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Incorporation of N15-labeled glycine and alanine into the proteins of developing sea urchin eggs
494
INCORPORATION OF N15-LABELED GLYCINE AND ALANINE INTO THE PROTEINS OF DEVELOPING SEA URCHIN EGGS TORE The Wenner-Gren
Institute
HULTIN
for Experimental Received
Biology,
December
University
of St&Jwlm,
Sweden
7, 1951
EGGmaterial:
Eggs from the sea urchin Psammechinus miliaris (Gmelin) were used in the experiments.’ Dilute suspensionsof eggswere fertilized and allowed to develop at 22” C in glasstrays as described by Lindahl (8). At least 90 per cent of the ripe eggs developed into normal plutei. Isotope incubations: Equal sampleswere taken from the egg suspensions1) just before fertilization, and 2) O-24 hours after fertilization at 4 hour intervals. The sampleswere washed by centrifugation and then transferred to sea water containing the isotope. All sampleswere incubated with isotope for 4 hours, In the glycine experiments I mg of glycine (61 per cent N15)in 5 ml of seawater (pH 8.0) was administered per 1 mg of total egg nitrogen. Higher glycine concentrations did not proportionally increase the rate of isotope incorporation (Fig. 1). In the alanine experiments a corresponding amount of DL-N15-alanine, 2.4 mg, was supplied. All incubations were made with jelly-free eggs. After the incubations, the eggs were washed by centrifuging once in sea water and twice in isotonic NaCl-solution containing 0.05 volumes of isotonic sodium citrate solution. The eggswere frozen and stored at -20” C. Preparation of protein samples: The frozen material was extracted 3 times with isotonic KCI-solution, and the soluble fraction as well as the residue were treated with 0.1 M carrier glycine or alanine for dilution of the free amino acid isotope. Both fractions were treated repeatedly with hot trichloroacetic acid and with lipid solvents. The nitrogen of the remaining, insoluble material was consideredas proteinnitrogen. All extrations were made by grinding the material with an air-driven, rotating glass-rod. Results: It seemslikely that there is not an unrestricted, free diffusion of amino acids into the cells of unfertilized or developing sea urchin eggs.Moreover, changes in cell permeability have been demonstrated during the fertilization (11). Therefore it would be desirable to relate the incorporation of isotope into the cell proteins with the total amount of labeled amino acid, actually taken up by the cells in each single experiment. Unfortunately this latter value can scarcely be established sinceit seems very problematic to satisfactorily free the cells from traces of highly labeled amino acid trapped within the fertilization membrane space, blastocoel, etc. Both in the glycine (Fig. 2) and alanine (Fig. 3) experiments the same general 1 The experiments 1950-1951.
were
performed
at the Zoological
Station
of Kristineberg,
Sweden,
during
Incorporation
Fig. Fig.
Fig.
of glycine
and alanine
into sea urchin
Fig.
1.
495
2.
1. Rates of isotope incorporation into the total protein nitrogen fraction after incubation of Psammechinus gastrulae for 4 hours with varying concentrations of Ni5-glycine (12-16 hours after fertilization). 2. Rates of isotope incorporation into the non-soluble (upper curve) and soluble (lower curve) cell proteins at different stages of development. The eggs were incubated with N’s-glycine for 4 hours. Mesenchyme cells appeared at 9 hours of development. Invagination started at 12 hours.
Fig.
3.
Fig.
4.
Fig. 3. Similar experiments as in Fig. 2 but isotope added in the form of Nib-DL-alanine. Fig. 4. The isotope incorporation rates of the soluble proteins at different stages related to the corresponding incorporation rates of the non-soluble proteins. Upper curve: alanine experiments; lower curve: glycine experiments.
picture obtained.
of
isotope
incorporation
into
the
cell
proteins
during
development
was
Sea urchin eggs contain large amounts of free glycine (7), which cause a considerable isotope dilution in glycine experiments. Probably also a higher metabolic activity of alanine contributes to the higher incorporation level found in the alanine experiments. In both glycine and alanine experiments the non-soluble proteins had a higher rate of incorporation than the soluble proteins. The rate of incorporation into the non-soluble proteins increased gradually soon after fertilization. After about 8 hours of development (blastula stage) the incorporation into the soluble proteins became intensified (Fig. 2, 3, 4). The present results are in close agreement with previous experiments (5), where the incorporation of labeled ammonia into the proteins of developing Paracentrofus eggs was studied. As will be shown in a subsequent report, the isotope in such experiments is mainly incorporated into the protein amides. If it is assumed that granular cell elements (mitochondria, microsomes) are preferentially concentrated in the non-soluble protein fraction, and that prominent features of cell fluid proteins are revealed by the soluble fraction, the present results are well suited to the same interpretation that was suggested in the previous study (5): As a result of fertiliza-
T. Hultin tion a progressive multiplication and growth (cf. 6) of small, granular cytoplasm constituents may be initiated. From the blastula stage onwards, such units become more and more capable of productive functions according to the actual demands of the differentiating organism. Labeled proteins are emitted (cf. 4) and appear in the cell fluid in increasing amounts (cf. 10). As will be shown in a later communication, other synthetic processes (syntheses of purines, pyrimidines and amino acids) are intensified during the same period. Increased enzymatic activities have also been reported to occur at the same time (9, 1, 2). Mitochondria reappear after the blastula stage in fertilized, mitochondria-free egg-quarters obtained by centrifugation (3). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
AUGUSTINSSON, K. B., and GUSTAFSSON, T., .7. Cellular Comp. Physiol., 32, 311 (1949). GUSTAFSSON, T., and HASSELBERG, I.,Expfl. Cell Res., 1, 371 (1950). HARVEY, E. B., J. Exptl. Zool., 102, 253 (1946). HULTIN, T., Expfl. Cell Res., 1, 376 (1950). ~ ibid, 1, 599 (1950). JEENER, R., Biochim. et Biophys. Acta, 2, 633 (1948). KUTSCHER, F., and ACKERMANN, D., Ann. Reu. Biochem., 5, 460 (1936). LINDAHL, P. E., Acta Zool., 17, 179 (1936). MAZIA, D., BLUMENTHAL, G., and BENSON, E., Biol. Bull.,95,250 (1948). PERLMANN, P., and GUSTAFSSON, T., Experientia, 4, 481 (1948). STEWART, D. FL, and JACOBS, M. IX., J. Cellular Comp. Physiol., 1, 83 (1932).
INCORPORATION OF N15-LABELED GLYCINE AND ALANINE INTO THE PROTEINS OF DEVELOPING SEA URCHIN EGGS TORE The Wenner-Gren
Institute
HULTIN
for Experimental Received
Biology,
December
University
of St&Jwlm,
Sweden
7, 1951
EGGmaterial:
Eggs from the sea urchin Psammechinus miliaris (Gmelin) were used in the experiments.’ Dilute suspensionsof eggswere fertilized and allowed to develop at 22” C in glasstrays as described by Lindahl (8). At least 90 per cent of the ripe eggs developed into normal plutei. Isotope incubations: Equal sampleswere taken from the egg suspensions1) just before fertilization, and 2) O-24 hours after fertilization at 4 hour intervals. The sampleswere washed by centrifugation and then transferred to sea water containing the isotope. All sampleswere incubated with isotope for 4 hours, In the glycine experiments I mg of glycine (61 per cent N15)in 5 ml of seawater (pH 8.0) was administered per 1 mg of total egg nitrogen. Higher glycine concentrations did not proportionally increase the rate of isotope incorporation (Fig. 1). In the alanine experiments a corresponding amount of DL-N15-alanine, 2.4 mg, was supplied. All incubations were made with jelly-free eggs. After the incubations, the eggs were washed by centrifuging once in sea water and twice in isotonic NaCl-solution containing 0.05 volumes of isotonic sodium citrate solution. The eggswere frozen and stored at -20” C. Preparation of protein samples: The frozen material was extracted 3 times with isotonic KCI-solution, and the soluble fraction as well as the residue were treated with 0.1 M carrier glycine or alanine for dilution of the free amino acid isotope. Both fractions were treated repeatedly with hot trichloroacetic acid and with lipid solvents. The nitrogen of the remaining, insoluble material was consideredas proteinnitrogen. All extrations were made by grinding the material with an air-driven, rotating glass-rod. Results: It seemslikely that there is not an unrestricted, free diffusion of amino acids into the cells of unfertilized or developing sea urchin eggs.Moreover, changes in cell permeability have been demonstrated during the fertilization (11). Therefore it would be desirable to relate the incorporation of isotope into the cell proteins with the total amount of labeled amino acid, actually taken up by the cells in each single experiment. Unfortunately this latter value can scarcely be established sinceit seems very problematic to satisfactorily free the cells from traces of highly labeled amino acid trapped within the fertilization membrane space, blastocoel, etc. Both in the glycine (Fig. 2) and alanine (Fig. 3) experiments the same general 1 The experiments 1950-1951.
were
performed
at the Zoological
Station
of Kristineberg,
Sweden,
during
Incorporation
Fig. Fig.
Fig.
of glycine
and alanine
into sea urchin
Fig.
1.
495
2.
1. Rates of isotope incorporation into the total protein nitrogen fraction after incubation of Psammechinus gastrulae for 4 hours with varying concentrations of Ni5-glycine (12-16 hours after fertilization). 2. Rates of isotope incorporation into the non-soluble (upper curve) and soluble (lower curve) cell proteins at different stages of development. The eggs were incubated with N’s-glycine for 4 hours. Mesenchyme cells appeared at 9 hours of development. Invagination started at 12 hours.
Fig.
3.
Fig.
4.
Fig. 3. Similar experiments as in Fig. 2 but isotope added in the form of Nib-DL-alanine. Fig. 4. The isotope incorporation rates of the soluble proteins at different stages related to the corresponding incorporation rates of the non-soluble proteins. Upper curve: alanine experiments; lower curve: glycine experiments.
picture obtained.
of
isotope
incorporation
into
the
cell
proteins
during
development
was
Sea urchin eggs contain large amounts of free glycine (7), which cause a considerable isotope dilution in glycine experiments. Probably also a higher metabolic activity of alanine contributes to the higher incorporation level found in the alanine experiments. In both glycine and alanine experiments the non-soluble proteins had a higher rate of incorporation than the soluble proteins. The rate of incorporation into the non-soluble proteins increased gradually soon after fertilization. After about 8 hours of development (blastula stage) the incorporation into the soluble proteins became intensified (Fig. 2, 3, 4). The present results are in close agreement with previous experiments (5), where the incorporation of labeled ammonia into the proteins of developing Paracentrofus eggs was studied. As will be shown in a subsequent report, the isotope in such experiments is mainly incorporated into the protein amides. If it is assumed that granular cell elements (mitochondria, microsomes) are preferentially concentrated in the non-soluble protein fraction, and that prominent features of cell fluid proteins are revealed by the soluble fraction, the present results are well suited to the same interpretation that was suggested in the previous study (5): As a result of fertiliza-
T. Hultin tion a progressive multiplication and growth (cf. 6) of small, granular cytoplasm constituents may be initiated. From the blastula stage onwards, such units become more and more capable of productive functions according to the actual demands of the differentiating organism. Labeled proteins are emitted (cf. 4) and appear in the cell fluid in increasing amounts (cf. 10). As will be shown in a later communication, other synthetic processes (syntheses of purines, pyrimidines and amino acids) are intensified during the same period. Increased enzymatic activities have also been reported to occur at the same time (9, 1, 2). Mitochondria reappear after the blastula stage in fertilized, mitochondria-free egg-quarters obtained by centrifugation (3). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
AUGUSTINSSON, K. B., and GUSTAFSSON, T., .7. Cellular Comp. Physiol., 32, 311 (1949). GUSTAFSSON, T., and HASSELBERG, I.,Expfl. Cell Res., 1, 371 (1950). HARVEY, E. B., J. Exptl. Zool., 102, 253 (1946). HULTIN, T., Expfl. Cell Res., 1, 376 (1950). ~ ibid, 1, 599 (1950). JEENER, R., Biochim. et Biophys. Acta, 2, 633 (1948). KUTSCHER, F., and ACKERMANN, D., Ann. Reu. Biochem., 5, 460 (1936). LINDAHL, P. E., Acta Zool., 17, 179 (1936). MAZIA, D., BLUMENTHAL, G., and BENSON, E., Biol. Bull.,95,250 (1948). PERLMANN, P., and GUSTAFSSON, T., Experientia, 4, 481 (1948). STEWART, D. FL, and JACOBS, M. IX., J. Cellular Comp. Physiol., 1, 83 (1932).