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The control of glycolysis in early embryogenesis
362
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25866 T H E CONTROL OF GLYCOLYSIS IN EARLY EMBRYOGENESIS L. S. MILMAN AND YU. G. Y U R O W I T Z K I
A. N. Severtzov Institute of Animal Morphology, Academy of Sciences of the U.£'.S.R. (Moscow) (Received March 13th, 1967) (Revised manuscript received June 2oth, 1967)
SUMMARY
An increase of glycolysis i s vivo at early stages of loach embryogenesis is related to the regulation of the level of fructose 1,6-diphosphate. The activity of enzymes of the system converting fructose 1,6-diphosphate into lactate remains constant in the course of development. An enhancement of glycolysis during the embryogenesis is not due to an increase of phosphofructokinase (EC 2.7.1.11 ) activity and cannot be attributed to changes of the level of intermediates controlling phosphofructokinase activity. The content of hexose monophosphates, citrate and adenylic nucleotides in loach embryos at early stages of development remains constant. A considerable decrease in fructose diphosphatase (EC 3.1.3.11) activity in early embryogenesis is reciprocal to an increase in the rate of glycolysis in intact eggs and to the level of fructose 1,6-diphosphate. The inhibition of phosphofructokinase activity by citrate and ATP and inhibition of fructose diphosphatase activity by AMP and fructose 1,6-diphosphate are assumed to control the level of fructose 1,6-diphosphate according to the feed-back type of regulation, depending on the level of intermediates. The decrease of fructose diphosphatase activity is regulated by the control mechanism of enzyme biosynthesis in the developing embryo.
INTRODUCTION
In early embryogenesis, a considerable (3-fold) increase in glycolysis occurs i~z vivo. This is related to the activation of the first steps of glycolysis at a constant
activity of the enzymes converting fructose 1,6-diphosphate to lactate 1-a. We have shown that the rate of glycolysis is determined by the fructose 1,6-diphosphate level in the cells of the embryo. It is not dependent on the concentration of hexose monophosphates and is directly proportional to fructose 1,6-diphosphate concentrations from o.I to 4 mM. Determinations of glycogen phosphorylase activity (EC 2.4.1.1) also confirms that its activity is not a glycolysis-limiting factor and does not change during early stages of embryogenesis. The fructose 1,6-diphosphate level is controlled by the activities of phosphofructokinase (EC 2.7.1. I I) and hexosediphosphatase (EC 3.1.3. II), i.e. enzymes taking part in the regulation of the Pasteur effect. Recently convincing evidence has been obtained from experiments on yeast, heart muscle, skeletal muscle and liver which Bioehim. Biophys. Acta, 148 (I967) 362-371
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
363
testify that the enzyme regulating glycolysis, phosphofructokinase, controls the Pasteur effect. PASSONEAU AND LOWRY4, MANSOUR 5, and other authors 6-9 have shown that the phosphofructokinase is inhibited by ATP and citrate. What is the relationship between the activity of phosphofructokinase and the increase of glycolysis in embryogenesis? Does an increase in phosphofructokinase activity occur simultaneously with an increase in glycolysis or is the control of increasing glycolysis in early embryogenesis brought about in some other way ? The solution of these questions can serve to clarify the more general problem of the relationship between mechanisms controlling the homeostasis in a cell (i.e. regulation of glycolysis of a cell 'in steady state') and systems controlling the general intensification of metabolism during the realization of genetic information in embryogenesis. No data on this problem have been reported yet. MATERIALS AND METHODS
Animals Experiments were performed on developing eggs of loach (Misgurnus fossilis). Mature eggs were obtained by injecting female loaches with 2o0 units of gonadotropic hormone 'Choriogonin' (Richter, Hungary). The stage of development was expressed in h at 21 ° according to NEIFAKHI°: 0--6 h = cleavage, 6- 9 h ~ blastula, 9-2o h = gastrulation, 20 h = blastopore closure. The isolated embryos (blastoderm) from loach eggs were separated from the yolk by means of centrifugation at 4000-50o0 × g on sucrose gradient after the trypsin treatment of tile eggs to remove the egg membrane 11. Determination of intermediates Assay of hexosephosphates and adenylic nucleotides were made in deproteinized HClO4-extracts of embryos according to the general procedure recommended by BERGMEYER12,17. The fixation of eggs or isolated embryos was made by immersion in liquid N 2. Hexose monophosphates were assayed in aliquots of deproteinized extracts by reduction of NADP + with 0.4 units of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and an excess of glucose-phosphate isomerase (EC 5.3.I.9) (ref. 12). Glucose 6-phosphate dehydrogenase was obtained by the method of KORNBERG AND HORRECKER from yeast 13. The enzyme was freed from phosphogluconate dehydrogenase (EC 1.1.1.44 ) activity. Fructose 1,6-diphosphate was assayed with a coupled system consisting of NADH and 3o-4o/~g of crystalline myogen A (isolated by the method of BARANOWSKY14,15) or with an excess of several times recrystallized aldolase (EC 4.1.2.13) and a-glycerophosphate dehydrogenase (EC 1.1.1.8). The above-mentioned enzymes were obtained according to the procedure of BEISENHERZ et al. 16. The loach embryos contained only a trace of phosphotrioses, which could be neglected. ADP was determined by the method of ADAM17 with NADH and an excess of lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.4o). AMP was determined in the same procedure after adding an excess of adenylate kinase. The citrate was determined in trichloroacetic acid-extracts by the method of Biochim. Biophys, Acta, 148 (1967) 362-371
364
L. S. M I L M A N , YU. G. Y U R O W I T Z K I
STERN~8; the Pl by the method of FISKE AND SUUBAROW19; the protein by the procedure of LowRY et al. ~°.
The preparation of homogenates For the assay of phosphofructokinase (EC 2.7.1.11) activity the loach eggs or isolated embryos were homogenized in an all-glass homogenizer in 50 mM KH2PO 4 (pH 7.5) containfng 5 mM/3-mercaptoethanol. For the assay of fructose diphosphatase, 50 mM Tris (pH 7.5) were employed instead of KH2PO 4. Extracts were obtained after 5 rain centrifugation at 2000 × g. In some experiments mitochondria-free extracts were obtained after 20 rain centrifugation at 18 ooo x g. The assay of enzyme activity Activity of phosphofructokinase was assayed by increasing the fructose 1,6diphosphate after 5 min incubation of an aliquot of extract in assay medium 21. The reaction was stopped by adding o.I ml 3o % HC10~. After centrifugation in the cold the supernatant was decanted and neutralized by KHCO a. The KC10 4 residue was discarded after the centrifugation. The amount of fructose 1,6-diphosphate in o. 4 nil aliquots of supernatant fluid was determined enzymatically (see above). The assay medimn (for maximal activity of phosphofructokinase) was comprised of 50 mM Tris-HC1 (pH 8.3), 25 mM KH2PO 4, 1,5 mM ATP, 2.5 mIVIMgSOa, 1.5 mM fructose 6-phosphate and 5 mM /3-mercaptoethanol. The second medium provided conditions for maximal effect of ATP and citrate and had a final concentration of 25 mM imidazole-HC1 (pH 7.2), 25 mM Pl (pH 7.2), 5 mM MgSO4, 0.5 mM fructose 6-phosphate and 5 mM/~-mercaptoethanol. Activity of fructose diphosphatase was determined by the increase in the amount of fructose 6-phosphate in the assay medium after Io rain incubation at 25 o. The assay medium was comprised of 50 mM Tris-HC1 (pH 7.5), I mM fructose 1,6diphosphate, IO mM MgSO,, 0.05 mM EDTA, 5 mM ¢/-mercaptoethanol. The increases of fructose 6-phosphate and glucose 6-phosphate were determined enzymatically (see above). In some experiments the release of Pt was determined according to the method of FISKE AND SUBBAI~OW19. The amount of released Pt in ~moles (with the increment for Pi released by non-specific phosphatases) corresponded to the sum of hexose monophosphates formed during the reaction. Preparation of partially purified fructose diphosphatase The fructose diphosphatase from loach embryos or rat liver was isolated according to BONSIGNORE et al.22,2a up to stage IV. The isolated preparations were free from activity of non-specific phosphatases, aldolase, phosphofructokinase, adenylate kinase, etc. Reagents Tris, fl-mercaptoethanol, NADH, ~-glycerophosphate were obtained from Light & Co (England); ATP, ADP, AMP: from Calbiochem (U.S.A.); glucose-0-phosphate: from Sigma (U.S.A.); NADP: from Lawson (England). Lactate dehydrogenase, phosphoenolpyruvate, pyru vatekinase and adenylate kinase were purchased from Boehringer (Germany). Commercially available barium salt of fructose 6-phosphate was Biochim. Biophys. dcta, 148 (1967) 362-371
365
CONTROL OF GLYCOLYSIS IN E M B R Y O G E N E S I S
completely purified to remove traces of fructose 1,6-diphosphate, glucose 6-phosphate and nucleotides. RESULTS
AND DISCUSSION
Determination of the activity of phosphofructokinase carried out under conditions providing for maximal activity of the enzyme has shown that the phosphofructokinase activity remains constant during development. In addition the presence of mitochondria was shown not to influence the results of the determination of phosphofructokinase activity. In both cases the activity of phosphofructokinase is equal to o.17 ~moles of fructose 1,6-phosphate formed during 5 min by ioo embryos. In the medium with a low concentration of fructose 6-phosphate (o.5 raM) in imidazole buffer, phosphofructokinase of loach embryos is inhibitited by ATP and citrate. A notable inhibition of the phosphofructokinase activity by an excess of ATP can be observed at ATP concentrations of 4 mM and higher (Table I). Yet we could not observe the described effect in the cases when Tris (pH 7.o) was substituted for imidazole. This confirms the data of UYEDA AND RACKER24. Citrate is the strongest inhibitor of phosphofructokinase. The inhibiting effect of citrate detected at higher concentrations of fructose 6-phosphate is slightly dependent on the type of buffer used. The given properties of phosphofructokinase (i.e. data of PASSONEAU AND L O W R Y 4, M A N S O U R , NEWSHOLM and others (refs. 5-9)) to a considerable extent account for the mechanism of the Pasteur effect. A decrease in the level both of ATP and citrate under anaerobic conditions as well as an increase in the level of ADP, AMP and Pl increase the intracellular activity of phosphofructokinase and inhibit the activity of an enzyme-antagonist, fructose diphosphatase. Alterations in the level of intermediates in loach embryos under aerobic and anaerobic conditions (Table II) completely confirmed the above-mentioned point of view. It is to be stressed that the relation between the levels of metabolites under aerobic and anaerobic conditions does not depend on the stage of development. The only exception is fructose 1,6-diphosphate. At the 7-h stage (blastula) the level of fructose 1,6-diphosphate is increased by 25 % due to anaerobic conditions, but when TABLE
I
INHIBITION OF PHOSPHOFRUCTOKINASE
ACTIVITY BY A T P AND CITRATE
T h e r e a c t i o n m i x t u r e c o n t a i n e d 25 m M i m i d a z o l e ( p H 7.2), 25 m M K H z P O 4 ( p H 7.2), 5 m M M g S O 4, 0.5 m M f r u c t o s e 6 - p h o s p h a t e , 5 m M f l - m e r c a p t o e t h a n o l a n d a n a l i q u o t of m i t o c h o n d r i a - f r e e e x t r a c t of i s o l a t e d l o a c h e m b r y o s . T h e a c t i v i t y of p h o s p h o f r u c t o k i n a s e e x p r e s s e d i n m # m o l e s of fructose 1,6-diphosphate formed during 5 rain per 5° isolated embryos.
d TP (mM)
Activity of phosphofruetokinase Control
0.6
63
1.2
7°
4 .0 6.0 8.0
55 35 17
in %
90 ioo 79 50 25
After z 5 min preincubation with 5o m M N a F
5 m M citrate added
-7° -35 17
3° 20 io 5 --
Bioehim. Biophys. Acta, 148 (1967) 3 6 2 - 3 7 1
366
L. S. MILMAN, YU. G. YUROWITZKI
TABLE 1I T H E A L T E R A T I O N S IN L E V E L S OF AND
ANAEROBIC
CONDITIONS
IN
ATP, ADP, AMP, ISOLATED
CITRATE AND HEXOSEPHOSPHATES
IN AEROBIC
LOACH EMBRYOS
Amounts of intermediates expressed in m/,moles per ioo embryos (The details of determinations see in M A T E R I A L A N D M E T H O D S ) . Intermediates
Stage of development
mltmoles of intermediates Per zoo isolated embryos
Per z m g of protein
Control
Co~trol
Fructose 1,6-diphosphate
Blastula The end of gastrulation
Hexosemonophosphates
Blastula The end of gastrulation
21 22
ADP
Blastula The end of gastrulation
AMP
Blastula The end of gastrulation
ATP
Blastula The end of gastrulation
Citrate
Blastula The end of gastrulation
Pi
Blastula The end of gastrulation
1 , 8 @ O.I 2. 3 ± o.i
A naerobiosis
I. 5 1.9
2.O 4.6
:~ o.9 ~ o.9
17.6
I7.6
2o.5 i o-5 19.6 :~ o.5
36.5 5 o.5 36.o -[ o.6
16.5 15.9
29.6 28.8
31.5 t- 0.5 31.3 --' o.5
68.o ~ o. 5 67.2 :~ o.5
27.o 27.2
55.0 54.2
28.5 24.0
8. 7 ii.2
~:::o.9 " o.9
ioo IOO 34 ± o.6 28.3 ~' 0.6 175 14o
2.4 ~ O.I 5.5 5 o.2
A naerobiosis
2o 21
6o 6o io :~ o. 3 13.8 ~ o. 3 230 19o
glycolysis is more intensive, the level of fructose 1,6-diphosphate is increased b y 5o % b y the end of gastrulation. As the a c t i v i t y of phosphofructokinase when det e r m i n e d u n d e r different conditions does not v a r y during development, the question arises of whether an intracellular a c t i v i t y of phosphofructokinase can be controlled in early embryogenesis b y alterations of the level of i n t e r m e d i a t e s of glycolysis a n d respiration. As was m e n t i o n e d above, such regulation is k n o w n as the P a s t e u r effect. The results of m e t a b o l i t e assays d u r i n g d e v e l o p m e n t (Table III) have shown t h a t the c o n t e n t of adenylic nucleotides a n d citrate, i.e. metabolites most strongly affecting the phosphofructokinase activity, does not v a r y in early embryogenesis, nor does a r e d i s t r i b u t i o n of metabolites between yolk a n d e m b r y o proper occur. The level of hexose m o n o p h o s p h a t e s reaches its m a x i m u m b y the 6-7-h stage (blastula) a n d then r e m a i n s c o n s t a n t d u r i n g further development. C o n s e q u e n t l y the m a x i m u m increase of glycolysis from the stage of b l a s t u l a to t h a t of 15 h (the middle of gastrulation) occurs at a c o n s t a n t level of hexose monophosphates. A n increase in the fructose 1,6-diphosphate level in general indicates the rate of increasing glycolysis i n vivo (Fig. 1). F l u c t u a t i o n s in the c o n t e n t of Pi in isolated embryos d u r i n g d e v e l o p m e n t are also negligible (Tables I I a n d III) a n d thus c a n n o t exert significant influence on the a c t i v i t y of phosphofructokinase. A n increase in the c o n t e n t of PI in developing loach eggs reported b y I{AFIANI AND TIMOFEEVA25 occurs not in the embryo proper b u t in the yolk which c o n t a i n s up to 9/lO of the Pt c o n t e n t of the whole egg. I n c o n t r a s t Biochim. Biophys. Aeta, 148 (z967) 362-371
% Y
0o
42
~"
III AND CITRATE IN DEVELOPING LOACH EMBRYOS
± 1.4
--
4°
± 0.7
31,2 ± 0.6
22,0 ± 0. 5
I00
33
2. 5 ± o . I
215o**
39
32
20
IOO
35
± 0.7
± 0,6
~= 0. 7
± 1.4
2.8 ~ o.15
± 1. 4
24oo**
38
0.6 ± o.9
31.6 i
20.8 ± 0. 4
I00"
35
3.1 ± o,15
**
T h e d a t a of I{AFIANI AND TIMOFEEVA 15.
* A b o u t 95 % of a d e u y l i c n u c l e o t i d e s w a s f o u n d in b l a s t o d e r m (i.e. i s o l a t e d e n l b r y o s ) .
16oo**
4°
Citrate
=t- 0.7
31.2 ± 0. 7
± 0. 5
=c 1.2
AMP
I00
14
2.1 ± o . I
22.0
pt
The end of gastrulation
-
± o.9
16o
33
0.6
~2 5
~ o.5
31.5 i
20.5 ± 0.5
-
2o
1.8 ± o . I
-
~_ I.O
I7o
33
0.7
q= 3
_q= 0. 5
32.5 !
19.5 ~ 0-5
-
2o
2.0 ± o . I
The beginnD~g of gastrulation
Blastula
The beginning of gastrulation
Cleavage
Blastula
Isolated embryos (blastoderm)
Developing eggs
Stages of development
ADP
ATP3*
fructose 6-phosphate
Glucose 6-phosphate +
Fructose 1,6-diphosphate
Intermediates
T h e a m o u n t s (the a v e r a g e s f r o m l O - 8 d e t e r m i n a t i o n s ) e x p r e s s e d in m / m l o l e s p e r i o o e m b r y o s .
THE CONTENT OF A T P , A D P , A M P , HEXOSI~PHOSPHATES
TABLE
-
~ I.I
I6o
3°
± 3
q- 0.8
31.5 =E 0.7
20.5 ± 0.5
-
22
2.3 ± o.15
The end of gastrulation
"q
o~
©
t~
db 0
Q
0
0
0
368
L . S . MILMAN, YU. G. YUROWITZKI
both the glycolytic activity and phosphofructokinase are mostly (2/3) localized in the embryo. The above statement is also supported by the fact that a considerable (more than two-fold) increase of glycolysis in isolated embryos in anaerobiosis a is followed b y a comparatively small (about 15 %) increase in the Pl level and by considerable increases (IOO % or more) in the content of adenylic nucleotides and citrate (Tables I I and III). Theoretically it cannot be ruled out that other mechanisms of regulation m a y be operative in the development of the embryo. It can be assumed that the control of certain processes in the embryo is performed by changing the relationship of the forms of enzymes with different kinetic properties. In this connection the data given by VII~!UELAet al. 26 are of considerable interest. The authors reported that in yeast two forms of phosphofructokinase had been found which had different susceptibilities to ATP (at the same fructose 6-phosphate concentration). According to VI~L'ELA et al. 2~ the conversion of a form of phosphofructokinase strongly inhibited by ATP into a form not susceptible to a high concentration of ATP can be observed at the incubation of a cytoplasmic fraction of yeast with o.I-O.O5 M NaF. After VII~UELA el al. an interconversion of the two forms of phosphofructokinase is responsible for the regulation of glycolysis in growing yeast. It is but natural to suggest that such regulation should occur in embryogenesis. Yet our results have not confirmed this view. As a matter of fact the presence of o.o3-o.1 M of N a F or preincubating extracts with fluoride keeps the activity of phosphofructokinase in the extracts constant (Table II). Homogenates prepared in o.o3-o.1 M N a F have the same activity as these prepared in phosphate buffer. The experiments carried out b y DE~,TON AND RANDLE27 on phosphofructokinase of adipose tissue also did not confirm the data of VII~'UELAet al. 26. It is necessary to point out that special experiments performed by us also did not show any differences in the mode of action of ATP and citrate on the phosphofructokinase in extracts of loach embryos at different stages of development. Consequently a replacement of phosphofructokinase with forms having different activities and different kinetic properties in early embryogenesis seems improbable. An increase in the amount of an active form of phosphofructokinase due to the activation of its latent form also seems to be unlikely as the content of adenylic nucleotides and magnesium ions remains constant in loach embryogenesis (see data of MANSOVI~ AND WAKII)28).Thus the activity of phosphofructokinase and the level of internlediates controlling its intracellular activity in early embryogenesis remain constant. Alongside the above-mentioned enzymes, the control of the fructose 1,6-diphosphate level is maintained b y fructose diphosphatase (EC 3.1.3. I I)*. This enzyme causes reconversion of fructose 1,6-diphosphate to fructose 6-phosphate thus providing gluconeogenesis and a pentose-phosphate cycle. A notable feature is the decrease in the activity of fructose diphosphatase during the embryogenesis since the moment of fertilization. The decrease of the fructose diphosphatase activity is in contrast to an increase in glycolysis and in the level of fructose 1,6-diphosphate in whole eggs (Fig. I). * i t was sh own b y us t h a t the level of a l d o l a s e in e a r l y e m b r y o g e n e s i s of l oa c h r e m a i n s cons t a n t a n d so t h e a c t i v i t y of a l d o l a s e is n o t r e s p o n s i b l e for t h e a l t e r a t i o n s of t h e level of f r u c t o s e 1,6-diphosphate during embryogenesis.
Biochim. Biophys. Acta, 148 (1967) 362-371
369
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
Fructose diphosphatase of loach embryos is similar to this enzyme of mammalian tissues in certain properties, e.g. both are inhibited by AMP and an excess of substrate (Figs. 2 and 3)- The activity of fructose-diphosphatase preparations (free of aldolase) increases as the concentration of fructose 1,6-diphosphate is increased to 4 raM. A further increase in the concentration of substrate inhibits the activity of the enzyme; at 15 mM substrate no activity can be detected. Substrate concentrations inhibiting fructose-diphosphatase activity in loach embryos are considerably
higher than those required for the inhibition of fructose diphosphatase in mammalian tissues. For example, SALAS, VII~UELA AND SOLS 29, and T A K E T A AND POGELL 3° have B.O- -1~ ~2
.c E lD
~3 x:l E ~2 © 0
© ©
d 2h !0 ,k
Y m
5 ~© la.
cleavage blastUlO gastrulo
organogenesis 2L
E,
o 5 lo 15 20 p5 ,3o Development time at 21°(h) Fig. i. R a t e of glycolysis in i n t a c t loach eggs (closed circles) a n d level of fructose 1 , 6 - d i p h o s p h a t e (open circles) in developing loach eggs, F r u c t o s e - d i p h o s p h a t a s e a c t i v i t y is d e s i g n a t e d b y triangles. T h e left ordinate, t h e rate of glycolysis in /,moles of l a c t a t e per iooo e m b r y o s a t I h; t h e r i g h t (inner ordinate), level of fructose 1 , 6 - d i p h o s p h a t e ill m f m o l e s per IOOO e m b r y o s ; t h e o u t e r o r d i n a t e (right), t h e a c t i v i t y of fructose d i p h o s p h a t a s e in ffmoles of fructose 6 - p h o s p h a t e released per iooo e m b r y o s at 5 rain. l//v 4~I
I I
100
I
I 1.ol
50
051 <
0.25
0.5
[AMP] (mM)
1.0
O 5 1/LFru-l.6-P~ (rnM)
10
0 5 10 [Fru-l,6-P~ (mM)
15
Fig. 2. T h e inhibition of fructose d i p h o s p h a t a s e of loach e m b r y o s b y AMP. E n z y m e a c t i v i t y w a s a s s a y e d b y f o r m a t i o n of Pt in all i n c u b a t i o n m e d i u m c o n t a i n i n g 50 m M Tris-HC1 (pH 7.5) io m M MgSO 4, 0.05 m M E D T A , 5 m M /~-mercaptoethanol a n d i m M fructose 1,6-diphosphate. Fig. 3- T h e relationship b e t w e e n t h e a c t i v i t y of p a r t i a l l y purified loach e m b r y o fructose diphosp h a t a s e a n d s u b s t r a t e c o n c e n t r a t i o n . T h e i n c u b a t i o n m e d i u m is t h e s a m e as described in t h e legend of Fig. 2 e x c e p t t h a t v a r i o u s c o n c e n t r a t i o n s of fructose 1 , 6 - d i p h o s p h a t e were added. Left, t h e plot of t h e reciprocal of t h e speed versus t h e reciprocal of t h e m M c o n c e n t r a t i o n of fructose 1,6-diphosphate. Biochim. Biophys. Acta, 148 (1967) 362-37z
37 °
L . S . MILMAN, YU. G. YUROWITZKI
shown that the inhibition of fructose diphosphatase by substrate in muscles and liver starts at a fructose-I,6-diphosphate concentration of o.I mM and more than 3/4 of the activity is inhibited at a substrate concentration of 2.0 mM. Fructose diphosphatase of loach embryos is also less susceptible to AMP than this enzyme of rat liver. For isolated fructose-diphosphatase preparations (free of AMP desaminase and adenylate kinase) 50 % inhibition was found at AMP concentrations higher than 0.2 raM. Fructose diphosphatase of rat liver is 7 ° % inhibited at o.16 mM of AMP and that of skeletal muscle at several /,M of AMP 29-31. Special experiments carried out by us have shown that fructose-diphosphatase susceptibility to inhibition by AMP and substrate renlains constant during embryogenesis. Does a decrease in the fructose-diphosphatase activity occur during the early stages of embryogenesis due to the conversion of some enzyme into an inactive form or a decrease in its activity due to some other mechanism? LuPPIS et al. 22 were the first to report that two forms of fructose diphosphatase in liver are likely to exist. Preincubation of dilute fructose-diphosphatase preparations in the presence of manganese ions and cysteine enhanced enzyme activity in some experiments performed by LuPPIS and his associates. Both the possible existence of a latent form of fructose diphosphatase, similar to that described by LuPPIS et al., and its role in the regulation of carbohydrate metabolism in an embryo are of great interest. Some attempts to discover the latent form of fructose diphosphatase in embryo extracts according to L•PPiS et al. 22 were not successful. Yet the effect of preincubation described by LuPPIS et al. 22 can be easily demonstrated on fructose-diphosphatase preparations isolated from loach embryos or from rat liver if the preparations were subjected to dialysis during the procedure of isolation. Thus we can suggest that the activity of fructose diphosphatase observed in extracts of loach embryos in optimal medium for the assay of fructose-diphosphatase activity should indicate the total activity of enzyme. Consequently the observed decrease in fructose-diphosphatase activity in early embryogenesis is caused by a decrease in the amount of enzyme.
REFERENCES 1 2 3 4 5 6 7 8 9 io II 12 13 14 15 I6
L. S. MILMAN, Zh. Obshch. Biol., 26 (1965) 237. A. COHEN, Physiol. Zool., 27 (1954) 128. Yu. G. •UROWITZKI AND L. S. MILMAN, Dokl. Akad. Nauk, S.S.S.R., 163 (1965) 781. J. V. PASSONEAU AND O. H. LOWRY, Biochem. Biophys. Res. Commun., 7 (1962) io. T. E. MANSOUR, 3r. Biol. Chem., 238 (1963) 2285. J. V. PASSONEAU AND O. H. LOWRY, Biochem. Biophys. Res. Commun., 13 (1963) 372. A. H. UNDERWOOD AND E. A. NEWSHOLM, Biochem. dr., 95 (1965) 868. A. PARMEGGIANI AND R. BOWMAN, Biochem. Biophys. Res. Commun., 12 (1964) 268. M. SALAS,E. VI~UELA, M. L. SALAS AND A. SOLS, Bioehem. Biophys. Res. Commun., 19 (1965) 371 . A. A. N1~IFAKH, Biokhimia, 25 (196o) 658. L. S. MILMAN AND YU. G. YUROWITZKI, Exptl. Cell Res., 42 (1966) 478. H. J. HOHORST, in H. U. BI~RGMEYER, Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, p. 131. A. KORNBERG AND B. HORRECKER,J. Biol. Chem., 182 (195 o) 805. T. BARANOWSKY,Z. Physiol. Chem., 260 (1939) 43. T. BARANOWSKY, J. Biol. Chem., 18o (1949) 535G. BEISENHERZ, H. J. BOLTZE, Z. H. GARBADE, TH. BOCHER AND R. CZOK Z. Naturforsch., 8b (1953) 555.
Bioehim. Biophys. Acta, i48 (1967) 362-371
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
371
17 H. ADAM,in H. U. BERGMEYER,Methoden der enzymatisehenAnalyse, VerlagChemie, Weinheim, 1962, P. 573. 18 J. H. STERN, in S. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 3, Academic, N e w York, 1957, p. 425 • 19 C. H. FISKE AND Y. SUBBAROW,J. Biol. Chem., 81 (1929) 629. 20 O. H. LowRY, M. ROSEBROUGH, A. FARR, AND R. RANDALL, J. Biol. Chem., 193 (1951) 265. 21 K. H. LING, W. L. BYRNE AND H. A. LARDY, in S. COLOWICK AND N. O. KAPLAN, IVIethods in Enzymology, Vol. I, Academic, N e w York, 1955, p. 306. 22 B. LupPIS, S. TRANIELLO, ~¢V.A. WOOD AND S. PONTREMOLLI, Biochem. Biophys. Res. Commun., 15 (I964) 458 . 23 A. BONSIGNORE, G. MANGIAROTTI, A. DE FLORA AND S. PONTREMOLI, J. Biol. Chem., 238 (1963) 3151 . 24 K. UYEDA AND IE. RACKER, J. Biol. Chem., 240 (1965) 4682. 25 K. A. I(AFIANY AND M. YA. TIMOFEEVA, Biohhimia, 27 (1962) 938. 26 ]L. VII~UELA, M. L. SALAS,M. SALAS AND A. SOLS, Biochem. Biophys. Res. Commun., 15 (1964) 243. 27 R. M. DENTON ANn P. T. RANDLE, Biochem. J., IOO (1966) 420. 28 T. E. MANSOUR AND N. M. WAKID, Biochem. Biophys. Res. Commun., 19 (1965) 721. 29 M. SALAS, E. \7II'~UELA, AND A. SOLS, Biochem. Biophys. Res. Commun., 17 (1964) 15o. 3 ° K. TAKETA AND B. M. POG:ELL, J. Biol. Chem., 240 (1965) 651. 31 H. A. KRI~gs AND M. WOODFORD, Biochem. J., 94 (1965) 436. 32 L. S. MILMAN AND YU. G. DANJUKOFF,Cytologia, 7 (I965) 732 (in Russian).
Biochim. Biophys. Acta, 148 (1967) 362-371
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25866 T H E CONTROL OF GLYCOLYSIS IN EARLY EMBRYOGENESIS L. S. MILMAN AND YU. G. Y U R O W I T Z K I
A. N. Severtzov Institute of Animal Morphology, Academy of Sciences of the U.£'.S.R. (Moscow) (Received March 13th, 1967) (Revised manuscript received June 2oth, 1967)
SUMMARY
An increase of glycolysis i s vivo at early stages of loach embryogenesis is related to the regulation of the level of fructose 1,6-diphosphate. The activity of enzymes of the system converting fructose 1,6-diphosphate into lactate remains constant in the course of development. An enhancement of glycolysis during the embryogenesis is not due to an increase of phosphofructokinase (EC 2.7.1.11 ) activity and cannot be attributed to changes of the level of intermediates controlling phosphofructokinase activity. The content of hexose monophosphates, citrate and adenylic nucleotides in loach embryos at early stages of development remains constant. A considerable decrease in fructose diphosphatase (EC 3.1.3.11) activity in early embryogenesis is reciprocal to an increase in the rate of glycolysis in intact eggs and to the level of fructose 1,6-diphosphate. The inhibition of phosphofructokinase activity by citrate and ATP and inhibition of fructose diphosphatase activity by AMP and fructose 1,6-diphosphate are assumed to control the level of fructose 1,6-diphosphate according to the feed-back type of regulation, depending on the level of intermediates. The decrease of fructose diphosphatase activity is regulated by the control mechanism of enzyme biosynthesis in the developing embryo.
INTRODUCTION
In early embryogenesis, a considerable (3-fold) increase in glycolysis occurs i~z vivo. This is related to the activation of the first steps of glycolysis at a constant
activity of the enzymes converting fructose 1,6-diphosphate to lactate 1-a. We have shown that the rate of glycolysis is determined by the fructose 1,6-diphosphate level in the cells of the embryo. It is not dependent on the concentration of hexose monophosphates and is directly proportional to fructose 1,6-diphosphate concentrations from o.I to 4 mM. Determinations of glycogen phosphorylase activity (EC 2.4.1.1) also confirms that its activity is not a glycolysis-limiting factor and does not change during early stages of embryogenesis. The fructose 1,6-diphosphate level is controlled by the activities of phosphofructokinase (EC 2.7.1. I I) and hexosediphosphatase (EC 3.1.3. II), i.e. enzymes taking part in the regulation of the Pasteur effect. Recently convincing evidence has been obtained from experiments on yeast, heart muscle, skeletal muscle and liver which Bioehim. Biophys. Acta, 148 (I967) 362-371
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
363
testify that the enzyme regulating glycolysis, phosphofructokinase, controls the Pasteur effect. PASSONEAU AND LOWRY4, MANSOUR 5, and other authors 6-9 have shown that the phosphofructokinase is inhibited by ATP and citrate. What is the relationship between the activity of phosphofructokinase and the increase of glycolysis in embryogenesis? Does an increase in phosphofructokinase activity occur simultaneously with an increase in glycolysis or is the control of increasing glycolysis in early embryogenesis brought about in some other way ? The solution of these questions can serve to clarify the more general problem of the relationship between mechanisms controlling the homeostasis in a cell (i.e. regulation of glycolysis of a cell 'in steady state') and systems controlling the general intensification of metabolism during the realization of genetic information in embryogenesis. No data on this problem have been reported yet. MATERIALS AND METHODS
Animals Experiments were performed on developing eggs of loach (Misgurnus fossilis). Mature eggs were obtained by injecting female loaches with 2o0 units of gonadotropic hormone 'Choriogonin' (Richter, Hungary). The stage of development was expressed in h at 21 ° according to NEIFAKHI°: 0--6 h = cleavage, 6- 9 h ~ blastula, 9-2o h = gastrulation, 20 h = blastopore closure. The isolated embryos (blastoderm) from loach eggs were separated from the yolk by means of centrifugation at 4000-50o0 × g on sucrose gradient after the trypsin treatment of tile eggs to remove the egg membrane 11. Determination of intermediates Assay of hexosephosphates and adenylic nucleotides were made in deproteinized HClO4-extracts of embryos according to the general procedure recommended by BERGMEYER12,17. The fixation of eggs or isolated embryos was made by immersion in liquid N 2. Hexose monophosphates were assayed in aliquots of deproteinized extracts by reduction of NADP + with 0.4 units of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and an excess of glucose-phosphate isomerase (EC 5.3.I.9) (ref. 12). Glucose 6-phosphate dehydrogenase was obtained by the method of KORNBERG AND HORRECKER from yeast 13. The enzyme was freed from phosphogluconate dehydrogenase (EC 1.1.1.44 ) activity. Fructose 1,6-diphosphate was assayed with a coupled system consisting of NADH and 3o-4o/~g of crystalline myogen A (isolated by the method of BARANOWSKY14,15) or with an excess of several times recrystallized aldolase (EC 4.1.2.13) and a-glycerophosphate dehydrogenase (EC 1.1.1.8). The above-mentioned enzymes were obtained according to the procedure of BEISENHERZ et al. 16. The loach embryos contained only a trace of phosphotrioses, which could be neglected. ADP was determined by the method of ADAM17 with NADH and an excess of lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.4o). AMP was determined in the same procedure after adding an excess of adenylate kinase. The citrate was determined in trichloroacetic acid-extracts by the method of Biochim. Biophys, Acta, 148 (1967) 362-371
364
L. S. M I L M A N , YU. G. Y U R O W I T Z K I
STERN~8; the Pl by the method of FISKE AND SUUBAROW19; the protein by the procedure of LowRY et al. ~°.
The preparation of homogenates For the assay of phosphofructokinase (EC 2.7.1.11) activity the loach eggs or isolated embryos were homogenized in an all-glass homogenizer in 50 mM KH2PO 4 (pH 7.5) containfng 5 mM/3-mercaptoethanol. For the assay of fructose diphosphatase, 50 mM Tris (pH 7.5) were employed instead of KH2PO 4. Extracts were obtained after 5 rain centrifugation at 2000 × g. In some experiments mitochondria-free extracts were obtained after 20 rain centrifugation at 18 ooo x g. The assay of enzyme activity Activity of phosphofructokinase was assayed by increasing the fructose 1,6diphosphate after 5 min incubation of an aliquot of extract in assay medium 21. The reaction was stopped by adding o.I ml 3o % HC10~. After centrifugation in the cold the supernatant was decanted and neutralized by KHCO a. The KC10 4 residue was discarded after the centrifugation. The amount of fructose 1,6-diphosphate in o. 4 nil aliquots of supernatant fluid was determined enzymatically (see above). The assay medimn (for maximal activity of phosphofructokinase) was comprised of 50 mM Tris-HC1 (pH 8.3), 25 mM KH2PO 4, 1,5 mM ATP, 2.5 mIVIMgSOa, 1.5 mM fructose 6-phosphate and 5 mM /3-mercaptoethanol. The second medium provided conditions for maximal effect of ATP and citrate and had a final concentration of 25 mM imidazole-HC1 (pH 7.2), 25 mM Pl (pH 7.2), 5 mM MgSO4, 0.5 mM fructose 6-phosphate and 5 mM/~-mercaptoethanol. Activity of fructose diphosphatase was determined by the increase in the amount of fructose 6-phosphate in the assay medium after Io rain incubation at 25 o. The assay medium was comprised of 50 mM Tris-HC1 (pH 7.5), I mM fructose 1,6diphosphate, IO mM MgSO,, 0.05 mM EDTA, 5 mM ¢/-mercaptoethanol. The increases of fructose 6-phosphate and glucose 6-phosphate were determined enzymatically (see above). In some experiments the release of Pt was determined according to the method of FISKE AND SUBBAI~OW19. The amount of released Pt in ~moles (with the increment for Pi released by non-specific phosphatases) corresponded to the sum of hexose monophosphates formed during the reaction. Preparation of partially purified fructose diphosphatase The fructose diphosphatase from loach embryos or rat liver was isolated according to BONSIGNORE et al.22,2a up to stage IV. The isolated preparations were free from activity of non-specific phosphatases, aldolase, phosphofructokinase, adenylate kinase, etc. Reagents Tris, fl-mercaptoethanol, NADH, ~-glycerophosphate were obtained from Light & Co (England); ATP, ADP, AMP: from Calbiochem (U.S.A.); glucose-0-phosphate: from Sigma (U.S.A.); NADP: from Lawson (England). Lactate dehydrogenase, phosphoenolpyruvate, pyru vatekinase and adenylate kinase were purchased from Boehringer (Germany). Commercially available barium salt of fructose 6-phosphate was Biochim. Biophys. dcta, 148 (1967) 362-371
365
CONTROL OF GLYCOLYSIS IN E M B R Y O G E N E S I S
completely purified to remove traces of fructose 1,6-diphosphate, glucose 6-phosphate and nucleotides. RESULTS
AND DISCUSSION
Determination of the activity of phosphofructokinase carried out under conditions providing for maximal activity of the enzyme has shown that the phosphofructokinase activity remains constant during development. In addition the presence of mitochondria was shown not to influence the results of the determination of phosphofructokinase activity. In both cases the activity of phosphofructokinase is equal to o.17 ~moles of fructose 1,6-phosphate formed during 5 min by ioo embryos. In the medium with a low concentration of fructose 6-phosphate (o.5 raM) in imidazole buffer, phosphofructokinase of loach embryos is inhibitited by ATP and citrate. A notable inhibition of the phosphofructokinase activity by an excess of ATP can be observed at ATP concentrations of 4 mM and higher (Table I). Yet we could not observe the described effect in the cases when Tris (pH 7.o) was substituted for imidazole. This confirms the data of UYEDA AND RACKER24. Citrate is the strongest inhibitor of phosphofructokinase. The inhibiting effect of citrate detected at higher concentrations of fructose 6-phosphate is slightly dependent on the type of buffer used. The given properties of phosphofructokinase (i.e. data of PASSONEAU AND L O W R Y 4, M A N S O U R , NEWSHOLM and others (refs. 5-9)) to a considerable extent account for the mechanism of the Pasteur effect. A decrease in the level both of ATP and citrate under anaerobic conditions as well as an increase in the level of ADP, AMP and Pl increase the intracellular activity of phosphofructokinase and inhibit the activity of an enzyme-antagonist, fructose diphosphatase. Alterations in the level of intermediates in loach embryos under aerobic and anaerobic conditions (Table II) completely confirmed the above-mentioned point of view. It is to be stressed that the relation between the levels of metabolites under aerobic and anaerobic conditions does not depend on the stage of development. The only exception is fructose 1,6-diphosphate. At the 7-h stage (blastula) the level of fructose 1,6-diphosphate is increased by 25 % due to anaerobic conditions, but when TABLE
I
INHIBITION OF PHOSPHOFRUCTOKINASE
ACTIVITY BY A T P AND CITRATE
T h e r e a c t i o n m i x t u r e c o n t a i n e d 25 m M i m i d a z o l e ( p H 7.2), 25 m M K H z P O 4 ( p H 7.2), 5 m M M g S O 4, 0.5 m M f r u c t o s e 6 - p h o s p h a t e , 5 m M f l - m e r c a p t o e t h a n o l a n d a n a l i q u o t of m i t o c h o n d r i a - f r e e e x t r a c t of i s o l a t e d l o a c h e m b r y o s . T h e a c t i v i t y of p h o s p h o f r u c t o k i n a s e e x p r e s s e d i n m # m o l e s of fructose 1,6-diphosphate formed during 5 rain per 5° isolated embryos.
d TP (mM)
Activity of phosphofruetokinase Control
0.6
63
1.2
7°
4 .0 6.0 8.0
55 35 17
in %
90 ioo 79 50 25
After z 5 min preincubation with 5o m M N a F
5 m M citrate added
-7° -35 17
3° 20 io 5 --
Bioehim. Biophys. Acta, 148 (1967) 3 6 2 - 3 7 1
366
L. S. MILMAN, YU. G. YUROWITZKI
TABLE 1I T H E A L T E R A T I O N S IN L E V E L S OF AND
ANAEROBIC
CONDITIONS
IN
ATP, ADP, AMP, ISOLATED
CITRATE AND HEXOSEPHOSPHATES
IN AEROBIC
LOACH EMBRYOS
Amounts of intermediates expressed in m/,moles per ioo embryos (The details of determinations see in M A T E R I A L A N D M E T H O D S ) . Intermediates
Stage of development
mltmoles of intermediates Per zoo isolated embryos
Per z m g of protein
Control
Co~trol
Fructose 1,6-diphosphate
Blastula The end of gastrulation
Hexosemonophosphates
Blastula The end of gastrulation
21 22
ADP
Blastula The end of gastrulation
AMP
Blastula The end of gastrulation
ATP
Blastula The end of gastrulation
Citrate
Blastula The end of gastrulation
Pi
Blastula The end of gastrulation
1 , 8 @ O.I 2. 3 ± o.i
A naerobiosis
I. 5 1.9
2.O 4.6
:~ o.9 ~ o.9
17.6
I7.6
2o.5 i o-5 19.6 :~ o.5
36.5 5 o.5 36.o -[ o.6
16.5 15.9
29.6 28.8
31.5 t- 0.5 31.3 --' o.5
68.o ~ o. 5 67.2 :~ o.5
27.o 27.2
55.0 54.2
28.5 24.0
8. 7 ii.2
~:::o.9 " o.9
ioo IOO 34 ± o.6 28.3 ~' 0.6 175 14o
2.4 ~ O.I 5.5 5 o.2
A naerobiosis
2o 21
6o 6o io :~ o. 3 13.8 ~ o. 3 230 19o
glycolysis is more intensive, the level of fructose 1,6-diphosphate is increased b y 5o % b y the end of gastrulation. As the a c t i v i t y of phosphofructokinase when det e r m i n e d u n d e r different conditions does not v a r y during development, the question arises of whether an intracellular a c t i v i t y of phosphofructokinase can be controlled in early embryogenesis b y alterations of the level of i n t e r m e d i a t e s of glycolysis a n d respiration. As was m e n t i o n e d above, such regulation is k n o w n as the P a s t e u r effect. The results of m e t a b o l i t e assays d u r i n g d e v e l o p m e n t (Table III) have shown t h a t the c o n t e n t of adenylic nucleotides a n d citrate, i.e. metabolites most strongly affecting the phosphofructokinase activity, does not v a r y in early embryogenesis, nor does a r e d i s t r i b u t i o n of metabolites between yolk a n d e m b r y o proper occur. The level of hexose m o n o p h o s p h a t e s reaches its m a x i m u m b y the 6-7-h stage (blastula) a n d then r e m a i n s c o n s t a n t d u r i n g further development. C o n s e q u e n t l y the m a x i m u m increase of glycolysis from the stage of b l a s t u l a to t h a t of 15 h (the middle of gastrulation) occurs at a c o n s t a n t level of hexose monophosphates. A n increase in the fructose 1,6-diphosphate level in general indicates the rate of increasing glycolysis i n vivo (Fig. 1). F l u c t u a t i o n s in the c o n t e n t of Pi in isolated embryos d u r i n g d e v e l o p m e n t are also negligible (Tables I I a n d III) a n d thus c a n n o t exert significant influence on the a c t i v i t y of phosphofructokinase. A n increase in the c o n t e n t of PI in developing loach eggs reported b y I{AFIANI AND TIMOFEEVA25 occurs not in the embryo proper b u t in the yolk which c o n t a i n s up to 9/lO of the Pt c o n t e n t of the whole egg. I n c o n t r a s t Biochim. Biophys. Aeta, 148 (z967) 362-371
% Y
0o
42
~"
III AND CITRATE IN DEVELOPING LOACH EMBRYOS
± 1.4
--
4°
± 0.7
31,2 ± 0.6
22,0 ± 0. 5
I00
33
2. 5 ± o . I
215o**
39
32
20
IOO
35
± 0.7
± 0,6
~= 0. 7
± 1.4
2.8 ~ o.15
± 1. 4
24oo**
38
0.6 ± o.9
31.6 i
20.8 ± 0. 4
I00"
35
3.1 ± o,15
**
T h e d a t a of I{AFIANI AND TIMOFEEVA 15.
* A b o u t 95 % of a d e u y l i c n u c l e o t i d e s w a s f o u n d in b l a s t o d e r m (i.e. i s o l a t e d e n l b r y o s ) .
16oo**
4°
Citrate
=t- 0.7
31.2 ± 0. 7
± 0. 5
=c 1.2
AMP
I00
14
2.1 ± o . I
22.0
pt
The end of gastrulation
-
± o.9
16o
33
0.6
~2 5
~ o.5
31.5 i
20.5 ± 0.5
-
2o
1.8 ± o . I
-
~_ I.O
I7o
33
0.7
q= 3
_q= 0. 5
32.5 !
19.5 ~ 0-5
-
2o
2.0 ± o . I
The beginnD~g of gastrulation
Blastula
The beginning of gastrulation
Cleavage
Blastula
Isolated embryos (blastoderm)
Developing eggs
Stages of development
ADP
ATP3*
fructose 6-phosphate
Glucose 6-phosphate +
Fructose 1,6-diphosphate
Intermediates
T h e a m o u n t s (the a v e r a g e s f r o m l O - 8 d e t e r m i n a t i o n s ) e x p r e s s e d in m / m l o l e s p e r i o o e m b r y o s .
THE CONTENT OF A T P , A D P , A M P , HEXOSI~PHOSPHATES
TABLE
-
~ I.I
I6o
3°
± 3
q- 0.8
31.5 =E 0.7
20.5 ± 0.5
-
22
2.3 ± o.15
The end of gastrulation
"q
o~
©
t~
db 0
Q
0
0
0
368
L . S . MILMAN, YU. G. YUROWITZKI
both the glycolytic activity and phosphofructokinase are mostly (2/3) localized in the embryo. The above statement is also supported by the fact that a considerable (more than two-fold) increase of glycolysis in isolated embryos in anaerobiosis a is followed b y a comparatively small (about 15 %) increase in the Pl level and by considerable increases (IOO % or more) in the content of adenylic nucleotides and citrate (Tables I I and III). Theoretically it cannot be ruled out that other mechanisms of regulation m a y be operative in the development of the embryo. It can be assumed that the control of certain processes in the embryo is performed by changing the relationship of the forms of enzymes with different kinetic properties. In this connection the data given by VII~!UELAet al. 26 are of considerable interest. The authors reported that in yeast two forms of phosphofructokinase had been found which had different susceptibilities to ATP (at the same fructose 6-phosphate concentration). According to VI~L'ELA et al. 2~ the conversion of a form of phosphofructokinase strongly inhibited by ATP into a form not susceptible to a high concentration of ATP can be observed at the incubation of a cytoplasmic fraction of yeast with o.I-O.O5 M NaF. After VII~UELA el al. an interconversion of the two forms of phosphofructokinase is responsible for the regulation of glycolysis in growing yeast. It is but natural to suggest that such regulation should occur in embryogenesis. Yet our results have not confirmed this view. As a matter of fact the presence of o.o3-o.1 M of N a F or preincubating extracts with fluoride keeps the activity of phosphofructokinase in the extracts constant (Table II). Homogenates prepared in o.o3-o.1 M N a F have the same activity as these prepared in phosphate buffer. The experiments carried out b y DE~,TON AND RANDLE27 on phosphofructokinase of adipose tissue also did not confirm the data of VII~'UELAet al. 26. It is necessary to point out that special experiments performed by us also did not show any differences in the mode of action of ATP and citrate on the phosphofructokinase in extracts of loach embryos at different stages of development. Consequently a replacement of phosphofructokinase with forms having different activities and different kinetic properties in early embryogenesis seems improbable. An increase in the amount of an active form of phosphofructokinase due to the activation of its latent form also seems to be unlikely as the content of adenylic nucleotides and magnesium ions remains constant in loach embryogenesis (see data of MANSOVI~ AND WAKII)28).Thus the activity of phosphofructokinase and the level of internlediates controlling its intracellular activity in early embryogenesis remain constant. Alongside the above-mentioned enzymes, the control of the fructose 1,6-diphosphate level is maintained b y fructose diphosphatase (EC 3.1.3. I I)*. This enzyme causes reconversion of fructose 1,6-diphosphate to fructose 6-phosphate thus providing gluconeogenesis and a pentose-phosphate cycle. A notable feature is the decrease in the activity of fructose diphosphatase during the embryogenesis since the moment of fertilization. The decrease of the fructose diphosphatase activity is in contrast to an increase in glycolysis and in the level of fructose 1,6-diphosphate in whole eggs (Fig. I). * i t was sh own b y us t h a t the level of a l d o l a s e in e a r l y e m b r y o g e n e s i s of l oa c h r e m a i n s cons t a n t a n d so t h e a c t i v i t y of a l d o l a s e is n o t r e s p o n s i b l e for t h e a l t e r a t i o n s of t h e level of f r u c t o s e 1,6-diphosphate during embryogenesis.
Biochim. Biophys. Acta, 148 (1967) 362-371
369
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
Fructose diphosphatase of loach embryos is similar to this enzyme of mammalian tissues in certain properties, e.g. both are inhibited by AMP and an excess of substrate (Figs. 2 and 3)- The activity of fructose-diphosphatase preparations (free of aldolase) increases as the concentration of fructose 1,6-diphosphate is increased to 4 raM. A further increase in the concentration of substrate inhibits the activity of the enzyme; at 15 mM substrate no activity can be detected. Substrate concentrations inhibiting fructose-diphosphatase activity in loach embryos are considerably
higher than those required for the inhibition of fructose diphosphatase in mammalian tissues. For example, SALAS, VII~UELA AND SOLS 29, and T A K E T A AND POGELL 3° have B.O- -1~ ~2
.c E lD
~3 x:l E ~2 © 0
© ©
d 2h !0 ,k
Y m
5 ~© la.
cleavage blastUlO gastrulo
organogenesis 2L
E,
o 5 lo 15 20 p5 ,3o Development time at 21°(h) Fig. i. R a t e of glycolysis in i n t a c t loach eggs (closed circles) a n d level of fructose 1 , 6 - d i p h o s p h a t e (open circles) in developing loach eggs, F r u c t o s e - d i p h o s p h a t a s e a c t i v i t y is d e s i g n a t e d b y triangles. T h e left ordinate, t h e rate of glycolysis in /,moles of l a c t a t e per iooo e m b r y o s a t I h; t h e r i g h t (inner ordinate), level of fructose 1 , 6 - d i p h o s p h a t e ill m f m o l e s per IOOO e m b r y o s ; t h e o u t e r o r d i n a t e (right), t h e a c t i v i t y of fructose d i p h o s p h a t a s e in ffmoles of fructose 6 - p h o s p h a t e released per iooo e m b r y o s at 5 rain. l//v 4~I
I I
100
I
I 1.ol
50
051 <
0.25
0.5
[AMP] (mM)
1.0
O 5 1/LFru-l.6-P~ (rnM)
10
0 5 10 [Fru-l,6-P~ (mM)
15
Fig. 2. T h e inhibition of fructose d i p h o s p h a t a s e of loach e m b r y o s b y AMP. E n z y m e a c t i v i t y w a s a s s a y e d b y f o r m a t i o n of Pt in all i n c u b a t i o n m e d i u m c o n t a i n i n g 50 m M Tris-HC1 (pH 7.5) io m M MgSO 4, 0.05 m M E D T A , 5 m M /~-mercaptoethanol a n d i m M fructose 1,6-diphosphate. Fig. 3- T h e relationship b e t w e e n t h e a c t i v i t y of p a r t i a l l y purified loach e m b r y o fructose diphosp h a t a s e a n d s u b s t r a t e c o n c e n t r a t i o n . T h e i n c u b a t i o n m e d i u m is t h e s a m e as described in t h e legend of Fig. 2 e x c e p t t h a t v a r i o u s c o n c e n t r a t i o n s of fructose 1 , 6 - d i p h o s p h a t e were added. Left, t h e plot of t h e reciprocal of t h e speed versus t h e reciprocal of t h e m M c o n c e n t r a t i o n of fructose 1,6-diphosphate. Biochim. Biophys. Acta, 148 (1967) 362-37z
37 °
L . S . MILMAN, YU. G. YUROWITZKI
shown that the inhibition of fructose diphosphatase by substrate in muscles and liver starts at a fructose-I,6-diphosphate concentration of o.I mM and more than 3/4 of the activity is inhibited at a substrate concentration of 2.0 mM. Fructose diphosphatase of loach embryos is also less susceptible to AMP than this enzyme of rat liver. For isolated fructose-diphosphatase preparations (free of AMP desaminase and adenylate kinase) 50 % inhibition was found at AMP concentrations higher than 0.2 raM. Fructose diphosphatase of rat liver is 7 ° % inhibited at o.16 mM of AMP and that of skeletal muscle at several /,M of AMP 29-31. Special experiments carried out by us have shown that fructose-diphosphatase susceptibility to inhibition by AMP and substrate renlains constant during embryogenesis. Does a decrease in the fructose-diphosphatase activity occur during the early stages of embryogenesis due to the conversion of some enzyme into an inactive form or a decrease in its activity due to some other mechanism? LuPPIS et al. 22 were the first to report that two forms of fructose diphosphatase in liver are likely to exist. Preincubation of dilute fructose-diphosphatase preparations in the presence of manganese ions and cysteine enhanced enzyme activity in some experiments performed by LuPPIS and his associates. Both the possible existence of a latent form of fructose diphosphatase, similar to that described by LuPPIS et al., and its role in the regulation of carbohydrate metabolism in an embryo are of great interest. Some attempts to discover the latent form of fructose diphosphatase in embryo extracts according to L•PPiS et al. 22 were not successful. Yet the effect of preincubation described by LuPPIS et al. 22 can be easily demonstrated on fructose-diphosphatase preparations isolated from loach embryos or from rat liver if the preparations were subjected to dialysis during the procedure of isolation. Thus we can suggest that the activity of fructose diphosphatase observed in extracts of loach embryos in optimal medium for the assay of fructose-diphosphatase activity should indicate the total activity of enzyme. Consequently the observed decrease in fructose-diphosphatase activity in early embryogenesis is caused by a decrease in the amount of enzyme.
REFERENCES 1 2 3 4 5 6 7 8 9 io II 12 13 14 15 I6
L. S. MILMAN, Zh. Obshch. Biol., 26 (1965) 237. A. COHEN, Physiol. Zool., 27 (1954) 128. Yu. G. •UROWITZKI AND L. S. MILMAN, Dokl. Akad. Nauk, S.S.S.R., 163 (1965) 781. J. V. PASSONEAU AND O. H. LOWRY, Biochem. Biophys. Res. Commun., 7 (1962) io. T. E. MANSOUR, 3r. Biol. Chem., 238 (1963) 2285. J. V. PASSONEAU AND O. H. LOWRY, Biochem. Biophys. Res. Commun., 13 (1963) 372. A. H. UNDERWOOD AND E. A. NEWSHOLM, Biochem. dr., 95 (1965) 868. A. PARMEGGIANI AND R. BOWMAN, Biochem. Biophys. Res. Commun., 12 (1964) 268. M. SALAS,E. VI~UELA, M. L. SALAS AND A. SOLS, Bioehem. Biophys. Res. Commun., 19 (1965) 371 . A. A. N1~IFAKH, Biokhimia, 25 (196o) 658. L. S. MILMAN AND YU. G. YUROWITZKI, Exptl. Cell Res., 42 (1966) 478. H. J. HOHORST, in H. U. BI~RGMEYER, Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, p. 131. A. KORNBERG AND B. HORRECKER,J. Biol. Chem., 182 (195 o) 805. T. BARANOWSKY,Z. Physiol. Chem., 260 (1939) 43. T. BARANOWSKY, J. Biol. Chem., 18o (1949) 535G. BEISENHERZ, H. J. BOLTZE, Z. H. GARBADE, TH. BOCHER AND R. CZOK Z. Naturforsch., 8b (1953) 555.
Bioehim. Biophys. Acta, i48 (1967) 362-371
CONTROL OF GLYCOLYSIS IN EMBRYOGENESIS
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17 H. ADAM,in H. U. BERGMEYER,Methoden der enzymatisehenAnalyse, VerlagChemie, Weinheim, 1962, P. 573. 18 J. H. STERN, in S. COLOWICK AND N. O. I~APLAN, Methods in Enzymology, Vol. 3, Academic, N e w York, 1957, p. 425 • 19 C. H. FISKE AND Y. SUBBAROW,J. Biol. Chem., 81 (1929) 629. 20 O. H. LowRY, M. ROSEBROUGH, A. FARR, AND R. RANDALL, J. Biol. Chem., 193 (1951) 265. 21 K. H. LING, W. L. BYRNE AND H. A. LARDY, in S. COLOWICK AND N. O. KAPLAN, IVIethods in Enzymology, Vol. I, Academic, N e w York, 1955, p. 306. 22 B. LupPIS, S. TRANIELLO, ~¢V.A. WOOD AND S. PONTREMOLLI, Biochem. Biophys. Res. Commun., 15 (I964) 458 . 23 A. BONSIGNORE, G. MANGIAROTTI, A. DE FLORA AND S. PONTREMOLI, J. Biol. Chem., 238 (1963) 3151 . 24 K. UYEDA AND IE. RACKER, J. Biol. Chem., 240 (1965) 4682. 25 K. A. I(AFIANY AND M. YA. TIMOFEEVA, Biohhimia, 27 (1962) 938. 26 ]L. VII~UELA, M. L. SALAS,M. SALAS AND A. SOLS, Biochem. Biophys. Res. Commun., 15 (1964) 243. 27 R. M. DENTON ANn P. T. RANDLE, Biochem. J., IOO (1966) 420. 28 T. E. MANSOUR AND N. M. WAKID, Biochem. Biophys. Res. Commun., 19 (1965) 721. 29 M. SALAS, E. \7II'~UELA, AND A. SOLS, Biochem. Biophys. Res. Commun., 17 (1964) 15o. 3 ° K. TAKETA AND B. M. POG:ELL, J. Biol. Chem., 240 (1965) 651. 31 H. A. KRI~gs AND M. WOODFORD, Biochem. J., 94 (1965) 436. 32 L. S. MILMAN AND YU. G. DANJUKOFF,Cytologia, 7 (I965) 732 (in Russian).
Biochim. Biophys. Acta, 148 (1967) 362-371