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Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs
DEVELOPMENTAL
56, 68-75
BIOLOGY
Compartmentation Eggs and Possible
of Thymidine Kinase in Unfertilized Sea Urchin Release of Thymidine Kinase from Particulates in Activated Eggs
MASARU Zoological
(1977)
Institute,
Received
July
NONAKA
AND HIROSHI
Faculty
of Science,
22,1976;
accepted
University in revised
TERAYAMA of Tokyo, form
October
Tokyo,
Japan
4, 1976
The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 3040%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammoniaactivated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 15008 pellet or even with the 2OOg pellet. Exposure of the 15OOg pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 15OOg pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization. INTRODUCTION
The unfertilized sea urchin eggs showing no appreciable DNA synthesis become an active system for DNA synthesis after fertilization (Hinegardner et al., 1964; Longo and Plunkett, 1973) or artificial activation of various kinds (von LedeburVilliger, 1972; Mazia and Ruby, 1974; Steinhardt and Epel, 1974; Vacquier and Brandriff, 1975). However, the mechanism for the initiation of DNA synthesis occurring immediately after fertilization or artificial activation has not yet been fully understood. Thymidine kinase is known as one of the key enzymes responsible for DNA synthesis and its regulation in various biological systems, including regenerating liver (Weissman et al., 1960; Sakuma and Terayama, 1967), in which the increase of thymidine kinase at the time of cellular proliferation has clearly been demonstrated.
The increase in the uptake of external thymidine and its phosphorylation in ouum has been reported to occur soon after fertilization of sea urchin eggs (Ord and Stocken, 1973; Nonaka and Terayama, 1975). In spite of the in viuo evidence showing possible activation of thymidine kinase in sea urchin eggs soon after fertilization, the in vitro evidence to show the increase in thymidine kinase activity at this critical stage of development has been lacking. The thymidine kinase activity of unfertilized egg homogenates reported so far was usually similar to that of the corresponding fertilized egg homogenates (Nonaka and Terayama, 1975; Nagano and Mano, 1968; Hansen-Delkeskamp and Duspiva, 1965). However, it should be noted that the media used for homogenizing sea urchin eggs in these earlier studies were rather hypotonic for marine invertebrates like sea urchins.
68 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
NONAKA
AND
TERAYAMA
Thymidine
In the present study, we used 1 M NaCl in most of the experiments to homogenize Hemicentrotus eggs, and consequently, we found in vitro evidence to show that thymidine kinase may actually be sequestered in some intracellular structures (possibly membrane-associated vesicles) and remain latent in the unfertilized eggs until it is released or solubilized as the result of fertilization or ammonia activation. MATERIALS
AND
METHODS
Eggs of sea urchins, Hemicentrotus pulcherrimus, were spawned by 0.55 M KC1 and washed with filtered sea water. Eggs, unfertilized or fertilized, were homogenized with 4 or 9 vol of 1 M NaCl in a Teflon-glass homogenizer for about 20 to 40 strokes by hand, or in a motor-driven homogenizer (1100 rpm) for about 5 strokes. The absence of unbroken eggs or large debris in the homogenates was confirmed each time prior to the assay of thymidine kinase. Sonication of egg homogenates or suspensions of particulate fractions prepared from them was carried out in a Branson sonifier B-12 for 10 set at 0°C (about 50 W). Assay of thymidine kinase of egg homogenates or fractions prepared from them was carried out as described previously (Nonaka and Terayama, 1975), using the DEAE-cellulose paper disc method (Bollum and Potter, 1959; Breitman, 1963) except that the tonicity of the reaction mixture was maintained hypertonic (usually, the reaction mixture fortified with about 1 M NaCl was used). Differential centrifugation of egg homogenates in 1 M NaCl was carried out by centrifugation at 1500g and 15,OOOg, each for 20 min, or at 2OOg, 6OOg, and 15OOg, each for 10 min. The bottom pellet at each centrifugation was resuspended in 1 M NaCl of a volume equal to that of the original egg homogenate. The volume of the final supernatant fraction was also adjusted to the original homogenate volume by adding 1 M NaCl. Aliquots (100 ~1) of
Kinase
Compartmentation
69
these fractions were assayed for thymidine kinase before and after sonication. Fertilized egg homogenates were prepared as follows. After insemination by the conventional method, the eggs were resuspended in sea water and incubated at 20°C with gentle shaking. Thirty minutes after insemination, eggs were collected by manual centrifugation, homogenized in 1 M NaCl, and assayed for thymidine kinase. In some cases, insemination was carried out in sea water containing 12 mM leupeptin (an antibiotic with the antiprotease activity) (Ikezawa et al., 1971) to prevent the formation of fertilization membranes, and hence, to facilitate homogenization of the fertilized eggs in 1 M NaCl. Artificial activation of sea urchin eggs by ammonia was carried out according to the procedure of Mazia (1974). Unfertilized eggs were exposed to ammonia-sea water (pH 9.0) for 60 min at 2o”C, washed with sea water, and resuspended in it. Homogenization of eggs was carried out 20 to 30 min after returning the eggs to normal sea water. Protein was assayed by the method of Lowry et al. (1951) with bovine serum albumin as a standard. [2J4ClThymidine (61 mCi/mmole) was purchased from the Radiochemical Centre, Amersham, England. Leupeptin (N-acetyl and/or N-propionyl-L-leucyl-L-leucyl-marginal) was a gift from the Institute for Microbial Chemistry, Tokyo. RESULTS
Unfertilized Hemicentrotus eggs were homogenized with deionized water (DW) or 1 M NaCl in a Teflon-glass homogenizer for various strokes by hand, and the homogenates thus prepared were mixed with an equal volume of 2 M NaCl or 1 M NaCl so that the final NaCl concentration was always 1 M. As shown in Fig. 1, the thymidine kinase activity of the DW homogenate was always higher than that of the 1 M NaCl homogenate. The former reached a higher plateau level after 3 strokes,
70
DEVELOPMENTAL
BIOLOGY
while the latter reached a lower plateau level after 5 strokes. It should be noted that no unbroken eggs were detected in the homogenates of more than 5 strokes except some cellular debris of various sizes and large membraneous organelles. However, the amount of larger cell debris was observed to decrease upon increasing the number of strokes in homogenization be-
04 0
5 Homogenmtion
I
I
( strokes
I
*
IO
1
FIG. 1. Thymidine kinase activities of unfertilized Hemicentrotus egg homogenates prepared in deionized water or 1 M NaCl after various strokes. One milliliter of the packed eggs was suspended in 4 ml of DW or 1 M NaCl and homogenized at 0°C for various strokes by hand. The homogenate in DW or that in 1 M NaCl was mixed with an equal volume of 2 M NaCl or 1 M NaCl, respectively, and assayed for thymidine kinase as described under Materials and Methods. 0 and l indicate the DW and the 1 M NaCl homogenates, respectively.
VOLUME
yond 5, suggesting that no direct relationship exists between the microscopically observed degradation of cell debris and thymidine kinase activity. A 0.3-ml packed cell volume of unfertilized Hemicentrotus eggs was homogenized with 1.2 ml of DW or various concentration NaCl for 20 strokes until no more large cell debris became discernible. The homogenates were then mixed with 1.5 ml of NaCl solutions of calculated concentrations so that the final NaCl concentration was 1 M, and then they were assayed for thymidine kinase activities before and after sonication. As summarized in Table 1, the thymidine kinase activity of the DW homogenate remained high and unaltered before and after sonication, while a considerable increase (more than 50%) in the enzyme activity by sonication was observed with the 1 M NaCl homogenate (Expt 1 in Table 1). As the concentration of NaCl in the homogenizing medium was decreased, the thymidine kinase activity before sonication tended to increase and the difference in thymidine kinase activity before and after sonication was found to decrease (Expt 2 in Table 1). It should be noted that the enzyme activities of the sonicated homogenates always remained high and similar to each other without regard to the NaCl concentration in the homogenization medium. The 1 M NaCl homogenate of unfertilized Hemicentrotus eggs was subjected to differential centrifugation as described un-
TABLE EFFECT OF SONICATION HOMOGENATE Expt
1
ON THE THYMIDINE KINASE ACTIVITY IN DEIONIZED WATER AND VARIOUS
Homogenizing
medium
Specific
Deionized 1 M NaCl
water
2
0.25 M NaCl 0.5 M NaCl 1.0 M NaCl
n Each specific activity is the mean from figures in parentheses indicate the percentage
duplicate activity.
OF UNFERTILIZED CONCENTRATIONS
thymidine
Before 1
56. 1977
kinase
activity
sonication
Hemicentrotus OF NaCl (cpm/mg After
EGG of protein)”
sonication
2380 + 40 (103) 1530 k 0 (66)
2320 f 30 (100) 2400 + 70 (103)
2070 2 10 (91) 1820 ? 10 (80) 1390 k 30 (61)
2270 + 20 (100) 2260 + 20 (100) 2060 k 20 (91)
determinations
it average
deviation.
Numerical
NONAKA
AND TERAYAMA
Thymidine
der Materials and Methods. As shown in Table 2 (Expt 11, almost all of the enzyme seemed to be recovered in the 15,000g supernatant when the enzyme was assayed without sonication. However, when the enzyme was assayed after sonication, we found that the enzyme corresponding to more than 20% of the total activity was retained in the 15OOg pellet (but not in the 15,OOOg pellet) and the rest of it in the 15,OOOg supernatant. Here again, the enzyme activity of the homogenate was shown to increase by 60% after sonication. In the second experiment (Table 2, Expt 2), differential centrifugation of the unfertilized egg homogenate in 1 M NaCl was carried out in a more finely graded scale. Quite interestingly, the latent thymidine kinase which is activated by sonication was shown to be actually associated with the 2OOg lo-min pellet. In order to know the density of intracellular structure(s) which may contain latent thymidine kinase, the 1500g pellet prepared from the 1 M NaCl homogenate was resuspended in 0.4 M sucrose-O.8 M NaCl, layered over a discontinuous su-
Expt
OF BOUND AND FREE THYMIDINE Hemicentrotus EGG HOMOGENATE
Thymidine Net
Homogenate 15OOg 20-min pellet 15,OOOg 20-min pellet 15,000g 20-min supernatant
2
Homogenate 200s lo-min pellet SOOg lo-min pellet 15OOg IO-min pellet 15OOg lo-min supernatant
2
KINASE AMONG FRACTIONS SEPARATED FROM THE UNFERTILIZED IN 1 M NaCl BY DIFFERENTIAL CENTRIFUGATION
Fractions
1
71
Compartmentation
crose density gradient (from the bottom to the top, 2.0 M sucrose-O M NaCl, 1.2 M sucrose-O.4 M NaCl, 1.0 M sucrose-O.5 M NaCl, 0.8 M sucrose-O.6 M NaCl, and 0.6 M sucrose-O.7 M NaCl, 4 ml for each layer), and centrifuged at 57,700g for 1 hr in a Spinco SW 25-l rotor. A pellet fraction at each interface was once washed with 1 M NaCl and assayed for thymidine kinase before and after sonication. The results (data not presented) showed that the latent thymidine kinase was most concentrated at the interface between 0.6 M sucrose-0.7 M NaCl and 0.8 M sucrose-O.6 M NaCl layers, suggesting that the density of the intracellular structure containing the latent thymidine kinase may be 1.12 to 1.13. In the following experiments, the comparison of latent thymidine kinase was made between the unfertilized and fertilized Hemicentrotus egg homogenates in 1 M NaCl. Unfertilized eggs were divided into two equal portions. Eggs in one portion were inseminated and those in another were not. Thirty minutes after insemination, the eggs in each group were
TABLE DISTRIBUTION
Kinase
activity
kinase
(cpm/50
~1)~
Without sonication
With
sonication
485 34.9 4.5 522
2 L k +
78 4.7 1.6 18
781 167 1.7 464
780 50 14 25 627
? k * k +
5 10 1 2 6
Protein (%I Specific activityb (cpm/mg of protein)
t + 2 k
115(100) 7 (21.3) 1.4 (0.2) 15 (59.4)
2120 1150 44.7 2410
100 42.1 11.7 54.3
1160 ” 235 k 20 + 4+-4 644 2
20 (100) 2 (20.3) 2 (1.7) (0.3) 35 (55.5)
2360 2010 440 200 2360
100 24.1 9.8 5.7 54.8
u The volume of each fraction in 1 M NaCl was made equal to that of the original homogenate aliquots were assayed for thymidine kinase before and after sonication. Values are means from determinations ? average deviations. Numerical figures in parentheses indicate the percentage * Specific activities after sonication.
and 50-~1 duplicate activity.
72
DEVELOPMENTAL
BIOLOGY
homogenized with 1 M NaCl in a PotterElvehjem homogenizer for about 40 strokes by hand because of the increased difficulty in homogenizing fertilized eggs in 1 A4 NaCl, probably due to the fertilization membranes (Expt 1). In order to prevent the formation of fertilization membranes, 12 m&f leupeptin was added at the time of insemination in the second experiment (Expt 2). In this case, homogenization of eggs (fertilized and unfertilized) was carried out for 20 strokes. In the third and fourth experiments, the eggs were activated by ammonia as described under Materials and Methods and the activated eggs and their controls were homogenized with 1 M NaCl for 20 strokes 20 min (Expt 3) or 30 min (Expt 4) after return to normal sea water. Thymidine kinase of each homogenate was assayed before and after sonication. As summarized in Fig. 2, the thymidine kinase activities of 1 M NaCl homogenates of unfertilized (or control) eggs were elevated markedly (more than 50%) by sonication throughout these experiments (Expts 1-4). On the other hand, the enzyme activities of the 1 it4 NaCl homogenates of the eggs inseminated in the absence of leupeptin (Expt 1) or in the presence of it (Expt 2>, or of the eggs activated by ammonia (Expts 3 and 4) were altered little by sonication, showing activities similar to those of the sonicated homogenates of the corresponding unfertilized or nonactivated controls. These results seem to suggest that the latent thymidine kinase which can be activated by sonication may not be present any longer after fertilization or ammonia activation of unfertilized eggs. The 1500g pellet isolated from the 1 M NaCl homogenates of fertilized eggs (Hemicentrotus) at 30 min after insemination showed only a trace of thymidine kinase activity, similar to the corresponding pellet from the unfertilized egg homogenate. However, sonication of the 1500g pellet from the fertilized egg homogenate did not
VOLUME
56, 1977
FIG. 2. Effect of sonication on the thymidine kinase activity of 1 M NaCl homogenates of unfertilized or activated Hemicentrotus eggs. One milliliter of packed eggs (unfertilized eggs and eggs activated as will be described later) were homogenized with 4 ml of 1 M NaCl for 20 strokes in Expt 2,3 and 4 and for 40 strokes in Expt 1. The homogenates were assayed for thymidine kinase before and after soni:ation as described under Materials and Methods. Different batches of eggs were used among the experiments, but the same batch of eggs was used in me experimental pair (“unfertilized” and “activated”). Expt 1: Eggs were inseminated in normal sea water (95% of the eggs elevated the fertilization membranes). Thirty minutes after insemination, the eggs (inseminated and unfertilized) were homogenized and thymidine kinase was assayed. Expt 3: Eggs were inseminated in sea water containing 12 mA4 leupeptin to prevent the elevation of fertilization membranes (the first cell division was observed In about 80% eggs). Thirty minutes insemination, :ggs were homogenized and thymidine kinase was assayed. Expt 3: Eggs were exposed to ammonia-sea sater (pH 9.0) at 20°C for 60 min and then returned ;o normal sea water (elevation of fertilization memcranes was not observed, but condensed chromosomes appeared in about 90% eggs). Twenty minutes after return to normal sea water, eggs were homogenized and thymidine kinase was assayed. Expt 4: The procedure was the same as in Expt 3 except that ?ggs were homogenized 30 min after return to nornal sea water (condensed chromosomes were observed in about 80% eggs). Solid bars correspond to ;hymidine kinase before sonication, while blank ,ars correspond to thymidine kinase after sonication Imeans from duplicate determinations 5 average leviations).
NONAKA
AND TERAYAMA
Thymidine
DISCUSSION
In the present study we have shown evidence that thymidine kinase in unfertilized sea urchin eggs (Hemicentrotus) may
TABLE OF THYMIDINE
KINASE
Medium
a Means
from
duplicate
3
FROM THE 15OOg PELLET Thymidine Without
1 M NaCl 1 M NaCl-0.2% Triton lMKC1 2 M sucrose 2 M glycerol 2 M propyleneglycol
X-100
determinations
182 2390 245 312 730 3830 + average
73
Compartmentation
gesting that the compartmentation may be destabilized by the lipophilic reagents. The variation of thymidine kinase activities observed among the sonicated incubates in Table 3 has been confirmed to be mainly due to the differnece in the effects of suspension media upon the enzyme activity. In the preliminary experiments searching for the factors possibly controlling the release of thymidine kinase from the particulates in ovum, effects of pH, redox reagents such as ferricyanide or dithiothreitol, Ca2+ ions, Ca-ionophore (A-23187), ATP, cyclic nucleotides such as CAMP or cGMP, ATP, thymidine, and phospholipases on the release of the latent thymidine kinase from the 1500g pellet suspended in 1 M NaCl or 0.5 M NaCl-1 M glycerol were investigated. So far, no remarkable effects of these reagents have been detected except in the cases of phospholipase C and snake venom (Crotalus atrox), where the accelerated release of thymidine kinase was clearly demonstrated (data not presented). Biological mechanisms for the release of latent thymidine kinase at fertilization as well as further characterization of the intracellular structure localizing the latent thymidine kinase are now under investigation in our laboratory.
cause enzyme activation at all in contrast to the about fivefold elevation of thymidine kinase activity of the 1500g pellet from the unfertilized egg homogenate. When the 1500g pellet was suspended in 1 M NaCl, sonicated, and then centrifuged at 1500g for 10 min, 80 to 90% of the thymidine kinase activity was recovered in the supernatant fraction, leaving only a small portion of the enzyme activity in the second 1500g pellet. Similarly, recentrifugation of the 1500g pellet suspended in DW resulted in a more than 90% recovery of thymidine kinase activity in the supernatant (data not presented). These findings seem to indicate that thymidine kinase bound to the 1500g pellet (particulates) is actually released or solubilized by exposure to sonication or DW. In order to get a clue to the mechanisms for the release of thymidine kinase from its sequestered loci in the eggs, the 1500g pellet prepared from 1 M NaCl homogenates of unfertilized eggs (Hemicentrotus) was incubated at 0°C for 30 min in various media such as 1 M NaCI, 1 M KCl, 1 M NaCl containing 0.2% Triton X-100, 2 M sucrose, 2 M glycerol, and 2 M propyleneglycol, and then thymidine kinase was assayed before and after sonication. As summarized in Table 3, thymidine kinase in the particulates seemed rather well retained in the hypertonic media such as 1 M NaCl, 1 M KCl, 2 M sucrose, and 2 M glycerol, while the enzyme seemed to be released completely in 1 M NaCl-0.2% Triton X-100 and 2 M propyleneglycol, sugRELEASE
Kinnse
IN VARIOUS
kinase activity protein)” sonication + k k k ? +
10 140 45 15 30 30
deviations.
MEDIA
(cpm/mg
With
(PC, of
30 min) Py--p.ts”epe
sonication
1020 2260 1220 1280 2420 3270
+ r * + k r
10 10 20 150 30 40
17.8 106 20.2 24.4 30.3 117
74
DEVELOPMENTAL
BIOLOGY
be at least partly sequestered in some intracellular structure(s) and can be isolated by centrifuging the 1 M NaCl homogenate of unfertilized eggs at low speeds (2OOg or 15OOg). The compartmentation of the latent thymidine kinase in the particulates seemed to be well maintained in various hypertonic media such as 1 M NaCl, 1 M KCl, 2 M sucrose, and 2 A4 glycerol. However, exposure of the particulates to hypotonic media or sonication resulted in immediate release of the latent enzyme into the media accompanied by an appreciable increase in the thymidine kinase activity. Furthermore, evidence seems to have been provided in the present study that thymidine kinase in the fertilized or ammonia-activated eggs may no longer be sequestered in the particulates, but may be released or solubilized in ovum. The finding that the thymidine kinase activity of the 15OOg pellet prepared from the fertilized egg homogenates in 1 M NaCl remains very low even when assayed after sonication may not only support the above hypothesis (together with the fact that the thymidine kinase activity of the homogenate of fertilized eggs is not affected by sonication), but also may eliminate the possibility for the contamination of unbroken eggs in the homogenates. In fact, we have examined every homogenate under a microscope prior to experiments to confirm that the homogenates do not contain any unbroken eggs or larger cell debris. It has been confirmed that about 30 to 50% of the total thymidine kinase in the unfertilized Hemicentrotus eggs can be recovered in the particulate fraction as the latent form under the present experimental conditions. However, the question of whether or not an almost complete recovery of thymidine kinase in the particulate fraction may be achieved if the homogenization conditions for the unfertilized eggs are improved further, still remains. Moreover, the recovery of thymidine kinase in the pellet fraction seemed to depend not only on the homogenization conditions but
VOLUME
56, 1977
also on the sea urchin species. In fact, we have found that the homogenates of unfertilized Anthocidaris crassispina eggs in 1 M NaCl show almost similar thymidine kinase levels before and after sonication. However, if 0.5 M NaCl-1 M glycerol was used as the homogenizing medium, about 40% of the total thymidine kinase was recovered in the 1500g pellet as the latent form. In this connection, it seems important to study on the homogenates of Pseudocentrotus eggs because we have noticed that the 0.25 M sucrose (hypotonic) homogenates of unfertilized Pseudocentrotus eggs often show the gradual elevation of thymidine kinase activity upon standing in the freshly prepared homogenates at 0°C for 30 min or so (Nonaka and Terayama, 1975). The 1500g pellet prepared from 1 M NaCl homogenates of unfertilized Hemicentrotus eggs usually showed a three- to fivefold increase in the specific activity. Larger extents of enzyme activation by sonication may be expected if the wellwashed pellet fractions are used. In fact, an elevenfold increase in the activity was observed for the pellet at the interface between the d = 1.12 and 1.13 sucrose layers in density gradient centrifugation. The fact that the specific activity of the sonicated pellet (15OOg pellet or 200g pellet in Table 2) was not greater than the specific activity of the sonicated whole egg homogenate may suggest that the pellet may contain as much extra-proteins other than thymidine kinase as the whole egg homogenate, at least at this stage of particulate fractionation. The latent thymidine kinase seems to be associated with quite large organelles. The densities of the organelles containing most of the latent thymidine kinase was about 1.12 to 1.13, as measured by sedimentation analysis in a sucrose density gradient. This density value seems to be somewhat smaller than the density (d = 1.16-1.18) of ordinary plasma membranes isolated from mammalian cells. Since the release of the enzyme is facili-
NONAKA
AND TERAYAMA
Th> fmidine
tated by exposure not only to sonication or hypotonic media, but also to lipophilic reagents (Triton X-100 and propyleneglycol) or to phospholipases (data not presented), the latent thymidine kinase seems be bound to or sequestered in the membranebound vesicles, although this must be substantiated by further experiments. REFERENCES BOLLUM, F. J., and POTTER, V. R. (1959). Nucleic acid metabolism in regenerating rat liver. IV. Soluble enzymes which convert thymidine to thymidine phosphate and DNA. Cancer Res. 19, 561565. BREITMAN, T. R. (1963). The feedback inhibition of thymidine kinase. Biochim. Biophys. Actu 67, 153-155. HANSEN-DELKESKAMP, E., and DUSPIVA, F. (1965). Aktivitatsverlauf der enzymatischen Phosphorylierung von Thymidin wlhrend der Entwicklung des Seeigels Psammechinus miliaris von der Befrechtung bis zum Zweizeller. Ezperientia 22,381382. HINEGARDNER, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res. 36, 53-61. IKEZAWA, H., AOYAGI, T., TAKEUCHI, T., and UMEZAWA, H. (19’71). Effect of protease inhibitors of Actinomycetes on lysosomal peptide-hydrolases from swine liver. J. Antibiotics Tokyo Ser. A 24, 488-490. LONGO, 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, Arbaciapunctulata. Develop. Biol. 30, 5667. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and
Kinase
Compartmentation
75
RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265275. MAZIA, D. (1974). Chromosome cycles turned on in unfertilized sea urchin eggs exposed to NH,OH. Proc. Nat. Acad. Sci. USA 71, 690-693. MAZIA, D., and RUBY, A. (1974). DNA synthesis turned on in unfertilized sea urchin eggs by treatment with NH,OH. Ezp. Cell Res. 85, 167-172. NAGANO, H., and MANO, Y. (1968). Thymidine kinase, thymidylate kinase and 32Pi and 14C thymidine incorporation into DNA during early embryogenesis of the sea urchin. Biochim. Biophys. Actu 157, 546-557. NONAKA, M., and TERAYAMA, H. (1975). Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization. Develop. Biol. 43, 322-332. ORD, M. G., and STOCKEN, L. A. (1973). Thymidine uptake by Purucentrotus eggs during the first cell cycle after fertilization. Exp. Cell Res. 83,411-414. SAKUMA, K., and TERAYAMA, H. (1967). Effects of adrenal hormone upon DNA synthesis in regenerating rat liver and tumors. J. Biochem. 61, 504511. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nut. Acad. Sci. USA 71, 1915-1919. VACQUIER, V. C., and BRANDRIFF, B. (1975). DNA synthesis in unfertilized sea urchin eggs can be turned on and turned off by the addition and removal of procaine hydrochloride. Develop. Biol. 47,12-31. VON LEDEBUR-VILLIGER, M. (1972). Cytology and nucleic acid synthesis of parthenogenetically activated sea urchin eggs. Exp. Cell Res. 72, 285-308. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine. Biochim. Biophys. Acta 45, 101-110.
56, 68-75
BIOLOGY
Compartmentation Eggs and Possible
of Thymidine Kinase in Unfertilized Sea Urchin Release of Thymidine Kinase from Particulates in Activated Eggs
MASARU Zoological
(1977)
Institute,
Received
July
NONAKA
AND HIROSHI
Faculty
of Science,
22,1976;
accepted
University in revised
TERAYAMA of Tokyo, form
October
Tokyo,
Japan
4, 1976
The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 3040%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammoniaactivated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 15008 pellet or even with the 2OOg pellet. Exposure of the 15OOg pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 15OOg pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization. INTRODUCTION
The unfertilized sea urchin eggs showing no appreciable DNA synthesis become an active system for DNA synthesis after fertilization (Hinegardner et al., 1964; Longo and Plunkett, 1973) or artificial activation of various kinds (von LedeburVilliger, 1972; Mazia and Ruby, 1974; Steinhardt and Epel, 1974; Vacquier and Brandriff, 1975). However, the mechanism for the initiation of DNA synthesis occurring immediately after fertilization or artificial activation has not yet been fully understood. Thymidine kinase is known as one of the key enzymes responsible for DNA synthesis and its regulation in various biological systems, including regenerating liver (Weissman et al., 1960; Sakuma and Terayama, 1967), in which the increase of thymidine kinase at the time of cellular proliferation has clearly been demonstrated.
The increase in the uptake of external thymidine and its phosphorylation in ouum has been reported to occur soon after fertilization of sea urchin eggs (Ord and Stocken, 1973; Nonaka and Terayama, 1975). In spite of the in viuo evidence showing possible activation of thymidine kinase in sea urchin eggs soon after fertilization, the in vitro evidence to show the increase in thymidine kinase activity at this critical stage of development has been lacking. The thymidine kinase activity of unfertilized egg homogenates reported so far was usually similar to that of the corresponding fertilized egg homogenates (Nonaka and Terayama, 1975; Nagano and Mano, 1968; Hansen-Delkeskamp and Duspiva, 1965). However, it should be noted that the media used for homogenizing sea urchin eggs in these earlier studies were rather hypotonic for marine invertebrates like sea urchins.
68 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
NONAKA
AND
TERAYAMA
Thymidine
In the present study, we used 1 M NaCl in most of the experiments to homogenize Hemicentrotus eggs, and consequently, we found in vitro evidence to show that thymidine kinase may actually be sequestered in some intracellular structures (possibly membrane-associated vesicles) and remain latent in the unfertilized eggs until it is released or solubilized as the result of fertilization or ammonia activation. MATERIALS
AND
METHODS
Eggs of sea urchins, Hemicentrotus pulcherrimus, were spawned by 0.55 M KC1 and washed with filtered sea water. Eggs, unfertilized or fertilized, were homogenized with 4 or 9 vol of 1 M NaCl in a Teflon-glass homogenizer for about 20 to 40 strokes by hand, or in a motor-driven homogenizer (1100 rpm) for about 5 strokes. The absence of unbroken eggs or large debris in the homogenates was confirmed each time prior to the assay of thymidine kinase. Sonication of egg homogenates or suspensions of particulate fractions prepared from them was carried out in a Branson sonifier B-12 for 10 set at 0°C (about 50 W). Assay of thymidine kinase of egg homogenates or fractions prepared from them was carried out as described previously (Nonaka and Terayama, 1975), using the DEAE-cellulose paper disc method (Bollum and Potter, 1959; Breitman, 1963) except that the tonicity of the reaction mixture was maintained hypertonic (usually, the reaction mixture fortified with about 1 M NaCl was used). Differential centrifugation of egg homogenates in 1 M NaCl was carried out by centrifugation at 1500g and 15,OOOg, each for 20 min, or at 2OOg, 6OOg, and 15OOg, each for 10 min. The bottom pellet at each centrifugation was resuspended in 1 M NaCl of a volume equal to that of the original egg homogenate. The volume of the final supernatant fraction was also adjusted to the original homogenate volume by adding 1 M NaCl. Aliquots (100 ~1) of
Kinase
Compartmentation
69
these fractions were assayed for thymidine kinase before and after sonication. Fertilized egg homogenates were prepared as follows. After insemination by the conventional method, the eggs were resuspended in sea water and incubated at 20°C with gentle shaking. Thirty minutes after insemination, eggs were collected by manual centrifugation, homogenized in 1 M NaCl, and assayed for thymidine kinase. In some cases, insemination was carried out in sea water containing 12 mM leupeptin (an antibiotic with the antiprotease activity) (Ikezawa et al., 1971) to prevent the formation of fertilization membranes, and hence, to facilitate homogenization of the fertilized eggs in 1 M NaCl. Artificial activation of sea urchin eggs by ammonia was carried out according to the procedure of Mazia (1974). Unfertilized eggs were exposed to ammonia-sea water (pH 9.0) for 60 min at 2o”C, washed with sea water, and resuspended in it. Homogenization of eggs was carried out 20 to 30 min after returning the eggs to normal sea water. Protein was assayed by the method of Lowry et al. (1951) with bovine serum albumin as a standard. [2J4ClThymidine (61 mCi/mmole) was purchased from the Radiochemical Centre, Amersham, England. Leupeptin (N-acetyl and/or N-propionyl-L-leucyl-L-leucyl-marginal) was a gift from the Institute for Microbial Chemistry, Tokyo. RESULTS
Unfertilized Hemicentrotus eggs were homogenized with deionized water (DW) or 1 M NaCl in a Teflon-glass homogenizer for various strokes by hand, and the homogenates thus prepared were mixed with an equal volume of 2 M NaCl or 1 M NaCl so that the final NaCl concentration was always 1 M. As shown in Fig. 1, the thymidine kinase activity of the DW homogenate was always higher than that of the 1 M NaCl homogenate. The former reached a higher plateau level after 3 strokes,
70
DEVELOPMENTAL
BIOLOGY
while the latter reached a lower plateau level after 5 strokes. It should be noted that no unbroken eggs were detected in the homogenates of more than 5 strokes except some cellular debris of various sizes and large membraneous organelles. However, the amount of larger cell debris was observed to decrease upon increasing the number of strokes in homogenization be-
04 0
5 Homogenmtion
I
I
( strokes
I
*
IO
1
FIG. 1. Thymidine kinase activities of unfertilized Hemicentrotus egg homogenates prepared in deionized water or 1 M NaCl after various strokes. One milliliter of the packed eggs was suspended in 4 ml of DW or 1 M NaCl and homogenized at 0°C for various strokes by hand. The homogenate in DW or that in 1 M NaCl was mixed with an equal volume of 2 M NaCl or 1 M NaCl, respectively, and assayed for thymidine kinase as described under Materials and Methods. 0 and l indicate the DW and the 1 M NaCl homogenates, respectively.
VOLUME
yond 5, suggesting that no direct relationship exists between the microscopically observed degradation of cell debris and thymidine kinase activity. A 0.3-ml packed cell volume of unfertilized Hemicentrotus eggs was homogenized with 1.2 ml of DW or various concentration NaCl for 20 strokes until no more large cell debris became discernible. The homogenates were then mixed with 1.5 ml of NaCl solutions of calculated concentrations so that the final NaCl concentration was 1 M, and then they were assayed for thymidine kinase activities before and after sonication. As summarized in Table 1, the thymidine kinase activity of the DW homogenate remained high and unaltered before and after sonication, while a considerable increase (more than 50%) in the enzyme activity by sonication was observed with the 1 M NaCl homogenate (Expt 1 in Table 1). As the concentration of NaCl in the homogenizing medium was decreased, the thymidine kinase activity before sonication tended to increase and the difference in thymidine kinase activity before and after sonication was found to decrease (Expt 2 in Table 1). It should be noted that the enzyme activities of the sonicated homogenates always remained high and similar to each other without regard to the NaCl concentration in the homogenization medium. The 1 M NaCl homogenate of unfertilized Hemicentrotus eggs was subjected to differential centrifugation as described un-
TABLE EFFECT OF SONICATION HOMOGENATE Expt
1
ON THE THYMIDINE KINASE ACTIVITY IN DEIONIZED WATER AND VARIOUS
Homogenizing
medium
Specific
Deionized 1 M NaCl
water
2
0.25 M NaCl 0.5 M NaCl 1.0 M NaCl
n Each specific activity is the mean from figures in parentheses indicate the percentage
duplicate activity.
OF UNFERTILIZED CONCENTRATIONS
thymidine
Before 1
56. 1977
kinase
activity
sonication
Hemicentrotus OF NaCl (cpm/mg After
EGG of protein)”
sonication
2380 + 40 (103) 1530 k 0 (66)
2320 f 30 (100) 2400 + 70 (103)
2070 2 10 (91) 1820 ? 10 (80) 1390 k 30 (61)
2270 + 20 (100) 2260 + 20 (100) 2060 k 20 (91)
determinations
it average
deviation.
Numerical
NONAKA
AND TERAYAMA
Thymidine
der Materials and Methods. As shown in Table 2 (Expt 11, almost all of the enzyme seemed to be recovered in the 15,000g supernatant when the enzyme was assayed without sonication. However, when the enzyme was assayed after sonication, we found that the enzyme corresponding to more than 20% of the total activity was retained in the 15OOg pellet (but not in the 15,OOOg pellet) and the rest of it in the 15,OOOg supernatant. Here again, the enzyme activity of the homogenate was shown to increase by 60% after sonication. In the second experiment (Table 2, Expt 2), differential centrifugation of the unfertilized egg homogenate in 1 M NaCl was carried out in a more finely graded scale. Quite interestingly, the latent thymidine kinase which is activated by sonication was shown to be actually associated with the 2OOg lo-min pellet. In order to know the density of intracellular structure(s) which may contain latent thymidine kinase, the 1500g pellet prepared from the 1 M NaCl homogenate was resuspended in 0.4 M sucrose-O.8 M NaCl, layered over a discontinuous su-
Expt
OF BOUND AND FREE THYMIDINE Hemicentrotus EGG HOMOGENATE
Thymidine Net
Homogenate 15OOg 20-min pellet 15,OOOg 20-min pellet 15,000g 20-min supernatant
2
Homogenate 200s lo-min pellet SOOg lo-min pellet 15OOg IO-min pellet 15OOg lo-min supernatant
2
KINASE AMONG FRACTIONS SEPARATED FROM THE UNFERTILIZED IN 1 M NaCl BY DIFFERENTIAL CENTRIFUGATION
Fractions
1
71
Compartmentation
crose density gradient (from the bottom to the top, 2.0 M sucrose-O M NaCl, 1.2 M sucrose-O.4 M NaCl, 1.0 M sucrose-O.5 M NaCl, 0.8 M sucrose-O.6 M NaCl, and 0.6 M sucrose-O.7 M NaCl, 4 ml for each layer), and centrifuged at 57,700g for 1 hr in a Spinco SW 25-l rotor. A pellet fraction at each interface was once washed with 1 M NaCl and assayed for thymidine kinase before and after sonication. The results (data not presented) showed that the latent thymidine kinase was most concentrated at the interface between 0.6 M sucrose-0.7 M NaCl and 0.8 M sucrose-O.6 M NaCl layers, suggesting that the density of the intracellular structure containing the latent thymidine kinase may be 1.12 to 1.13. In the following experiments, the comparison of latent thymidine kinase was made between the unfertilized and fertilized Hemicentrotus egg homogenates in 1 M NaCl. Unfertilized eggs were divided into two equal portions. Eggs in one portion were inseminated and those in another were not. Thirty minutes after insemination, the eggs in each group were
TABLE DISTRIBUTION
Kinase
activity
kinase
(cpm/50
~1)~
Without sonication
With
sonication
485 34.9 4.5 522
2 L k +
78 4.7 1.6 18
781 167 1.7 464
780 50 14 25 627
? k * k +
5 10 1 2 6
Protein (%I Specific activityb (cpm/mg of protein)
t + 2 k
115(100) 7 (21.3) 1.4 (0.2) 15 (59.4)
2120 1150 44.7 2410
100 42.1 11.7 54.3
1160 ” 235 k 20 + 4+-4 644 2
20 (100) 2 (20.3) 2 (1.7) (0.3) 35 (55.5)
2360 2010 440 200 2360
100 24.1 9.8 5.7 54.8
u The volume of each fraction in 1 M NaCl was made equal to that of the original homogenate aliquots were assayed for thymidine kinase before and after sonication. Values are means from determinations ? average deviations. Numerical figures in parentheses indicate the percentage * Specific activities after sonication.
and 50-~1 duplicate activity.
72
DEVELOPMENTAL
BIOLOGY
homogenized with 1 M NaCl in a PotterElvehjem homogenizer for about 40 strokes by hand because of the increased difficulty in homogenizing fertilized eggs in 1 A4 NaCl, probably due to the fertilization membranes (Expt 1). In order to prevent the formation of fertilization membranes, 12 m&f leupeptin was added at the time of insemination in the second experiment (Expt 2). In this case, homogenization of eggs (fertilized and unfertilized) was carried out for 20 strokes. In the third and fourth experiments, the eggs were activated by ammonia as described under Materials and Methods and the activated eggs and their controls were homogenized with 1 M NaCl for 20 strokes 20 min (Expt 3) or 30 min (Expt 4) after return to normal sea water. Thymidine kinase of each homogenate was assayed before and after sonication. As summarized in Fig. 2, the thymidine kinase activities of 1 M NaCl homogenates of unfertilized (or control) eggs were elevated markedly (more than 50%) by sonication throughout these experiments (Expts 1-4). On the other hand, the enzyme activities of the 1 it4 NaCl homogenates of the eggs inseminated in the absence of leupeptin (Expt 1) or in the presence of it (Expt 2>, or of the eggs activated by ammonia (Expts 3 and 4) were altered little by sonication, showing activities similar to those of the sonicated homogenates of the corresponding unfertilized or nonactivated controls. These results seem to suggest that the latent thymidine kinase which can be activated by sonication may not be present any longer after fertilization or ammonia activation of unfertilized eggs. The 1500g pellet isolated from the 1 M NaCl homogenates of fertilized eggs (Hemicentrotus) at 30 min after insemination showed only a trace of thymidine kinase activity, similar to the corresponding pellet from the unfertilized egg homogenate. However, sonication of the 1500g pellet from the fertilized egg homogenate did not
VOLUME
56, 1977
FIG. 2. Effect of sonication on the thymidine kinase activity of 1 M NaCl homogenates of unfertilized or activated Hemicentrotus eggs. One milliliter of packed eggs (unfertilized eggs and eggs activated as will be described later) were homogenized with 4 ml of 1 M NaCl for 20 strokes in Expt 2,3 and 4 and for 40 strokes in Expt 1. The homogenates were assayed for thymidine kinase before and after soni:ation as described under Materials and Methods. Different batches of eggs were used among the experiments, but the same batch of eggs was used in me experimental pair (“unfertilized” and “activated”). Expt 1: Eggs were inseminated in normal sea water (95% of the eggs elevated the fertilization membranes). Thirty minutes after insemination, the eggs (inseminated and unfertilized) were homogenized and thymidine kinase was assayed. Expt 3: Eggs were inseminated in sea water containing 12 mA4 leupeptin to prevent the elevation of fertilization membranes (the first cell division was observed In about 80% eggs). Thirty minutes insemination, :ggs were homogenized and thymidine kinase was assayed. Expt 3: Eggs were exposed to ammonia-sea sater (pH 9.0) at 20°C for 60 min and then returned ;o normal sea water (elevation of fertilization memcranes was not observed, but condensed chromosomes appeared in about 90% eggs). Twenty minutes after return to normal sea water, eggs were homogenized and thymidine kinase was assayed. Expt 4: The procedure was the same as in Expt 3 except that ?ggs were homogenized 30 min after return to nornal sea water (condensed chromosomes were observed in about 80% eggs). Solid bars correspond to ;hymidine kinase before sonication, while blank ,ars correspond to thymidine kinase after sonication Imeans from duplicate determinations 5 average leviations).
NONAKA
AND TERAYAMA
Thymidine
DISCUSSION
In the present study we have shown evidence that thymidine kinase in unfertilized sea urchin eggs (Hemicentrotus) may
TABLE OF THYMIDINE
KINASE
Medium
a Means
from
duplicate
3
FROM THE 15OOg PELLET Thymidine Without
1 M NaCl 1 M NaCl-0.2% Triton lMKC1 2 M sucrose 2 M glycerol 2 M propyleneglycol
X-100
determinations
182 2390 245 312 730 3830 + average
73
Compartmentation
gesting that the compartmentation may be destabilized by the lipophilic reagents. The variation of thymidine kinase activities observed among the sonicated incubates in Table 3 has been confirmed to be mainly due to the differnece in the effects of suspension media upon the enzyme activity. In the preliminary experiments searching for the factors possibly controlling the release of thymidine kinase from the particulates in ovum, effects of pH, redox reagents such as ferricyanide or dithiothreitol, Ca2+ ions, Ca-ionophore (A-23187), ATP, cyclic nucleotides such as CAMP or cGMP, ATP, thymidine, and phospholipases on the release of the latent thymidine kinase from the 1500g pellet suspended in 1 M NaCl or 0.5 M NaCl-1 M glycerol were investigated. So far, no remarkable effects of these reagents have been detected except in the cases of phospholipase C and snake venom (Crotalus atrox), where the accelerated release of thymidine kinase was clearly demonstrated (data not presented). Biological mechanisms for the release of latent thymidine kinase at fertilization as well as further characterization of the intracellular structure localizing the latent thymidine kinase are now under investigation in our laboratory.
cause enzyme activation at all in contrast to the about fivefold elevation of thymidine kinase activity of the 1500g pellet from the unfertilized egg homogenate. When the 1500g pellet was suspended in 1 M NaCl, sonicated, and then centrifuged at 1500g for 10 min, 80 to 90% of the thymidine kinase activity was recovered in the supernatant fraction, leaving only a small portion of the enzyme activity in the second 1500g pellet. Similarly, recentrifugation of the 1500g pellet suspended in DW resulted in a more than 90% recovery of thymidine kinase activity in the supernatant (data not presented). These findings seem to indicate that thymidine kinase bound to the 1500g pellet (particulates) is actually released or solubilized by exposure to sonication or DW. In order to get a clue to the mechanisms for the release of thymidine kinase from its sequestered loci in the eggs, the 1500g pellet prepared from 1 M NaCl homogenates of unfertilized eggs (Hemicentrotus) was incubated at 0°C for 30 min in various media such as 1 M NaCI, 1 M KCl, 1 M NaCl containing 0.2% Triton X-100, 2 M sucrose, 2 M glycerol, and 2 M propyleneglycol, and then thymidine kinase was assayed before and after sonication. As summarized in Table 3, thymidine kinase in the particulates seemed rather well retained in the hypertonic media such as 1 M NaCl, 1 M KCl, 2 M sucrose, and 2 M glycerol, while the enzyme seemed to be released completely in 1 M NaCl-0.2% Triton X-100 and 2 M propyleneglycol, sugRELEASE
Kinnse
IN VARIOUS
kinase activity protein)” sonication + k k k ? +
10 140 45 15 30 30
deviations.
MEDIA
(cpm/mg
With
(PC, of
30 min) Py--p.ts”epe
sonication
1020 2260 1220 1280 2420 3270
+ r * + k r
10 10 20 150 30 40
17.8 106 20.2 24.4 30.3 117
74
DEVELOPMENTAL
BIOLOGY
be at least partly sequestered in some intracellular structure(s) and can be isolated by centrifuging the 1 M NaCl homogenate of unfertilized eggs at low speeds (2OOg or 15OOg). The compartmentation of the latent thymidine kinase in the particulates seemed to be well maintained in various hypertonic media such as 1 M NaCl, 1 M KCl, 2 M sucrose, and 2 A4 glycerol. However, exposure of the particulates to hypotonic media or sonication resulted in immediate release of the latent enzyme into the media accompanied by an appreciable increase in the thymidine kinase activity. Furthermore, evidence seems to have been provided in the present study that thymidine kinase in the fertilized or ammonia-activated eggs may no longer be sequestered in the particulates, but may be released or solubilized in ovum. The finding that the thymidine kinase activity of the 15OOg pellet prepared from the fertilized egg homogenates in 1 M NaCl remains very low even when assayed after sonication may not only support the above hypothesis (together with the fact that the thymidine kinase activity of the homogenate of fertilized eggs is not affected by sonication), but also may eliminate the possibility for the contamination of unbroken eggs in the homogenates. In fact, we have examined every homogenate under a microscope prior to experiments to confirm that the homogenates do not contain any unbroken eggs or larger cell debris. It has been confirmed that about 30 to 50% of the total thymidine kinase in the unfertilized Hemicentrotus eggs can be recovered in the particulate fraction as the latent form under the present experimental conditions. However, the question of whether or not an almost complete recovery of thymidine kinase in the particulate fraction may be achieved if the homogenization conditions for the unfertilized eggs are improved further, still remains. Moreover, the recovery of thymidine kinase in the pellet fraction seemed to depend not only on the homogenization conditions but
VOLUME
56, 1977
also on the sea urchin species. In fact, we have found that the homogenates of unfertilized Anthocidaris crassispina eggs in 1 M NaCl show almost similar thymidine kinase levels before and after sonication. However, if 0.5 M NaCl-1 M glycerol was used as the homogenizing medium, about 40% of the total thymidine kinase was recovered in the 1500g pellet as the latent form. In this connection, it seems important to study on the homogenates of Pseudocentrotus eggs because we have noticed that the 0.25 M sucrose (hypotonic) homogenates of unfertilized Pseudocentrotus eggs often show the gradual elevation of thymidine kinase activity upon standing in the freshly prepared homogenates at 0°C for 30 min or so (Nonaka and Terayama, 1975). The 1500g pellet prepared from 1 M NaCl homogenates of unfertilized Hemicentrotus eggs usually showed a three- to fivefold increase in the specific activity. Larger extents of enzyme activation by sonication may be expected if the wellwashed pellet fractions are used. In fact, an elevenfold increase in the activity was observed for the pellet at the interface between the d = 1.12 and 1.13 sucrose layers in density gradient centrifugation. The fact that the specific activity of the sonicated pellet (15OOg pellet or 200g pellet in Table 2) was not greater than the specific activity of the sonicated whole egg homogenate may suggest that the pellet may contain as much extra-proteins other than thymidine kinase as the whole egg homogenate, at least at this stage of particulate fractionation. The latent thymidine kinase seems to be associated with quite large organelles. The densities of the organelles containing most of the latent thymidine kinase was about 1.12 to 1.13, as measured by sedimentation analysis in a sucrose density gradient. This density value seems to be somewhat smaller than the density (d = 1.16-1.18) of ordinary plasma membranes isolated from mammalian cells. Since the release of the enzyme is facili-
NONAKA
AND TERAYAMA
Th> fmidine
tated by exposure not only to sonication or hypotonic media, but also to lipophilic reagents (Triton X-100 and propyleneglycol) or to phospholipases (data not presented), the latent thymidine kinase seems be bound to or sequestered in the membranebound vesicles, although this must be substantiated by further experiments. REFERENCES BOLLUM, F. J., and POTTER, V. R. (1959). Nucleic acid metabolism in regenerating rat liver. IV. Soluble enzymes which convert thymidine to thymidine phosphate and DNA. Cancer Res. 19, 561565. BREITMAN, T. R. (1963). The feedback inhibition of thymidine kinase. Biochim. Biophys. Actu 67, 153-155. HANSEN-DELKESKAMP, E., and DUSPIVA, F. (1965). Aktivitatsverlauf der enzymatischen Phosphorylierung von Thymidin wlhrend der Entwicklung des Seeigels Psammechinus miliaris von der Befrechtung bis zum Zweizeller. Ezperientia 22,381382. HINEGARDNER, R. T., RAO, B., and FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Exp. Cell Res. 36, 53-61. IKEZAWA, H., AOYAGI, T., TAKEUCHI, T., and UMEZAWA, H. (19’71). Effect of protease inhibitors of Actinomycetes on lysosomal peptide-hydrolases from swine liver. J. Antibiotics Tokyo Ser. A 24, 488-490. LONGO, 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, Arbaciapunctulata. Develop. Biol. 30, 5667. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and
Kinase
Compartmentation
75
RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265275. MAZIA, D. (1974). Chromosome cycles turned on in unfertilized sea urchin eggs exposed to NH,OH. Proc. Nat. Acad. Sci. USA 71, 690-693. MAZIA, D., and RUBY, A. (1974). DNA synthesis turned on in unfertilized sea urchin eggs by treatment with NH,OH. Ezp. Cell Res. 85, 167-172. NAGANO, H., and MANO, Y. (1968). Thymidine kinase, thymidylate kinase and 32Pi and 14C thymidine incorporation into DNA during early embryogenesis of the sea urchin. Biochim. Biophys. Actu 157, 546-557. NONAKA, M., and TERAYAMA, H. (1975). Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization. Develop. Biol. 43, 322-332. ORD, M. G., and STOCKEN, L. A. (1973). Thymidine uptake by Purucentrotus eggs during the first cell cycle after fertilization. Exp. Cell Res. 83,411-414. SAKUMA, K., and TERAYAMA, H. (1967). Effects of adrenal hormone upon DNA synthesis in regenerating rat liver and tumors. J. Biochem. 61, 504511. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nut. Acad. Sci. USA 71, 1915-1919. VACQUIER, V. C., and BRANDRIFF, B. (1975). DNA synthesis in unfertilized sea urchin eggs can be turned on and turned off by the addition and removal of procaine hydrochloride. Develop. Biol. 47,12-31. VON LEDEBUR-VILLIGER, M. (1972). Cytology and nucleic acid synthesis of parthenogenetically activated sea urchin eggs. Exp. Cell Res. 72, 285-308. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine. Biochim. Biophys. Acta 45, 101-110.