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 Singh Department of Biochemistry. Banaras Hindu Uni versity,

Varanasi 22 1 005, India

Received 24 April 2000; revised alld accepled 16 Jllly 2001

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 in acti 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 ac ti vity at di fferent pH (6.5-S) suggests that the proton behaves formally as an "uncompetitive inh ibitor". A basic group of the enzyme (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 enzymati cally inact ive 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 Zn~+, Cd2+, C02+ and Ca2+ ions brings about part ial recovery. Other metal ions Fe2+, Cu2+ and N i ~+ are ineffective.

NADP+-linked isoc itrate dehydrogenase (NADP+­lin ked [CDH, EC 1.1. 1. 42) has been studied in many prokaryoti c and eukaryotic organisms. It has been purified and characteri zed from several bacteri al and mammali an species l

-7

. Although the presence of th is enzy me in plant cytosoI8.

9.1O

, mitochondri a l o and chloroplasts II is known there are only a few reports of isolation and characteri zati on of thi s enzyme from plant ti ssues')· 12-15. We have isolated the enzy me from germinating pea seeds and studi ed some properti es, which have direct bearing on the structural symmetry characteri sti cs and partial identi fication of the constituents of the active site.

Matedals and Methods Threo Ds(+)i socitric acid , NADP+ (di sodium salt),

trichloroacetic acid, bovine serum albumin , Folin­Ciocalteau's phenol reagent , acrylamide, N,N- methy­lene bis acryl amide, ribofl avin , N,N,N,N-tetramethyl­ethylene di amine, sodiumdodecyl-sulphate, 2-mercap­toethanol , tris(hydroxymethyl ) aminomethane and DEAE cellulose (coarse mesh) were from Sisco Research Laboratories Pvt. Ltd. Bombay.

Dialysis tubing from Arthur H. Thomas, Philadelphia, P.A. US A, Bio-gel P200 from Bio-Rad Laboratories, Richmond, Californ ia USA, ovalbumin , glyceraldehyde 3-phosphate dehydrogenase, catalase and y-globulin from Sigma Chemicals Co, St Loui s, USA were used. Commercial-rectified spirit (95%

* Author for correspondence

ethanol) was reflu xed over silver oxide and di stilled twice before use. Other chemicals were of analytica l reagent grade. All solutions were prepared in double disti lied water.

Plallllllaleria! Pea seeds (Pisum sativlIIII ) (Azad-PI) obtained

from Department of Ag;-onomy lAS (B HU) were washed thoroughl y wi th double distilled water and soaked fo r 12 hr, spread over moist bed of washed sand fo r germ ination for 72 hr at 30°C and the germinated seeds were coll ected (1 50 g).

Extraction alld purificatioll of ell zyme All operations were carried out at 0-4°C unless

stated otherwi se.

Preparatioll of acetolle powder Germinated seeds were washed with cold double

di stilled 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 disso 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 supernatant (18 ml) with stirring to bring it to 50% saturation. The pH was maintained constant at 7.8 with 10-fold diluted liquid ammonia and the solution was centrifuged. The enzymically inactive precipitate 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 enzymically 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 DEAE­

cellulose 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

16 - ' 2 ·0 -Z 12

;/ I

<II 1·5 _

!:: Q, z => E

~ 8 1.2 z W

~ .... .... 0 u

~o co

<t 4 0·8 a.

0 0·4 20 30 40 50 50 70 80

ELUTI ON VOLUME (ml I

Fig. I-NADP+-linked isocitrate dehydrogenase of germin:lIing pea seeds after DEAE-cellulose column chromatography at 0-4°C. [The enzyme was e luted with 60 mM phosphate 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

Reisfeld et a1. 17• 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 et al. 16

. The dilution factors of standard proteins and pea seed enzyme were calculated by the following 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 required to NADPH/min.

defined as the amount of enzyme transform 1 )..l11101e NADP+ to

The specific activity is expressed in

SRIVASTAVA & SINGH: NADP+-LINKED ISOCITRATE DEHYDROGENASE FROM P. SATIVUM 337

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.

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 equal to 1.31 and gives single 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 germinating pea seeds (150 g) Step Volume Total unit Total Protein Sp. Activity Fold % Recovery

(ml) (mg) Units!mg purification

Acetone powder 280 403 Ethanol ex tract 18.0 352 Ammoniulll sulphate 5.40 280 (50-75 % saturation) Dialysis 5.70 256 DEAE cellulose column 1.30 97.0 chromatography (Protein, 0-90% Ammon ium sulphate saturation)

..... ... .... ·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]

protein

3298 0.122 545 0.645 54.6 5.13

7.50 12.9

5.29 42.0

106

+

87.3 69.5

63.5 24 .1

Fig. 3--PAGE of purified NADP+-linked isoc itrate 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 6-B 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.

Addit ion of NAD+ instead of NAOP+ to the solution 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+.

Moleclllar weight The molecul ar weight of the enzy me estimated by

,· 0 ,-----------------,

E c o (])

'" \:i UJ u ~ 0 5 CD a: o <fl <D «

COLU I~N : SUPEROSE 68 ( FP Le i

BUFFER : No-PHOSPHATE 60mM, pH 7-B

FLOW : 0·15 ml I min RATE

15 0 180

comparing its dilution factor with those of known 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 monomers of NAOP+­linked isocitrate dehydrogenase of germinating 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, myoglobin , G-3P­dehydrogenase and BSA. Results of such experiments are shown in Fig. 6. The subunit molecular weight for isocitrate dehydrogenase is found to be 36,000.

E cc

0·85

~ 0 ·75 ...J

<D o ::E 0 .65 w > .... <t

~ 0 ·4 5 cc

RIBONUCLEASE

o MYOGLOBIN

ISOCITRATE - - DEH YDROGEN ASE

G-3P DEHYDRO-GE NASE

~SA L-_~~_-J~_--L-_~ __ ~--,

4· ' 4 ·3 4 ·5 4 ·7 4 .9 5 .1

LOG SUB UNIT MOLECULAR WE IG HT

Fig. 6--Subunit mo lecul ar weight for the purified NADP+- lin ked i ocitrate dehydrogenase

TIME ( min. ) 100 ...-------.,-..".,.,cc---------, >-

~ Fig. .+--FPLC o f purifi ed NADP+-linked isoc itrate r

dehydroge nase. I Pre- made superose 6 B column (27x I em) o f FPLC (Pharmacia L K B) was used at wave length 280 nm in 60 rnM sodium phosphate buffer at p H 7.8]

8 r------------------------------,

a: o

6

OVALBUMIN

t; BSAO <t: 1L. 4 z o I-::J

g 2

ISOCITRATE - - DEHYDROGENASE

y-GLOBU LIN

CATALASE

OL--L----~----~--~~--~~----~ 5·0 5·2 46 4 ·8

LOG MOLECULAR WEIGHT

Fig. 5--Mo lecular wei ght of purified NADP+ -linked isocitrate dehydrogenase

t: >

>-f-:; I-U

;= 1· B

~ U <t

<i. ' · 6 => Q

iDH a: ..

• 8 ' · 2 ..J

<t: ....J <t:

0 1.0 0 0 20 GO 60 80

::J 40 0 (/)

w a:

0

20 ;;--

~"' 0

0 0 20 40 60 80 100

TIME (m in)

Fig. 7-Thermal inactivation of purified NADP+- li nked isocitrate dehydrogenase at 4SOC and SO°C in 60 mM sodium phosphate buffer, pH 7.8. [The protein concentrat ion in each case was 0.15 mg/ml. The enzyme so lution was incubated at 4S oC, (. -. ) and SO°C, (0-0). Aliquots were withdrawn at different intervals o f time and assayed immediately for the act ivity o f enzy me at 366 nm]

SRIV ASTA V A & SING H: NADP+-L1NK ED ISOCrrRATE DEHYDROGENASE FROM P. SATI VUM 339

Then-nal inactivation Fig. 7 shows the time-dependent inacti vation of the

puri fied enzy me at 4S oC and SO°c. The inactivation fo llows simple first order kinetics which is evident in the semi log plot of the same data shown as the inset of Fig. 7. The rate constants at 4SoC and SO°C are shown in Table 2. The first order kinetics for thermal inacti vation suggests absence of site-site hetero­geneity, indicating that all subunits behave in similar manner.

Effect of pH Optimum pH of the enzy me is found to be 7.S to 8.0 which is identical with that reported for the

enzy me from BOlllbyx /I /O ri2o and is close to the pH optima fo r the enzymes from bass li ver21, Tlziobacillus

II 21 . II ? 3 d . /l ove us -, mai ze scute um-' an matunng castor bean seeds I' . At pH below 7.S and above 8.0 and at high substrate concentration , there is strong inhibi tion of the enzyme acti vity. The double reciprocal plot (Fig. 8) shows that between pH 6. S and 8 the proton behaves as an "uncompetitive inhibitor". This suggests that a bas ic group (presumably at the active site) is protonated in thi s pH range in the presence of substrate only. From the data in Fig. 8, the pKa value

Table 2-Kinetics o f inacti vation of native and apo-enzy 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 ternall y added magnesium ions (3. 75 mM)l

Temperature (OC)

45 50

-- --- -

Rate constants k (min"l) Nati ve enzyme Apo-enzy me

20

'7c:. 15 E o ~ 10

0 .017 0.03 1

10

0 .139 0 .231

20 30

1/ {r~ o'; trate] (mM- ' 1

40

Fig. 8--Double rec iprocal plot for the e ffect o f pH on the Km and Vmux of isocitrate for the NADP+-linked isoc itrate dehydrogenase from pea seeds. [The concentration of 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 enzyme concentration was 10.2 ~g/ml. The rate of reaction is expressed in terms of rate of absorbance change at 366 nm]

of thi s "masked" bas ic group is found to be 6.78, which correspond to the 'masked' bas ic group in the enzyme-substrate complex and suggests the 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

activity in the presence of Mg2+ or Mn2+ 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 the enzyme against EDTA and removal of excess EDTA by di alysis against sodium phosphate buffer (60 mM , pH 7.8) at O°C results in loss of enzy me acti vity in the absence of externally added Mg2+. In addi ti on to Mg2+, Mn2+, Zn2+, Cd2+, C02+ and Ca2+ at 3.7S mM are effective. Cu2+, Fe2+ and Ni2+ are not effecti ve at th is concentration (Table 3). EDTA dialyzed enzy me on thermal inactivation shows the same kineti c properties as the untreated "native" enzyme in presence of Mg:!+ (Fig. 10). The rate constants of the EDT A dialyzed enzyme at different temperatures are given in the Table 2.

Discussion Gel filtrati on and gel electrophoresis (with and

without SOS) and FPLC show that the puri fied enzyme is near homogeneous. The enzyme protein is

0;-c E ci 6 <l ~ -

300

250

200

15 0

100

a 5

Illsocitrate ( m M-')

•

10

Fi g. 9--Kinetics 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 dehydrogenase 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

Nati ve enzy me

Dialysed against EDTA and phosphate buffer (60 mM pH 7.8)

Metal ions added (3.75 mM)

None

M 2+ n M,, 2+

0

Zn2+ Cd2+ C02+ Ca2+ Cu2+ Fe2+ Ni2+

Sp. activity EU/mg protein

6.42

0.00

5.72 3.24 2.94 2.38 2.34 0.23 Nil Nil Nil

a tet rameric molecule made up of apparently identical monomers. The first order kinetics for thermal inactivation of the enzy me suggests that there is no si te-site heterogeneity, indicating that all subunits are similar and isocitrare dehydrogenase has a regular te trameric structure. The kinetics of thermal inactivation of EDTA-dialysed enzyme also exhibits fi rst order kinetics with high rate constants as observed with the native enzyme (Table 2) and comparison of the rate constants shows that the over­all structure of the enzyme does not change significantly on removal 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 isocitrate dehydrogenase protein but the stabi lity of this enzyme decreases on removal of the bound metal ions. Thus the metal ions are required for acti vity and do not affect the structural symmetry characteri stics of the enzyme. Since no other known effector or specific li gand was present in these experiments, it is concluded that the symmetry is an inherent property of the enzyme protein and 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 protonated 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

100 .---- --.---

B 12 16

TIME (l1)in . )

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 phosphate buffer, pH 7.8 at des ired temperature. Aliquots were wlthdrnwn at different time interva ls and assayed for enzy me activity in presence of externally 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 must be well below 6.5. We believe that this group has an important role in catalysis. It is noteworthy that the requirements of basic form of an essential ionizable group in the enzyme substrate complex with pKa values 6.9, 6. 5.5 have been reported in isocitrate dehydrogenase from different sources i.e., Cephalosporillll/ acrelllollillll /

24

b f h 25 d h 26· ' ea eart an uman heart respectively. We have also checked and confirmed the modification of this group by diethylpyro carbonate (DEPC) and detailed 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 Mn2

+ (3.75 mM). It is likely that the enzyme preparation has some 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 Mn2+ and partially restored in the presence of some divalent metal ions at same concentration of Mg2+, Z02+, Cd2+, C02+ but Fe2+ and Ni2+ 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 significant ly 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 341

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 acknowledged.

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