Vol. 66, No. 1, 1976
BIOCHEMICAL
KINETIC
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PROPERTIES OF PYRUVATE KINASE ISOLATED FROM RAT HEPATIC TUMOURS
Michael
G. Irving'
and John
F. Williams2
1Institute
of Radiotherapy, The Prince of Wales Hospital Randwick, N.S.W. 2031, Australia. 2 Department of Biochemistry, School of General Studies, The Australian National University, Canberra, A.C.T. 2600, Australia. (Address all correspondence to Professor
Received
October
J.F.
Williams)
16,1975
demonstrate the existence of L and M forms SUMMARY. The results --of pyruvate kinase in rat hepatomas. Tumours were induced by feeding The kinetic properties of the L-type tumour N-Nitrosodiethylamine. enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE celluloseSephadex 6200 chromatography (Sp. activity 41 units/mg). MgADP-/ADP'of 20/l gave optimum activity for both the intrinsic and F1,6di-P ATP did not inhibit the enzyme. Alanine (2.5 mM) stimulated reactions. caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or Fl,Gdi-P stimulated reactions with PEP concentrations as high as 10 ti. INTRODUCTION: hepatomas, different using
it
Following
from
either
in certain
in foetal Few studies
kinase
isolated
kinase
of the
different
from
rat
observed
electrophoresis hepatomas
rapidly
that that
kinase
from
Novikoff
enzyme may be a new isozyme, forms
a unique A number
of the enzyme.
pyruvate
kinase
of investigators an isozyme
migrated
like
Criss isozyme
(3,4,5)
of pyruvate the
form
(2) in have
kinase
of this
enzyme
liver.
have been reported from
this
or muscle
hepatomas.
shown by means of gel
found
liver
focusing
differentiated
was present
the
of pyruvate
(1) that
was suggested
isoelectric
poorly
studies
liver
tumours.
growing
the L-form
of the
hepatoma
enzyme found
kinetic
properties
of
(6)
the
It was reported
3924A had kinetic in normal
liver
that
pyruvate pyruvate
properties and also
from
the
BIOCHEMICAL
Vol. 68, No. 1,1976
normal
muscle
was not possible not
Tumours
activated induced
in
rats
hepatic
rats
increases
in the
by the
hepatic
this
concentration
pathway.
paper
reports
the isolation
tumours
induced
in rats
hepatic
The unusual kinase
kinetic
response
to increasing
the altered lower
administered
Male
for
16 weeks,
the
animals
were
involved tumour
Wistar
liver
procedures in these
were
The results specific
et aZ.
(9).
as described
following reported
using
in Table
1 show that
(6)
that
the
of the L-form
DEAE-cullulose
of the
rat
liver
kinase
tumour
pyruvate
suggests flux
150-2009)
of 50 mg/l
tumours
were
that
in the
tissue
features
of tumours
All
of the experimental
by Irving
was a 320-fold
G-200
of pyruvate
tumour
examined
by the
& Williams
increase pyruvate
in the kinase It
from
used
pyruvate
by Irving
isolated
of
procedures
chromatography.
kinase
285
were
tumour
there
non-
preparation
hepatic
described
liver
before
before
from
(10).
procedures
of rat
diet,
& Williams
of rat
were
in the drinking
dissected
of necrotic
and Sephadex
the L-form
of pyruvate
on a carcinogen-free
Two forms
isolated
activity
suggesting
N-Nitrosodiethylamine.
body weight
Hepatic
RESULTS AND DISCUSSION: kinase
and
of the glycolytic
enzyme may increase
by 6 weeks
Histological
were
in the
(8),
segment
of two forms
(initial
at O°C and freed
studies
decreases
occurred
lower
at a level
sacrificed.
of SchmZhl
In
pathway.
rats
followed
homogenate.
in the
of this
N-Nitrosodiethylamine
water
marked
(7).
of phosphoenolpyruvate
of the glycolytic
METHODS:
N-Nitrosodie-
and triose-phosphates
of the L-form
properties
it
enzyme was
of glycolysis
administered
concentrations
kinetic
segment
rate
of lactate
of an enzyme or enzymes
from
tumour
carcinogen
of phosphoenolpyruvate
hepatic
this
carcinogen,
the activation This
that
enzyme
1,6-diphosphate.
to have a high
administered
concentrations
of the tumour
conclusively by fructose
have been shown
preneoplastic
of the instability
to demonstrate
allosterically
thylamine
Because
enzyme.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
had been the trans-
(10).
Vol. 68, No. 1, 1976
Table
1.
BIOCHEMICAL
Purification
AND BIOPHYSICAL
of Rat Liver
Tumour
RESEARCH COMMUNICATIONS
Pyruvate
Kinase
Extraction, isolation and assay procedures were as described by Irving & Williams (1973). The enzyme reaction was started with the addition of phosphoenolpyruvate to a final concentration of 1.0 mM. Values are for an extract obtained from 28 g of tumour tissue.
Volume (ml)
Step Crude
extract
O-45%-satd. fraction
(NH,)~SO~
(NH~)~S~~
fractionation
DEAE-cellulose Sephadex
Morris
hepatoma
at 37'C),
and it
was for
fraction
was deleted
addition
of sucrose
solutions
appeared
purification
536
0.7
72%
5.4
3.0
305
105
2.9
59%
22.3
17
163
5
32.6
31%
251
9
105
41.5
20%
319
5
30
60
0.5
6%
3.8
5
90
64.2
1.4
17%
10.8
fractions.
of the
This
of the enzyme DEAE-cellulose
until
The activation
with
unstable
that
heat
rabbit
used here
fraction from
rabbit
liver
the pH was raised phosphoenolpyruvate of the L-form
of rat
286
(NH4)2S04
However,
the
mM) to all
as was seen
buffer the
in the partial
(10).
sedimented showed
(tLZ = 4 min
to the enzyme because
as marked enzyme
1
of the
(10). (0.25
some protection
liver
in solution treatment
M) and dithiothreitol
of the enzyme
isolated
2.53
the procedure
was not
enzyme
kinetics
reason
to afford
The M-fraction
Menten
375
3924A was very
from
of enzyme activity
100%
Purification
20
this
(0.25
0.13
Yield %
4000
(NH4)2S04
plantable
Specific Activity (units /vi)
520
(NH~)~s~~
55-65%-satd. fraction
Total Protein 1 (mg)
130
G-200
4:,,5,5;T;;td.
loss
Total Activit (units
in the 45-65%-satd.
similar in that to 8.5, as the liver
properties it
did
and it variable tumour
(NH4)2S04
to the M-form not absorb
exhibited
to
Michaelis-
substrate. pyruvate
kinase
by
BIOCHEMICAL
Voi.68,No.1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
43 &I LPhosphoenolpyruvatel
(mM)
Fig. 1. Influence of Fructose 1,6-Diphosphate on the Relationship Between the L-Form of Rat Liver Tumour Pyruvate Kinase Activity and Phosphoenolpyruvate Concentrations at a Constant MgADP- Concentration (4 mM-MgADP-, MgADP-IADPL= 5O:l) The reaction mixture including enzyme was incubated at 30°C for 10 min, and the reaction was started by the addition of phosphoenolpyruvate and fructose 1,6-diphosphate. All concentrations of phosphoenolpyruvate were studied separately, and were not due to incremental additions of phosphoenolpyruvate to the assay mixture. Each experimental point represents quadruplicate determinations for each phosphoenolpyruvate concentration. 0, no fructose 1,6-diphosphate; 0, 0.1 mM-fructose 1,6-diphosphate. Standard errors for each experimental point are shown.
Mg2+ and ADP2- was studied A MgADP-/ADP2-
ratio
6.0 mM gave optimum
of 2O:l activity
diphosphate-stimulated using
was also
and 0.36
1,6-diphosphate-stimulated
a final
The effect on the
velocity
being
concentration
were
The effect
MgADP-/MgADP2' were
obtained reactions,
recorded.
All
of increasing
concentrations
of the L-form
of rat
liver
287
ratio for
of 1,6-
carried
out
of increasing
both
with
of 5O:l the intrinsic
Km values
experiments
of phosphoenolpyruvate
(10).
and fructose
experiments
1 mM and 4 n+l.
curves
& Williams Mg2+ concentration
the intrinsic
These
of
Saturation
mM respectively,
using
both
reactions.
of Irving
to a total
of MgADP- at a fixed
studied.
and fructose
the method
equivalent for
MgADP- concentrations
the concentration
out
using
were
of 0.25
of 0.4 carried
mM.
of phosphoenolpyruvate tumour
pyruvate
kinase
is
shown
Vol.68,No.1,1976
in Fig. at low
1.
BIOCHEMICAL
A sigmoid
response
concentrations
of the rabbit
liver
were
pattern
enzyme,
not
observed
coefficients
and between
rat
the intrinsic
the
for
for
enzyme
It
capacity
reflected
in activation liver
a Km value
This
reaction
of the enzyme.
were
for
in excess
1,6-diphosphate-stimulated
mM and Hill These
the
reaction.
occurred
of 2.9
indicate
that
of the
to catabolize
of the enzyme the
tumour
phosphoenolpyruvate lower
segment
with
both
with
Km
and 1.1 there rat
of phosphoenolpyruvate
that
kinase
reactions;
of the L-form
appear
reaction,
of pyruvate
kinetics
coefficients
of the L-form
of the
intrinsic
of the L-form
results
response
would
of 5 mM reaction.
the fructose
saturation
kinetic
is
liver
a marked tumour
in comparison isolated
from
enzyme has a greatly and this
may be
of the glycolytic
pathway
tumours.
The response concentrations
and saturation
obtained
properties
and fructose
1,6-diphosphate-stimulated
concentrations
liver.
to increase
1,6-diphosphate-stimulated
kinetic
the L-form
intrinsic
3 and 4 were
recorded.
increased
in rat
intrinsic
revealed
to increasing
unlike
preparations
that
in the
rat
different
liver
being
the
of the enzyme
10 mM-phosphoenolpyruvate.
parameters
the kinetic
normal
of both
of the kinetic
of 0.7 mM and 0.15
difference
the L-form
continued
the fructose
and fructose
respectively
with
for
1 and 2.5
normal
values
four
between
Reinvestigation from
with
with
Km values
as was the Km value Hill
for
However
the activity reactions
was obtained
Approximate
was obtained
of phosphoenolpyruvate.
1,6-diphosphate-stimulated kinetics
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the M-form
of the
of phosphoenolpyruvate of 0.54
mM.
tumour
enzyme to increasing
gave Michaelis-Menten
No activation
by fructose
kinetics
1,6-diphosphate
with was
observed. Study the
rat
liver
concentration acitivitywas
of the effect tumour of 0.25 recorded
of pH on the
pyruvate
kinase
mM revealed at pH 6.5,
that
activity
of the L and M forms
at a constant for
phosphenolpyruvate
both
enzyme forms
and the addition
of fructose
288
of
the maximum 1,6-diphosphate
BIOCHEMICAL
Vol. 68, No. 1, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 2. Effect of ATP and Alanine an the Relationship Between the L-Form of Rat Liver Tumour Pyruvate Kinase Activity and Phosphoenolpyruvate Concentrations in the Presence and Absence of Fructose 1,6-Diphosphate The reaction mixture, including enzyme, was preincubated at 300C for 5 min, and then ATP or alanine was added to the cuvette and allowed to incubate at 300C for a further 5 min before the reaction was commenced by the addition of phosphoenolpyruvate or phosphoenolpyruvate and fructose 1,6-diphosphate. All concentrations of phosphoenolpyruvate were studied separately and were not due to incremental additions of phosphoenolpyruvate to the assay mixture. Each experimental point represents triplicate determinations for each phosphoenolpyruvate concentration. 0, phosphoenolpyruvate with no fructose 1,6-diphosphate; 0, phosphoenolpyruvate plus 0.1 mM-fructose 1,6-diphosphate; A, phosphoenolpyruvate plus 2.5 mM-ATP; A, phosphoenolpyruvate, 2.5 mM-ATP plus I, phosphoenolpyruvate plus 2.5 mMtIiInNMfructose 1,6-diphosphate; ; a , phosphoenolpyruvate, 2.5 mM-alanine plus 0.1 mM-fructose Reaction conditions w$ye as described in the 1,6-diphosphate. Experimental Section except that the Mg concentration was doubled to give a total value of 33 mM.
did
not
affect
raised,
the pH optimum
the shapes
of the curves
became increasingly but
differed (10)
where coefficient
increased
fructose
not
was marked observed
with
for
the
it
of the enzyme.
intrinsic
become more susceptible
observed
with
was observed for
concentrations. normal that
rat
liver
reaction
of the
L-form
These (11)
results
and normal
increased,
rabbit
the value and there
as the pH increased.
1,6-diphosphate-stimulation
the L-form
pH was
1,6-diphosphate
as the pH was raised,
the intrinsic
As the
nH = 2.2 at pH 7.7)
to fructose
1,6-diphosphate-stimulation fructose
reaction
(nH = 1.9 at pH 6.5;
phosphoenolpyruvate
those
Hill
there
not
at low from
liver the
sigmoidal
the enzyme did
activation
of the L-form
of pyruvate 289
kinase
either
was Furthermore,
at pi-l 6.5 which isolated
of
from
was normal
Vol. 68, No. 1, 1976
rat
or rabbit
the
tumour
response with
BIOCHEMICAL
liver.
These
enzyme of the
from
non-saturating
reported
solely
adjusted
show that L-form
that
tumour
liver
L-form
of the
(10).
activation
(0.25 occurring
Alanine
also
stimulated
in the
of the tumour
The kinetic
inhibition pyruvate Alanine
patterns
in muscle
reaction
caused
that
the
some steric
a full
occurred
the L-form
of normal
inhibitor
of the phosphoenolpyruvate
was relievedwith concentration
of the fructose
appear
some (1 mM).
1,6-diphosphate-
presence hindrance
expression
pyruvate
of either
ATP or
to the L-form
of the fructose
1,6-
caused
liver
by interactions
tumour kinase
(13)
pyruvate
of ATP and alanine kinase
and the M-form
were
identical
of rabbit
with to those
liver
Even in the presence caused
kinase was still
the
enzyme activity.
of the rat
(10).
would
for
2
A slight
at low
inhibition
in Fig.
some activation
(1 mM).
a 60% inhibition this
effecters
reaction
and in fact
with
by
of all
intrinsic
phosphoenolpyruvate
enzyme preventing
diphosphate-stimulated
the M-form
causing
assay mixture
the
control
inhibition
The results
mM) was an effective
a 10% inhibition It
an inadequate
to prevent
assay.
not observed
mM), although
reaction.
with
by ATP may be
1,6-diphosphate-stimulated
at higher
caused
kinase
concentrations
(2.5
enzyme
10 mM-phosphoenolpyruvate.
and solutions
kinase,
a feature
Alanine
tumour
concentrations
kinase
pyruvate
(4%) of the fructose
rabbit
concentrations
of Mg2+ was added
phosphoenolpyruvate
of ATP,
phosphoenolpyruvate
together
pH before
distinguished to the unique
with
of Mg2+,
also
related
of pyruvate
mM) in no way inhibited
liver
in the presence
found
inhibition
which
are probably
observed
by the chelation
at high
inhibition
alanine
being
to the experimental
of rat
occurred
enzyme
to increasing
amount
ATP (2.5
properties
of Mg2+ by ATP,
An equivalent
ATP caused were
enzyme
to chelation
of pH.
normal
kinetics
Wood (12) attributed
two kinetic
the
tumour
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by chelation was inhibited
pyruvate
of additional Mg2+ to counteract any 2+ of Mg , the M-form of rat liver tumour
by ATP (26% at 1 mM-phosphoenolpyruvate).
the more effective
inhibitor
290
causing
a 43% inhibition
BIOCHEMICAL
Vol. 68, No. 1,1976
in the presence did
of
not alleviate
the
of pyruvate
inhibition
by either
report
referred
kinase
could
chromatography
was not observed
enzyme studied
here.
enzyme are consistent capacity
J.F.
However with
to catabolize
regulation
of
its
Fructosel,6-diphosphate
1 mM-phosphoenolpyruvate.
The unpublished isozymes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or ATP.
to by Weinhouse be separated
(14)
the
those
kinetic
properties
of a new isozyme
by changes
the
three
of tumour
of the rat which
and is not in the concentration
ACKNOWLEDGEMENTS: This investigation Williams from the Australian Research
that
by DEAE-cellulose
in any of the preparations
phosphoenolpyruvate,
activity
alanine
liver
tumour
has an increased as sensitive
to
of effecters.
was supported by grants Grants Committee.
to
REFERENCES: 1.
1:: 11.
F.A., Adelman, R.C., Lo, C.H., Morris, H.P. & Weinhouse, S. Cancer Res. 28: 1897-1900 (1968). Criss, W.E. Biochem. Biophys. Res. Commun. 35: 901-905 (1969). Bonney, R.J. & Potter, V.R. Fed. Proc. 31:1618 (1972). Knox, W.E., Farron, F. & Hsu, H.H.J. PrE. Amer. Assoc. Cancer Res. 13:135 (1972). Walker, P.R. & Potter, V.R. Advan. Enzyme Regul. 10: 339-364 (1972). Taylor, C.B., Morris, H.P. & Weber, G. Life Sci. 8:635-644 (1969). Heise, E. & Garlich, M. Exptl. Cell Res. 33: 289-300 (1964). Irving, M.G. Ph.D. Thesis, University of Ew South Wales, Australia. (1973). SchmZhl, D., Preussman, R. & Hamperl, H. Naturwisenschaften 47: l-2 (1960). Irving, M.G. & Williams, J.F. Biochem. J. -.131: 287-301 (1973r Rozengurt, E., Jimenez De Asda, L. & Carminatti, H. J. Biol. Chem.
12. 13.
Wood, T. Biochem. Biophys. Reynard, A.M., Hass, L.F.,
2.
3. 4. 5. ;:
8.
14.
Farina,
244: 3142-3147 (1969). __ 236: 2277-2283 (1961). __
Weinhouse,
S. Cancer
Res. Commun. 31: 779-785 (1968). Jacobsen, 0.0.X Boyer, P.D. J. Biol.
Res. -32: 2007-2016
291
(1972).
Chem.
BIOCHEMICAL
KINETIC
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PROPERTIES OF PYRUVATE KINASE ISOLATED FROM RAT HEPATIC TUMOURS
Michael
G. Irving'
and John
F. Williams2
1Institute
of Radiotherapy, The Prince of Wales Hospital Randwick, N.S.W. 2031, Australia. 2 Department of Biochemistry, School of General Studies, The Australian National University, Canberra, A.C.T. 2600, Australia. (Address all correspondence to Professor
Received
October
J.F.
Williams)
16,1975
demonstrate the existence of L and M forms SUMMARY. The results --of pyruvate kinase in rat hepatomas. Tumours were induced by feeding The kinetic properties of the L-type tumour N-Nitrosodiethylamine. enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE celluloseSephadex 6200 chromatography (Sp. activity 41 units/mg). MgADP-/ADP'of 20/l gave optimum activity for both the intrinsic and F1,6di-P ATP did not inhibit the enzyme. Alanine (2.5 mM) stimulated reactions. caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or Fl,Gdi-P stimulated reactions with PEP concentrations as high as 10 ti. INTRODUCTION: hepatomas, different using
it
Following
from
either
in certain
in foetal Few studies
kinase
isolated
kinase
of the
different
from
rat
observed
electrophoresis hepatomas
rapidly
that that
kinase
from
Novikoff
enzyme may be a new isozyme, forms
a unique A number
of the enzyme.
pyruvate
kinase
of investigators an isozyme
migrated
like
Criss isozyme
(3,4,5)
of pyruvate the
form
(2) in have
kinase
of this
enzyme
liver.
have been reported from
this
or muscle
hepatomas.
shown by means of gel
found
liver
focusing
differentiated
was present
the
of pyruvate
(1) that
was suggested
isoelectric
poorly
studies
liver
tumours.
growing
the L-form
of the
hepatoma
enzyme found
kinetic
properties
of
(6)
the
It was reported
3924A had kinetic in normal
liver
that
pyruvate pyruvate
properties and also
from
the
BIOCHEMICAL
Vol. 68, No. 1,1976
normal
muscle
was not possible not
Tumours
activated induced
in
rats
hepatic
rats
increases
in the
by the
hepatic
this
concentration
pathway.
paper
reports
the isolation
tumours
induced
in rats
hepatic
The unusual kinase
kinetic
response
to increasing
the altered lower
administered
Male
for
16 weeks,
the
animals
were
involved tumour
Wistar
liver
procedures in these
were
The results specific
et aZ.
(9).
as described
following reported
using
in Table
1 show that
(6)
that
the
of the L-form
DEAE-cullulose
of the
rat
liver
kinase
tumour
pyruvate
suggests flux
150-2009)
of 50 mg/l
tumours
were
that
in the
tissue
features
of tumours
All
of the experimental
by Irving
was a 320-fold
G-200
of pyruvate
tumour
examined
by the
& Williams
increase pyruvate
in the kinase It
from
used
pyruvate
by Irving
isolated
of
procedures
chromatography.
kinase
285
were
tumour
there
non-
preparation
hepatic
described
liver
before
before
from
(10).
procedures
of rat
diet,
& Williams
of rat
were
in the drinking
dissected
of necrotic
and Sephadex
the L-form
of pyruvate
on a carcinogen-free
Two forms
isolated
activity
suggesting
N-Nitrosodiethylamine.
body weight
Hepatic
RESULTS AND DISCUSSION: kinase
and
of the glycolytic
enzyme may increase
by 6 weeks
Histological
were
in the
(8),
segment
of two forms
(initial
at O°C and freed
studies
decreases
occurred
lower
at a level
sacrificed.
of SchmZhl
In
pathway.
rats
followed
homogenate.
in the
of this
N-Nitrosodiethylamine
water
marked
(7).
of phosphoenolpyruvate
of the glycolytic
METHODS:
N-Nitrosodie-
and triose-phosphates
of the L-form
properties
it
enzyme was
of glycolysis
administered
concentrations
kinetic
segment
rate
of lactate
of an enzyme or enzymes
from
tumour
carcinogen
of phosphoenolpyruvate
hepatic
this
carcinogen,
the activation This
that
enzyme
1,6-diphosphate.
to have a high
administered
concentrations
of the tumour
conclusively by fructose
have been shown
preneoplastic
of the instability
to demonstrate
allosterically
thylamine
Because
enzyme.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
had been the trans-
(10).
Vol. 68, No. 1, 1976
Table
1.
BIOCHEMICAL
Purification
AND BIOPHYSICAL
of Rat Liver
Tumour
RESEARCH COMMUNICATIONS
Pyruvate
Kinase
Extraction, isolation and assay procedures were as described by Irving & Williams (1973). The enzyme reaction was started with the addition of phosphoenolpyruvate to a final concentration of 1.0 mM. Values are for an extract obtained from 28 g of tumour tissue.
Volume (ml)
Step Crude
extract
O-45%-satd. fraction
(NH,)~SO~
(NH~)~S~~
fractionation
DEAE-cellulose Sephadex
Morris
hepatoma
at 37'C),
and it
was for
fraction
was deleted
addition
of sucrose
solutions
appeared
purification
536
0.7
72%
5.4
3.0
305
105
2.9
59%
22.3
17
163
5
32.6
31%
251
9
105
41.5
20%
319
5
30
60
0.5
6%
3.8
5
90
64.2
1.4
17%
10.8
fractions.
of the
This
of the enzyme DEAE-cellulose
until
The activation
with
unstable
that
heat
rabbit
used here
fraction from
rabbit
liver
the pH was raised phosphoenolpyruvate of the L-form
of rat
286
(NH4)2S04
However,
the
mM) to all
as was seen
buffer the
in the partial
(10).
sedimented showed
(tLZ = 4 min
to the enzyme because
as marked enzyme
1
of the
(10). (0.25
some protection
liver
in solution treatment
M) and dithiothreitol
of the enzyme
isolated
2.53
the procedure
was not
enzyme
kinetics
reason
to afford
The M-fraction
Menten
375
3924A was very
from
of enzyme activity
100%
Purification
20
this
(0.25
0.13
Yield %
4000
(NH4)2S04
plantable
Specific Activity (units /vi)
520
(NH~)~s~~
55-65%-satd. fraction
Total Protein 1 (mg)
130
G-200
4:,,5,5;T;;td.
loss
Total Activit (units
in the 45-65%-satd.
similar in that to 8.5, as the liver
properties it
did
and it variable tumour
(NH4)2S04
to the M-form not absorb
exhibited
to
Michaelis-
substrate. pyruvate
kinase
by
BIOCHEMICAL
Voi.68,No.1,1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
43 &I LPhosphoenolpyruvatel
(mM)
Fig. 1. Influence of Fructose 1,6-Diphosphate on the Relationship Between the L-Form of Rat Liver Tumour Pyruvate Kinase Activity and Phosphoenolpyruvate Concentrations at a Constant MgADP- Concentration (4 mM-MgADP-, MgADP-IADPL= 5O:l) The reaction mixture including enzyme was incubated at 30°C for 10 min, and the reaction was started by the addition of phosphoenolpyruvate and fructose 1,6-diphosphate. All concentrations of phosphoenolpyruvate were studied separately, and were not due to incremental additions of phosphoenolpyruvate to the assay mixture. Each experimental point represents quadruplicate determinations for each phosphoenolpyruvate concentration. 0, no fructose 1,6-diphosphate; 0, 0.1 mM-fructose 1,6-diphosphate. Standard errors for each experimental point are shown.
Mg2+ and ADP2- was studied A MgADP-/ADP2-
ratio
6.0 mM gave optimum
of 2O:l activity
diphosphate-stimulated using
was also
and 0.36
1,6-diphosphate-stimulated
a final
The effect on the
velocity
being
concentration
were
The effect
MgADP-/MgADP2' were
obtained reactions,
recorded.
All
of increasing
concentrations
of the L-form
of rat
liver
287
ratio for
of 1,6-
carried
out
of increasing
both
with
of 5O:l the intrinsic
Km values
experiments
of phosphoenolpyruvate
(10).
and fructose
experiments
1 mM and 4 n+l.
curves
& Williams Mg2+ concentration
the intrinsic
These
of
Saturation
mM respectively,
using
both
reactions.
of Irving
to a total
of MgADP- at a fixed
studied.
and fructose
the method
equivalent for
MgADP- concentrations
the concentration
out
using
were
of 0.25
of 0.4 carried
mM.
of phosphoenolpyruvate tumour
pyruvate
kinase
is
shown
Vol.68,No.1,1976
in Fig. at low
1.
BIOCHEMICAL
A sigmoid
response
concentrations
of the rabbit
liver
were
pattern
enzyme,
not
observed
coefficients
and between
rat
the intrinsic
the
for
for
enzyme
It
capacity
reflected
in activation liver
a Km value
This
reaction
of the enzyme.
were
for
in excess
1,6-diphosphate-stimulated
mM and Hill These
the
reaction.
occurred
of 2.9
indicate
that
of the
to catabolize
of the enzyme the
tumour
phosphoenolpyruvate lower
segment
with
both
with
Km
and 1.1 there rat
of phosphoenolpyruvate
that
kinase
reactions;
of the L-form
appear
reaction,
of pyruvate
kinetics
coefficients
of the L-form
of the
intrinsic
of the L-form
results
response
would
of 5 mM reaction.
the fructose
saturation
kinetic
is
liver
a marked tumour
in comparison isolated
from
enzyme has a greatly and this
may be
of the glycolytic
pathway
tumours.
The response concentrations
and saturation
obtained
properties
and fructose
1,6-diphosphate-stimulated
concentrations
liver.
to increase
1,6-diphosphate-stimulated
kinetic
the L-form
intrinsic
3 and 4 were
recorded.
increased
in rat
intrinsic
revealed
to increasing
unlike
preparations
that
in the
rat
different
liver
being
the
of the enzyme
10 mM-phosphoenolpyruvate.
parameters
the kinetic
normal
of both
of the kinetic
of 0.7 mM and 0.15
difference
the L-form
continued
the fructose
and fructose
respectively
with
for
1 and 2.5
normal
values
four
between
Reinvestigation from
with
with
Km values
as was the Km value Hill
for
However
the activity reactions
was obtained
Approximate
was obtained
of phosphoenolpyruvate.
1,6-diphosphate-stimulated kinetics
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the M-form
of the
of phosphoenolpyruvate of 0.54
mM.
tumour
enzyme to increasing
gave Michaelis-Menten
No activation
by fructose
kinetics
1,6-diphosphate
with was
observed. Study the
rat
liver
concentration acitivitywas
of the effect tumour of 0.25 recorded
of pH on the
pyruvate
kinase
mM revealed at pH 6.5,
that
activity
of the L and M forms
at a constant for
phosphenolpyruvate
both
enzyme forms
and the addition
of fructose
288
of
the maximum 1,6-diphosphate
BIOCHEMICAL
Vol. 68, No. 1, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Fig. 2. Effect of ATP and Alanine an the Relationship Between the L-Form of Rat Liver Tumour Pyruvate Kinase Activity and Phosphoenolpyruvate Concentrations in the Presence and Absence of Fructose 1,6-Diphosphate The reaction mixture, including enzyme, was preincubated at 300C for 5 min, and then ATP or alanine was added to the cuvette and allowed to incubate at 300C for a further 5 min before the reaction was commenced by the addition of phosphoenolpyruvate or phosphoenolpyruvate and fructose 1,6-diphosphate. All concentrations of phosphoenolpyruvate were studied separately and were not due to incremental additions of phosphoenolpyruvate to the assay mixture. Each experimental point represents triplicate determinations for each phosphoenolpyruvate concentration. 0, phosphoenolpyruvate with no fructose 1,6-diphosphate; 0, phosphoenolpyruvate plus 0.1 mM-fructose 1,6-diphosphate; A, phosphoenolpyruvate plus 2.5 mM-ATP; A, phosphoenolpyruvate, 2.5 mM-ATP plus I, phosphoenolpyruvate plus 2.5 mMtIiInNMfructose 1,6-diphosphate; ; a , phosphoenolpyruvate, 2.5 mM-alanine plus 0.1 mM-fructose Reaction conditions w$ye as described in the 1,6-diphosphate. Experimental Section except that the Mg concentration was doubled to give a total value of 33 mM.
did
not
affect
raised,
the pH optimum
the shapes
of the curves
became increasingly but
differed (10)
where coefficient
increased
fructose
not
was marked observed
with
for
the
it
of the enzyme.
intrinsic
become more susceptible
observed
with
was observed for
concentrations. normal that
rat
liver
reaction
of the
L-form
These (11)
results
and normal
increased,
rabbit
the value and there
as the pH increased.
1,6-diphosphate-stimulation
the L-form
pH was
1,6-diphosphate
as the pH was raised,
the intrinsic
As the
nH = 2.2 at pH 7.7)
to fructose
1,6-diphosphate-stimulation fructose
reaction
(nH = 1.9 at pH 6.5;
phosphoenolpyruvate
those
Hill
there
not
at low from
liver the
sigmoidal
the enzyme did
activation
of the L-form
of pyruvate 289
kinase
either
was Furthermore,
at pi-l 6.5 which isolated
of
from
was normal
Vol. 68, No. 1, 1976
rat
or rabbit
the
tumour
response with
BIOCHEMICAL
liver.
These
enzyme of the
from
non-saturating
reported
solely
adjusted
show that L-form
that
tumour
liver
L-form
of the
(10).
activation
(0.25 occurring
Alanine
also
stimulated
in the
of the tumour
The kinetic
inhibition pyruvate Alanine
patterns
in muscle
reaction
caused
that
the
some steric
a full
occurred
the L-form
of normal
inhibitor
of the phosphoenolpyruvate
was relievedwith concentration
of the fructose
appear
some (1 mM).
1,6-diphosphate-
presence hindrance
expression
pyruvate
of either
ATP or
to the L-form
of the fructose
1,6-
caused
liver
by interactions
tumour kinase
(13)
pyruvate
of ATP and alanine kinase
and the M-form
were
identical
of rabbit
with to those
liver
Even in the presence caused
kinase was still
the
enzyme activity.
of the rat
(10).
would
for
2
A slight
at low
inhibition
in Fig.
some activation
(1 mM).
a 60% inhibition this
effecters
reaction
and in fact
with
by
of all
intrinsic
phosphoenolpyruvate
enzyme preventing
diphosphate-stimulated
the M-form
causing
assay mixture
the
control
inhibition
The results
mM) was an effective
a 10% inhibition It
an inadequate
to prevent
assay.
not observed
mM), although
reaction.
with
by ATP may be
1,6-diphosphate-stimulated
at higher
caused
kinase
concentrations
(2.5
enzyme
10 mM-phosphoenolpyruvate.
and solutions
kinase,
a feature
Alanine
tumour
concentrations
kinase
pyruvate
(4%) of the fructose
rabbit
concentrations
of Mg2+ was added
phosphoenolpyruvate
of ATP,
phosphoenolpyruvate
together
pH before
distinguished to the unique
with
of Mg2+,
also
related
of pyruvate
mM) in no way inhibited
liver
in the presence
found
inhibition
which
are probably
observed
by the chelation
at high
inhibition
alanine
being
to the experimental
of rat
occurred
enzyme
to increasing
amount
ATP (2.5
properties
of Mg2+ by ATP,
An equivalent
ATP caused were
enzyme
to chelation
of pH.
normal
kinetics
Wood (12) attributed
two kinetic
the
tumour
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by chelation was inhibited
pyruvate
of additional Mg2+ to counteract any 2+ of Mg , the M-form of rat liver tumour
by ATP (26% at 1 mM-phosphoenolpyruvate).
the more effective
inhibitor
290
causing
a 43% inhibition
BIOCHEMICAL
Vol. 68, No. 1,1976
in the presence did
of
not alleviate
the
of pyruvate
inhibition
by either
report
referred
kinase
could
chromatography
was not observed
enzyme studied
here.
enzyme are consistent capacity
J.F.
However with
to catabolize
regulation
of
its
Fructosel,6-diphosphate
1 mM-phosphoenolpyruvate.
The unpublished isozymes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or ATP.
to by Weinhouse be separated
(14)
the
those
kinetic
properties
of a new isozyme
by changes
the
three
of tumour
of the rat which
and is not in the concentration
ACKNOWLEDGEMENTS: This investigation Williams from the Australian Research
that
by DEAE-cellulose
in any of the preparations
phosphoenolpyruvate,
activity
alanine
liver
tumour
has an increased as sensitive
to
of effecters.
was supported by grants Grants Committee.
to
REFERENCES: 1.
1:: 11.
F.A., Adelman, R.C., Lo, C.H., Morris, H.P. & Weinhouse, S. Cancer Res. 28: 1897-1900 (1968). Criss, W.E. Biochem. Biophys. Res. Commun. 35: 901-905 (1969). Bonney, R.J. & Potter, V.R. Fed. Proc. 31:1618 (1972). Knox, W.E., Farron, F. & Hsu, H.H.J. PrE. Amer. Assoc. Cancer Res. 13:135 (1972). Walker, P.R. & Potter, V.R. Advan. Enzyme Regul. 10: 339-364 (1972). Taylor, C.B., Morris, H.P. & Weber, G. Life Sci. 8:635-644 (1969). Heise, E. & Garlich, M. Exptl. Cell Res. 33: 289-300 (1964). Irving, M.G. Ph.D. Thesis, University of Ew South Wales, Australia. (1973). SchmZhl, D., Preussman, R. & Hamperl, H. Naturwisenschaften 47: l-2 (1960). Irving, M.G. & Williams, J.F. Biochem. J. -.131: 287-301 (1973r Rozengurt, E., Jimenez De Asda, L. & Carminatti, H. J. Biol. Chem.
12. 13.
Wood, T. Biochem. Biophys. Reynard, A.M., Hass, L.F.,
2.
3. 4. 5. ;:
8.
14.
Farina,
244: 3142-3147 (1969). __ 236: 2277-2283 (1961). __
Weinhouse,
S. Cancer
Res. Commun. 31: 779-785 (1968). Jacobsen, 0.0.X Boyer, P.D. J. Biol.
Res. -32: 2007-2016
291
(1972).
Chem.
