Indian Journal of Biochemistry & Biophysics
Vol. 38, October 2001 , pp. 335-34 1
Isolation and characterization of NADP+ -linked
isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)
P K Sri vastava* and 0 S Sin gh
Department of Biochemistry. Banaras Hindu Uni versity,
Varanasi 22 1 005, India
Received 24 April 2000; revised alld accepled 16 Jllly 200 1
NA DP+- li nked isocitrate dehydrogenase (E.C. I. I. 1.42) has been purified to homogeneity from germinating pea seeds.
The enzyme is a tetra meric protein (mol wt. about 146,000) made up of apparently identical monomers (subunit mol wI.
about 36.000). Thermal inacti vation of purified enzyme at 45° and 50°C shows simple fi rst order kineti cs. The enzyme
shows opti mum acti vity at pH range 7.5-S . Effect of substrate [SI on enzyme acti vity at di fferent pH (6.5-S) suggests th at
the proton behaves formally as an "uncompetitive inh ibitor". A basic group of the enzy me (site) is protonated in th is pH
range in the presence of substrate only. with a pKa equal to 6.7S. On successive dialysis against EDT A and phosphate
buffer. pH 7.S at O°C, yields an enzy mati cally inactive protein showing ki netics of thermal inactivati on identical to the
untreated (nati ve) enzyme. Maximum enzy me activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM ). Addit ion
of Z n ~+, Cd2+, C02+and Ca2+ions brings about part ial recovery. Other metal ions Fe2+, Cu2+and N i ~+ are ineffective.
NADP+-lin ked isoc itrate dehydrogenase (NADP+lin ked [CDH , EC 1. 1. 1. 42) has been stu died in many
prokaryoti c and euk aryotic organisms. It has been
purifi ed and characteri zed fro m several bacteri al and
ma mm ali an species l -7 . Although the presence of th is
enzy me in plant cytosoI8.9. 1O, mitochondri a l o and
chl oropl asts II is known there are onl y a few reports of
isolati on and characteri zati on of thi s enzym e fro m
plant tissues')· 12-15. We have isolated the enzy me fro m
germinating pea seeds and studi ed so me properti es,
whi ch have direct bearin g on the structural symm etry
characteri sti cs and parti al identi fication of the
co nstituents of the active site.
ethanol) was reflu xed over silver oxide and di still ed
twice before use. Other chemi cals were of anal ytica l
reagent grade. All soluti ons were prepared in double
disti lied water.
Plallllllaleria!
Pea seeds (Pis um sativlIIII ) (Azad-PI) obtained
from Department of Ag;-onomy lAS (B HU ) were
washed thoroughl y wi th double di still ed water and
soa ked fo r 12 hr, spread over moist bed of washed
sand fo r ge rm ination for 72 hr at 30°C and the
ge rminated seeds we re coll ected (1 50 g).
Extraction alld purificatioll of ell zyme
Matedals and Methods
Threo Ds(+)i socitric acid , NADP+ (di sodium salt),
tri chloroacetic acid, bovine serum albumin , FolinCiocalteau's phenol reagent, acrylamide, N,N- methylene bis acryl amide, ribofl avin , N,N,N,N-tetrameth ylethylene di amine, sodiumdodecyl-sulphate, 2-mercaptoethanol , tris(hydrox ymethyl ) amin omethane and
DEAE cellul ose (coarse mesh) were from Si sco
Research Laboratori es Pvt. Ltd. Bombay.
Di alysis tubing from Arthur H. Thomas,
Philadelphi a, P.A. US A, Bio-gel P200 from Bio-Rad
Laboratories, Richmond, Californ ia US A, ovalbu min ,
glyceraldehyde 3-phosphate dehydrogenase, catalase
and y-globulin from Sigma Chemicals Co, St Loui s,
USA were used. Commercial-rectifi ed spirit (95 %
*Author for correspondence
All operations were carri ed out at 0-4°C unl ess
stated otherwi se.
Preparatioll of acetolle p owder
Germin ated seeds were washed with cold doubl e
di still ed water and blotted dry with filter paper. The
seeds (ISO g) were homogenized in 300 ml precooled
acetone in waring blender fo r one min , filtered
through four layers of cheese cloth, excess liquid was
pressed out and the process was repeated twice with
the residual materi al keeping the volume of fresh
cooled acetone the same. The residue was pressed and
spread on filter paper at room temperature and
allowed to dry rapidly .
An average yield of 140 g of acetone powder from
150 g of germinating pea seeds was noted which was
next,mixed with 300 ml of 40 InM sodium phosphate
336
INDIAN J. BIOCHEM. BIOPHYS .. VOL. 38, OCTOBER 2001
buffer at pH 7.8 and stirred slowly for 30 min at O°C.
The suspension was centrifuged at 15000 rpm for 20
min and the clear supernatant solution was used .
Extraction with ethanol
To the above supernatant which was cooled to
- 10°C, precooled (-18 °C) ethanol (25mll 100 ml
extract) was added slowly with gentle stirring. The
mixture was allowed to stand for 5 min and
centrifuged at 15000 rpm for 10 min . The precipitate
was discarded. To the supernatant, more of precooled
ethanol (60 mill 00 ml of supernatant) was added.
After keeping at -18°C for 5 min, the suspension was
centrifuged at 15000 rpm for 5 min, the precipitate
was di sso lved in phosphate buffer (60 mM, pH 7.8)
and again centrifuged to get a clear solution (18 ml).
Allllllonilllli sulphate fractionation
Solid ammonium sulphate was added gradually to
the above supern atant (18 ml) with stirring to bring it
to 50% saturation. The pH was maintained constant at
7.8 with 10-fold dil uted liquid ammonia and the
solution was centrifuged. The enzymically inactive
prec ipitate was di scarded and the supernatant was
brought to approximately 75 % saturation with solid
ammonium sulphate. The stirring was continued for
30 min and suspension was centrifuged at 15000 rpm
for 20 min. The e nzymically active precipitate was
collected and suspended in 60 mM sodium phosphate
buffer, pH 7.8 and centrifuged to obtain a clear
solution (5.4 ml ).
Dialysis
The clear solution obtained was dialysed against
precooled phosphate buffer (60 mM, pH 7.8) at 0-4°C
with 3-5 repeated change of same buffer till complete
removal of ammonium ions was confirmed on
checking with Nessler's reagent.
DEAE cellulose colulIln chromatography
The dialysed solution was loaded onto DEAEcellulose column (2.5x25 em) previously equilibrated
with 60 mM phosphate buffer pH 7.8, and at a flow
rate of 45-50 mllhr, and using the same buffer for
elution, fractions of about 60 ml were collected.
Fractions showing NADP+-linked isocitrate dehydrogenase activity were pooled together and the protein
was precipitated at 0-90% ammonium sulphate
saturation. col\ected by centrifugation and dissolved
in a final volume (1 .3 ml) of phosphate buffer. The
elution profile is shown in Fig 1.
20
-
Z
16
12
<II
!::
z
=>
~
~
....
u
<t
8
4
0
- ' 2 ·0
I
1·5 _
;/
20
Q,
E
1.2 z
W
....
0
~o
co
0·8
a.
0·4
30
40
50
50
70
80
ELUTI ON VOLUME (ml I
Fig. I-NADP+-linked isocitrate dehydrogenase o f germin:lIing
pea seeds after DEAE-cellulose column chromatog raphy at 0-4°C.
[The enzyme was e luted with 60 mM phospha te buffer, pH 7. 8.
Each fracti on was tested for ICDH activity (0-0) and protein ,
(.-.)I
Polyacrylamide gel electrophoresis (PAGE)
. PAGE was carried out at pH 8.3 according to
17
Reisfeld et a1. • SDS-PAGE was carried out at pH 7.0
by the method of Weber and Osborn 18. Protein bands
were stained with Coomassie blue.
Gel filtrat ion
The molecular weight of the enzyme was
determined by the method of Ezzeddine and AIKhalidi 19 using Biogel-P2oo (0 .5-0.6 g) column
(2.5xl.5cm) and 60 mM sodium phosphate buffer, pH
7.8. Protein was estimated by the method of Lowry
16
et al. . The dilution factors of standard proteins and
pea seed enzyme were calculated by the followin g
formula:
Dilution factor=
Concentration of protein in the solution in the column
Concentration of protein in the filtrat e
Ovalbumin, BSA, y-globulin and catalase were
used as markers.
Enzyme assay
The rate of formation of NADPH by the oxidation
of isocitrate was monitored at 366 nm for the enzyme
assai 3. The reaction mixture contained isocitrate (2.5
mM) NADP+ (0.625 mM) and MgCh (3.75 mM) in 60
mM sodium phosphate buffer pH 7 .8 at 30°C. The
reaction was started by the addition of appropriate
amount of diluted enzyme solution. The CNADPH at 366
nm was found to be 3.llxlO3 MI cm-I . A unit of the
enzyme was defined as the amount of enzyme
required to transform 1 )..l11101e NADP+ to
NADPH/min. The specific activity is expressed in
337
SRIVASTAVA & SINGH: NADP+-LINKED ISOCITRATE DEHYDROGENASE FROM P. SATIVUM
terms of enzyme units per mg protein. Protein was
estimated by the method of Lowry et al. 16.
Thermal inactivatioll
The enzyme solution of appropriate dilution in
suspension buffer was kept in a thermostat at desired
temperature. Aliquots of 0.05 ml were withdrawn at
different intervals of time, chilled, and tested
immediately for enzyme activity at 30°C.
Step
Results
From Table 1, it is seen that about 106-fold
purification with about 24% recovery of the enzyme
has been achieved in the present work. The purified
enzyme shows maximum absorbance at 280 nm with
an A28r/ A260 ratio equa l to 1.31 and gives sing le
protein band on PAGE, both in presence and absence
of SDS (Fig. 2, 3). The purified enzyme shows one
Table I- Purification of NADP+ -linked isocitrate dehydrogenase from ge rminatin g pea seeds (150 g)
Volume
Total unit
Total Protein
Sp. Activity
Fold
(ml)
(mg)
Units!mg
purification
protein
Acetone powder
Ethanol ex tract
Ammoniulll sulphate
(50-75 % saturation)
Dialysis
DEAE cellulose column
chromatography (Protein,
0-90% Ammon ium sulphate
saturation)
% Recovery
280
18.0
5.40
403
352
280
3298
545
54.6
0.122
0.645
5.13
5.29
42.0
87.3
69.5
5.70
1.30
256
97.0
7.50
12.9
106
63.5
24 . 1
... . ... ....
·A-
+
Fig.
2-PAGE
of
purified
NADP+-linked
isocitrate
dehydrogenase [Protein (100 Ilg) was applied in the presence of
2% SDS at pH 7.0]
+
3--PAGE
of
purified
NADP+-linked
isoc itrate
Fig.
dehydrogenase in the presence of SDS. [Protein (50 /lg) was
applied at pH 8.3]
338
INDIAN J . BIOCHEM. BIOPHYS. , VOL. 38, OCTOBER 200 1
major and some minor peaks on pre made superose 6B column (27 x I cm) of FPLC ( Pharmacia LKB) at
wavelength 280 nm in 60 mM sodium phosphate
buffer at p H 7.8 (Fig. 4) . The specific activity is 12.9
units/mg protein.
Addition of NAD+ instead of NAOP+ to the
soluti on of isocitrate dehydrogenase in the assay
system does not show any activity, suggesting that the
enzyme preparati on is free from NAO+- linked
isocitrate dehydrogenase and its speci fi city towards
NAOP+.
comparing its dilution factor with those of know n
proteins (Fig. 5) has been found to be 146,000. PAGE
yields a single protein band in presence of SOS
(Fig. 2), suggesting that all the mono mers of NAOP+linked isocitrate dehydrogenase of germinatin g pea
seeds are of the same molecular weight. A rough
estimate of the size of the monomer was obtained by
comparing its electrophoretic mobility in presence of
SOS, with the mobi lities of known proteins or their
subunits, namely ribonuclease, myog lobin , G-3Pdehydrogenase and BSA. Results of such ex periments
are shown in Fig. 6. The subunit molecular weight for
isocitrate dehydrogenase is found to be 36,000.
Moleclllar weig ht
The molec ul ar weight of the enzy me estimated by
,· 0 , - - - - - - - -- - - - - - - - - ,
COL U I~N :
E
c
0·85
E
SUPEROS E 68
( FP Le i
o(])
BUFFER : No-PHOSPHATE
60mM, pH 7-B
'"\:i
FLOW
RATE
cc
~
RIBONUCLEASE
o
0 · 75
MYOG LOBI N
...J
<D
o
: 0·15 ml I min
::E
w
UJ
0 . 65
ISOCITRATE
- - DEH YDROGEN AS E
>
u
....
~ 05
G-3P DEHYDROGE NASE
<t
~
CD
a:
cc
o
0 ·4 5
<fl
~ SA
L-_~~_-J~_--L-_~
<D
«
4· '
4 ·3
4 ·5
4 ·7
_ _~--,
4 .9
5 .1
LOG SUB UNIT MO LECULA R WE IG HT
Fig. 6--Subunit mo lec ul ar weight fo r the purified NADP+- lin ked
i ocitrate dehydroge nase
15 0
180
TIME ( min. )
100 ...-------.,-..".,.,cc---------,
>-
Fig.
.+--FPLC
of
purifi ed
NADP+-linked
isoc itrate
de hydroge nase. I Pre- made superose 6 B column (27x I em) o f
FPLC (Pharmacia L K B) was used at wa ve le ng th 280 nm in 60
rnM sodium phosp hate buffe r at p H 7.8]
U
<t
<i.
=>
r------------------------------,
iDH
a:
f-
:;
•
<t:
0
....J
0
<t:
::J
OVALBUMIN
a:
0
w
a:
0
t;
<t:
1L.
;;--
BSAO
' ·2
..J
1.0
0
~
20
GO
60
80
20
0
4
z
o
ISOCITRATE
- - DEHYDROGENASE
I::J
g
..
8
~"'
40
(/)
6
o
' ·6
Q
>-
IU
8
~
t:
>
;= 1· B
r
y-GLOBU LIN
2
CATALASE
OL--L----~----~--~~--~~----~
46
5·0
5·2
4 ·8
LOG MOLECULAR WEIGHT
Fig. 5--Mo lec ular wei ght of purified NADP+-linked isocitrate
de hydroge nase
0
0
20
40
60
TIME (m i n)
80
100
Fig. 7-Thermal in activati o n of purified NADP+- li nked isocitrate
de hydroge nase at 4SOC a nd SO°C in 60 mM sodium phosphate
buffer, pH 7.8. [The protein conce ntrat io n in eac h case wa s 0.15
mg/ml. Th e enzy me so lution was inc ubated at 4S oC, (. -. ) and
SO°C, (0-0). Aliquots were withdrawn at different inte rva ls o f
time and assayed immediately for th e act ivity o f e nzy me at 366
nm]
339
S RIV ASTA V A & SIN G H: NADP+- L1NK ED ISOC rrR ATE DEHYDROGENASE FRO M P. SATI VUM
Then-nal inactivation
Fig. 7 shows the time-dependent inacti vati on of the
puri fied enzy me at 4S oC and SO°c. The inacti vation
fo llows simple first order kineti cs which is evident in
the semi log plot of the same data shown as th e inset
of Fig. 7. The rate constants at 4S oC and SO°C are
show n in Table 2. The first order kinetics for thermal
inacti vati on suggests absence of site-site heterogeneity, indicating th at all subunits behave in simil ar
manner.
Effect of pH
Optimum pH of the enzy me is found to be 7.S to
8.0 which is identical with th at reported for the
enzy me from BOlllbyx /I /O ri 2o and is close to th e pH
optima fo r the enzy mes from bass li ver21, Tlziobacillus
?3 and matunn
. g castor
/l oveII us 21-, mai. ze scute II um-'
bean seeds I' . At pH below 7.S and above 8.0 and at
hi gh substrate concentration , th ere is stron g inhibition
of the enzy me acti vity. The doubl e reciprocal plot
(Fig. 8) shows that betwee n pH 6. S and 8 the proton
behaves as an "uncompetitive inhibitor". This
suggests th at a bas ic group (pres umably at the acti ve
site) is protonated in thi s pH range in the presence of
substrate onl y. From the data in Fi g. 8, the pKa value
Table 2-Kinetics o f inacti vati o n o f native and apo-e nzy me at
45 ° and 50°C in 60 mM sodium phosphate buffer, pH 7.8
[The enzyme acti vity of native and apo-enzy me were
determined by ex tern all y added mag nesium ions (3. 75 mM)l
of thi s "masked" bas ic group is found to be 6. 78,
which correspond to the 'mas ked' bas ic group in th e
enzyme-substrate complex and suggests th e poss ible
presence of a histidine residue present at the active
site of pea seed enzyme.
Role of metal iOl/s
The purified pea seed enzyme shows max imal
acti vity in the presence of Mg2+ or Mn 2+ ions. In the
absence of Mg2+, the activity is reduced to one-third of
that in the presence of Mg2+. EDT A acts as a
competiti ve inhibitor (Fig. 9). Dialys is of th e enzyme
aga inst EDTA and removal of excess EDTA by
di alysis against sodium phos phate buffer (60 mM , p H
7.8) at O°C results in loss of enzy me acti vity in the
absence of externall y added Mg2+. In addi ti on to
Mg2+, Mn2+, Zn 2 +, Cd 2+, C02+ and Ca2 + at 3.7S mM are
effective. Cu 2+, Fe2+ and Ni 2+ are not effecti ve at th is
concentrati on (Table 3). EDTA di alyzed enzy me on
thermal inactiv ation shows the same kineti c properties
as th e untreated "native" enzyme in presence of Mg:!+
(Fi g. 10). The rate constants of the EDT A di alyzed
enzyme at different temperatures are given in the
Table 2.
Discussion
Gel filtrati on and gel electrophoresis (with and
without SOS) and FPLC show th at the puri fied
enzyme is near homogeneous. The enzyme protein is
Rate constants k (min"l)
Nati ve e nzy me
Apo-enzy me
Temperature
(OC)
45
50
0 .017
0.03 1
300
•
250
0 . 139
0 .231
200
0;c
E
20
ci
E
<l
~
o
~ 10
-- --- -
15 0
6
'7c:. 15
10
20
1/
{r~ o'; trate]
30
100
40
(mM- ' 1
a
Fig. 8--Doubl e rec iproca l plot for the e ffect o f pH on the Km and
Vmux of isocitrate for the NADP+-linked isoc itrate dehydroge nase
from pea seeds. [The concentrati on o f substrate (isocitrate) was
varied and p H of assay system was fi xed i.e., 8.0, 7.0 and 6.5 for
curve I, 2 and:) respecti vely. The e nzy me concentrati o n was 10.2
~g/ml. The rate of reacti on is expressed in terms o f rate of
absorbance change at 366 nm]
5
Illsocitrate
10
( m M-')
Fi g. 9--Kineti cs of inhibition of NADP+-linked isoc itrate
dehydrogenase from pea seeds with EDT A (0.09 111M) in 60 mM
sodium phosphate buffer, pH 7.8 at 30°C [Concentration of the
enzyme was 12 ~g/ml. The acti vity was estimated in the absence
of MgCI21
340
INDIAN 1. I310CII EM. BIOPHYS. , VOL. 38, OCTOBER 2001
Table 3--Effect of different di va lent metal ions on the activity
of NADP+- linked isocitrate dehydroge nase of pea seeds
IThe enzy me ac ti vi ty was estimated in 60 mM phosphate
buffe r. pH 7.8 at 30°C by adding different chloride sa lt of
metal ions to the "apo- isoc itrate dehydrogenase"l
Enzyme
Na ti ve enzy me
Dialysed again st EDTA
and phos phate buffer
(60 mM pH 7.8)
Metal ions added
(3.75 mM)
None
--.---
Sp. activity
EU/mg
protein
B
12
16
TIME (l1)in . )
6.42
0.00
M n2+
M,, 2+
0
Zn2+
Cd 2+
C0 2+
Ca 2+
Cu2+
Fe 2+
Ni2+
100 . -- --
5.72
3.24
2.94
2.38
2.34
0.23
Nil
Nil
Nil
a tet rameric mo lec ule made up of appare ntly identical
monomers. Th e first order kinetics for thermal
inactivation of the e nzy me suggests that there is no
si te-site heterogeneity, indicating that all subunits are
similar and isoc itrare dehydrogenase has a regular
te trameric structure. Th e kineti cs of thermal
inactivati o n of EDTA-dialysed enzyme also exhibits
fi rst ord er kinetics with hi gh rate constants as
observed with th e native enzyme (Tab le 2) and
co mpari so n of th e rate constants shows that the overall structure of the enzyme does not change
significa ntly on re moval of the metal ions from native
and apo-enzyme. Thi s suggests that the endogenous
metal ions do not contribute to large extent to the over
all structure of the isoc itrate dehydrogenase protein
but the stabi lity of thi s enzyme decreases on removal
of the bound metal ions. Thus the metal ions are
required for acti vity and do not affect the structural
sy mmetry characteri stics of the enzyme. Since no
other known effector or specific li gand was present in
these ex periments, it is concluded that the symmetry
is an inherent property of the enzyme protein a nd not
induced by any ligand. There are no reports on the
kinetics of thermal inactivation of isocitrate dehydrogenase from other sources .
The uncompetitive inhibit ion by protons (Fig. 8)
suggests the presence of a " masked" basic group at
the active site. In the free enzyme, this group is not
proton ated in the p H range 6.5 -8. However, the
presence of substrate facilitates protonation. The pKa
apparent in the presence of substrate is found to be
o
TI ME (m,n . )
Fig. IO--Kinetics of thermal inac ti va ti on of the apo-enzyme at
45°C, (0-0) and 50°C, (e-e ) in 60 mM sod ium phosphate buffer.
pH 7.8. [The apo-enzyme (0.21 mg/ml ) was incu bated in 60 mM
phos phate buffer, pH 7. 8 at des ired temperature. Aliquots were
wlthdrn wn at different time interva ls and assayed for enzy me
ac tivity in presence of externall y added Mg2+ (3.75 mM). A semi
log plot of the data is show n in the inset of the fi gure]
6.78. In the free enzyme thi s mu st be well belo w 6.5.
We believe that this group has an important role in
catalysis. It is notewo rthy that the requirements of
basic form of an essential io nizabl e gro up in the
enzyme substrate complex with pKa valu es 6.9, 6. 5.5
have been reported in isoc itrate dehydrogenase from
24
different so urces i.e., Cephalosporillll/ acr elll ollillll /
6·
respectively.
We have'
bea f heart 25 an d human heart2
also checked and confirmed the mod ification of thi s
group by diethylpyro carbonate (DEPC) a nd detail ed
kinetic studies are in progress.
The enzyme shows some activity in the absence of
externally added metal ions. T he act ivity increases
about two-fold on addition of Mn 2+ (3.75 mM). It is
likely that the enzyme preparation has so me
endogenous metal ions, which are responsible for the
residual activity in the absence of ex ternally added
metal ions. This was confirmed by carrying out
dialysis of the enzyme against EDTA and removal of
excess EDT A. The resulting protein solution is
completely devoid of NADP+-linked ICDH activity.
Full activity is restored on the addition of Mn 2+ and
partially restored in the presence of some divalent
metal ions at same concentration of Mg2+, Z02+, Cd 2+,
C0 2+ but Fe2+ and Ni 2+ are ineffecti ve. NADP+-linked
isocitrate dehydrogenases from other sources have
been known to require divalent metal ions 1.9.13.24. It
has already been shown earlier that the metal ions do
not contribute significantly to the over all structure of
isocitrate dehydrogenase. But ahe stability of this
enzyme decreases on removal of the bound metal
ions. Therefore, it may be concluded that, the
SRIV ASTA V A & SINGH: NADP+-L1NKED ISOCITRATE DEHYDROGENASE FROM P. SA T1VUM
endogenous metal ions (and poss ibly the externally
added Mg2+ ions) are directly involved in substrate
binding and/or catalytic steps. The molecular
sy mmetry apparent from the single exponential loss of
activity of enzyme (native as well as apo-enzyme)
must be a consequence of the regular tetrameric
structure of the NADP+-linked isocitrate dehydrogenase.
Acknowledgement
Authors are thankful to Prof 0 P Malhotra for
helpful suggestions. Financial ass istance in the form
of Research Fellowship from Banaras Hindu
University is gratefully acknow ledged.
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2
3
4
5
6
7
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