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Activation of amino acids during sea urchin development
AR(!HIVES
OF
BIOCHEMISTRY
Activation
AND
BIOPHYSICS
of Amino
Acids
R. MAGGIO From the Laboratory
103, 164-168 (1963)
during
Sea Urchin
Development
AND C. CATALAN02
of Comparative
Anatomy,
The University
of Palermo,
rtaly
Received July 10, 1963 INTRODUCTION Previous work from this laboratory has demonstrated that unfertilized sea urchin eggs have very little, if any, ability to incorporate in vivo labelled amino acids into their proteins. Incorporation begins immediately following fertilization but its rate becomes especially high between blastula and midgastrula (1, 10, 12). Experiments carried out in vitro (4, 5) indicate that the ribosomes isolated from unfertilized eggs are unable to incorporate amino acids into proteins but acquire this ability within 30 minutes after fertilization. However, the ribosomes from unfertilized eggs can be induced in vitro to synthesize polyphenylalanine by the addition of synthetic polyuridylic acid (13, 16, 17). These results seem, therefore, to warrant the suggestion that the lack of synthetic ability on the part of the ribosomes of the unfertilized egg may depend on the nonavailability of messenger RNA. That a new synthesis of RNA begins shortly after fertilization is indicated by the recent work of Gross and Cousineau (2). Another important step, and indeed the first one, in the process of protein synthesis to which, in the case of the sea urchin embryo, almost no attention has thus far been paid is the activation of amino acids. 1This work has been supported by grants from the Consiglio Nazionale delle Ricerche (Research Group on the Problem of Differentiation) and the National Institute of Health, U.S. Public Health Service (RGO6211) to the Laboratory of Comparative Anatomy, 2 Holder of a research fellowship from the Ministry of Educat,ion. 164
That amino acid activating enzymes are present in the unfertilized sea urchin egg was shown by Scarano and Maggio (14). The results to be described in this paper show that the activity of these enzymes undergoes a sudden increase at the blastula stage, i.e., coinciding with the increase in the rate of protein synthesis. MATERIALS
PREPARATION
AND
METHODS
OF THE ENZYMATIC EXTRACT
Eggs of Paracentrotus lividus were collected as described (9). Each batch was divided into two parts, one of which was fertilized and allowed to develop at 1%20°C. In the unfertilized eggs the jelly coat was removed by treatment with acid sea water. The eggs and embryos were homogenized in the cold, using two different media: Medium 9, containing 0.25 M sucrose, 0.008 iM MgC12, 0.04 M KF, and 0.05 Jf Tris buffer, pH 7.5, was used for the ATP-PPa2 exchange reaction; Medium B was a 0.05 M Na phosphate buffer at pH 7.38 which was employed for the preparations to be used for the hydroxamate reaction. The homogenates were centrifuged at 4°C. for 20 minutes at 15.OOOgand the supernatant was recentrifuged at 105OOOg for 60 minutes to remove all particulates. A great difficulty encountered in the study of amino acid activation in sea urchin eggs and embryos is the presence of a very large pool of free amino acids (Kavanau, 1954). Extensive removal of the free amino acids has been obtained by filtering the final supernatants through a Sephadex column. A Sephadex G 25 column (23 X 1 cm.) was equilibrated with 0.05 M Tris buffer, pH 7.5 (for filtration of extracts in medium A), or with Na-phosphate buffer, pH 7.38 (when medium B was used). Usually about 7 ml. of final supernatant, containing lOO-200 mg. of proteins, were submitted to filtration; all the operations were carried out in the cold room at 3°C. The first 5 ml.
ACTIVATION
OF AMINO
of effluent were used for the enzymatic assay. The protein concentration of this fraction (as determined by the method of Lowry et al., 1951) is very close to that of the original supernatant. On the other hand, the amino acid concentration (ninhydrin method according to Moore and Stein, 1948) was about 2 pM per 10 mg. of proteins, as against 55-58 &f in the original supernatant.
ASSAY OF THE ENZYMATIC ACTIVITY
ATP-PP32 Exchange Reaction PPa2 was synthesized by condensation of Paz according to the method of Kornberg and Pricer (7). Aliquots of Sephadex-filtered supernatant, containing lo-20 mg. of protein were used for each experiment. The reaction mixture and the conditions of each experiment are described in the explanations given in the tables. The addition of fluoride to the reaction mixture is particularly imperative owing to the presence of a very active pyrophosphatase. At the end of the incubation,
the ATP was isolated and its concentration radioactivity were determined as previously scribed (14).
“;g.
Hydroxamic Acid Reaction
RESULTS
52=
Unfert
Early
59d
60”
AND
I
PP32-ATP EXCHANGE REACTION E~~BRYOS OF PARACENTROTUS
. eggs
blast.
Mesenchyme
Mesenchyme
blast.
blast.
DISCUSSION
The data in Table I show that extracts of sea urchin eggs operate an ATP-PP32
ACID-DEPENDENT
stage
and de-
Salt-free NHZOH was prepared according to the method already described (15). Aliquots containing 8-16 mg. of proteins of filtered supernatant were added to a reaction mixture containing: 10 pM MgC12, 10 FM KF, about 1000 PM NHsOH, the proper amount of amino acids, and 100 PM Tris buffer pH 7.5 in a total volume of 1.7 ml. and then incubated for 1 hr. at 30°C. The reaction was stopped by the addition of 1 volume of FeCh solution, according to Hoagland et al. (3), and left in the cold for some hours. After centrifugation, the absorbancy of the clear supernatant was read at 520 mp using glycinhydroxamate as a standard.
TABLE L-GLUTAMIC
165
ACIDS
Substrate CUM)
IN EGGS AND
cpm/sM ATPa
% exchange”
None + n-glutamic + n-glutamic
acid, 28 acid, 85
2560 2330 3620
5.05 4.60 7.15
None + L-glutamic + n-glutamic
acid, 28 acid, 85
2170 3240 5250
4.34 6.48 10.50
none -I- n-glutamic + n-glutamic
acid, 85 acid, 85
5850 10,250 5400
6.88 12.00 6.35
none + n-glutamic + n-glutamic + n-glutamic
acid, 80 acid, 80 acid, 80 + Paz
5628 10,221 5575 176
10.20 18.57 10.10 0.3
n As calculated for 10 mg. of proteins of the enzymatic extract. Values subtracted from the zero time. b y0 exchange = (specific activity ATP”)/(0.5 X specific activity PP) X 100. c Conditions for Expt. 52: ATP, 22.84 FM; PP3?, 18.27 /AM (101,500 cpm per FM and 100,000 cpm per pM, respectively, for the unfertilized eggs and blastula). Enzymatic extract: 19.2 mg. protein for the unfertilized eggs and 16.0 mg. protein for the blastula. d Conditions for Expt. 59: ATP, 8.96 pM; PP3*, 11.13 pM (170,000 cpm per PM). Enzymatic extract: 7.66 mg. protein. 8 Conditions for Expt. 60: ATP, 8.96 pM; PP 32, 11.13 pM (110,000 cpm per MM); P3*, 12 pM (116,400 cpm per plM). Enzymatic extract: 11.3 mg. protein. To the reaction mixture were also added: MgC12, 10 pM; KF, 10 pM; Tris buffer, 0.05 M, final concentration, pH 7.5, final volume, 2 ml. Incubation for 20 minutes at 30°C.
166
MAGGIO
AND TABLE
PP32-ATP
EXCHANGE
REACTION
Expt. No.
54h
Unfert.
Unfert
58d
gastrula
. eggs
Mesenchyme
ACIDS
IN THE SEA URCHIN
DEVELOPMENT
Substrate b.W
eggs
Swimming
II
BY AMINO
stage
Early
55”
CATALYZED
CATALAKO
blast.
blast.
% exchange
none + L-phenylalanine, + L-leucine, GO
GO
3250 1635 3410
5.75 2.89 6.04
none + L-phenylalanine, f L-leucine, 60
GO
8380 8450 15,700
15.50 15.08 29.00
none + L-histidine, 60 + glycine, 100
GO80 6400 6300
10.10 10.80 10.50
none + L-histidine, GO + glycine, 100
5960 7560 8550
10.70 13.70 15.50
none + L-isoleucine, 80 + L-serine, 80
10,800 21,900 16,650
a As for Table I. 6 Conditions for Expt. 54: 7 &f ATP; 6.55 p&Z PP32 (113,000 cpm per PM and 108,000 respectively, for the unfertilized eggs and gastrula). Enzymatic extract: 9.6 mg. protein and 11.3 mg. protein gastrula. c Conditions for Expt. 55: 11.42 &I4 ATP; 11.8 pjl4 PP3* (120,000 cpm per &1 and 110,300 respectively, for the unfertilized eggs and blastula). Enzymatic extract: 23.20 mg. protein eggs and 18.95 mg. protein blastula. d Conditions for Expt. 58: 8.96 PM ATP; 11.13 PM PPa2 (210,000 cpm/MiM). Enzymatic mg. protein. The general experimental condition is described in Table I.
exchange reaction catalyzed by glutamic acid; the percentage of exchange increases as development proceeds. Actually, all throughout cleavage, and indeed until the blastula stage is reached, there is very little, if any, increase as compared with the unfertilized egg, while from the blastula stage onward, activity is considerably higher. The high blank value (incorporation in the ATP in the absence of added substrate) is likely to be due to the stimulation caused by the small amounts of free amino acids not removed by Sephadex filtration. Obviously this becomes particularly noticeable in the case of the developmental stages in which the activity of the enzyme system is higher. It must be added that control experiments have also shown that during incubation a small release of amino acids
10.3 20.8 15.9
cpm per &f, unfertilized cpm per PM, unfertilized extract:
8.74
(l-2 PM per 10 mg. of proteins both in the unfertilized eggs and in the developmental stages) takes place, and this may add to the above stimulation. However, since glutamic acid represents but a small percentage of the whole free amino acid pool [about 2%; (S)] we may feel confident that the stimulation observed when glutamic acid is added is entirely due to the substrate. As shown in experiments 39 and 60, n-glutamic acid does not stimulate ATP-PP32 exchange; if anything, it causes a slight inhibition. Furthermore, the exchange takes place only in the presence of pyrophosphate (experiment 60). Experiments in which the activation was estimated by the hydroxamate formation duplicate these results. In Table II the results of the exchange
ACTIVATION
OF AMINO
reaction catalyzed by some other amino acids are presented. The result obtained with phenylalanine is worth mentioning. The addition of this amino acid to extracts of unfertilized eggs brings about a considerable inhibition in the endogenous exchange reaction, while in the embryos phenylalanine causes a slight but definite reaction. The exchange reaction catalyzed by leucine, hystidine, glycine, serine, and isoleucine also undergoes an increase at the blastula stage. This is especially remarkable in the case of leucine. Although this work has been concerned with a limited number of amino acids, we feel that we may be entitled to make a few comments as to the role of the amino acid activating enzymes during the development of the sea urchin egg. As previously mentioned, amino acid incorporation into proteins, as tested in vivo, begins immediately following fertilization; its rate somewhat increases until about the 64.cell stage and then slightly declines until just before the early blastula. On the other hand, the ribosomes, as tested in vitro, appear to be fully activated within half an hour or so of fertilization. The observations presented in this paper show that the activity of the amino acid activating enzymes does not exhibit any significant increase until the early blastula. This may suggest that until the blastula stage the activity of the amino acid activating enzymes already present in the unfertilized egg is sufficient to meet the requirements of the relatively low rate of protein synthesis. When, however, this becomes very high, as it is between blastula and midgastrula, a greater activity of the amino acid activating enzymes is required as well. Taken together these facts indicate that the amino acid activating system, while not involved in the activation of protein synthesis following fertilization may be the rate limiting factor of protein synthesis in the early development of the sea urchin embryo.
167
ACIDS
A final point worth mentioning is the demonstration of the strong activation of L-glutamic acid by sea urchin eggs and embryos. To our knowledge, in no other biological system has the activation of this amino acid been so clearly demonstrated. ACKNOWLEDGMENTS The authors wish to express their gratitude to Professor A. Monroy for his helpful suggestions and advice in the course of this work. They also thank Dr. E. Scarano for his comments. REFERENCES M. L., AND MONROY, 1. GIUDICE, G., VITTORELLI, A. Acta Embryol. Morphol. Exptl. 6, 113 (1962). 2. GROSS, P. R., AND COUSINE.QU, G. H., Biochem. Biophys. Res. Communs. 10, 321 (1963). M. B., KELLER, E. B., AND 3. HOAGL~ND, ZAMECNIH, P. C., J. Biol. Chem. 218, 345 (1956). T., Exptl. Cell Res. 26, 405 (1961). 4. HULTIN, T., AND BERGSTR.~ND, A., Develop. 5. HULTIN, Biol. 2, 61 (1960). J. L., Exptl. Cell Res. 7, 530 (1954). 6. KAV.~NAU, A., BND PRICER, W. E., J. Biol. 7. KORNBERG, Chem. 191, 535 (1951). N. J., FARR, 8. LOWRY, 0. H., ROSEBROUGH, A. L., AND RAND.\LL, R. J., J. Biol. Chem. 193, 265 (1951). M. L., BND GUAR9. MONROY, A., VITTORELLI, NERI, R., Acta Embryol. Morphol. Exptl. 4, 77 (1961). M. L., J. Cell. 10. MONROY, A., AND VITTORELLI, Comp. Physiol. 60, 285 (1962). 11. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948). 12. NAKANO, E., AND MONROY, A., Exptl. Cell Res. 14, 236 (1958). 13. NEMER, M., Biochem. Biophys. Res. Communs. 8, 511 (1962). 14. SCARANO, E., .~ND MAGGIO, R., Exptl. Cell Res. 12, 403 (1957). 15. SCARANO, E., AND MaGGIO, R., Giorn. Biochim. 8, 98 (1959). 16 TYLER, A., Proc. Conf. Immuno Reproduction, The Population Council 13 (1962). 17. WILT, F. H., AND HULTIN, T., Biochem. Biophys. Res. Communs. 9, 313 (1962).
OF
BIOCHEMISTRY
Activation
AND
BIOPHYSICS
of Amino
Acids
R. MAGGIO From the Laboratory
103, 164-168 (1963)
during
Sea Urchin
Development
AND C. CATALAN02
of Comparative
Anatomy,
The University
of Palermo,
rtaly
Received July 10, 1963 INTRODUCTION Previous work from this laboratory has demonstrated that unfertilized sea urchin eggs have very little, if any, ability to incorporate in vivo labelled amino acids into their proteins. Incorporation begins immediately following fertilization but its rate becomes especially high between blastula and midgastrula (1, 10, 12). Experiments carried out in vitro (4, 5) indicate that the ribosomes isolated from unfertilized eggs are unable to incorporate amino acids into proteins but acquire this ability within 30 minutes after fertilization. However, the ribosomes from unfertilized eggs can be induced in vitro to synthesize polyphenylalanine by the addition of synthetic polyuridylic acid (13, 16, 17). These results seem, therefore, to warrant the suggestion that the lack of synthetic ability on the part of the ribosomes of the unfertilized egg may depend on the nonavailability of messenger RNA. That a new synthesis of RNA begins shortly after fertilization is indicated by the recent work of Gross and Cousineau (2). Another important step, and indeed the first one, in the process of protein synthesis to which, in the case of the sea urchin embryo, almost no attention has thus far been paid is the activation of amino acids. 1This work has been supported by grants from the Consiglio Nazionale delle Ricerche (Research Group on the Problem of Differentiation) and the National Institute of Health, U.S. Public Health Service (RGO6211) to the Laboratory of Comparative Anatomy, 2 Holder of a research fellowship from the Ministry of Educat,ion. 164
That amino acid activating enzymes are present in the unfertilized sea urchin egg was shown by Scarano and Maggio (14). The results to be described in this paper show that the activity of these enzymes undergoes a sudden increase at the blastula stage, i.e., coinciding with the increase in the rate of protein synthesis. MATERIALS
PREPARATION
AND
METHODS
OF THE ENZYMATIC EXTRACT
Eggs of Paracentrotus lividus were collected as described (9). Each batch was divided into two parts, one of which was fertilized and allowed to develop at 1%20°C. In the unfertilized eggs the jelly coat was removed by treatment with acid sea water. The eggs and embryos were homogenized in the cold, using two different media: Medium 9, containing 0.25 M sucrose, 0.008 iM MgC12, 0.04 M KF, and 0.05 Jf Tris buffer, pH 7.5, was used for the ATP-PPa2 exchange reaction; Medium B was a 0.05 M Na phosphate buffer at pH 7.38 which was employed for the preparations to be used for the hydroxamate reaction. The homogenates were centrifuged at 4°C. for 20 minutes at 15.OOOgand the supernatant was recentrifuged at 105OOOg for 60 minutes to remove all particulates. A great difficulty encountered in the study of amino acid activation in sea urchin eggs and embryos is the presence of a very large pool of free amino acids (Kavanau, 1954). Extensive removal of the free amino acids has been obtained by filtering the final supernatants through a Sephadex column. A Sephadex G 25 column (23 X 1 cm.) was equilibrated with 0.05 M Tris buffer, pH 7.5 (for filtration of extracts in medium A), or with Na-phosphate buffer, pH 7.38 (when medium B was used). Usually about 7 ml. of final supernatant, containing lOO-200 mg. of proteins, were submitted to filtration; all the operations were carried out in the cold room at 3°C. The first 5 ml.
ACTIVATION
OF AMINO
of effluent were used for the enzymatic assay. The protein concentration of this fraction (as determined by the method of Lowry et al., 1951) is very close to that of the original supernatant. On the other hand, the amino acid concentration (ninhydrin method according to Moore and Stein, 1948) was about 2 pM per 10 mg. of proteins, as against 55-58 &f in the original supernatant.
ASSAY OF THE ENZYMATIC ACTIVITY
ATP-PP32 Exchange Reaction PPa2 was synthesized by condensation of Paz according to the method of Kornberg and Pricer (7). Aliquots of Sephadex-filtered supernatant, containing lo-20 mg. of protein were used for each experiment. The reaction mixture and the conditions of each experiment are described in the explanations given in the tables. The addition of fluoride to the reaction mixture is particularly imperative owing to the presence of a very active pyrophosphatase. At the end of the incubation,
the ATP was isolated and its concentration radioactivity were determined as previously scribed (14).
“;g.
Hydroxamic Acid Reaction
RESULTS
52=
Unfert
Early
59d
60”
AND
I
PP32-ATP EXCHANGE REACTION E~~BRYOS OF PARACENTROTUS
. eggs
blast.
Mesenchyme
Mesenchyme
blast.
blast.
DISCUSSION
The data in Table I show that extracts of sea urchin eggs operate an ATP-PP32
ACID-DEPENDENT
stage
and de-
Salt-free NHZOH was prepared according to the method already described (15). Aliquots containing 8-16 mg. of proteins of filtered supernatant were added to a reaction mixture containing: 10 pM MgC12, 10 FM KF, about 1000 PM NHsOH, the proper amount of amino acids, and 100 PM Tris buffer pH 7.5 in a total volume of 1.7 ml. and then incubated for 1 hr. at 30°C. The reaction was stopped by the addition of 1 volume of FeCh solution, according to Hoagland et al. (3), and left in the cold for some hours. After centrifugation, the absorbancy of the clear supernatant was read at 520 mp using glycinhydroxamate as a standard.
TABLE L-GLUTAMIC
165
ACIDS
Substrate CUM)
IN EGGS AND
cpm/sM ATPa
% exchange”
None + n-glutamic + n-glutamic
acid, 28 acid, 85
2560 2330 3620
5.05 4.60 7.15
None + L-glutamic + n-glutamic
acid, 28 acid, 85
2170 3240 5250
4.34 6.48 10.50
none -I- n-glutamic + n-glutamic
acid, 85 acid, 85
5850 10,250 5400
6.88 12.00 6.35
none + n-glutamic + n-glutamic + n-glutamic
acid, 80 acid, 80 acid, 80 + Paz
5628 10,221 5575 176
10.20 18.57 10.10 0.3
n As calculated for 10 mg. of proteins of the enzymatic extract. Values subtracted from the zero time. b y0 exchange = (specific activity ATP”)/(0.5 X specific activity PP) X 100. c Conditions for Expt. 52: ATP, 22.84 FM; PP3?, 18.27 /AM (101,500 cpm per FM and 100,000 cpm per pM, respectively, for the unfertilized eggs and blastula). Enzymatic extract: 19.2 mg. protein for the unfertilized eggs and 16.0 mg. protein for the blastula. d Conditions for Expt. 59: ATP, 8.96 pM; PP3*, 11.13 pM (170,000 cpm per PM). Enzymatic extract: 7.66 mg. protein. 8 Conditions for Expt. 60: ATP, 8.96 pM; PP 32, 11.13 pM (110,000 cpm per MM); P3*, 12 pM (116,400 cpm per plM). Enzymatic extract: 11.3 mg. protein. To the reaction mixture were also added: MgC12, 10 pM; KF, 10 pM; Tris buffer, 0.05 M, final concentration, pH 7.5, final volume, 2 ml. Incubation for 20 minutes at 30°C.
166
MAGGIO
AND TABLE
PP32-ATP
EXCHANGE
REACTION
Expt. No.
54h
Unfert.
Unfert
58d
gastrula
. eggs
Mesenchyme
ACIDS
IN THE SEA URCHIN
DEVELOPMENT
Substrate b.W
eggs
Swimming
II
BY AMINO
stage
Early
55”
CATALYZED
CATALAKO
blast.
blast.
% exchange
none + L-phenylalanine, + L-leucine, GO
GO
3250 1635 3410
5.75 2.89 6.04
none + L-phenylalanine, f L-leucine, 60
GO
8380 8450 15,700
15.50 15.08 29.00
none + L-histidine, 60 + glycine, 100
GO80 6400 6300
10.10 10.80 10.50
none + L-histidine, GO + glycine, 100
5960 7560 8550
10.70 13.70 15.50
none + L-isoleucine, 80 + L-serine, 80
10,800 21,900 16,650
a As for Table I. 6 Conditions for Expt. 54: 7 &f ATP; 6.55 p&Z PP32 (113,000 cpm per PM and 108,000 respectively, for the unfertilized eggs and gastrula). Enzymatic extract: 9.6 mg. protein and 11.3 mg. protein gastrula. c Conditions for Expt. 55: 11.42 &I4 ATP; 11.8 pjl4 PP3* (120,000 cpm per &1 and 110,300 respectively, for the unfertilized eggs and blastula). Enzymatic extract: 23.20 mg. protein eggs and 18.95 mg. protein blastula. d Conditions for Expt. 58: 8.96 PM ATP; 11.13 PM PPa2 (210,000 cpm/MiM). Enzymatic mg. protein. The general experimental condition is described in Table I.
exchange reaction catalyzed by glutamic acid; the percentage of exchange increases as development proceeds. Actually, all throughout cleavage, and indeed until the blastula stage is reached, there is very little, if any, increase as compared with the unfertilized egg, while from the blastula stage onward, activity is considerably higher. The high blank value (incorporation in the ATP in the absence of added substrate) is likely to be due to the stimulation caused by the small amounts of free amino acids not removed by Sephadex filtration. Obviously this becomes particularly noticeable in the case of the developmental stages in which the activity of the enzyme system is higher. It must be added that control experiments have also shown that during incubation a small release of amino acids
10.3 20.8 15.9
cpm per &f, unfertilized cpm per PM, unfertilized extract:
8.74
(l-2 PM per 10 mg. of proteins both in the unfertilized eggs and in the developmental stages) takes place, and this may add to the above stimulation. However, since glutamic acid represents but a small percentage of the whole free amino acid pool [about 2%; (S)] we may feel confident that the stimulation observed when glutamic acid is added is entirely due to the substrate. As shown in experiments 39 and 60, n-glutamic acid does not stimulate ATP-PP32 exchange; if anything, it causes a slight inhibition. Furthermore, the exchange takes place only in the presence of pyrophosphate (experiment 60). Experiments in which the activation was estimated by the hydroxamate formation duplicate these results. In Table II the results of the exchange
ACTIVATION
OF AMINO
reaction catalyzed by some other amino acids are presented. The result obtained with phenylalanine is worth mentioning. The addition of this amino acid to extracts of unfertilized eggs brings about a considerable inhibition in the endogenous exchange reaction, while in the embryos phenylalanine causes a slight but definite reaction. The exchange reaction catalyzed by leucine, hystidine, glycine, serine, and isoleucine also undergoes an increase at the blastula stage. This is especially remarkable in the case of leucine. Although this work has been concerned with a limited number of amino acids, we feel that we may be entitled to make a few comments as to the role of the amino acid activating enzymes during the development of the sea urchin egg. As previously mentioned, amino acid incorporation into proteins, as tested in vivo, begins immediately following fertilization; its rate somewhat increases until about the 64.cell stage and then slightly declines until just before the early blastula. On the other hand, the ribosomes, as tested in vitro, appear to be fully activated within half an hour or so of fertilization. The observations presented in this paper show that the activity of the amino acid activating enzymes does not exhibit any significant increase until the early blastula. This may suggest that until the blastula stage the activity of the amino acid activating enzymes already present in the unfertilized egg is sufficient to meet the requirements of the relatively low rate of protein synthesis. When, however, this becomes very high, as it is between blastula and midgastrula, a greater activity of the amino acid activating enzymes is required as well. Taken together these facts indicate that the amino acid activating system, while not involved in the activation of protein synthesis following fertilization may be the rate limiting factor of protein synthesis in the early development of the sea urchin embryo.
167
ACIDS
A final point worth mentioning is the demonstration of the strong activation of L-glutamic acid by sea urchin eggs and embryos. To our knowledge, in no other biological system has the activation of this amino acid been so clearly demonstrated. ACKNOWLEDGMENTS The authors wish to express their gratitude to Professor A. Monroy for his helpful suggestions and advice in the course of this work. They also thank Dr. E. Scarano for his comments. REFERENCES M. L., AND MONROY, 1. GIUDICE, G., VITTORELLI, A. Acta Embryol. Morphol. Exptl. 6, 113 (1962). 2. GROSS, P. R., AND COUSINE.QU, G. H., Biochem. Biophys. Res. Communs. 10, 321 (1963). M. B., KELLER, E. B., AND 3. HOAGL~ND, ZAMECNIH, P. C., J. Biol. Chem. 218, 345 (1956). T., Exptl. Cell Res. 26, 405 (1961). 4. HULTIN, T., AND BERGSTR.~ND, A., Develop. 5. HULTIN, Biol. 2, 61 (1960). J. L., Exptl. Cell Res. 7, 530 (1954). 6. KAV.~NAU, A., BND PRICER, W. E., J. Biol. 7. KORNBERG, Chem. 191, 535 (1951). N. J., FARR, 8. LOWRY, 0. H., ROSEBROUGH, A. L., AND RAND.\LL, R. J., J. Biol. Chem. 193, 265 (1951). M. L., BND GUAR9. MONROY, A., VITTORELLI, NERI, R., Acta Embryol. Morphol. Exptl. 4, 77 (1961). M. L., J. Cell. 10. MONROY, A., AND VITTORELLI, Comp. Physiol. 60, 285 (1962). 11. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948). 12. NAKANO, E., AND MONROY, A., Exptl. Cell Res. 14, 236 (1958). 13. NEMER, M., Biochem. Biophys. Res. Communs. 8, 511 (1962). 14. SCARANO, E., .~ND MAGGIO, R., Exptl. Cell Res. 12, 403 (1957). 15. SCARANO, E., AND MaGGIO, R., Giorn. Biochim. 8, 98 (1959). 16 TYLER, A., Proc. Conf. Immuno Reproduction, The Population Council 13 (1962). 17. WILT, F. H., AND HULTIN, T., Biochem. Biophys. Res. Communs. 9, 313 (1962).