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Separation of the fertilization membrane in Urechis and sea urchin eggs as a phenomenon caused by breakage of hydrogen bonds
467
Experimental Cell Research 41, 467-472 (1966)
SEPARATION
OF THE
URECHIS AND CAUSED
FERTILIZATION
SEA URCHIN
BY BREAKAGE
IV. D E N A T U R I N G
EGGS
MEMBRANE
IN
AS A PHENOMENON
OF HYDROGEN
ACTION OF PROTEOLYTIC
BONDS ENZYMES
S. I S A K A
Biological Institute, Chiba University, Konakadai, Chiba, Japan Received July 8, 1965
LUNDBLADr e p o r t e d
that s o m e proteolytic activity was f o u n d in extracts of sea u r c h i n s p e r m a t o z o a [4], a n d B u n n s t r 6 m [61 a n d Moore [5] o b s e r v e d that p u r e trypsin c a u s e d the elevation of the fertilization m e m b r a n e in unfertilized sea u r c h i n eggs. T h e s e results h a v e led the a u t h o r to investigate the effect of several proteolytic e n z y m e s on m e m b r a n e elevation in some m a r i n e ova. Although trypsin causes m e m b r a n e elevation in Urechis eggs, this e n z y m e has less effect on eggs of the sea urchin, Anthocidaris crassispina; m e m b r a n e elevation occurs only sporadically. H o w e v e r , it was f o u n d that some proteolytic e n z y m e s like p a p a i n are v e r y effective in elevating the fertilization m e m b r a n e in eggs of Urechis, starfish a n d some sea urchins. Proteolytic e n z y m e s are generally divided into three categories. T h o s e belonging to the first are inhibited b y diiso-propyl p h o s p h o f l u o r i d a t e a n d not inhibited b y thiol reagents. T h e second category includes several enzymes, like p a p a i n a n d cathepsins, w h o s e activity d e p e n d s on the p r e s e n c e of one or m o r e thiol groups a n d w h i c h m a y b e inactivated b y various reagents w h i c h c a n r e a c t with thiol groups. Metalloproteinases are in the third category; they are f o u n d to be almost inactive in m e m b r a n e elevation. H o w e v e r , c r u d e p a p a i n causes full m e m b r a n e elevation in all the eggs of Anthocidaris crassispina after the addition of s u c h activators as Versene a n d thioglycolate. As c r u d e papai~n is k n o w n to contain l y s o z y m e , ficin a n d c h y m o p a p a i n , a n e x p e r i m e n t w a s c a r r i e d out with crystalline papain; it was f o u n d that p u r e p a p a i n v e r y effectively p r o d u c e s m e m b r a n e elevation in the p r e s e n c e of activators, while no m e m b r a n e elevation was o b s e r v e d w h e n p a p a i n alone was applied. In this investigation the effect on m e m b r a n e elevation of the proteolytic e n z y m e s in the s e c o n d category was m a i n l y studied in m a r i n e ova. It is 3 1 - 661802
Experimenlal Cell Research 41
468
S. Isaka
s u g g e s t e d t h a t t h e d e n a t u r i n g a c t i o n of t h e s e e n z y m e s is r e s p o n s i b l e f o r m e m b r a n e e l e v a t i o n : s o m e p r o t e i n a s e s s e e m to b r e a k h y d r o g e n b o n d s i n t h e v i t e l l i n e m e m b r a n e a f t e r e n t e r i n g t h e m e m b r a n e a n d also to c u t h y d r o gen bonding between the vitelline and plasma membrane p a r t h e n o g e n e t i c a g e n t s , as r e p o r t e d p r e v i o u s l y I21. MATERIAL
just like other
AI~ID M E T H O D
Unfertilized eggs of sea urchins were collected b y the usual potassium chloride m e t h o d a n d the following species were used as e x p e r i m e n t a l materials: Anthocidaris crassispina, Echinostrephus aciculatus, Astriclgpeus manni, Temnopleurus toreumaticus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus. Eggs of the starfish, Asterias amurensis, were collected b y the injection of its nerve extract [3]. Urechis eggs were o b t a i n e d b y the m e t h o d reported previously [2]. A b o u t 0.1 m l of crystalline p a p a i u suspension (N.B.C.) was dissolved in 5 ml of deionized water, a n d 0.2 ml of this solution was diluted with 3 ml of sea w a t e r c o n t a i n i n g Versene a n d thioglycolate, b o t h in 3 x 10-5 M at p H 8.2. The protein c o n t e n t of this p a p a i n - s e a water, as d e t e r m i n e d with F o l i n ' s phenol reagent, was made e q u i v a l e n t to 40 7 bovine serum a l b u m i n . This solution was usually used to check the observations made on eggs. C h y m o p a p a i n was p a r t i a l l y purified in this laboratory [1]. Crystalline ficin a n d crude bromeline (N.B.C.) were gifts from Dr M. Akino. These were used in the same way as papain. Crystalline t r y p s i n a n d chymot r y p s i n (N.B.C.) were used as 0.01 0.02 per cent sea water solution. BAL (2,3d i m e r c a p t o - l - p r o p a n o l ) was useful instead of Versene a n d thioglycolate, a n d cysteine could serve instead of thioglycolate. Other conditions of the e x p e r i m e n t were the same as previously reported [2]. RESULTS
Anthocidaris crassispina A c t i v a t e d p a p a i n i n sea w a t e r c a u s e d all t h e eggs to e l e v a t e f e r t i l i z a t i o n m e m b r a n e s w i t h i n a f e w m i n u t e s o v e r t h e r a n g e of p H 6.5 8.5. T h e f o r m a t i o n of t h e h y a l i n e l a y e r w a s s u c c e e d e d b y n u c l e a r a c t i v a t i o n . A n i n c r e a s e d a m o u n t of t h i o g l y c o l a t e (10 -2 M ) s u p p r e s s e d m e m b r a n e e l e v a t i o n b u t n o t f o r m a t i o n of t h e h y a l i n e l a y e r . W i t h o u t a c t i v a t o r s , t h e e n z y m e c a u s e d o n l y t h e f o r m a t i o n of a t h i n h y a l i n e l a y e r a n d o f t e n r o d s w e r e e x t r u d e d a r o u n d t h e eggs.
Figs. 1 and 2: Elevation and contraction of a thin membrane in papain-treated eggs. Fig. 1.--(a) 2 rain after exposure of Anthocidaris egg to papain-NaC1 solution at pH 8.2; (b) 5 min after exposure; (c) 8 rain; (d) 35 rain. Membrane began to elevate again within 20 min after exposure, as the hyaline layer formed. Fig. 2.--(a) 1 rain after immersion of AstricIgpeus egg in papain-sea water at pH 8.2; (b) 2 rain after immersion; (c) 8 rain (d) 3 hr. Experimental Cell Research 41
Activation of eggs bg hydrogen bond breakage. I V
469
Experimental Cell Research4L
470
S. Isaka
Th e activated papai n was also very effective in isotonic sodium chloride and in potassium chloride solutions at pH 8.2. In this case, the fully elevated thin m e m b r a n e was smoothly retracted to the egg surface in about 5 min and then re-elevated on formation of the hyaline layer. T he activated enzyme caused only blisters on the egg surface in isotonic magnesium chloride and in calcium chloride solutions at pH 8.2. C h y m o p a p a i n was quite similar in activity to papain. Echinostrephus eggs b eh av ed like Anthocidaris eggs in p a p a i n - s e a water and c h y m o p a p a i n sea water solutions.
Astriclgpeus manni Activated p a pai n in sea water brought about the elevation of a thin m em brane, which reversibly contracted within a few rain. In about 20 min, the hyaline layer was f or m e d and the m e m b r a n e gradually elevated again in a wrinkled f or m and disappeared while kept in this solution. Clgpeaster, Temnopleurus and Pseudocenlrotus eggs behaved similarly to Aslriclgpeus eggs, although the p a p a i n caused m e m b r a n e elevation in only 20-30 per cent of the eggs. Activated c h y m o p a p a i n had more effect on Pseudocenlrotus eggs than papain. However, Hemicenlrolus eggs were sensitive only to bromelin in about 15 per cent.
Asterias amurensis T r y p s i n and chymotrypsin in sea water caused the elevation of a low membrane, whereas papai n and c h y m o p a p a i n in activated form elevated a norm al fertilization m e m b r a n e in all the eggs. However, activators alone brought a b o u t m e m b r a n e elevation in 20-30 per cent of eggs. Urechis eggs were equally sensitive to trypsin, chymotrypsin, papain and c h y m o p a p a i n . In the starfish and Urechis eggs, the reversible contraction of the m e m b r a n e was not observed. This m a y be ascribed to the thickness of the vitelline m e m b r a n e . DISCUSSION
It was formerly believed that proteolyfic enzymes only attack protein when it occurs in an unfolded or denatured form (except synthetic simple peptides). However, data now exist for the kinetics of hydrolysis of peptide bonds by proteinases in native and denatured proteins. According to these data, the peptide bond is hydrolyzed more slowly in a native molecule than in a denatured protein. As hydrolysis of a native molecule proceeds slowly, there should be a disruption of the internal hydrogen bonds prior to hydro-
Experimental Cell Research 41
Activation of eggs bg hgdrogen bond breakage. I V
471
lysis; this disruption is characterized as "proteolytic explosion". Recent experiments clearly demonstrate that a proteolytic enzyme often behaves as a denaturase [7]. W h e n sea urchin eggs kept in p a p a i n - s e a water are inseminated, a fertilization m e m b r a n e is elevated normally according to Tyler [8]. However, it gradually retracts to the egg surface. In the present experiment, without the intervention of sperm, a similar p h e n o m e n o n is observed. This m a y be explained as follows. Although there is a possibility that p a p a i n dissolves the vitelline m e m b r a n e , ~he author is of the opinion that this enzyme first enters into the vitelline m e m b r a n e and then acts as a denaturase to break the hydrogen bonds in this m e m b r a n e and those between the vitelline and plasma membranes. Moreover, Runnstr6m [61 has suggested that the elevation of the vitelline m e m b r a n e in Echinocardium eggs by trypsin might be due to a breaking of the links between the molecules of the m e m b r a n e , and that this change might be a c c om pani ed by unfolding of the more complicated molecules. The denaturing action of p a p a i n is thought to be very weak and mild, because the elevated m e m b r a n e smoothly retracts to the egg surface. T he contracted m e m b r a n e often elevates again, and this re-elevation might be due to an osmotically active substance p r o d u c e d by the explosion of the cortical granules. This seems to be supported by microscopic observations which reveal that most granules r e m a i n e d intact during the first m e m b r a n e elevation. The granules gradually exploded after the contraction of the membrane, which led to the formation of the hyaline layer and the re-elevation of the m e m b r a n e . As shown in the present experiment unactivated, pure pap ain only caused the formation of a thin hyaline layer, even after contact for several hours with Anthocidaris eggs. However, m e m b r a n e elevation was always observed within a few minutes when activated papai n was applied. The unfolded p a p a i n might cause disintegration of the vitelline m e m b r a n e f r o m the outside and then bring about explosion of the granules. On the other hand, the enzyme, regularly folded u n d e r the influence of activators, would penetrate through the vitelline m e m b r a n e onto the plasma m e m b r a n e , where it would act as a denaturase. Th e reason w h y the once-elevated m e m b r a n e later contracts m a y be explained as follows. T he explosion of the granules is m u c h delayed in this case. Thus, the hardening of the elevated m e m b r a n e cannot occur during this first elevation. T he presence of activators results in weakening of the rigidity of the m e m b r a n e . In isotonic magnesium chloride and calcium chloride solutions, activated
Experimental Cell Research41
472
S. ~ a k a
p a p a i n only causes the f o r m a t i o n of blisters on the s u r f a c e of A n t h o c i d a r i x eggs. This m a y be b e c a u s e (1) the m a g n e s i u m or c a l c i u m ions h a r d e n the vitelline m e m b r a n e , p r e v e n t i n g its elevation, or (2) the activated e n z y m e c a n n o t w o r k effectively in s u c h solutions containing a large a m o u n t of divalent ions. T h e e x p e r i m e n t s h o w e d that the s a m e p r o t e i n a s e is not ahvays e q u a l l y effective in m e m b r a n e elevation a m o n g the m a r i n e ova e x a m i n e d . T h e r e m a y well be specific d e n a t u r a s e s to attack the p r o t e i n o u s configurations of the vitelline m e m b r a n e s characteristic of v a r i o u s species.
SUMMARY
W h e n sea u r c h i n , starfish a n d U r e c h i s eggs are i m m e r s e d in sea w a t e r c o n t a i n i n g activated p a p a i n or c h y m o p a p a i n , they elevate a fertilization m e m b r a n e a n d f o r m a t i o n of the h y a l i n e l a y e r as well as n u c l e a r activation is b r o u g h t about. T h e e n z y m e s cause s u c h m e m b r a n e elevation in sea u r c h i n eggs in isotonic solutions of s o d i u m chloride a n d of p o t a s s i u m chloride. T h e elevated m e m b r a n e often retracts to the egg surface a n d then separates again. T h e first elevation of the m e m b r a n e is t h o u g h t to be b r o u g h t a b o u t b y the b r e a k a g e of h y d r o g e n b o n d s b e t w e e n the vitelline a n d p l a s m a m e m b r a n e s a n d of those within the vitelline m e m b r a n e , owing to the d e n a t u r i n g action of the proteolytic e n z y m e . T h e s e c o n d elevation is c a u s e d b y the h y d r o lytie action of the e n z y m e , w h i c h causes extrusion of the cortical granules. T h e m e m b r a n e is elevated owing to an osmotically active s u b s t a n c e w h i c h releases these granules. H e m i c e n t r o t u s eggs only r e s p o n d to activated b r o m e line. I n a d d i t i o n to p a p a i n a n d c h y m o p a p a i n , t r y p s i n a n d e h y m o t r y p s i n are effective in elevating the m e m b r a n e of U r e c h i s a n d starfish eggs. The author thanks staffs of the Misaki Marine Biological Station for use of facilities and Dr H. Kanatani and Dr J. Dan for reading the manuscript.
REFERENCES
1. 2. 3. 4. 5.
COL0WICI<,S. P. and N. O. KAPLAN., Methods in Enzgmol. 2, 61 (1963). ISAKA,S. and AIKAWA,T., Exptl Cell Res. 30, 139 (1963). KANATANI,H. and NOUMURA,T., J. Fac. Sci. Univ. Tokgo sect. I V . 9, 397 (1962). LUNDBLAD,G., Arkio Kemi 7: 19~ 169 (1954). MOORE,A. R., Exptl Celt Bes. 1, 284 (1950). 6. RUNNSTROM,J., Arkiv Zool. 40 A: 17 1(948). 7. SCHERAGA,H. A., Protein Structure, p. 77. Academic Press. New York, 1961. 8. TYLER, A. and SPIEGEL, M., Biol. Bull. 110, 196 (1956). Experimental Cell Research 41
Experimental Cell Research 41, 467-472 (1966)
SEPARATION
OF THE
URECHIS AND CAUSED
FERTILIZATION
SEA URCHIN
BY BREAKAGE
IV. D E N A T U R I N G
EGGS
MEMBRANE
IN
AS A PHENOMENON
OF HYDROGEN
ACTION OF PROTEOLYTIC
BONDS ENZYMES
S. I S A K A
Biological Institute, Chiba University, Konakadai, Chiba, Japan Received July 8, 1965
LUNDBLADr e p o r t e d
that s o m e proteolytic activity was f o u n d in extracts of sea u r c h i n s p e r m a t o z o a [4], a n d B u n n s t r 6 m [61 a n d Moore [5] o b s e r v e d that p u r e trypsin c a u s e d the elevation of the fertilization m e m b r a n e in unfertilized sea u r c h i n eggs. T h e s e results h a v e led the a u t h o r to investigate the effect of several proteolytic e n z y m e s on m e m b r a n e elevation in some m a r i n e ova. Although trypsin causes m e m b r a n e elevation in Urechis eggs, this e n z y m e has less effect on eggs of the sea urchin, Anthocidaris crassispina; m e m b r a n e elevation occurs only sporadically. H o w e v e r , it was f o u n d that some proteolytic e n z y m e s like p a p a i n are v e r y effective in elevating the fertilization m e m b r a n e in eggs of Urechis, starfish a n d some sea urchins. Proteolytic e n z y m e s are generally divided into three categories. T h o s e belonging to the first are inhibited b y diiso-propyl p h o s p h o f l u o r i d a t e a n d not inhibited b y thiol reagents. T h e second category includes several enzymes, like p a p a i n a n d cathepsins, w h o s e activity d e p e n d s on the p r e s e n c e of one or m o r e thiol groups a n d w h i c h m a y b e inactivated b y various reagents w h i c h c a n r e a c t with thiol groups. Metalloproteinases are in the third category; they are f o u n d to be almost inactive in m e m b r a n e elevation. H o w e v e r , c r u d e p a p a i n causes full m e m b r a n e elevation in all the eggs of Anthocidaris crassispina after the addition of s u c h activators as Versene a n d thioglycolate. As c r u d e papai~n is k n o w n to contain l y s o z y m e , ficin a n d c h y m o p a p a i n , a n e x p e r i m e n t w a s c a r r i e d out with crystalline papain; it was f o u n d that p u r e p a p a i n v e r y effectively p r o d u c e s m e m b r a n e elevation in the p r e s e n c e of activators, while no m e m b r a n e elevation was o b s e r v e d w h e n p a p a i n alone was applied. In this investigation the effect on m e m b r a n e elevation of the proteolytic e n z y m e s in the s e c o n d category was m a i n l y studied in m a r i n e ova. It is 3 1 - 661802
Experimenlal Cell Research 41
468
S. Isaka
s u g g e s t e d t h a t t h e d e n a t u r i n g a c t i o n of t h e s e e n z y m e s is r e s p o n s i b l e f o r m e m b r a n e e l e v a t i o n : s o m e p r o t e i n a s e s s e e m to b r e a k h y d r o g e n b o n d s i n t h e v i t e l l i n e m e m b r a n e a f t e r e n t e r i n g t h e m e m b r a n e a n d also to c u t h y d r o gen bonding between the vitelline and plasma membrane p a r t h e n o g e n e t i c a g e n t s , as r e p o r t e d p r e v i o u s l y I21. MATERIAL
just like other
AI~ID M E T H O D
Unfertilized eggs of sea urchins were collected b y the usual potassium chloride m e t h o d a n d the following species were used as e x p e r i m e n t a l materials: Anthocidaris crassispina, Echinostrephus aciculatus, Astriclgpeus manni, Temnopleurus toreumaticus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus. Eggs of the starfish, Asterias amurensis, were collected b y the injection of its nerve extract [3]. Urechis eggs were o b t a i n e d b y the m e t h o d reported previously [2]. A b o u t 0.1 m l of crystalline p a p a i u suspension (N.B.C.) was dissolved in 5 ml of deionized water, a n d 0.2 ml of this solution was diluted with 3 ml of sea w a t e r c o n t a i n i n g Versene a n d thioglycolate, b o t h in 3 x 10-5 M at p H 8.2. The protein c o n t e n t of this p a p a i n - s e a water, as d e t e r m i n e d with F o l i n ' s phenol reagent, was made e q u i v a l e n t to 40 7 bovine serum a l b u m i n . This solution was usually used to check the observations made on eggs. C h y m o p a p a i n was p a r t i a l l y purified in this laboratory [1]. Crystalline ficin a n d crude bromeline (N.B.C.) were gifts from Dr M. Akino. These were used in the same way as papain. Crystalline t r y p s i n a n d chymot r y p s i n (N.B.C.) were used as 0.01 0.02 per cent sea water solution. BAL (2,3d i m e r c a p t o - l - p r o p a n o l ) was useful instead of Versene a n d thioglycolate, a n d cysteine could serve instead of thioglycolate. Other conditions of the e x p e r i m e n t were the same as previously reported [2]. RESULTS
Anthocidaris crassispina A c t i v a t e d p a p a i n i n sea w a t e r c a u s e d all t h e eggs to e l e v a t e f e r t i l i z a t i o n m e m b r a n e s w i t h i n a f e w m i n u t e s o v e r t h e r a n g e of p H 6.5 8.5. T h e f o r m a t i o n of t h e h y a l i n e l a y e r w a s s u c c e e d e d b y n u c l e a r a c t i v a t i o n . A n i n c r e a s e d a m o u n t of t h i o g l y c o l a t e (10 -2 M ) s u p p r e s s e d m e m b r a n e e l e v a t i o n b u t n o t f o r m a t i o n of t h e h y a l i n e l a y e r . W i t h o u t a c t i v a t o r s , t h e e n z y m e c a u s e d o n l y t h e f o r m a t i o n of a t h i n h y a l i n e l a y e r a n d o f t e n r o d s w e r e e x t r u d e d a r o u n d t h e eggs.
Figs. 1 and 2: Elevation and contraction of a thin membrane in papain-treated eggs. Fig. 1.--(a) 2 rain after exposure of Anthocidaris egg to papain-NaC1 solution at pH 8.2; (b) 5 min after exposure; (c) 8 rain; (d) 35 rain. Membrane began to elevate again within 20 min after exposure, as the hyaline layer formed. Fig. 2.--(a) 1 rain after immersion of AstricIgpeus egg in papain-sea water at pH 8.2; (b) 2 rain after immersion; (c) 8 rain (d) 3 hr. Experimental Cell Research 41
Activation of eggs bg hydrogen bond breakage. I V
469
Experimental Cell Research4L
470
S. Isaka
Th e activated papai n was also very effective in isotonic sodium chloride and in potassium chloride solutions at pH 8.2. In this case, the fully elevated thin m e m b r a n e was smoothly retracted to the egg surface in about 5 min and then re-elevated on formation of the hyaline layer. T he activated enzyme caused only blisters on the egg surface in isotonic magnesium chloride and in calcium chloride solutions at pH 8.2. C h y m o p a p a i n was quite similar in activity to papain. Echinostrephus eggs b eh av ed like Anthocidaris eggs in p a p a i n - s e a water and c h y m o p a p a i n sea water solutions.
Astriclgpeus manni Activated p a pai n in sea water brought about the elevation of a thin m em brane, which reversibly contracted within a few rain. In about 20 min, the hyaline layer was f or m e d and the m e m b r a n e gradually elevated again in a wrinkled f or m and disappeared while kept in this solution. Clgpeaster, Temnopleurus and Pseudocenlrotus eggs behaved similarly to Aslriclgpeus eggs, although the p a p a i n caused m e m b r a n e elevation in only 20-30 per cent of the eggs. Activated c h y m o p a p a i n had more effect on Pseudocenlrotus eggs than papain. However, Hemicenlrolus eggs were sensitive only to bromelin in about 15 per cent.
Asterias amurensis T r y p s i n and chymotrypsin in sea water caused the elevation of a low membrane, whereas papai n and c h y m o p a p a i n in activated form elevated a norm al fertilization m e m b r a n e in all the eggs. However, activators alone brought a b o u t m e m b r a n e elevation in 20-30 per cent of eggs. Urechis eggs were equally sensitive to trypsin, chymotrypsin, papain and c h y m o p a p a i n . In the starfish and Urechis eggs, the reversible contraction of the m e m b r a n e was not observed. This m a y be ascribed to the thickness of the vitelline m e m b r a n e . DISCUSSION
It was formerly believed that proteolyfic enzymes only attack protein when it occurs in an unfolded or denatured form (except synthetic simple peptides). However, data now exist for the kinetics of hydrolysis of peptide bonds by proteinases in native and denatured proteins. According to these data, the peptide bond is hydrolyzed more slowly in a native molecule than in a denatured protein. As hydrolysis of a native molecule proceeds slowly, there should be a disruption of the internal hydrogen bonds prior to hydro-
Experimental Cell Research 41
Activation of eggs bg hgdrogen bond breakage. I V
471
lysis; this disruption is characterized as "proteolytic explosion". Recent experiments clearly demonstrate that a proteolytic enzyme often behaves as a denaturase [7]. W h e n sea urchin eggs kept in p a p a i n - s e a water are inseminated, a fertilization m e m b r a n e is elevated normally according to Tyler [8]. However, it gradually retracts to the egg surface. In the present experiment, without the intervention of sperm, a similar p h e n o m e n o n is observed. This m a y be explained as follows. Although there is a possibility that p a p a i n dissolves the vitelline m e m b r a n e , ~he author is of the opinion that this enzyme first enters into the vitelline m e m b r a n e and then acts as a denaturase to break the hydrogen bonds in this m e m b r a n e and those between the vitelline and plasma membranes. Moreover, Runnstr6m [61 has suggested that the elevation of the vitelline m e m b r a n e in Echinocardium eggs by trypsin might be due to a breaking of the links between the molecules of the m e m b r a n e , and that this change might be a c c om pani ed by unfolding of the more complicated molecules. The denaturing action of p a p a i n is thought to be very weak and mild, because the elevated m e m b r a n e smoothly retracts to the egg surface. T he contracted m e m b r a n e often elevates again, and this re-elevation might be due to an osmotically active substance p r o d u c e d by the explosion of the cortical granules. This seems to be supported by microscopic observations which reveal that most granules r e m a i n e d intact during the first m e m b r a n e elevation. The granules gradually exploded after the contraction of the membrane, which led to the formation of the hyaline layer and the re-elevation of the m e m b r a n e . As shown in the present experiment unactivated, pure pap ain only caused the formation of a thin hyaline layer, even after contact for several hours with Anthocidaris eggs. However, m e m b r a n e elevation was always observed within a few minutes when activated papai n was applied. The unfolded p a p a i n might cause disintegration of the vitelline m e m b r a n e f r o m the outside and then bring about explosion of the granules. On the other hand, the enzyme, regularly folded u n d e r the influence of activators, would penetrate through the vitelline m e m b r a n e onto the plasma m e m b r a n e , where it would act as a denaturase. Th e reason w h y the once-elevated m e m b r a n e later contracts m a y be explained as follows. T he explosion of the granules is m u c h delayed in this case. Thus, the hardening of the elevated m e m b r a n e cannot occur during this first elevation. T he presence of activators results in weakening of the rigidity of the m e m b r a n e . In isotonic magnesium chloride and calcium chloride solutions, activated
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p a p a i n only causes the f o r m a t i o n of blisters on the s u r f a c e of A n t h o c i d a r i x eggs. This m a y be b e c a u s e (1) the m a g n e s i u m or c a l c i u m ions h a r d e n the vitelline m e m b r a n e , p r e v e n t i n g its elevation, or (2) the activated e n z y m e c a n n o t w o r k effectively in s u c h solutions containing a large a m o u n t of divalent ions. T h e e x p e r i m e n t s h o w e d that the s a m e p r o t e i n a s e is not ahvays e q u a l l y effective in m e m b r a n e elevation a m o n g the m a r i n e ova e x a m i n e d . T h e r e m a y well be specific d e n a t u r a s e s to attack the p r o t e i n o u s configurations of the vitelline m e m b r a n e s characteristic of v a r i o u s species.
SUMMARY
W h e n sea u r c h i n , starfish a n d U r e c h i s eggs are i m m e r s e d in sea w a t e r c o n t a i n i n g activated p a p a i n or c h y m o p a p a i n , they elevate a fertilization m e m b r a n e a n d f o r m a t i o n of the h y a l i n e l a y e r as well as n u c l e a r activation is b r o u g h t about. T h e e n z y m e s cause s u c h m e m b r a n e elevation in sea u r c h i n eggs in isotonic solutions of s o d i u m chloride a n d of p o t a s s i u m chloride. T h e elevated m e m b r a n e often retracts to the egg surface a n d then separates again. T h e first elevation of the m e m b r a n e is t h o u g h t to be b r o u g h t a b o u t b y the b r e a k a g e of h y d r o g e n b o n d s b e t w e e n the vitelline a n d p l a s m a m e m b r a n e s a n d of those within the vitelline m e m b r a n e , owing to the d e n a t u r i n g action of the proteolytic e n z y m e . T h e s e c o n d elevation is c a u s e d b y the h y d r o lytie action of the e n z y m e , w h i c h causes extrusion of the cortical granules. T h e m e m b r a n e is elevated owing to an osmotically active s u b s t a n c e w h i c h releases these granules. H e m i c e n t r o t u s eggs only r e s p o n d to activated b r o m e line. I n a d d i t i o n to p a p a i n a n d c h y m o p a p a i n , t r y p s i n a n d e h y m o t r y p s i n are effective in elevating the m e m b r a n e of U r e c h i s a n d starfish eggs. The author thanks staffs of the Misaki Marine Biological Station for use of facilities and Dr H. Kanatani and Dr J. Dan for reading the manuscript.
REFERENCES
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COL0WICI<,S. P. and N. O. KAPLAN., Methods in Enzgmol. 2, 61 (1963). ISAKA,S. and AIKAWA,T., Exptl Cell Res. 30, 139 (1963). KANATANI,H. and NOUMURA,T., J. Fac. Sci. Univ. Tokgo sect. I V . 9, 397 (1962). LUNDBLAD,G., Arkio Kemi 7: 19~ 169 (1954). MOORE,A. R., Exptl Celt Bes. 1, 284 (1950). 6. RUNNSTROM,J., Arkiv Zool. 40 A: 17 1(948). 7. SCHERAGA,H. A., Protein Structure, p. 77. Academic Press. New York, 1961. 8. TYLER, A. and SPIEGEL, M., Biol. Bull. 110, 196 (1956). Experimental Cell Research 41