Purification and characterization of an allosteric fructose-1,6- bisphosphate aldolase from germinating mung beans (Vigna radiata)

Article abstract of DOI:10.1016/j.phytochem.2005.03.009

Cytosolic fructose-1,6-P₂ (FBP) aldolase (ALD_c) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 μmol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD _c)-IgG. The enzyme's native M_r was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg²⁺ had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65°C. It demonstrated: (i) a temperature coefficient (Q₁₀) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD_c is bifunctional for FBP and sedoheptulose-1,7-P₂ (K_m ≈ 17 μM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K_i values = 2.2, 3.1 and 7.5 mM, respectively). By contrast, the addition of 2 mM ATP: (i) reduced V_max by about 2-fold, (ii) increased K_m(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h = 1.0 and 2.6 in the absence and presence of 2 mM ATP, respectively). Potent feedback inhibition of ALD_c by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.

Full text of DOI:10.1016/j.phytochem.2005.03.009

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 968–974
www.elsevier.com/locate/phytochem
Purification and characterization of an allosteric
fructose-1,6-bisphosphate aldolase from germinating
mung beans (Vigna radiata)
a,1
Ashish Lal , William C. Plaxton , Arvind M. Kayastha
b
a,
*
a
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India
Departments of Biology and Biochemistry, QueenÕs University, Kingston, Ont., Canada K7L 3N6
b
Received 22 October 2004; received in revised form 10 March 2005
Available online 26 April 2005
Abstract
Cytosolic fructose-1,6-P
homogeneity and a final specific activity of 15.1 lmol FBP cleaved/min per mg of protein. SDS–PAGE of the final preparation
revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD )-IgG. The enzymeÕs
native M was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This
2 c
(FBP) aldolase (ALD ) from germinated mung beans has been purified 1078-fold to electrophoretic
c
r
2
+
ALD is a class I ALD, since EDTA or Mg had no effect on its activity, and was relatively heat-stable losing 0–25% of its activity
when incubated for 5 min at 55–65 ꢀC. It demonstrated: (i) a temperature coefficient (Q10) of 1.7; (ii) an activation energy of 9.2
c
kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD is bifunctional for FBP and sedoheptulose-
1
enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K values = 2.2, 3.1 and 7.5 mM, respectively). By con-
,7-P (Km ꢀ 17 lM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified
i
2
m
trast, the addition of 2 mM ATP: (i) reduced Vmax by about 2-fold, (ii) increased K (FBP) by about 4-fold, and (iii) shifted the FBP
saturation kinetic plot from hyperbolic to sigmoidal (h = 1.0 and 2.6 in the absence and presence of 2 mM ATP, respectively). Potent
feedback inhibition of ALD by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and
c
respiration in germinating mung beans.
2005 Elsevier Ltd. All rights reserved.
ꢁ
Keywords: Aldolase; Plant glycolysis; Metabolic control; Carbohydrate metabolism (seed); Vigna radiata
1
. Introduction
Fructose-1,6-bisphosphate (FBP ) aldolase (ALD; D-
Fru-1,6-bisphosphate D-glyceraldehyde-3-P-lyase; E.C.
4.1.2.13) is a ubiquitous and abundant glycolytic enzyme
that catalyzes the reversible aldol cleavage of FBP into
glyceraldehyde-3-P and dihydroxyacetone-P. ALDs are
either dependent upon divalent cations for activity (Class
II ALD) or not (Class I ALD). Class II ALDs occur in
2
*
Corresponding author. Tel.: + 91 542 2368331; fax: +91 542
fungi and prokaryotes and are homodimers with M s of
r
2368693.
about 80 kDa, whereas the enzymes from animals and
vascular plants fall into Class I and are homotetramers
E-mail address: kayastha@fastmail.ca (A.M. Kayastha).
Laboratory of Cellular and Molecular Biology, GRC, NIA-IRP,
1
National Institutes of Health, 5600, Nathan Shock Drive, Baltimore,
MD 21224, USA.
(
M approx. 160 kDa). In mammals, three tissue-specific
r
ALD isozymes have been identified in muscle, liver or
kidney, and brain (Gefflaut et al., 1995). These isozymes
have slightly different physical features, and the muscle
and liver ALDs also vary in their substrate specificities.
2
Abbreviations: ALD, fructose-1,6-P
2
aldolase; ALD
aldolase, respec-
; FPLC, fast protein liquid chromatogra-
phy; SBP, sedoheptulose-1,7-P
c p
and ALD ,
cytosolic and plastidic isozymes of fructose-1,6-P
tively; FBP, fructose-1,6-P
2
2
2
.
0
031-9422/$ - see front matter ꢁ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.03.009

A. Lal et al. / Phytochemistry 66 (2005) 968–974
969
The occurrence of tissue-specific ALD isozymes in
vascular plants has not yet been established. However,
cytosolic and plastidic ALD isozymes (ALDc and
et al., 1994; Pontremoli et al., 1979). ALD was one of
c
seven different glycolytic enzymes reported to be physi-
cally associated with the cytosolic face of the outer
membrane of mitochondria isolated from Arabidopsis
thaliana suspension cells (Gieg e´ et al., 2003). The en-
zyme has also been implicated as a key ÔscaffoldingÕ en-
zyme during the formation of a glycolytic complex on
actin-containing filaments in contracting mammalian
muscle tissues (Ov a´ di and Srere, 2000). In the present
ALD , respectively) are expressed in photosynthetic
p
and non-photosynthetic plant tissues and are encoded
by separate nuclear genes that likely evolved from dupli-
cation of a common ancestral gene (Plaxton, 1996).
ALD and ALD differ somewhat in their respective
c
p
thermal stability, net charge, amino acid composition,
immunological properties and subunit size. In photosyn-
thetic tissues ALD plays a pivotal role in the C reduc-
study we report the purification of ALD from germi-
c
nated mung beans and characterize the enzymeÕs physi-
cal, immunological and kinetic properties. Potent
allosteric inhibition of the enzyme by ATP is suggested
to contribute to the fine metabolic control of glycolysis
and respiration in this tissue.
p
3
tive pentose-P (Calvin–Benson) cycle and represents as
much as 95% of the total leaf ALD activity (Kr u¨ ger
and Schnarrenberger, 1983; Lebherz et al., 1984). Puri-
fied ALD and ALD preparations from various leaves
p
c
have been characterized (Kr u¨ ger and Schnarrenberger,
983; Lebherz et al., 1984; Marsh et al., 1989; Schnar-
1
renberger and Kr u¨ ger, 1986). Comparatively little is
known about the ALDs from non-photosynthetic plant
tissues. However, the contribution of ALD to the total
2. Result and discussion
2.1. Enzyme purification
c
ALD activity of non-green tissues appears to be much
greater than in green leaves and often accounts for more
than 50% of the total ALD activity present (Kr u¨ ger and
Schnarrenberger, 1985; Nishimura and Beevers, 1981;
Botha and OÕKennedy, 1989; Moorhead and Plaxton,
The specific ALD activity in clarified extracts of ger-
minated mung beans gradually increased to a maximum
in 30 h to about 0.014 U/mg, following imbibition.
Thereafter, ALD specific activity showed a marked de-
cline (results not shown). A similar developmental pro-
file has been noted for several other glycolytic enzymes
of germinated mung beans (Kumar and Malhotra,
1992; Malhotra and Kayastha, 1990; Malhotra et al.,
1979). As outlined in Table 1, ALD from 30 h germi-
nated mung beans was purified 1078-fold to a final spe-
cific activity of 15.1 U/mg and an overall yield of 21%.
About 35% of the total ALD activity applied to the
phosphocellulose column consistently failed to absorb
to this matrix, even when the resin bed volume was in-
creased by 200%. Similarly, ALD , but not ALD , from
1
2
990; Hodgson and Plaxton, 1998; Schwab et al.,
001). The physical, immunological and kinetic proper-
ties of homogeneous ALDc preparations from non-
green plant tissues have thus far only been reported
for the germinating Phaseolus vulgaris and Ricinus com-
munis seed and carrot storage root enzymes (Botha and
OÕKennedy, 1989; Moorhead and Plaxton, 1990; Hodg-
son and Plaxton, 1998).
It is generally believed that a key glycolytic control
site is the conversion of Fructose-6-P to FBP, a reaction
catalyzed in the plant cytosol by the ATP- or PPi-depen-
dent phosphofructokinase (Plaxton, 1996). Although
ALD catalyzes a readily reversible reaction and is not
generally considered to be an important regulatory en-
zyme, it may exert significant metabolic control in vivo
p
c
spinach leaves (Kr u¨ ger and Schnarrenberger, 1983) and
germinated P. vulgaris seeds (Botha and OÕKennedy,
1989) do not bind to phosphocellulose, thereby enabling
easy separation from the corresponding ALDc.
since: (i) moderate reductions in ALD activity by anti-
p
sense techniques markedly inhibited photosynthetic CO2
fixation and growth of transgenic potato plants (Haake
et al., 1998), and (ii) the in vitro activity of several puri-
fied plant and animal ALDs is significantly modulated
by physiologically relevant concentrations of various
metabolite effectors (Botha and OÕKennedy, 1989;
Moorhead and Plaxton, 1990; Hodgson and Plaxton,
2.2. Physical and immunological properties
SDS–PAGE of the final ALD preparation revealed a
single protein-staining polypeptide of about 40 kDa that
strongly cross-reacted with rabbit anti-(carrot ALD )-
c
IgG (Fig. 1A and B). Plant and animal ALD s exhibit
c
an identical subunit M of 40 kDa, whereas vascular
r
1
Furthermore, ALD has been suggested to physically
998; Akkerman, 1985; MacDonald and Storey, 2002).
plant ALD has a slightly smaller subunit M of about
p
r
38 kDa (Lebherz et al., 1984; Botha and OÕKennedy,
1989; Moorhead and Plaxton, 1990; Hodgson and Plax-
ton, 1998; Schwab et al., 2001). Homogeneity of the final
preparation was confirmed by non-denaturing PAGE
which generated a single protein-staining polypeptide
that co-migrated with ALD activity (Fig. 2). No cross-
reactivity was observed when an immunoblot of the
c
interact in vivo with other functionally related glycolytic
or gluconeogenic enzymes, including cytosolic ATP- and
PPi-dependent phosphofructokinases in carrot storage
roots (Moorhead and Plaxton, 1992), as well as cytosolic
FBPase during gluconeogenesis in the mammalian liver
and germinating R. communis endosperm (Moorhead

970
A. Lal et al. / Phytochemistry 66 (2005) 968–974
Table 1
Purification of cytosolic aldolase from 100 g of 30-h germinated mung beans
Step
Volume (ml)
Activity (U)
Protein (mg)
Specific activity (U/mg)
Purification (fold)
Yield (%)
Clarified extract
Acid step (pH 5.5)
120
110
7
35
35
2510
1000
120
0.014
0.035
0.15
–
–
2.5
11
100
53
25
21
(
NH
4
)
2
SO
4
fractionation
Phosphocellulose chromatography
18.5
8.8
7.4
12
0.70
0.49
12.6
15.1
893
1071
a
DEAE-cellulose chromatography
0.5
a
Concentrated pooled fractions.
Fig. 1. SDS–PAGE and immunoblot analysis of ALD
nated mung beans. (A) SDS–PAGE (12% separating gel) of 5 lg of
purified mung bean ALD stained with Coomassie Blue R-250. (B)
Immunoblot analysis was performed using affinity-purified rabbit anti-
carrot storage root ALD )-IgG (Moorhead and Plaxton, 1990).
c
from germi-
c
Fig. 2. Non-denaturing PAGE of purified mung bean ALD . The
c
electrophoresis was carried out in a 7.5% (w/v) resolving gel. (A)
Protein staining was performed with Coomassie Blue R-250. (B)
Distribution of ALD activity; activity was determined as described in
the Materials and Methods. The protein-stained lane contains 3 lg of
protein, whereas the lane used to detect ALD activity contains 7 lg of
protein. O, origin; TD, tracking dye front.
(
c
Immunoreactive polypeptides were detected using an alkaline phos-
phatase-conjugated secondary antibody; phosphatase staining was for
4
min at 30 ꢀC. Lane 1 contains 50 ng of purified mung bean ALD
whereas lane 2 contains 7.5 lg of the unbound protein fraction eluting
from the phosphocellulose column. The indicated M is based upon the
c
,
r
mobility of SDS–PAGE protein standards. O, origin; TD, tracking dye
front.
mentioned results demonstrate that the purified mung
bean ALD corresponds to ALD rather than ALD .
c
p
unbound protein fraction eluting from the phosphocel-
lulose column was probed with the anti-(carrot
ALD )-IgG (Fig. 1B, lane 2). The same anti-(carrot
The native M of mung bean ALD was estimated to
r c
be 160 kDa by gel filtration FPLC of the final prepara-
tion on a calibrated Superose 6 HR 10/30 column. Thus,
as with all other plant and animal ALD s studied to
c
ALD )-IgG specifically cross-reacted with ALD , but
c
c
c
not ALD , from spinach leaves (Moorhead and Plaxton,
p
date, the native mung bean enzyme appears to exist as
a homotetramer.
1
990).
The purified ALD was relatively heat stable, losing
0
5
%, 10% and 25% of its activity when incubated for
min at 55, 60 and 65 ꢀC, respectively. Heat stability ap-
2.3. Kinetic properties
pears to be a characteristic shared by all plant ALD s
c
2.3.1. Effect of temperature
The temperature coefficient (Q ) for the reaction
examined to date (Lebherz et al., 1984; Botha and
OÕKennedy, 1989; Moorhead and Plaxton, 1990; Hodg-
son and Plaxton, 1998; Schwab et al., 2001). By con-
trast, the heat-labile plant ALDp is completely
inactivated by similar heat treatments (Lebherz et al.,
1
0
catalyzed by mung bean ALD was 1.7. An Arrhenius
c
plot was linear over the range 4–40 ꢀC (Fig. 3). The
activation energy (E ) for the enzyme was calculated
from the Arrhenius equation to be 9.2 kcal/mol active
site.
a
1
984; Schwab et al., 2001). Taken together, the afore-

A. Lal et al. / Phytochemistry 66 (2005) 968–974
971
Table 2
Kinetic parameters for substrates of cytosolic aldolase from germi-
nated mung beans
-
-
-
-
0.6
0.8
1.0
À1
À1
À1
)
Substrate
Vmax (lmol
substrate
kcat (s
)
K
m
(lM)
kcat/K
m
(s lM
cleaved/min/
mg protein)
v
FBP
SBP
15
12.5
40
33.3
16.7
16.7
2.4
2.0
All values are the mean of at least three independent determinations
and are reproducible to within ±10% (SE) of the mean value.
1.2
3
.25
3.30
3.35
3.40
3.45
3.50
3.55
results imply that ALD might play a bifunctional role
c
-1
1
000 (K
T
)
in catalyzing the aldol cleavage of FBP and SBP in the
cytosol of the germinated mung bean. While the bifunc-
tional role of class I ALD for the reversible FBP and
p
Fig. 3. Arrhenius plot (logv versus 1000/T) for the effect of temper-
ature on aldolase activity. Assay conditions are described in the
experimental section. The rate of the reaction (v) has been expressed as
the rate of absorbance decrease per min.
SBP condensation in the reductive pentose-P cycle of
vascular plants is well known (Flechner et al., 1999), this
has yet to be firmly established for plant ALD . Neither
c
fructose-1-P nor fructose-6-P (10 mM each) served as
substrates for mung bean ALD . This was also reported
for purified carrot ALDc (Moorhead and Plaxton,
2
.3.2. Effect of pH
Similar to ALD s from other plant tissues (Lebherz
c
c
1
990), but differs from pea, wheat, and corn leaf ALDcs,
et al., 1984; Schnarrenberger and Kr u¨ ger, 1986; Botha
and OÕKennedy, 1989; Moorhead and Plaxton, 1990;
Hodgson and Plaxton, 1998; Schwab et al., 2001) the
as well as castor bean ALD , which all showed activity
c
at high fructose-1-P concentrations (Schnarrenberger
and Kr u¨ ger, 1986; Hodgson and Plaxton, 1998).
purified ALD demonstrated a broad pH/activity profile
c
with a maximum occurring at about pH 7.5 (results not
shown). All subsequent kinetic studies were carried out
at pH 7.5.
2.6. Metabolite effectors
A variety of compounds were tested for effects on
mung bean ALD activity at subsaturating (20 lM)
2
.4. Effect of cations
c
FBP. The following substances had no effect (±10% con-
trol rate) on enzyme activity: dithiothreitol, Pi, P-enol-
pyruvate, 3-P-gluconate, citrate, glucose, glucose-1-P,
glucose-6-P, fructose, ribose, arabinose, adenosine and
ALD activity was assayed in the absence and pres-
c
ence of 1 mM MgCl or 1 mM EDTA. No difference
2
in activity was detected with or without MgCl2 or
EDTA. Thus, as is the case with ALDs from other
eukaryotic sources, the mung bean ALD is a class I
sucrose (5 mM each); and fructose-2,6-P (50 lM). None
2
of the compounds tested served as activators. However,
ribose-5-P, AMP, ADP, and particularly ATP were
effective inhibitors (Table 3). Inhibition by ribose-5-P,
ADP and AMP was further evaluated and uniformly
determined to be competitive (Fig. 4) with corresponding
c
ALD (non-metal-requiring).
2
.5. Substrate specificity
Similar to other plant ALD s (Botha and OÕKennedy,
K values of 2.2, 3.1 and 7.5 mM, respectively.
i
c
1
989; Moorhead and Plaxton, 1990; Hodgson and Plax-
Notably, the presence of physiological concentrations
of ATP (2 mM) (Kobr and Beevers, 1971; Podest a´ and
Plaxton, 1991; Siegnthaler and Douet-Orhant, 1994)
markedly influenced FBP binding by the enzyme such
ton, 1998) the mung bean enzyme catalyzed the aldol
cleavage of FBP and SBP. Table 2 shows the Vmax, kcat
K , and specificity constants (k /K ) obtained with
,
m
cat
m
FBP and SBP as substrates. Notable are the relatively
low catalytic-centre activities for FBP and SBP, respec-
tively (Table 2). In both cases, Michaelis–Menten satu-
ration kinetics were observed. Although the Km values
were equivalent, the Vmax, kcat, and corresponding spec-
ificity constant values with FBP were slightly (20%)
greater than those obtained with SBP. In contrast, plant
ALD generally exhibit significantly lower K (SBP) val-
that Vmax was lowered by about 2-fold, K (FBP) in-
m
creased by about 4-fold, and the FBP saturation plot
shifted from hyperbolic to sigmoidal (Fig. 5). This was
not an artifact of the coupled assay system since the
À1
addition of extra coupling enzymes (5 U mL each of
triose-P isomerase and glycerol-3-P dehydrogenase) or
0.1 mM NADH at the completion of each assay had
no influence on the degree of ALD inhibition that
p
m
c
ues than their corresponding K (FBP) values (Kr u¨ ger
m
and Schnarrenberger, 1983; Flechner et al., 1999). Our
was observed in the presence of 2 mM ATP. Hill plots
of the FBP saturation kinetic data obtained in the

972
A. Lal et al. / Phytochemistry 66 (2005) 968–974
Table 3
Influence of several metabolites on the activity of purified ALD
germinated mung beans
c
from
1
1
1
4
2
0
8
6
4
2
0
Addition
Relative activity (%)
m
No additions (K = 17 M, h = 1.0)
5
5
5
2
mM Ribose-5-P
mM AMP
mM ADP
49
76
60
6
+2 mM ATP (S0.5 = 60 µM, h = 2.6)
mM ATP
1.2
No additions
0.8
0.4
Standard assay conditions were used except that the FBP concentra-
tion was subsaturating (20 lM). Activities are expressed relative to the
control determined in the absence of any additions and set at 100%. All
values are the mean of at least 3 independent determinations and are
reproducible to within ±10% (SE) of the mean value.
-v
0
.0
-0.4
0.8
-1.2
mxa
V
v
+
2 mM ATP
2.0
-
1.0
1.5
log[FBP] (µM)
0
20 40 60 80 100 200
400
600
800 1000
(
(
(
A) Ribose-5-P
inhibition
[
FBP] (µM)
0.45
Fig. 5. Influence of ATP on the FBP saturation kinetics of purified
ALD from germinated mung beans. The inset shows the Hill plot of
c
0.30
the data.
spect to FBP binding. To the best of our knowledge this
is the first time that any ALD has been demonstrated to
exhibit sigmoidal FBP saturation kinetics. As ALD cat-
alyzes a reaction that is close to equilibrium in vivo
(Plaxton, 1996), the direction of flux through the enzyme
will largely be dictated by FBP: triose-P mass action ra-
0.15
B) ADP
inhibition
0.30
tios. However, our results imply that ALD may partic-
c
ipate in the control of glycolytic flux in the cytosol of
germinated mung beans. Inhibition of the enzyme by
elevated cytosolic ATP concentrations may exert signif-
icant respiratory control in vivo. Further studies are re-
0.15
quired to fully assess the potential role of ALD in the
c
control of plant glycolysis and respiration.
C) AMP
0.30
inhibition
3
. Experimental
3
.1. Chemicals and plant material
0.15
Biochemicals, coupling enzymes, and protein stan-
dards were purchased from Sigma–Aldrich Corporation.
All other reagents were of analytical grade and were sup-
plied by BDH Chemicals or Sisco Research Laboratories.
All solutions were prepared using double distilled water.
Mung beans (Vigna radiata, cv. Wilczek) were gener-
ously provided by the Department of Genetics and Plant
Breeding, Institute of Agricultural Sciences, Banaras
Hindu University, and imbibed for 8 h in water at
-
0.05
0.00
0.05
1
0.10
-
1
/[FBP] ( M)
Fig. 4. Inhibition of mung bean ALDc by ribose-5-P (A), ADP (B),
and AMP (C). Enzyme activity was determined at varying concentra-
tions of FBP in the presence of 0 mM (d), 3 mM (j) or 6 mM (m) in
case of (A); 0 mM (d), 4 mM (j) or 6 mM (m) in case of (B) and
2
5 ꢀC, and germinated in the dark at 30 ꢀC by spreading
0
mM (d), 5 mM (j) or 10 mM (m) in case of (C). Activity is
À1
À1
the imbibed seeds over moist filter paper on a moist sand
bed. For enzyme purification, 30-h germinated seeds
(containing radical and cotyledons) were used.
expressed as lmol FBP cleaved min mg protein.
presence and absence of 2 mM ATP are shown in Fig. 5
(
inset). Corresponding Hill coefficients (h) were deter-
mined to be 2.6 and 1.0 in the presence and absence of
mM ATP, respectively. These data indicate that the
3.2. Enzyme and protein assays, and kinetic studies
2
ATP binding promotes a conformational change caus-
ing the enzyme to exhibit positive cooperativity with re-
The ALD reaction was routinely coupled with the
triose-P isomerase and glycerol-3-P dehydrogenase

A. Lal et al. / Phytochemistry 66 (2005) 968–974
973
reactions and assayed in the forward (glycolytic)
direction at 30 ꢀC by continuously monitoring NADH
utilization at 340 nm using a Spectronic 1001 spectropho-
tometer. Standard assay conditions for ALD were:
3.4.3. Phosphocellulose chromatography
The dialysate was absorbed at 0.5 ml/min onto a col-
umn (1.5 · 21 cm) of phosphocellulose (Whatman P11)
pre-equilibrated with buffer B. The column was washed
until the A280 decreased to baseline, then ALD eluted as
a single peak with buffer B containing 1 mM FBP (frac-
tion size 3 ml).
5
1
0 mM Tris–HCl (pH 7.5), 1 mM FBP, 0.1 mM NADH,
0 U triose-P isomerase and 1 U glycerol-3-P dehydroge-
nase in a final volume of 1 ml. Coupling enzymes were de-
salted before use. All assays were initiated by the addition
of enzyme preparation and were corrected for NADH
oxidase activity, when necessary, by omitting FBP from
the reaction mixture. One unit of activity is defined as
the amount of enzyme resulting in cleavage of 1 lmol
substrate/min at 30 ꢀC, equivalent to the oxidation of
3.4.4. DEAE-cellulose chromatography
Pooled peak phosphocellulose fractions were ab-
sorbed at 0.5 ml/min onto a column (1 · 7 cm) of
DEAE-cellulose (Sigma–Aldrich Corporation) pre-
equilibrated with buffer B. The column was washed
with buffer B containing 50 mM KCl until the A280
decreased to baseline, and ALD eluted with buffer B
containing 100 mM KCl (fraction size 1 ml). Pooled
peak activity fractions were concentrated to about
0.5 ml by dialysis against solid sucrose, divided into
20 ll aliquots, and stored at À20 ꢀC. The purified en-
zyme was stable for at least 3 weeks when stored at
À20 ꢀC.
2
lmol NADH in the case of FBP and of 1 lmol NADH
in the case of sedoheptulose-1,7-P (SBP). In all cases the
2
rate of reaction was proportional to concentration of en-
zyme assayed and remained linear with respect to time.
Apparent K , S0.5, and h (Hill coefficient) values were
m
determined from the Hill equation using Sigma Plot.
Competitive inhibition constants (K values) were deter-
i
mined from Dixon plots. All parameters are the mean
of triplicate determinations from three independent prep-
arations of the purified enzyme and are reproducible to
within ±10% SE of the mean value.
3.5. Electrophoresis and immunoblotting
Protein concentration was estimated by the Lowry
et al. (1951) procedure using Bovine Serum Albumin
(BSA) as the standard protein. Specific activity is ex-
pressed as enzyme U/mg protein.
Non-denaturing and SDS–PAGE, subunit M esti-
r
mations, and immunoblotting were performed using
the Bio-Rad mini-gel apparatus as previously described
(Moorhead and Plaxton, 1992; Hodgson and Plaxton,
1998). Immunological specificities were confirmed by
3
.3. Buffers used in aldolase purification
performing immunoblots in which rabbit pre-immune
serum was substituted for the affinity-purified rabbit
anti-(carrot ALD )-IgG. To detect ALD activity fol-
lowing non-denaturing PAGE, a lane was sliced into
2 mm segments and each slice was incubated in
Buffer A: 50 mM Tris–HCl (pH 8.5) containing
0 mM b-mercaptoethanol. Buffer B: 20 mM Tris–HCl
c
1
(
pH 7.3) containing 10 mM b-mercaptoethanol.
0
.15 ml of 50 mM Tris–HCl (pH 7.5), containing
0.1 mM-NADH, 10 units of triose-P isomerase and
of glycerol-3-P dehydrogenase (final volume
3
.4. Enzyme purification
1
U
All purification procedures were carried out at 4 ꢀC.
1 ml). Relative ALD activity was determined by moni-
toring the decrease in A340 following a 15 min incuba-
tion at 30 ꢀC.
Germinated mung beans (100 g) were homogenized
using Waring blender in 1 volume of buffer A, filtered
through four layers of cheesecloth, and centrifuged at
2
1,000g for 15 min at 4 ꢀC.
3.6. Estimation of native molecular mass
3
.4.1. Acidification
The clarified extract was adjusted to pH 5.5 with 7 M
This was done by FPLC on a Superose 6 HR 10/30
column, using 100 ll sample volumes and 25 mM
potassium phosphate (pH 7.5) containing 15% (v/v)
glycerol, 200 mM KCl, and 10 mM b-mercaptoethanol
as the column buffer. Fractions (0.3 ml) were collected
with a flow rate of 0.2 ml/min and assayed for protein
acetic acid, gently stirred for 5 min, and centrifuged at
1,000g for 5 min. The supernatant was adjusted to
pH 7.5 with 15 M NH OH. This treatment removed pig-
2
4
mented material which otherwise interfered with subse-
quent purification steps.
and/or ALD activity. The native M of ALD was deter-
r
mined from a plot of KD (partition coefficient) versus
log Mr for the following protein standards: BSA
(66 kDa), rabbit muscle glyceraldehyde-3-P dehydroge-
nase (145 kDa), horse liver alcohol dehydrogenase
(150 kDa), rabbit muscle aldolase (160 kDa) and cata-
lase (232 kDa).
3
.4.2. Ammonium sulphate fractionation
Proteins precipitating in the range 37–50% (satura-
tion) ammonium sulphate were collected by centrifuga-
tion, dissolved in buffer A, and dialyzed against buffer
A for 2 h with four buffer changes (500 ml each).

974
A. Lal et al. / Phytochemistry 66 (2005) 968–974
Acknowledgements
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.
Protein measurement with the folin phenol regent. J. Biol. Chem.
93, 265–275.
1
Financial assistance of the University Grants Com-
mission, New Delhi (Junior and Senior Fellowships to
A.L.) is gratefully acknowledged. We are thankful to
Prof. O.P. Malhotra, Department of Biochemistry, Fac-
ulty of Science, Banaras Hindu University, for helpful
discussions.
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glyceraldehyde 3-phosphate dehydrogenase from germinating
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