VoI. 72, No. 3, 1976
CYCLIC
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
NUCLEOTIDE- r PYRUVATE-
PYRUVATE
Theo
J.C.
KINASE
ACTIVITY
van Berkel,
Johan
Received
July
CHANGES
IN
RAT H E P A T O C Y T E S
Johan
F. K o s t e r
and
C. H ~ i s m a n n
of B i o c h e m i s t r y versity
IN I S O L A T E D
K. K r u i j t ,
Willem
Department
AND HORMONE-INDUCED
I, F a c u l t y
Rotterdam,
of M e d i c i n e ,
Erasmus
Uni-
The Netherlands
27,1976
SUMMARY. I n c u b a t i o n of i s o l a t e d rat h e p a t o c y t e s w i t h g l u c a g o n (10 -6 M), d b - c A M P (0.I mM) and d b - c G M P (0.I mM) c a u s e s a d e c r e a s e in p y r u v a t e k i n a s e a c t i v i t y of 46, 49 and 34% r e s p e c t i v e l y , w h e n m e a s u r e d at 1 mM Mg~ • + ree and s u b o p t i m a l s u b s t r a t e ( P - e n o l p y r u v a t e ) c o n c e n t r a t i o n s , w h 1 ~ e the Vma x is u n l n f l u e n c e d . An i n c r e a s e in a c t i v i t y (25%) is n o t i c e d w h e n the cells are i n c u b a t e d w i t h 1 mM p y r u v a t e . The g l u c a g o n i n a c t i v a t e d e n z y m e (Lb) shows a d e c r e a s e d a f f i n i t y for the s u b s t r a t e P - e n o l p y r u v a t e and for the a l l o s t e r i c a c t i v a t o r F r u - l , 6 - P 2 as c o m p a r e d to the a c t i v a t e d form (La). The n a t u r e of the h o r m o n e and c y c l i c n u c l e o t i d e - i n d u c e d c h a n g e s in p y r u v a t e k i n a s e is d i s c u s s e d . It is c o n c l u d e d that the P - e n o l p y r u v a t e cycle is u n d e r c o m p a r a b l e acute h o r m o n a l c o n t r o l as the F D P a s e - P F K cycle. Both cycles are l i n k e d by the c o m m o n e f f e c t o r F r u - l , 6 - P 2 m a k i n g not only d i r e c t but also i n d i r e c t h o r m o n a l control of p y r u v a t e k i n a s e flux p o s s i b l e . Pyruvate catalyses in the
kinase
the
last
regulation
step
two
hepatocytes
enzyme
From kinetic perties
sequence vator
only
studies
enzyme
during
of the high
itself
can
proposed. changes
influence
parenchymal Taunton
et
types
the
concluded
could
affinity
of the
It was
suggested
its
affinity
al.
to test
(7,8)
in Vma x of p y r u v a t e
not
Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any iorrn reserved.
enzyme
917
that
that
lead
this
may
(I),
enzyme to i n a c t i v a t e (3-6).
the k n o w n
pro-
to the p r e s e n c e
of
This
was
mainly
the
con-
for
its
allosteric
acti
"A change and
possibility
recently be
gly-
uncertain
for F r u - l , 6 - P 2 "
showed
kinase
This
is still
and kinase
the m e c h a n i s m
gluconeogenesis.
cells
(2).
2.7.1.40)
is i m p o r t a n t
of p y r u v a t e
L-type but
it was
kinase
activity
EC
gluconeogenesis
conditions
in v i t r o
pyruvate
F r u - l , 6 - P 2 (6).
isolated
contain
properties
gluconeogenic
of L - t y p e
an i n a c t i v e
contains
regulatory
under
Its
liver
isolated
the
of g l y c o l y s i s .
between
Although
many
phosphotransferase,
of the b a l a n c e
colysis.
possesses
(ATP:pyruvate
that
induced
by
in the the
was
enzyme
use
of
therefore
short
term
alteration
of
Vol. 72, No. 3, 1976
the h o r m o n a l that
state
cyclic-AMP
in the vate
intact
kinase
effect ation we
of the
induces
liver
was
of the
ratio
can be
by
kinase
of F D P a s e
of p y r u v a t e both
while
Rognstad
of the p y r u v a t e
Howeverp
a direct
Clark flux
as
suggested
activities.
direct
which
and
flux
rate
on p y r u -
an i n d i r e c t of an a l t e r -
In the p r e s e n t effects
suggests
indirect
showed
effect
a consequence
and h o r m o n e - i n d u c e d
kinase
(9)
kinase
hormonal
(I0) w h o rate
and PFK
nucleotide-
under
animal,
inhibition
questioned
cyclic
tic p r o p e r t i e s
intact
cell.
on the p y r u v a t e
report
flux
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
that
hormonal
paper
on the k i n e -
pyruvate
kinase
control.
MATERIALS AND METHODS. I s o l a t e d rat l i v e r h e p a t o c y t e s w e r e p r e p a r e d f r o m fed a n i m a l s e s s e n t i a l l y as d e s c r i b e d by B e r r y and F r i e n d (11), m o d i f i e d as d e s c r i b e d p r e v i o u s l y (12). The p a r e n c h y mal cells w e r e i n c u b a t e d at 37°C in 50 ml E r l e n m e y e r f l a s k s s t o p p e r e d w i t h r u b b e r caps. The i n c u b a t i o n m e d i u m was 10 ml of K r e b s - R i n g e r b u f f e r c o n t a i n i n g the a d d i t i o n s as i n d i c a t e d in the f i g u r e s . At the i n d i c a t e d times 2 ml s a m p l e s w e r e w i t h d r a w n , h o m o g e n i z e d i m m e d i a t e l y and put in i c e - w a t e r . Zero time c o n t r o l s c o n t a i n e d all the a d d i t i o n s but no i n c u b a t i o n at 37°C was p e r f o r m e d . A f t e r t e r m i n a t i o n of the i n c u b a t i o n (the total time did not e x c e e d 50 min) the h o m o g e n i z e d cells w e r e c e n t r i f u g e d for 10 m i n at 10.000 g and the s u p e r n a t a n t was i m m e d i a t e l y t e s t e d for p y r u v a t e k i n a s e a c t i v i t y by a d d i n g 0.5 ml s u p e r n a t a n t to a c u v e t t e c o n t a i n i n g 24 m M T r i s - H C l pH 7.5, 200 m M KCI, l m M ADP 0.1 mg l a c tate d e h y d r o g e n a s e , 0.4 m M N A D H and l mM free Mg 2+. The free Mg 2+ c o n c e n t r a t i o n was c a l c u l a t e d as d e s c r i b e d e a r l i e r (13). The r e a c t i o n was s t a r t e d by a d d i n g p h o s p h o e n o l p y r u v a t e (final c o n c e n t r a t i o n 2.5 mM) a f t e r 5 min p r e i n c u b a t i o n . This s e q u e n c e of a d d i tions is e s s e n t i a l b e c a u s e b o u n d F r u - 1 , 6 - P 2 w i l l be split off u n d e r the a p p l i e d h i g h salt c o n c e n t r a t i o n in the a b s e n c e of P - e n o l p y r u v a t e (12,14). L i n e a r c u r v e s w e r e o b t a i n e d by this a d d i t i o n s e q u e n c e t h r o u g h o u t this study. W h e n i n d i c a t e d F r u - l , 6 - P 2 (0.5 m M final c o n c e n t r a t i o n ) was a d d e d as s e c o n d a d d i t i o n .
RESULTS Fig.
1 shows
of p y r u v a t e
kinase
l mM pyruvate. kinase
causes
when
the
It can be
activity
incubation. AMP
the e f f e c t
The a 50%
at
presence
that
This
of
decrease
cyclic
isolated
seen
10 min.
of d i b u t y r y l - c y c l i c - G M P
of
liver
alter
alone
+ 0.1
kinase the
V
on the
are
however,
l mM pyruvate
not
cells
pyruvate
effect,
of p y r u v a t e
does
nucleotides
activity
incubated
raises
the p y r u v a t e
disappears mM
on f u r t h e r
dibutyryl-cyclic-
activity.
effect
with
The
addition
of d b - c A M P .
When
v a l u e s are m e a s u r e d at s a t u r a t i n g F r u - l , 6 - P 2 c o n c e n t r a t i o n s max n e i t h e r d b - c A M P a l o n e nor its c o m b i n a t i o n w i t h d b - c G M P h a v e an effect
on the m a x i m a l
enzymatic
activity.
918
V o l . 7 2 , N o . 3, 1 9 7 6
BIOCHEMICAL AND
B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
-1 -1 nmoles m~n mg 125
1
1 mM pyr. + db-cAMP + db-cGMP
1 mM pyr. + db-cAMP
mM pyr.
100
75
50
•
0
25
lo
2o
4'o
I
I
I
10
20
40
,0
2'o
4o
time (m~nutes)
Fig. ] I n f l u e n c e of p y r u v a t e , p y r u v a t e + d b - c A M P (0°I mM) and pyruvate+db-cAMP (0.1 mM) + d b - c G M P (0.I mM) on the p y r u v a t e k i n a s e a c t i v i t y of i s o l a t e d h e p a t o c y t e s . The a d d i t i o n s to inc u b a t i o n s of i n t a c t cells are i n d i c a t e d in the figure. For f u r t h e r i n c u b a t i o n s see M A T E R I A L S AND M E T H O D S . The open symbols r e p r e s e n t the p ~ u v a t e k i n a s e a c t i v i t y at 2.5 mM P - e n o l p y r u v a t e and 1 m M M g ~ r e e . The c l o s e d s y m b o l s r e p r e s e n t the a c t i v i t y a f t e r s u b s e q u e n t a d d i t i o n of F r u - 1 , 6 - P 2 (0.5 mM).
nmoles rain-1 mg-1
125
10 mM glucose + insulin
1 mM pyr. + glucagon
150
1
100 75
A
50' ~
~
25 0
~
,
i
5
10
1S
I
0
5
i
I
10
15
I
5
I
I
10 15 time (minutes)
Fig. 2 I n f l u e n c e of p y r u v a t e + d b - c G M P (O.l mM), p y r u v a t e + g l u c a g o n (lO -6 M) and g l u c o s e + i n s u l i n (4 mU/ml) on the p y r u v a t e k i n a s e a c t i v i t y of i s o l a t e d h e p a t o c y t e s . The additions to i n c u b a t i o n s of intact cells are i n d i c a t e d in the figure. For f u r t h e r i n c u b a t i o n c o n d i t i o n s see M A T E R I A L S AND M E T H O D S . The open s y m b o l s r e p r e s e n t the p y r u v a t e k i n a s e activ i t y at 2,5 mM P - e n o l p y r u v a t e + l mM M g ~ e e . The c l o s e d symbols r e p r e s e n t the a c t i v i t y a f t e r s u b s e q u e n t a d d i t i o n of F r u - l , 6 - ~ 2 (0.5 mM).
919
Vol. 72, No. 3, 1976
TABLE
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
EFFECT
OF M g
2+
A N D F R U - I , 6 - P 2 ON T H E
HORMONE-INDUCED
CHANGES
IN P Y R U V A T E
CYCLIC NUCLEOTIDEKINASE
Additions
to i n c u b a t i o n s
Activity
of i n t a c t
cells
1 m.MMg2+ free
i mid pyr.
AND
ACTIVITY
+i0 m M M g 2+
at
+I0
mM Mg
+0.5 mM Fru1,6-P 2
72.8
138.4
164.3
i m M pyr.
+ 0.i m M d b - c A M P
33.7
103.0
160.9
I m M pyr.
+ 0.i m M d b - c G M P
42.6
109.7
158.9
i m M pyr.
+ 10 -6 M g l u c a g o n
i0 m M g l u c o s e i0 m M g l u c o s e
When
shown
added been
+ insulin (4 m U / m l )
to be p r e s e n t
to the
incubated
first
column.
Fig. pyruvate
for
2 shows kinase
that
0.I
activity.
the
cleotides
causes
a decrease
taken
place
liver
cell
forms
of p y r u v a t e
the
enzymes
nucleotides
in the
thiol and
the h o m o g e n a t e s
is not
involved
and T i t a n j i
133.4
158.7
the
intact We
hand,
kinase
exist
groups
(5,14).
were
prepared
suggesting
in the o b s e r v e d et
al.
(]6)
the
the
than
TABLE
of
I);
that Fig.
cyclic
Insulin
that
upon the
cells
2
nu-
in the p r e -
shown
that
compounds
the
redox
effects
to the
state
of the
unaltered
or p r e s e n c e
of e s s e n t i a l
Recently that
had
interconvertible
were
absence
effects.
changes
several
oxidation
920
smaller
also
of the
However,
have
is
as well.
earlier
in the
decreases
instead
depending
that
in the
m g -I p r o t e i n .
also
(see
had
is i n e f f e c t i v e .
added
on i n t a c t
cells
mentioned
effect
the n a t u r e
reported
of g l u c a g o n
its
intact
m i n -I
itself
in a c t i v i t y
cell we
the
substances
in n m o l e s
of d b - c A M P
other
0.5 m M F r u - l , 6 - P 2 w e r e
after
of g l u c a g o n
to e x p l o r e
homogenate.
mercaptoethanol,
(15)
addition
of g l u c o s e s on the
In an a t t e m p t
64.6
However,
concentration
sence
159.6
mM db-cGMP
also
that
162.4
expressed
of an e q u i m o l a r shows
99.5 129.1
prepared
I0 m i n w i t h
Activity
37.7 62.4
i0 m M M g 2+ a n d / o r
cell homogenates
2+
under
cyclic
whether
of
10 m M
-SH
groups
Ljungstrom certain
of
et al.
condi-
Vol. 72, No. 3, 1976
tions
liver p y r u v a t e
panied
kinase
by a d e c r e a s e
optimal other
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
can be p h o s p h o r y l a t e d ,
of activity,
P-enolpyruvate
especially
concentrations
(16).
which
is accom-
when measured
at sub-
The p h o s p h o r y l a t i o n
of
enzymes,
bation
such as p y r u v a t e d e h y d r o g e n a s e is r e v e r s e d by incu2+ at high Mg c o n c e n t r a t i o n s (17,18) w h i c h activates p h o s p h o -
protein
phosphatases.
concentrations
TABLE
I illustrates
on the enzyme
a c t i v i t y • At
the effect
of high Mg 2+
! mM M gfree 2+
the effects
of cyclic n u c l e o t i d e s and hormones are maximal, a d d i t i o n of 10 mM 2+ Mg causes a prompt (within 10 sec) s t i m u l a t i o n of p y r u v a t e kinase activity. cyclic
However,
even
nucleotides
1,6-P 2 relieves TABLE
and h o r m o n e s
these
the enzyme
by g l u c a g o n obtained
the changes treatment
after
w h i ~ L a is the enzyme
pyruvate
(I mM)
TABLE
alone.
of k i n e t i c
of the
incubation
glucagon
in affinity
may be observed.
the effects o f
Addition
of Fru-
differences.
II summarizes
vate kinase
at high Mg 2+ c o n c e n t r a t i o n s
It can be
for the s u b s t r a t e
liver
of the
obtained seen
properties cells•
cells
from cells
that besides
P-enolpyruvate,
for
of pyru-
L b stands
10 min with
incubated
with
the d i f f e r e n c e
the L b form also
II
COMPARISON
OF T H E K I N E T I C
PARAMETERS
OF P Y R U V A T E
KINASE
TYPES
L a AND L b
La K0. 5
P-enolpyruvate
K0.5a Fru-l,6-P 2 K0.5a Fru-l,6-P 2 in p r e s e n c e of A T P and a l a n i n e
L b was
prepared
+ glucagon
(10 -6 M)
L a was p r e p a r e d 1 mM pyruvate
Lb
2.2 m M
4.1 m M
0.6 ~M
1.0 ~ M
4.2 ~ M
9.8 ~M
(2 mM) (i mM)
by i n c u b a t i o n
of the cells w i t h
1 mM pyruvate
for i0 m i n
by i n c u b a t i o n
of the cells
alone.
921
for
for i0 m i n w i t h
Vol. 72, No. 3, 1976
possesses
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
a lower
apparent
Fru-l,6-P 2 especially tions
of the
affinity
for
in the p r e s e n c e
allosteric
inhibitors
the
allosteric
activator
of p h y s i o l o g i c a l
ATP
(2 mM)
and
concentra-
alanine
(I mM)
(6,19).
DISCUSSION Taunton on the
enzymatic
pyruvate firmed
kinase
can
(21).
L-type from
zyme
should
be
as
fully
enzyme 2+ Mgfree
inactivated
enzyme
form
kinase,
enzyme.
will
gluconeogenic
(6).
The
The
the
The
of
1 mM
lower
conversions
may
and
was
con-
and
isolated to
studies
hepato-
study with
the
iso-
concluded
by e x t r a p o l a -
situation
that
both
glycolytic
show
with
that
properties
forms of
the
liver
distinct
cell from
an the
of the L b f o r m of p y r u v a t e
for
the
liver
allosteric kinase cells
of the
identical
summarized
gluconeo-
parameters
in the
of p y r u v a t e of
the en-
at the p h y s i o l o g i c a l
properties
affinity
and
interconvertible kinetic
to an a c t i v a t i o n
be
(20)
that
as d e t e r m i n e d
properties
FDPase This
we
Incubation
leads
kinetic
effects
in v i v o
that
a decrease
conditions.
rapid
as a m o d e l
earlier
reported
(22),
described
the
our
under (6)
show
(6),
(2,5,6,19) to
glucagon,
enable
surprisingly
form possesses
data
From
can be p r e s e n t
especially
Fru-l~6-P2,
earlier
suggested
by
concentration
purified
kinase
and B l a i r
experiments
kinase.
in vivo.
to o b s e r v e
of h o r m o n e s .
Cimbala
activated
We
be p r e s e n t
(Lb)
by
suggested
in v i t r o
conditions.
first
of p h o s p h o f r u c t o k i n a s e ,
The p r e s e n t
should
(l mM)
kinase
pyruvate
the
the
the a d m i n i s t r a t i o n
of p y r u v a t e
tion
genie
activities by
serve,
regulation lated
(7,8) w e r e
for p y r u v a t e
Friedrichs cytes
et al.
activator
activity
with
under
pyruvate
enzyme.
This
La enzyme
to the p u r i f i e d
as f o l l o w s :
glucagon db-cAMP (db-cGMP) La x (active form) The m e c h a n i s m clear the
of the
at the m o m e n t .
thiol
groups
into
the L a form.
transition
Regulation
of the
of the L b f o r m w i t h
Lb (inactive
pyruvate
enzyme
between
by m e a n s seems
Recently
it was
922
shown
L b and L a f o r m
of the
less
10 m M m e r c a p t o e t h a n o l
form)
reduction
probable did not
that
as
of
incubation
convert
pyruvate
is not state
this
kinase
form
from
Vol. 72, No. 3, 1976
rat
liver
into
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
(15,16)
and
chicken
a phosphorylated,
catalysed present
by a cAMP
paper
that
is d i m i n i s h e d enzyme
in intact
by g l u c a g o n
it is possible
endogenous rylationp binding et al.
of other (24)
This
vitro
it is only
zyme.
that
of such
link hormone
activity
tion
recently through tate
to both
under
pyruvate
for p y r u v a t e
the F D P a s e - P F K
the k i n e t i c
cyclic
gluconeogenic at high
for g l u c o n e o g e n e s i s ~ no effect
(27)
pyruvate
state
that with
necessary
for the t r a n s f e r
(28).
occurrence
The
that with
of p y r u v a t e direct
FDPase-PFK
couple,
can be
tides,
which
Fru-Ip6-P2~
effect
(lO) who
kinase
pyruvate kinase
kinase
activity
could
in
(25).
to the en-
(26).
and
the
The
possibly
the p y r u v a t e
Rognstad cAMP
(9)
showed the f l u x
With
the p y r u v a t e
of cAMP
is noticed.
equivalents cycle
in rela-
inhibited
concentrations.
as s u b s t r a t e
of hormones
on p y r u v a t e
lackinase
Katz
cycle
and is
to the cytosol
is s t r e n g t h e n e d
by our
for g l u c o n e o g e n e s i s
of PFK
on p y r u v a t e
an indirect kinase.
directly
to an enzyme
by g l u c a g o n
and the
dependent
923
kinase
influence,
Our data
form with
that under p h y s i o l o g i c a l is largely
of p y r u v a t e
is a t t r a c t i v e
the
increases.
suggests
the product
earlier
acids
the P - e n o l p y r u v a t e
of reducing
of such a useful
influenced lead
showed
Pilkis
although
properties
however,
low and c o n s e q u e n t l y
ed by Clark
cycle
conditions
pyruvate
flux was
activity
kinase
the
inside
concentrations fatty
nucleotides
Rognstad
The
Recently
of
phospho-
influencing
kinase,
high
of free
of liver m e t a b o l i s m .
kinase
as s u b s t r a t e
observation
the b r e a k d o w n Besides
for p h o s p h o f r u c t o k i n a s e
to influence and/or
regulation
that
to be d e t e r m i n e d ~
Ca 2+ t r a n s p o r t
of p y r u v a t e
on
efficiency
cycle.
by hormones
to the
compounds.
promotes
kinase
be based
a lower
prevent
action by
at r e l a t i v e l y
a mechanism
The p o s s i b ~ i t y kinase
their
is the b i n d i n g
suggested
actions
remains
added will
weight
glucagon
effective
has been
occurrence
exert
is an i n h i b i t o r
possibility
of cGMP
indeed
has
is
of the
of p y r u v a t e
could
which
findings
of p h o s p h o d i e s t e r a s e .
low m o l e c u l a r
cation
This
kinase
an effect
also
then
in vitro
phosphorylation The
the a c t i v i t y
db-cGMP,
db-cGMP
This
kinase.
and db-cAMP
that
may
showed
cell.
A further
cells
cAMP by i n h i b i t i o n hormones
form.
protein
Whether
represents
(23) may be c o n v e r t e d
active,
dependent
phosphorylation.
than db-cAMPp since
less
liver
show
and/or
a lowered
substrate
this
is q u e s t i o n by the
that p y r u v a t e cyclic
affinity
nucleofor
for FDPase.
conditions
upon
flux
mediated
We
the p y r u v a t e
compound.
This
in-
Vol. 72, No. 3, 1976
dicates
that
glucagon
a relation
can regulate
influencing encing
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
the
exists
between
the pyruvate
affinity
kinase
for Fru-1,6-P2,
the Fru-l,6-P 2 c o n c e n t r a t i n n
FDPase-PFK
the two cycles,
itself
flux both
in which
directly,
and indirectly through
by
by influ-
the balance
of
activities.
ACKNOWLEDGEMENTS. Miss A.C. Hanson is thanked for her help in the p r e p a r a t i o n of the manuscript. The Netherlands Foundation for Fundamental Medical Research (FUNGO) is acknowledged for partial financial support (grant 13-39-18).
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