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Binding sites of -SH reagents in dividing sea urchin egg
Printed in Sweden Copyright 0 1973 by Acodrmic Al/ right,7 of rrprodurrion in my
Press,
form
Inc. rescrord
Experimental
BINDING
Cell Research 82 (1973) 325-334
SITES OF -SH REAGENTS
IN DIVIDING Y. OKAZAKF, Department
SEA URCHIN
I. MABUCHP,
I. KIMURA
EGG and H. SAKAl
of Biophysics and Biochemistry, Faculty sf’ Science, University of Tokyo, Tokyo, Japan
SUMMARY Inhibition of cleavage of sea urchin eggs by - SH reagents was examined by varying their concentration, and the time and stage of the treatment during the first cleavage cycle. It was found that more than half of - SH groups in the ‘cortex protein’, localized in the cortical layer, reacted with p-chloromercuribenzoate (PCMB) when cleavage was suppressed, while other protein fractions of eggs revealed only a little binding of PCMB. The amount of non-protein -SH groups (NPSH) in the egg decreased to a level 50 % that of the intact cell when cytokinesis was completely blocked by 1 mM PCMB. Even at lower PCMB cont., which did not block cell division, the NPSH level decreased to 60% and remained there.In pulse-treatment with 0.1 mM N-ethylmaleimide (NEM), which did not block cell division, NPSH groups reacted quickly with the reagent to protect protein - SH groups from the alkylation. However, if the concentration was increased, the pulse-treatment resulted in suppression of cleavage and alkylation of protein - SH groups, especially of cortex protein, accompanied by decrease in ATPase activity involved in the cortex protein fraction.
Ever since an absence of fluctuation in the amount of glutathione has been demonstrated during cleavage cycles in sea urchin eggs [19, 211, it has been postulated that a structural protein found in the cortical layer of the sea urchin egg plays a role in cytokinesis. Evidence has been presented demonstrating that -SH groups in the cortex protein fluctuated in close association with cleavage [22, 231 and that inhibition of cleavage by ether-sea water was accompanied by cessation of the fluctuation [24]. Later, the cyclic change in -SH content of the cortex protein was found to be associated with a mirror image fluctuation of -SH groups in a calcium-insoluble protein fraction [25]. This 1 Present address: Department of Biochemical Pharmacology, Institutes -of Biological Science, Mitsui Pharmaceuticals, Co., Mobara, Chiba. 2 Present address: Biological Institute, University of Tokyo, Meguro-ku, Tokyo.
kind of thiol-disulfide exchange reaction has been elucidated as being a requisite for the initiation of cytokinesis [28]. Recent demonstrations have shown that highly purified cortex protein of sea urchin eggs was an active hydrogen donor or acceptor in the thiol-disulfide exchange reaction with tubulin [13, 161, catalysed by a transhydrogenase [27]. Furthermore, the cortex protein functions in activation as well as stabilization of cortical ATPase, properties of which have most recently been established by Mabuchi [17]. Although reactivities of -SH groups have been well known in proliferating tissues and cells (reviewed by Brachet [3-5]), in the mitotic center of the sea urchin egg [I I], and inhibition of cleavage by -SH reagents has frequently been reported with various cells [3, 81, the blocking sites of the reagents within the cell has not yet been clarified. Exptl Cell Res 82 (1973)
326
Y. Okazaki et al.
Our results, described below, confirm that inhibition of cleavage by -SH reagents primarily indicates the binding of the reagents to the cortex protein. Additional information is presented concerning the effect of the reagents on ATPase in the cortical layer of the sea urchin egg. MATERIALS AND METHODS Most of the experiments were carried out with the sea urchins, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina which were collected at the Misaki Marine Biological Station. Gametes were obtained by the injection of acetylcholine into the body cavity of the animals 191.The removal of fertilization membranes was performed by the use of 1 M urea [16].
Treatment of eggs with -SH
reagents
Eggs were treated with 100 vol of sea water containing -SH reagents. PCMB was dissolved in a small amount of 0.1 N NaOH and was added to Ca-free sea water (CFSW) to avoid precipitation of PCMB. pH was adjusted to 8.2. p-Chloromercuriphenyl sulphonate (PCMPS), iodoacetamide (IAA), and N-ethylmaleimide (NEM) were dissolved directly in CFSW or ordinary sea water. Under those conditions. 0.1 mM -SH reagent was 0.66 and 1.7 times equivalent to total - SH groups (NPSH + protein - SH) or to NPSH groups, respectively. The inhibition of cleavage was determined 1 h after control eggs had divided by counting the number of divided and non-divided eggs out a total of more than 300 and expressed as % of cells not divided.
Isolation of cortical layer of sea urchin eggs The isolation was carried out bv the nrocedure reported by Sakai [22]. When eggs were treated with PCMB. 5 mM CaCl, was used as an isolation medium instead’of 0.1 M MgCl,.
Fractionation
of protein components
Temperatures between 0 and 4°C were maintained throughout these fractionation orocedures as renorted previously [16]. As is shown in fig. 1, eggs were-gently homogenized in 5 mM CaCL or 0.1 M M&I, and centrifuged briefly. The supernatant was -used as ‘cytonlasmic fraction’. After washing repeatedly with the isolation medium, the appearances of cortical hulls isolated by 5 mM CaCl, were usually similar to those isolated by 0.1 M MgC&; these procedures allowed preparations of masses of isolated cortices with little contamination of unbroken cells. Isolated cortices were first extracted with 10 mM phosphate buffer (pH 7.0) containing 1 mM ATP overnight, followed by centrifugation at 15 000 g for 10 min. The supernatant was called ‘ATP-extract’. From the precipitate, cortical proteins were extracted Exptl Cell Res 82 (1973)
with 0.6 M KC1 containing 10 mM nhosphate buffer (pH 7.0) or 10 mM Tris-HCl (pH 8.4) ‘for 1 h and centrifuged at 15 000 R for 10 min. The nrecinitate was once more extracted with 0.6 M K-Cl and an insoluble material, ‘membrane fraction’, was obtained after centrifugation. After suspending in 0.6 M KCI, a number of cell membranes and few cytoplasmic particles were observed under a phase microscope. This fraction consisted mostly of cellular and some vesicular membranes when viewed by an electron microscope [16]. The first KCl-extract was adjusted to 55 % saturation with respect to ammonium sulfate and precipitates thus formed, “55 % SAS fr”, were collected by centrifugation at 10000 g for 10 min, followed bv dissolution and dialysis against 10 mM phosphate -buffer (pH 7.0) to - precipitate ‘cortex protein’.
Determination of PCMB bound to proteins The principle of the determination was based on the fact that organic mercurials which bound to protein -SH groups could easily be dissociated in acidified organic solvents such as acetone or alcohol [29]. The reacted protein was precipitated by addition of acetone (final 90%) and washed once with pure acetone. To the final orecioitate. washed 3 times with 95 % ethanol, was added acidified ethanol (one drop of concentrated HCl in 5 ml 95 % ethanol) to 5 ml to dissociate PCMB from protein. The amount of PCMB was assaved bv titration with alutathione Il. 21. In several series of preliminary det&minations‘with excess protein and known amount of PCMB, a recovery of lOOf4.9 % was obtained. Since sea urchin eggs contain as much as 6 mM glutathione [21], bound PCMB in the cortex might be dissociated in the course of isolation of cortices. This possibility was checked as follows. First, cytoplasmic bulk protein of sea urchin eggs was incubated with a known amount of PCMB to react completely. Since eggs were homogenized in 9 vol of isolation medium, the glutathione cont. in the homogenate was in the range of 0.6 mM. Therefore, 1.0 mM glutathione was mixed with the PCMB-bound protein and kept standing for 15 min at room temperature, followed by dissociation and determination of PCMB. The recovery of PCMB was less than 50 % depending on the incubation time as expected. Since Ca or Mg ions inhibit the reactivity of thiol groups, incubation of the PCMB-bound protein was performed in the presence of 1 mM glutathione plus 5 to 20 mM CaCl, or plus 0.1 M MgCI,. In those cases, recoveries of PCMB were 100 + 4.9 (6 series of determinations) or 68 + 9.2 % (8 se& of determinations). respectively. Therefore, 5 mM CaCl, was used throughout the present experiments to prevent release of bound PCMB by intracellular glutathione during the isolation of cortices. In the experiments with other reagents, the isolation of cortices was performed by the original procedure [22].
Assay of cortical ATPase activity The standard assay was carried out in a total volume of 2.0 ml (17), which included: 20 mM Tris-HCl buffer (pH 7.0), 10 mM MgCl,, 0.15 M KCI, 0.5
Binding sites qf’
e\rracted
with
I mM
ATP.
IO mM
phosphate
buffer.
SH wagents
327
pH 7.0
PClkl cxlracted phosphate wntrlfuged.
with 0.6 M KCI. IO mM buffer, pH 7.0, I h 15 000 @ IO mill
Pellet washed wth centrifuged,
d~s\ol\ed d!al>\ed
I” 0.1 M KU, ;!e;un\t IO mM
IO mM phosphate phosphate buffer.
0.6 M KCI 15000 g IO ,n,~n
buffer pH 7.0
mM ATP and about 0.5 mg protein of enzyme fraction. Incubations were performed at 25°C for 15 min. The reactions were stopped with 0.5 ml of 30% trichloroacetic acid (TCA). After removal of precipitates by centrifugation, inorganic phosphate in the supernatant was determined by the method of Fiske & Subarrow [7]. Controls were determined by incubating without addition of the enzyme fraction and were subtracted from the total Pi liberated in the presence of the enzyme fractions.
Determination of protein - SH and NPSH groups Total protein -SH groups were assayed by the use of mercury orange [29]. For the determination of NPSH groups in eggs (mostly glutathione [21]) treated with PCMB, aliquots of unfertilized or fertilized eggs were quickly washed twice with CFSW to remove free PCMB. To the packed egg mass were added 5 ml of 10% TCA containing 4 mM EDTA to extract NPSH and allowed to stand for 30 min at 0°C. The suspensions was centrifuged at 1 000 g for 5 min and the pH of aliquots of the supernatant was adjusted exactly to 8.0 with NaOH; NPSH groups were measured by Ellman’s method [6]. The same extraction was conducted in the case of treatment with NEM. Thereafter, the TCA-extract was directly assayed for NPSH groups by iodometric titration after oxidation of ascorbic acid by 2,6-dichlorophenol indophenol [21].
Fig. 1. Fractionation of protein components of isolated cortices.
Determination of radioactivity After fertilized eggs were pulse-treated with 0.1 and 0.3 mM NEM-2,3J*C (1.6 mCi/nmole; The Biochemical Centre, Amersham), egg cortices were isolated and protein components were separated. Proteins were precipitated in 90% acidified acetone (one drop of concentrated HCl in 5 ml of acetone), collected onto glass filters (Whatman, GF 83), and washed 3 times with HCl-acetone. In order to determine materials of low mol. wt which are bound with NEM, an aliquot of an equilibrated dialysate of the cytoplasmic fraction (fig. 1) was adsorbed in the glass filters and dried. The amount of radioactivity bound to the dried filters was determined in a toluene scintillation mixture (4 g PPO, 0.2 g Me,POPOP in 1 1 toluene). The counting efficiency for 14C was 86 U0 with an Aloka LSC 501 scintillation counter. The data shown in all figures and tables are based on assays which were carried out in duplicate.
RESULTS AND DISCUSSIONS Inhibition
of cleavage by ~ SH reagents
Two series of experiments were carried out to determine the effect of both continuous and pulse treatment with -SH reagents. In the first, the critical cont. of -SH reagents for Exptl Cell Res 82 (1973)
328 Y. Okazaki et al. what stage of the cell cycle was pulse-treated. It could be recognized that the egg became rather resistant before cleavagewhen exposed to lower cont. of the inhibitor as shown in fig. 3. After these experiments, the shortest duration of the pulse-treatment for 100% inhibition was determined. The concentration of the inhibitors, 1 mM IAA for Pseudocentrotus and 0.3 mM NEM for Anthocidaris Fig. 2. Abscissa: cont. - SH reagents (mM); ordinate: eggs, were chosen from the experiments % cells not cleaved. n, PCMB; 0, PCMPS; 0, IAA; l , NEM. Cont. mentioned above. As shown in fig. 4, when -SH reagents for inducing inhibition of cleavage treated at any stage of the cell cycle, full by treating eggs continuously after fertilization (Pseudocentrotus). inhibition of cleavage was caused by a pulsetreatment as short as 3 min with NEM. One inhibition of cleavage was determined. Sea mM IAA, on the other hand, exhibited 100% urchin eggs were continuously exposed to the inhibition of cleavage after 7 min treatment. inhibitors from 5 min after fertilization. The However, the sea urchin specieswas different results are shown in fig. 2.0.14 mM alkylating from that used in the experiments with reagents blocked cleavage completely, while NEM. the same PCMB or PCMPS cont. showed no inhibition. In cases of such mercaptide Change in NPSH level by the treatment with forming reagents, a cont. higher than 0.5 - SH reagents mM was required for the inhibition. It was our expectation that -SH reagents Secondly, fertilized eggswere pulse-treated react faster with intracellular glutathione with inhibitors for 5 or 10 min at regular than with protein-SH groups because of the intervals as indicated in fig. 3a, b, One mM high glutathione cont. within the cell. When PCMB or PCMPS showed no delay of cleav- fertilized eggs at the late monaster stage age. On the other hand, 1 mM IAA or 0.3 mM (about 30 min after fertilization at 20°C) NEM completely blocked cleavage no matter were exposed to 1.5 mM PCMB, NPSH a
-t------t------
h
a---
‘-+ Cf
Fig. 3. Abscissa: time after insemination (min), stages of pulse treatment are indicated beneath; ordinate: % cells not cleaved. Inhibition of cleavage by pulse treatment with alkylating reagents. Fertilized eggs were pulse-treated for 5 and 10 min at regular intervals indicated along the abscissa with (a) IAA, 1 mM (0) and 0.3 mM (0) for 10 min (Pseudocentrotus); (b) NEM, 1 mM (o), 0.3 mM (0), 0.1 mM ( x ), 0.03 mM (A), and 0.01 mM (0) for 5 min (Anthocidaris). Exptl Cell Res 82 (1973)
Binding sites of ~- SH reagents
groups decreased to half the original level after a 10 min treatment (fig. 5~). This treatment routinely produced a complete block of cleavage. A similar time course for the decreasein NPSH groups was observed with 0.3 mM PCMB, which did not inhibit the first cleavage at all. In both cases,the NPSH level remained stationary after 10 min treatment. Quite similar patterns in the decrease of NPSH groups were observed in Hemicentrofus eggs also. These were rather intriguing observations, because PCMB was 25 times equivalent to intracellular glutathione under the present experimental conditions. This may be explained by assuming the existence of barriers which prevent further diffusion of PCMB into the cell. Fig. 5b shows the effect of NEM on the NPSH level of sea urchin eggs. It was shown that when pulse-treated with 0.1 mM NEM for 3 min, the level in metaphase cells decreasedto 40 ‘%,of that of intact cells (fig. 5b, II); no inhibition of cleavage being observed. However, further decreasesin NPSH groups were detected after a 5 min treatment with 0.3 mM NEM; cleavage no longer occurred (fig. 5 b, III). Similar results were obtained by determining bound NEM with the radioactive reagent. When metaphaseeggs
:’
20. 0
329
I
2
3
4
5
6
7
8
9
IO
4. Abscissa: time of pulse treatment (min); ordinate: 96 cells not cleaved. Inhibition of cleavage by pulse treatment with alkylating reagents. Metaphase cells were pulsetreated for the time indicated with 0.3 mM NEM (0) for Anthociduris and 1.O mM IAA (:I ) for
Fig.
Pseudocentrotus.
were pulse-treated with 0.1 and 0.3 mM for 3 min, the amount of radioactive NEM which was assessed to bind with NPSH groups within the cell corresponded to 55 and 85 % level of total NPSH groups, respectively. These values were similar to the data, 64, and 80 %, obtained from the measurement of NPSH groups. In all cases,the amount of NPSH groups remained constant after the pulse treatment, indicating no synthesis of glutathione thereafter. Comparing the data shown in fig. 5b with those of fig. 5a, it was evident that NEM penetrated into the egg faster than PCMB. Furthermore, it was clear from fig. 5b, curve
Fig. 5. Abscissa: time after beginning of treatment; ordinate:
pmoles NPSH as glutathione/egg. (a) Change in NPSH groups of fertilized eggs treated continuously with 0.3 mM (0) and 1.5 mM ( l ) PCMB from 30 min after fertilization (Anthocidaris); (b) change in NPSH groups of eggs treated with NEM. I, unfertilized eggs, 0.3 mM, 5 min; II, metaphase eggs, 0.1 mM, 3 min; III, metaphase eggs, 0.3 mM, 5 min. Arrow NEM and SW indicate beginning and end of pulse treatment, respectively. Exptl Cell Res 82 (1973)
330
Y. Okazaki et al.
Table 1. Time course of PCMB-binding
in the cortex (Hemicentrotus)
10, 20, 30, and 40 min after the beginning of the treatment, eggs were collected and washed with CFSW to remove free PCMB, followed by the isolation of cortices Concentration of PCMB at 0.4 mM Time of treatment with PCMB (min) 0 10 20 30 40
1.0 mM
nmoles PCMB bound/mg protein
Blocked SHa calculated ( %)
0
0
3.5 T.7
5.8
-11.1
nmoles PCMB bound/mg protein
Blocked SHa calculated ( %)
0
0
::: 20.4 13.2
15.1 10.7 33.8 21.8
a Total protein - SH groups in the cortex at the beginning of treatment: 60.5 nmoles/mg protein.
I that unfertilized eggs were more resistant than fertilized eggs to the penetration of the reagent (cf curve III). This may be due to the well known fact that the membrane of the unfertilized egg is less permeable than that of the fertilized egg. A considerable number of such pulse-treated unfertilized eggs could be fertilized, but did not develop. Binding sites of PCMB and inhibition of cleavage
It is necessary to remove fertilization membranes for the isolation of cortices and the procedure requires about 20 min for replacing eggsfrom 1 M urea to CFSW. Therefore, the PCMB-treatment started at 30 min after
insemination. Preliminary experiments showed that when eggs were continuously treated with 1 mM PCMB from 30 min after fertilization, cleavage was inhibited, while with 0.4 mM PCMB no inhibition was observed. It was found that bound PCMB gradually increased during the treatment (table 1). Assuming that PCMB binds specifically with the -SH groups of cortices under the experimental conditions, 10 % of total - SH groups was blocked after 40 min-treatment with 0.4 mM PCMB; under these conditions, no inhibition of cleavage was observed. On the other hand, more than 30 % of the ~ SH groups found in the isolated cortices were
Table 2. Binding of PCMB to protein components of cortices (Hemicentrotus) Fertilized eggs were treated with 1 mM PCMB in CFSW for 40 min, washed with CFSW to remove free PCMB and isolation of cortices and fractionation of protein components were carried out as described in Method
Fraction
Protein (md
nmoles PCMB bound
ATP-extract Cortex protein Membrane fraction
1.25 1.65 5.95
9.4 77.5 16.5
a Values measured at the beginning of treatment. Exptl Cell Res 82 (1973)
nmoles PCMB/mg protein (A)
nmolesa SH/mg protein (B)
A/B Xl00
7.5 47.0 2.8
78.0 82.0 57.0
9.6 57.3 4.9
Binding sites uj’ - SH reagents
33 I
Table 3. Decrease in - SH groups in protein fractions of cortices by pulse treatment with NEM qf metaphase eggs (Anthocidaris) (expressed as nmoles - SH per mg protein) Treatment with Fraction Cytoplasmic fraction Control Treated ATP-extract Control Treated Cortex protein (55“<,SAS fr) Control Treated Membrane fraction Control Treated
0.1 mM, 3 min
0.3 mM, 3 min
0.3 mM, 5 min
0.3
53.3 53.7 (~t1.3)
48.5 45.8
53.3 47.6
( 10.7)
41.9 41.7
( 10.9)
74.0 73.0(-1.4)
74.9 68.8 (-8.1)
74.0 6X.5
( ~~7.4)
71.8 53.0
( 26.2,
69.8 68.5 (-1.9)
69.8 52.5
(- 24.8)
69.8 46.4
( 33.5)
71.8 37.3
( 48.0)
35.2 35.5
40.0 37.9
(-- 5.3)
35.2 31.6
(-- 10.2)
36.1 28.2
(- 21.9)
(7 0.9)
( -5.6)
mM, 7 min
Values in parenthesesindicate decreasein - SH groups in percentage.
blocked when the cont. was increased up to 1 mM, which caused 100 Y0inhibition of cleavage. The conditions of these experiments were similar to those in fig 5a. Therefore, the cleavage inhibition is not due to the decrease of NPSH, but to that of protein-SH groups in the cortex to the extent of 30%. The binding sites of PCMB were further analysed by fractionation of the protein components of the isolated cortices. As shown in table 2, more than half of the PCMB bound in the cortex was detected in the cortex protein fraction. The membrane fraction did not have so many sites for binding PCMB and the ATP-extract, in which tubulin might be involved at metaphase [17], did not show a significant amount of binding under the present experimental conditions. The highest binding of PCMB was found in the cortex protein fraction, when calculating the magnitude of block of -SH groups as shown in table 2. Therefore, it was strikingly apparent that the binding of PCMB to the cortex was mainly due to the reactivity of the cortex protein fraction.
Binding sites of NEM in suppression
qf cleaaage
A similar experiment was designed to establish the action of NEM on cleavage inhibition. The inhibitory effect of alkylating reagents on cell division was much stronger than that of mercaptide forming reagents as described in figs 2 and 3. Furthermore, only a short pulse-treatment was necessary for 100 O: inhibition of cleavage (fig. 4). Preliminary experiments with unfertilized eggs indicated that -SH groups decreased only in the cortex protein fraction after a 5 min pulse-treatment with 0.3 mM NEM. On the contrary, the ATP-extract and the the membrane fraction revealed little change in their -SH amount. This was probably due to the free reactivity of -SH groups of the cortex protein in unfertilized eggs. It should also be mentioned that the -SH groups of the cytoplasmic proteins did not change in amount by the treatment. This may mean that the reagents were trapped one after another by a ‘cortical ~~SH barrier’ and could not penetrate into the cytoplasm or that NPSH groups, that is those of 6 mM Exptl Cell Res 82 (1973)
332 Y. Okazaki et al. glutathione within the cell, reacted quickly with NEM to prevent alkylation of the cytoplasmic proteins. To clarify which possibility was responsible, the level of NPSH in unfertilized eggs was assayed before and after the pulse-treatment with 0.3 mM NEM. The data shown in fig. 5b supported the latter possibility. In general, the cell is rather resistant after the formation of the mitotic apparatus to anti-mitotic agents and to various reagents which inhibit respiration, oxidative phosphorylation and so on. The same held true with low cont. of alkylating reagents. However, as the cont. was increased, cell division was completely blocked even if the eggs were treated after the completion of nuclear division. In order to determine the minimal binding of alkylating reagent necessary to induce inhibition of cleavage, the NEM cont. and the duration of pulse treatment were selected as 0.3 mM and 3 min, respectively, from the data shown in fig. 4. For comparison, treatments with 0.1 mM, 3 min; 0.3 mM, 5 min; and 0.3 mM, 7 min were also chosen. The results are shown in table 3, where a significant decrease in -SH groups of the cortex protein fraction is shown when eggs are pulse-treated for 3 min. The cytoplasmic bulk protein, the ATP-extract and the membrane fraction showed little change in their -SH amount. Assuming that -SH groups in the cortex protein fraction available for the reaction with NEM were about 70 % of total -SH groups, including masked ones [23], alkylation of 36 % of the available -SH groups results in 100% inhibition of cleavage. After increasing the time of the pulsetreatment (5 and 7 min), a considerable amount of NEM bound to all of the fractions. On the other hand, 0.1 mM, 3 min treatment resulted in no inhibition of cleavage and little change in the -SH content of all Exptl
Cell Res 82 (1973)
fractions. This is apparent from the data shown in fig. 5b, II. That is, in the case of 0.1 mM NEM, the intracellular glutathione quickly trapped the incoming NEM to protect the cortical proteins from alkylation during the 3 min pulse treatment. This was confirmed by the fact that the 0.1 mM reagent was only 1.7 times equivalent to NPSH groups in the sea urchin egg under the conditions of these experiments (see Method). To confirm the results on the effect of NEM, bound NEM was directly assayed by the use of 14C-NEM. Consistent with the results as documented above, little binding of the reagent was detected in all the fractions when eggs were pulse-treated for 3 min with 0.1 mM radioactive NEM. When the cont. was raised to 0.3 mM, on the other hand, the radioactivities in the cortex protein fraction were the most significant. Among them, the membrane fraction showed very high activities. This is apparently different from the data obtained from the experiments with PCMB. This discrepancy may be explained by the lower specificity of NEM for thiols [30] than PCMB. The treatment with NEM, therefore, might also involve binding of the reagent to sites other than -SH groups. Effect of NEM on ATPase activity in the cortical layer An ATPase activity in the cortex of the sea urchin egg was first reported by Miki [18] and a fluctuation of the activity was found closely associated with the division cycle. Later, it was demonstrated that addition of ATP induced the progress of furrowing in a glycerinated dividing sea urchin egg [15] similar to a telophase model of fibroblast [lo, 141.Furthermore, it was recently demonstrated that an ATPase extractable concomitantly with the cortex protein from the isolated cortices could be separated from the cortex protein and partially purified by
Binding sites of
-~ SH reagents
333
Table 4. Change in ATPase activities in protein fractions of isolated cortices b), the treatmrnt of metaphaseeggs with NEM (Anthocidaris) (expressedas ,umoles Pijmg proteinlmin) Treatment with Fraction ATP-extract Control Treated Cortex protein (55 “b SASfr) Control Treated Membrane fraction Control Treated
0.3 mM, 3 min
0.3 mM, 7 min
0.0060 0.0078 0.0071 (-I- 18.3 :h) 0.0046 (--41.0%) 0.016 0.013 (- 18.7)
0.026 0.012 ( 53.8)
0.0089 0.0110 ( t- 23.6)
0.0089 0.0070 (-21.3)
Values in parentheses indicate percentage decrease or increase in spec. act.
sucrose density gradient centrifugation [17]. The enzyme (cortical ATPase) possessed dynein-like properties and was activated by the cortex protein through the formation of a complex. Other ATPases were also detected in both the ATP-extract and the membrane fraction, which have been considered to be ‘cytoplasmic’ and ‘membrane’ ATPases, respectively. Those facts led to the experiments to examine the effect of NEM on the ATPases involved in the three protein fractions of the cortical layer. After metaphase, eggs were pulse-treated and each protein fraction of the isolated cortices was subjected to the assay for ATPase activity as shown in table 4. The results indicated that the enzyme activity in the cortex protein fraction decreased about 20 % consistently, while the activities in other fractions slightly increased when pulsetreated with 0.3 mM NEM for 3 min. The decreases in activity were due to blocking of -SH groups in the cortical ATPase beyond a certain level [ 171. The increases of the activities in the ATP-extract and the membrane fraction could also be explained by the fact that ATPases are activated if treated with a low concentration of -SH reagents [12, 17,
201. This is consistent with the fact that cortical components obtained as ATP extract and membrane fraction were less susceptible to -SH reagents than the cortex protein. Furthermore, activities in all the fractions decreased when the treatment was longer (7 min), possibly because of further loss of their -SH content (table 3). It can be said that the ATPase which comes into solution with the cortex protein is the most sensitive to the action of NEM in situ. It was previously demonstrated that the cortical ATPase was activated by the purified cortex protein rich in -SH groups and inactivated by the same protein when poor in -SH content [17]. Furthermore, the fluctuation of the activity was quite coincident with that of the -SH content in the cortex protein fraction during the division cycle (cf ref [ 181 with [23]). Therefore, the fluctuation of the ATPase activity found by Miki might be explained by an interaction between the cortical ATPase and the cortex protein [17]. This was further suspected from the fact that both the proteins formed complexes at the ionic strength of 0.1 in vitro. The cortex protein seemed to be localized underneath the cell membrane [16]. It is likely that the Exptl Cell Res 82 (1973)
334
Y. Okazaki et al.
cortical ATPase exhibits the same behavior concerning its localization. This situation could be one of the reasons why the cortex protein and the cortical ATPase were preferentially attacked by -SH reagents. At the critical treatment to induce inhibition of cleavage (0.3 mM NEM for 3 min), the inhibition of the ATPase activity was rather small (20%). This may indicate that the binding of -SH reagents to the cortex protein affects the interaction between the cortex protein and the ATPase more profoundly than either protein alone inducing a modification which leads to failure of cytokinesis. CONCLUSION
treatment could be mainly attributed to a modification in the cortical layer. This might involve alteration in the mode of interaction between the cortex protein and cortical ATPase. REFERENCES 1. Benesch, R & Benesch, R E, Meth biochem anal 10 (1962) 43. 2. Boyer, P D, J Am them sot 76 (1954) 4331. 3. Brachet, J, Chemical embryology, p. 170. Interscience, New York (1950). 4. Brachet, J, Biochemical cytology, p. 194. Academic Press, New York (1957). 5. Brachet, J. The biochemistry of development. n. 104. Pergamon Press, London (1960). ^ 6. Ellman. G L, Arch biochem bionhvs _ - 82 (1959) . , 70. I. Fiske, C H & Subarrow, Y, J biol them 66 (1925) 375. 8. Harris, J W, Allen, N P & Teng, S S, Exptl cell res 68 (1971) 1. 9. Hinegardner, R T, Biol bull 137 (1969) 465. 10. Hoffmann-Berling, H, Biochim biophys acta 15 (1954) 226. 11. Kawamura, N & Dan, K, J biophys biochem cytol 4 (1958) 615. 12. Kielley, W W & Bradley, L B, J biol them 218 (1956) 653. 13. Kimura, I, Exptl cell res 79 (1973) 445. 14. Kinoshita, S, Benjamin, S, & Hoffmann-Berling, H, Biochim biophys acta 79 (1964) 88. 15. Kinoshita, S & Yazaki, I, Exptl cell res 47 (1967) 449. 16. Mabuchi, I & Sakai, H, Development growth & differentiation 14 (1972) 247. 17. Mabuchi, I, Biochim biophys acta 297 (1973) 317. 18. Miki. T, Exptl cell res 33 (1964) 575. 19. Neufeld; E F & Mazia, D,‘Exptl cell res 13 (1957) 622. 20. Ogawa, K & Mohri, H, Biochim biophys acta 256 (1972) 142. 21. Sakai, H & Dan, K, Exptl cell res 16 (1959) 24. 22. Sakai, H, J biophys biochem cytol 8 (1960) 603. 23. - Ibid 8 (1960) 609. 24. - Exptl cell res 32 (1963) 391. 25. - Biochim biophys acta 102 (1965) 235. 26. - Ibid 112 (1966) 132. 27. - J biol them 242 (1967) 1458. 28. - Int rev cytol 23 (1968) 89. - Anal biochem 26 (1968) 269. ii: Smyth, D G, Nagamatsu, A & Fruton, J S, J Am them sot 82 (1960) 4600.
It has been established that when sea urchin eggs were exposed to -SH reagents, NPSH groups quickly reacted with them to protect cortical proteins from mercaptide formation or alkylation. When the modifications of the cortical proteins was negligible during the treatment, cell division was never inhibited even when the level of NPSH decreased to less than half of the original level. However, when the treatment was stronger, excess reagents, which were no longer sequestered by NPSH groups, reacted preferentially with the -SH groups of the cortex protein in the cortical layer, resulting in suppression of cleavage. PCMB binds to more than half of the -SH groups in the cortex protein under such a condition. Furthermore, alkylation of cortical proteins and consequent decreasein cortical ATPase activity resulted in a irreversible block of cytokinesis. Since nuclear division was not affected under the present experimental condition with NEM, Received March 9, 1973 the inhibition of cytokinesis by the pulse Revised version received July 3, 1973
Exptl Cell Res 82 (1973)
Press,
form
Inc. rescrord
Experimental
BINDING
Cell Research 82 (1973) 325-334
SITES OF -SH REAGENTS
IN DIVIDING Y. OKAZAKF, Department
SEA URCHIN
I. MABUCHP,
I. KIMURA
EGG and H. SAKAl
of Biophysics and Biochemistry, Faculty sf’ Science, University of Tokyo, Tokyo, Japan
SUMMARY Inhibition of cleavage of sea urchin eggs by - SH reagents was examined by varying their concentration, and the time and stage of the treatment during the first cleavage cycle. It was found that more than half of - SH groups in the ‘cortex protein’, localized in the cortical layer, reacted with p-chloromercuribenzoate (PCMB) when cleavage was suppressed, while other protein fractions of eggs revealed only a little binding of PCMB. The amount of non-protein -SH groups (NPSH) in the egg decreased to a level 50 % that of the intact cell when cytokinesis was completely blocked by 1 mM PCMB. Even at lower PCMB cont., which did not block cell division, the NPSH level decreased to 60% and remained there.In pulse-treatment with 0.1 mM N-ethylmaleimide (NEM), which did not block cell division, NPSH groups reacted quickly with the reagent to protect protein - SH groups from the alkylation. However, if the concentration was increased, the pulse-treatment resulted in suppression of cleavage and alkylation of protein - SH groups, especially of cortex protein, accompanied by decrease in ATPase activity involved in the cortex protein fraction.
Ever since an absence of fluctuation in the amount of glutathione has been demonstrated during cleavage cycles in sea urchin eggs [19, 211, it has been postulated that a structural protein found in the cortical layer of the sea urchin egg plays a role in cytokinesis. Evidence has been presented demonstrating that -SH groups in the cortex protein fluctuated in close association with cleavage [22, 231 and that inhibition of cleavage by ether-sea water was accompanied by cessation of the fluctuation [24]. Later, the cyclic change in -SH content of the cortex protein was found to be associated with a mirror image fluctuation of -SH groups in a calcium-insoluble protein fraction [25]. This 1 Present address: Department of Biochemical Pharmacology, Institutes -of Biological Science, Mitsui Pharmaceuticals, Co., Mobara, Chiba. 2 Present address: Biological Institute, University of Tokyo, Meguro-ku, Tokyo.
kind of thiol-disulfide exchange reaction has been elucidated as being a requisite for the initiation of cytokinesis [28]. Recent demonstrations have shown that highly purified cortex protein of sea urchin eggs was an active hydrogen donor or acceptor in the thiol-disulfide exchange reaction with tubulin [13, 161, catalysed by a transhydrogenase [27]. Furthermore, the cortex protein functions in activation as well as stabilization of cortical ATPase, properties of which have most recently been established by Mabuchi [17]. Although reactivities of -SH groups have been well known in proliferating tissues and cells (reviewed by Brachet [3-5]), in the mitotic center of the sea urchin egg [I I], and inhibition of cleavage by -SH reagents has frequently been reported with various cells [3, 81, the blocking sites of the reagents within the cell has not yet been clarified. Exptl Cell Res 82 (1973)
326
Y. Okazaki et al.
Our results, described below, confirm that inhibition of cleavage by -SH reagents primarily indicates the binding of the reagents to the cortex protein. Additional information is presented concerning the effect of the reagents on ATPase in the cortical layer of the sea urchin egg. MATERIALS AND METHODS Most of the experiments were carried out with the sea urchins, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, and Anthocidaris crassispina which were collected at the Misaki Marine Biological Station. Gametes were obtained by the injection of acetylcholine into the body cavity of the animals 191.The removal of fertilization membranes was performed by the use of 1 M urea [16].
Treatment of eggs with -SH
reagents
Eggs were treated with 100 vol of sea water containing -SH reagents. PCMB was dissolved in a small amount of 0.1 N NaOH and was added to Ca-free sea water (CFSW) to avoid precipitation of PCMB. pH was adjusted to 8.2. p-Chloromercuriphenyl sulphonate (PCMPS), iodoacetamide (IAA), and N-ethylmaleimide (NEM) were dissolved directly in CFSW or ordinary sea water. Under those conditions. 0.1 mM -SH reagent was 0.66 and 1.7 times equivalent to total - SH groups (NPSH + protein - SH) or to NPSH groups, respectively. The inhibition of cleavage was determined 1 h after control eggs had divided by counting the number of divided and non-divided eggs out a total of more than 300 and expressed as % of cells not divided.
Isolation of cortical layer of sea urchin eggs The isolation was carried out bv the nrocedure reported by Sakai [22]. When eggs were treated with PCMB. 5 mM CaCl, was used as an isolation medium instead’of 0.1 M MgCl,.
Fractionation
of protein components
Temperatures between 0 and 4°C were maintained throughout these fractionation orocedures as renorted previously [16]. As is shown in fig. 1, eggs were-gently homogenized in 5 mM CaCL or 0.1 M M&I, and centrifuged briefly. The supernatant was -used as ‘cytonlasmic fraction’. After washing repeatedly with the isolation medium, the appearances of cortical hulls isolated by 5 mM CaCl, were usually similar to those isolated by 0.1 M MgC&; these procedures allowed preparations of masses of isolated cortices with little contamination of unbroken cells. Isolated cortices were first extracted with 10 mM phosphate buffer (pH 7.0) containing 1 mM ATP overnight, followed by centrifugation at 15 000 g for 10 min. The supernatant was called ‘ATP-extract’. From the precipitate, cortical proteins were extracted Exptl Cell Res 82 (1973)
with 0.6 M KC1 containing 10 mM nhosphate buffer (pH 7.0) or 10 mM Tris-HCl (pH 8.4) ‘for 1 h and centrifuged at 15 000 R for 10 min. The nrecinitate was once more extracted with 0.6 M K-Cl and an insoluble material, ‘membrane fraction’, was obtained after centrifugation. After suspending in 0.6 M KCI, a number of cell membranes and few cytoplasmic particles were observed under a phase microscope. This fraction consisted mostly of cellular and some vesicular membranes when viewed by an electron microscope [16]. The first KCl-extract was adjusted to 55 % saturation with respect to ammonium sulfate and precipitates thus formed, “55 % SAS fr”, were collected by centrifugation at 10000 g for 10 min, followed bv dissolution and dialysis against 10 mM phosphate -buffer (pH 7.0) to - precipitate ‘cortex protein’.
Determination of PCMB bound to proteins The principle of the determination was based on the fact that organic mercurials which bound to protein -SH groups could easily be dissociated in acidified organic solvents such as acetone or alcohol [29]. The reacted protein was precipitated by addition of acetone (final 90%) and washed once with pure acetone. To the final orecioitate. washed 3 times with 95 % ethanol, was added acidified ethanol (one drop of concentrated HCl in 5 ml 95 % ethanol) to 5 ml to dissociate PCMB from protein. The amount of PCMB was assaved bv titration with alutathione Il. 21. In several series of preliminary det&minations‘with excess protein and known amount of PCMB, a recovery of lOOf4.9 % was obtained. Since sea urchin eggs contain as much as 6 mM glutathione [21], bound PCMB in the cortex might be dissociated in the course of isolation of cortices. This possibility was checked as follows. First, cytoplasmic bulk protein of sea urchin eggs was incubated with a known amount of PCMB to react completely. Since eggs were homogenized in 9 vol of isolation medium, the glutathione cont. in the homogenate was in the range of 0.6 mM. Therefore, 1.0 mM glutathione was mixed with the PCMB-bound protein and kept standing for 15 min at room temperature, followed by dissociation and determination of PCMB. The recovery of PCMB was less than 50 % depending on the incubation time as expected. Since Ca or Mg ions inhibit the reactivity of thiol groups, incubation of the PCMB-bound protein was performed in the presence of 1 mM glutathione plus 5 to 20 mM CaCl, or plus 0.1 M MgCI,. In those cases, recoveries of PCMB were 100 + 4.9 (6 series of determinations) or 68 + 9.2 % (8 se& of determinations). respectively. Therefore, 5 mM CaCl, was used throughout the present experiments to prevent release of bound PCMB by intracellular glutathione during the isolation of cortices. In the experiments with other reagents, the isolation of cortices was performed by the original procedure [22].
Assay of cortical ATPase activity The standard assay was carried out in a total volume of 2.0 ml (17), which included: 20 mM Tris-HCl buffer (pH 7.0), 10 mM MgCl,, 0.15 M KCI, 0.5
Binding sites qf’
e\rracted
with
I mM
ATP.
IO mM
phosphate
buffer.
SH wagents
327
pH 7.0
PClkl cxlracted phosphate wntrlfuged.
with 0.6 M KCI. IO mM buffer, pH 7.0, I h 15 000 @ IO mill
Pellet washed wth centrifuged,
d~s\ol\ed d!al>\ed
I” 0.1 M KU, ;!e;un\t IO mM
IO mM phosphate phosphate buffer.
0.6 M KCI 15000 g IO ,n,~n
buffer pH 7.0
mM ATP and about 0.5 mg protein of enzyme fraction. Incubations were performed at 25°C for 15 min. The reactions were stopped with 0.5 ml of 30% trichloroacetic acid (TCA). After removal of precipitates by centrifugation, inorganic phosphate in the supernatant was determined by the method of Fiske & Subarrow [7]. Controls were determined by incubating without addition of the enzyme fraction and were subtracted from the total Pi liberated in the presence of the enzyme fractions.
Determination of protein - SH and NPSH groups Total protein -SH groups were assayed by the use of mercury orange [29]. For the determination of NPSH groups in eggs (mostly glutathione [21]) treated with PCMB, aliquots of unfertilized or fertilized eggs were quickly washed twice with CFSW to remove free PCMB. To the packed egg mass were added 5 ml of 10% TCA containing 4 mM EDTA to extract NPSH and allowed to stand for 30 min at 0°C. The suspensions was centrifuged at 1 000 g for 5 min and the pH of aliquots of the supernatant was adjusted exactly to 8.0 with NaOH; NPSH groups were measured by Ellman’s method [6]. The same extraction was conducted in the case of treatment with NEM. Thereafter, the TCA-extract was directly assayed for NPSH groups by iodometric titration after oxidation of ascorbic acid by 2,6-dichlorophenol indophenol [21].
Fig. 1. Fractionation of protein components of isolated cortices.
Determination of radioactivity After fertilized eggs were pulse-treated with 0.1 and 0.3 mM NEM-2,3J*C (1.6 mCi/nmole; The Biochemical Centre, Amersham), egg cortices were isolated and protein components were separated. Proteins were precipitated in 90% acidified acetone (one drop of concentrated HCl in 5 ml of acetone), collected onto glass filters (Whatman, GF 83), and washed 3 times with HCl-acetone. In order to determine materials of low mol. wt which are bound with NEM, an aliquot of an equilibrated dialysate of the cytoplasmic fraction (fig. 1) was adsorbed in the glass filters and dried. The amount of radioactivity bound to the dried filters was determined in a toluene scintillation mixture (4 g PPO, 0.2 g Me,POPOP in 1 1 toluene). The counting efficiency for 14C was 86 U0 with an Aloka LSC 501 scintillation counter. The data shown in all figures and tables are based on assays which were carried out in duplicate.
RESULTS AND DISCUSSIONS Inhibition
of cleavage by ~ SH reagents
Two series of experiments were carried out to determine the effect of both continuous and pulse treatment with -SH reagents. In the first, the critical cont. of -SH reagents for Exptl Cell Res 82 (1973)
328 Y. Okazaki et al. what stage of the cell cycle was pulse-treated. It could be recognized that the egg became rather resistant before cleavagewhen exposed to lower cont. of the inhibitor as shown in fig. 3. After these experiments, the shortest duration of the pulse-treatment for 100% inhibition was determined. The concentration of the inhibitors, 1 mM IAA for Pseudocentrotus and 0.3 mM NEM for Anthocidaris Fig. 2. Abscissa: cont. - SH reagents (mM); ordinate: eggs, were chosen from the experiments % cells not cleaved. n, PCMB; 0, PCMPS; 0, IAA; l , NEM. Cont. mentioned above. As shown in fig. 4, when -SH reagents for inducing inhibition of cleavage treated at any stage of the cell cycle, full by treating eggs continuously after fertilization (Pseudocentrotus). inhibition of cleavage was caused by a pulsetreatment as short as 3 min with NEM. One inhibition of cleavage was determined. Sea mM IAA, on the other hand, exhibited 100% urchin eggs were continuously exposed to the inhibition of cleavage after 7 min treatment. inhibitors from 5 min after fertilization. The However, the sea urchin specieswas different results are shown in fig. 2.0.14 mM alkylating from that used in the experiments with reagents blocked cleavage completely, while NEM. the same PCMB or PCMPS cont. showed no inhibition. In cases of such mercaptide Change in NPSH level by the treatment with forming reagents, a cont. higher than 0.5 - SH reagents mM was required for the inhibition. It was our expectation that -SH reagents Secondly, fertilized eggswere pulse-treated react faster with intracellular glutathione with inhibitors for 5 or 10 min at regular than with protein-SH groups because of the intervals as indicated in fig. 3a, b, One mM high glutathione cont. within the cell. When PCMB or PCMPS showed no delay of cleav- fertilized eggs at the late monaster stage age. On the other hand, 1 mM IAA or 0.3 mM (about 30 min after fertilization at 20°C) NEM completely blocked cleavage no matter were exposed to 1.5 mM PCMB, NPSH a
-t------t------
h
a---
‘-+ Cf
Fig. 3. Abscissa: time after insemination (min), stages of pulse treatment are indicated beneath; ordinate: % cells not cleaved. Inhibition of cleavage by pulse treatment with alkylating reagents. Fertilized eggs were pulse-treated for 5 and 10 min at regular intervals indicated along the abscissa with (a) IAA, 1 mM (0) and 0.3 mM (0) for 10 min (Pseudocentrotus); (b) NEM, 1 mM (o), 0.3 mM (0), 0.1 mM ( x ), 0.03 mM (A), and 0.01 mM (0) for 5 min (Anthocidaris). Exptl Cell Res 82 (1973)
Binding sites of ~- SH reagents
groups decreased to half the original level after a 10 min treatment (fig. 5~). This treatment routinely produced a complete block of cleavage. A similar time course for the decreasein NPSH groups was observed with 0.3 mM PCMB, which did not inhibit the first cleavage at all. In both cases,the NPSH level remained stationary after 10 min treatment. Quite similar patterns in the decrease of NPSH groups were observed in Hemicentrofus eggs also. These were rather intriguing observations, because PCMB was 25 times equivalent to intracellular glutathione under the present experimental conditions. This may be explained by assuming the existence of barriers which prevent further diffusion of PCMB into the cell. Fig. 5b shows the effect of NEM on the NPSH level of sea urchin eggs. It was shown that when pulse-treated with 0.1 mM NEM for 3 min, the level in metaphase cells decreasedto 40 ‘%,of that of intact cells (fig. 5b, II); no inhibition of cleavage being observed. However, further decreasesin NPSH groups were detected after a 5 min treatment with 0.3 mM NEM; cleavage no longer occurred (fig. 5 b, III). Similar results were obtained by determining bound NEM with the radioactive reagent. When metaphaseeggs
:’
20. 0
329
I
2
3
4
5
6
7
8
9
IO
4. Abscissa: time of pulse treatment (min); ordinate: 96 cells not cleaved. Inhibition of cleavage by pulse treatment with alkylating reagents. Metaphase cells were pulsetreated for the time indicated with 0.3 mM NEM (0) for Anthociduris and 1.O mM IAA (:I ) for
Fig.
Pseudocentrotus.
were pulse-treated with 0.1 and 0.3 mM for 3 min, the amount of radioactive NEM which was assessed to bind with NPSH groups within the cell corresponded to 55 and 85 % level of total NPSH groups, respectively. These values were similar to the data, 64, and 80 %, obtained from the measurement of NPSH groups. In all cases,the amount of NPSH groups remained constant after the pulse treatment, indicating no synthesis of glutathione thereafter. Comparing the data shown in fig. 5b with those of fig. 5a, it was evident that NEM penetrated into the egg faster than PCMB. Furthermore, it was clear from fig. 5b, curve
Fig. 5. Abscissa: time after beginning of treatment; ordinate:
pmoles NPSH as glutathione/egg. (a) Change in NPSH groups of fertilized eggs treated continuously with 0.3 mM (0) and 1.5 mM ( l ) PCMB from 30 min after fertilization (Anthocidaris); (b) change in NPSH groups of eggs treated with NEM. I, unfertilized eggs, 0.3 mM, 5 min; II, metaphase eggs, 0.1 mM, 3 min; III, metaphase eggs, 0.3 mM, 5 min. Arrow NEM and SW indicate beginning and end of pulse treatment, respectively. Exptl Cell Res 82 (1973)
330
Y. Okazaki et al.
Table 1. Time course of PCMB-binding
in the cortex (Hemicentrotus)
10, 20, 30, and 40 min after the beginning of the treatment, eggs were collected and washed with CFSW to remove free PCMB, followed by the isolation of cortices Concentration of PCMB at 0.4 mM Time of treatment with PCMB (min) 0 10 20 30 40
1.0 mM
nmoles PCMB bound/mg protein
Blocked SHa calculated ( %)
0
0
3.5 T.7
5.8
-11.1
nmoles PCMB bound/mg protein
Blocked SHa calculated ( %)
0
0
::: 20.4 13.2
15.1 10.7 33.8 21.8
a Total protein - SH groups in the cortex at the beginning of treatment: 60.5 nmoles/mg protein.
I that unfertilized eggs were more resistant than fertilized eggs to the penetration of the reagent (cf curve III). This may be due to the well known fact that the membrane of the unfertilized egg is less permeable than that of the fertilized egg. A considerable number of such pulse-treated unfertilized eggs could be fertilized, but did not develop. Binding sites of PCMB and inhibition of cleavage
It is necessary to remove fertilization membranes for the isolation of cortices and the procedure requires about 20 min for replacing eggsfrom 1 M urea to CFSW. Therefore, the PCMB-treatment started at 30 min after
insemination. Preliminary experiments showed that when eggs were continuously treated with 1 mM PCMB from 30 min after fertilization, cleavage was inhibited, while with 0.4 mM PCMB no inhibition was observed. It was found that bound PCMB gradually increased during the treatment (table 1). Assuming that PCMB binds specifically with the -SH groups of cortices under the experimental conditions, 10 % of total - SH groups was blocked after 40 min-treatment with 0.4 mM PCMB; under these conditions, no inhibition of cleavage was observed. On the other hand, more than 30 % of the ~ SH groups found in the isolated cortices were
Table 2. Binding of PCMB to protein components of cortices (Hemicentrotus) Fertilized eggs were treated with 1 mM PCMB in CFSW for 40 min, washed with CFSW to remove free PCMB and isolation of cortices and fractionation of protein components were carried out as described in Method
Fraction
Protein (md
nmoles PCMB bound
ATP-extract Cortex protein Membrane fraction
1.25 1.65 5.95
9.4 77.5 16.5
a Values measured at the beginning of treatment. Exptl Cell Res 82 (1973)
nmoles PCMB/mg protein (A)
nmolesa SH/mg protein (B)
A/B Xl00
7.5 47.0 2.8
78.0 82.0 57.0
9.6 57.3 4.9
Binding sites uj’ - SH reagents
33 I
Table 3. Decrease in - SH groups in protein fractions of cortices by pulse treatment with NEM qf metaphase eggs (Anthocidaris) (expressed as nmoles - SH per mg protein) Treatment with Fraction Cytoplasmic fraction Control Treated ATP-extract Control Treated Cortex protein (55“<,SAS fr) Control Treated Membrane fraction Control Treated
0.1 mM, 3 min
0.3 mM, 3 min
0.3 mM, 5 min
0.3
53.3 53.7 (~t1.3)
48.5 45.8
53.3 47.6
( 10.7)
41.9 41.7
( 10.9)
74.0 73.0(-1.4)
74.9 68.8 (-8.1)
74.0 6X.5
( ~~7.4)
71.8 53.0
( 26.2,
69.8 68.5 (-1.9)
69.8 52.5
(- 24.8)
69.8 46.4
( 33.5)
71.8 37.3
( 48.0)
35.2 35.5
40.0 37.9
(-- 5.3)
35.2 31.6
(-- 10.2)
36.1 28.2
(- 21.9)
(7 0.9)
( -5.6)
mM, 7 min
Values in parenthesesindicate decreasein - SH groups in percentage.
blocked when the cont. was increased up to 1 mM, which caused 100 Y0inhibition of cleavage. The conditions of these experiments were similar to those in fig 5a. Therefore, the cleavage inhibition is not due to the decrease of NPSH, but to that of protein-SH groups in the cortex to the extent of 30%. The binding sites of PCMB were further analysed by fractionation of the protein components of the isolated cortices. As shown in table 2, more than half of the PCMB bound in the cortex was detected in the cortex protein fraction. The membrane fraction did not have so many sites for binding PCMB and the ATP-extract, in which tubulin might be involved at metaphase [17], did not show a significant amount of binding under the present experimental conditions. The highest binding of PCMB was found in the cortex protein fraction, when calculating the magnitude of block of -SH groups as shown in table 2. Therefore, it was strikingly apparent that the binding of PCMB to the cortex was mainly due to the reactivity of the cortex protein fraction.
Binding sites of NEM in suppression
qf cleaaage
A similar experiment was designed to establish the action of NEM on cleavage inhibition. The inhibitory effect of alkylating reagents on cell division was much stronger than that of mercaptide forming reagents as described in figs 2 and 3. Furthermore, only a short pulse-treatment was necessary for 100 O: inhibition of cleavage (fig. 4). Preliminary experiments with unfertilized eggs indicated that -SH groups decreased only in the cortex protein fraction after a 5 min pulse-treatment with 0.3 mM NEM. On the contrary, the ATP-extract and the the membrane fraction revealed little change in their -SH amount. This was probably due to the free reactivity of -SH groups of the cortex protein in unfertilized eggs. It should also be mentioned that the -SH groups of the cytoplasmic proteins did not change in amount by the treatment. This may mean that the reagents were trapped one after another by a ‘cortical ~~SH barrier’ and could not penetrate into the cytoplasm or that NPSH groups, that is those of 6 mM Exptl Cell Res 82 (1973)
332 Y. Okazaki et al. glutathione within the cell, reacted quickly with NEM to prevent alkylation of the cytoplasmic proteins. To clarify which possibility was responsible, the level of NPSH in unfertilized eggs was assayed before and after the pulse-treatment with 0.3 mM NEM. The data shown in fig. 5b supported the latter possibility. In general, the cell is rather resistant after the formation of the mitotic apparatus to anti-mitotic agents and to various reagents which inhibit respiration, oxidative phosphorylation and so on. The same held true with low cont. of alkylating reagents. However, as the cont. was increased, cell division was completely blocked even if the eggs were treated after the completion of nuclear division. In order to determine the minimal binding of alkylating reagent necessary to induce inhibition of cleavage, the NEM cont. and the duration of pulse treatment were selected as 0.3 mM and 3 min, respectively, from the data shown in fig. 4. For comparison, treatments with 0.1 mM, 3 min; 0.3 mM, 5 min; and 0.3 mM, 7 min were also chosen. The results are shown in table 3, where a significant decrease in -SH groups of the cortex protein fraction is shown when eggs are pulse-treated for 3 min. The cytoplasmic bulk protein, the ATP-extract and the membrane fraction showed little change in their -SH amount. Assuming that -SH groups in the cortex protein fraction available for the reaction with NEM were about 70 % of total -SH groups, including masked ones [23], alkylation of 36 % of the available -SH groups results in 100% inhibition of cleavage. After increasing the time of the pulsetreatment (5 and 7 min), a considerable amount of NEM bound to all of the fractions. On the other hand, 0.1 mM, 3 min treatment resulted in no inhibition of cleavage and little change in the -SH content of all Exptl
Cell Res 82 (1973)
fractions. This is apparent from the data shown in fig. 5b, II. That is, in the case of 0.1 mM NEM, the intracellular glutathione quickly trapped the incoming NEM to protect the cortical proteins from alkylation during the 3 min pulse treatment. This was confirmed by the fact that the 0.1 mM reagent was only 1.7 times equivalent to NPSH groups in the sea urchin egg under the conditions of these experiments (see Method). To confirm the results on the effect of NEM, bound NEM was directly assayed by the use of 14C-NEM. Consistent with the results as documented above, little binding of the reagent was detected in all the fractions when eggs were pulse-treated for 3 min with 0.1 mM radioactive NEM. When the cont. was raised to 0.3 mM, on the other hand, the radioactivities in the cortex protein fraction were the most significant. Among them, the membrane fraction showed very high activities. This is apparently different from the data obtained from the experiments with PCMB. This discrepancy may be explained by the lower specificity of NEM for thiols [30] than PCMB. The treatment with NEM, therefore, might also involve binding of the reagent to sites other than -SH groups. Effect of NEM on ATPase activity in the cortical layer An ATPase activity in the cortex of the sea urchin egg was first reported by Miki [18] and a fluctuation of the activity was found closely associated with the division cycle. Later, it was demonstrated that addition of ATP induced the progress of furrowing in a glycerinated dividing sea urchin egg [15] similar to a telophase model of fibroblast [lo, 141.Furthermore, it was recently demonstrated that an ATPase extractable concomitantly with the cortex protein from the isolated cortices could be separated from the cortex protein and partially purified by
Binding sites of
-~ SH reagents
333
Table 4. Change in ATPase activities in protein fractions of isolated cortices b), the treatmrnt of metaphaseeggs with NEM (Anthocidaris) (expressedas ,umoles Pijmg proteinlmin) Treatment with Fraction ATP-extract Control Treated Cortex protein (55 “b SASfr) Control Treated Membrane fraction Control Treated
0.3 mM, 3 min
0.3 mM, 7 min
0.0060 0.0078 0.0071 (-I- 18.3 :h) 0.0046 (--41.0%) 0.016 0.013 (- 18.7)
0.026 0.012 ( 53.8)
0.0089 0.0110 ( t- 23.6)
0.0089 0.0070 (-21.3)
Values in parentheses indicate percentage decrease or increase in spec. act.
sucrose density gradient centrifugation [17]. The enzyme (cortical ATPase) possessed dynein-like properties and was activated by the cortex protein through the formation of a complex. Other ATPases were also detected in both the ATP-extract and the membrane fraction, which have been considered to be ‘cytoplasmic’ and ‘membrane’ ATPases, respectively. Those facts led to the experiments to examine the effect of NEM on the ATPases involved in the three protein fractions of the cortical layer. After metaphase, eggs were pulse-treated and each protein fraction of the isolated cortices was subjected to the assay for ATPase activity as shown in table 4. The results indicated that the enzyme activity in the cortex protein fraction decreased about 20 % consistently, while the activities in other fractions slightly increased when pulsetreated with 0.3 mM NEM for 3 min. The decreases in activity were due to blocking of -SH groups in the cortical ATPase beyond a certain level [ 171. The increases of the activities in the ATP-extract and the membrane fraction could also be explained by the fact that ATPases are activated if treated with a low concentration of -SH reagents [12, 17,
201. This is consistent with the fact that cortical components obtained as ATP extract and membrane fraction were less susceptible to -SH reagents than the cortex protein. Furthermore, activities in all the fractions decreased when the treatment was longer (7 min), possibly because of further loss of their -SH content (table 3). It can be said that the ATPase which comes into solution with the cortex protein is the most sensitive to the action of NEM in situ. It was previously demonstrated that the cortical ATPase was activated by the purified cortex protein rich in -SH groups and inactivated by the same protein when poor in -SH content [17]. Furthermore, the fluctuation of the activity was quite coincident with that of the -SH content in the cortex protein fraction during the division cycle (cf ref [ 181 with [23]). Therefore, the fluctuation of the ATPase activity found by Miki might be explained by an interaction between the cortical ATPase and the cortex protein [17]. This was further suspected from the fact that both the proteins formed complexes at the ionic strength of 0.1 in vitro. The cortex protein seemed to be localized underneath the cell membrane [16]. It is likely that the Exptl Cell Res 82 (1973)
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cortical ATPase exhibits the same behavior concerning its localization. This situation could be one of the reasons why the cortex protein and the cortical ATPase were preferentially attacked by -SH reagents. At the critical treatment to induce inhibition of cleavage (0.3 mM NEM for 3 min), the inhibition of the ATPase activity was rather small (20%). This may indicate that the binding of -SH reagents to the cortex protein affects the interaction between the cortex protein and the ATPase more profoundly than either protein alone inducing a modification which leads to failure of cytokinesis. CONCLUSION
treatment could be mainly attributed to a modification in the cortical layer. This might involve alteration in the mode of interaction between the cortex protein and cortical ATPase. REFERENCES 1. Benesch, R & Benesch, R E, Meth biochem anal 10 (1962) 43. 2. Boyer, P D, J Am them sot 76 (1954) 4331. 3. Brachet, J, Chemical embryology, p. 170. Interscience, New York (1950). 4. Brachet, J, Biochemical cytology, p. 194. Academic Press, New York (1957). 5. Brachet, J. The biochemistry of development. n. 104. Pergamon Press, London (1960). ^ 6. Ellman. G L, Arch biochem bionhvs _ - 82 (1959) . , 70. I. Fiske, C H & Subarrow, Y, J biol them 66 (1925) 375. 8. Harris, J W, Allen, N P & Teng, S S, Exptl cell res 68 (1971) 1. 9. Hinegardner, R T, Biol bull 137 (1969) 465. 10. Hoffmann-Berling, H, Biochim biophys acta 15 (1954) 226. 11. Kawamura, N & Dan, K, J biophys biochem cytol 4 (1958) 615. 12. Kielley, W W & Bradley, L B, J biol them 218 (1956) 653. 13. Kimura, I, Exptl cell res 79 (1973) 445. 14. Kinoshita, S, Benjamin, S, & Hoffmann-Berling, H, Biochim biophys acta 79 (1964) 88. 15. Kinoshita, S & Yazaki, I, Exptl cell res 47 (1967) 449. 16. Mabuchi, I & Sakai, H, Development growth & differentiation 14 (1972) 247. 17. Mabuchi, I, Biochim biophys acta 297 (1973) 317. 18. Miki. T, Exptl cell res 33 (1964) 575. 19. Neufeld; E F & Mazia, D,‘Exptl cell res 13 (1957) 622. 20. Ogawa, K & Mohri, H, Biochim biophys acta 256 (1972) 142. 21. Sakai, H & Dan, K, Exptl cell res 16 (1959) 24. 22. Sakai, H, J biophys biochem cytol 8 (1960) 603. 23. - Ibid 8 (1960) 609. 24. - Exptl cell res 32 (1963) 391. 25. - Biochim biophys acta 102 (1965) 235. 26. - Ibid 112 (1966) 132. 27. - J biol them 242 (1967) 1458. 28. - Int rev cytol 23 (1968) 89. - Anal biochem 26 (1968) 269. ii: Smyth, D G, Nagamatsu, A & Fruton, J S, J Am them sot 82 (1960) 4600.
It has been established that when sea urchin eggs were exposed to -SH reagents, NPSH groups quickly reacted with them to protect cortical proteins from mercaptide formation or alkylation. When the modifications of the cortical proteins was negligible during the treatment, cell division was never inhibited even when the level of NPSH decreased to less than half of the original level. However, when the treatment was stronger, excess reagents, which were no longer sequestered by NPSH groups, reacted preferentially with the -SH groups of the cortex protein in the cortical layer, resulting in suppression of cleavage. PCMB binds to more than half of the -SH groups in the cortex protein under such a condition. Furthermore, alkylation of cortical proteins and consequent decreasein cortical ATPase activity resulted in a irreversible block of cytokinesis. Since nuclear division was not affected under the present experimental condition with NEM, Received March 9, 1973 the inhibition of cytokinesis by the pulse Revised version received July 3, 1973
Exptl Cell Res 82 (1973)