorescence
emission spectra of GAPDHs with excitation at 280 nm

Protein concentration was 2
μmol/L in 5 mmol/L borate buffer, pH 7.5, containing 1 mmol/L EDTA.

Thermal
denaturation curves followed by fluorescence show that the temperature of
denaturation is slightly different in for the two proteins, being 57 ℃ and 60 ℃ for the euthermic protein and
hibernating one respectively (Fig.3).

Fig.3       Thermal
denaturation of GAPDHs followed by fluorescence

The
intrinsic protein fluorescence was monitored at an emission wavelength of 320
nm (excitation at 280 nm) as temperature of the cuvette increased in the
indicated range. GAPDH was in borate buffer 5 mmol/L, pH 7.5, containing 1
mmol/L EDTA. Protein concentration was 2 μmol/L. (A) Euthermic animal. (B)
Hibernating animal.

Their CD spectra
are presented in Fig.4. The far- UV region shows a broad peak that is in
principle consistent with the presence of α helices, as observed for other
GAPDHs[20]. The near-UV region, which reflects the asymmetric environment of
aromatic residues in the folded enzyme molecule, is similar for the euthermic
and hibernating GAPDH. The similarities of the CD spectra suggest that
hibernation do not markedly change either secondary or the tertiary structure
of these proteins. We notice, however, a difference in the 230 nm region than
can be related to a different contribution of aromatic residues in the two
proteins to the far UV CD spectra[21]. These results reinforce the fluorescence
study and indicate that the environment of tryptophan residues in the two
enzymes is different.

Fig.4       Circular
dichroism spectra of GAPDHs from euthermic and hibernating jerboa

Circular dichroism spectra of GAPDHs from euthermic and hibernating
jerboa were carried out using a Jasco CD spectropolarimeter, with a constant
temperature cuvette holder. (A) Near UV. (B) Far UV. Enzyme concentration was 1
μmol/L in 5 mmol/L borate buffer, pH 7.5, containing 1 mmol/L EDTA. The CD
spectra were scanned at room temperature.

The study of the
thermal denaturation of both proteins by near-UV CD shows that the tertiary
structure of the euthermic GAPDH sharply decreases above 60 ℃ while that of the hibernating
protein seems to start to disappear at around 80 ℃ (Fig.5). These results agree with the observed higher residual
activity of the hibernating enzyme. The non coincidence of the transition
temperatures as monitored by fluorescence and by near-UV CD, may reflect that
the unfolding transition of GAPDH is not two-state[22], although this needs to
be confirmed when using other buffer and protein concentration. The higher
temperature stability of the hibernating enzyme showed by both the residual
activities and the higher unfolding temperatures is intriguing, but the
physical cause remains unknown so far. It seems that both enzymes are quite
similar at room temperature, because their spectra, particularly the near-UV CD
spectrum, are very similar. As discussed before, some differences in the
fluorescence spectra and in the 230 nm region of the far UV CD spectra suggest
that there could be a difference in either the content or the environment of
tryptophan residues that deserves further investigation.

Fig.5       Thermal
denaturation followed by CD spectroscopy of euthermic and hibernating GAPDHs

The
enzymes, final concentration 1 μmol/L, were dissolved in 5 mmol/L borate
buffer, pH 7.5, containing 1 mmol/L EDTA. The temperature of the samples was
subsequently increased in the cuvette, and the ellipticity was recorded at 273
nm or 270 nm for GAPDH from euthermic or hibernating tissue, respectively.

For a
hibernating animal, central metabolism is strongly reduced, and a lower enzyme
activity is an essential need. Then one might wonder why hibernating GAPDH is
comparatively more stable at high temperature. Although several explanations
can be suggested, we point out that a higher stability of a protein at high
temperature may be accompanied by a lower one at low temperature due to the
bell shape of protein stability curves[23]. If this were the case, the
hibernating jerboa would use a GAPDH enzyme with the stability curve shifted
towards higher temperatures so that, at the hibernating environmental
temperature, the enzyme would be conformationally unstable, thus giving rise to
lower activity levels. If this hypothesis were correct, a rise in temperature
could very rapidly result in a high activity level since the already
synthesized GAPDH enzyme would become active upon correct folding or assembly.
In agreement with this proposal, hibernating and euthermic GAPDHs were first
incubated at 25 ℃, then at 4 ℃, and finally again at 25 ℃, aliquots were withdrawn for the
activity assays at different intervals. The resulted t_1/2
values of the reactivation curves were <1 min and 17 min for the hibernating and euthermic GAPDH, respectively (Fig.6). The kinetics of cold inactivation and subsequent reactivation clearly show a faster and more efficient recovery of the activity of the hibernating GAPDH. These activity changes may be ascribed to subunit dissociation, as was reported for GAPDHs from other sources[6,24]. An efficient and fast recovery from cold inactivation should be an adaptative advantage for the hibernating jerboa, since in this way the central carbon metabolism would be ready to function immediately upon animal arousal. Whether this physiological adaptation is directly related with the conformational changes reported here for hibernating muscle GAPDH requires further studies.

Fig.6       Kinetics
of cold inactivation and subsequent reactivation of euthermic and hibernating
GAPDHs

Incubation mixtures (1.5 μmol/L of both euthermic and
hibernating tissue enzyme in 50 mmol/L Tris-HCl buffer, pH 7.5, containing 1
mmol/L EDTA) were first incubated at 25 ℃, then at 4 ℃ and finally again
at 25 ℃. At the indicated
times aliquots were withdrawn for the activity assays. The ordinate measures
changes in the percentage specific enzyme activity. The estimated t1/2 values
of the reactivation curves was <1 min and 17 min for the hibernating and euthermic muscle GAPDHs, respectively.

Acknowledgements            The
authors thank Prof. M. Losada (grant PB 97－1135) for encouragement and help

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_______________________________________

Received: May
27, 2003;Accepted: June 13,
2003

This work was supported by AECI (Spain), a
collaborative grant of the Andalusian Regional Government (Convenio Colaboración Univ. Marroquíes, grupo PAI CVI-261), and grants
BMC 2001-252 and P120/2001 from the Spanish Ministry of Education and the DGA

*Corresponding author: Tel,
(212)22230680/84; Fax: (212)22230674; e-mail: [email protected]

Updated at: 2003-10-09