Plant Physiol. (1998) 118: 249-255
Purification and Characterization of NAD-Isocitrate Dehydrogenase
from Chlamydomonas reinhardtii1
José M. Martínez-Rivas2, * and
José
M. Vega
Instituto de Bioquímica Vegetal y Fotosíntesis,
Centro de Investigaciones Isla de la Cartuja, Universidad de
Sevilla-Consejo Superior de Investigaciones Científicas,
Avenida Américo Vespucio s/n, 41092-Sevilla, Spain
 |
ABSTRACT |
NAD-isocitrate
dehydrogenase (NAD-IDH) from the eukaryotic microalga
Chlamydomonas reinhardtii was purified to
electrophoretic homogeneity by successive chromatography steps on
Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and
Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to
be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation
or inhibition by specific reagents for arginine or lysine. NADH was a
competitive inhibitor (Ki, 0.14 mM) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mM) with respect to
NAD+. Citrate and adenine nucleotides at concentrations
less than 1 mM had no effect on the activity, but 10 mM citrate, ATP, or ADP had an inhibitory effect. In
addition, NAD-IDH was inhibited by inorganic monovalent anions, but
L-amino acids and intermediates of glycolysis and the
tricarboxylic acid cycle had no significant effect. These data support
the idea that NAD-IDH from photosynthetic organisms may be a key
regulatory enzyme within the tricarboxylic acid cycle.
| |
INTRODUCTION |
IDH catalyzes the oxidative decarboxylation of isocitrate to
produce 2-oxoglutarate. According to the specificity for the electron
acceptor, two enzymes with IDH activity are known, NAD-IDH (EC
1.1.1.41) and NADP-IDH (EC 1.1.1.42) (Chen and Gadal, 1990a
).
In photosynthetic organisms NADP-IDH has been detected in the cytosol,
chloroplasts, mitochondria, and peroxisomes. Cytosolic NADP-IDH has
been purified from higher plants (Chen et al., 1988) and eukaryotic
algae (Martínez-Rivas et al., 1996), and its cDNA has been
cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et
al., 1993), potato (Fieuw et al., 1995), and tobacco (Gálvez et
al., 1996). This 80-kD isoenzyme is a dimer, and it is likely to be
involved in the synthesis of NADPH for biosynthetic purposes in the
cytosol (Chen et al., 1988), in the synthesis of 2-oxoglutarate for
ammonium assimilation (Chen and Gadal, 1990b), and in the cycling,
redistribution, and export of amino acids (Fieuw et al., 1995).
Chloroplastic NADP-IDH has been studied in higher plants (Gálvez
et al., 1994) and eukaryotic algae (Martínez-Rivas and Vega,
1994). It is a 154-kD dimer that has been proposed to be involved in
the supply of NADPH for biosynthetic reactions in the chloroplast when
photosynthetic NADPH production is low (Gálvez et al., 1994). The
mitochondrial NADP-IDH of higher plants may have a physiological role
in the production of NADPH, which can be converted to NADH by a
transhydrogenase or used to reduce glutathione in the mitochondrial
matrix (Rasmusson and Møller, 1990). NADP-IDH activity has also been
detected in peroxisomes from spinach leaves (Yamazaki and Tolbert,
1970).
NAD-IDH is localized exclusively in the mitochondria in association
with the TCA cycle. This enzyme has been purified from several
nonphotosynthetic eukaryotes such as fungi (Keys and McAlister-Henn, 1990; Alvarez-Villafañe et al., 1996) and animals (Giorgio et al., 1970), in which it appears to be a 300-kD octamer. Its key regulatory role in the TCA cycle is well documented. The NAD-IDH from
yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963),
whereas the animal enzyme is activated by ADP and citrate (Cohen and
Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from
yeast (Cupp and McAlister-Henn, 1991, 1992) and animals (Nichols et
al., 1995; Zeng et al., 1995). In these organisms, the enzyme is
composed of two (yeast) or more (animals) different subunits encoded by
different genes.
To our knowledge, no NAD-IDH from photosynthetic organisms has yet been
purified to homogeneity, mainly because of the low stability of the
enzyme (Oliver and McIntosh, 1995). However, partial purifications have
been reported from pea (Cox and Davies, 1967; Cox, 1969; McIntosh
and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al., 1996),
and the eukaryotic microalga Chlamydomonas reinhardtii
(Martínez-Rivas and Vega, 1994). Matrix and membrane forms of
the enzyme have been detected in potato (Tezuka and Laties, 1983) and
pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits
sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967;
McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et
al., 1990), the regulatory importance of NAD-IDH in photosynthetic
organisms is still under debate.
To elucidate the regulatory significance of NAD-IDH in photosynthetic
organisms and its apparent contribution to the 2-oxoglutarate supply for ammonium assimilation, we have purified and characterized the NAD-IDH from C. reinhardtii.
| |
MATERIALS AND METHODS |
Chemicals
Metabolites, standard proteins, PDP, and PGL were purchased from
Sigma. NAS and BTD were supplied by ICN Biomedicals. Phenyl-Sepharose, Blue-Sepharose, DEAE-Sephacel, and Sephacryl S-300 were from Pharmacia Biotech. Chemicals for electrophoresis were purchased from Bio-Rad. All
other chemicals were supplied by Merck (Darmstadt, Germany).
Organism and Culture Conditions
The eukaryotic microalga Chlamydomonas reinhardtii,
wild-type strain 21gr, was grown at 25°C in liquid medium
(Martínez-Rivas et al., 1991) with 10 mM
KNO3 as the nitrogen source. The cultures were
flushed with air supplemented with 5% (v/v) CO2
and continuously illuminated with white light from fluorescent lamps
(50 W m
2).
Enzyme Assay and Protein Determination
The NAD-IDH activity was determined in a 1-mL reaction containing
50 mM potassium phosphate buffer, pH 7.5, 0.5 mM MnCl2, 1.5 mM
NAD+, 4 mM
D,L-isocitrate, and the appropriate amount of
enzyme. The reaction was started by the addition of isocitrate and
followed by the increase in A340. One unit
of activity was defined as the amount of enzyme that catalyzed the
production of 1 µmol NADH min1.
Protein was estimated by the method of Bradford (1976) using
protein reagent dye (Bio-Rad) with BSA as a standard.
Enzyme Purification
Cells were harvested during the logarithmic phase of growth, and
were then broken by freezing in liquid nitrogen for 2 min and thawing
in buffer A (10 mM potassium phosphate buffer, pH 7.5, and
14 mM 2-mercaptoethanol) including 1 mM PMSF.
The homogenate was centrifuged at 16,000g for 30 min at
4°C, and the supernatant was used as a crude extract. All subsequent
steps were carried out at 4°C.
A solution of 2% (w/v) protamine sulfate, pH 7.5, was slowly added to
the crude extract up to 0.16% (w/v) final concentration. After 10 min
of incubation with gentle stirring, the suspension was centrifuged at
16,000g for 30 min and the pellet was discarded.
A Phenyl-Sepharose column (1.6 × 40 cm) was equilibrated at a
flow rate of 30 mL h1 with buffer A adjusted to
15% saturation with solid
(NH4)2SO4. The column was loaded with the supernatant previously adjusted to
similar ionic strength, and then washed with 300 mL of the same buffer.
The NADP-IDH activity was eluted with 300 mL of buffer A containing
(NH4)2SO4
to a saturation of 5%. After another washing with 300 mL of buffer A,
the NAD-IDH activity was eluted with 300 mL of this buffer, including
50% (v/v) ethylene glycol. Fractions (3 mL) containing enzyme activity
were pooled and diluted with buffer A to 10% (v/v) ethylene glycol
(buffer B).
The resulting NAD-IDH preparation was applied onto a Blue-Sepharose
column (1.6 × 12 cm) preequilibrated with buffer B at a flow rate
of 15 mL h1. The column was washed with 60 mL
of this buffer, and the enzyme was eluted with 60 mL of buffer B
containing 50 mM KCl. Active fractions (fraction size, 1 mL) were pooled and dialyzed against 10 mM Tris-HCl, pH
8.5, containing 14 mM 2-mercaptoethanol and 10% (v/v)
ethylene glycol (buffer C) overnight.
The dialysate was loaded onto a DEAE-Sephacel column (0.7 × 6 cm), which had been previously equilibrated with buffer C at a flow
rate of 15 mL h1. The column was washed with 20 mL of buffer C. Bound protein was eluted with a 20-mL linear gradient
from 0 to 100 mM KCl in buffer C. Fractions (0.5 mL)
containing NAD-IDH activity were pooled and concentrated to 2 mL by
ultrafiltration.
The DEAE-Sephacel-purified NAD-IDH was applied onto a Sephacryl S-300
column (1.6 × 100 cm) equilibrated with 10 mM
potassium phosphate buffer, pH 7.5, at a flow rate of 15 mL
h1 and calibrated with blue dextran, ferritin
(364 kD), catalase (240 kD), alcohol dehydrogenase (150 kD), BSA (66 kD), ovalbumin (45 kD), and Cyt c (12 kD). The enzyme was
eluted with the same buffer and 1-mL fractions were collected. The
pooled peak-activity fractions were concentrated by ultrafiltration and
kept at 4°C for immediate use.
Gel Electrophoresis
Nondenaturing PAGE and SDS-PAGE were performed as described
previously (Laemmli, 1970), using 5% and 12% polyacrylamide gels, respectively. Proteins were located in the gel by staining with 0.1%
(w/v) Coomassie brilliant blue R-250 in 25% (v/v) ethanol and 10%
(v/v) acetic acid. NAD-IDH activity was located by submerging the gel
in a reaction mixture containing 100 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 7 mM
NAD+, 40 mM
D,L-isocitrate, 0.05% (w/v) nitroblue
tetrazolium, and 0.005% (w/v) phenazine methosulfate. After 30 min at
room temperature in the dark, the activity was shown by a blue band.
| |
RESULTS AND DISCUSSION |
Purification of NAD-IDH from C. reinhardtii
Cells of C. reinhardtii contain two IDH
enzymes specific for either NAD+ or NADP+,
which can be separated by hydrophobic chromatography on
Phenyl-Sepharose. In addition, the NADP-IDH preparation can be further
resolved in two different isoenzymes by affinity chromatography on
Blue-Sepharose (Martínez-Rivas and Vega, 1994). The main
problem in achieving complete purification of the NAD-IDH from
photosynthetic organisms has been its low stability (McIntosh and
Oliver, 1992). To stabilize the NAD-IDH activity from C. reinhardtii, we found that it was critical to keep ethylene
glycol in the buffer after elution from the Phenyl-Sepharose
chromatography step. The high concentration of ethylene glycol (50%)
required for the elution of the enzyme showed its high hydrophobicity.
Before it was applied to the Blue-Sepharose column, we reduced the
concentration of ethylene glycol in the preparation from 50% to 10%
to avoid its interaction with the gel matrix. A typical profile
corresponding to this chromatography step is shown in Figure
1A. The eluate obtained in this step was
dialyzed against 10 mM Tris-HCl, pH 8.5, containing 14 mM 2-mercaptoethanol and 10% (v/v) ethylene glycol to
change the pH of the buffer. This was required to bind the NAD-IDH
activity to the DEAE-Sephacel matrix, in contrast to previous studies
in which the enzyme was partially purified using a similar approach but
keeping the pH of the buffer at 7.5. The enzyme was eluted with a salt
concentration of 75 mM KCl in a linear gradient from 0 to
100 mM, and separated from other proteins that eluted at
different ionic strengths (Fig. 1B). The combination of the affinity
chromatography with the anion-exchange matrix allowed us to obtain a
preparation of high purity. Finally, we used gel-filtration
chromatography on Sephacryl S-300 to remove some minor contaminant
proteins of low Mr (Fig. 1C).
View larger version (20K):
[in this window]
[in a new window]
| Figure 1.
Purification of NAD-IDH from C. reinhardtii by column chromatography. NAD-IDH activity ( )
was measured using the standard assay conditions and protein ( ) was
monitored by A280. A, Affinity
chromatography on Blue-Sepharose. B, Anion-exchange chromatography on
DEAE-Sephacel. C, Gel-filtration chromatography on Sephacryl S-300.
|
|
The purification procedure summarized in Table
I produced 0.12 mg of NAD-IDH from
148 g of cells, with a total purification of about 922-fold, a
yield of 6%, and a specific activity of 16.6 units
mg1 protein. Comparable specific activities
have been reported for the purified preparations of NAD-IDH from
Phycomyces blakesleeanus (Alvarez-Villafañe et al.,
1996) and Saccharomyces cerevisiae (Keys and McAlister-Henn,
1990).
The purified enzyme migrated as a single band in a nondenaturing
gel stained for NAD-IDH activity (Fig.
2A). The homogeneity of the preparation
was also confirmed by SDS-PAGE (Fig. 2B). According to the purification
method described here, isolation of the mitochondrial fraction was not
required to purify the NAD-IDH from C. reinhardtii, as was
reported previously for the plant enzyme (Tezuka and Laties, 1983;
McIntosh and Oliver, 1992). In contrast to the NAD-IDH from pea, where
35% to 50% of the activity was found tightly associated with the
membrane (McIntosh, 1997), no NAD-IDH activity was detected in the
membrane pellet obtained in the crude extract step after solubilization
with 0.5% Triton X-100. However, we cannot discount that a membrane
form was also present but was denatured during the treatment with the
detergent.
View larger version (55K):
[in this window]
[in a new window]
| Figure 2.
Nondenaturing PAGE and SDS-PAGE of purified
NAD-IDH from C. reinhardtii. A, Five percent
polyacrylamide gel stained for NAD-IDH activity. B, SDS-12%
polyacrylamide gel stained with Coomassie blue. Five-microgram samples
of protein were loaded in each well.
|
|
Structural Characterization of NAD-IDH from C. reinhardtii
The native molecular mass of NAD-IDH from C. reinhardtii, as estimated by gel-filtration chromatography on
Sephacryl S-300, was 320 kD. A subunit molecular mass of 45 kD was
deduced from SDS-PAGE (Fig. 2B). Therefore, the enzyme in its native
form appears to be an octamer, but from our data we cannot determine if
it is composed of identical or different subunits. The octameric structure and the native molecular mass value are consistent with those
reported for the NAD-IDH from bovine heart (333 kD; Giorgio et al.,
1970) and from P. blakesleeanus (338 kD;
Alvarez-Villafañe et al., 1996), and for the matrix (300 kD; McIntosh and Oliver, 1992) and membrane forms (320 kD;
McIntosh, 1997) from pea.
McIntosh and Oliver (1992) reported the correlation between the loss of
activity during subsequent purification steps and a change in the
native molecular mass of the protein from pea. However, we did not
detect any dissociation of the C. reinhardtii NAD-IDH
protein during the purification. To check this, preparations from each
purification step were analyzed by gel-filtration chromatography, in
which a single activity peak corresponding to 320 kD was always observed, and by nondenaturing PAGE followed by activity staining, in
which a single band with the same electrophoretic mobility was always
observed (data not shown).
The heat-inactivation profile of NAD-IDH from C. reinhardtii
is shown in Figure 3. The temperature at
which 50% of the enzyme activity was recovered after 10 min of
incubation was 42°C. These data are similar to those from the
cytosolic NADP-IDH isoenzyme (Martínez-Rivas et al., 1996) and
the NAD-IDH from spruce (Cornu et al., 1996). However, in this plant,
mitochondrial NADP-IDH was more resistant than NAD-IDH to heat
inactivation. As reported earlier for the NADP-IDH isoenzymes
(González-Villaseñor and Powers, 1985;
Martínez-Rivas et al., 1996), the presence of the pyridin
nucleotide, isocitrate, and, especially, isocitrate plus divalent
cation, protects NAD-IDH activity against thermal inactivation. The
binding of the substrate to the enzyme may produce a conformational modification that increases the enzyme's resistance to heat
inactivation.
View larger version (20K):
[in this window]
[in a new window]
| Figure 3.
Heat-inactivation profiles of NAD-IDH from
C. reinhardtii. Enzyme samples were incubated at the
indicated temperature in 10 mM potassium phosphate buffer,
pH 7.5, and the indicated substrates at a 5 mM final
concentration in a final volume of 0.3 mL. After 10 min the samples
were cooled rapidly on ice and assayed for NAD-IDH activity (100%
activity = 0.1 unit mL1).
|
|
The essential nature of amino acids with sulfhydryl, amino, and
carboxyl side groups was demonstrated previously for the NAD-IDH activity from C. reinhardtii (Martínez-Rivas and
Vega, 1994). To better characterize those observations, we tested the
effects of specific reagents for Arg (PGL or BTD) and Lys (PDP or NAS) on the NAD-IDH activity. The enzyme was almost completely inactivated in the presence of any of these compounds at a concentration of 1 mM (Fig. 4). The simultaneous
presence of isocitrate and Mn2+, but not
isocitrate alone, prevented this inactivation. These results indicate
that the true substrate for the enzyme is the complex formed by
isocitrate and the divalent cation, as has been demonstrated for the
NAD-IDH from pea leaves (Duggleby and Dennis, 1970a), pig heart (Cohen
and Colman, 1974), and P. blakesleeanus (Alvarez-Villafañe et al., 1996).
View larger version (45K):
[in this window]
[in a new window]
| Figure 4.
Effect of specific reagents on the NAD-IDH
activity from C. reinhardtii. The enzyme was incubated
at 4°C in 10 mM potassium phosphate buffer, pH 7.5, containing 1 mM specific reagents for Arg (PGL or BTD) or
Lys (PDP or NAS), and the indicated substrate at 5 mM final
concentration in a final volume of 0.3 mL. After 2 h, NAD-IDH
activity was measured (100% activity = 0.13 unit
mL1).
|
|
In addition, these data show the involvement of Arg and Lys residues in
the active site for the isocitrate-divalent cation substrate, as has
been reported previously for the NAD-IDH from bovine heart (Fan and
Plaut, 1974). However, these amino acids are not involved in the active
site for NAD+, because this substrate did not
protect against inactivation. Arg and Lys residues are conserved in the
deduced amino acid sequences of the corresponding NAD-IDH cDNA clones
from yeast (Cupp and McAllister-Henn, 1991, 1992) and animals (Nichols
et al., 1995; Zeng et al., 1995). The presence of these residues in the
active site for the isocitrate-divalent cation substrate, but not for NADP+, has been well established for the NADP-IDH
from Escherichia coli (Hurley et al., 1989).
Kinetic and Regulatory Properties of NAD-IDH from C. reinhardtii
In a previous study (Martínez-Rivas and Vega, 1994), we
found that the NAD-IDH from C. reinhardtii showed
sigmoidal kinetics for isocitrate (S0.5 = 0.37 mM, n = 1.82, k = 0.16), but standard Michaelis-Menten kinetics for
NAD+ (Km = 0.15 mM) and Mn2+
(Km = 0.03 mM). Other kinetic
parameters were also determined: optimum pH (8.0), optimum temperature
(40°C), and activation energy (78.1 kJ mol1).
In the present study we investigated the kinetic properties of this
enzyme to search for possible regulatory mechanisms.
NAD-IDH from C. reinhardtii was not inhibited by an excess
of isocitrate, NAD+, or
NADP+, but 10 mM 2-oxoglutarate, the
product of the reaction, inhibited it slightly (Martínez-Rivas
and Vega, 1994). This metabolite had no effect on the enzyme from swede
(Coultate and Dennis, 1969) and spruce (Cornu et al., 1996). In
contrast, NADH was a competitive inhibitor
(Ki = 0.14 mM) with respect to
NAD+ (Fig. 5A), as
has been reported for the NAD-IDH from pea (Duggleby and Dennis, 1970b;
McIntosh and Oliver, 1992; McIntosh, 1997) and swede
(Coultate and Dennis, 1969). The inhibition by NADH may be an important
regulatory mechanism of the TCA cycle. NADPH was also an inhibitor of
NAD-IDH activity, but it was noncompetitive (Ki = 0.42 mM) with respect to
NAD+ (Fig. 5B). The same effect was reported for
the matrix form (McIntosh and Oliver, 1992) and the membrane form
(McIntosh, 1997) of the enzyme from pea, and these authors suggested
that there is a separate binding site for NADPH on the NAD-IDH enzyme
that is not the catalytic site for NAD+ and NADH
binding. A physiological role for the NADPH inhibition has been
suggested in the control and balance of NADH and NADPH production
(McIntosh and Oliver, 1992).
View larger version (15K):
[in this window]
[in a new window]
| Figure 5.
Effect of NADH (A) and NADPH (B) on the NAD-IDH
activity from C. reinhardtii. Reaction mixtures were as
described for the standard assay except that the concentration of
NAD+ was varied as indicated in the figure. NADH or NADPH
was added to a 0.2 mM final concentration. Initial rates
(V1) are expressed as micromoles of NADH produced per
minute.
|
|
Unlike previous studies using the NAD-IDH from pea (Cox and Davies,
1967) and swede (Coultate and Dennis, 1969), the enzyme from
C. reinhardtii was not activated by citrate at
concentrations less than 1 mM (Table
II), and 1 mM citrate did not
produce a change in the kinetics with respect to isocitrate from
sigmoidal to Michaelis-Menten (data not shown). These differences could be explained by the fact that we always used freshly purified enzyme,
thus avoiding problems of subunit dissociation caused by freezing and
thawing of the enzyme preparation (McIntosh and Oliver, 1992). At
concentrations greater than 1 mM, citrate inhibited C. reinhardtii NAD-IDH activity. This effect was caused by
the chelation of the divalent cation (Coultate and Dennis, 1969) and the competitive inhibition with respect to isocitrate (Cox and Davies,
1969). However, the high concentration required for it to act as an
inhibitor indicates that the regulatory role of citrate may be very
limited.
View this table:
[in this window]
[in a new window]
|
Table II.
Effect of citrate and nucleotides on the NAD-IDH
from C. reinhardtii
Reaction mixtures were as described for the standard assay except that
the corresponding metabolite was included. One hundred percent NAD-IDH
activity was 0.58 unit
mL1.
|
|
In contrast to the NAD-IDH from yeast or animals, no activation was
observed when the enzyme was incubated with AMP or ADP (Table II). In
contrast, ATP or ADP at concentrations greater than 1 mM
showed an inhibitory effect at least partially attributable to the
formation of a complex with the divalent cation (Omran and Dennis,
1971). Similar results were reported for this enzyme from swede
(Coultate and Dennis, 1969), potato (Tezuka and Laties, 1983), pea
(McIntosh and Oliver, 1992), and spruce (Cornu et al., 1996). However,
this inhibition is considered to be of little regulatory significance
(Meixner-Monori et al., 1986). Incubation of the enzyme with 10 mM GTP or GDP brought about a 30% inactivation of the
enzyme (data not shown).
Glycolysis intermediates were checked for inhibitory effects. Pyruvate,
PEP, dihydroxyacetone phosphate, Fru-1,6-bisP, Fru-6-P, Glc-6-P, and
Glc-1-P at a concentration of 10 mM had a slight or no
inhibitory effect on the NAD-IDH activity from C. reinhardtii (data not shown). TCA intermediates such as
acetyl-CoA, cis-aconitate, succinate, fumarate,
L-malate, and oxaloacetate also had no significant effect.
In addition, no significant inhibition was observed when any of the
proteinogenic amino acids were tested at a final concentration of 10 mM. On the other hand, NAD-IDH activity from C. reinhardtii was strongly inhibited by monovalent inorganic anions
such as chloride, iodide, and nitrate at a concentration of 100 mM. No significant effect was observed when divalent anions
such as sulfate or arsenate were tested at the same concentration (data
not shown). Similar results were obtained by Cox and Davies (1967) with
the pea enzyme. It has been proposed that the monovalent inorganic anions may cause a conformational change in NAD-IDH (Coultate and
Dennis, 1969), but such an effect would have no physiological regulatory significance because of the high concentrations required.
| |
CONCLUSIONS |
The molecular properties of NAD-IDH from C. reinhardtii
(molecular mass, number of subunits, and configuration of the active site) are similar to those reported from nonphotosynthetic eukaryotes such as S. cerevisiae (Keys and McAlister-Henn, 1990) or pig
heart (Giorgio et al., 1970). However, the NAD-IDH from C. reinhardtii did not exhibit the same regulatory properties, since
citrate, ADP, and AMP did not act as effectors. The main differences
between the NAD-IDH enzyme from unicellular and multicellular
photosynthetic organisms were: (a) the requirement for a pH change to
retain the C. reinhardtii NAD-IDH on an anion-exchange
column, thereby showing different electrostatic properties, and (b) the
high hydrophobicity of the algal enzyme.
The most important regulatory effect was the strong inhibition by NADH
and NADPH, which, together with the concentration of the divalent
cations (Duggleby and Dennis, 1970a), seem to be the main regulators.
Therefore, NAD-IDH from photosynthetic organisms can be considered a
key regulatory point in the carbon flow through the TCA cycle.
Furthermore, because of its low in vitro activity, it has been
suggested that NAD-IDH catalyzes the rate-limiting step in the TCA
cycle in these organisms (Møller and Palmer, 1984). On the other hand,
the participation of NAD-IDH in the 2-oxoglutarate supply for ammonium
assimilation cannot be discounted. The TCA cycle is stimulated under
conditions in which there is a high demand for this metabolite (Weger
et al., 1988), and NAD-IDH activity increases under nitrogen starvation
(Martínez-Rivas and Vega, 1993). Future studies, including the
cloning of the gene, are required to understand the molecular basis of
the structure and the regulatory mechanisms of NAD-IDH.
| |
FOOTNOTES |
1
This work was supported by research grant no.
PB96-1367 from Dirección General de Investigación
Científica y Técnica, Spain. J.M.M.-R. was the recipient
of a postdoctoral contract from the Ministerio de Educación y
Ciencia, Spain.
2
Present address: Institut für Allgemeine
Botanik, Universität Hamburg, Ohnhorststrasse 18, 22609-Hamburg,
Germany.
*
Corresponding author; e-mail mrivas{at}cica.es; fax
49-40-822-82-254.
Received April 8, 1998;
accepted June 19, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BTD, 2,3-butanedione.
IDH, isocitrate
dehydrogenase.
NAS, N-acetyl-succinimide.
PDP, pyridoxal
5
-phosphate.
PGL, phenylglyoxal.
TCA, tricarboxylic acid.
| |
ACKNOWLEDGMENT |
We thank Dr. Jodi Scheffler for revising the English version of
the manuscript.
| |
LITERATURE CITED |
Alvarez-Villafañe E,
Soler J,
del Valle P,
Busto F,
de Arriaga D
(1996)
Two NAD+-isocitrate dehydrogenase forms in Phycomyces blakesleeanus: induction in response to acetate growth and characterization, kinetics, and regulation of both enzyme forms.
Biochemistry
35:
4741-4752
[Medline]
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Chen RD,
Gadal P
(1990a)
Structure, functions and regulation of NAD and NADP dependent isocitrate dehydrogenases in higher plants and in other organisms.
Plant Physiol Biochem
28:
411-427
Chen RD,
Gadal P
(1990b)
Do the mitochondria provide the 2-oxoglutarate needed for glutamate synthesis in higher plant chloroplasts?
Plant Physiol Biochem
28:
141-145
[ISI]
Chen RD,
LeMaréchal P,
Vidal J,
Jacquot JP,
Gadal P
(1988)
Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves.
Eur J Biochem
175:
565-572
[ISI][Medline]
Cohen PF,
Colman RF
(1972)
Diphosphopyridine nucleotide dependent isocitrate dehydrogenase from pig heart: characterization of the active substrate and modes of regulation.
Biochemistry
11:
1501-1508
[CrossRef][Medline]
Cohen PF,
Colman RF
(1974)
Role of manganous ion in the kinetics of pig-heart NAD-specific isocitrate dehydrogenase.
Eur J Biochem
47:
35-45
[Medline]
Cornu S,
Pireaux JC,
Gerard J,
Dizengremel P
(1996)
NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings.
Physiol Plant
96:
312-318
[CrossRef]
Coultate TP,
Dennis DT
(1969)
Regulatory properties of a plant NAD:isocitrate dehydrogenase: the effect of inorganic ions.
Eur J Biochem
7:
153-158
[Medline]
Cox GF
(1969)
Isocitrate dehydrogenase (NAD-specific) from pea mitochondria.
Methods Enzymol
13:
47-51
Cox GF,
Davies DD
(1967)
Nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria: purification and properties.
Biochem J
105:
729-734
[ISI][Medline]
Cox GF,
Davies DD
(1969)
The effects of pH and citrate on the activity of nicotinamide-adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria.
Biochem J
113:
813-820
[Medline]
Cupp JR,
McAlister-Henn L
(1991)
NAD+-dependent isocitrate dehydrogenase: cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae.
J Biol Chem
266:
22199-22205
[Abstract/Free Full Text]
Cupp JR,
McAlister-Henn L
(1992)
Cloning and characterization of the gene encoding the IDH1 subunit of NAD+-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.
J Biol Chem
267:
16417-16423
[Abstract/Free Full Text]
Duggleby RG,
Dennis DT
(1970a)
Nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase from a higher plant: the requirement for free and metal-complexed isocitrate.
J Biol Chem
245:
3745-3750
[Abstract/Free Full Text]
Duggleby RG,
Dennis DT
(1970b)
Regulation of the nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase from a higher plant: the effect of reduced nicotinamide adenine dinucleotide and mixtures of citrate and isocitrate.
J Biol Chem
245:
3751-3754
[Abstract/Free Full Text]
Fan CC,
Plaut GWE
(1974)
Functional groups of diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.
J Biol Chem
249:
4839-4845
[Abstract/Free Full Text]
Fieuw S,
Müller-Röber B,
Gálvez S,
Willmitzer L
(1995)
Cloning and expression analysis of the cytosolic NADP+-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism.
Plant Physiol
107:
905-913
[Abstract]
Gálvez S,
Bismuth E,
Sarda C,
Gadal P
(1994)
Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from mixotrophic tobacco cells. Comparison with the cytosolic enzyme.
Plant Physiol
105:
593-600
[Abstract]
Gálvez S,
Hodges M,
Decottignies P,
Bismuth E,
Lancien M,
Sangwan RS,
Dubois F,
LeMaréchal P,
Crétin C,
Gadal P
(1996)
Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase.
Plant Mol Biol
30:
307-320
[CrossRef][ISI][Medline]
Giorgio NA,
Yip JAT,
Fleming J,
Plaut GWE
(1970)
Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart.
J Biol Chem
245:
5469-5477
[Abstract/Free Full Text]
González-Villaseñor LI,
Powers DA
(1985)
A multilocus system for studying tissue and subcellular specialization: the three NADP-dependent isocitrate dehydrogenase isoenzymes of the fish Fundulus heteroclitus.
J Biol Chem
260:
9106-9113
[Abstract/Free Full Text]
Hathaway JA,
Atkinson DE
(1963)
The effect by adenylic acid on yeast nicotinamide adenine dinucleotide isocitrate dehydrogenase: a possible metabolic control mechanism.
J Biol Chem
238:
2875-2881
[Free Full Text]
Hurley JH,
Thorsness PE,
Ramalingam V,
Helmers NH,
Koshland DE Jr,
Stroud RM
(1989)
Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.
Proc Natl Acad Sci USA
86:
8635-8639
[Abstract/Free Full Text]
Keys DA,
McAlister-Henn L
(1990)
Bacteriol
172:
4280-4287
[Abstract/Free Full Text]
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Laties GG
(1983)
Membrane-associated NAD-dependent isocitrate dehydrogenase in potato mitochondria.
Plant Physiol
72:
953-958
[Abstract/Free Full Text]
Martínez-Rivas JM,
García-Díaz MT,
Vega JM
(1996)
Purification and properties of cytosolic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii.
Plant Physiol Biochem
34:
673-676
Martínez-Rivas JM,
Vega JM
(1993)
Plant
88:
599-603
Martínez-Rivas JM,
Vega JM
(1994)
Studies on the isoforms of isocitrate dehydrogenase from Chlamydomonas reinhardtii.
J Plant Physiol
143:
129-134
Martínez-Rivas JM,
Vega JM,
Márquez AJ
(1991)
Differential regulation of the nitrate-reducing and ammonium-assimilatory systems in synchronous cultures of Chlamydomonas reinhardtii.
FEMS Microbiol Lett
78:
85-88
[CrossRef]
McIntosh CA
(1997)
Partial purification and characteristics of membrane-associated NAD+-dependent isocitrate dehydrogenase activity from etiolated pea mitochondria.
Plant Sci
129:
9-20
[CrossRef]
McIntosh CA,
Oliver DJ
(1992)
NAD+-linked isocitrate dehydrogenase: isolation, purification, and characterization of the protein from pea mitochondria.
Plant Physiol
100:
69-75
[Abstract/Free Full Text]
Meixner-Monori B,
Kubicek CP,
Harrer W,
Schreferl G,
Rohr M
(1986)
NADP-specific isocitrate dehydrogenase from the citric acid-accumulating fungus Aspergillus niger.
Biochem J
236:
549-557
[Medline]
Møller IM,
Palmer JM
(1984)
Regulation of the tricarboxylic acid cycle and organic acid metabolism.
Soc Exp Biol Semin Ser
20:
105-122
Nichols BJ,
Perry ACF,
Hall L,
Denton RM
(1995)
Molecular cloning and deduced amino acid sequences of the 
- and 
-subunits of mammalian NAD+-isocitrate dehydrogenase.
Biochem J
310:
917-922
Oliver DJ, McIntosh CA (1995) The biochemistry of the
mitochondrial matrix. In CS Levings III, IK Vasil, eds, The
Molecular Biology of Plant Mitochondria, Vol 3: Advances in Cellular
and Molecular Biology of Plants. Kluwer Academic Publishers, Dordrecht,
The Netherlands, pp 237-280
Omran RG,
Dennis DT
(1971)
Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from a higher plant. Isolation and characterization.
Plant Physiol
47:
43-47
[Abstract/Free Full Text]
Rasmusson AG,
Møller IM
(1990)
NADP-utilizing enzymes in the matrix of plant mitochondria.
Plant Physiol
94:
1012-1018
[Abstract/Free Full Text]
Shorrosh BS,
Dixon RA
(1992)
Molecular characterization and expression of an isocitrate dehydrogenase from alfalfa (Medicago sativa L.).
Plant Mol Biol
20:
801-807
[Medline]
Tezuka T,
Laties GG
(1983)
Isolation and characterization of inner membrane- associated and matrix NAD-specific isocitrate dehydrogenase in potato mitochondria.
Plant Physiol
72:
959-963
[Abstract/Free Full Text]
Tezuka T,
Yamamoto Y,
Kondo N
(1990)
Activation of O2 uptake and NAD-specific isocitrate dehydrogenase in mitochondria isolated from cotyledons of castor bean by cis,trans-abscisic acid.
Plant Physiol
92:
147-150
[Abstract/Free Full Text]
Udvardi MK,
McDermott TR,
Kahn ML
(1993)
Isolation and characterization of a cDNA encoding NADP+-specific isocitrate dehydrogenase from soybean (Glycine max).
Plant Mol Biol
21:
739-752
[CrossRef][Medline]
Weger HG,
Birch DG,
Elrifi IR,
Turpin DH
(1988)
Ammonium assimilation requires mitochondrial respiration in the light.
Plant Physiol
86:
688-692
[Abstract/Free Full Text]
Yamazaki RK,
Tolbert NE
(1970)
Enzymic characterization of leaf peroxisomes.
J Biol Chem
245:
5137-5144
[Abstract/Free Full Text]
Zeng Y,
Weiss C,
Yao TT,
Huang J,
Siconolfi-Baez L,
Hsu P,
Rushbrook JI
(1995)
Isocitrate dehydrogenase from bovine heart: primary structure of subunit 3/4.
Biochem J
310:
507-516
Top
Abstract
Introduction