Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates

J. Biosci., Vol. 1, Number 1, March 1979, pp. 13-25. © Printed in India.
Studies on glutamine synthetase. Purification of the enzyme from
mung bean (Phaseolus aureus) seedlings and modulation of the
enzyme-antibody reaction by the substrates
S. SEETHALAKSHMI and N. APPAJI RAO
Department of Biochemistry, Indian Institute of Science, Bangalore 560012
MS received 6 November 1978
Abstract.. Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2)
from Phaseolus aureus (mung bean) seedlings was purified to homogeneity by
ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex
G-200 gel filtration and affinity chromatography on histidine-Sepharose. The
enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of
identical subunits with an approximate subunit molecular weight of 50,000. Hyper-
bolic saturation curves were obtained with the substrates, glutamate, ATP and
hydroxylamine.
Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely
inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate
and ATP, prior to the addition of the antibody, partially protected the enzyme
against inhibition. The Km values of this enzyme-antibody complex and the native
enzyme were identical (glutamate, 2 . 5 m M ; ATP, 1 m M ; hydroxylamine, 0·5 mM).
The Km values of the partially inhibited enzyme (the enzyme pretreated with anti-
body prior to the addition of substrates) were 2-fold higher than those of the native
enzyme. These results suggested that the substrate-induced conformational changes
in the enzyme were responsible for the protection against inhibition of the enzyme
activity by the antibody.
Keywords. Mung bean; glutamine synthetase; antibody interactions; Phaseolus
aureus.
Introduction
Earlier results from this laboratory (Seethalakshmi, 1979) showed that the activity
of mung bean glutamine synthetase was regulated by the amino acid and nucleo-
tide end products of glutamine metabolism, in a manner similar to that reported
in Escherchia coli by Stadtman (1973). The ?-glutamyl transferase reaction catalysed
by this enzyme proceeded by a ping pong mechanism (Seethalakshmi et al., 1977).
Glutamine synthetase, catalyses a ter ter biosynthetic reaction shown below, and
13

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14 S. Seethalakshmi and N. Appaji Rao
therefore, serves as an excellent model for understanding the substrate-induced
conformational changes. In this study, the enzyme-antibody reaction has
M e 2 +
Glutamate + ATP + NH4+ g Glutamine + ADP + P4
been used to probe the conformational changes in the enzyme.
Materials and methods
Materials
The following chemicals were purchased from Sigma Chemical Company,
St. Louis, MO, USA : imidazole, ß-mercaptoethanol, ATP, diethylaminoethyl
(DEAE)-cellulose, sodium L-glutamate, hydroxylamine hydrochloride, EDTA,
Coomassie brilliant blue R, sodium dodecyl sulphate, ?, ?' methylene bis acryl
amide, ?, ?, ?', N' tetramethylene
diamine
(TEMED),
riboflavin,
tris
(hydroxymethyl) aminomethane, bovine serum albumin, chymotrypsin, bovine
liver glutamate hydrogenase, catalase, Escherichia coli ß-galactosidase, l-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride, hexamethylenediamine,
ferritin and the amino acids. Sepharose-4B, Sephadex G-200 and Sephadex
G-25 were purchased from Pharmacia, Uppsala, Sweden. Agar and Freund's
complete adjuvant were obtained from Difco Lab., Detroit, MC, USA. All
other reagents used in this investigation were of the analytical reagent grade.
Mung bean seeds were purchased from the local market.
Methods
Enzyme assay: Glutamine synthetase activity was measured by estimating the
?-glutamylhydroxamate formed in the reaction mixture (Rowe et al., 1970). The
reaction mixture (1·0 ml) contained the following components : 80 mM imida-
zole hydrochloride buffer (pH 7·2), 10 mM MgCl2, 10 mM ß-mercaptoethanol
100 mM glutamate, 10 mM ATP, 10 mM hydroxylamine (freshly neutralised to
pH 7 ·2) and an appropriate amount of the enzyme. After incubation at 37° C for
15min, the reaction was stopped by the addition of 1·5 ml of ferric chloride
reagent. The precipitated proteins were removed by centrifugation. The absor-
bance of the supernatant solution was measured at 535 nm in a Pye Unicam
SP-500 spectrophotometer.
Preparation of histidine-Sepharose: Aminohexane-Sepharose (20 ml), prepared
as described by March et al. (1974), was washed thoroughly with glass-distilled
water. Histidine hydrochloride (2 mmol, pH 4·5) was added to the gel and the
mixture was gently stirred. The carboxylate moiety of histidine was coupled to
the amino group of the gel by the addition of 0·1 ? carbodiimide and the mix
ture was gently stirred for 24 h at 4° C. This preparation was then washed with
water and stored at 4°C.
Protein estimation: Protein content was determined according to the method
of Lowry et al. (1951) using bovine serum albumin as the standard,

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Mung bean glutamine synthetase 15
Disc gel electrophoresis: Analytical polyacrylamide gel electrophoresis in 5%
gel was performed as described by Davis (1964). Electrophoresis of the sodium
dodecyl sulphate dissociated proteins was carried out as described by Weber
and Osborn (1969).
Double diffusion test: The antibody to the enzyme was raised by repeated injec
tion of mung bean glutamine synthetase (step 6, table 1) into a male rabbit. Anti
serum was obtained by centrifuging the blood collected by puncturing the margi
nal ear vein. The ? globulin fraction isolated by ammonium sulphate fractiona
tion of the antiserum was used in this study. The Ouchterlony plates were pre
pared with 1·5% agar in 0·01 ? sodium phosphate buffer (pH 7·5) containing
0·14 ? sodium chloride and 0·02% sodium azide. After placing the antigen
and antibody in the wells cut into agar plates, the plates were developed in a
humid chamber at 0 5° C for 24 h.
Purification of the enzyme: All operations were carried out at 0 4° C and all
centrifugations at 12,000 g for 10 min in a Sorvall RC 2B centrifuge.
Mung bean seedlings (800 g; germinated for 24 h at 37° C) were washed and
homogenised for 2 min at 30 sec intervals in 500 ml of 0 · 1 ? imidazole hydro
chloride buffer (pH 7·2) in a precooled Waring blendor. The
homogenate was
squeezed through cheese cloth and centrifuged. The supernatant solution was
designated as the crude extract (step 1, table 1). Protamine sulphate (2% solution)
was added with constant stirring to the crude extract such that the protamine
sulphate to protein ratio was 1 : 5. After 15 min, the precipitated nucleoproteins
were removed by centrifugation (step 2). Solid ammonium sulphate was added
to the supernatant solution to obtain 0·45 of saturation. After
10
min, the preci
pitate was removed by centrifugation. The supernatant fraction was raised to 0 · 6
of saturation by a further addition of solid ammonium sulphate and the preci
pitate was dissolved in a small volume of 0·02 ? imidazole hydrochloride buffer
Table 1. Purification of glutamine synthetase from the germinated seedlings of
Phaseolus aureus (mung bean).
* µmol ? glutamylhydroxamate formed/min.

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16 S. Seethalakshmi and N. Appaji Rao
(pH 7·2) and desalted by passage through a Sephadex G-25 column (1·5 × 80 cm)
previously equilibrated with 0·02 ? imidazole hydrochloride buffer (pH 7·2)
(step 3). Alumina C? gel was prepared as described by Willstatter and Kraut
(1923). The enzyme fraction was added to alumina C? gel (0·4mg of gel per
mg of protein) and gently stirred for 15 min. The
supernatant solution obtained
on centrifugation was designated as alumina C? gel supernatant (step 4).
The enzyme fraction (420
mg) was loaded onto a DEAE cellulose column
(1·4 × 65 cm) equilibrated with 0·02 ? imidazole hydrochloride buffer (pH 7·2)
containing 10mM ? mercaptoethanol, 0 · l m M EDTA, 0·1 mM ADP and
0·075 ? sodium chloride. The column was washed with the same buffer until
the absorbance of the eluate at 280 nm was reduced to 0·05. ? linear sodium
chloride gradient (0·075 0·2 M) with a total volume of 500
ml was applied.
Fractions (2 ml) were collected, and assayed for enzyme activity. The enzyme
was eluted between 0· 15 0·19 ? sodium chloride. The active fractions were
pooled and the enzyme was precipitated by the addition of solid ammonium sul
phate to 0·60 of saturation. The
precipitate was dissolved in 0·02 ? imidazole
hydrochloride buffer (pH 7·2) containing 10 mM ? mercaptoethanol, 0 · l m M
EDTA, 0·1 mM ADP and 0·1 ? sodium chloride (step 5).
The enzyme obtained from the previous step was applied on a Sephadex G 200
column (1·4 × 65 cm) which had been previously equilibrated with 0 · 02 ?
imidazole hydrochloride buffer (pH 7·2) containing 10 mM ? mercaptoethanol,
0·1 mM EDTA, 0 · l m M ADP and 0·1 ? sodium chloride. The enzyme
was
eluted with the same buffer. Fractions (1 ml) were collected and assayed for
enzyme activity. The enzyme activity appeared immediately after the void
volume. The
fractions with high specific activity (> 4 · 5) were pooled and used
for further purification (step 6).
Histidine Sepharose was packed into a column (1 × 10 cm) and equilibrated
with 0·02 ? imidazole hydrochloride buffer (pH 7·2) containing 10
mM
ß mercaptoethanol, 0·1 mM ADP, 0·1 mM EDTA and 0·05 ? sodium chloride.
The enzyme fraction from step 6 was diluted with the buffer to reduce the sodium
chloride concentration to 0·05 ? and loaded onto the column. The
column was
washed with the equilibrating buffer till the absorbance of the eluants was less than
0·05. The
enzyme was eluted from the column with the same buffer containing
50 mM glutamate, 2 mM ATP and 2 mM magnesium chloride. Fractions (1 ml)
were collected, assayed for enzyme activity and pooled. A
summary of the puri
fication procedure is given in table 1, This procedure resulted in a 1000 fold
purification with 3% recovery of the enzyme activity.
One unit of enzyme activity is defined as the amount of enzyme required to
produce 1 µmol of ? glutamylhydroxamate per min at pH 7·2 and 37° C.
Results
Criteria of purity
The purified enzyme gave a single band when subjected to electrophoresis in 5%
gel in Tris glycine buffer (pH 8·6) (figure 1). The purity of the enzyme prepa
ration was confirmed by the Ouchterlony double diffusion test. Figure 2 shows
a single antigen antibody precipitin band. Two faint bands, in addition to the

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Mung bean glutamine synthetase
17
Figure 1. Polyacrylamide gel electrophoresis of mung bean glutamine synthetase
Electrophoresis was carried out in 5% acrylamide gel in 0·1 ? Tris glycine buffer
(pH 8·6) for 1 h. The migration of the protein (50 µg) was from top (cathode)
to the bottom (anode). A single band was obtained even when 100 µg of the protein
was used.
Figure 2. Ouchterlony double diffusion analysis of mung bean glutamine synthe
tase against its anitbody.
The centre well contained the ??globulin fraction of the rabbit antiserum. Well 1:
enzyme obtained from step 6 (table 1). Well 2: enzyme obtained from step 7
(table 1).
Figure 3. Sodium dodecyl sulphate gel electrophoresis of mung bean glutamine
synthetase.
Mung bean glutamine synthetase (50 µg) was subjected to denaturation in the
presence of sodium dodecyl sulphate and ß-mercaptoethanol and the electro-
phoresis was carried out according to the method of Weber and Osborn (1969).

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Mung bean glutamine synthetase 19
major precipitin band, were observed when the enzyme from step 6 was used.
These bands are not clear in the photographs.
Molecular weight
The molecular weight of the enzyme was determined by gel filtration on Sepharose-
4B. The marker proteins used in this analysis were ß-galactosidase (520,000),
ferritin (480,000), catalase (240,000) and glutamate dehydrogenase (350,000).
From the standard curve (not shown), the molecular weight of mung bean gluta-
mine synthetase was calculated to be 775,000 ± 25,000.
Subunits
The subunit composition of the enzyme was determined by sodium dodecyl
sulphate polyacrylamide gel electrophoresis. Bovine serum albumin, cyto-
chrome c, ovalbumin, chymotrypsin were used as the marker proteins. Figure 3
depicts the migration of the single subunit of mung bean glutamine synthetase.
From the calibration curve (not shown), the molecular weight of the single subunit
was found to be approximately 50,000.
Properties of the enzyme
The enzyme reaction was linear with time upto 15 min and enzyme concentration
up to 20µ g. The enzyme was optimally active at pH 7·2 and 45° C. Hyperbolic
saturation curves were obtained with glutamate, ATP and hydroxylamine. The
Km values for glutamate, hydroxylamine and ATP, calculated from the linear
Lineweaver-Burk plots, were 2·5, 0·5 and 1 mM, respectively (data not shown).
Inhibition of mung bean glutamine synthetase activity by its antibody
Figure 4a depicts the inhibition of mung bean glutamine synthetase activity on
increasing the concentration of the antibody. Amounts of antibody greater
than 200/µg almost completely inhibited the enzyme activity. A value of 0·01
was obtained for the intercept on the ?-axis in the plot of reciprocal of per cent
inhibition against reciprocal of antibody concentration (inset figure 4), indicating
complete inhibition. Values greater than 0·01 would indicate partial inhibition.
Protection of glutamine synthetase activity against inhibition by antibody
It can be seen from table 2 that glutamate in the presence of ATP affords partial
protection against inhibition by antibody. However, these substrates when
present alone in the preincubation medium were not effective in protecting the
enzyme against inhibition. Hydroxylamine was not effective either when present
alone or in combination with either ATP or glutamate. The enzyme pretreated with
glutamate and ATP is designated as the " protected, enzyme ".
The protected enzyme was preincubated with varying amounts of antibody
indicated in figure 4 (curve b). The maximum amount of inhibition of the
protected enzyme was only 70%.
In view of the protection afforded by saturating concentrations of glutamate
and ATP, it was of interest to determine the minimum concentration of glutamate
and ATP required for protection. The results shown in tables 3 and 4 suggested
that concentrations of ATP (0·05mM) and glutamate (2mM) less than their Km
values (1 and 2·5 mM, respectively) were able to maximally protect the enzyme
against inhibition by the antibody. The protection could be due to a conformational
J.B.–3

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20 S. Seethalakshmi and N. Appaji Rao
Figure 4. Effect of antibody on the activity of the native and the protected mung
bean glutamine synthetase.
(a) The enzyme (5 6 µg) was preincubated with varying amounts of ??globulin
fraction for 5 min at 37° C and the reaction was started by the addition of
the saturating concentrations of the substrates (glutamate, 100
mM; hydroxyl
amine, 10mM; ATP, 10mM).
The velocity was determined by estimating
the ? glutamylhydroxamate formed in the reaction mixture, (b) The enzyme
(5 6 µg) was preincubated with saturating concentrations of glutamate (100 mM)
and ATP (10 mM) for 5 min at 37° C. This enzyme was designated as the protected
enzyme. This was followed by the second pre incubation with varying amounts of
??globulin fraction for 5 min at 37° C. The reaction was started by the addition
of saturating concentration of hydroxylamine (10 mM). The
velocity was deter
mined by estimating the ? glutamylhydroxamate formed in the reaction mixture.
Inset: Double reciprocal plot of per cent inhibition of the enzyme activity versus
the concentration of antibody.
( ? ? ) Protected enzyme ; (-·-·-) Native enzyme. In: per cent
inhibition.
change in the enzyme resulting in decreased ability of the antibody to
interact with the enzyme or due to the possibility that antigen antibody complex
may be partially active. It
was,
therefore, of interest to determine the kinetic para
meters of the three enzyme species, viz., native, protected and inhibited glutamine
synthetase.
Comparison of kinetic parameters of the native, protected and the inhibited
glutamine synthetase
The ? values of the three forms of the enzyme, for glutamate, ATP and hydroxyl
m
amine were determined. The
concentrations of the antibody were chosen such
that the " p r o t e c t e d " and the inhibited enzymes had identical V
values.
max

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Mung bean glutamine synthetase 21
Table 2. . Protection by substrates of mung bean glutamine synthetase activity
against inhibition by its antibody.
* The enzyme activity in the absence of antibody.
** The enzyme preincubated with the antibody (150 µg) for 5 min at 37° C prior to a second
preincubation with the substrates.
The enzyme (5-6 µg) was preincubated separately with the saturating concentrations of
a. glutamate and ATP, b. ATP (10 mM), c. glutamate (100 mM), d. hydroxylamine
(10 mM), e. glutamate and hydroxylamine, f. hydroxylamine and ATP, for 5 min at
37° C. This was followed by a second preincubation with the antibody (150 µg) under
identical conditions. The reaction was started by the addition of substrate(s) which was
not present in the preincubation medium. The enzyme in the absence of antibody pre-
incubated as above served as the control and the activity was normalised to 100.
Table 3. Minimum concentration of glutamate required for protection of glutamine
synthetase against inhibition by antibody.
* Enzyme activity in the absence of antibody, normalised to 100. The enzyme
was preincubated with a mixture of ATP (10 mM, saturating) and glutamate (0·1 to
100 mM) for 5 min at 37° C followed by a second preincubation with the antibody
(50 µg). The enzyme activity was measured at saturating concentrations of gluta-
mate and hydroxylamine,

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22 S. Seethalakshmi and N. Appaji Rao
Table 4. Minimum concentration of ATP required for protection of glutamine
synthetase activity against inhibition by antibody.
* Enzyme activity in the absence of antibody normalised to 100. The
enzyme was
preincubated with a mixture of glutamate (100 mM) and ATP (0·05 mM 10 mM) for
5 min at 37° C prior to preincubation with the antibody (50 µg). The
enzyme
acti
vity was measured at saturating concentrations of ATP and hydroxylamine.
Figures 5, 6 and 7 depict the Lineweaver Burk plots for glutamate, ATP and
hydroxylamine, respectively. The K values of the inhibited enzyme for glutamate
m
(6·25 mM) was 2 · 5 fold higher than that for the native enzyme while the K values
m
for ATP and hydroxylamine were two fold greater than those for the native
Figure 5. Double reciprocal plot of velocity versus glutamate concentration.
( ··) Native enzyme; (5 6µg); ( ? ? ); Protected enzyme.
The enzyme preincubated with 2 mM glutamate and 10 mM ATP prior to the
addition of antibody (30 µg). Glutamate concentration was varied in the reaction
mixture from 2 50 mM and the reaction was started by the addition of saturating
concentration of hydroxylamine. ( p p ) The enzyme was preincubated with
the antibody for 5 min at 37° C followed by a second preincubation with glutamate
concentrations indicated in figures and ATP (10 mM). The reaction was started
by the
addition
of
saturating concentration of hydroxylamine,

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Mung bean glutamine synthetase
23
Figure 6. Double
reciprocal plot of velocity versus ATP concentration.
(- = - = -) Native e n z y m e ; (- O - O -) Protected enzyme.
The enzyme was preincubated with 100 mM glutamate and 0·1 mM ATP prior to
the addition of antibody (30 µg). ATP concentration was varied in the reaction
mixture from 0·5 10 mM and the reaction was started by the addition of saturating
concentration of hydroxylamine (10 mM). (- p- p-). The enzyme was
preincubated with the antibody for 5 min at 37° C followed by a second preincu
bation with ATP (concentrations indicated in figure 6) and glutamate (100
mM).
The reaction was started by the addition of saturating concentration of hydroxyl
amine.
Figure 7. Determination of K value for hydroxylamine.
m
(- = - = -) Native enzyme
;
(

?

?

)
Protected enzyme.
[The enzyme preincubated with saturating concentrations of glutamate and ATP
prior to the addition of antibody]. The
reaction
was started by the addition of
varying amounts of hydroxylamine indicated in the figure. ( - p
- p
- )
inhibited
enzyme. [The enzyme preincubated with the antibody prior to the addition of the
substrates]. The
reaction was started by the addition of varying amounts of hydro
xylamine indicated in the figure.

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