- Email: [email protected]
Enzyme activities in the glycolysis system of Urechis eggs with special reference to the activation of its rate-limiting enzymes following fertilization
Printed in Sweden Copyright © 1977 by Academic Press, lnc. All rights of reproduction in any form reserved ISSN 0014-4827
Experimental Cell Research 108 (1977) 191-199
ENZYME ACTIVITIES IN THE GLYCOLYSIS SYSTEM OF
URECHIS EGGS WITH SPECIAL REFERENCE ACTIVATION
O F ITS R A T E - L I M I T I N G
FOLLOWING
TO T H E
ENZYMES
FERTILIZATION
E. TAZAWA and I. YASUMASU
Biological Institute, Faculty of Literature and Science, Yokohama City University, Yokohama 236, and Department of Biology, School of Education, Waseda University, Shinjiku-Ku, Tokyo 160, Japan
SUMMARY In fertilized eggs of Urechis unicinctus, the activities of phosphorylase, phosphofructokinase, and pyruvate kinase are markedly higher than those in unfertilized eggs. Fertilization results in a remarkable change in these enzyme activities which catalyse their respective reactions at the rate-limiting steps in the glycolysis system. The phosphorylase activity in unfertilized eggs is stimulated by AMP and is enhanced after incubation in the presence of ATP, Ca 2+, and cAMP. The phosphofructokinase activity is stimulated by cAMP, Ca 2+, and AMP, and is inhibited by ATP. cAMP, Ca ~+ and fructose 1,6-diphosphate cause an elevation of the pyruvate kinase activity. Since the cAMP concentration in the eggs increases for up to 40 min after fertilization, the activation of these enzymes will be, in a part at least, ascribed to the increase in cAMP concentration following fertilization. The activities of the other enzymes in the glycolysis system, such as hexokinase, phosphoglucomutase, phosphoglucoisomerase, fructose 1,6-diphosphatase, aldolase, glyceroaldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase hardly change upon fertilization, c~-l,4-Glucosidase and glucose 6-phosphate dehydrogenase activity do not change at all following fertilization.
In a previous paper [l] it was hypothesized that the flux rate in the glycolysis system of Urechis eggs increases after fertilization due to the elevation of activity of three ratelimiting enzymes, i.e. phosphorylase, phosphofructokinase and pyruvate kinase. This hypothesis is based on the displacement of the apparent mass action ratio from the equilibrium constant at each step of the reaction in the glycolysis system. The mass action ratio of each reaction was calculated from the concentrations of glycolytic intermediates and adenine nucleotides in the eggs. Then the mass action ratios, at 13-771801
the respective reaction steps catalysed by phosphorylase, phosphofructokinase and pyruvate kinase were found to be widely d!splaced from their respective equilibrium constants. Thus, it was concluded that the reaction steps in unfertilized and fertilized eggs were not in an equilibrium state, and the activities of the enzymes which catalysed these reactions were not sufficient to bring the reactions to equilibrium. The displacement of the mass action ratios in these reaction steps from their respective equilibrium constants becomes slighter after fertilization. This means an increase in the flux Exp CellRes 108 (1977)
192
Tazawa and Yasumasu
Table 1. Activity and distribution of the enzymes in 1.0 M sucrose homogenate of Urechis eggs Activities (nmoles/min/mg protein) Unfertilized
Fertilized (30 min)
Enzyme
Whole
Super-
Residue
Whole
Super-
Residue
P-rylase (-AMP)* P-rylase (+AMP)* c~-Glucosidase* PGluM HK G6P-DH GPIxl02 PFK (0.1 mM ATP) PFK (1 mM ATP) F-diphosphatase Aldolase GA3P-DH Enolase PK LDH
1.39+0.20 2.95_+0.40 4.33_+0.64 1.44_+0.28 2.59_+0.49 16.18_+1.20 6.63_+0.54 6.06_+0.72 1.41_+0.53 9.87+ 1.03 11.63_+2.20 15.91-+1.18 8.75_+ 1.97 12.58_+ 1.97 6.54_+1.89
1.21_+0.13 2.72_+0.43 3.86_+0.72 1.43_+0.33 2.53_+0.48 15.23_+0.92 2.29_+0.08 5.64_+0.52
0.16_+0.13 0.27+0.04 0.67_+0.05 0.05_+0.04 0.03_+0.03 1.06_+0.73 4.08+0.96 0.45_+0.27
2.19_+0.09 3.22_+0.21 3.52_+0.23 1.41_+0.38 2.17_+0.43 15.74_+0.60 2.31+0.57 6.62_+0.40
0.21_+0.03 0.44-+0.10 0.63_+0.15 0.04_+0.03 0.06_+0.03 0.95_+0.43 3.91_+0.43 0.66_+0.25
9.72_+1.12 10.61_+2.14 15.63_+1.28 8.20_+2.19 11.95_+2.25 3.86_+0.56
0.15_+0.10 1.54_+0.40 0.29+0.11 0.45_+0.22 0.75_+0.26 2.74_+1.28
2.38_+0.09 3.67_+0.10 4.41_+0.41 1.43_+0.37 2.80_+0.47 16.69_+0.91 6.15_+0.80 7.20_+0.18 1.88_+0.26 9.78_+ 1.24 11.15_+2.12 15.87_+1.82 8.26_+2.61 22.22_+2.97 6.59_+1.82
9.70_+1.22 9.75+1.35 15.32_+1.35 7.61_+2.63 19.76_+3.57 3.80_+0.96
0.13-+0.09 1.42-+0.79 0.49_+0.53 0.63_+0.29 2.47_+0.56 2.77_+1.11
The enzyme activities are expressed as nmoles/min/mg protein except those marked by *: nmoles/20 min/mg protein. Each value is mean + S.E.M. obtained in three separate experiments. Abbreviations: P-rylase, phosphorylase; a-glucosidase, exo-c~-l,4-glucosidase; PGIuM, phosphoglucomutase; HK, hexokinase; G6P-DH, glucose-6-phosphate dehydrogenase; GPI, glucose phosphate isomerase; PFK, phosphofructokinase; F-diphosphatase, fructose 1,6-diphosphatase; aldolase, fructose diphosphate aldolase; GA3P-DH, glyceraldehyde-3-phosphate dehydrogenase; enolase, phosphopyruvatehydratase; PK, pyruvate kinase; LDH, lactate dehydrogenase.
rate of glycolytic intermediates and probably suggests an activation of these three enzymes catalysing rate-limiting steps in glycolysis system. In sea urchin eggs, aldolase [2], glucose-6-phosphate dehydrogenase [3], B-1,3-glucanohydrolase [4], and phosphorylase [5] are reportedly released from the bound state following fertilization, and consequently the latent activities of these enzymes are assumed to be changed into an activated state. In Urechis eggs, such a redistribution of the enzymes has not been investigated. Hence, it is necessary to investigate the fertilization-induced change in the intracellular distribution and activities of the enzymes in the glycolysis system in order to elucidate the mechanism which modulates the rate of glycolysis in Urechis eggs. Exp Cell Res 108 (1977)
In the present study, the intracellular distribution and activities of the enzymes in the glycolysis system were studied in fertilized and unfertilized eggs of Urechis. Studies were also carried out on the effect of several known modulators of these enzymes such as Ca 2+ and cAMP on the activity of phosphorylase, phosphofructokinase and pyruvate kinase in Urechis eggs. MATERIAL AND M E T H O D S The echiuroid, Urechis unicinctus, collected at Choshi, Chiba Prefecture, was reared in an aerated and temperature-controlled sea water tank until used. Gametes of Urechis were obtained from the segmental organ which was removed from the body cavity. The eggs were washed twice with filtered sea water. After an aliquot of the egg suspension was removed for an unfertilized egg sample, the eggs were inseminated and allowed to develop at 20°C. At the indicated time, the eggs were collected in a hand-driven centrifuge. Then, the eggs were washed and suspended in a 1 M sucrose
Enzyme activities in the glycolysis system 150
t00
j
~o
'
UF
II
'
'
30
60
Fig. 1. Abscissa: time after fertilization (min); ordinate: percent activation (%). Changes in the activity of O, phosphorylase a: O, phosphofructokinase; and x, pyruvate kinase in Urechis eggs following fertilization. Each point represents the mean of the values obtained in three experiments, on different enzyme sources. Vertical range bar shows S.E.M.
solution. The egg suspension was homogenized in an ice bath with a glass homogenizer equipped with a motor-driven Teflon pestle. One part of the homogenate was stored to measure the activities of the enzymes in whole homogenate, and the other part was centrifuged at 105 000 g for 60 min at 2--4°C. The precipitate obtained was suspended in an amount of 1 M sucrose, sufficient to give the same volume as that of the original egg homogenate. All enzyme activities were estimated at 25°C. Activities of phosphorylase, c~-l,4-glucosidase, hexokinase, phosphoglucomutase, phosphoglucoisomerase, fructose diphosphatase, phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase were estimated according to the methods described in a previous paper [6]. Glyceroaldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase activity were also estimated by enzymatic methods [7, 8]. Two ml of the mixture for the estimation of the 3-phosphoglycerate dehydrogenase activity contained 10 /xmoles 3-phosphoglycerate; 60/zmoles Tris buffer, pH 7.2; 10/zmoles MgSO4; 36/xmoles NADH~; and an appropriate concentration of the enzyme source. The reaction was initiated at 25°C by addition of 3-phosphoglycerate, and the decrease in absorbance at 340 nm was recorded on a spectrophotometer (Hitachi 124 or Shimadzu UV-200) equipped with a recorder (Hitachi QPD 134 or Shimadzu U-125 MU). Two ml of the reaction mixture for estimation of the phosphoglycerate kinase activity contained 10 /~moles 1,3-diphosphoglycerate; 10/xmoles ADP; 5 ~g 3-phosphoglycerate dehydrogenase; 10 /zmoles MgSO4; 36 ~moles NADH~; 60 /zmoles Tris buffer pH 7.2; and an ap-
193
propriate concentration of the enzyme source. The reaction was initiated by adding 1,3-diphosphoglycerate, and the decrease in absorbance at 340 nm was recorded. Except for phosphorylase and c~-l,4-glucosidase, the enzyme activities were expressed as nmoles of the respective products per min/mg protein of the whole egg homogenate. The activity of phosphorylase and glucosidase were expressed as nmoles of the product per 20 min/mg protein of whole egg homogenate. Protein estimation was made by the method of Lowry et al. [9]. Estimation of the cAMP concentration was made using the method of Gilman [10], based on the competition of cold cAMP in the binding of 3H-cAMP to a known amount of cAMPbinding protein. The pellet was obtained by centrifugation at 2000 g for 2 min, and was frozen by liquid nitrogen. Ten percent perchloric acid was added in an amount equivalent to frozen egg pellet, and the pellet was homogenized in a mortar chilled by liquid nitrogen. After centrifugation of the egg homogenate at 105 000 g for 30 min, the aliquot obtained was neutralized with saturated K~CO3 and centrifuged again at 105000 g for 1 h. The supernatant was then analysed for cAMP. Radioactivity of 3H-cAMP was estimated on a liquid scintillation counter (Nihon Musen Co., Tokyo). All enzymes and co-enzymes were the products of Boehringer Mannheim Co., Germany. The kit for cAMP estimation was also purchased from Boehringer Mannheim Co.
RESULTS Table ! shows the activity of phosphorylase, ~-glucosidase, phosphoglucomutase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phospho-
I 0
I 50
I
OlO
Fig. 2. Abscissa: AMP conc. (mM); ordinate: phosphorylase activity, expressed as nmoles of the G1P produced per 20 min/mg protein. The activity of phosphorylase at various AMP concentrations in Urechis unfertilized eggs. Exp Cell Res 108 (1977)
194
Tazawa and Yasumasu
0
]
F
I
0 10 -5
I
10-4
I
10-3
Fig. 3. Abscissa: Ca ~+ conc. (M); ordinate: phosphorylase activity, expressed as nmoles of the G1P produced per 20 min/mg protein. The values show cAMP concentrations (M). Effects of Ca 2+ and cAMP on the activity of phosphorylase in Urechis unfertilized eggs. The enzyme activity was estimated after 10 rain preincubation in the presence of cAMP, Ca 2+ and ATP.
fructokinase, fructose 1,6-diphosphate aldolase, glyceroaldehyde 3-phosphate dehydrogenase, enolase, pyruvate kinase, and lactate dehydrogenase in unfertilized and fertilized eggs of Urechis unicinctus. The eggs collected after 30 min of insemination are identified as fertilized eggs hereafter. unless specified. All enzyme activities in fertilized eggs except phosphorylase, phosphofructokinase, and pyruvate kinase activity were very similar to those in unfertilized eggs. The glucosidase and hexokinase activity in fertilized eggs seems to be slightly higher than that of unfertilized eggs but the difference between them is not statistically significant. Among the glycolytic enzymes in Urechis eggs the phosphoglucoisomerase activity is very high, though it is not enhanced upon fertilization. The same has been found in sea urchin eggs [6]. The activity of all enzymes except phosphoglucoisomerase and lactate dehydrogenase are found in the soluble fractions in both unfertilized and fertilized Urechis eggs (table 1). More than 50% of the phosphoE.rp Cell Res 108 (1977)
glucoisomerase activity is found in the particulate fraction, but the intracellular distribution of the enzyme activity does not change upon fertilization. About 50 % of the lactate dehydrogenase activity is also found in the particulate, but redistribution of the enzyme activity does not occur following fertilization. The same has been found in sea urchin eggs [6]. Redistribution of several enzyme activities, such as phosphorylase [5], G6P dehydrogenase [3] and aldolase [2], from the particulate to the soluble fraction has been found in sea urchin eggs upon fertilization. However, all enzyme activities in Urechis eggs are found in a soluble fraction, and there is no redistribution of any enzyme activity upon fertilization. Fig. 1 shows the change in activity of phosphorylase a, phosphofructokinase, and pyruvate kinase in Urechis eggs following fertilization. The phosphorylase a activity increases at first, and the pyruvate kinase activity is slightly enhanced after fertiliza-
7&. 5
17
1
0
0.5
1
Fig. 4. Abscissa: ATP conc. (raM); ordinate: phosphofructokinase activity, expressed as nmoles of FDP produced per min/mg protein. The values show the cAMP concentrations (M). Effects of cAMP and ATP on the.._activity of ph(?sphofructokinase in Urechis unfertilized eggs.
Enzyme activities in the glycolysis system
Table 2. Effect of adenine nucleotides and Ca 2+on phosphofructokinase activity in unfertilized eggs of Urechis P h o s p h o f r u c t o k i n a s e activity (nmole F D P formed/ rain/rag protein) A T P conc. Addition
Conc. (M)
None
0.1 m M
1 mM
5.10
0.97
10-5 10 4 10-z 5 x 10 -3
5.21 5.40 6.05 6.07
0.98 1.33 1.89 1.77
AMP
10 4 10 3
6.71 6.90
1.90 2.20
ADP
10 4 10-3
5.09 5.12
0.98 0.91
CaC12
tion. Then the activity of phosphofructokinase suddenly increases. The sequential changes in these enzyme activities are well in agreement with findings reported previously [1] in which sequential activation of these enzymes has been assumed, based on the change in the apparent mass action ratios following fertilization in these reaction steps. Fig. 2 shows the activity of phosphorylase estimated in the presence of AMP. The maximum activity of the enzyme in unfertilized eggs was observed in the presence of AMP at concentrations above 25 raM. A considerable amount of phosphorylase b seems to be present in unfertilized eggs. Fig. 3 shows the effects of Ca z+ and cAMP on the activity of phosphorylase. After the homogenate of unfertilized eggs containing 2 mM ATP and 5 mM MgC12 was incubated for 10 min at 25°C in the presence of cAMP or Ca 2÷, the phosphorylase a activity was enhanced, but was not activated without ATP. Preincubation of the enzyme source with 2 mM ATP, 5 mM MgClz and cAMP results in an increase in phosphorylase a
195
activity in the enzyme source. Addition of Ca ~+ to a preincubation mixture containing cAMP cause a further increase in the enzyme activity. The activity which has been enhanced by preincubation in the presence of adequate concentrations of cAMP and Ca 2+ is higher than that in fertilized eggs. These results indicate that phosphorylase b changes to a-form due to phosphorylase kinase, a Ca2+-dependent kinase whose activity is itself regulated by cAMP through the action of cAMP-dependent protein kinase. Activation of phosphorylase activity by phosphorylation of the enzyme has been demonstrated in mammalian tissues [11]. The same has been found in the enzyme activity of sea urchin eggs [12]. Fig. 4 shows the effect of cAMP and ATP on the activity of phosphofructokinase in unfertilized Urechis eggs. At ATP concentrations lower than 0.02 raM, the enzyme activity in Urechis eggs is related to the ATP concentration, one of the substrates of the enzyme, but the enzyme activity be-
50
0
--s 10 -7
10-6
10-5
10-4
10-3
Fig. 5. Abscissa: concentrations of c A M P (M), of Ca 2+ (M), and of fructose 1,6-diphosphate (M); ordinate: percent increase in pyruvate kinase activity. P e r c e n t increase is the e n z y m e activity in the presence of c A M P (©), in the p r e s e n c e of Ca 2+ (O), and in the presence of F D P ( x ) . Effect of c A M P , Ca, and F D P on the activity of p y r u v a t e kinase in the Urechis unfertilized eggs. Exp Cell Res 108 (1977)
196
Tazawa and Yasumasu
50 ¸
3O
10
0
r
I£
UF
'
'
30
60
Fig. 6. Abscissa: time after fertilization (min); ordinate: concentration of c A M P (pmoles/10 a eggs). Concentration of c A M P in the Urechis eggs before and after fertilization. Vertical range bars represent S.E.M.
comes lowered in the presence of the substrate at higher concentrations than 0.02 mM. Hence, it is probable that ATP is not only the substrate but also the negative modulator of phosphofructokinase in Urechis eggs. ATP inhibition of the enzyme activity has been found in mammalian cells [13]. The same has also been found in sea urchin eggs [14]. cAMP causes a reversal of ATP inhibition of the enzyme activity. It seems that cAMP not only causes reversal of the ATP inhibition, but also elevation of the enzyme activity. As shown in table 2, Ca 2+ slightly stimulates the enzyme activity. ADP hardly causes any enzyme activation, while AMP causes an increase in the enzyme activity. Phosphofructokinase activity in the unfertilized egg homogenate, estimated in the presence of 0.5 mM Ca 2+ and cAMP at a concentration above 10.5 M, is highest among those examined and higher than that in fertilized eggs. As shown in fig. 5, the pyruvate kinase activity is enhanced by cAMP, FDP, and Ca 2+. FDP Exp Cell Res 108 (1977)
exerts the highest stimulating effect on the enzyme activity among these compounds. The FDP concentration in the eggs increases to a level high enough to stimulate the enzyme activity at 20 min after fertilization [ 1]. As shown in fig. 6, the cAMP level began to increase 10 rain and reached its maximum concentration 40 min after insemination. The change in Ca 2+ concentration should also be observed in order to elucidate the changes in enzyme activity in Urechis eggs following fertilization. But this has not yet been studied. Since it has been pointed out that procaine, a narcotics, probably stimulates the release of bound Ca 2+ [15], it is likely that these Ca2+-sensitive enzymes are enhanced in procaine-treated eggs. The activities of the enzymes in the glycolysis system were estimated in unfertilized eggs which had been kept in sea water containing 10 mM procaine for 20 min at 20°C. During the treatment, the germinal vesicle disappeared in 38.5+ 10.4% of the unfertilized eggs. As shown in table 3, the activity of phosphorylase, phosphofructokinase and pyruvate kinase in procaine-treated eggs are higher than those in intact unfertilized eggs, while other enzyme activities remain the same as in intact unfertilized eggs. The increase in the activities of these three enzymes may be supposed to be due to parthenogenetic activation induced by procaine treatment, since about one-third of the procainetreated eggs show germinal vesicle breakage. It has been reported that the parthenogenetic activation of sea urchin eggs with procaine treatment is due to the release of protein from the surface of the eggs [16]. However, it is also probable that procaine causes an increase in Ca 2+ concentration in Urechis eggs following parthenogenic activation. The Ca ~+ concentration can be
Enzyme activities in the glycolysis system
197
Table 3. Effect of procaine on the enzyme activities in the glycolysis system in unfertilized eggs of Urechis Procaine conc.
Enzymes
None
10-3 M
P-rylase c~-glucosidase PGluM HK G6P-DH GPIxl02 P F K (0.1 mM ATP) F-diphosphatase Aldolase GA3P-DH Enolase PK LDH
1.40+0.31 4.21+0.39 1.25+0.33 2.47+0.59 16.06+1.33 6.82+0.72 6.02_+0.10 10.31___2.12 9.52+2.45 14.95+_2.36 8.61_+1.04 11.79+1.92 7.38+2.42
1.98+0.15 3.98* 1.35" 2.39* 16.72" 7.13" 6.11+0.19 9.50* 7.39* 14.02" 8.98* 14.17+2.21 8.13"
% increase +41.4
+ 0.01
+20.2
5 x 10-3 M
% increase
10-2 M
% increase
2.39+0.30 4.22+0.35 1.20+0.29 2.50+0.51 15.87+3.20 6.54+0.43 7.21+0.29 10.91+2.72 9.43+2.25 14.62_+2.98 8.70+2.04 16.09+3.12 7.89+2.98
+70.7 + 0 - 0.4 + 0.1 - 0.01 - 0.04 +19.7 + 0.01 - 0.01 - 0.02 + 0.01 +36.5 + 6.9
2.21+0.11 4.19+0.50 1.29+0.42 2.43+0.24 15.89+2.29 6.79+0.81 7.20_+0.43 9.95_+1.15 9.60+2.32 14.72_+4.29 8.59+1.10 16.12-+4.92 7.32+1.96
+57.8 - 0.05 + 0.3 - 0.02 - 0.01 - 0.04 +19.6 - 0.03 + 0.01 - 0.01 - 0.01 +36.7 - 0.01
Procaine treatment of unfertilized eggs was performed for 20 min at 20°C. All enzyme activities are expressed as in table 1. Abbreviations, see table 1. Values shown are the ±S~E.M. of three separate experiments except *. *, One experiment. The percentage increase in the enzyme activity is shown in parentheses.
hypothesized to increase in procainetreated Urechis eggs, since the percent increase in phosphorylase activity, the most Ca2+-sensitive enzyme among these is highest and that of phosphofructokinase activity, the least sensitive enzyme, is lowest in procaine-treated eggs (figs 3, 4; table 3). In procaine-treated eggs, cAMP did not increase during 20 min of treatment (0.9 pmoles/106 eggs at 20 min of 10-2 M procaine treatment). Based on the hypothesis that procaine stimulates the release of bound Ca 2+, as has been hypothesized in sea urchin eggs [17], the activation of these enzymes seems to be due to the increase in Ca ~+ concentration in procaine-treated unfertilized eggs.
DISCUSSION Enzyme activities in the glycolysis system of sea urchin eggs have been reported previously [6]. In Urechis eggs, each enzyme activity in the glycolysis seems to be very
similar to that in sea urchin eggs, but the intracellular distribution of several enzymes in Urechis eggs differs from that in sea urchin eggs. In both Urechis and sea urchin eggs, the phosphoglucoisomerase activity is very high as compared with other enzyme activities in the glycolysis system. However, more than 50 % of the enzyme activity is associated with the particulate in Urechis eggs, while it has been found in the soluble fraction in sea urchin eggs [6]. In both organisms, about 50% of the lactate dehydrogenase activity is detectable in the particulate fraction. Change in intracellular distribution of these enzymes does not occur following fertilization, and the reactions catalysed by these enzymes seem to be in an equilibrium state [1, 18]. Hence, the enzyme activities in the partfculate fraction are not supposed to be in a latent state. Pyruvate kinase activity in sea urchin eggs can be also detected in the particulate fraction and the activity in Urechis eggs is present in the soluble fraction. Though the flux Exp Cell Res 108 (1977)
198
Tazawa and Yasumasu
rate in this reaction step increases following fertilization [1, 18], no redistribution occurs in sea urchin [6] as well as in Urechis eggs. Aldolase [2, 6] and phosphorylase [5] activity have been found in the particulate fraction in unfertilized sea urchin eggs, but those can only be detected in the soluble fraction in unfertilized and fertilized eggs of Urechis. It has been reported that the aldolase and phosphorylase activities in sea urchin eggs become apparent following fertilization as a consequence of the redistribution of the enzymes [2, 5]. In the aldolase reaction in unfertilized and fertilized sea urchin eggs [18] and Urechis eggs [1], the reaction step has been found to be in a quasi-equilibrium state. Hence, the apparent activation of the aldolase activity in the consequence of the enzyme redistribution in sea urchin eggs hardly causes any increase in the flux rate at the reaction step. In the phosphorylase step, the reaction has been demonstrated to be in a regulated state both in sea urchin eggs [18] and in Urechis eggs [1] and is assumed to be one of the rate-limiting steps in glycolysis. The release of phosphorylase activity from its bound state following fertilization reported in the eggs of the sea urchins Sphaerechinus granularis and Paracentrotus lividus [5] seems to account for the increase in the flux rate at the reaction step catalyzed by phosphorylase in sea urchin fertilized eggs [18]. However, in unfertilized as well as in fertilized eggs of Urechis, phosphorylase a activity is detected only in the soluble fraction, although the reaction is still apparently rate-limiting [1]. Thus the mechanism responsible for the increased phosphorylase a activity after fertilization in Urechis must not involve redistribution of the enzyme but perhaps instead an increase in the calcium concentration, as has been found for sea Exp Cell Res 108 (1977)
urchin eggs [19, 20]. This is supported by the ability of procain to increase the Urechis phosphorylase a activity, possibly due to its proposed effects on the calcium levels [17]. The possibility that AMP could affect the phosphorylase b activity in Urechis eggs at fertilization seems unlikely, since AMP levels do not change after fertilization and the AMP concentration is too low to stimulate the phosphorylase b activity [1]. Similarly, cAMP does not increase early enough (fig. 5) to account for the rapid increase in phosphorylase a activity after fertilization (fig. 1). As in the case of phosphorylase a activity, a small but rapid increase in the pyruvate kinase activity in Urechis eggs following fertilization (fig. l) can be ascribed to an increase in the Ca 2+ level. The sudden increase in the phosphofructokinase activity a while after fertilization seems to be due to an increase in the cAMP level. A further increase in the pyruvate kinase activity keeping pace with that in phosphofructokinase activity is probably due to the increased cAMP level and/or to the FDP level, which raises following activation of the phosphofructokinase activity. Although this study was prompted by the finding that subcellular redistribution of enzymes occurs after fertilization in sea urchins [2, 5], no such redistribution is evident in most sea urchin species, most of the phosphorylase a activity being completely soluble [6, 12]. The results in this paper suggest that Urechis is more like the predominant form of sea urchin, with phosphorylase a regulated more likely by Ca 2+ and phosphorylation than by changes in the subcellular distribution of the enzyme. The similarity of these Urechis results to most sea urchin work is supported by the similarity in the two organisms of the profile of activity changes following fertilization of
E n z y m e a c t i v i t i e s in the g l y c o l y s i s s y s t e m phosphorylase a, phosphofructokinase, and p y r u v a t e k i n a s e [6, 12, 14] a n d b y th e f a c t t h a t in b o t h o r g a n i s m s t h e s e t h r e e e n z y m e s are a p p a r e n t l y r a t e - l i m i t i n g f o r g l y c o l y s i s b o t h b e f o r e a n d a f t e r f e r t i l i z a t i o n [1, 18] a n d ar e also r e g u l a t e d b y C a 2+ a n d c A M P [19-22]. REFERENCES 1. Yasumasu, I, Tazawa, E & Fujiwara, A, Exp cell res 93 (1975) 166. 2. Ishihara, K, J fac sci univ Tokyo sect IV 8 (1957) 71. 3. Isono, N, Tsusaka, A & Nakano, E, J fac sci univ Tokyo sect IV 10 (1963) 55. 4. Epel, D, Weaver, A M, Muchmo~e, A V & Schimke, R T, Science 163 (1969) 294. 5. Bergami, M, Mansour, T E & Scarano, E, Exp cell res 49 (1968) 650. 6. Yasumasu, I, Fujiwara, A, Shoger, R L & Asami, K, Exp cell res 92 (1975)444. 7. Bergmeyer, H-U, Gawehn, K & Grassl, M, Methods of enzymatic analysis (ed H-U Bergmeyer) vol. 1, p. 468. Academic Press, New York (1974). 8. - - Ibid vol. 1, p. 502 (1974).
199
9. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 10. Gilman, A G, Proc natl acad sci US 64 (1969) 367. 11. Fischer, E H, Pocker, A & Saari, J C, Essays in biochemistry (ed P N Campbell & F Dickens) vol. 6, p. 23. Academic Press, London (1970). 12. Shoger, R L, Asami, K, Yasumasu, I & Fujiwara, A, Exp cell res 82 (1973) 375. 13. Epel, D, Pressman, B C, Elsaesser, S & Weaver, A M, The cell cycle (ed G M Padilla, G L Whitson & I L Cameron) p. 297. Academic Press, New York (1969). 14. Asami, K, Fujiwara, A, Shoger, R L & Yasumasu, I, Cell structure and function 1 (1975) 43. 15. Seeman, P, Pharmacol rev 24 (1972) 583. 16. Johnson, J D & Epel, D, Proc natl acad sci US 72 (1975) 4474. 17. Vacquier, V & Brandriff, B, Dev biol 47 (1975) 12. 18. Yasumasu, I, Asami, K, Shoger, R L & Fujiwara, A, Exp cell res 80 (1973) 361. 19. Mazia, D, J cell comp physiol 10 (1937) 291. 20. Nakamura, M & Yasumasu, I, J gen physiol 63 (1974) 374. 21. Nath, J & Rebhun, L I, Exp cell res 77 (1973) 319. 22. Yasumasu, I, Fujiwara, A & Ishida, K, Biochem biophys res commun 54 (1973) 628.
Received December 8, 1976 Accepted March 25, 1977
Exp Cell Res 108 (1977)
Experimental Cell Research 108 (1977) 191-199
ENZYME ACTIVITIES IN THE GLYCOLYSIS SYSTEM OF
URECHIS EGGS WITH SPECIAL REFERENCE ACTIVATION
O F ITS R A T E - L I M I T I N G
FOLLOWING
TO T H E
ENZYMES
FERTILIZATION
E. TAZAWA and I. YASUMASU
Biological Institute, Faculty of Literature and Science, Yokohama City University, Yokohama 236, and Department of Biology, School of Education, Waseda University, Shinjiku-Ku, Tokyo 160, Japan
SUMMARY In fertilized eggs of Urechis unicinctus, the activities of phosphorylase, phosphofructokinase, and pyruvate kinase are markedly higher than those in unfertilized eggs. Fertilization results in a remarkable change in these enzyme activities which catalyse their respective reactions at the rate-limiting steps in the glycolysis system. The phosphorylase activity in unfertilized eggs is stimulated by AMP and is enhanced after incubation in the presence of ATP, Ca 2+, and cAMP. The phosphofructokinase activity is stimulated by cAMP, Ca 2+, and AMP, and is inhibited by ATP. cAMP, Ca ~+ and fructose 1,6-diphosphate cause an elevation of the pyruvate kinase activity. Since the cAMP concentration in the eggs increases for up to 40 min after fertilization, the activation of these enzymes will be, in a part at least, ascribed to the increase in cAMP concentration following fertilization. The activities of the other enzymes in the glycolysis system, such as hexokinase, phosphoglucomutase, phosphoglucoisomerase, fructose 1,6-diphosphatase, aldolase, glyceroaldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase hardly change upon fertilization, c~-l,4-Glucosidase and glucose 6-phosphate dehydrogenase activity do not change at all following fertilization.
In a previous paper [l] it was hypothesized that the flux rate in the glycolysis system of Urechis eggs increases after fertilization due to the elevation of activity of three ratelimiting enzymes, i.e. phosphorylase, phosphofructokinase and pyruvate kinase. This hypothesis is based on the displacement of the apparent mass action ratio from the equilibrium constant at each step of the reaction in the glycolysis system. The mass action ratio of each reaction was calculated from the concentrations of glycolytic intermediates and adenine nucleotides in the eggs. Then the mass action ratios, at 13-771801
the respective reaction steps catalysed by phosphorylase, phosphofructokinase and pyruvate kinase were found to be widely d!splaced from their respective equilibrium constants. Thus, it was concluded that the reaction steps in unfertilized and fertilized eggs were not in an equilibrium state, and the activities of the enzymes which catalysed these reactions were not sufficient to bring the reactions to equilibrium. The displacement of the mass action ratios in these reaction steps from their respective equilibrium constants becomes slighter after fertilization. This means an increase in the flux Exp CellRes 108 (1977)
192
Tazawa and Yasumasu
Table 1. Activity and distribution of the enzymes in 1.0 M sucrose homogenate of Urechis eggs Activities (nmoles/min/mg protein) Unfertilized
Fertilized (30 min)
Enzyme
Whole
Super-
Residue
Whole
Super-
Residue
P-rylase (-AMP)* P-rylase (+AMP)* c~-Glucosidase* PGluM HK G6P-DH GPIxl02 PFK (0.1 mM ATP) PFK (1 mM ATP) F-diphosphatase Aldolase GA3P-DH Enolase PK LDH
1.39+0.20 2.95_+0.40 4.33_+0.64 1.44_+0.28 2.59_+0.49 16.18_+1.20 6.63_+0.54 6.06_+0.72 1.41_+0.53 9.87+ 1.03 11.63_+2.20 15.91-+1.18 8.75_+ 1.97 12.58_+ 1.97 6.54_+1.89
1.21_+0.13 2.72_+0.43 3.86_+0.72 1.43_+0.33 2.53_+0.48 15.23_+0.92 2.29_+0.08 5.64_+0.52
0.16_+0.13 0.27+0.04 0.67_+0.05 0.05_+0.04 0.03_+0.03 1.06_+0.73 4.08+0.96 0.45_+0.27
2.19_+0.09 3.22_+0.21 3.52_+0.23 1.41_+0.38 2.17_+0.43 15.74_+0.60 2.31+0.57 6.62_+0.40
0.21_+0.03 0.44-+0.10 0.63_+0.15 0.04_+0.03 0.06_+0.03 0.95_+0.43 3.91_+0.43 0.66_+0.25
9.72_+1.12 10.61_+2.14 15.63_+1.28 8.20_+2.19 11.95_+2.25 3.86_+0.56
0.15_+0.10 1.54_+0.40 0.29+0.11 0.45_+0.22 0.75_+0.26 2.74_+1.28
2.38_+0.09 3.67_+0.10 4.41_+0.41 1.43_+0.37 2.80_+0.47 16.69_+0.91 6.15_+0.80 7.20_+0.18 1.88_+0.26 9.78_+ 1.24 11.15_+2.12 15.87_+1.82 8.26_+2.61 22.22_+2.97 6.59_+1.82
9.70_+1.22 9.75+1.35 15.32_+1.35 7.61_+2.63 19.76_+3.57 3.80_+0.96
0.13-+0.09 1.42-+0.79 0.49_+0.53 0.63_+0.29 2.47_+0.56 2.77_+1.11
The enzyme activities are expressed as nmoles/min/mg protein except those marked by *: nmoles/20 min/mg protein. Each value is mean + S.E.M. obtained in three separate experiments. Abbreviations: P-rylase, phosphorylase; a-glucosidase, exo-c~-l,4-glucosidase; PGIuM, phosphoglucomutase; HK, hexokinase; G6P-DH, glucose-6-phosphate dehydrogenase; GPI, glucose phosphate isomerase; PFK, phosphofructokinase; F-diphosphatase, fructose 1,6-diphosphatase; aldolase, fructose diphosphate aldolase; GA3P-DH, glyceraldehyde-3-phosphate dehydrogenase; enolase, phosphopyruvatehydratase; PK, pyruvate kinase; LDH, lactate dehydrogenase.
rate of glycolytic intermediates and probably suggests an activation of these three enzymes catalysing rate-limiting steps in glycolysis system. In sea urchin eggs, aldolase [2], glucose-6-phosphate dehydrogenase [3], B-1,3-glucanohydrolase [4], and phosphorylase [5] are reportedly released from the bound state following fertilization, and consequently the latent activities of these enzymes are assumed to be changed into an activated state. In Urechis eggs, such a redistribution of the enzymes has not been investigated. Hence, it is necessary to investigate the fertilization-induced change in the intracellular distribution and activities of the enzymes in the glycolysis system in order to elucidate the mechanism which modulates the rate of glycolysis in Urechis eggs. Exp Cell Res 108 (1977)
In the present study, the intracellular distribution and activities of the enzymes in the glycolysis system were studied in fertilized and unfertilized eggs of Urechis. Studies were also carried out on the effect of several known modulators of these enzymes such as Ca 2+ and cAMP on the activity of phosphorylase, phosphofructokinase and pyruvate kinase in Urechis eggs. MATERIAL AND M E T H O D S The echiuroid, Urechis unicinctus, collected at Choshi, Chiba Prefecture, was reared in an aerated and temperature-controlled sea water tank until used. Gametes of Urechis were obtained from the segmental organ which was removed from the body cavity. The eggs were washed twice with filtered sea water. After an aliquot of the egg suspension was removed for an unfertilized egg sample, the eggs were inseminated and allowed to develop at 20°C. At the indicated time, the eggs were collected in a hand-driven centrifuge. Then, the eggs were washed and suspended in a 1 M sucrose
Enzyme activities in the glycolysis system 150
t00
j
~o
'
UF
II
'
'
30
60
Fig. 1. Abscissa: time after fertilization (min); ordinate: percent activation (%). Changes in the activity of O, phosphorylase a: O, phosphofructokinase; and x, pyruvate kinase in Urechis eggs following fertilization. Each point represents the mean of the values obtained in three experiments, on different enzyme sources. Vertical range bar shows S.E.M.
solution. The egg suspension was homogenized in an ice bath with a glass homogenizer equipped with a motor-driven Teflon pestle. One part of the homogenate was stored to measure the activities of the enzymes in whole homogenate, and the other part was centrifuged at 105 000 g for 60 min at 2--4°C. The precipitate obtained was suspended in an amount of 1 M sucrose, sufficient to give the same volume as that of the original egg homogenate. All enzyme activities were estimated at 25°C. Activities of phosphorylase, c~-l,4-glucosidase, hexokinase, phosphoglucomutase, phosphoglucoisomerase, fructose diphosphatase, phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase were estimated according to the methods described in a previous paper [6]. Glyceroaldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase activity were also estimated by enzymatic methods [7, 8]. Two ml of the mixture for the estimation of the 3-phosphoglycerate dehydrogenase activity contained 10 /xmoles 3-phosphoglycerate; 60/zmoles Tris buffer, pH 7.2; 10/zmoles MgSO4; 36/xmoles NADH~; and an appropriate concentration of the enzyme source. The reaction was initiated at 25°C by addition of 3-phosphoglycerate, and the decrease in absorbance at 340 nm was recorded on a spectrophotometer (Hitachi 124 or Shimadzu UV-200) equipped with a recorder (Hitachi QPD 134 or Shimadzu U-125 MU). Two ml of the reaction mixture for estimation of the phosphoglycerate kinase activity contained 10 /~moles 1,3-diphosphoglycerate; 10/xmoles ADP; 5 ~g 3-phosphoglycerate dehydrogenase; 10 /zmoles MgSO4; 36 ~moles NADH~; 60 /zmoles Tris buffer pH 7.2; and an ap-
193
propriate concentration of the enzyme source. The reaction was initiated by adding 1,3-diphosphoglycerate, and the decrease in absorbance at 340 nm was recorded. Except for phosphorylase and c~-l,4-glucosidase, the enzyme activities were expressed as nmoles of the respective products per min/mg protein of the whole egg homogenate. The activity of phosphorylase and glucosidase were expressed as nmoles of the product per 20 min/mg protein of whole egg homogenate. Protein estimation was made by the method of Lowry et al. [9]. Estimation of the cAMP concentration was made using the method of Gilman [10], based on the competition of cold cAMP in the binding of 3H-cAMP to a known amount of cAMPbinding protein. The pellet was obtained by centrifugation at 2000 g for 2 min, and was frozen by liquid nitrogen. Ten percent perchloric acid was added in an amount equivalent to frozen egg pellet, and the pellet was homogenized in a mortar chilled by liquid nitrogen. After centrifugation of the egg homogenate at 105 000 g for 30 min, the aliquot obtained was neutralized with saturated K~CO3 and centrifuged again at 105000 g for 1 h. The supernatant was then analysed for cAMP. Radioactivity of 3H-cAMP was estimated on a liquid scintillation counter (Nihon Musen Co., Tokyo). All enzymes and co-enzymes were the products of Boehringer Mannheim Co., Germany. The kit for cAMP estimation was also purchased from Boehringer Mannheim Co.
RESULTS Table ! shows the activity of phosphorylase, ~-glucosidase, phosphoglucomutase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phospho-
I 0
I 50
I
OlO
Fig. 2. Abscissa: AMP conc. (mM); ordinate: phosphorylase activity, expressed as nmoles of the G1P produced per 20 min/mg protein. The activity of phosphorylase at various AMP concentrations in Urechis unfertilized eggs. Exp Cell Res 108 (1977)
194
Tazawa and Yasumasu
0
]
F
I
0 10 -5
I
10-4
I
10-3
Fig. 3. Abscissa: Ca ~+ conc. (M); ordinate: phosphorylase activity, expressed as nmoles of the G1P produced per 20 min/mg protein. The values show cAMP concentrations (M). Effects of Ca 2+ and cAMP on the activity of phosphorylase in Urechis unfertilized eggs. The enzyme activity was estimated after 10 rain preincubation in the presence of cAMP, Ca 2+ and ATP.
fructokinase, fructose 1,6-diphosphate aldolase, glyceroaldehyde 3-phosphate dehydrogenase, enolase, pyruvate kinase, and lactate dehydrogenase in unfertilized and fertilized eggs of Urechis unicinctus. The eggs collected after 30 min of insemination are identified as fertilized eggs hereafter. unless specified. All enzyme activities in fertilized eggs except phosphorylase, phosphofructokinase, and pyruvate kinase activity were very similar to those in unfertilized eggs. The glucosidase and hexokinase activity in fertilized eggs seems to be slightly higher than that of unfertilized eggs but the difference between them is not statistically significant. Among the glycolytic enzymes in Urechis eggs the phosphoglucoisomerase activity is very high, though it is not enhanced upon fertilization. The same has been found in sea urchin eggs [6]. The activity of all enzymes except phosphoglucoisomerase and lactate dehydrogenase are found in the soluble fractions in both unfertilized and fertilized Urechis eggs (table 1). More than 50% of the phosphoE.rp Cell Res 108 (1977)
glucoisomerase activity is found in the particulate fraction, but the intracellular distribution of the enzyme activity does not change upon fertilization. About 50 % of the lactate dehydrogenase activity is also found in the particulate, but redistribution of the enzyme activity does not occur following fertilization. The same has been found in sea urchin eggs [6]. Redistribution of several enzyme activities, such as phosphorylase [5], G6P dehydrogenase [3] and aldolase [2], from the particulate to the soluble fraction has been found in sea urchin eggs upon fertilization. However, all enzyme activities in Urechis eggs are found in a soluble fraction, and there is no redistribution of any enzyme activity upon fertilization. Fig. 1 shows the change in activity of phosphorylase a, phosphofructokinase, and pyruvate kinase in Urechis eggs following fertilization. The phosphorylase a activity increases at first, and the pyruvate kinase activity is slightly enhanced after fertiliza-
7&. 5
17
1
0
0.5
1
Fig. 4. Abscissa: ATP conc. (raM); ordinate: phosphofructokinase activity, expressed as nmoles of FDP produced per min/mg protein. The values show the cAMP concentrations (M). Effects of cAMP and ATP on the.._activity of ph(?sphofructokinase in Urechis unfertilized eggs.
Enzyme activities in the glycolysis system
Table 2. Effect of adenine nucleotides and Ca 2+on phosphofructokinase activity in unfertilized eggs of Urechis P h o s p h o f r u c t o k i n a s e activity (nmole F D P formed/ rain/rag protein) A T P conc. Addition
Conc. (M)
None
0.1 m M
1 mM
5.10
0.97
10-5 10 4 10-z 5 x 10 -3
5.21 5.40 6.05 6.07
0.98 1.33 1.89 1.77
AMP
10 4 10 3
6.71 6.90
1.90 2.20
ADP
10 4 10-3
5.09 5.12
0.98 0.91
CaC12
tion. Then the activity of phosphofructokinase suddenly increases. The sequential changes in these enzyme activities are well in agreement with findings reported previously [1] in which sequential activation of these enzymes has been assumed, based on the change in the apparent mass action ratios following fertilization in these reaction steps. Fig. 2 shows the activity of phosphorylase estimated in the presence of AMP. The maximum activity of the enzyme in unfertilized eggs was observed in the presence of AMP at concentrations above 25 raM. A considerable amount of phosphorylase b seems to be present in unfertilized eggs. Fig. 3 shows the effects of Ca z+ and cAMP on the activity of phosphorylase. After the homogenate of unfertilized eggs containing 2 mM ATP and 5 mM MgC12 was incubated for 10 min at 25°C in the presence of cAMP or Ca 2÷, the phosphorylase a activity was enhanced, but was not activated without ATP. Preincubation of the enzyme source with 2 mM ATP, 5 mM MgClz and cAMP results in an increase in phosphorylase a
195
activity in the enzyme source. Addition of Ca ~+ to a preincubation mixture containing cAMP cause a further increase in the enzyme activity. The activity which has been enhanced by preincubation in the presence of adequate concentrations of cAMP and Ca 2+ is higher than that in fertilized eggs. These results indicate that phosphorylase b changes to a-form due to phosphorylase kinase, a Ca2+-dependent kinase whose activity is itself regulated by cAMP through the action of cAMP-dependent protein kinase. Activation of phosphorylase activity by phosphorylation of the enzyme has been demonstrated in mammalian tissues [11]. The same has been found in the enzyme activity of sea urchin eggs [12]. Fig. 4 shows the effect of cAMP and ATP on the activity of phosphofructokinase in unfertilized Urechis eggs. At ATP concentrations lower than 0.02 raM, the enzyme activity in Urechis eggs is related to the ATP concentration, one of the substrates of the enzyme, but the enzyme activity be-
50
0
--s 10 -7
10-6
10-5
10-4
10-3
Fig. 5. Abscissa: concentrations of c A M P (M), of Ca 2+ (M), and of fructose 1,6-diphosphate (M); ordinate: percent increase in pyruvate kinase activity. P e r c e n t increase is the e n z y m e activity in the presence of c A M P (©), in the p r e s e n c e of Ca 2+ (O), and in the presence of F D P ( x ) . Effect of c A M P , Ca, and F D P on the activity of p y r u v a t e kinase in the Urechis unfertilized eggs. Exp Cell Res 108 (1977)
196
Tazawa and Yasumasu
50 ¸
3O
10
0
r
I£
UF
'
'
30
60
Fig. 6. Abscissa: time after fertilization (min); ordinate: concentration of c A M P (pmoles/10 a eggs). Concentration of c A M P in the Urechis eggs before and after fertilization. Vertical range bars represent S.E.M.
comes lowered in the presence of the substrate at higher concentrations than 0.02 mM. Hence, it is probable that ATP is not only the substrate but also the negative modulator of phosphofructokinase in Urechis eggs. ATP inhibition of the enzyme activity has been found in mammalian cells [13]. The same has also been found in sea urchin eggs [14]. cAMP causes a reversal of ATP inhibition of the enzyme activity. It seems that cAMP not only causes reversal of the ATP inhibition, but also elevation of the enzyme activity. As shown in table 2, Ca 2+ slightly stimulates the enzyme activity. ADP hardly causes any enzyme activation, while AMP causes an increase in the enzyme activity. Phosphofructokinase activity in the unfertilized egg homogenate, estimated in the presence of 0.5 mM Ca 2+ and cAMP at a concentration above 10.5 M, is highest among those examined and higher than that in fertilized eggs. As shown in fig. 5, the pyruvate kinase activity is enhanced by cAMP, FDP, and Ca 2+. FDP Exp Cell Res 108 (1977)
exerts the highest stimulating effect on the enzyme activity among these compounds. The FDP concentration in the eggs increases to a level high enough to stimulate the enzyme activity at 20 min after fertilization [ 1]. As shown in fig. 6, the cAMP level began to increase 10 rain and reached its maximum concentration 40 min after insemination. The change in Ca 2+ concentration should also be observed in order to elucidate the changes in enzyme activity in Urechis eggs following fertilization. But this has not yet been studied. Since it has been pointed out that procaine, a narcotics, probably stimulates the release of bound Ca 2+ [15], it is likely that these Ca2+-sensitive enzymes are enhanced in procaine-treated eggs. The activities of the enzymes in the glycolysis system were estimated in unfertilized eggs which had been kept in sea water containing 10 mM procaine for 20 min at 20°C. During the treatment, the germinal vesicle disappeared in 38.5+ 10.4% of the unfertilized eggs. As shown in table 3, the activity of phosphorylase, phosphofructokinase and pyruvate kinase in procaine-treated eggs are higher than those in intact unfertilized eggs, while other enzyme activities remain the same as in intact unfertilized eggs. The increase in the activities of these three enzymes may be supposed to be due to parthenogenetic activation induced by procaine treatment, since about one-third of the procainetreated eggs show germinal vesicle breakage. It has been reported that the parthenogenetic activation of sea urchin eggs with procaine treatment is due to the release of protein from the surface of the eggs [16]. However, it is also probable that procaine causes an increase in Ca 2+ concentration in Urechis eggs following parthenogenic activation. The Ca ~+ concentration can be
Enzyme activities in the glycolysis system
197
Table 3. Effect of procaine on the enzyme activities in the glycolysis system in unfertilized eggs of Urechis Procaine conc.
Enzymes
None
10-3 M
P-rylase c~-glucosidase PGluM HK G6P-DH GPIxl02 P F K (0.1 mM ATP) F-diphosphatase Aldolase GA3P-DH Enolase PK LDH
1.40+0.31 4.21+0.39 1.25+0.33 2.47+0.59 16.06+1.33 6.82+0.72 6.02_+0.10 10.31___2.12 9.52+2.45 14.95+_2.36 8.61_+1.04 11.79+1.92 7.38+2.42
1.98+0.15 3.98* 1.35" 2.39* 16.72" 7.13" 6.11+0.19 9.50* 7.39* 14.02" 8.98* 14.17+2.21 8.13"
% increase +41.4
+ 0.01
+20.2
5 x 10-3 M
% increase
10-2 M
% increase
2.39+0.30 4.22+0.35 1.20+0.29 2.50+0.51 15.87+3.20 6.54+0.43 7.21+0.29 10.91+2.72 9.43+2.25 14.62_+2.98 8.70+2.04 16.09+3.12 7.89+2.98
+70.7 + 0 - 0.4 + 0.1 - 0.01 - 0.04 +19.7 + 0.01 - 0.01 - 0.02 + 0.01 +36.5 + 6.9
2.21+0.11 4.19+0.50 1.29+0.42 2.43+0.24 15.89+2.29 6.79+0.81 7.20_+0.43 9.95_+1.15 9.60+2.32 14.72_+4.29 8.59+1.10 16.12-+4.92 7.32+1.96
+57.8 - 0.05 + 0.3 - 0.02 - 0.01 - 0.04 +19.6 - 0.03 + 0.01 - 0.01 - 0.01 +36.7 - 0.01
Procaine treatment of unfertilized eggs was performed for 20 min at 20°C. All enzyme activities are expressed as in table 1. Abbreviations, see table 1. Values shown are the ±S~E.M. of three separate experiments except *. *, One experiment. The percentage increase in the enzyme activity is shown in parentheses.
hypothesized to increase in procainetreated Urechis eggs, since the percent increase in phosphorylase activity, the most Ca2+-sensitive enzyme among these is highest and that of phosphofructokinase activity, the least sensitive enzyme, is lowest in procaine-treated eggs (figs 3, 4; table 3). In procaine-treated eggs, cAMP did not increase during 20 min of treatment (0.9 pmoles/106 eggs at 20 min of 10-2 M procaine treatment). Based on the hypothesis that procaine stimulates the release of bound Ca 2+, as has been hypothesized in sea urchin eggs [17], the activation of these enzymes seems to be due to the increase in Ca ~+ concentration in procaine-treated unfertilized eggs.
DISCUSSION Enzyme activities in the glycolysis system of sea urchin eggs have been reported previously [6]. In Urechis eggs, each enzyme activity in the glycolysis seems to be very
similar to that in sea urchin eggs, but the intracellular distribution of several enzymes in Urechis eggs differs from that in sea urchin eggs. In both Urechis and sea urchin eggs, the phosphoglucoisomerase activity is very high as compared with other enzyme activities in the glycolysis system. However, more than 50 % of the enzyme activity is associated with the particulate in Urechis eggs, while it has been found in the soluble fraction in sea urchin eggs [6]. In both organisms, about 50% of the lactate dehydrogenase activity is detectable in the particulate fraction. Change in intracellular distribution of these enzymes does not occur following fertilization, and the reactions catalysed by these enzymes seem to be in an equilibrium state [1, 18]. Hence, the enzyme activities in the partfculate fraction are not supposed to be in a latent state. Pyruvate kinase activity in sea urchin eggs can be also detected in the particulate fraction and the activity in Urechis eggs is present in the soluble fraction. Though the flux Exp Cell Res 108 (1977)
198
Tazawa and Yasumasu
rate in this reaction step increases following fertilization [1, 18], no redistribution occurs in sea urchin [6] as well as in Urechis eggs. Aldolase [2, 6] and phosphorylase [5] activity have been found in the particulate fraction in unfertilized sea urchin eggs, but those can only be detected in the soluble fraction in unfertilized and fertilized eggs of Urechis. It has been reported that the aldolase and phosphorylase activities in sea urchin eggs become apparent following fertilization as a consequence of the redistribution of the enzymes [2, 5]. In the aldolase reaction in unfertilized and fertilized sea urchin eggs [18] and Urechis eggs [1], the reaction step has been found to be in a quasi-equilibrium state. Hence, the apparent activation of the aldolase activity in the consequence of the enzyme redistribution in sea urchin eggs hardly causes any increase in the flux rate at the reaction step. In the phosphorylase step, the reaction has been demonstrated to be in a regulated state both in sea urchin eggs [18] and in Urechis eggs [1] and is assumed to be one of the rate-limiting steps in glycolysis. The release of phosphorylase activity from its bound state following fertilization reported in the eggs of the sea urchins Sphaerechinus granularis and Paracentrotus lividus [5] seems to account for the increase in the flux rate at the reaction step catalyzed by phosphorylase in sea urchin fertilized eggs [18]. However, in unfertilized as well as in fertilized eggs of Urechis, phosphorylase a activity is detected only in the soluble fraction, although the reaction is still apparently rate-limiting [1]. Thus the mechanism responsible for the increased phosphorylase a activity after fertilization in Urechis must not involve redistribution of the enzyme but perhaps instead an increase in the calcium concentration, as has been found for sea Exp Cell Res 108 (1977)
urchin eggs [19, 20]. This is supported by the ability of procain to increase the Urechis phosphorylase a activity, possibly due to its proposed effects on the calcium levels [17]. The possibility that AMP could affect the phosphorylase b activity in Urechis eggs at fertilization seems unlikely, since AMP levels do not change after fertilization and the AMP concentration is too low to stimulate the phosphorylase b activity [1]. Similarly, cAMP does not increase early enough (fig. 5) to account for the rapid increase in phosphorylase a activity after fertilization (fig. 1). As in the case of phosphorylase a activity, a small but rapid increase in the pyruvate kinase activity in Urechis eggs following fertilization (fig. l) can be ascribed to an increase in the Ca 2+ level. The sudden increase in the phosphofructokinase activity a while after fertilization seems to be due to an increase in the cAMP level. A further increase in the pyruvate kinase activity keeping pace with that in phosphofructokinase activity is probably due to the increased cAMP level and/or to the FDP level, which raises following activation of the phosphofructokinase activity. Although this study was prompted by the finding that subcellular redistribution of enzymes occurs after fertilization in sea urchins [2, 5], no such redistribution is evident in most sea urchin species, most of the phosphorylase a activity being completely soluble [6, 12]. The results in this paper suggest that Urechis is more like the predominant form of sea urchin, with phosphorylase a regulated more likely by Ca 2+ and phosphorylation than by changes in the subcellular distribution of the enzyme. The similarity of these Urechis results to most sea urchin work is supported by the similarity in the two organisms of the profile of activity changes following fertilization of
E n z y m e a c t i v i t i e s in the g l y c o l y s i s s y s t e m phosphorylase a, phosphofructokinase, and p y r u v a t e k i n a s e [6, 12, 14] a n d b y th e f a c t t h a t in b o t h o r g a n i s m s t h e s e t h r e e e n z y m e s are a p p a r e n t l y r a t e - l i m i t i n g f o r g l y c o l y s i s b o t h b e f o r e a n d a f t e r f e r t i l i z a t i o n [1, 18] a n d ar e also r e g u l a t e d b y C a 2+ a n d c A M P [19-22]. REFERENCES 1. Yasumasu, I, Tazawa, E & Fujiwara, A, Exp cell res 93 (1975) 166. 2. Ishihara, K, J fac sci univ Tokyo sect IV 8 (1957) 71. 3. Isono, N, Tsusaka, A & Nakano, E, J fac sci univ Tokyo sect IV 10 (1963) 55. 4. Epel, D, Weaver, A M, Muchmo~e, A V & Schimke, R T, Science 163 (1969) 294. 5. Bergami, M, Mansour, T E & Scarano, E, Exp cell res 49 (1968) 650. 6. Yasumasu, I, Fujiwara, A, Shoger, R L & Asami, K, Exp cell res 92 (1975)444. 7. Bergmeyer, H-U, Gawehn, K & Grassl, M, Methods of enzymatic analysis (ed H-U Bergmeyer) vol. 1, p. 468. Academic Press, New York (1974). 8. - - Ibid vol. 1, p. 502 (1974).
199
9. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. 10. Gilman, A G, Proc natl acad sci US 64 (1969) 367. 11. Fischer, E H, Pocker, A & Saari, J C, Essays in biochemistry (ed P N Campbell & F Dickens) vol. 6, p. 23. Academic Press, London (1970). 12. Shoger, R L, Asami, K, Yasumasu, I & Fujiwara, A, Exp cell res 82 (1973) 375. 13. Epel, D, Pressman, B C, Elsaesser, S & Weaver, A M, The cell cycle (ed G M Padilla, G L Whitson & I L Cameron) p. 297. Academic Press, New York (1969). 14. Asami, K, Fujiwara, A, Shoger, R L & Yasumasu, I, Cell structure and function 1 (1975) 43. 15. Seeman, P, Pharmacol rev 24 (1972) 583. 16. Johnson, J D & Epel, D, Proc natl acad sci US 72 (1975) 4474. 17. Vacquier, V & Brandriff, B, Dev biol 47 (1975) 12. 18. Yasumasu, I, Asami, K, Shoger, R L & Fujiwara, A, Exp cell res 80 (1973) 361. 19. Mazia, D, J cell comp physiol 10 (1937) 291. 20. Nakamura, M & Yasumasu, I, J gen physiol 63 (1974) 374. 21. Nath, J & Rebhun, L I, Exp cell res 77 (1973) 319. 22. Yasumasu, I, Fujiwara, A & Ishida, K, Biochem biophys res commun 54 (1973) 628.
Received December 8, 1976 Accepted March 25, 1977
Exp Cell Res 108 (1977)