Actomyosin-like ATPase activity at the surface of fish eggs

Experimental Cell Research 71 (1972) 460-464

ACTOMYOSIN-LIKE

ATPase ACTIVITY OF FISH EGGS

AT THE SURFACE

N.-C. JIZIRGENSEN’ Zoophysiological Laboratory B & C, University of Copenhagen, Denmark

SUMMARY A membranous structure could be isolated by manual dissection from the surface of egg cells from a freshwater fish. This structure has a thickness of about 0.1-0.2 pm and is located round the egg cell underneath the so-called chorion. The isolated material showed a (Caa+/Mg*+)dependent ATPase activity with a specific activity of 1.5 nmoles Pi per mg protein per min. pH-optimum was about 6.3. The ATPase was activated by Ca*+, the Cae+-activation being counteracted by Mga+. Without the addition of Ca 8+, Mg*+ was slightly activating. The effects of Cae+ and Mge+ were dependent upon the ionic strength of the reaction mixture, Mgz+ being inhibitory at a KCl-concentration of 600 mM, where Ca*+ was still activating although to a lesser degree. Na+ was inhibitory. The enzymic activity was very sensitive to SH-inhibitors, more sensitive to mercurials than to inhibitors of the alkylating type, while the heart glycoside ouabain was without effect. The ATPase activity is compared with the ATPase activity of actomyosin to which it shows several similarities. The presence of an actomyosin-like ATPase activity at the surface of egg cells might support the theory that an actomyosin-like system located at the cell surface is involved in the changes of shape.and the cleavage of egg cells.

In recent years growing evidencehas indicated that some of the fundamental properties of the contractile mechanism in myofibrils are common to other systemsof motility as well. One example is cytokinesis, which has mainly been investigated in the case of cleaving egg cells. Addition of ATP to sea urchin eggs, glycerine-extracted during the stage of cytokinesis, was shown to induce the continuation of cytoplasmic cleavage [8], and from a homogenate of sea urchin eggs an actin-like protein was isolated [5]. The intracellular localization of this protein has not been determined. Cortical fibres, however, have 1 Present address: Zoophysiological Laboratory C, August Krogh Institute, University of Copenhagen, 13, Universitetsparken, 2100 Copenhagen 0, Denmark.

Enptl Cell Res 71

been found in several cell types such as fertilized sea urchin eggs [3], and these fibres have some structural resemblance to filaments of the actin-like protein isolated from eggs of sea urchins [5]. Furthermore, indirect evidence has been presented of actinlike filaments in the cleavage furrow of the newt egg [13]. Other observations indicate that the force of cleavage could be associated with the cell surface. In responseto mechanical stimulation the surface layer of fertilized sturgeon and amphibian eggs undergoes a contraction, suppressed by the same inhibitors which arrest egg cleavage [17J.A similar contractile response at the surface of egg cells from both amphibia and sea urchins was shown to be dependent upon Ca2+ [2]. ATPase activity has been found in cortical material of sea urchin eggs, and it reaches a

ATPase activity at egg cell surface

maximum at the metaphase of the division cycle [9]. This ATPase activity, however, was not further characterized. The present paper describesa (Ca2+/Mg2+)dependent ATPase activity located at the periphery of egg cells from a freshwater fish (E’iplatys dageti). The ATPase activity is characterized in regard to pH-optimum, effects of inorganic salts, and sensitivity to inhibitors, and shows several similarities to the ATPase activity of actomyosin, thus supporting the theory that an actomyosinlike system

located

at the ce]] surface might

be active in the cleavage of egg cells [lo]. MATERIAL

AND METHODS

Mature unfertilized eggs of Epiplatys dageti were used, squeezed out from anesthetized fishes (M. S. 222, Sandoz, 1: 2000). The eggs were centrifuged (45 min, 2”C, 10000 g) in a 60% solution of Ficoll (&,400 000; Pharmacia, Sweden) in 300 mM sucrose, in which the eggs were not damaged during the centrifugation. By this procedure a packing of particulate material inside the eggs is obtained. By manual dissection under a stereozoom microscope (magnification x 90) the so-called chorion [16] was removed from the centrifuged eggs, whereafter a membranous structure could be isolated from the surface of the egg cells. The membranous structure has a thickness of about 0.1-0.2 pm and a clear appearance at x 1 300 in phase contrast. The dissection was carried out in the following medium: 300 mM sucrose, 4 mM glutathione, 20 mM histidine/HCl, pH 6.3, which is iso-osmotic with the eggs. The isolated membranous material was washed and centrifuged three times (2”C, 1 750 g) in the same medium as used for dissection. The sediment was homogenized by ultrasound (MSE ultrasonic disintegrator 100 watt model, 6 x 1 min, amplitude: 8 pm, N,-bubbling through the medium, cooling in ice water) with the same medium used above. During the homogenization glass globules (Ballatini no. 14) were added. The preparation was stored at -30°C until use, at most for 4 days. After thawing it was rehomogenized 3 x 1 min under the same conditions as above.

Enzyme assay ATPase activity was determined by measuring the inorganic phosphate released from ATP during 60 min at 3O”C, pH 6.3. The reaction mixture was 50 ~112 mM ATP (disodium salt), 5O,ul80 mM histidine/ HCl buffer, and 100 ~1 enzyme preparation. The final ATP concentration was 3 mM. The reaction medium wasvaried by addition of salts or inhibitors to the buffer solution. Inorganic salts were present as

461

chlorides. The following inhibitors were used: ouabain, p-chloromercuribenzoate (PCMB), salyrgan (sodium salt of o-[(3-hydroxymercuri-2-methoxypropyl) carbamoyl] phenoxyacetic acid), iodoacetamide, and N-ethylmaleimide (NEM). When inhibitors were used glutathione was not added to the enzyme preparation. The pH-dependence of the enzymic activity was determined in the pa-interval 4.5-9.5 in a reaction mixture composed of 50 yl 12 mM ATP (disodium salt), 50 ~1 120 mM buffer with varying amounts of KC1 but the same quantity of CaCl, added, and 100 ,ul enzyme preparation. The final concentration of CaCl, was 20 mM and of KC1 100 mM in all assays. Buffers used were: acetic acid/KOH (pH 4.5-6.0), glycyl-glycine/KOH (pH 6.5-&O), and glycine/KOH (pH 8.5-9.5). Inorganic phosphate was determined calorimetrically by the method of Baginsky et al. [l] adapted to an assay volume of 200 ~1 and absorbance was read at the absorbance maximum at 860 nm. All experiments were carried out in duplicate. ATPase activity is expressed as nmoles P,/ml enzyme extract per hour. One ml enzyme extract contained the membranous material from 20 eggs. Dry matter and protein: Dry matter was determined on enzyme samples dialysed 120 h against glass distilled water, and protein in the dry matter was estimated by micro Kjeldahl (protein factor: 6.25). All reagents were commercial samples of analytical grade. Glass distilled water was consistently used.

RESULTS When Kf and Ca2+ were present in the medium the pH-optimum was between 6.0 and 6.5 with an average of 6.3 from 10 assays. In all experiments the enzymic activity showed a rather steep decline towards the basic side, seefig. 1. Enzyme preparations from membrane material of 20 eggs in a total of 1 ml extraction medium gave ATPase activities of about 12.9 nmoles P,/ml per hour (from 6 to 33) corresponding to 32.4 nmoles/mg dry matter per h or 1.5 nmoles/mg protein per min. These activities were assayed without salt added to the reaction medium. The results given in table 1 show that while Ca2+ has a pronounced activating effect on the ATPase activity Mg2+ is much less effective at the concentrations used. At Mg2+-concentrations below 10 mM there is a slight activation. It varies from 7 % to 39 %, which is outside the variation found in Exptl Cell Res 71

462

N.-C. Jwgensen

100 - /*. I\ i

Table 2. Influence of the concentration of KC1 on the Ca2+- andMg2+-activation of an ATPase present in the surface of fish eggs Reaction mixture: 3 mM ATP, 20 mM histidine/HCI buffer and different concentrations of KCI, KC1 plus

l

10 mM CaCI, or KC1 plus 10 mM MgCI,. Reaction time: 1 h at pH 6.3 and 30°C. ATPase activity: nmoles P,/ml enzyme extract. All figures are averages from 2 enzyme assays

5o-,

5

,

1,

6

.7

nmoles Pi/ml enzyme extract

.

Expt

8

Fig. I. Abscissa: pH; ordinate: %maximalactivity. Effect of pH on the activity of an ATPase present

in the surface of fish eggs.Experimental conditions are describedin text.

double assays(below 5 % in all experiments). On the other hand the activation by Ca2+ isWclearly blocked by the presence of Mg2+ (table 1). As seen in table 2 the effects of Ca2+ and Mg2+ on the enzymic activity depend upon the ionic strength of the reaction mix-

ture. The Ca2+-activation decreases with Table 1. Influence of Ca2+ and Mg2+ on the activity of an ATPase present in the surface of fish eggs Reaction mixture: 3 mM ATP, 20 mM histidine/HCl buffer and different concentrations of CaCl, and MgCl,. Reaction time: 1 h at pH 6.3 and 30°C. ATPase activity: nmoles P, per ml enzyme extract. All figures are averages from 2 enzyme assays nmoles Pi/ml enzyme extract Expt. 0 5 mM 10 mM 15 mM 20 mM 0 5 mM 10 mM 15 mM

1

2

10.5 Ca*+ 11.9 Caa+ 16.8 Cae+ 18.9 Caa+ MgB+ MgS+ MgS+

5 mM Ca’f 10 mM Mg”+ Exptl Cell Res 71

18.6 19.0 20.6 20.6

3

4

8.9 12.4 19.5 29.3

8.8 30.4 13.6 43.4 15.8 56.5 16.7 63.7 18.9 8.8 30.4 11.0 31.9 11.0 36.2 10.1 33.3

33.4 41.0 43.1 44.2

31.9

32.3

8.9 12.4 9.8 8.9

5

6

33.4 35.6 32.0 32.0

0

10 mM 100mM 600 mM 10 mM 100mM 10 mM 600mM 10 mM 10 mM 100mM 10 mM 600 mM

Cae+ KC1 KC1 Ca*+ KC1 Ca*+ KC1 Mg2+ Mg*+ KC1 Mgs+ KC1

1

2

12.8 37.3 12.8 11.0 27.5

6.3 17.1 8.9 7.6 15.2

18.9

13.3

15.3 14.1

7.6 8.9

0

3.2

increasing ionic strength, whereas Mg2+ at high ionic strength (KC1 concentration 600 mM) inhibits the enzymic activity. From table 3 it appears that the ATPase activity

is inhibited

by NaCl.

KC1 in the

same concentration interval was without significant effect. The enzymic activity turned out to be very sensitive to SH-inhibitors, whereas the heart glycoside ouabain, efficient against the (Na+/ K+)-activated ATPase, was here without any effect. The results of the inhibitor experiments are given in table 4. The enzyme was preincubated with the inhibitor for 1 h before starting the reaction by addition of ATP. In these experiments the reaction medium did not contain glutathione, which appeared to counteract the effect of the SH-inhibitors applied. Taking into consideration

the con-

ATPase activity at egg cell surface

Table 3. Influence of NaCZ on the activity of an ATPase present in the surface of fish eggs Reaction mixture: 3 mM ATP, 20 mM histidine/HCl buffer and different concentrations of NaQ. Reaction time: 1 h at pH 6.3 and 30°C. ATPase activity: nmoles Pi/ml enzyme extract. All figures are averages from 2 enzyme assays nmoles Pi/ml enzyme extract Expt

1

0

10.6 6.9 6.9 6.9 3.8

20 mM 40 mM 80 mM 160 mM

NaCl NaCl NaCl NaCl

centrations of the inhibitors used, salyrgan has the strongest effect and NEM the weakest. Na+glycerophosphate, which is a substrate for the unspecific phosphatase, could only to a small extent serve as a substrate for the enzymic preparation, as reaction mixtures with 3 mM Na-/?-glycerophosphate and the same reaction conditions as for determination of ATPase activity only showed 20% of the activity shown towards ATP. It was not possible to demonstrate an ATPase activity having these properties in Table 4. Inhibition by different enzyme inhibitors of the activity of an ATPasepresent in the surface of fish eggs Reaction mixture: 3 mM ATP, 20 mM histidine/HCl buffer and inhibitor. Preincubation with inhibitor: 1 h. Reaction time: 1 h at pH 6.3 and 30°C. ATPase activity: nmoles Pi/ml enzyme extract. All figures are averages from 2 enzyme assays nmoles P,/ml enzyme extract Expt 0 2 mM NEM 2 mM iodoacetamide

1O-4M PCMB lo-” M salyrgan 1O-4M ouabain

1

2

10.5 9.3 4.3

8.5 5.8 3.2 4.8 3.2 8.5

7.4

5.0 10.5

preparations made from the cytoplasm (ovoplasm) of the egg cells used, although another ATPase activity inhibited by Ca2+ was easily detected in the cytoplasm. It is therefore unlikely that the ATPase activity measured should be due to cytoplasmic contamination. DISCUSSION

2 28.8 23.0 23.0 21.6 18.1

463

The ATPase activity present in the isolated membranesis very labile. The specific activity is also very small: 1.5 nmoles P,/mg protein per min (without activating salts added to the reaction medium) compared with the specific activity of the isolated Cae+-activated ATPase from striated muscle (0.257 pmoles P,/mg protein per min [4]). It is, however, of the same order of magnitude as the actomyosin-like ATPase isolated from tumour cells (0.0027 ,umoles Pi/mg protein per min

Fl). The pH-optimum determined (6.3) is in fairly good agreement with the pH-optimum of the ATPase activity of actomyosin as demonstrated under similar experimental conditions (6.2-6.5 [ll]). Other properties common with actomyosin are: The strong activation by Ca2+ and a slightly activating effect of Mg2+ at the lower concentrations [4] (table l), the strong inhibition of Ca2+-activation by Mg2+ [l l] (table l), the divergent effects of Ca2+ and Mg2+ at high ionic strength, particularly the different effects of Mg2+ at low concentration of KC1 and at 600 mM KCl, so characteristic for the ATPase activity of actomyosin [4] (table 2), the inhibiting effect of KC1 on the activation by Ca2+ [4] (table 2), and the sensitivity to SH-inhibitors in connection with lacking reaction to ouabain [15] (table 4). It is also remarkable that the strongest inhibitory effect was obtained by salyrgan, claimed to be one of the most specific inhibitors for enzymes of the actomyosin type [6]. FurtherExptl Cell Res 71

464 N.-C. Jmgensen more the inhibitor was used in the sameconcentration as is active against actomyosin. The much smaller effect of the alkylating inhibitors used (NEM and iodoacetamide) as compared with the mercurials (salyrgan and PCMB) is also of some interest as myosin SH-groups react readily with mercurials but are rather resistant to alkylation [15]. The pronounced inhibitory effect of Naf is also worth noting as Na+ inhibits the K+-activated activity of myosin [14]. The lacking effect of ouabain compared with the influence of salts on the ATPase activity described here makes the presence of any appreciable (Mg2+/Na+/K+)-ATPase activity in the enzyme preparation investigated rather unlikely. Of course it might be eclipsed by the (Ca2+/Mg2+)-dependentATPase present. The membrane material used for the preparation should certainly contain the plasma membrane of the egg cell but only as a minor part. In addition to this the presence of an active (Na+/K+)-pump in the plasma membrane of mature egg cells from a freshwater fish is not very likely as these cells have practically no exchange with Naf and Kf in the outer medium [7]. In further investigations of the biological significance of the ATPase activity other possibilities ought to be considered in addition to its possible action in the changes of shape and the cleavage of the egg cell. A participation in the so-called fertilization impulse [16] might be considered. This fertilization impulse cannot be started without the presence of Ca2+ in the outer medium, Ca2+ apparently being released from the cortex of the egg cell on stimulation [16],

Exptl Cell Res 71

and some indirect evidence suggests the participation of ATP in the fertilization impulse [12]. A possible role in the release of the contents of the cortical alveoli during the activation of the egg cell might also be taken into consideration. A closer electronmicroscopic investigation of the isolated membranous structure including a more precise localization of the ATPase activity has been taken up. The author wishes to thank Professor S. 0. Andersen for valuable help in preparing the manuscript. His thanks are also due to Mrs Birgit Blytmann Jorgensen and Miss Winnie Taagerup for excellent technical assistance.

REFERENCES 1. Baginsky, E S, Foa, P P & Zak, B, Clin chim acta 15 (1967) 155. 2. Gingell, D, J embryo1 exptl morph01 23 (1970) 583. 3. Harris, P, Exptl cell res 52 (1968) 677. 4. Hasselbach, W, Z Naturforsch 76 (1952) 163. 5. Hatano, S, Kondo, H & Miki-Notimura, T, Exptl cell r-es55 (1969) 275. 6. Hoffmann-Berling, H, Biochim biophys acta 19 (1956) 453. Jorgensen, N-C. Unpublished results. I: Kinoshita, S & Yazaki, I, Exptl cell res 47 (1967) 449. 9. Miki, T, Exptl cell res 33 (1964) 575. 10. Miki-Noumura, T & Kondo, H, Exptl cell res 61 (1970) 31. 11. Mommaerts, W F H M & Seraidarian, K, J gen physiol 30 (1947) 401. 12. Okazaki, R, Exptl cell res 10 (1956) 476. 13. Perry, M M, John, H A & Thomas, N S T, Exptl cell res 65 (1971) 249. Seidel, J C, Biochim biophys acta 189 (1969) 162. :I Singer, T P & Barron, E S G, Proc sot exptl biol med 56 (1944) 120. 16. Yamamoto, T, Int rev cytol 12 (1961) 361. 17. Zotin, A I, J embryo1 exptl morph01 12 (1964) 247. Received August 9, 1971 Revised version received October 14, 1971