Indian Journal of Biochemis try & Bi ophys ics
Vol. 39, October 2002, pp. 332-34 1
Purification and characterization of 3-phosphoglycerate kinase from Ehrlich
ascites carcinoma cells
Kasturi Mukheljee l , Swapna Ghosh l , Manju Ray' * and Subhankar Ra /
'Department of Bi olog ica l Chem istry. Indian Assoc iati on for th e Cult iva ti on of Sc ience, Kolkata 700032. Ind ia
'Departmcnt of Biochemi stry, Universit y College of Science, Uni versit y of Calc ulla, Kolkaw 7000 19, India
Rece il'ed 30 Mav 2002; rel'ised al/d accep led 29 Jllly 2002
3- Phosphog ly.:era te kinase (3- PGK ) has been purified to ap parent homogeneit y from Ehrli ch asc ites carcinoma (EAC)
ccll s by (N H4),S04 prcc ipitati on, ge l filtration and ion-exc hange chromatography. The cnzy me has bee n partially
charactcri zed and compared with th e characteristi cs of thi s enzy mc of oth er normal and malignant ce ll s. The EAC cell 3PGK is composed of a single subunit of 47 kDa . It has a broad pH optimum (p H 6.0-7.5) for it s enzy matic ac ti vity. The
apparent K,,, va lues of 3-phosphog lyce rate (3-PGA) and ATP for 3- PGK ha ve been found out to be 0.25 mM and 0.1 IWV/
res pectivel y. Similar to 3- PGK of oth er cells, th e EAC enzyme requires either Mg 2+ or Mn 2+ for full acti vity; thc optimu m
conce ntrati ons of Mg2+ and Mn 2 + are 0.8 mM and 0.5 mM respec ti ve ly. When ATP and 3-PGA act as substrates. ADP. the
reacti on prod uct of 3- I'GK -catalyzed reaction has been found to inhibit thi s enzyme. Kinetic studies wcre madc on the
inhibition of ADP in presence of th e substrates ATP and 3-PGA. Allempt s to hybridize 3-PGK and glyceral de hyde-3phosphate dehydrog.:nase of EAC ce ll s by NAD or glutaraldehyde were un successfu l.
Glyceraldehyde-3-pl osphate dehydrogenase (GAPDH )
(EC 1.2.1.12), an important enzyme of the glycol ytic
pathway has been recently purified in our laboratory
from a rapidl y grow ing, hi ghl y dedifferentiated
mali gnant cell, Ehrl ich ascites carcinoma (EAC) cel li.
Investigators from di fferent laboratories including our
ow n, for th e last severa l years had implicated thi s
enzy me with mali gna nt aberrations ' -~. Moreove r, it
had been obse rved th at several properties of thi s
enzy me of EAC and other mali gnant cell s are
strikingly differen t from thi s enzyme of oth er normal
ce ll sl. s .~ . One peculiar property of EAC cell GAPDH
is that it is a heterodi mer of M, 87,000 ± 3,000
co ntainin g two subunit s of M, 54,000 ± 2,000 and
33.000 ± 1,000; whereas thi s enzyme of several
normal so urces is a hOll1otetram er of M, 146,000 1. The
cata lytic activity of the mali gnant cell enzyme had
al so bee n found to be much hi gher th an that of thi s
enzyme from other normat so urces I. Several
investi ga tors had sugges ted that GAPDH and 3phosphog lycera te kina se (3 -PGK) (EC 2.7.2.3), the
nex t enzy me of the glyco lytic pathway mi ght be
assoc iated in several ce ll s res ulting in signifi ca ntl y
"' Aut hor for correspondence
033 473 590~: Fax: 033 473 2805
e.ma il: bClllr@mah en dra.iacs .res. in
AblJ/"('\'iario/ls
/l sed: GA PDI-I.
glyceraldehyde-3-phosphatc
dehydroge nase: 3- PGK . 3-p hosphog lyccrat e kinase: 3-PGA, 3pho s phog l yce r ~l t e; GAP, D-g lyccraldehyde-3-p hos phate.
T~ l ep h o ll e:
..
' activity
h·tg I1er cata IyttC
0
f these two enzymes 10- 1'\-. rt
had been speculated that as a res ult of th is association
of th ese two enzymes th e overall glyco lytic flux wi ll
be enhanced. That enhanced g lyco lys is is a very
important property of mali gnant cell s had been known
for a long time. It is pertinent here to mention th at th e
mo lec ular weight of 3-PGK of various sources is
around 50,000" " which is close to th e molecular
weight of th e large r subunit of GAPDH purified from
EAC ce ll s '.
In glyco lys is, the combined action of th e enzymes
GAPDH and 3- PGK cata lyze one of th e two ATP
ge neratin g steps.
The
l ,l-b isphos phog lycerate
ge nerated in GAPDH catalyzed reac tion is co nve rted
to 3-phos phoglyce rate (3- PG A) by 3- PGK with th e
co nco mitant formation of ATP.
The enzy me 3- PGK had been purified from man y
.zecl l~ - ' R . S ev ra l tn
' vestlgators
.
so urces ancI C1<lracten
I
had sugges ted so me ro le of 3-PGK in ca ncer cell s I9 -21 .
Moreover, it had been proposed that excess i ve AT P
formation in cell s may lead to mali gnanc/ 2. Howeve r
this enzy me, had been purifi ed from only one type of
malignant cell i.e. human leukemi c granulocy te 'x .
The prese nt paper desc ribes the puri ficati on of 3PGK from a hi ghl y dedifferentiated rapidl y growing
malignant cell , EAC cell and so me pro perti es of thi s
enzy me. It has also bee n in ves ti gated whether
GAPDH and 3- PGK of EAC cells are indeed
MUKHERJEE
el al.:
3-PHOSPHOGLYCE RATE KINASE FROM EHRLICH ASCITES CARC INOMA CELLS
associated resulting in overall
g lyco lytic flux and A TP synthesis.
increase
In
the
Materials and Methods
Materials
Unless mentioned otherwise, all the biochemicals,
enzymes, marker enzymes and DEAE-Sephacell were
purchased from Sigma Chemical Co., USA Sephacryl
S-200, Sephadex G-100 and CM-cellulose were
products of Pharmacia Fine Chemicals, Sweden .
Centricon 30 membrane filter was a product of
Amicon Inc., U .S.A. The coupling enzyme GAPDH
purified either from EAC cells or rabbit muscle was
used for the assay of 3-PGK. The EAC enzyme was
puri fied in our laboratory' and the rabbi t muscle was
purchased from Sigma. The GAPDH of both these
sources indicated a single band in PAGE. All other
chemicals were of analytical grade and were obtained
from local manufacturers.
An aqueous solution of D,L-glyceraldehyde-3phosphate was prepared from the water insoluble
barium salt of o,L-glyceraJdehyde-3-phosphate
diethylacetal as described previously. The concentration of specifically o-glyceraldehyde-3-phosphate
(GAP) was determined by enzymatic assay'.
Animals and transplantation of tumors
The EAC cells were maintained in and harvested
from intraperitoneal cavity of Swiss albino mice as
described previously'. The EAC cells were then
homogenized for purification of GAPDH and/or 3PGK as described below.
Assay ofGAPDH
Unless mentioned otherwise, GAPDH was
routinely assayed in triethanolamine-HCI buffer, pH
8.51.23. To monitor the reaction, the increase in
absorbance at 340 nm due to the formation of NADH
from NAD was noted at 30-s intervals, the values
remained almost linear for 3 min (M, 0 .0250.060/min) . The assay mixture contained in a total
volume of I ml, 50 Ilmol triethanolamine buffer, 50
Ilmol Na2HP04, 0 .2 Ilmol EDT A, I Ilmol NAD and
0.5 Ilmol of GAP. The reaction was started by the
addition of an appropriate amount of a solution of
o,L-glyceraldehyde-3-phosphate
(prepared
as
described above) which contained the requi site
amount (0.5 Ilmol) of GAP. The enzyme GAPDH was
also assayed similarly but using arsenate instead of Pi .
In that case, 10 Ilmol of sodium arsenate was added to
the above reaction mixture in stead of 50 Ilmol
Na2HPOl3.
333
The enzyme was also assayed by the reverse
reaction. The ATP-dependent phosphorylation of 3PGA was catalysed by 3-PGK; then
1,3bisphosphoglycerate formed was reduced by NADH
to GAP by GAPDH. The oxidation of NADH was
monitored at 340 nm. The rate of this reaction was
about 1.2 times higher than that of the forward
reaction with GAP as substratel.24.
One unit of activity of GAPDH is defined as the
amount of the enzyme required to convert I Ilmol of
NAD to NADH per min under standard conditions of
assay. The specific activity is defined as the units of
activity per mg of protein .
Assay of 3-PGK
3-Phosphoglycerate kinase was routinely assayed in
the backward reaction leading to the formation of 1,3bisphosphoglycerate from 3-PGA as originally
described by Bucher25 . 3-PGK catalyzed the ATPdependent phosphorylation of 3-PGA; then 1,3bisphosphoglycerate ' formed was reduced and
dephosphorylated to GAP by GAPDH, and NADH
was oxidized to NAD. The oxidation of NADH was
monitored at 340 nm at 1 min interval for 4 min . The
assay mixture contained in a total volume of 1 ml, 40
mM triethanolamine-HCI buffer pH 7.4, MgS04 (0.8
mM for EAC cell 3-PGK and 2 mM for rabbit mu sc le
3-PGK), 1 mM ATP, 0.15 mM NADH, 2.5 mM 3PGA and 0 .3 units of GAPDH purified from either
EAC cells or rabbit muscle.
One unit of the enzyme activity is defined as the
amount of the enzyme required for the formation of
1 jlmol of GAP or NAD+ per minute at 25°C. Specific
activity is expressed in units/mg protein . The assays
to determine the optimum pH for the enzymatic
activity were done in four different buffers: 50 mM
sodium acetate-acetic acid (PH 4.0-5.4), 50 mM
imidazole-HCI (PH 6.2-8.0), 50 mM Tris-HCI (PH
7.4-8 .5) and 50 mM triethanolamine-HCI (PH 7.4-8 .8).
Estimation of protein
Using BSA as a standard, protein was estimated
using the method of either Lowry et al. or Warburg
and Christian as outlined by Layne 26 . Appropriate
control was maintained with triethanolamine buffer to
correct for the interference of this compound,
particularly with the method of Lowry et al.
PAGE and SDS-PAGE
Polyacrylamide gel electrophoresis was carried out
according to the method of Davis 27 with minor
334
INDIAN J. BIOCHEM. BIOPHYS., VOL. 39, OCTOBER 2002
modi fication. The elec troph oresis was carried out at
15°C with Tris-glycine buffer, pH 8.8 as rese rvoir
buffer. For SOS-PAGE, the method of Laemmli was
used2R.
Deterlllination of 1II0/ecular weight of 3-PGK by gel
filtratioll
The Mr of EAC cell 3-PGK was determined by ge l
fi ltration using separately Sephacryl S-200 and
Sephadex G-IOO columns. The reference marker
proteins were BSA (M" 66,000), ovalbumin (M r,
45 ,000), chymotripsinogen
A
(M r, 25,000),
cytoc hrome c (M" 13,000). The buffer for gel
filtration was 50 mM triethanolamine-HCI , pH 7.4
containing 10 mM ~-me rcap toet h ano l and 10 mM
EDTA .
Deterlllillation of SlIbllllil lIIoleclllar weiglil of EAC
cell 3-PGK by SDS-PAGE
Subunit molecular we ight of EAC cell 3-PGK was
determined by SDS-PAGE llsing carbon ic anhydrase
(M r, 29,000), rabbit muscle GAPDH (Mr. 36,000)
ovalb umin (M r, 45,000) and BSA (M" 66,000) as
reference marker proteins .
Purification of 3-PGK from EAC cells
Extractioll oltlie ellZVllle
Packed EAC cell s (8 g) were suspended in 10 ml of
25 mM triethanolamine-HCI buffer, pH 7.4 co ntaining
5 mM ~-mercapto-ethanol and 5 mM EDT A and kept
at room temperature fo r 30 min . The suspension was
then coo led to 4°C and homogeni zed in a tightly
fitt ing glass- teflon Poller Elvehjem homogenizer with
25 up and down strokes . All operations we re ca rri ed
out at OAoC subsequ entl y unless mentioned
otherw ise . To the homogenate was added 8 ml of 50
mM triethanolamine-HCI buller, pH 7.4 co ntainin g 5
mM EDTA and 5 mM ~-mercaptoetha n o l (hereafte r
referred as "buller A") and cen tri fuged at 18,000xg
for 20 min. The supernatant was retained. The pell et
was re-su spended in 8 ml of buffer A and
homoge ni zed and centri fuged as abo ve. The pellet
was rejec ted. This supernatant was comb ined with the
previo us supernatant.
A JIll/ lUll iU/JI sulpliate fmcl iOlla IiOIl
The pool ed supernatant was subj ected to
(N H-/)2S0-l fracti onation . In thi s and in all oth er
(N H-l)2S0-j fractionation steps, th e pH of the
suspe nsion was maintai ned at 7.4 by dropwise
additio n of water diluted ( 1:1 ) NH-l OH. The protein
that precipitated at 55-90% (N H-l)2S0q satu ration was
dissolved in a minimum vo lume of buffer A and
dialyzed for six hI' in th e same buffer. The dialyzed
protei n was centri fuged to remove some sedi menl.
The supernatant was then subj ected to anoth er round
of (N H-lhSO-l fractionation. The preci pitate which
appeared at 55-90% (N H4 h SO-l saturati on was
dissolved in a minimum volume of buffer A.
Gel fillralioll
A portion of th e enzyme (0.7 ml) fraction was
applied on a Sephacryl S-200 column (75x 1.6 cm)
eq uilibrated prev iously with buffer A. The most
active fraction s wi th hi gh specific activ ity of the
enzy me were pooled and subj ected to (N H-lhSO-l
precipitation (0-90% saturati on). The precipitated
protein was dissolved in 1.2 ml of buffe r A and
filtered through a Sephadex G-100 co lumn (80 x 1.8
cm) equilibrated previously with buffer A. The most
acti ve fractions with hi gh specific activ ity of the
enzy me were pooled and th e vo lume was red uced to
1.1 ml by passing the enzyme so luti on through a
centri con 30 membrane filter. The enzy me fraction
was aga in app lied on a Sephadex G-IOO column
(80x 1.8 cm) eq uili brated prev iously with buffer A.
The most active fractions were pooled and
co nce ntrated to 1.3 1111 by centricon-30-membran e
filter.
l on- excha nge ch rolllOlograpllY
The enzy me fraction was applied on a DE AESephacell column (3 x 1. 2 em) equilib rated previou sly
wit h buffer A. The most active fractions of th e eluent
we re co nce ntrated by ce ntricon-30 me mbran e fi lter
and passed aga in through anothe r DEAE-S ephacell
co lumn (3 x 1.2 cm) prev ious ly equ ilibrated with
buffer A. The enzy me passed unadsorbed throu gh th e
DEAE-Sephacell column and the fractions containing
hi ghest spec ific activity of th e enzy me were poo led.
To the pooled enzyme fraction 50 mM Na-phosphate
buffer, pH 7.0 containing 2 mM EDTA and 2 mM ~­
mercaptoethanol were added and concentrated
through centricon 30 membrane filter. This was
repeated 3-4 times until th e enzyme beca me
eq uilibrated in the buffer. The vo lume of th e enzyme
so luti on after was hin g in thi s buffer was concentrated
to 0.6 ml and app li ed on a CM-cellu lose col umn (3 x
I cm) prev iously eq uilibrated in 50 mM Na-phosphate
buffer, pH 7 con taining 2 mM EDT A and 2 mM ~ ­
mercaptoethan ol. The enzyme also passed unadsorbed
through th e CM-ce llulose co lumn . The enzyme
335
MUKHERJEE ef al.: 3-PHOSPHOGLYCERATE KINASE FROM EHRLICH ASCITES CARCINOMA CELLS
11
fractions with hi ghest specific actlYlty were pooled
and concentrated by passing through a centricon 30
membrane filter and kept at -20 D C with 10% glycerol
for further studies.
Results and Discussion
The purification procedure for 3-PGK as described
in detail in 'Materials and Methods' and summarized
in Table 1 could be conveniently reproduced using
different batches of EAC cells . The enzyme fraction
purified after CM-cellulose column step was purified
876-fold as compared to the crude extract. The
specific activity at this stage was 169 (Table 1).
The purified 3-PGK showed a single band in nondenaturing PAGE (Fig. 1).
The EAC cell GAPDH was also separated during
this purification process specially during Sephacryl S200 column step. When the contents of the tubes
containing maximum specific activity of 3-PGK were
pooled and assayed for GAPDH, no activity of
GAPDH was found . The absence of GAPDH in these
tubes was further confirmed by addition of standard
GAPDH to the assay mixture and significant
reduction of NAD was observed in presence of GAP.
Similarly the eluents containing maximum specific
activity of GAPDH were also assayed for 3-PGK and
absence of the latter enzyme was observed. This was
further confirmed by the enzymatic activity of 3-PGK
on ly by the addition of known 3-PGK externally to
the assay mixture, which contained the eluent of
purified GAPDH.
Studies to investigate the possible hybridization of
GAPDH with 3-PGK
It had been shown previously that GAPDH and 3PGK of mung bean formed a complex, which
assumed a molecular weight of 86,000 1 Similarly,
these two enzymes of rabbit muscle formed a
°.
complex of molecular weight 82,000 • It had also
been reported that formation of GAPDH-3-PGK
complex was facilitated in presence of GAP, NAD
and Pi, and each of these compounds can individually
stabilize the GAPDH-3-PGK complex . So, to
investigate whether EAC cell GAPDH and 3-PGK
could be covalently linked, experiments were
performed in which protein cross-linking reagent
glutaraldehyde and also NAD were used.
Fig. 1 - PAGE in non-denaturing condition of 3-PGK purified
from EAC cells [Tubes 1 and 2 show PAGE in 7.5 % gel. Protein
applied on tubes I and 2 were 16!lg and 12 !lg respectivel y. In all
the tubes, the faint bands at the lower portion indicate the dye
front. The stain used was Coomassie brilliant blue. Migrati o n was
from top (cathode) to bottom (anode)]
Table I- Purification of 3-PGK from EAC cells
Steps
Crude
First (NH 4 hS04
Second (NH 4hS04
Sephacryl S-2OO
1st Sephadex G-I 00
2nd Sephadex G-loo
1st DEAE-Sephacell
2nd DEAE-Sephacell
CM -Ce llul ose
Total activity
(units)
Total protein
(mg)
Sp. activity
(units/mg)
Puri fication fold
Yi eld
(%)
116.7
603
52
0.193
1.83
2.78
5.85
17.24
23.8
52.4
63.1
169
1
9.48
14.4
30.31
89.3
123
272
327
876
100
8 1.4
7 1.4
57.1
42 .8
30.6
22.9
15.7
14.5
95
83.3
66.7
50
35.7
26.7
18.3
16.9
30
11.4
2.9
1.5
0.51
0.29
0.10
336
IND IAN.I. 1310C H EM. BIOPHYS., VOL 3<), OCTOBE I{ 1002
For this experiment, 25 ~tg eac h of purifi ed
GA POH and 3-PGK from same batch of EAC ce ll s
were incubated in presence o f 0.05 % gl utaraldeh yde
for 120 min at -1 0c. Artcr th e incubat ion , PAG E was
perfonned in a non-denatu rin g cond iti on wi th an
app ropria te aliquot from the incubat ion mi xture as
al so with GAPDH and 3- PGK. It had been observed
that two dis tin ct band s were present in th e lane on
whic h the aliqu ot from th e inc uba ti on mi xture wa s
applied . These two band s corresponded w i th
respec ti ve band s of GAPOI-! and 3- PG K indicating
that no assoc iation took pl ace (Fig. 2).
Poss ibl e cross- linking o f EAC ce ll GAPO I-! and 3PGK wi th 0.02 mM of AD was also in ves ti gated in
a similar fashion as performed w ith gl utaraldehyde
and it had been observed that no hy brid izati on of
th ese two enzy mes of EAC ce ll s oCC UlTed (Fi gure not
presented).
Mo lecular weight of3-PGK
The molecular we ight o f purified EAC cell 3- PGK
was determin cd to be 47 ,000±2.000 by gel filtrati on
separat ely using Sep hacry l S-200 and Scphadex G100 co lu mns in three different buffers. Fig. 3 sho ws
thi s determina ti on In Sephadex G- 100 co lu mn.
Studies described in th e literature sho w that normal
ce llular 3- PGK I~ as we ll as 3- PGK purified fmlll
leukemi c granul ocy tes IS ha ve a molcc tIi ar wei ght of
47, 000 ± 3,000.
NUlllb er ofsubllllit(s) alld subullit lIlolecular weight
of3-PGK ofEAC cells
1n SOS - PAGE, th e EAC ce ll 3- PGK apparentl y
i ndicated th e presence o f a single subu nit (Fig . 4)
w hi ch is similar to that of 3- PGK pu rifi ed from oth er
so urcesl ~.
Th e subunit molecular weight of EAC cell 3-PG K
was also determin ed by SOS-PAGE from the pl ot of
log of mol ecular weig ht ve r slls relati ve mobility o f
2'4 r - - - - - - - -- -- - - -- - ---,
Cytochrome C
Chymotrypsinogen A
OvalblJmin
--EAC 3-PGK
BSA
~
1'4
I ' 0 ~--:-'-:-----'--_---'----'_---1. _ _ _.L.---l
4 '0
4'2
4'4
4 '6
4 'a
Log of mol. wt.
Fig. 2 - PAGE in non-denaturing co nditi on to investi gate th e
poss ible crosslinking betwee n GAPDH and 3- PGK in presence of
0.05 % glutaraldehyde [All the tubes show non-denaturing PAGE
in 7.5% ge l. Tube J shows GAPDH and 3-PGK which we re
mi xed and in cubated in presence of 0.05 % glutara ldehyde, tube 2
and tu be 3 show GAPDH and 3- PGK respec ti ve ly w ithout
glutaraldehyde. Th e protein appli ed on tubes J, 2 and 3 we re 25
fi g. ! 0 ~t g and 5 ~l g respec ti ve ly. The band s at th e bottom indi ca te
th e dye front. Th e stain used was Coomassie brilliant blue.
Migrati on was from top (cath ode) to bottom (anode)]
Fig. 3 Determin ati on of molec ular we ight of 3-PGK by
Sephadex G-IOO colul11n [0.8 ml of 3- PGK so luti on conta ining
about 5 units of 3-PGK was applied on a Sephadex G- I 00 co lumn
(80 cm x 1. 8 cm), previously equ ilibrated with buffer A. The tl ow
rate was maintained at I 1111/22 min . Th e voi d vo lume (Yo) of the
co lumn was prev iously determined w ith blue dextran. T he rat io of
elution vo lume (Y c ) of th e respec ti ve proteins and the void
vo lume ( Yo) were pl otted against log of molecular weight of
reference marker proteins. The reference marker protein s used
we re
BSA
(M"
66,000),
ova lbumin
(M"
45 ,000),
chymot rypsinogen A (M " 25,000) and cytochrome c (M" J 3,000) J
337
M UKH ERJE E (' I III. ] -PHOSP HOGLYC ERATE KIN ASE FROM EHRLI C H ASC ITES CA RC INOMA CE LLS
..
2
"
5 ' O ~--------------------'
3 : '.
BSA
'3
~
....
,-
- - - - - - - ---
EAC 3'PGK
Ovalbumin
o 4 '6
E
~.
APDH (rabbi t
muscle)
'0
g
/
4 ·4
Carbonic
anhydrase
-'
:.J
4' 2
4~.L4---0-'·5---0J.·6---0.L
· 7L---O·L
8 ----'O''9---:-':I
:·O
Relative mobility
Fig. ~ - Subunit co mpositi on o f CAC cell ]- PGK rSDS- PAG E
was pcrformed on a sheet of 7.5 % po lyacrylamide gG I. In all the
lanes, the thin luwer bands indicate th e dye fro nt. The stain used
lVas Cou mass ie brill iant blue. The mi g ratio n was frum top
(cath ode) to bo tt o m ('lJ1 ude) . Lane I. GSA (to p band), ova lbumi n
(middle ba nd ). ca rbunic anh yd rase (bottom band): lane 2. ] -PGK
purified (6 pg prote in ) fro m EAC ed ls: lane ] . purified GA PDH
( 12 p g prote in ) from EAC cell s l
refere nce mark er prote ins (Fig. 5). The subunit
ITlo lec ular weigh t was detcrmined to be 47 ,OOO± 1,000
whi ch is similar to that or thi s enzy me purifi ed from
variou s oth er so urces I ~ .
Fig. 5 - Determin ati o n o f subunit mol. wt. u f EAC ce ll 3- PGK
ITh t! Illolec ular weigh t o f 3- PGK was determin ed from the plot of
log of mo l. wt. I'e r.w s re lati ve Illobilit y o f respec ti ve reference
mark er pro te ins. T he marker prote ins and purifi ed 3-PGK we re
ap pli ed on separate lanes in SDS-pol yacrylamide gel (7.5 '7< ) and
th e ir re lat ive mobilities with respect tLl th e dye front were
de termin ed . The reference marker prote in s used were I3SA (M ,.
66.0(0). ova lbumin (M,. 45.000), ra bbit Illu sc le GAPDH (M ,.
]6.000) and carbo ni c anh ydrase (M ,. 29.000)]
70
60
..,
0
x
pH OptilJlulJI
Fig. 6 shows th e activit y of EAC ce ll 3- PGK as a
fun cti on of pH . Thc EAC ce ll enzy me shows
maximuIll activity at pH 6.0-7 .5. At pH 5.-J., the
enzy me showed acti vit y whi ch was about 30% o r that
shown at pH 6.5 and 7.4 . At pH 8.0, th e enzy me
showcd acti vity whi ch was about 60% of that show n
at pH 6.5 and 7.4.
As reported in th e literature a trun cated p H acti vity
prorile with maximal acti vit y at pH 7.2-9.0 was
obse rved in case o r human 3-PGK puriricd rrom
normal R£3 C t7 . The pH pro ril es o r rabbit skel etal
mu scle and yeast 3-PG K showcd a broad optimum
bel ween pH 6.0 and 9. 225 .
A pparellt K,I/ valu es qj' EAC cell 3-PGK for th e
substrates 3-PGA alld A TP
Th e apparent Kill of' EA C cell 3-PGK was
dete rmin ed for 3- PG A and 1\ TP at pH 6.5 with 50
mM imidazo le- HCl buller. For thi s expe rim ent ,
purifi ed EAC cell 3-PGK after eM -cellul ose co lum n
50
c
E 40
.......
<t
<l
30
~
0
a:
-=
OL-__~____~__~L-__~__~~__~L-_ _
5'2
5'7
6 '2
6'7
7'2
7'7
8' 2
8'7
pH
Fi g. 6 - Ac ti, ity of Ei\C ce ll .1- PG K as a fun l' ti ll n of !II'
IOpti mum pH of 3- PCiK lVas detCl'mined lI sillg purifi ed EA C ee l.
3 ·PCi K after C M-cellul use puri fi cati on ste p (T abl e I).
(----J.
(t.- t. ). (e-_ ) and (O-D) represe nt th e acti vity of 3-PGK in
sodiu m ,.lCetate-acGtic acid . imidazo le- HC I, tri ethan o lamine- HCI.
Tri s-HC I buffers. respecti vel y al th e indi cat ed p H. Other
co ndition s of the assay an~ desc ribed in tlK "M,ll crials an d
Methods" secti on I
INDIAN 1. BIOC HEM. BIOPHYS., VO L. 39 , OCTO BE R 2002
338
step (Tabl e I ) was used. The substrate saturation
c urve for both the substrates (for 3-PGA and ATP,
Fi g. 7 A and B respectively) follo wed a typical
Mi c haelis Me nten kine tics. The apparent Km from the
Linewea ver Burk plot was determined to be 0.25 mM
fo r 3-PGA (7 A, inset) and 0. 1 mM for ATP (Fig. 78 ,
inset).
The re ported Kill values of 3-PGA and ATP for the
yeast en zyme at pH 6 .9 was 0.20 mM and 0. 11 mM
res pecti vely. The apparent KI11 of 3-PGA and ATP for
th e rabb it skeletal muscl e en zyme at pH 7 was 1.22
s
mM a nd 0.48 mM respecti vell .
activ ity of 3-PGK at different co ncentrations of
MgS04 and MnS04. The acti vity of E AC cell 3-PGK
was neg ligible w ithout Mg2+ co mpared to their
activity at optimum concentration of M g2+, which is
0.8 mM for EAC cell.
The effect of other divalent ions Zn 2 + and C0 2+ on
EAC cell 3-PGK could not be studi ed because of
inhibition of EAC ce ll GAPDH , the co up ling enzy me
used for assay of 3-PGK was in acti vated by C0 2+ and
2
Zn +. With I mM ZnS04 almost complete in hibition
of EAC cell GAPDH was observed and 65 %
inhibiti on of EAC cell GAPDH was observed w ith
I mMCoCb.
f3-
Stability of EAC cell 3-PGK ill presellce of
mercaptoethalloi alld EDTA
Effect of A DP 011 EAC ceIl3-PGK
We have sys te mati cally investi gated the stability of
3-PGK in prese nce of vari ous reagents and fo und that
the e nzy me was optimall y stable in presence o f I mM
E DT A a nd 5 mM ~-m e rcaptoeth a nol.
Effect of MgS04 alld MIlS04 Oil 3-PGK
It had bee n reported th at 3-PGK requires the
presence of either Mg2+ or Mn 2+ for full activiti 4.
Both mu sc le and yeast kinases require Mg2+ or Mn 2+
(app rox . 2 mM)
for full
acti vity.
H igher
co nce ntrati o ns of th e meta l io ns were sli ghtly
inhib itorl 9 . So, the effec ts of Mg2+ and Mn 2+ on 3PGK fro m EA C ce ll were studi ed . Tab le 2 shows th e
Since ADP is a product of 3-PGK catalyzed
reacti on of g lyco lysis from reverse directio n and thi s
enzy me is frequ ently assayed from thi s directi on, we
have tes ted the effec t o f this purin e nucl eotide on th e
acti vity of 3-PGK of E AC cells. Other inves ti gators
had also prev iously stud ied the effec t o f A DP and
other nucl eotides on thi s enzyme puri fied from other
sources IS.2S .
Fi g . 8 presents the effect of diffe re nt co nce ntrati o ns
of ADP at fix ed concentrations of the substrates ATP
and 3-PGK and also of MgS04. At concentrati ons of
0.1 mM and 0.6 mM ADP, the ac ti vity of th e e nzy me
was found to be inh ibited to the ex te nt o f 26% and
IT)
40
60
(A)
IS?
Ir--,
50
n')
a
.....
30
><:
40
'"
~
""t
I
-3
0 '12
0 '08
\l 0 '04
.... ".b
Q::
'PS?
20
ON!
.... >.c;
0 ' /5
·s
0 '12
~
0'09
.......
-.3
0 '06
Q::
1 ~ 0'03
..... '1l
1.---..
~
~
'-'
<U
ru 10
Q::
0
/
20
40
I
3-P6 A
0
0 '02
0 '0 4
0 '06
3 -PGA (mM)
60
(8)
0 '/ 6
·S
~
"
·s
0 '2 0
.... >.c;
80 100
0
20
1
ATP
30
40
60
80
(m MF'
•
20
10
(MT'
m
0 ' 08
0
0 '02
0 '04
0 '06
0 '08
0 '10
0 '/2
A TP (mM )
Fig . 7 - De te rmin at io n o f Kill o f 3-PGA and AT P for th e e nzy me 3-PG K of EAC ce lls [Assays were do ne us ing 50 mM imidazo le-HC I
bulTe r, p H 6 .5 in presence o f op timum co nce ntra ti o ns o f MgS 0 4 (0. 8 mM). (A): With va ry in g co nccntrati o ns o f 3-PGA and satu ratin g
co ncentra lio n o f I mM ATP. (B ): With vary ing co ncentratio ns o f ATP a nd saturating conce ntrati o n o f 2.5 mM 3-PGA. III both A a nd B.
Ih e inse t shows Lin ewea ver-Burk pl o t fo r th e sa ille d ata. T he res ults are ave rages o f fi ve sets o f experim e nt s]
MUKHERJEE el al. : 3-PHOSPHOGL YCERATE KINASE FROM EHRLICH ASCITES CARCINOMA CELLS
Table 2 MgS04 or MnS04
(mM)
339
Effect of various concentrations of MgS04 on the activity of EAC cell 3-PGK
Activity of EAC cell 3-PGK
with MgS04 (M/min) x lO'
% Activity (EAC cell
3-PGK)" with MgS04
Activity of EAC cell 3-PGK
with MnS04 (M/min) x l0 3
% Activity (EAC ce ll
3-PGK)b with MnS04
0
3
4
3
3
0.1
24
31
NOc
NO c
0.2
46
59
6
7
0.5
70
90
92
100
0.8
78
100
88
96
67
86
80
87
2
59
76
63
68
5
53
68
23
25
8
38
49
17
18
10
30
38
II
12
This experiment was carried out with purified 3-PGK of EAC cells (Table I). Assay of 3-PGK and other experimental conditions are
described in "Materials and Methods" section.
" The maximum ac tivity of EAC cell 3-PG K at 0.8 mM ofMgS04 was considered as 100%.
b The maximum activity of EAC cell 3-PGK at 0.5 mM of MnS04 was considered as 100%.
C NO - Not determined.
The K; of ADP for EAC cell 3-PGK was calculated
from the intersect of the extrapolated lines to the
abscissa and found to be 0.175 mM. A similar K;
value was obtained for ADP with 3-PGA using a
Dixon plot (Fig. 9Aii) and from the plot of slope of
the reciprocal plot versus concentration of ADP (Fig.
9Aiii). The non-competitive nature of the binding
suggest s that ADP does not bind to 3-PGA binding
site.
60
...
0
50
)(
c:
40
E
"-
«
30
<l
~
~
20
a
0::
10
0
0'3
ADP(mM)
Fig. 8 - Effect of vary ing concentrations of AOP on the activity
of EAC cell 3-PGK at fixed concen trati ons of ATP, 3-PGA and
MgS04 lThe assay co nditi on was that of usual assay mixture for
3- PGK as de sc ribed in "Material s and Methods" secti o n]
55 % respectively. The inhibitory effect of ADP was
further pronounced in presence of IIlcreasing
concentration of MgS04 (data not presented).
Fig. 9Bi shows the competitive nature of the
inhibition of ADP with respect to ATP at pH 6.5. The
K; of ADP for EAC cell 3-PGK was calculated from
the intersect of the extrapolated lines to the abscissa
and found to be 0.08 mM. A similar K; value was
obtained for ADP with ATP using Dixon plot (Fig.
9Bii) and from the plot of slope of the reciprocal plot
versus concentration of ADP (Fig. 9Biii). The
competitive nature of the binding suggests that ADP
binds to the ATP binding site.
/!.jfect of AMP and NAD Oil 3-PGK of EAC cell
Kinetics of inhibition of EAC cell 3-PGK by ADP
with various cOllcentrations of the substrates
Since ADP has significant inhibitory effect on EAC
cell 3-PGK, we studied th e effect of different
concentrations of ADP on thi s enzyme at various
co ncentrations of 3-PGA and ATP, the substrates in
the reverse reaction of glycolysi s.
Fig. 9Ai shows the non-competi tive nature of th e
inhibition of ADP with respect to 3-PGA at pH 6.5.
Since we have observed that ADP has significant
inhibitory effect on 3-PGK of EAC cells at low
concentrations, we were interested in studying the
effect of other two nucleotides AMP and NAD on
EAC cell 3-PGK. At lower concentration of I mM,
AMP and NAD were found to inhibit the enzymatic
activity to the ex tent of 3% and 6% respecti vely. At
hi gher concentration of 10 mM AM P, 50% inhibition
of EAC cell 3-PGK was noted. A similar inhibitory
IND IAN J. BIOC HEM. BI O PHY S., VOL. :1<). OCTOB ER 20()2
340
ADP ( mM )
018
(A)
(il
ADP (mMl
(B1
(0'4)
(0 4 )
(i)
0"6
014
010
0 '04
'0
0"02
x
~
30
40
50
) -PGA(mM)
(0'0 2)
( i i)
0" 8
'-e
0 "6
"'' 0
0 '/40"2
0u
(0'04)
0'10
e
«
<l
-I~
6
2
1fY
0
/'
(0'06)
0
0 '0 6
Vi
o
o
Ci..
-0
0 '08
0
QI
Q.
(iii)
0 "4
0'/0
4
(003)
0 '/ 6
~
-5
0 '0 4
0"2
0'3
0 '4
ADP(mM )
0
0 ',
0"2
0 '3
o
u
~
'"
.c
6
/
'"
0-
Vi
0"4
ADP(mMl
u
e
.9-
o
0'
0"
-,
ATP(mM)
0"12
u
40
30
(mM)
( ii)
o 'B
.9QI
20
1
ATP
"-
(iii)
0
a.
'0
E
X
u...
0 '/0
-AD?
1
J-PGA (mM)
E
-I]
0 '/ 2 -
(0'0 5)
'"' 0
20
'-
<t
<l
(0' 1 )
~
10
c
0'/4
~
0'0 8
0'06
"l
(02)
~
0"12
0',
02
0 '3
0 '4
AD P( mM )
0 '1
ADP( mM)
l'ig . 9 - Dctc: rll1in ati on o f K, o f AD !' for CAC ce ll :I- I'GK 1(1\ ) : Inhihiti o n by ADP wit h vary ing co nce ntrati ons o f :I-pC;A: In ( i)
ac ti \' ities of :\ -PGK at AD P co nce ntrati ons 0 (0 - 0). O.OS 111M (0-0 ). 0. 1 111M (~-6). 0.2 111M (e--e ). and 0.4 111M ( ....- .... ). (ii ) and
(iii ) show the Di xo n pltli and slope o f the rec iproca l plot \ '(' 1".1" /1.1" ADP co nce nt rati o ns rcs pec ti ve ly fOl" ob ta ining K,. The co nce ntr:lIi o ns of
AT!' and MgSO.1 were I 111M and O.X Ill M respecti ve ly. ( B): Inh ibiti on by /\DP wit h v:lryin g clHlCc ntrat io ns of /\T I' : In Ii). acti vit ies o f :\I'CiK at 1\1)1' conce lllr;lIions 0 (e ---e ). O.OS 111M (~-~) , 0. 1 111M (0 - 0 ),0. 2 111 M ( .... - .... ) and 0.4 111M (0 - 0 ). (ii ) and (ii i) ,how the
Di xo n plot and slope of the rec iprocal plot \·('/"S/I.I" AD!' co nce ntrati ons res pecti ve ly for ohta ining K,. T he ClHlCelllr;lli l)nS of :\ -PCl A allli
l\ll gS0., werc 2.S 111M and O. X 111M re specti\'c ly. All assays were don e at fi ll ().S. The rcsul ts arc ;Iwrages o f fi \'e sC ls o f e.\pe rillle nt-;.
Othcr co ndit ion s of th e assay arc dcscribed in "Materi al s and Meth ods" sec ti o nl
elTeci Or AMP and NA D had been obse rved in th e
2
case o r 3- PG K of ra bb it skeletal mu sc le "'.
Several
glyco ly ti c
intermediates,
g lucose-6ph osphate,
rru ctose-6-phosphate,
rru ctose- I,6bi phosphate, phosphoeno l pyru vate, pyruva te and
lactate having structural simil arities w it h and/or
rclated Illelabolites or one or the substrates or 3- PGK
i .e . 3- PG A were tested ror th e poss ible crrcct on th e
activit y or thi s enzy me. However, none or these
l1letabolites had any errcct on th e acti vit y or 3- PGK at
a conce ntration or I mM. It had been observed
previou sly that none or th ese metabolites up to
co nce ntrati oil of 20 IllM had any effect on yeast and
25
rabbi t l1lu sc le 3- PG K
Ci trat e. the ri rst reacti on
product of the TCA cyc le al so d id not have any erfect
on the activit y of CAC ce l l 3- PG K at phys iolog ical
pH of 7.4 up to a concent rati on of 25 IllM. However,
about 40% inhibiti on o f acti vit y was noted a( 50 IllM
con centrati on of ci trate.
Th e present paper describes th e purificati on o r 3PGK from EAC cc ll , a rapidl y grow ing, hi ghl y
dedilTerentiated mali gnant ce ll. The limited stud y th at
has been made he re on th e propert ie s o f thi s enzyme
ane! al so previou s studi es of thi s eli zYllle of se veral
lH
sho\\' no
normal cell s and olle malignant ce lJ
important di rrerence in the propcrti e~ of til is enzyn1L'
or normal and malignant ce l ls. In so me malignan t
ce ll s. thc presence of an isozy me or 3- PGK had bcen
reported I') HO\vever, th ere was no report in th e
literature th at th e properties of thi s isozyme an:
significantl y different from that oj 3-PG K purifi ed
from variou s normal ce ll s.
Th e res ults presented i n th is PJP'r also clearly
indi cat e that in EAC ce l ls, the two giyco ly tic enzymes
GA PDH and 3- PGK arc not associated.
MUKHERJEE el III.: :1- I'II 0S PIIOC; L YCERATE K I ASE f'IWM EIIRU C II ASC ITE S CA RC INOM ;\ CELLS
From the res ults or all th ese studi es i t can be
co ncluded th at 3- PG K ha s no maj or ro le in mal ignant
aberrati on ane! thi s enzy me is pos sibl y not upregulatee!lo synthesi ze hi gher amount o r ATP in these
cells. On th e oth er hand, it seems likely that enhan ced
catalyti c activity or GAPD H or mali gnant ce ll s
provides hi gher am ount o r th e reducing equi va lent
NAD H . Sin ce NADH IS a substrat e o f bo th
mitochond ri al complex I and lactic dehydrogenase,
thi s hi gher amount or
ADH may sti mulate both
mitocho ndrial
ox ida ti ve
phosphoryla tion
and
glyco lys is 111 mali gnant ce ll s thu s au gmenting th eir
ATP pool.
Acknowled ge ment
This wo rk was spon sored by gran ts f ro m Council
or Sc ientifi c & Indu stri al Resea rch, Gov!. o f India,
India.
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