- Email: [email protected]
Distribution of proteolytic enzymes in protein fractions from non-fertilized and fertilized eggs of the sea urchin Paracentrotus lividus
The
pan and contents were then dried to constant weight at 100 C: and the rlrh of the animals so found. The water content was calculated by difference and expressed as a percentage of the wet weight. From three such rlcterminations, a mean value of RO.:I per cent w/w was obtained (Std. De\,. :( per cent). This corresponds to 92.0 per cent v/v. From the above results it is seen that the two methods show a considerable discrepancy, the reason for which is not known. There is little doubt that the second method is preferable as a technique, requiring far less manipulation than the heavywater method. r\ further and more serious objection to the use of heavy water is the need for extrapolating the graph, since a very small error in the position of the is line would cause a large error in the reading at zero time. At the present stage, there is little point in speculating whether the different results yielded by the two methods have any biological significance. Shortage of time precluded any further investigation of this; in the subsequent work on potassium distribution, a figure of 90 per cent \V/W was taken as sufficiently accurate for the purpose. weight
I am grateful to Dr. J. X. Kitching, F.R.S., for advice and criticism of this work, and to the Department of Scientific and Industrial Research for the provision of a maintenance grant. REFERENCES 1. HOLTER,
H. and ZEUTHEN, E., Corn@. Rend. True. Cnrlsberg Ser. Chim. 26, 277 S., J. Esptl. Zool. 145, 139 (1960). S. and PIGON, A.? Compt. Rend. Tmo. Carlsberg Ser. Chim. 28, 1 (1951). 4. LUMS~EN,~. E.and ROBINSON,C.V., Ibid.28,358 (1953). 5. PRESCOTT, D. RI. and ZEUTHEN, E., A& Physiol. Stand. 28, 77 (1953). 6. RIDDLE, J., Espfl. Cell Research 26, 158 (1962).
(1918).
2. LOVTRUP, 3. LBVTRUP,
DISTRIBUTION
OF PROTEOLYTIC
FRACTIONS
FROM
NON-FERTILIZED
EGGS OF THE SEA URCHIN G. LUNDBLAD National
Bacteriological Laboratory. Stockholm, Sweden, and The for Experimental Biology, University of Stockholm,
BEsums cathepsin egg with
II three different the pH-optima
1 This investigation was supported Council and from the Swedish Canrer at Stazione Zoologica, Naples. Experimenlnl
AND
PARACENTROTUS
Cell Research
27
IN PROTEIN FERTILIZED LIVIDUS’
and .J. RUNNSTRijM
Received
sea urchin
ENZYMES
March
Wenner-Gren Sweden
Institute
20, 1962
proteolytic enzymes were demonstrated in the (cf. [3, 41). The) 6.7, -7.0 and 7.X respectively
bv grants from the Swedish Soc‘ietg. The experimental part
Natural Sciences Research of the work was carried out
Proteolytic
enzymes in protein
fractions
329
from sea urchin
were designated El, E2 and E3. El and E3 are activated by SH-compounds like cystein, whereas E2 is inactivated by these compounds. These enzymes have a low activity in the unfertilized egg but immediately after fertilization they become activated. The sequence of activation is probably E2 > El >E3. Their activity soon declines, however. About 15 min after fertilization (sperm aster stage), they have again a low activity (cf. [3] Fig. 3). The importance of this enzyme activation in
Fig.
1.
Fig.
2.
Fractionation on columns of DE.AE-Sephades of homogenates from unfertilized, fertilized eggs (10 min after fertilization), cf. Fig. 2. Further data in text.
cf. Fig.
1, and
fertilization is confirmed by the fact that inhibitors like ovomucoid and soybean trypsin inhibitor seriously interfere with the cortical changes normally occurring upon attachment of the effective spermatozoon [l]. As the previous studies were carried out at a time when chromatography of proteins was not yet developed, a reexamination and extension of the previous results with modern methods seemed desirable. Methods.---~The eggs of Paracentrotus lividus were filtered through bolting silk and thoroughly washed in sea water. The jelly coats were removed by exposure to sea water acidified by hydrochloric acid to pH 5.5. Thereafter the eggs were washed three times in sea water: 6 ml 0.02 d1 Tris buffer pH 7.4 was added to 6 ml sediment of unfertilized eggs; the suspensionwas passedsix times through a glassfilter G2 in order to bring about homogenization of the eggs. The homogenate was centrifuged for 12 min at 8000 rpm in an angle centrifuge by which the coarser particulate components were removed. The procedure for fertilized eggs was essentially the same; 8 min after insemination the volume of the suspensionof 100 per cent fertilized eggs was brought from 200 to 30 ml; 10 min after insemination 30 ml Tris buffer was added. The ensuing procedure was as indicated above. Samplesof 5 ml were applied each time to 10 g of DEAE-Sephadexl A25 (columns 30 ;- 1.8 cm), equilibrated with 0.02 LIf Tris (pH 7.4). The change of eluents is indicated by arrows in Figs. 1 and 2. They were as follows: (1) 0.02 LV Tris pH 7.4; (2) 1 l\Ianufactured
by *\B
Pharmacia,
Lrppsala,
Sweden. Exprrimentul
Cell
Research
27
0.1 31 NaCl Tris; (3) 0.2 JI NaCl Tris: (.4) 0.5 31 NaCl Tris; (5) 1.0 31 NaCl Tris. The effluent was collected in 1 ml fractions at 60 ml/hr. The solid lines in Figs. 1 and 2 represent estinctions at 280 m/c. The roman figures above the curves indicate the main maxima of extinction. The fractions corresponding to these maxima will be referred to below as fraction I, II, etc. (the arahic figures below the abscissae of the curves give the consecutive numbers of the collection tubes). The fractions I, II, etc. were tested with respect to their action on the viscosity of a gelatine solution (cf. [2,3] ). A biological test was also used. This is based on the fact that exposure of unfertilized eggs to trypsin of varying doses (time ...concentration) causes a rather well defined gamut of changes in the eg g. These changes are observable particularly after the fertilization [6, 7, 81. Low closes of trypsin (e.g. exposure to 5 1O-5 per cent tryst trypsin for 10 min) bring about an increase in retardation of the fertilization membrane, measured in Leitz polarization microscope provided with a Koehler compensator. Increasing doses (5 i 1O-5 --lo-” per cent for 15 min) cause inhibition of the elevation of the fertilization membrane and impairment of cleavage. At higher doses ( b lo-$ for 15 min) a dissolution of the vitelline membrane of the unfertilized egg occurred; as a consequence no continuous fertilization membrane appeared after insemination. The effects were similar after exposure of eggs to certain homogenate fractions. Before being used in the biological test the fractions were dialyzed over night in cold room against sea water. After exposure for different lengths of time (e.g. 1 fr, 1, 7 hr) to the differently diluted homogenate fractions the eggs were transferred to normal sea water and inseminated. A final insemination was carried out with eggs still remaining in the homogenate fractions. Addition of glutathione to a final concentration of 0.0023 A1 should decide whether a certain action is due to E2 (inhibitory effect) or to El, E3 (enhancing effect). Hesults and discLlssion--~Fraction I from homogenate of unfertilized eggs (cf. Fig. 1) forms a very conspicuous peak. The fraction is strongly colored. According to the gelatine test it contains cathepsin II. =\fter fertilization fraction I decreases considerably. It loses its color, which is now found in fraction Y. Even after this shift the color was found to be accompanied by cathepsin II. The activities of El-E3 were lower and did not lend themselves as well for a stud] with the gelatine test. Certain modification of the methods of preparation will certainly give more active fractions. Nevertheless the gelatine test indicated that a proteolytic activity was present in fraction I from urlfertilized eggs at pH 6.7 (El). In homogenate from fertilized eggs this El activity was shifted to fraction II. A certain proteolgtic activity was foulid in fraction III of homogenates from fertilized eggs. This should, however, rather be ascribed to E2 (pH-optimumN7.0, inhibition by reduced glutathione). The biological test proved to be more sensitive than the gelatine test, even if it was sometimes more ambiguous. Eggs pretreated with fraction I from unfertilized eggs for 80-180 min, presented after washing and insemination a retardation of the fertilization membrane that was raised by about 50 per cent (from about 12 to 18.5 mr). On addition of glutathione during the pretreatment this effect was removed; the eggs pretreated without glutathione presented also a condensed jelly coat in which spermatozoa were entangled. Sometimes the jelly coat had a marked exterior outline. Spermatozoa adjacent to the eggs often showed a tail agglutination. This is characteristic of the effects obtained after pretreatment of the eggs with low trypsin doses. Experimental
Cell Research
27
Proteolytic
enzymes in protein
fractions
331
from sea urchin
They have already been ascribed to an E2 action induced by trypsin [6]. The removal of the effect by glutathione is in favor of this assumption. In no other fraction tested did an increase in retardation of the membrane occur. When the pretreatment with fraction I lasted for 94 hr and the eggs were fertilized in the presence of the fraction an adhering fertilization membrane appeared that had the higher retardation. No or only some steps of cleavage were taken. In the presence of glutathione, however, a cleavage of the eggs occurred but the membrane was often broken up at least in one region and presented the lower retardation. These data clearly point to the presence of E2 in fraction I in homogenate of unfertilized eggs. They do not exclude the possibility that El or E3 or both are present (cf. the effect of the addition of glutathione on the membrane, etc.). After fertilization the E2 effect resided in fraction III, which is in agreement with the results of the gelatine test. In fraction II from fertilized eggs a pronounced hydrolytic activity was found, as demonstrated by the failure to form a continuous fertilization membrane. The hydrolytic action was promoted by addition of glutathione to fraction II. This hydrolytic action may be again due to El or E3 or to a combination of both. The enzyme E2 gives the appearance of having a more thrombin-like effect breaking up certain “masking” linkages (cf. [9]). It was evident in the present experiments that an activation of the proteolytic enzymes (El, E3) took place upon fertilization and still persisted 10 min after insemination. These preliminary data tend to show that several proteolytic enzymes along with a pigment are collected in fraction I from unfertilized eggs. After fertilization only a residue of fraction I remains; a part of its content is distributed over several fractions. This may mean that before fertilization several enzymes are bound to some inhibitory substance that is removed upon fertilization. The consequence is a release and redistribution of the enzymes that in this way become activated. The results give promise of further insight into the mechanism of enzyme activation that according to the opinion of these writers occurs upon fertilization (cf. [2, 3, 4, 51). This investigation will be continued.
REFERENCES 1. HAGSTRBM, 2. LUNDBLAD, 3. ~ Arkio
ES., drkio Zool. (2) 10, 307 (1956). G., Exptl. Cell Research 1, 264 (1950). Kemi 7, 127 (1951).
4. __ 5. 6. 7. 8. 9.
Proteolytic Activity in Sea Urchin Gametes. Ahnquist & Wiksell, Uppsala, RUNNSTR~M, J., Erptl. Cell Research Suppl. I, 169 (1919). __ Exptl. Cell Research 22, 576 (196li~~ Unpuhlished. RUNNSTH~M, J. and KRISZAT, G., drkio Zool. (2) 13, 95 (1960). SCHERAGA, H. A. and LASKOWSKI, JR., Aduances in Protein Chem. 12, 2 (1957).
Experimental
Cell
1954.
Research
27
pan and contents were then dried to constant weight at 100 C: and the rlrh of the animals so found. The water content was calculated by difference and expressed as a percentage of the wet weight. From three such rlcterminations, a mean value of RO.:I per cent w/w was obtained (Std. De\,. :( per cent). This corresponds to 92.0 per cent v/v. From the above results it is seen that the two methods show a considerable discrepancy, the reason for which is not known. There is little doubt that the second method is preferable as a technique, requiring far less manipulation than the heavywater method. r\ further and more serious objection to the use of heavy water is the need for extrapolating the graph, since a very small error in the position of the is line would cause a large error in the reading at zero time. At the present stage, there is little point in speculating whether the different results yielded by the two methods have any biological significance. Shortage of time precluded any further investigation of this; in the subsequent work on potassium distribution, a figure of 90 per cent \V/W was taken as sufficiently accurate for the purpose. weight
I am grateful to Dr. J. X. Kitching, F.R.S., for advice and criticism of this work, and to the Department of Scientific and Industrial Research for the provision of a maintenance grant. REFERENCES 1. HOLTER,
H. and ZEUTHEN, E., Corn@. Rend. True. Cnrlsberg Ser. Chim. 26, 277 S., J. Esptl. Zool. 145, 139 (1960). S. and PIGON, A.? Compt. Rend. Tmo. Carlsberg Ser. Chim. 28, 1 (1951). 4. LUMS~EN,~. E.and ROBINSON,C.V., Ibid.28,358 (1953). 5. PRESCOTT, D. RI. and ZEUTHEN, E., A& Physiol. Stand. 28, 77 (1953). 6. RIDDLE, J., Espfl. Cell Research 26, 158 (1962).
(1918).
2. LOVTRUP, 3. LBVTRUP,
DISTRIBUTION
OF PROTEOLYTIC
FRACTIONS
FROM
NON-FERTILIZED
EGGS OF THE SEA URCHIN G. LUNDBLAD National
Bacteriological Laboratory. Stockholm, Sweden, and The for Experimental Biology, University of Stockholm,
BEsums cathepsin egg with
II three different the pH-optima
1 This investigation was supported Council and from the Swedish Canrer at Stazione Zoologica, Naples. Experimenlnl
AND
PARACENTROTUS
Cell Research
27
IN PROTEIN FERTILIZED LIVIDUS’
and .J. RUNNSTRijM
Received
sea urchin
ENZYMES
March
Wenner-Gren Sweden
Institute
20, 1962
proteolytic enzymes were demonstrated in the (cf. [3, 41). The) 6.7, -7.0 and 7.X respectively
bv grants from the Swedish Soc‘ietg. The experimental part
Natural Sciences Research of the work was carried out
Proteolytic
enzymes in protein
fractions
329
from sea urchin
were designated El, E2 and E3. El and E3 are activated by SH-compounds like cystein, whereas E2 is inactivated by these compounds. These enzymes have a low activity in the unfertilized egg but immediately after fertilization they become activated. The sequence of activation is probably E2 > El >E3. Their activity soon declines, however. About 15 min after fertilization (sperm aster stage), they have again a low activity (cf. [3] Fig. 3). The importance of this enzyme activation in
Fig.
1.
Fig.
2.
Fractionation on columns of DE.AE-Sephades of homogenates from unfertilized, fertilized eggs (10 min after fertilization), cf. Fig. 2. Further data in text.
cf. Fig.
1, and
fertilization is confirmed by the fact that inhibitors like ovomucoid and soybean trypsin inhibitor seriously interfere with the cortical changes normally occurring upon attachment of the effective spermatozoon [l]. As the previous studies were carried out at a time when chromatography of proteins was not yet developed, a reexamination and extension of the previous results with modern methods seemed desirable. Methods.---~The eggs of Paracentrotus lividus were filtered through bolting silk and thoroughly washed in sea water. The jelly coats were removed by exposure to sea water acidified by hydrochloric acid to pH 5.5. Thereafter the eggs were washed three times in sea water: 6 ml 0.02 d1 Tris buffer pH 7.4 was added to 6 ml sediment of unfertilized eggs; the suspensionwas passedsix times through a glassfilter G2 in order to bring about homogenization of the eggs. The homogenate was centrifuged for 12 min at 8000 rpm in an angle centrifuge by which the coarser particulate components were removed. The procedure for fertilized eggs was essentially the same; 8 min after insemination the volume of the suspensionof 100 per cent fertilized eggs was brought from 200 to 30 ml; 10 min after insemination 30 ml Tris buffer was added. The ensuing procedure was as indicated above. Samplesof 5 ml were applied each time to 10 g of DEAE-Sephadexl A25 (columns 30 ;- 1.8 cm), equilibrated with 0.02 LIf Tris (pH 7.4). The change of eluents is indicated by arrows in Figs. 1 and 2. They were as follows: (1) 0.02 LV Tris pH 7.4; (2) 1 l\Ianufactured
by *\B
Pharmacia,
Lrppsala,
Sweden. Exprrimentul
Cell
Research
27
0.1 31 NaCl Tris; (3) 0.2 JI NaCl Tris: (.4) 0.5 31 NaCl Tris; (5) 1.0 31 NaCl Tris. The effluent was collected in 1 ml fractions at 60 ml/hr. The solid lines in Figs. 1 and 2 represent estinctions at 280 m/c. The roman figures above the curves indicate the main maxima of extinction. The fractions corresponding to these maxima will be referred to below as fraction I, II, etc. (the arahic figures below the abscissae of the curves give the consecutive numbers of the collection tubes). The fractions I, II, etc. were tested with respect to their action on the viscosity of a gelatine solution (cf. [2,3] ). A biological test was also used. This is based on the fact that exposure of unfertilized eggs to trypsin of varying doses (time ...concentration) causes a rather well defined gamut of changes in the eg g. These changes are observable particularly after the fertilization [6, 7, 81. Low closes of trypsin (e.g. exposure to 5 1O-5 per cent tryst trypsin for 10 min) bring about an increase in retardation of the fertilization membrane, measured in Leitz polarization microscope provided with a Koehler compensator. Increasing doses (5 i 1O-5 --lo-” per cent for 15 min) cause inhibition of the elevation of the fertilization membrane and impairment of cleavage. At higher doses ( b lo-$ for 15 min) a dissolution of the vitelline membrane of the unfertilized egg occurred; as a consequence no continuous fertilization membrane appeared after insemination. The effects were similar after exposure of eggs to certain homogenate fractions. Before being used in the biological test the fractions were dialyzed over night in cold room against sea water. After exposure for different lengths of time (e.g. 1 fr, 1, 7 hr) to the differently diluted homogenate fractions the eggs were transferred to normal sea water and inseminated. A final insemination was carried out with eggs still remaining in the homogenate fractions. Addition of glutathione to a final concentration of 0.0023 A1 should decide whether a certain action is due to E2 (inhibitory effect) or to El, E3 (enhancing effect). Hesults and discLlssion--~Fraction I from homogenate of unfertilized eggs (cf. Fig. 1) forms a very conspicuous peak. The fraction is strongly colored. According to the gelatine test it contains cathepsin II. =\fter fertilization fraction I decreases considerably. It loses its color, which is now found in fraction Y. Even after this shift the color was found to be accompanied by cathepsin II. The activities of El-E3 were lower and did not lend themselves as well for a stud] with the gelatine test. Certain modification of the methods of preparation will certainly give more active fractions. Nevertheless the gelatine test indicated that a proteolytic activity was present in fraction I from urlfertilized eggs at pH 6.7 (El). In homogenate from fertilized eggs this El activity was shifted to fraction II. A certain proteolgtic activity was foulid in fraction III of homogenates from fertilized eggs. This should, however, rather be ascribed to E2 (pH-optimumN7.0, inhibition by reduced glutathione). The biological test proved to be more sensitive than the gelatine test, even if it was sometimes more ambiguous. Eggs pretreated with fraction I from unfertilized eggs for 80-180 min, presented after washing and insemination a retardation of the fertilization membrane that was raised by about 50 per cent (from about 12 to 18.5 mr). On addition of glutathione during the pretreatment this effect was removed; the eggs pretreated without glutathione presented also a condensed jelly coat in which spermatozoa were entangled. Sometimes the jelly coat had a marked exterior outline. Spermatozoa adjacent to the eggs often showed a tail agglutination. This is characteristic of the effects obtained after pretreatment of the eggs with low trypsin doses. Experimental
Cell Research
27
Proteolytic
enzymes in protein
fractions
331
from sea urchin
They have already been ascribed to an E2 action induced by trypsin [6]. The removal of the effect by glutathione is in favor of this assumption. In no other fraction tested did an increase in retardation of the membrane occur. When the pretreatment with fraction I lasted for 94 hr and the eggs were fertilized in the presence of the fraction an adhering fertilization membrane appeared that had the higher retardation. No or only some steps of cleavage were taken. In the presence of glutathione, however, a cleavage of the eggs occurred but the membrane was often broken up at least in one region and presented the lower retardation. These data clearly point to the presence of E2 in fraction I in homogenate of unfertilized eggs. They do not exclude the possibility that El or E3 or both are present (cf. the effect of the addition of glutathione on the membrane, etc.). After fertilization the E2 effect resided in fraction III, which is in agreement with the results of the gelatine test. In fraction II from fertilized eggs a pronounced hydrolytic activity was found, as demonstrated by the failure to form a continuous fertilization membrane. The hydrolytic action was promoted by addition of glutathione to fraction II. This hydrolytic action may be again due to El or E3 or to a combination of both. The enzyme E2 gives the appearance of having a more thrombin-like effect breaking up certain “masking” linkages (cf. [9]). It was evident in the present experiments that an activation of the proteolytic enzymes (El, E3) took place upon fertilization and still persisted 10 min after insemination. These preliminary data tend to show that several proteolytic enzymes along with a pigment are collected in fraction I from unfertilized eggs. After fertilization only a residue of fraction I remains; a part of its content is distributed over several fractions. This may mean that before fertilization several enzymes are bound to some inhibitory substance that is removed upon fertilization. The consequence is a release and redistribution of the enzymes that in this way become activated. The results give promise of further insight into the mechanism of enzyme activation that according to the opinion of these writers occurs upon fertilization (cf. [2, 3, 4, 51). This investigation will be continued.
REFERENCES 1. HAGSTRBM, 2. LUNDBLAD, 3. ~ Arkio
ES., drkio Zool. (2) 10, 307 (1956). G., Exptl. Cell Research 1, 264 (1950). Kemi 7, 127 (1951).
4. __ 5. 6. 7. 8. 9.
Proteolytic Activity in Sea Urchin Gametes. Ahnquist & Wiksell, Uppsala, RUNNSTR~M, J., Erptl. Cell Research Suppl. I, 169 (1919). __ Exptl. Cell Research 22, 576 (196li~~ Unpuhlished. RUNNSTH~M, J. and KRISZAT, G., drkio Zool. (2) 13, 95 (1960). SCHERAGA, H. A. and LASKOWSKI, JR., Aduances in Protein Chem. 12, 2 (1957).
Experimental
Cell
1954.
Research
27