BIOCHIMICAET BIOPHYSICAACTA
763
BBA 66554
T H E I N F L U E N C E OF GLUCOSE 1 , 6 - D I P H O S P H A T E ON T H E
ENZYMATIC A C T I V I T Y OF P Y R U V A T E K I N A S E
j . F. K O S T E R a, R. G. S L E E % G. E. J. S T A A L b AND TH. J. C. VAN B E R K E L a
aDepartment of Biochemistry I, Medical Faculty, Rotterdam (The Netherlands) and bHaematological
Department, State University Hospital, Utrecht (The Netherlands)
(Received N o v e m b e r 2 5 t h , 1971)
SUMMARY
I. The influence of Glc-I,6-P 2 on hepatic and red blood cell pyruvate kinase
phosphotransferase, EC 2.7.1.4o ) is quite similar to that of Fru-i,6P v The hexose diphosphates can replace each other in stimulating pyruvate kinase;
after maximal stimulation by one of the compounds, the other is not capable of further stimulation.
2. The regulatory role of Fru-I,6-P2 on the activity of pyruvate kinase is discussed in view of the results obtained.
(ATP: pyruvate
INTRODUCTION
A large number of studies have been devoted to the regulation of pyruvate
kinase (ATP: pyruvate phosphotransferase, EC 2.7.I.4O), one of the key enzymes in
glycolysis. I t was found that there are two types 1, a M (muscle) type and a L (liver)
type. I t turned out that the L type exhibits allosteric properties allowing regulatory
function. The glycolytic intermediate F r u - i , 6 - P 2 is able to stimulate the activity of
the L type and changes the S-shaped relationship between substrate concentration
and velocity into a normal hyperbolic one 2. The [Fru-I,6-P21 of the liver fluctuates
with the nutritional state and, therefore, it was assumed that Fru-I,6-P 2 has a
regulatory influence on pyruvate kinase. Furthermore, it was found that alanine
inhibits the hepatic pyruvate kinase reaction 3, which can be of importance during
gluconeogenesis. In our studies4, 5 on hepatic and erythrocytic pyruvate kinase it was
found that the phosphorylated hexoses are able to stimulate the pyruvate kinase
reaction and to overcome the ATP inhibition. In this study we compare the influences
of Glc-I,6-P~ and F r u - i , 6 - P 2 on the enzymatic reaction.
MATERIALS AND METHODS
Wistar rats, maintained On a normal diet and water ad libitum, were decapitated
and the livers removed. Liver homogenates were prepared in o.15 M NaC1 containing
Abbreviation: PEP, phosphoenolpyruvate.
Biochim. Biophys. ~'tcta, 258 (i972) 763-768
764
j.F. KOSTER el al.
TABLE
i
THE EFFECT OF G L C - I . 6 - P t (0.2 nlM) AND F R U - I , 6 - P , a (0.2 raM) ON THE ACTIVITY OF HEPATIC
PYRUVATE KIN.a.SE IN (0.25 M) T R I s - H C 1 BUFFER ( p H 7.5) WITH [ P E P i - - 0. 5 m M AND ! A D P ] 0.3 m M
Addition
Activity ( % )
None
(;lc-I,6-Po
Fru-I,6-Pz
ioo
218
228
I mM mercaptoethanol and centrifuged for 6o min at ioo ooo X g. From the supernatant, the partially purified L type preparations were obtained according to the
method of PASSERON et al2. The preparations were finally dissolved in 0.25 M Tris
HC1 buffer (pH 7.5) containing I mM mercaptoethanol. Red blood cell pyruvate
kinase was isolated from human erythrocytes according the isolation procedure
described previously 5. The enzymatic activity was assayed by following the decrease
of absorbance at 340 nm in the reaction coupled with lactate dehydrogenase as
described by BOCHER AND PFLEIDERER ~. The activities shown are initial reaction
rates.
ADP, P E P (phosphoenolpyruvate), NADH, Fru-I,6-P~ and Glc-I,6-P 2 were
obtained from Boehringer (Mannheim, Germany). Glc-I,6-P 2 was free of Fru-I,6-P 2,
when analysed by the method of B(~CHER AND HOHORST8. All other reagents were of
analytical grade purity.
v(pmole/mln ling
protein)
• controJ
• with GIc-I,6-P 2
0.4
0.3
0.2
0.1
J
I
1.0
I
2.0
I
3.0
[PEP]
I
4.0
I
5.0
(raM)
F i g . I. T h e v versus ~ P E P ] p l o t f o r h e p a t i c p y r u v a t e
(0--0)
a n d p r e s e n c e o f 0.2 m M G l c - i , 6 - P z ( A - - & ) .
k i n a s e a t E A D P ] ~ o.3 m M , in t h e a b s e n c e
RESULTS
Table I shows that Glc-i,6-P 2 is able to stimulate the hepatic pyruvate kinase
reaction. This stimulation is about equal to the stimulation obtained with Fru-i,6-P~
(Table I), at least under these conditions.
Biochim. Biophys. Acta, 2 5 8 (1972) 7 6 3 - 7 6 8
INFLUENCE OF
Glc-I,6-P 2 ON PYRUVATE KINASE
765
v (A~mln)
0.4
• control
• with GIc-I,6-P 2
0.3
0.2
0.1
I
I
1.0
2.0
[PEP]
I
I
I
3.0
4.0
5.0
I
6.0
(raM)
Fig. 2. The v v e r s u s [PEP] plot for red blood cell p y r u v a t e kinase at [ADP] = 2 mM, in the absence (l~--O) and presence of o.2 mM Glc-I,6-P 2 (st--st), 20/~g of protein added.
% activity
A
hepatic pyr. klnase
I//~
B
~-: Cw°tTr~lc'l'6"P2
1.0
2.0
3.0
red blood cell pyr.klnase
I/~
4.0
[ATP]
:-'~ Cw°trhr~k:'1'6-P2
1.0
2.0
3.0
4.0
(raM)
Fig. 3A. The ATP inhibition plots for hepatic p y r u v a t e kinase at [PEP] = 0. 5 mM and [ADP]
0.5 mM. O---O, control; st--A, with Glc-x,6-P.s (o.z mM).
Fig. 3 B. The A T P inhibition plot for red blood cell pyruvate kinase at [PEP] = 0. 5 mM and
[ADP] = 0. 5 raM; O---O, control; s t - - s t , with Glc-I,6-P 2 (o.2 mM), 20 #g protein added.
B i o c h i m . B i o p h y s . A c t a , 258 (1972) 763-768
j.F. I~OST~Ct~et al.
766
Glc-I,6-P2, not only stimulates the pyruvate kinase reaction, but is also able to
transform the S-shaped curve into a normal hyperbolic one (Fig. I). These results are
quite similar to the results obtained with the ligand Fru-I,6-P2 (refs. I, 2). At pH 5.9
the effects of Fru-I,6-P2 disappear. The same results were obtained with Glc-I,6-P2;
no stimulation occurs at all. Fig. 2 shows the influence of Glc-I,6-P 2 on the red blood
cell pyruvate kinase reaction at pH 7.6. These results are similar to the results obtained with the hepatic pyruvate kinase,
Similar to Fru-I,6-P~, Glc-I,6-P 2 is able to reverse the ATP inhibition on the
pyruvate kinase reaction (Fig. 3A). This is also the case with red blood cell pyruvate
kinase (Fig. 3B).
It is clear from these data that the effects of Glc-I,6-P 2 and Fru-I,6-P~ are
quite similar. In liver both ligands have a synergistic effect only at low (<5o#IVl)
concentration (Fig. 4)- Fig. 5 shows that with the red blood cell pyruvate kinase at
% activity
200- ~ ' ~
•
control
-
t
-
160-
120-
80
40-
,I
I
I
I
I
0.2
0.4
0.6
0,8
1.0
GIc°I,6-P2 (raM)
Fig. 4. T h e v v e r s u s [Glc-I,6-Pe] for h e p a t i c p y r u v a t e kinase in t h e presence a n d a b s e n c e of
F r u - I , 6 - P 2 ; I ~ - - O , control; • - - • ,
w i t h Fru-l,6-P,,. (0.2 mM), [ P E P ] = 0. 5 m M a n d [ADPI~ =
0. 3 mM.
5oo/~M, Glc-I,6-P 2 Fru-I,6-P~, does not further stimulate, but that in the absence of
F r u - I , 6 - P 2 half-maximal stimulation by Glc-I,6-P~ is obtained at 60/~M.
At a [Fru-I,6-P~l of 50 #M the hepatic pyruvate kinase is practically fully
stimulated; however, at IO/~M the stimulation is already about 90% of the maximal
obtainable. For Glc-I,6-P 2 the concentration necessary for full stimulation is about
50 #M ; at a concentration of IO/~M the stimulation is about 60% of maximal obtained.
For the red blood cell pyruvate kinase tile situation is somewhat different. At io yM
of F r u - I , 6 - P 2 the reaction is fully stimulated but for Glu-I,6-P2 the pyruvate kinase
reaction is still not fully stimulated at a concentration of 200 #M.
B i o c h i m . B i o p h y s . A c t a , 258 (1972) 763-768
I N F L U E N C E OF
GIc-I,6-P 2 ON PYRUVATE KINASE
767
v ( ",A/r,,in)
•
L&
0.20'
0.15
A
control
Fru-l,6-P 2
added
0.10
0.~
i
0.05
i
O. lO
i
0.15
l
0.20
l
0.25
i
05
[GIc'I,6-P2 ] (mM)
Fig. 5. T h e v v e r s u s [GIc-I,6-P2] for red blood cell p y r u v a t e k i n a s e in t h e a b s e n c e a n d presence of
F r u - I , 6 - P 2 ; O---O, control; A - - A , w i t h F r u - I , 6 - P 2 (o. 5 mM). [ P E P ! = 0. 5 m M a n d CADPI =
0. 5 mM, 20 # g o f p r o t e i n added.
DISCUSSION
From the data presented it is clear that the effects of F r u - I , 6 - P 2 and Glc-I,6-P 2
are quite similar. The concentration of Fru-I,6-P 2 in the liver fluctuates during
feeding and fasting (5-20/~M) 9. However, the EGlc-I,6,P2] remains constant at about
I5 #M 1°. At this concentration of Glc-I,6-P 2 the hepatic pyruvate kinase is more than
80% stimulated, while at 5/~M F r u - I , 6 - P 2 pyruvate kinase is already stimulated for
50%. This means that F r u - I , 6 - P 2 cannot have any regulatory function on hepatic
pyruvate kinase. For red blood cell pyruvate kinase it was shown that Fru-i,6-P~ has
already a fully stimulating effect at IO/zM, while for Glc-I,6-P 2 the concentration has
to be more than 200 #M. However, the tGlc-I,6-P2~ in the red blood cell is 300/*M 11.
According to B A R T L E T T 11, the red blood cell EFru-I,6-P2~ is 200/zM. With these concentrations the pyruvate kinase in the erythrocyte is fully stimulated. This means that
the enzyme is in the R state 5. For the red blood cell it is rather doubtfull if a regulation
of pyruvate kinase is necessary. The rate of glycolysis is governed by the activities of
hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1 ) and phosphofructokinase (ATP: D-fructose-6-phosphate I-phosphotransferase, EC 2.7.I.II ), in which
the concentrations of adenine nucleotides also play an important role1,5,12,13.
Although there will be some wasting of ATP during the gluconeogenesis due to
cycling 9, the p y r u v a t e kinase reaction must be inhibited in order to increase the P E P
concentration which is necessary for the gluconeogenesis. However, this regulation
cannot be done by a fluctuating F r u - I , 6 - P 2 concentration of the liver.
B i o c h i m . 13iophys. Mcta, 258 (1972) 763-768
768
J. t~. KOST~I~et al.
ACKNOWLEDGEMENT
The authors are indebted to Dr. W. C. Hfilsmann and Dr. M. C. Verloop for
help and suggestions. The Foundation for Fundamental Medical Research (Fungo) is
acknowledged for partial financial support.
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Biochim. Biophys. Acta, 258 (1972) 763-768