DEVELOPMENTAL
BIOLOGY
Thymidine
43,
322-332
(1975)
Kinase Activation Homogenization
in Unfertilized
Sea Urchin
Eggs by
and Fertilization
MASARU NONAKA AND HIROSHI TERAYAMA Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan Accepted
December
9, 1974
Kinetics of in oiuo phosphorylation of 3H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated 8H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100%of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization. In contrast to the in oiuo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase. Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs. INTRODUCTION
possible activation of thymidine kinase or other DNA key enzymes in sea urchin eggs after fertilization, although the uptake of thymidine, its phosphorylation and its incorporation into DNA in the egg are known to be markedly increased after fertilization (Suzuki and Mano, 1974, Ord and Stocken, 1973, Hinegardner et al., 1964). One of the puzzling problems in assuming the activation of thymidine kinase upon fertilization seems to be the fact that the enzyme activity in unfertilized sea urchin egg homogenates is in general as high as that in fertilized ones. In the present paper we wish to present some evidence showing that thymidine kinase may actually be activated not only by fertilization but also by homogenization of unfertilized eggs.
Thymidine kinase (EC 2.7.1.21) is known as one of the key enzymes responsible for the DNA synthesis and its regulation in various tissues such as regenerating liver (Bollum and Potter, 1959, Maley et al., 1965, Weissman et al., 1960), virusinfected cells (Bresnick and Rapp, 1969), hormone-stimulated cells (Masui and Garren, 1971), PHA-stimulated lymphocytes (Pegoraro and Bernengo, 1971) and so on. It is reported that the thymidine kinase activity in the synchronous cell culture shows a periodic change within a cell cycle, starting to increase upon entering the Sphase (Stubblefield and Murphree, 1967, Adams, 1969). Similar periodical changes in thymidine kinase, thymidylate kinase as well as DNA polymerase during a cell cycle MATERIALS AND METHODS have also been reported for the cleavage stage embryos of sea urchins (HansenSea urchins, Hemicentrotus pulcherDelkeskamp and Duspiva, 1965, Nagano rimus, Anthocidaris crassispina and and Mano, 1968). However, little evidence Pseudocentrotus depressus, were obtained has been presented so far to support the at the Misaki Marine Biological Station of 322 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
NONAKA
AND TERAYAMA
Activation
the University of Tokyo and used throughout the present study. Unfertilized eggs were collected by KCl-induced spawning and washed three times with filtered sea water. In vitro assay of thymidine kinase was carried out by the DEAE-cellulose paper disk method (Bollum and Potter, 1959, Breitman, 1963). One volume of packed eggs was homogenized with 10 vol of cold 0.25 M sucrose in a motor-driven Teflon-glass homogenizer for five strokes within 1 min and used for the assay of thymidine kinase immediately or after standing at 0°C for certain periods of time. The complete reaction mixture of a final volume of 150 ~1 contained 30 mM Tris-HCl buffer, pH 8.0, 1 mM MgClz, 10 mM ATP, 0.05 mM [2-“Clthymidine (5 mCi/mmole: Radiochemical Centre, U.K.), about 200 mM sucrose and the enzyme (egg homogenate equivalent to 300-600 pg protein). The enzyme, usually in 100 ~1, was added last and the reaction mixture was incubated at 20°C for 10 min except otherwise specified. After incubation the reaction was stopped by putting the reaction tube in a boiling water bath for 2 min and then cooled. A 50+1 aliquot of the heated reaction mixture was applied onto a DEAE-cellulose paper disk (Whatman DE81), washed three times with 0.01 M ammonium formate, once with water and lastly with methanol. After drying in the air, the radioactivity retained in the disk was measured in a liquid scintillation spectrophotometer using 8 ml of the toluene-PPO-POPOP scintillation fluid. In vivo uptake and phosphorylation of thymidine by eggs (unfertilized and fertilized) were measured as follows: Unfertilized or fertilized eggs of Anthocidaris crassispina of about 0.5-ml packed cell volume were suspended in 2 ml of sea water containing 1 PC1 of methyl-3H-thymidine (5 Ci/mmole; The Radiochemical Centre, U.K.) at the concentration of 0.2 PM. At various time points during incubation at 20°C the eggs were spun down in a manual centrifuge, quickly washed twice with cold
of Egg Thymidine
Kinase
323
sea water and then suspended in 1.25 ml of cold 5% PCA. The suspension was immediately sonicated at 0°C for 20 sec. The sonicate was centrifuged at 3000 rpm for 10 min to separate the supernatant from the pellet. The pellet was washed once with 1 ml of cold 5% PCA and was referred to as the acid-insoluble fraction. The supernatant and wash were combined and then centrifuged at 105,000 g for 1 hr. The supernatant thus obtained was neutralized with 3 N KOH and centrifuged at 3000 rpm for 10 min. The final supernatant thus prepared was referred to as the acid-soluble fraction. Ten microliter of the acid-soluble fraction mixed with adequate amounts of nonlabelled carriers such as thymidine, thymidylate and fl-aminoisobutyrate (a catabolite) were subjected to paper chromatography (descending) using a solvent system of n-butanol:ethyl acetate:methanol: cont. ammonium hydroxide (7:4:3:4 v/v) according to the procedure of Shimizu et al. (1969). Spots corresponding to thymidine (fast migration), fl-amino-isobutyrate (intermediate migration) and phosphorylated thymidine derivatives (near origin) were visualized under U.V. irradiation or by spraying ninhydrin, cut off and extracted with water. Radioactivities in the extracts were counted in a liquid scintillation spectrophotometer using toluene-triton X-PPO as scintillator. Radioactivity of the acid-insoluble fraction was measured in a liquid scintillation spectrophotometer using toluene-ethylcellosolve-PPO scintillator after it had been oxidized with 0.2 ml of 60% PCA and 0.4 ml of 30% H,Oz at 80-90°C for 30-60 min according to Mahin and Lofberg (1966). Protein was assayed according to the, method of Lowry et al. (1951) with bovine serum albumin as standard. RESULTS
1. Preliminary assessments for the assay thymidine kinase of sea urchin egg homogenates. Since the thymidine kinase activity of unfertilized Hemicentrotus egg of
324
DEVELOPMENTALBIOLOGY
homogenates was almost equal to that of fertilized ones, the unfertilized egg homogenates of Hemicentrotus pulcherrimus were used for establishing the standard conditions for assaying thymidine kinase in sea urchin egg homogenates. It was found that the amount of phosphorylated derivatives of thymidine produced during 10 min of incubation at 20°C in the complete reaction mixture increased with increasing enzyme concentrations within a range of enzyme levels below 1.9-2.1 mg protein per 150 ~1 of the reaction mixture. The thymidine kinase activity surveyed in a range of pH 6.0-9.0 showed a broad pH-activity curve having a peak at 7.5. Addition of external Mg2+ up to 1 mM stimulated the enzyme activity slightly but inhibited it beyond that concentration. Addition of Ca2+ or Znzf inhibited the enzyme activity markedly as evidenced in Fig. 1. On the other hand, EDTA added at concentrations up to 1 mM stimulated the enzyme activity but inhibited it at higher concentrations as also shown in Fig. 1. This biphasic effect of EDTA may be ascribed to the elimination of inhibitory metal ions such as Ca2+ or others at lower EDTA
VOLUME 43, 1975
concentrations and to the elimination of Mg2+ ions at higher ones. The enzyme kinetics was usually found to be linear with time within 60 min of incubation except in case of freshly prepared homogenates (used immediately after homogenization) of Pseudocentrotus depressus eggs. With the unfertilized egg homogenates of Pseudocentrotus, the enzyme kinetics was in many cases rather complicated, showing an accelerating or activating tendency during the first 20 min incubation as illustrated in Fig. 2. This interesting finding has led us to investigate further on the possible activation of thymidine kinase occurring in the unfertilized sea urchin egg homogenates. 2. Elevation of thymidine kinase activity in freshly prepared egg homogenate upon standing at 0°C. During the course of
preliminary investigations on the stability of thymidine kinase in sea urchin egg homogenates we noticed that the enzyme activity of freshly prepared homogenates of unfertilized eggs of Hemicentrotus pulcherrimus or Anthocidaris crassispina is sometimes enhanced a little (by 10-20s at the best) when the homogenates stand at
0 0
20 Concentration
-*--.--
znsoq Cdl2
-
EDTA
40 of
EDTA
or
60 divalent
catlons
( mM )
FIG. 1. Effects of EDTA or divalent cations on thymidine kinase of sea urchin egg (unfertilized) CaC12, the control activity was 1600 cpm/mg protein/l0 min (Anthocidaris homogenate. --O--O--: crussispinu). .O.. .O.. .: ZnSO,, the control activity was 1500 cpm/mg protein/l0 min (Hemicentrotus pulcherrimus). -A-A-: EDTA, the control activity was 1300 cpm/mg protein/IO min (Anthocidaris crassispina).
NONAKA
Activation
AND TERAYAMA
0°C for 30 min or so, but later on the enzyme activity tends to decrease gradually even at 0°C. The increase in thymidine kinase during the first 30 min of standing at 0°C was observed more markedly and more often with the freshly prepared Pseudocentrotus depressus egg homogenates. As evidenced in Fig. 3 the enzyme activity increased several fold within 30 min of standing at 0°C. When the enzyme
of Egg Thymidine
325
Kinase
activity after homogenization was followed in both the unfertilized and fertilized (12 min after insemination) eggs starting from the same batch of Pseudocentrotus unfertilized eggs, it was found that the enzyme activity of the fertilized egg homogenate was high and unchanged while that of the unfertilized one was low at the beginning, but reached the level in the fertilized egg homogenate after 30 min of standing at 0°C as evidenced in Fig. 4. These results described above may suggest that thymidine kinase in unfertilized eggs may be activated during and/or after homogenization of the eggs. The Pseudocentrotus eggs may be understood to be slowest in the rate of activation among the species examined. 3. Possible involvement of redox reaction in the homogenization-induced thymidine kinuse activation. In the preliminary
2. Time course of thymidine phosphorylation by freshly prepared unfertilized egg homogenates of Hemicentrotus and Pseudocentrotus. The reaction mixture was same as described in Materials and Methods. -O-O-: Hemicentrotus pulcherrimus. --O-O-: Pseudocentrotus depressus. FIG.
investigations we have found that thymidine kinase in sea urchin egg homogenates can be inhibited by some oxidants such as potassium ferricyanide or ferric chloride and that the inhibition by these oxidants can be reversed by the addition of gluta-
20 Time
40 after
homogenization
60 ( min
)
FIG. 3. Rise in thymidine kinase activity of freshly prepared homogenate of unfertilized eggs of Pseudocentrotus depressus and Hemicentrotus pulcherrimus upon standing at 0°C. The unfertilized egg homogenate was kept at 0°C after homogenization and aliquots were assayed for thymidine kinase activity from time to time as described in Materials and Method (at 20°C for 10 min). 0 and 0 indicate two different batches of unfertilized eggs of Pseudocentrotus depressus. A and A indicate two different batches of unfertilized eggs of Hemicentrotus pulcherrimus.
326
DEVELOPMENTALBIOLOGY VOLUME43, 1975
.a....~
40
20 Time
: Fert.
after
60
homogenization
( min
)
FIG. 4. Change in thymidine kinase activities of unfertilized and fertilized egg homogenates during standing at 0°C. Unfertilized eggs were obtained from one female Pseudocentrotus depressus and divided into two groups: one was fertilized but another was not fertilized. The fertilized eggs (12 min after insemination) and the unfertilized eggs were homogenized and the freshly prepared homogenates were kept at 0°C. At various time points after homogenization, aliquots of the homogenates were used for the thymidine kinase assay in the same way as in the legend for Fig. 3. 04 unfertilized; 0. .O fertilized.
4GQ-
*----a I’ I’
-3oo-
--a---
---__ *
--‘GSH
!
s z.. .Z
-____ --e--*.‘---
----:j,TT
:’ : :
4
4
2 Concentration
of
reducing
6 agents
( rd.4 )
FIG. 5. Effects of GSH and D’IT on thymidine kinase activity of freshly prepared homogenates of unfertilized sea urchin eggs. Various concentrations of GSH (-) and DTI’ (-----) were added to the standard complete reaction mixture. -A-A--: Hemicentrotus pulcherrimus; the control activity was 3100 cpmlmg protein/l0 min. -A-A--: Pseudocentrotus depressus; the control activity was 950 cpm/mg protein/l0 min. - -0- -0- -: Pseudocentrotus depressus; the control activity was 2400 cpm/mg protein/l0 min. - -O- -O- -: Pseudocentrotw depressus; the control activity was 1050 cpm/mg protein/l0 min.
thion (GSH) or dithiothreitol (DTT). GSH or DTT itself, when added to the assay system containing the fully activated homogenates, for instance Pseudocentrotus egg homogenate after 30 min of standing at 0°C did not stimulate the enzyme activity
to any considerable extents. However, as shown in Fig. 5, when GSH or DTT was added to the freshly prepared and thus seemingly only partially activated homogenates of Pseudocentrotus eggs, a marked elevation of enzyme activity was observed.
NONAKA AND TERAYAMA
Activation
DTI’ at concentration as low as 1 mA4 or GSH at 3-4 mM seems to be enough to give a maximum activation. The activation of thymidine kinase of the egg homogenates of Hemicentrotus or Anthocidaris by GSH or DTT was scarcely observed probably because of the enzyme may be almost fully activated in the freshly prepared homogenates of eggs. These results seem to suggest that the activation of thymidine kinase may be accompanied by reduction of disulfide to sulfhydryl groups. In order to confirm this point further, effects of SH-blockers were also investigated. As shown in Fig. 6, the enzyme activity of the freshly prepared egg homogenate of Hemicentrotus and also of was inhibited by pAnthocidaris chloromercuribenzoate (PCMB) in a rather simple fashion upon increasing the concentration of PCMB. On the other hand, the enzyme activity of freshly prepared Pseudocentrotus egg homogenate was found to be activated by PCMB at concen-
ofEgg Z’hymidine
Kinase
327
trations lower than 1.5 x 1O-5 M but to be inhibited beyond that concentration of PCMB. When PCMB was added to the fully activated Pseudocentrotus egg homogenate, no stimulation was observed similarly to the other species of sea urchin egg homogenates. Monoiodoacetate and N-ethylmaleimide were also found to be inhibitory to thymidine kinase in Hemicentrotus egg homogenates. The biphasic effect of PCMB on thymidine kinase in freshly prepared homogenates of unfertilized Pseudocentrotus eggs will be discussed later. 4. In vivo evidence for tion of thymidine kinase shortly after fertilization. of Anthocidaris were
the possible elevain sea urchin eggs
Unfertilized eggs divided into two groups. Eggs in one group were inseminated and 10 min later SH-thymidine was added (1 &i/ml; 0.2 p&f), while eggs in another group were not inseminated but 3H-thymidine was added similarly. At 1, 5, and 9 min after the addition of 3H-thymidine, radioactivities in the acid-soluble and acid-insoluble fractions as well as radioactivity distribution among the acidsoluble components (intact thymidine, j?aminoisobutyrate and phosphorylated thymidine derivatives) were measured as described in Materials and Methods. As summarized in Table 1, the uptake of total radioactivity (sum of acid-soluble and acid-insoluble radioactivities) increased almost linearly with increasing time of incubation in both unfertilized and fertilized eggs although there was a great difference in the rate of uptake between the unfertilized and fertilized eggs. The rate of thymidine uptake in the fertilized eggs was 35-40 I times as large as that in the unfertilized 1 2 3 eggs. About 90% of the total radioactivity ConcentratKm of PCMB ( X105t4 ) taken up by the eggs were found in the FIG. 6. Effect of PCMB on thymidine kinase activity of freshly prepared homogenates of unfertilized acid-soluble fraction in both groups. The eggs. Various concentrations of PCMB were added to distribution of radioactivity of the acidthe standard complete reaction mixture. -Q-O-: soluble fraction among the subcomponents Hemicentrotus pulcherrimus; the control activity was was, however, shown to vary characteristi3400 cpm/mg protein/l0 min. -O--O-: Pseudocencally between the fertilized and unfertiltrotus depressus; the control activity was 920 cpm/mg protein/l0 min. ized groups. It seems obvious that the
328
DEVELOPMENTAL
VOLUME 43, 1975
BIOLOGY
bated for 10 min with 3H-thymidine of 1 pC!i/ml but of different net concentrations
percentage of phosphorylated thymidine increases with increasing incubation time in the unfertilized eggs while it remains unchanged and holds very high values near 100% in the fertilized eggs in spite of a tremendous increase in the net influx of thymidine. The above results seem to suggest that somehow the capacity of eggs to uptake thymidine is enlarged with accompanied increase in the enzyme(s) responsible for phosphorylating thymidine. In order to examine to what extent the thymidine-phosphorylating enzyme(s) supposed to be activated in the fertilized eggs can cope with the increasing influx of thymidine, another series- of experiments was carried out, in which Anthocidaris eggs at 10 min after insemination were incu-
(0.2-200 /.&I.
As summarized in Table 2, the uptake of thymidine increased with increasing net thymidine concentrations in the medium, while the percentage of phosphorylated thymidine in the acid-soluble radioactivity remained nearly unchanged at thymidine concentrations less than 2 pA4, but then tended to decrease upon increasing thymidine concentrations beyond that concentration. The results seem to suggest that the level of thymidine kinase activated in the fertilized eggs may be high enough to cope with the increased influx of thymidine up to a concentration of 2 PM thymidine added in the medium. Beyond that concen-
TABLE UPTAKE
AND PHOSPHORYLATION
OF 3H-T~~~~~~~~
1
BY UNFERTILIZED
AND FERTILIZED
EGGS OF
Anthocidaris
crassispina
Eggs
Incubation time (min)
Radioactivity (lO-S x dpm/mg protein)” Acid-insol.
Unfertilized
1 5 9
Fertilized
1 5 9
Distribution (%) of acid-sol. radioactivity among
Acid-sol.
0.046 0.488 0.119
Tbymidine
fl-AIBAb
Nucleotides
82 41 15
0 16 24
18 43 61
0.844 1.53 2.58
2.42 6.75 12.8
29.3 61.3 104
0.5 0.1 2.2
2.0 1.7 1.7
97.5 98.2 96.1
DRadioactivity was expressed per mg protein of eggs. b j3-Aminoisobutyrate (thymidine catabolite). TABLE EFFECT OF 8H-T~~~~~~~
Acid-insoluble fraction (lOmax dpm)
0.2 2.0 20 200
Acid-soluble fraction
(lo-’ x nmole) (lo-$ x dpm)
3.42 4.40 2.34 0.846
a Radioactivity of W-thymidine a series of experiments.
0.302 4.00 21.1 76.9
35.4 44.8 28.4 12.5
added to the incubation
BY FERTILIZED
EGGS
% Distribution of acid-sol. radioactivity among
‘H-Thymidine uptaken by eggs (per mg protein)
Cont.
of ‘H-thymidine” (nmole/ml)
2
CONCENTRATION ON UPTAKE AND PHOSPHOR~ATION OF THYMIDINE OF Anthocidaris crassispina DURING 10 MIN OF INCUBATION
Thymidine
&AIBA
Nucleotides
4.2 4.1 16.5 29.7
56.4 50.9 50.5 41.1
39.4 45.0 33.0 29.2
(lo-’ x nmole) 3.22 40.8 259 1130
medium was kept constant at 1 &i/ml
throughout
NONAKA AND TERAKAMA
Activation of Egg Thymidine
329
Kinase
TABLE 3 FATE OF 3H-T~~~~~~~~ TAKEN UP BY Anthocidaris crasskpina (UNFERTILIZED AND FERTILIZED) DURING THE COURSE OF SECOND INCUBATION IN THE ABSENCE OF SH-T~~~~~~~~ Eggs
Incubation time” (min)
Radioactivity (10e3 dpm/mg protei# Acid-insol.
Acid-sol.
Unfertilized
0 5 15 25
0.078 0.055 0.067 0.066
7.39 11.2 10.5 8.93
Fertilized
0 5 15 25
1.66 2.15 3.79 4.14
64.7 50.8 46.7 83.5
Distribution (o/o)of acid-sol. radioactivity among Thymidine 13 12 13 12 1.4 2.3 2.5 3.8
j3-AIBA
Nucleotides
19 21 23 20
68 67 64 68
3.6 7.8 4.3 4.4
95.0 89.9 93.2 91.8
“Unfertilized and fertilized (10 min after insemination) eggs were incubated with sH-thymidine (1 &i/ml, 0.2 PM) for 5 min, followed by second incubation in the absence of ‘H-thymidine. bRadioactivity was expressed per mg protein of eggs.
phosphorylation of thymidine and uridine in the eggs 15 min after fertilization. Our results presented in this paper seem to be in accord with these reports and seem to suggest that thymidine kinase activity elevates during a short period of time after fertilization. Longo and Plunkett (1973) also suggested thymidine kinase activation after fertilization based on the measurement of TTP formed in the eggs. Piatigorsky and Whiteley (1965) considered that uridine uptake may be coupled with its phosphorylation. However, such uptake-phosphorylation coupling does not seem to be held for thymidine. The datum shown in Table 2 indicates the increase in the ratio of thymidine to nucleotides with increasing concentration of added thymidine and suggests that the uptake of thymidine may not be limited by the intracellular phosphorylation capacity. In contrast to the apparent elevation of DISCUSSION thymidine kinase activity in sea urchin There are many papers reporting the eggs occurring soon after fertilization as increase in the capacity to uptake exter- assumed from the in uiuo results, the total nally supplied nucleosides into sea urchin thymidine kinase activity in egg extract or eggs soon after fertilization (Nemer, 1962, homogenate was found to be unchanged Piatigorsky and Whiteley, 1965, Pasternak, before and after fertilization a’s shown in 1973, Ord and Stocken, 1973). Ord and the present paper. Almost similar results Stocken (1973) also reported the increased are also presented by Suzuki and Mano tration, however, the phosphorylation capacity appears to become unable to catch up the increasing influx of thymidine. In order to investigate the fate of 3Hthymidine once taken up by sea urchin eggs, unfertilized and fertilized (10 min after insemination) eggs of Anthocidaris were first exposed to SH-thymidine (1 &i/ ml; 0.2 PM) for 5 min at 25”C, and then washed and cultured in the absence of thymidine for another 25-min period. At various points of time during the second incubation, aliquots of the suspensions were removed and assayed for the radioactivity distribution. The results summarized in Table 3 seem to indicate that the distribution of acid-soluble radioactivity among thymidine, P-aminoisobutyrate and phosphorylated thymidine derivatives (nucleotides) remains almost unchanged throughout the second incubation course.
330
DEVELOPMENTALBIOLOGY VOLIJME43, 19%
(1974) although thymidine kinase activity may fluctuate a bit depending on time after insemination. This puzzling discrepancy between the in uiuo and in vitro results on thymidine kinase activity has not yet been answered up to the present time. Similar discrepancy was also reported by Epel (1964) and Blomquist (1973) about nicotinamide adenine dinucleotide (NAD) kinase. Though the stimulation of NAD kinase after insemination was expected from the marked increase in NADP+ and NADPH content with a concomitant decrease in NAD+ occurring soon after insemination, no difference in the NAD kinase activity was detected between the homogenates of unfertilized and fertilized eggs. Blomquist (1973) discussed that the increase in NADP+ may not be the results of new synthesis of NAD kinase because of the rapidity of the enzyme response after fertilization. This seems also to be held for thymidine kinase. As shown in this paper thymidine kinase appears to be fully activated within the first 10 min after insemination. In this paper we have shown that thymidine kinase in unfertilized sea urchin eggs may be activated even at 0°C during or after homogenization of the eggs. This may account for the apparently similar levels of thymidine kinase activity in both fertilized and unfertilized egg homogenates. Activation of the enzyme after homogenization was more markedly and reproducibly observed with unfertilized Pseudocentrotus eggs as compared with the other ones. The enzyme in the eggs of latter species seems to be fully activated during homogenization, showing rather high enzyme activity even in the freshly prepared homogenates. Reasons for the apparent difference in the rate of homogenization-induced enzyme activation among species are not yet clear. The results presented in this paper seem to indicate that thymidine kinase of sea urchin eggs may be a SH-enzyme similarly
to that of mammalian tumors (Bresnick and Thompson, 1965, Hashimoto et al., 1972) or of bacteria (Durham and Ives, 1971). The enzyme activity was inhibited by PCMB or ferricyanide. GSH and DTT were effective in reversing the inhibition by PCMB or ferricyanide. When unfertilized eggs were homogenized with ferricyanide, the thymidine kinase activity of homogenate was very low even with Hemicentrotus or Anthocidaris eggs. Addition of GSH, DTT or even NADH to the ferricyanidehomogenate was found to activate the enzyme gradually within 30 min of incubation at 0°C. GSH and DTT were also effective in stimulating the thymidine kinase in the freshly prepared Pseudocentrotus egg homogenate as evidenced in this paper. These results suggest the possible involvement of some redox reaction in the mechanism of thymidine kinase activation by homogenization. Although added GSH, DTT and NADH are effective in activating thymidine kinase in the homogenates, the endogenous reductant which may actually be responsible for activating thymidine kinase in the eggs has not been clarified. PCMB at low concentrations (below 1.5 x 1O-5 M) added to freshly prepared homogenate of unfertilized Pseudocentrotus eggs, in which thymidine kinase may be only partially activated, did stimulate the enzyme activity. PCMB showed only an inhibitory effect when given to homogenates with fully activated enzyme such as freshly prepared Hemicentrotus or Anthocidaris egg homogenates. These results suggest that there may be a thymidine kinase inhibitor in the eggs, which may also require SH group(s) to interact with thymidine kinase and is more sensitive to PCMB than thymidine kinase itself. The final conclusion including other possibilities about the activation mechanism of thymidine kinase has to be waited until further experimental evidence will be accumulated. Details of the in vitro activation
NONAKA AND TERAYAMA
Actioation
mechanism for thymidine kinase in unfertilized eggs by homogenation as well as the problem how it may be related to the enzyme activation occurring in the eggs after fertilization are now under investigation. This study was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Japan. REFERENCES ADAMS, R. L. P. (1969). Phosphorylation of tritiated thymidine by L 929 mouse fibroblasts. Exp. Cell Res. 56, 49-54. BLOMQUIST, C. H. (1973). Partial purification and characterization of nicotinamide adenine dinucleotide kinase from sea urchin eggs. J. Biol. Chem. 248, 7044-7048. BOLLUM, F. J., and POTTER, V. R. (1959). Nucleic acid metabolism in regenerating rat liver. VI. Soluble enzymes which convert thymidine to thymidine phosphate and DNA. Cancer Res. 19, 561-565. BREITMAN, T. R. (1963). The feedback inhibition of thymidine kinase. Biochim. Biophys. Acta 67, 153-155. BRESNICK, E., and THOMPSON, U. B. (1965). Properties of deoxythymidine kinase partially purified from animal tumors. J. Biol. Chem. 240, 39673974. BRESNICK, E., and RAPP, F. (1969). Thymidine kinase activity in cells abortively and productively infected with human adenoviruses. Virology 34, 799802. DURHAM, J. P., and IVES, D. H. (1971). The metabolism of deoxyribonucleosides in Lactobacillus acidphilus; Regulation of deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine kinase activities by nucleotides. Biochim. Biophys. Acta 228, 9-25. EKER, P. (1965). Activities of thymidine kinase and thymine deoxyribonucleoside phosphate during growth of cells in tissue culture. J. Biol. Chem. 240, 2607-2611. EPEL, D. (1964). A primary metabolic change of fertilization; Interconversion of pyridine nucleotides. Biochem. Biophys. Res. Commun. 17, 62-68. HANSEN-DELKESKAMP, E., and DUSPIVA, F. (1965). Aktivititsverlauf der enzymatischen Phosphorylierung von Thymidin wahrend der Entwicklung des Seeigels Psammechinus miliaris von der Befrechtung bis zum Zweizeller. Experientia 22, 381382. HASHIMOTO, T., ARIMA, T., OKUDA, H., and FUJII, S. (1972). Purification and properties of deoxythymidine kinase from the Yoshida sarcoma. Cancer Res. 32, 67-73.
o/Egg Thymidine
Kinase
331
HINEGARDNER, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of sea urchin egg. Exp. Cell Res. 36, 53-61. LONCO, F. J., and PLUNKETT, W. (1973). The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata. Deuelop. Biol. 30, 56-67. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MAHNIN, D. T., and LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14 or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biothem. 16, 500-509. MALEY, G. F., LORENSON,M. G., and MALEY, F. (1965). Inhibitors of protein synthesis; Effect on the levels of deoxycytidylate deaminase, thymidylate synthetase and thymidine kinase in regenerating rat liver. Biochem. Biophys. Res. Commun. 18, 364-370. MASUI, H., and GARREN, L. D. (1971). On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution. J. Biol. Chem. 246, 5407-5413. NAGANO, H., and MANO, Y. (1968). Thymidine kinase, thymidylate kinase and 32Pi and “C thymidine incorporation into DNA during early embryogenesis of the sea urchin. Biochim. Biophys. Acta 157, 546-557. NEMER, M. (1962). Characteristics of the utilization of nucleosides by embryos of Puracenfrotus liuidus. J. Biol. Chem. 237, 143-149. ORD, M. G., and STOCKEN, L. A. (1973). Thymidine uptake by Paracentrotus eggs during the first cell cycle after fertilization. Erp. Cell Res. 83, 411-414. PASTERNAK, C. A. (1973). Phospholipid synthesis in cleaving sea urchin eggs; Model for specific membrane assembly. Deuelop. Biol. 30, 403-410. PEGORARO, L., and BERNENGO, M. G. (1971). Thymidine kinase, deoxycytidine kinase and deoxycytidylate deaminase activities in phytohaemagglutinin-stimulated human lymphocytes. Exp. CelI Res. 68, 283-290. PIATKORSKY, J., and WHITELEY, A. H. (1965). A change in permeability and uptake of ‘“C uridine in response to fertilization in Strongylocentrotus purpuratus eggs. Biochim. Biophys. Acta 108, 404-418. SHIMIZC, H., DALY, J. W., and CREVELING, C. R. (1969). A radioisotopic method for measuring the formation of adneosine 3’,5’-cyclic monophosphate in incubated slices of brain. J. Neurochem. 16, 1609-1619.
332
DEVELOPMENTALBIOLOGY
STUBBLEFIELD, E., and MURPHREE, S. (1967). Synchronized mammalian cell cultures. II. Thymidine kinase activity in colcemid synchronized fibroblasts.
Exp. Cell Res. 48, 652-656. SUZUKI, N., and MANO, Y. (1974). Phosphorylation of deoxyribonucleosides and DNA synthesis in early cleaving embryos of the sea urchin. J. Biochem.
VOLUME 43, 1975
75, 1349-1362. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. Biochim. BioThe phosphorylation of thymidine. ph.~s. Acta 45, 101-110.
BIOLOGY
Thymidine
43,
322-332
(1975)
Kinase Activation Homogenization
in Unfertilized
Sea Urchin
Eggs by
and Fertilization
MASARU NONAKA AND HIROSHI TERAYAMA Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan Accepted
December
9, 1974
Kinetics of in oiuo phosphorylation of 3H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated 8H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100%of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization. In contrast to the in oiuo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase. Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs. INTRODUCTION
possible activation of thymidine kinase or other DNA key enzymes in sea urchin eggs after fertilization, although the uptake of thymidine, its phosphorylation and its incorporation into DNA in the egg are known to be markedly increased after fertilization (Suzuki and Mano, 1974, Ord and Stocken, 1973, Hinegardner et al., 1964). One of the puzzling problems in assuming the activation of thymidine kinase upon fertilization seems to be the fact that the enzyme activity in unfertilized sea urchin egg homogenates is in general as high as that in fertilized ones. In the present paper we wish to present some evidence showing that thymidine kinase may actually be activated not only by fertilization but also by homogenization of unfertilized eggs.
Thymidine kinase (EC 2.7.1.21) is known as one of the key enzymes responsible for the DNA synthesis and its regulation in various tissues such as regenerating liver (Bollum and Potter, 1959, Maley et al., 1965, Weissman et al., 1960), virusinfected cells (Bresnick and Rapp, 1969), hormone-stimulated cells (Masui and Garren, 1971), PHA-stimulated lymphocytes (Pegoraro and Bernengo, 1971) and so on. It is reported that the thymidine kinase activity in the synchronous cell culture shows a periodic change within a cell cycle, starting to increase upon entering the Sphase (Stubblefield and Murphree, 1967, Adams, 1969). Similar periodical changes in thymidine kinase, thymidylate kinase as well as DNA polymerase during a cell cycle MATERIALS AND METHODS have also been reported for the cleavage stage embryos of sea urchins (HansenSea urchins, Hemicentrotus pulcherDelkeskamp and Duspiva, 1965, Nagano rimus, Anthocidaris crassispina and and Mano, 1968). However, little evidence Pseudocentrotus depressus, were obtained has been presented so far to support the at the Misaki Marine Biological Station of 322 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
NONAKA
AND TERAYAMA
Activation
the University of Tokyo and used throughout the present study. Unfertilized eggs were collected by KCl-induced spawning and washed three times with filtered sea water. In vitro assay of thymidine kinase was carried out by the DEAE-cellulose paper disk method (Bollum and Potter, 1959, Breitman, 1963). One volume of packed eggs was homogenized with 10 vol of cold 0.25 M sucrose in a motor-driven Teflon-glass homogenizer for five strokes within 1 min and used for the assay of thymidine kinase immediately or after standing at 0°C for certain periods of time. The complete reaction mixture of a final volume of 150 ~1 contained 30 mM Tris-HCl buffer, pH 8.0, 1 mM MgClz, 10 mM ATP, 0.05 mM [2-“Clthymidine (5 mCi/mmole: Radiochemical Centre, U.K.), about 200 mM sucrose and the enzyme (egg homogenate equivalent to 300-600 pg protein). The enzyme, usually in 100 ~1, was added last and the reaction mixture was incubated at 20°C for 10 min except otherwise specified. After incubation the reaction was stopped by putting the reaction tube in a boiling water bath for 2 min and then cooled. A 50+1 aliquot of the heated reaction mixture was applied onto a DEAE-cellulose paper disk (Whatman DE81), washed three times with 0.01 M ammonium formate, once with water and lastly with methanol. After drying in the air, the radioactivity retained in the disk was measured in a liquid scintillation spectrophotometer using 8 ml of the toluene-PPO-POPOP scintillation fluid. In vivo uptake and phosphorylation of thymidine by eggs (unfertilized and fertilized) were measured as follows: Unfertilized or fertilized eggs of Anthocidaris crassispina of about 0.5-ml packed cell volume were suspended in 2 ml of sea water containing 1 PC1 of methyl-3H-thymidine (5 Ci/mmole; The Radiochemical Centre, U.K.) at the concentration of 0.2 PM. At various time points during incubation at 20°C the eggs were spun down in a manual centrifuge, quickly washed twice with cold
of Egg Thymidine
Kinase
323
sea water and then suspended in 1.25 ml of cold 5% PCA. The suspension was immediately sonicated at 0°C for 20 sec. The sonicate was centrifuged at 3000 rpm for 10 min to separate the supernatant from the pellet. The pellet was washed once with 1 ml of cold 5% PCA and was referred to as the acid-insoluble fraction. The supernatant and wash were combined and then centrifuged at 105,000 g for 1 hr. The supernatant thus obtained was neutralized with 3 N KOH and centrifuged at 3000 rpm for 10 min. The final supernatant thus prepared was referred to as the acid-soluble fraction. Ten microliter of the acid-soluble fraction mixed with adequate amounts of nonlabelled carriers such as thymidine, thymidylate and fl-aminoisobutyrate (a catabolite) were subjected to paper chromatography (descending) using a solvent system of n-butanol:ethyl acetate:methanol: cont. ammonium hydroxide (7:4:3:4 v/v) according to the procedure of Shimizu et al. (1969). Spots corresponding to thymidine (fast migration), fl-amino-isobutyrate (intermediate migration) and phosphorylated thymidine derivatives (near origin) were visualized under U.V. irradiation or by spraying ninhydrin, cut off and extracted with water. Radioactivities in the extracts were counted in a liquid scintillation spectrophotometer using toluene-triton X-PPO as scintillator. Radioactivity of the acid-insoluble fraction was measured in a liquid scintillation spectrophotometer using toluene-ethylcellosolve-PPO scintillator after it had been oxidized with 0.2 ml of 60% PCA and 0.4 ml of 30% H,Oz at 80-90°C for 30-60 min according to Mahin and Lofberg (1966). Protein was assayed according to the, method of Lowry et al. (1951) with bovine serum albumin as standard. RESULTS
1. Preliminary assessments for the assay thymidine kinase of sea urchin egg homogenates. Since the thymidine kinase activity of unfertilized Hemicentrotus egg of
324
DEVELOPMENTALBIOLOGY
homogenates was almost equal to that of fertilized ones, the unfertilized egg homogenates of Hemicentrotus pulcherrimus were used for establishing the standard conditions for assaying thymidine kinase in sea urchin egg homogenates. It was found that the amount of phosphorylated derivatives of thymidine produced during 10 min of incubation at 20°C in the complete reaction mixture increased with increasing enzyme concentrations within a range of enzyme levels below 1.9-2.1 mg protein per 150 ~1 of the reaction mixture. The thymidine kinase activity surveyed in a range of pH 6.0-9.0 showed a broad pH-activity curve having a peak at 7.5. Addition of external Mg2+ up to 1 mM stimulated the enzyme activity slightly but inhibited it beyond that concentration. Addition of Ca2+ or Znzf inhibited the enzyme activity markedly as evidenced in Fig. 1. On the other hand, EDTA added at concentrations up to 1 mM stimulated the enzyme activity but inhibited it at higher concentrations as also shown in Fig. 1. This biphasic effect of EDTA may be ascribed to the elimination of inhibitory metal ions such as Ca2+ or others at lower EDTA
VOLUME 43, 1975
concentrations and to the elimination of Mg2+ ions at higher ones. The enzyme kinetics was usually found to be linear with time within 60 min of incubation except in case of freshly prepared homogenates (used immediately after homogenization) of Pseudocentrotus depressus eggs. With the unfertilized egg homogenates of Pseudocentrotus, the enzyme kinetics was in many cases rather complicated, showing an accelerating or activating tendency during the first 20 min incubation as illustrated in Fig. 2. This interesting finding has led us to investigate further on the possible activation of thymidine kinase occurring in the unfertilized sea urchin egg homogenates. 2. Elevation of thymidine kinase activity in freshly prepared egg homogenate upon standing at 0°C. During the course of
preliminary investigations on the stability of thymidine kinase in sea urchin egg homogenates we noticed that the enzyme activity of freshly prepared homogenates of unfertilized eggs of Hemicentrotus pulcherrimus or Anthocidaris crassispina is sometimes enhanced a little (by 10-20s at the best) when the homogenates stand at
0 0
20 Concentration
-*--.--
znsoq Cdl2
-
EDTA
40 of
EDTA
or
60 divalent
catlons
( mM )
FIG. 1. Effects of EDTA or divalent cations on thymidine kinase of sea urchin egg (unfertilized) CaC12, the control activity was 1600 cpm/mg protein/l0 min (Anthocidaris homogenate. --O--O--: crussispinu). .O.. .O.. .: ZnSO,, the control activity was 1500 cpm/mg protein/l0 min (Hemicentrotus pulcherrimus). -A-A-: EDTA, the control activity was 1300 cpm/mg protein/IO min (Anthocidaris crassispina).
NONAKA
Activation
AND TERAYAMA
0°C for 30 min or so, but later on the enzyme activity tends to decrease gradually even at 0°C. The increase in thymidine kinase during the first 30 min of standing at 0°C was observed more markedly and more often with the freshly prepared Pseudocentrotus depressus egg homogenates. As evidenced in Fig. 3 the enzyme activity increased several fold within 30 min of standing at 0°C. When the enzyme
of Egg Thymidine
325
Kinase
activity after homogenization was followed in both the unfertilized and fertilized (12 min after insemination) eggs starting from the same batch of Pseudocentrotus unfertilized eggs, it was found that the enzyme activity of the fertilized egg homogenate was high and unchanged while that of the unfertilized one was low at the beginning, but reached the level in the fertilized egg homogenate after 30 min of standing at 0°C as evidenced in Fig. 4. These results described above may suggest that thymidine kinase in unfertilized eggs may be activated during and/or after homogenization of the eggs. The Pseudocentrotus eggs may be understood to be slowest in the rate of activation among the species examined. 3. Possible involvement of redox reaction in the homogenization-induced thymidine kinuse activation. In the preliminary
2. Time course of thymidine phosphorylation by freshly prepared unfertilized egg homogenates of Hemicentrotus and Pseudocentrotus. The reaction mixture was same as described in Materials and Methods. -O-O-: Hemicentrotus pulcherrimus. --O-O-: Pseudocentrotus depressus. FIG.
investigations we have found that thymidine kinase in sea urchin egg homogenates can be inhibited by some oxidants such as potassium ferricyanide or ferric chloride and that the inhibition by these oxidants can be reversed by the addition of gluta-
20 Time
40 after
homogenization
60 ( min
)
FIG. 3. Rise in thymidine kinase activity of freshly prepared homogenate of unfertilized eggs of Pseudocentrotus depressus and Hemicentrotus pulcherrimus upon standing at 0°C. The unfertilized egg homogenate was kept at 0°C after homogenization and aliquots were assayed for thymidine kinase activity from time to time as described in Materials and Method (at 20°C for 10 min). 0 and 0 indicate two different batches of unfertilized eggs of Pseudocentrotus depressus. A and A indicate two different batches of unfertilized eggs of Hemicentrotus pulcherrimus.
326
DEVELOPMENTALBIOLOGY VOLUME43, 1975
.a....~
40
20 Time
: Fert.
after
60
homogenization
( min
)
FIG. 4. Change in thymidine kinase activities of unfertilized and fertilized egg homogenates during standing at 0°C. Unfertilized eggs were obtained from one female Pseudocentrotus depressus and divided into two groups: one was fertilized but another was not fertilized. The fertilized eggs (12 min after insemination) and the unfertilized eggs were homogenized and the freshly prepared homogenates were kept at 0°C. At various time points after homogenization, aliquots of the homogenates were used for the thymidine kinase assay in the same way as in the legend for Fig. 3. 04 unfertilized; 0. .O fertilized.
4GQ-
*----a I’ I’
-3oo-
--a---
---__ *
--‘GSH
!
s z.. .Z
-____ --e--*.‘---
----:j,TT
:’ : :
4
4
2 Concentration
of
reducing
6 agents
( rd.4 )
FIG. 5. Effects of GSH and D’IT on thymidine kinase activity of freshly prepared homogenates of unfertilized sea urchin eggs. Various concentrations of GSH (-) and DTI’ (-----) were added to the standard complete reaction mixture. -A-A--: Hemicentrotus pulcherrimus; the control activity was 3100 cpmlmg protein/l0 min. -A-A--: Pseudocentrotus depressus; the control activity was 950 cpm/mg protein/l0 min. - -0- -0- -: Pseudocentrotus depressus; the control activity was 2400 cpm/mg protein/l0 min. - -O- -O- -: Pseudocentrotw depressus; the control activity was 1050 cpm/mg protein/l0 min.
thion (GSH) or dithiothreitol (DTT). GSH or DTT itself, when added to the assay system containing the fully activated homogenates, for instance Pseudocentrotus egg homogenate after 30 min of standing at 0°C did not stimulate the enzyme activity
to any considerable extents. However, as shown in Fig. 5, when GSH or DTT was added to the freshly prepared and thus seemingly only partially activated homogenates of Pseudocentrotus eggs, a marked elevation of enzyme activity was observed.
NONAKA AND TERAYAMA
Activation
DTI’ at concentration as low as 1 mA4 or GSH at 3-4 mM seems to be enough to give a maximum activation. The activation of thymidine kinase of the egg homogenates of Hemicentrotus or Anthocidaris by GSH or DTT was scarcely observed probably because of the enzyme may be almost fully activated in the freshly prepared homogenates of eggs. These results seem to suggest that the activation of thymidine kinase may be accompanied by reduction of disulfide to sulfhydryl groups. In order to confirm this point further, effects of SH-blockers were also investigated. As shown in Fig. 6, the enzyme activity of the freshly prepared egg homogenate of Hemicentrotus and also of was inhibited by pAnthocidaris chloromercuribenzoate (PCMB) in a rather simple fashion upon increasing the concentration of PCMB. On the other hand, the enzyme activity of freshly prepared Pseudocentrotus egg homogenate was found to be activated by PCMB at concen-
ofEgg Z’hymidine
Kinase
327
trations lower than 1.5 x 1O-5 M but to be inhibited beyond that concentration of PCMB. When PCMB was added to the fully activated Pseudocentrotus egg homogenate, no stimulation was observed similarly to the other species of sea urchin egg homogenates. Monoiodoacetate and N-ethylmaleimide were also found to be inhibitory to thymidine kinase in Hemicentrotus egg homogenates. The biphasic effect of PCMB on thymidine kinase in freshly prepared homogenates of unfertilized Pseudocentrotus eggs will be discussed later. 4. In vivo evidence for tion of thymidine kinase shortly after fertilization. of Anthocidaris were
the possible elevain sea urchin eggs
Unfertilized eggs divided into two groups. Eggs in one group were inseminated and 10 min later SH-thymidine was added (1 &i/ml; 0.2 p&f), while eggs in another group were not inseminated but 3H-thymidine was added similarly. At 1, 5, and 9 min after the addition of 3H-thymidine, radioactivities in the acid-soluble and acid-insoluble fractions as well as radioactivity distribution among the acidsoluble components (intact thymidine, j?aminoisobutyrate and phosphorylated thymidine derivatives) were measured as described in Materials and Methods. As summarized in Table 1, the uptake of total radioactivity (sum of acid-soluble and acid-insoluble radioactivities) increased almost linearly with increasing time of incubation in both unfertilized and fertilized eggs although there was a great difference in the rate of uptake between the unfertilized and fertilized eggs. The rate of thymidine uptake in the fertilized eggs was 35-40 I times as large as that in the unfertilized 1 2 3 eggs. About 90% of the total radioactivity ConcentratKm of PCMB ( X105t4 ) taken up by the eggs were found in the FIG. 6. Effect of PCMB on thymidine kinase activity of freshly prepared homogenates of unfertilized acid-soluble fraction in both groups. The eggs. Various concentrations of PCMB were added to distribution of radioactivity of the acidthe standard complete reaction mixture. -Q-O-: soluble fraction among the subcomponents Hemicentrotus pulcherrimus; the control activity was was, however, shown to vary characteristi3400 cpm/mg protein/l0 min. -O--O-: Pseudocencally between the fertilized and unfertiltrotus depressus; the control activity was 920 cpm/mg protein/l0 min. ized groups. It seems obvious that the
328
DEVELOPMENTAL
VOLUME 43, 1975
BIOLOGY
bated for 10 min with 3H-thymidine of 1 pC!i/ml but of different net concentrations
percentage of phosphorylated thymidine increases with increasing incubation time in the unfertilized eggs while it remains unchanged and holds very high values near 100% in the fertilized eggs in spite of a tremendous increase in the net influx of thymidine. The above results seem to suggest that somehow the capacity of eggs to uptake thymidine is enlarged with accompanied increase in the enzyme(s) responsible for phosphorylating thymidine. In order to examine to what extent the thymidine-phosphorylating enzyme(s) supposed to be activated in the fertilized eggs can cope with the increasing influx of thymidine, another series- of experiments was carried out, in which Anthocidaris eggs at 10 min after insemination were incu-
(0.2-200 /.&I.
As summarized in Table 2, the uptake of thymidine increased with increasing net thymidine concentrations in the medium, while the percentage of phosphorylated thymidine in the acid-soluble radioactivity remained nearly unchanged at thymidine concentrations less than 2 pA4, but then tended to decrease upon increasing thymidine concentrations beyond that concentration. The results seem to suggest that the level of thymidine kinase activated in the fertilized eggs may be high enough to cope with the increased influx of thymidine up to a concentration of 2 PM thymidine added in the medium. Beyond that concen-
TABLE UPTAKE
AND PHOSPHORYLATION
OF 3H-T~~~~~~~~
1
BY UNFERTILIZED
AND FERTILIZED
EGGS OF
Anthocidaris
crassispina
Eggs
Incubation time (min)
Radioactivity (lO-S x dpm/mg protein)” Acid-insol.
Unfertilized
1 5 9
Fertilized
1 5 9
Distribution (%) of acid-sol. radioactivity among
Acid-sol.
0.046 0.488 0.119
Tbymidine
fl-AIBAb
Nucleotides
82 41 15
0 16 24
18 43 61
0.844 1.53 2.58
2.42 6.75 12.8
29.3 61.3 104
0.5 0.1 2.2
2.0 1.7 1.7
97.5 98.2 96.1
DRadioactivity was expressed per mg protein of eggs. b j3-Aminoisobutyrate (thymidine catabolite). TABLE EFFECT OF 8H-T~~~~~~~
Acid-insoluble fraction (lOmax dpm)
0.2 2.0 20 200
Acid-soluble fraction
(lo-’ x nmole) (lo-$ x dpm)
3.42 4.40 2.34 0.846
a Radioactivity of W-thymidine a series of experiments.
0.302 4.00 21.1 76.9
35.4 44.8 28.4 12.5
added to the incubation
BY FERTILIZED
EGGS
% Distribution of acid-sol. radioactivity among
‘H-Thymidine uptaken by eggs (per mg protein)
Cont.
of ‘H-thymidine” (nmole/ml)
2
CONCENTRATION ON UPTAKE AND PHOSPHOR~ATION OF THYMIDINE OF Anthocidaris crassispina DURING 10 MIN OF INCUBATION
Thymidine
&AIBA
Nucleotides
4.2 4.1 16.5 29.7
56.4 50.9 50.5 41.1
39.4 45.0 33.0 29.2
(lo-’ x nmole) 3.22 40.8 259 1130
medium was kept constant at 1 &i/ml
throughout
NONAKA AND TERAKAMA
Activation of Egg Thymidine
329
Kinase
TABLE 3 FATE OF 3H-T~~~~~~~~ TAKEN UP BY Anthocidaris crasskpina (UNFERTILIZED AND FERTILIZED) DURING THE COURSE OF SECOND INCUBATION IN THE ABSENCE OF SH-T~~~~~~~~ Eggs
Incubation time” (min)
Radioactivity (10e3 dpm/mg protei# Acid-insol.
Acid-sol.
Unfertilized
0 5 15 25
0.078 0.055 0.067 0.066
7.39 11.2 10.5 8.93
Fertilized
0 5 15 25
1.66 2.15 3.79 4.14
64.7 50.8 46.7 83.5
Distribution (o/o)of acid-sol. radioactivity among Thymidine 13 12 13 12 1.4 2.3 2.5 3.8
j3-AIBA
Nucleotides
19 21 23 20
68 67 64 68
3.6 7.8 4.3 4.4
95.0 89.9 93.2 91.8
“Unfertilized and fertilized (10 min after insemination) eggs were incubated with sH-thymidine (1 &i/ml, 0.2 PM) for 5 min, followed by second incubation in the absence of ‘H-thymidine. bRadioactivity was expressed per mg protein of eggs.
phosphorylation of thymidine and uridine in the eggs 15 min after fertilization. Our results presented in this paper seem to be in accord with these reports and seem to suggest that thymidine kinase activity elevates during a short period of time after fertilization. Longo and Plunkett (1973) also suggested thymidine kinase activation after fertilization based on the measurement of TTP formed in the eggs. Piatigorsky and Whiteley (1965) considered that uridine uptake may be coupled with its phosphorylation. However, such uptake-phosphorylation coupling does not seem to be held for thymidine. The datum shown in Table 2 indicates the increase in the ratio of thymidine to nucleotides with increasing concentration of added thymidine and suggests that the uptake of thymidine may not be limited by the intracellular phosphorylation capacity. In contrast to the apparent elevation of DISCUSSION thymidine kinase activity in sea urchin There are many papers reporting the eggs occurring soon after fertilization as increase in the capacity to uptake exter- assumed from the in uiuo results, the total nally supplied nucleosides into sea urchin thymidine kinase activity in egg extract or eggs soon after fertilization (Nemer, 1962, homogenate was found to be unchanged Piatigorsky and Whiteley, 1965, Pasternak, before and after fertilization a’s shown in 1973, Ord and Stocken, 1973). Ord and the present paper. Almost similar results Stocken (1973) also reported the increased are also presented by Suzuki and Mano tration, however, the phosphorylation capacity appears to become unable to catch up the increasing influx of thymidine. In order to investigate the fate of 3Hthymidine once taken up by sea urchin eggs, unfertilized and fertilized (10 min after insemination) eggs of Anthocidaris were first exposed to SH-thymidine (1 &i/ ml; 0.2 PM) for 5 min at 25”C, and then washed and cultured in the absence of thymidine for another 25-min period. At various points of time during the second incubation, aliquots of the suspensions were removed and assayed for the radioactivity distribution. The results summarized in Table 3 seem to indicate that the distribution of acid-soluble radioactivity among thymidine, P-aminoisobutyrate and phosphorylated thymidine derivatives (nucleotides) remains almost unchanged throughout the second incubation course.
330
DEVELOPMENTALBIOLOGY VOLIJME43, 19%
(1974) although thymidine kinase activity may fluctuate a bit depending on time after insemination. This puzzling discrepancy between the in uiuo and in vitro results on thymidine kinase activity has not yet been answered up to the present time. Similar discrepancy was also reported by Epel (1964) and Blomquist (1973) about nicotinamide adenine dinucleotide (NAD) kinase. Though the stimulation of NAD kinase after insemination was expected from the marked increase in NADP+ and NADPH content with a concomitant decrease in NAD+ occurring soon after insemination, no difference in the NAD kinase activity was detected between the homogenates of unfertilized and fertilized eggs. Blomquist (1973) discussed that the increase in NADP+ may not be the results of new synthesis of NAD kinase because of the rapidity of the enzyme response after fertilization. This seems also to be held for thymidine kinase. As shown in this paper thymidine kinase appears to be fully activated within the first 10 min after insemination. In this paper we have shown that thymidine kinase in unfertilized sea urchin eggs may be activated even at 0°C during or after homogenization of the eggs. This may account for the apparently similar levels of thymidine kinase activity in both fertilized and unfertilized egg homogenates. Activation of the enzyme after homogenization was more markedly and reproducibly observed with unfertilized Pseudocentrotus eggs as compared with the other ones. The enzyme in the eggs of latter species seems to be fully activated during homogenization, showing rather high enzyme activity even in the freshly prepared homogenates. Reasons for the apparent difference in the rate of homogenization-induced enzyme activation among species are not yet clear. The results presented in this paper seem to indicate that thymidine kinase of sea urchin eggs may be a SH-enzyme similarly
to that of mammalian tumors (Bresnick and Thompson, 1965, Hashimoto et al., 1972) or of bacteria (Durham and Ives, 1971). The enzyme activity was inhibited by PCMB or ferricyanide. GSH and DTT were effective in reversing the inhibition by PCMB or ferricyanide. When unfertilized eggs were homogenized with ferricyanide, the thymidine kinase activity of homogenate was very low even with Hemicentrotus or Anthocidaris eggs. Addition of GSH, DTT or even NADH to the ferricyanidehomogenate was found to activate the enzyme gradually within 30 min of incubation at 0°C. GSH and DTT were also effective in stimulating the thymidine kinase in the freshly prepared Pseudocentrotus egg homogenate as evidenced in this paper. These results suggest the possible involvement of some redox reaction in the mechanism of thymidine kinase activation by homogenization. Although added GSH, DTT and NADH are effective in activating thymidine kinase in the homogenates, the endogenous reductant which may actually be responsible for activating thymidine kinase in the eggs has not been clarified. PCMB at low concentrations (below 1.5 x 1O-5 M) added to freshly prepared homogenate of unfertilized Pseudocentrotus eggs, in which thymidine kinase may be only partially activated, did stimulate the enzyme activity. PCMB showed only an inhibitory effect when given to homogenates with fully activated enzyme such as freshly prepared Hemicentrotus or Anthocidaris egg homogenates. These results suggest that there may be a thymidine kinase inhibitor in the eggs, which may also require SH group(s) to interact with thymidine kinase and is more sensitive to PCMB than thymidine kinase itself. The final conclusion including other possibilities about the activation mechanism of thymidine kinase has to be waited until further experimental evidence will be accumulated. Details of the in vitro activation
NONAKA AND TERAYAMA
Actioation
mechanism for thymidine kinase in unfertilized eggs by homogenation as well as the problem how it may be related to the enzyme activation occurring in the eggs after fertilization are now under investigation. This study was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Japan. REFERENCES ADAMS, R. L. P. (1969). Phosphorylation of tritiated thymidine by L 929 mouse fibroblasts. Exp. Cell Res. 56, 49-54. BLOMQUIST, C. H. (1973). Partial purification and characterization of nicotinamide adenine dinucleotide kinase from sea urchin eggs. J. Biol. Chem. 248, 7044-7048. BOLLUM, F. J., and POTTER, V. R. (1959). Nucleic acid metabolism in regenerating rat liver. VI. Soluble enzymes which convert thymidine to thymidine phosphate and DNA. Cancer Res. 19, 561-565. BREITMAN, T. R. (1963). The feedback inhibition of thymidine kinase. Biochim. Biophys. Acta 67, 153-155. BRESNICK, E., and THOMPSON, U. B. (1965). Properties of deoxythymidine kinase partially purified from animal tumors. J. Biol. Chem. 240, 39673974. BRESNICK, E., and RAPP, F. (1969). Thymidine kinase activity in cells abortively and productively infected with human adenoviruses. Virology 34, 799802. DURHAM, J. P., and IVES, D. H. (1971). The metabolism of deoxyribonucleosides in Lactobacillus acidphilus; Regulation of deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine kinase activities by nucleotides. Biochim. Biophys. Acta 228, 9-25. EKER, P. (1965). Activities of thymidine kinase and thymine deoxyribonucleoside phosphate during growth of cells in tissue culture. J. Biol. Chem. 240, 2607-2611. EPEL, D. (1964). A primary metabolic change of fertilization; Interconversion of pyridine nucleotides. Biochem. Biophys. Res. Commun. 17, 62-68. HANSEN-DELKESKAMP, E., and DUSPIVA, F. (1965). Aktivititsverlauf der enzymatischen Phosphorylierung von Thymidin wahrend der Entwicklung des Seeigels Psammechinus miliaris von der Befrechtung bis zum Zweizeller. Experientia 22, 381382. HASHIMOTO, T., ARIMA, T., OKUDA, H., and FUJII, S. (1972). Purification and properties of deoxythymidine kinase from the Yoshida sarcoma. Cancer Res. 32, 67-73.
o/Egg Thymidine
Kinase
331
HINEGARDNER, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of sea urchin egg. Exp. Cell Res. 36, 53-61. LONCO, F. J., and PLUNKETT, W. (1973). The onset of DNA synthesis and its relation to morphogenetic events of the pronuclei in activated eggs of the sea urchin, Arbacia punctulata. Deuelop. Biol. 30, 56-67. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MAHNIN, D. T., and LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14 or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biothem. 16, 500-509. MALEY, G. F., LORENSON,M. G., and MALEY, F. (1965). Inhibitors of protein synthesis; Effect on the levels of deoxycytidylate deaminase, thymidylate synthetase and thymidine kinase in regenerating rat liver. Biochem. Biophys. Res. Commun. 18, 364-370. MASUI, H., and GARREN, L. D. (1971). On the mechanism of action of adrenocorticotropic hormone. The stimulation of thymidine kinase activity with altered properties and changed subcellular distribution. J. Biol. Chem. 246, 5407-5413. NAGANO, H., and MANO, Y. (1968). Thymidine kinase, thymidylate kinase and 32Pi and “C thymidine incorporation into DNA during early embryogenesis of the sea urchin. Biochim. Biophys. Acta 157, 546-557. NEMER, M. (1962). Characteristics of the utilization of nucleosides by embryos of Puracenfrotus liuidus. J. Biol. Chem. 237, 143-149. ORD, M. G., and STOCKEN, L. A. (1973). Thymidine uptake by Paracentrotus eggs during the first cell cycle after fertilization. Erp. Cell Res. 83, 411-414. PASTERNAK, C. A. (1973). Phospholipid synthesis in cleaving sea urchin eggs; Model for specific membrane assembly. Deuelop. Biol. 30, 403-410. PEGORARO, L., and BERNENGO, M. G. (1971). Thymidine kinase, deoxycytidine kinase and deoxycytidylate deaminase activities in phytohaemagglutinin-stimulated human lymphocytes. Exp. CelI Res. 68, 283-290. PIATKORSKY, J., and WHITELEY, A. H. (1965). A change in permeability and uptake of ‘“C uridine in response to fertilization in Strongylocentrotus purpuratus eggs. Biochim. Biophys. Acta 108, 404-418. SHIMIZC, H., DALY, J. W., and CREVELING, C. R. (1969). A radioisotopic method for measuring the formation of adneosine 3’,5’-cyclic monophosphate in incubated slices of brain. J. Neurochem. 16, 1609-1619.
332
DEVELOPMENTALBIOLOGY
STUBBLEFIELD, E., and MURPHREE, S. (1967). Synchronized mammalian cell cultures. II. Thymidine kinase activity in colcemid synchronized fibroblasts.
Exp. Cell Res. 48, 652-656. SUZUKI, N., and MANO, Y. (1974). Phosphorylation of deoxyribonucleosides and DNA synthesis in early cleaving embryos of the sea urchin. J. Biochem.
VOLUME 43, 1975
75, 1349-1362. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. Biochim. BioThe phosphorylation of thymidine. ph.~s. Acta 45, 101-110.
