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Amino acid component in the fish egg
DEVELOPMENTAL
BIOLOGY
28.
528-530 (1972)
BRIEF Amino
NOTES
Acid Component
in the Fish Egg
EIZO NAKANO AND SADAAKI YAMAMOTO Biological
Institute
and Radioisotope
Centre, Nagoya University,
Accepted January
Nagoya 464, Japan
24, 1972
The free amino acids and the amino acids of peptides and proteins in Oryzias quantitatively determined by the amino acid analyzer. Among the free amino acid, serine, and alanine occur in large amounts and glycine is present in a Among the protein amino acids, asparticand glutamic acids, proline, alanine, tine are dominant. There is some evidence for the existence of peptide pools, alanine, aspartic, and glutamic acids occur in large amounts. INTRODUCTION
In studies of protein metabolism using fish eggs as material, it is often of considerable value for the investigator to have the data on the amino acid component of the egg. Experiments with labeled methionine and leucine demonstrated the existence of amino acid pools in Or&as eggs (Monroy et al., 1961; Nakano and IshidaYamamoto, 1968), but the amount of these amino acids in the native eggs has been uncertain. The present report is for the purpose of giving some information on the amino acid pools and the amino acid components of the yolk protein in Oryzias
eggs. MATERIALS
AND
METHODS
The material used was the orange-red variety of the medaka, Oryzias latipes. Fertilized eggs were raised until the late gastrula stage in 0.1 M Ringer’s solution. After several washings with water, the embryos were crushed in a glass mortar and the chorions were removed by filtration with bolting silk. The filtrate was precipitated with ice-cold 5% TCA. The precipitate was allowed to stand for 30 min at 6”C, centrifuged, suspended in 5% TCA and centrifuged again. The precipi-
tate was hydrolyzed with 6 N HCl at 105°C for 22 hr in a sealed tube and an aliquot was analyzed for its amino acid content on a Beckman Model 120 autoanalyzer. The TCA supernatant, together with the washings, was shaken three times with ether to remove TCA and then methanol was added to give a final concentration of 90%. The mixture was allowed to stand overnight in a refrigerator and centrifuged. The precipitate was hydrolyzed with 6 N HCl as described above and used for the determination of amino acids. The supernatant was concentrated and directly submitted to the automatic analyzer. Eggs labeled with 14C-leucine were obtained as described in the previous paper (Monroy et al., 1961). After washing with water containing cold leucine, the eggs were homogenized in ice-cold 5% TCA containing cold leucine. The precipitate was removed by centrifugation and the supernatant was shaken three times with ether to remove TCA, and concentrated under reduced pressure. An aliquot was used for paper chromatography. The solvent used was tertiary butanol-formic acid-water (70 : 15 : 15) and the radioactivity on paper chromatograms was counted by the use of a paper chromatogram scanner (Aloka JPC-102).
528 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
eggs have been acids, glutamic small quantity. valine, and leuin which serine,
BRIEF
RESULTS
AND
DISCUSSION
Table 1 demonstrates the amounts of amino acids found in the three fractions. The TCA-insoluble, TCA-soluble-methanol-insoluble, and methanol-soluble fractions are referred to as protein, peptide, and free amino acids, respectively. The results show that most of the amino acids exist as protein, which may be mainly contained in the yolk. Among them, aspartic and glutamic acids, proline, alanine, valine, and leucine occur in large amounts, comprising more than half of the protein amino acids. The free amino acid pools and peptide pools are of roughly the same size, and two orders of magnitude smaller than the protein amino acids. Since glutamic acid, serine, and alanine are dominant in the free amino acid pools, these amino acids are not appropriate to use as tracers for studying protein metabolism in Oryzias eggs. Methionine and leucine, which were used in the previous experiments (Monroy et al., 1961; Nakano and Ishida-Yamamoto, 1968) are present only in small amounts, making them suitable for isotope experiments. Glycine occurs in low concentration among both free and peptide amino acids. In this respect, Oryzias eggs are quite different from sea urchin eggs (Kavanau, 1954; Silver and Comb, 1966) and other marine invertebrate eggs (Morrill, 1963; Reverberi et al., 1964). The presence of peptide pools in Oryzias eggs should be emphasized. This is in good agreement with the fact that when the eggs are labeled with 14C-leucine, the radioactivity of the TCA-soluble fraction is mainly concentrated in the peptide fraction and only small amounts are found as free amino acids (Fig. 1). The only distinct peaks of radioactivity were found at the origin and in the position of leucine. However, a continuous zone of radioactivity extends between these two points. When these peptide fractions were eluted, hydrolyzed and
529
NOTES
rechromatographed, the radioactive peak matched the chromatographic position of leucine very closely. The role of peptide pools in Oryzias eggs is not clear. The investigation of this point will be a subject of future research. TABLE 1 AMINO ACIDS IN THE Oryzias EG@ Amino acid
Free
Peptide
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.35 0.02
0.39
“Values tru1a.
0.19
0.23 0.16 0.53 0.60 0.29 0.11 0.53 0.36 0.06 0.29 0.34 0.12 0.09
expressed
38.3 13.3 22.3 50.3 22.3 20.3 56.3 54.3 30.3 49.8 5.8 41.0 8.5 31.5 52.3 3.5 20.3
0.20 0.95 1.64 0.49 1.55 1.41 Trace
0.26 1.78 0.74 0.06 0.18 0.30 0.22
in pmoles/103
1 0.83
Protein
eggs. Late gas-
0 Rf
FIG. 1. Distribution of radioactivity on the paper chromatogram of the TCA-soluble fraction of Oryzias eggs labeled with “C-leucine.
REFERENCES KAVANAU, J. L. (1954). Amino acid metabolism in the early development of the sea urchin Parocentrotus hi&s. Exp. Cell Res. 7,530-557. MONROY, A., ISHIDA, M., and NAKANO, E. (1961). The
530
DEVELOPMENTAL
BIOLOGY
pattern of transfer of the yolk material to the embryo during the development of the teleostean fish, Orytias lntipes. Embryologia 6, 151-158. MORRILL, J. B. (1963). Bound amino acids of egg albumen and free amino acids in the larval and adult Limnaea palustris. Acta Embryol. Morphol. Exp. 6, 339-343. NAKANO, E., and ISHIDA-YAMAMOTO, M. (1968). Uptake and incorporation of labeled amino acids in
VOLUME
28.
1972
fish oocytes. Acta Embryol. Morphol. Exp. 10, 109-116. REVERBERI, G., MALIARO, M., and METAFORA, S. (1964). Free amino acids in the development of the Aplysia egg. Acta Embryol. Morphol. Exp. 7, lOl105. SILVER, D. J., and COMB, D. G. (1966). Free amino acid pools in the developing sea urchin Lytechinus variegatus. Exp. Cell Res. 43, 699-700.
BIOLOGY
28.
528-530 (1972)
BRIEF Amino
NOTES
Acid Component
in the Fish Egg
EIZO NAKANO AND SADAAKI YAMAMOTO Biological
Institute
and Radioisotope
Centre, Nagoya University,
Accepted January
Nagoya 464, Japan
24, 1972
The free amino acids and the amino acids of peptides and proteins in Oryzias quantitatively determined by the amino acid analyzer. Among the free amino acid, serine, and alanine occur in large amounts and glycine is present in a Among the protein amino acids, asparticand glutamic acids, proline, alanine, tine are dominant. There is some evidence for the existence of peptide pools, alanine, aspartic, and glutamic acids occur in large amounts. INTRODUCTION
In studies of protein metabolism using fish eggs as material, it is often of considerable value for the investigator to have the data on the amino acid component of the egg. Experiments with labeled methionine and leucine demonstrated the existence of amino acid pools in Or&as eggs (Monroy et al., 1961; Nakano and IshidaYamamoto, 1968), but the amount of these amino acids in the native eggs has been uncertain. The present report is for the purpose of giving some information on the amino acid pools and the amino acid components of the yolk protein in Oryzias
eggs. MATERIALS
AND
METHODS
The material used was the orange-red variety of the medaka, Oryzias latipes. Fertilized eggs were raised until the late gastrula stage in 0.1 M Ringer’s solution. After several washings with water, the embryos were crushed in a glass mortar and the chorions were removed by filtration with bolting silk. The filtrate was precipitated with ice-cold 5% TCA. The precipitate was allowed to stand for 30 min at 6”C, centrifuged, suspended in 5% TCA and centrifuged again. The precipi-
tate was hydrolyzed with 6 N HCl at 105°C for 22 hr in a sealed tube and an aliquot was analyzed for its amino acid content on a Beckman Model 120 autoanalyzer. The TCA supernatant, together with the washings, was shaken three times with ether to remove TCA and then methanol was added to give a final concentration of 90%. The mixture was allowed to stand overnight in a refrigerator and centrifuged. The precipitate was hydrolyzed with 6 N HCl as described above and used for the determination of amino acids. The supernatant was concentrated and directly submitted to the automatic analyzer. Eggs labeled with 14C-leucine were obtained as described in the previous paper (Monroy et al., 1961). After washing with water containing cold leucine, the eggs were homogenized in ice-cold 5% TCA containing cold leucine. The precipitate was removed by centrifugation and the supernatant was shaken three times with ether to remove TCA, and concentrated under reduced pressure. An aliquot was used for paper chromatography. The solvent used was tertiary butanol-formic acid-water (70 : 15 : 15) and the radioactivity on paper chromatograms was counted by the use of a paper chromatogram scanner (Aloka JPC-102).
528 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
eggs have been acids, glutamic small quantity. valine, and leuin which serine,
BRIEF
RESULTS
AND
DISCUSSION
Table 1 demonstrates the amounts of amino acids found in the three fractions. The TCA-insoluble, TCA-soluble-methanol-insoluble, and methanol-soluble fractions are referred to as protein, peptide, and free amino acids, respectively. The results show that most of the amino acids exist as protein, which may be mainly contained in the yolk. Among them, aspartic and glutamic acids, proline, alanine, valine, and leucine occur in large amounts, comprising more than half of the protein amino acids. The free amino acid pools and peptide pools are of roughly the same size, and two orders of magnitude smaller than the protein amino acids. Since glutamic acid, serine, and alanine are dominant in the free amino acid pools, these amino acids are not appropriate to use as tracers for studying protein metabolism in Oryzias eggs. Methionine and leucine, which were used in the previous experiments (Monroy et al., 1961; Nakano and Ishida-Yamamoto, 1968) are present only in small amounts, making them suitable for isotope experiments. Glycine occurs in low concentration among both free and peptide amino acids. In this respect, Oryzias eggs are quite different from sea urchin eggs (Kavanau, 1954; Silver and Comb, 1966) and other marine invertebrate eggs (Morrill, 1963; Reverberi et al., 1964). The presence of peptide pools in Oryzias eggs should be emphasized. This is in good agreement with the fact that when the eggs are labeled with 14C-leucine, the radioactivity of the TCA-soluble fraction is mainly concentrated in the peptide fraction and only small amounts are found as free amino acids (Fig. 1). The only distinct peaks of radioactivity were found at the origin and in the position of leucine. However, a continuous zone of radioactivity extends between these two points. When these peptide fractions were eluted, hydrolyzed and
529
NOTES
rechromatographed, the radioactive peak matched the chromatographic position of leucine very closely. The role of peptide pools in Oryzias eggs is not clear. The investigation of this point will be a subject of future research. TABLE 1 AMINO ACIDS IN THE Oryzias EG@ Amino acid
Free
Peptide
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
0.35 0.02
0.39
“Values tru1a.
0.19
0.23 0.16 0.53 0.60 0.29 0.11 0.53 0.36 0.06 0.29 0.34 0.12 0.09
expressed
38.3 13.3 22.3 50.3 22.3 20.3 56.3 54.3 30.3 49.8 5.8 41.0 8.5 31.5 52.3 3.5 20.3
0.20 0.95 1.64 0.49 1.55 1.41 Trace
0.26 1.78 0.74 0.06 0.18 0.30 0.22
in pmoles/103
1 0.83
Protein
eggs. Late gas-
0 Rf
FIG. 1. Distribution of radioactivity on the paper chromatogram of the TCA-soluble fraction of Oryzias eggs labeled with “C-leucine.
REFERENCES KAVANAU, J. L. (1954). Amino acid metabolism in the early development of the sea urchin Parocentrotus hi&s. Exp. Cell Res. 7,530-557. MONROY, A., ISHIDA, M., and NAKANO, E. (1961). The
530
DEVELOPMENTAL
BIOLOGY
pattern of transfer of the yolk material to the embryo during the development of the teleostean fish, Orytias lntipes. Embryologia 6, 151-158. MORRILL, J. B. (1963). Bound amino acids of egg albumen and free amino acids in the larval and adult Limnaea palustris. Acta Embryol. Morphol. Exp. 6, 339-343. NAKANO, E., and ISHIDA-YAMAMOTO, M. (1968). Uptake and incorporation of labeled amino acids in
VOLUME
28.
1972
fish oocytes. Acta Embryol. Morphol. Exp. 10, 109-116. REVERBERI, G., MALIARO, M., and METAFORA, S. (1964). Free amino acids in the development of the Aplysia egg. Acta Embryol. Morphol. Exp. 7, lOl105. SILVER, D. J., and COMB, D. G. (1966). Free amino acid pools in the developing sea urchin Lytechinus variegatus. Exp. Cell Res. 43, 699-700.