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Incorporation of 14 C-Amino Acids into Egg Proteins by the Laying Hen1,2,3
ULTIMOBRANCHIAL EXTRACT
calcemic effect of thyrocalcitonin. Federation Proc. 26: 368-370. Palmeri, G. M. A., J. S. Thompson and L. P. Eliel, 1969. Hypomagnesemic effect of thyrocalcitonin. Endocrinology, 84: 1509-1511. Pearse, A. G. E., and A. F. Carvalheira, 1967. Cytochemical evidence for an ultimobranchial origin of rodent thyroid "C" cells. Nature (London), 214: 929-930. Tauber, S. D., 1967. The ultimobranchial origin of thyrocalcitonin. Proc. National Academy Sci. 58: 1684-1687. Urist, M. R., 1967. Avian parathyroid physiology: Including a special comment on calcitonin. Amer. Zoologist, 7: 883-895.
Incorporation of 14C-Amino Acids into Egg Proteins by the Laying Hen1'2'3 J. HASSELL, J. D. YAGER AND N. W. KLEIN Department of Animal Genetics, University of Connecticut, Storrs, Connecticut 06268 (Received for publication March 9, 1970)
L
AYING hens have been fed and in' jected with various radioactive compounds in order to investigate the synthesis of egg constituents as well as to produce labeled egg materials for use in subsequent experimentation. Mandeles and Ducay (1962) injected laying hens with "C-lysine and 14C-glutamic acid and examined the radioactivity in the conalbumin, ovalbumin, and lysozyme protein fractions of the egg white. Following a single injection of the two amino acids, peak activities in 1 Scientific contribution No. 395 oi the Agricultural Experiment Station, University of Connecticut. Contribution No. 184 of the Institute of Cellular Biology. 2 This work was supported in part by U.S. Atomic Energy Commission Contract No. AT(3O-l)4030 and the National Science Foundation (GB-5129). 3 This study was initiated at the Biology Department, Marquette University, Milwaukee, Wisconsin.
the egg white proteins were observed in the second egg produced after injection. The rates at which labeled amino acids were incorporated into the conalbumin and ovalbumin fractions were similar, but higher than for the lysozyme fraction. These observations in conjunction with results obtained with oviduct minces (Mandeles and Ducay, 1962) suggested that the time or rate of synthesis for these proteins varied during the egg laying cycle and that the oviduct was the common site of synthesis. Kritchevsky et al. (1951) fed 14C-sodium acetate to a laying hen over a ten day period and determined total uptake into the egg components (Kritchevsky et al., 1951) as well as incorporation into specific yolk fractions (Kritchevsky and Kirk, 1951). As might be expected with the particular precursor used, the yolks contained considerably more activity than either the white or shell and the specific activities of the
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
Hirsch, P. F., and P. L. Munson, 1969. Thyrocalcitonin. Physiological Reviews, 49: 548-611. Kenny, A. D., and C. A. Heiskell, 1965. Effect of crude thyrocalcitonin on calcium and phosphorus metabolism in rats. Proc. Soc. Exp. Biol. Med. 120: 269-270. Kenny, A. D., 1964. Discussion. Recent Progress in Hormone Research, 20: 84. Kraintz, L., and K. Instcher, 1969. Effect of calcitonin on the domestic fowl. Can. J. Physiol. Pharmacol. 47: 313-3 IS. Lloyd, J. W., and W. E. Collins, 1969. Hypomagnesemic effect of avian calcitonin. Poultry Sci. 49: 446-448. Orimo, H., 1967. Influences of age on the hypo-
1121
1122
J. HASSELL, J. D. YAGER AND N. W. KLEIN
MATERIALS AND METHODS A White Leghorn hen was injected with one mCi. of a 14C-labeled amino acid mixture* into the wing vein. In an attempt to maintain a high level of circulating radioactivity, one half of the injection (0.5 ml.) was administered 30 minutes after the first egg of a new clutch was laid and the second half was injected 10 hours later. Egg collections began the following morning and were continued for 36 eggs. The data presented will be limited to the first five whites and the first 10 yolks. Eggs were collected at * Schwarz Bioresearch, Inc., reconstituted protein hydrolysate with the following specific activities in mCi./m mole.: alanine, 90; arginine, 143; aspartic acid, 120; glutamic acid, 130; isoleucine, 140; leucine, 140; lysine, 135; phenylalanine, 250; proline, 109; serine, 105; threonine, 119; tyrosine, 240; valine, 120.
daily intervals except for day five when an egg was not produced. The whites were withdrawn through a hole made in the small end of the eggs with a syringe attached to a 13 gauge Horseley needle. One ml. of sterile chick Ringer's solution was used to rinse the yolk and inner shell surface and was combined with the white. The basic procedure of Rhodes et al. (1958) was used to isolate the white proteins. Whites were stirred for five hours at 4°C, dialized against starting buffer (0.1 M acetic acid-ammonium hydroxide, pH 4.3) for 18 to 24 hours, and then centrifuged at 30,000 X g for 15 minutes to remove insoluble materials. Carboxymethylcellulose (Sigma Chemical Co.) was rinsed successively with N NaOH, H 2 0, M NaCl, H 2 0, N HC1, H 2 0, and starting buffer. A refrigerated (4°C.) column 1.9 X 30 cm. was used for the separations and was repacked for each white. The white proteins were eluted at a flow rate of 1.5 ml. per minute with stepwise pH changes from 4.3 to 10.0. Following the elution of the pH 9.5 globulin fraction, a pH 10.0 buffer with 0.025 M Na 2 C0 3 was used to elute the avidin and then a pH 10.0 buffer with 0.2 M Na 2 C0 3 was used to elute the lysozme. Optical density changes at 280 mp,. were monitored at three minute intervals with a recording spectrophotometer (Gilford Instrument Laboratories, Inc.). The volume of each white fraction was reduced with 20 M Carbowax (Union Carbide Corp.) dialized exhaustively against water, lyophilized to dryness, and weighed to the nearest mg. Yolks were rinsed thoroughly with chick Ringer's solution to remove traces of adhering white and then freed from the vitelline membrane by compressing the yolks in a fold of aluminum foil. The basic procedure of Bernandi and Cook (1960) was used to separate the yolks into three fractions. Two volumes of 0.45 M MgS04 were first added to the yolks and then solid
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
yolk cholesterol fractions were much greater than that of the yolk proteins. Peak activities for the various yolk fractions occurred in the fifth to seventh egg laid after the initiation of labeled acetate feeding. The significance of these peaks to the process of yolk synthesis can not be adequately considered because the labeled material was administered over a period of several days. In present investigation labeled amino acids were administered into a laying hen and the radioactivities contained in all the major white protein fractions as well as the proteins of three yolk fractions were subsequently determined. The data obtained confirm certain other studies dealing with yolk and white formation and should provide a basis for investigators interested in preparing labeled egg components. However, it should be noted that a single hen was employed in these studies and that the possibility of individual variations in the utilization of labeled amino acids was not evaluated.
1123
AMINO ACIDS IN EGGS
Approximately one mg. of each of the white fractions was dissolved in one ml. of water and aliquots were taken for protein determinations (Lowry et al., 1951) and for platings on stainless steel planchettes. Aliquots of 0.01 ml. of each yolk fraction were extracted successively with 5% trichloroacetic acid, 95% alcohol, chloroformethanol (70°C), 95% alcohol, and 5% perchloric acid (70°C). The remaining protein precipitates were dissolved in N NaOH at 60 °C. and aliquots were taken
for protein determinations (Lowry et al., 1951) and platings on planchettes. All planchette counting was done with a Nuclear Chicago low-background counter and the counts obtained were corrected for background and self absorption. RESULTS AND DISCUSSION
The quantities of the various protein fractions recovered basically agreed with the results reported by Rhodes et al. (1958) (Table 1). Polyacrylamide gel electrophoresis of the egg white fractions indicated that complete purification for all the fractions was not achieved by one passage through the column. For example, although ovalbumin Aa exhibited a single electrophoretic band, ovalbumin A3 was heavily contaminated with ovalbumins Ai, A2, and globulin. The high degree of purity reported by Rhodes et al. (1958) in contrast to these observations may indicate that a more sensitive analytical procedure was employed in the present study. Peak specific activity values for all egg white protein fractions were observed in the white of the second egg laid following injection (Table 1). This was expected as the second half of the labeled material was
TABLE 1.—The incorporation of radioactivity into egg white protein fractions following the administration of a uC-amino acid mixture into a hen Egg number Fraction
Elution PH
1
2
3
4
5
Weight/fraction 1 mg.
c.p.m./mg. c.p.m./mg. c.p.m./mg. c.p.m./mg. c.p.m./mg. Ovomucoid+ Flavoprotein Ovalbumin Ai Ovalbumin A2 Ovalbumin A3 Globulin Conalbumin Conalbumin Globulin Globulin Avidin Lysozyme 1
4.3 4.55 4.75 5.0 5.5 6.3 7.0 8.5 9.5 10.0 10.0
Mean ± standard error.
1,342 1,178 1,118 1,005 583 758 1,430 1,250 1,984 966 711
5,972 6,875 5,714 6,084 5,183 6,128 5,656 4,145 5,278 3,438 3,850
1,028 964 1,590 560 858 710 1,227 561 816
—
614
333 382 460 424 513 539 512 1,000 848 455 324
344 327 338 345 404 415 663 293 336 368
—
630+ 59 1,658 + 107 421+ 45 170+ 22 105+ 15 177+ 70 678+ 98 27+ 7 29+ 4 18+ 1 98+ 8
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
MgS0 4 was added to adjust the final concentration to 0.45 M. The solutions consisting of approximately 40 ml. were stirred for one hour at 4°C. and centrifuged for 24 hours at 30,000 r.p.m. using a type 30 rotor in a Beckman Model L Ultracentrifuge. The bottoms of the tubes were punctured and the yellow high density fractions (HDF) of approximately 19 ml. and the white buffy intermediate fractions (IF) of approximately 6 ml. were successively collected. The gel-like low density fractions (LDF) remaining at the tops of the tubes were collected with the aid of a spatula and dissolved in 0.45 M MgS0 4 . The volume of this fraction prior to the addition of the salt solution was approximately 15 ml.
1124
J. HASSELL, J. D. YAGER AND N. W. KLEIN
TABLE 2.—The incorporation of radioactivity into
yolk protein fractions following the administration of a uC-amino acid mixture into a hen Day after Egg injection number
High density Low density Intermediate fraction fraction fraction cp.m./rag. c.p.m./mg. c.p.m./mg. protein protein protein
1
125 948 1,211 1,295 1,608 1,459 888 655 420 254 376.5±31.1
Mean ± standard error.
administered after the formation of the first egg white but before the formation of the second (Romanoff and Romanoff, 1949). The increase in specific activities of the various white protein fractions between the first and second eggs ranged from approximately three to nine fold. This was followed by a decrease in the fraction specific activities which was most striking between eggs two and three. The decrease between eggs two and three ranged from 72.2% to 90.8% but between eggs three and four this value was between 40.2% and 71.1% with the exceptions of the pH 8.5 globulin which approximately doubled in specific activity and the pH 9.5 globulin which essentially did not change. The relationship between the various specific activities of the fractions in one egg did not hold for the other eggs. However, the differences in labeling previously reported by Mandeles and Ducay (1962) were confirmed. Thus, in the second egg the ovalbumin and conalbumin fractions had activities which were similar and higher than the lysozyme fraction. The differences in activities of the fractions may simply reflect differences in the amino acid compositions of the proteins or that the white proteins are synthesized at different periods during the egg laying cycle (Mandeles and Ducay, 1962). Alternatively, the
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
1 1 97 0 2 2 1,030 895 3 3 1,270 888 4 4 1,480 1,240 6 5 1,630 1,150 7 6 1,510 1,300 8 7 1,050 1,080 9 8 724 268 10 9 473 609 11 10 411 356 mgprotein/fraction 1 1,152.5±65.8 474.8+35.4
various proteins may be formed at sites within the oviduct which differed in labeled precursor concentrations. The observations that specific white proteins are synthesized by different cell types within the oviduct support this latter possibility (O'Malley et al, 1967; Kohler et al., 1968). The specific activities of the yolk proteins also increased rapidly between the first and second eggs (Table 2). However, unlike the white proteins, the yolk proteins continued to increase in specific activity reaching a peak for the HDF and IF in the fifth egg formed following injection (sixth day) and in the sixth egg for the LDF. Althrough previous observations have indicated that the greatest proportion of yolk was deposited during the last four days prior to ovulation (Romanoff and Romanoff, 1949), these data indicate that yolk proteins were deposited at the highest rate either on the fourth or fifth day prior to ovulation (an egg was not laid on day five). The rate at which specific activities declined in the yolk proteins following peak activities was more gradual and continued for a longer period than was observed for the white. For example, the specific activity of the HDF fraction was reduced by 71% only after four additional eggs had been laid. The specific activities for the HDF and IF of the first ten eggs were quite similar and slightly higher than the LDF. Polyacrylamide gel electrophoresis of these fractions indicated considerable similarity although the LDF contained fewer protein components. Autoradiographic analysis of the HDF gels indicated no major differences in the labeling of the various proteins comprising this fraction. Thus, unlike the white proteins, the yolk proteins may be formed as a unit from a common precursor pool within a limited period of time. Recovery values were calculated on the basis that a full millicurie was administered and a counting efficiency of 22%. The
1125
AMINO ACIDS IN EGGS
white of eggs one to five contained 7.8% of the injected radioactivity with 5.3% in egg two alone. The yolks from eggs one to ten combined contained only 3.9%. Thus, the second white alone accounted for about one-half of the total radioactivity recovered from the first five white and ten yolks combined. SUMMARY
ACKNOWLEDGMENT
The authors wish to thank Mrs. K. Hagedorn for her technical assistance, Dr. P. Abramoff for injecting the hen, and Dr. C. R. Grau for his helpful suggestions.
Kohler, P. 0., P. M. Grimley and B. W. O'Malley, 1968. Protein synthesis: differential stimulation of cell-specific proteins in epithelial cells of chick oviduct. Science, 160: 86-87. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Mandeles, S., and E. D. Ducay, 1962. Site of egg white protein formation. J. Biol. Chem. 237: 3196-3199. O'Malley, B. W., W. L. McGuire and S. G. Korenman, 1967. Estrogen stimulation of synthesis of specific proteins and RNA polymerase activity in the immature chick oviduct. Biochim. Biophys. Acta, 145: 204-207. Rhodes, M. B., P. R. Azari and R. E. Feeney, 1958. Analysis, fractionation, and purification of egg white proteins with cellulose-cation exchanger. J. Biol. Chem. 230: 399-408. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons, Inc., New York.
NEWS AND NOTES (Continued from page 1093) was greatest for the modulus of elasticity and least for the shear values. There was no significant difference in modulus of toughness between raw and cooked muscle. Universal testing instrument measurements and the shear values all showed a significant difference between breast and thigh muscles for both raw and cooked turkeys. The shear values for raw and cooked muscle, and the modulus of toughness for raw also indicated a significant difference between the outer two sample depths of the breast muscle. It is thought
here that this is most likely the result of differences in structural characteristics between surface and deeper layers of the muscle. The second slice from the breast muscle had the lowest instrument values and the thigh the highest. Where sex exerted a significant effect, toms had the higher values with one exception. Where groups were significant, heavier birds were found to have larger shear and modulus of toughness values, but lighter birds had the larger values for maximum stress and modulus of elasticity. Age, where signif-
(Continued on page 1131)
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
A laying hen was injected with a total of one mCi. of a "C-labeled amino acid mixture over a ten hour period. Peak protein activities were observed in the second white, and the fifth and sixth yolks. Differences were observed in the specific activities of the individual white proteins from a single egg whereas the specific activities of two yolk fractions were similar and somewhat higher than that of a third fraction. A total of 11.7% of the administered radioactivity was located in the proteins of the whites of the first five eggs plus the yolk proteins of the first ten eggs with approximately one-hlaf of this total located in the white proteins of the second egg.
REFERENCES Bernardi, G., and W. H. Cook, 1960. An electrophoretic and ultracentrifugal study of the proteins of the high density fraction of egg yolk. Biochem. Biophys. Acta, 44: 86-96. Kritchevsky, D., C. R. Grau, B. M. Tobert and B. J. Kruecker, 1951. Distribution of radioactivity in the egg after feeding sodium acetate1-C". Proc. Soc. Exp. Biol. Med. 76: 741-743. Kritchevsky, D., and M. R. Kirk, 1951. Radioactive eggs. II. Distribution of radioactivity in the yolks. Proc. Soc. Exp. Biol. Med. 78: 200202.
calcemic effect of thyrocalcitonin. Federation Proc. 26: 368-370. Palmeri, G. M. A., J. S. Thompson and L. P. Eliel, 1969. Hypomagnesemic effect of thyrocalcitonin. Endocrinology, 84: 1509-1511. Pearse, A. G. E., and A. F. Carvalheira, 1967. Cytochemical evidence for an ultimobranchial origin of rodent thyroid "C" cells. Nature (London), 214: 929-930. Tauber, S. D., 1967. The ultimobranchial origin of thyrocalcitonin. Proc. National Academy Sci. 58: 1684-1687. Urist, M. R., 1967. Avian parathyroid physiology: Including a special comment on calcitonin. Amer. Zoologist, 7: 883-895.
Incorporation of 14C-Amino Acids into Egg Proteins by the Laying Hen1'2'3 J. HASSELL, J. D. YAGER AND N. W. KLEIN Department of Animal Genetics, University of Connecticut, Storrs, Connecticut 06268 (Received for publication March 9, 1970)
L
AYING hens have been fed and in' jected with various radioactive compounds in order to investigate the synthesis of egg constituents as well as to produce labeled egg materials for use in subsequent experimentation. Mandeles and Ducay (1962) injected laying hens with "C-lysine and 14C-glutamic acid and examined the radioactivity in the conalbumin, ovalbumin, and lysozyme protein fractions of the egg white. Following a single injection of the two amino acids, peak activities in 1 Scientific contribution No. 395 oi the Agricultural Experiment Station, University of Connecticut. Contribution No. 184 of the Institute of Cellular Biology. 2 This work was supported in part by U.S. Atomic Energy Commission Contract No. AT(3O-l)4030 and the National Science Foundation (GB-5129). 3 This study was initiated at the Biology Department, Marquette University, Milwaukee, Wisconsin.
the egg white proteins were observed in the second egg produced after injection. The rates at which labeled amino acids were incorporated into the conalbumin and ovalbumin fractions were similar, but higher than for the lysozyme fraction. These observations in conjunction with results obtained with oviduct minces (Mandeles and Ducay, 1962) suggested that the time or rate of synthesis for these proteins varied during the egg laying cycle and that the oviduct was the common site of synthesis. Kritchevsky et al. (1951) fed 14C-sodium acetate to a laying hen over a ten day period and determined total uptake into the egg components (Kritchevsky et al., 1951) as well as incorporation into specific yolk fractions (Kritchevsky and Kirk, 1951). As might be expected with the particular precursor used, the yolks contained considerably more activity than either the white or shell and the specific activities of the
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
Hirsch, P. F., and P. L. Munson, 1969. Thyrocalcitonin. Physiological Reviews, 49: 548-611. Kenny, A. D., and C. A. Heiskell, 1965. Effect of crude thyrocalcitonin on calcium and phosphorus metabolism in rats. Proc. Soc. Exp. Biol. Med. 120: 269-270. Kenny, A. D., 1964. Discussion. Recent Progress in Hormone Research, 20: 84. Kraintz, L., and K. Instcher, 1969. Effect of calcitonin on the domestic fowl. Can. J. Physiol. Pharmacol. 47: 313-3 IS. Lloyd, J. W., and W. E. Collins, 1969. Hypomagnesemic effect of avian calcitonin. Poultry Sci. 49: 446-448. Orimo, H., 1967. Influences of age on the hypo-
1121
1122
J. HASSELL, J. D. YAGER AND N. W. KLEIN
MATERIALS AND METHODS A White Leghorn hen was injected with one mCi. of a 14C-labeled amino acid mixture* into the wing vein. In an attempt to maintain a high level of circulating radioactivity, one half of the injection (0.5 ml.) was administered 30 minutes after the first egg of a new clutch was laid and the second half was injected 10 hours later. Egg collections began the following morning and were continued for 36 eggs. The data presented will be limited to the first five whites and the first 10 yolks. Eggs were collected at * Schwarz Bioresearch, Inc., reconstituted protein hydrolysate with the following specific activities in mCi./m mole.: alanine, 90; arginine, 143; aspartic acid, 120; glutamic acid, 130; isoleucine, 140; leucine, 140; lysine, 135; phenylalanine, 250; proline, 109; serine, 105; threonine, 119; tyrosine, 240; valine, 120.
daily intervals except for day five when an egg was not produced. The whites were withdrawn through a hole made in the small end of the eggs with a syringe attached to a 13 gauge Horseley needle. One ml. of sterile chick Ringer's solution was used to rinse the yolk and inner shell surface and was combined with the white. The basic procedure of Rhodes et al. (1958) was used to isolate the white proteins. Whites were stirred for five hours at 4°C, dialized against starting buffer (0.1 M acetic acid-ammonium hydroxide, pH 4.3) for 18 to 24 hours, and then centrifuged at 30,000 X g for 15 minutes to remove insoluble materials. Carboxymethylcellulose (Sigma Chemical Co.) was rinsed successively with N NaOH, H 2 0, M NaCl, H 2 0, N HC1, H 2 0, and starting buffer. A refrigerated (4°C.) column 1.9 X 30 cm. was used for the separations and was repacked for each white. The white proteins were eluted at a flow rate of 1.5 ml. per minute with stepwise pH changes from 4.3 to 10.0. Following the elution of the pH 9.5 globulin fraction, a pH 10.0 buffer with 0.025 M Na 2 C0 3 was used to elute the avidin and then a pH 10.0 buffer with 0.2 M Na 2 C0 3 was used to elute the lysozme. Optical density changes at 280 mp,. were monitored at three minute intervals with a recording spectrophotometer (Gilford Instrument Laboratories, Inc.). The volume of each white fraction was reduced with 20 M Carbowax (Union Carbide Corp.) dialized exhaustively against water, lyophilized to dryness, and weighed to the nearest mg. Yolks were rinsed thoroughly with chick Ringer's solution to remove traces of adhering white and then freed from the vitelline membrane by compressing the yolks in a fold of aluminum foil. The basic procedure of Bernandi and Cook (1960) was used to separate the yolks into three fractions. Two volumes of 0.45 M MgS04 were first added to the yolks and then solid
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
yolk cholesterol fractions were much greater than that of the yolk proteins. Peak activities for the various yolk fractions occurred in the fifth to seventh egg laid after the initiation of labeled acetate feeding. The significance of these peaks to the process of yolk synthesis can not be adequately considered because the labeled material was administered over a period of several days. In present investigation labeled amino acids were administered into a laying hen and the radioactivities contained in all the major white protein fractions as well as the proteins of three yolk fractions were subsequently determined. The data obtained confirm certain other studies dealing with yolk and white formation and should provide a basis for investigators interested in preparing labeled egg components. However, it should be noted that a single hen was employed in these studies and that the possibility of individual variations in the utilization of labeled amino acids was not evaluated.
1123
AMINO ACIDS IN EGGS
Approximately one mg. of each of the white fractions was dissolved in one ml. of water and aliquots were taken for protein determinations (Lowry et al., 1951) and for platings on stainless steel planchettes. Aliquots of 0.01 ml. of each yolk fraction were extracted successively with 5% trichloroacetic acid, 95% alcohol, chloroformethanol (70°C), 95% alcohol, and 5% perchloric acid (70°C). The remaining protein precipitates were dissolved in N NaOH at 60 °C. and aliquots were taken
for protein determinations (Lowry et al., 1951) and platings on planchettes. All planchette counting was done with a Nuclear Chicago low-background counter and the counts obtained were corrected for background and self absorption. RESULTS AND DISCUSSION
The quantities of the various protein fractions recovered basically agreed with the results reported by Rhodes et al. (1958) (Table 1). Polyacrylamide gel electrophoresis of the egg white fractions indicated that complete purification for all the fractions was not achieved by one passage through the column. For example, although ovalbumin Aa exhibited a single electrophoretic band, ovalbumin A3 was heavily contaminated with ovalbumins Ai, A2, and globulin. The high degree of purity reported by Rhodes et al. (1958) in contrast to these observations may indicate that a more sensitive analytical procedure was employed in the present study. Peak specific activity values for all egg white protein fractions were observed in the white of the second egg laid following injection (Table 1). This was expected as the second half of the labeled material was
TABLE 1.—The incorporation of radioactivity into egg white protein fractions following the administration of a uC-amino acid mixture into a hen Egg number Fraction
Elution PH
1
2
3
4
5
Weight/fraction 1 mg.
c.p.m./mg. c.p.m./mg. c.p.m./mg. c.p.m./mg. c.p.m./mg. Ovomucoid+ Flavoprotein Ovalbumin Ai Ovalbumin A2 Ovalbumin A3 Globulin Conalbumin Conalbumin Globulin Globulin Avidin Lysozyme 1
4.3 4.55 4.75 5.0 5.5 6.3 7.0 8.5 9.5 10.0 10.0
Mean ± standard error.
1,342 1,178 1,118 1,005 583 758 1,430 1,250 1,984 966 711
5,972 6,875 5,714 6,084 5,183 6,128 5,656 4,145 5,278 3,438 3,850
1,028 964 1,590 560 858 710 1,227 561 816
—
614
333 382 460 424 513 539 512 1,000 848 455 324
344 327 338 345 404 415 663 293 336 368
—
630+ 59 1,658 + 107 421+ 45 170+ 22 105+ 15 177+ 70 678+ 98 27+ 7 29+ 4 18+ 1 98+ 8
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
MgS0 4 was added to adjust the final concentration to 0.45 M. The solutions consisting of approximately 40 ml. were stirred for one hour at 4°C. and centrifuged for 24 hours at 30,000 r.p.m. using a type 30 rotor in a Beckman Model L Ultracentrifuge. The bottoms of the tubes were punctured and the yellow high density fractions (HDF) of approximately 19 ml. and the white buffy intermediate fractions (IF) of approximately 6 ml. were successively collected. The gel-like low density fractions (LDF) remaining at the tops of the tubes were collected with the aid of a spatula and dissolved in 0.45 M MgS0 4 . The volume of this fraction prior to the addition of the salt solution was approximately 15 ml.
1124
J. HASSELL, J. D. YAGER AND N. W. KLEIN
TABLE 2.—The incorporation of radioactivity into
yolk protein fractions following the administration of a uC-amino acid mixture into a hen Day after Egg injection number
High density Low density Intermediate fraction fraction fraction cp.m./rag. c.p.m./mg. c.p.m./mg. protein protein protein
1
125 948 1,211 1,295 1,608 1,459 888 655 420 254 376.5±31.1
Mean ± standard error.
administered after the formation of the first egg white but before the formation of the second (Romanoff and Romanoff, 1949). The increase in specific activities of the various white protein fractions between the first and second eggs ranged from approximately three to nine fold. This was followed by a decrease in the fraction specific activities which was most striking between eggs two and three. The decrease between eggs two and three ranged from 72.2% to 90.8% but between eggs three and four this value was between 40.2% and 71.1% with the exceptions of the pH 8.5 globulin which approximately doubled in specific activity and the pH 9.5 globulin which essentially did not change. The relationship between the various specific activities of the fractions in one egg did not hold for the other eggs. However, the differences in labeling previously reported by Mandeles and Ducay (1962) were confirmed. Thus, in the second egg the ovalbumin and conalbumin fractions had activities which were similar and higher than the lysozyme fraction. The differences in activities of the fractions may simply reflect differences in the amino acid compositions of the proteins or that the white proteins are synthesized at different periods during the egg laying cycle (Mandeles and Ducay, 1962). Alternatively, the
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on May 16, 2015
1 1 97 0 2 2 1,030 895 3 3 1,270 888 4 4 1,480 1,240 6 5 1,630 1,150 7 6 1,510 1,300 8 7 1,050 1,080 9 8 724 268 10 9 473 609 11 10 411 356 mgprotein/fraction 1 1,152.5±65.8 474.8+35.4
various proteins may be formed at sites within the oviduct which differed in labeled precursor concentrations. The observations that specific white proteins are synthesized by different cell types within the oviduct support this latter possibility (O'Malley et al, 1967; Kohler et al., 1968). The specific activities of the yolk proteins also increased rapidly between the first and second eggs (Table 2). However, unlike the white proteins, the yolk proteins continued to increase in specific activity reaching a peak for the HDF and IF in the fifth egg formed following injection (sixth day) and in the sixth egg for the LDF. Althrough previous observations have indicated that the greatest proportion of yolk was deposited during the last four days prior to ovulation (Romanoff and Romanoff, 1949), these data indicate that yolk proteins were deposited at the highest rate either on the fourth or fifth day prior to ovulation (an egg was not laid on day five). The rate at which specific activities declined in the yolk proteins following peak activities was more gradual and continued for a longer period than was observed for the white. For example, the specific activity of the HDF fraction was reduced by 71% only after four additional eggs had been laid. The specific activities for the HDF and IF of the first ten eggs were quite similar and slightly higher than the LDF. Polyacrylamide gel electrophoresis of these fractions indicated considerable similarity although the LDF contained fewer protein components. Autoradiographic analysis of the HDF gels indicated no major differences in the labeling of the various proteins comprising this fraction. Thus, unlike the white proteins, the yolk proteins may be formed as a unit from a common precursor pool within a limited period of time. Recovery values were calculated on the basis that a full millicurie was administered and a counting efficiency of 22%. The
1125
AMINO ACIDS IN EGGS
white of eggs one to five contained 7.8% of the injected radioactivity with 5.3% in egg two alone. The yolks from eggs one to ten combined contained only 3.9%. Thus, the second white alone accounted for about one-half of the total radioactivity recovered from the first five white and ten yolks combined. SUMMARY
ACKNOWLEDGMENT
The authors wish to thank Mrs. K. Hagedorn for her technical assistance, Dr. P. Abramoff for injecting the hen, and Dr. C. R. Grau for his helpful suggestions.
Kohler, P. 0., P. M. Grimley and B. W. O'Malley, 1968. Protein synthesis: differential stimulation of cell-specific proteins in epithelial cells of chick oviduct. Science, 160: 86-87. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Mandeles, S., and E. D. Ducay, 1962. Site of egg white protein formation. J. Biol. Chem. 237: 3196-3199. O'Malley, B. W., W. L. McGuire and S. G. Korenman, 1967. Estrogen stimulation of synthesis of specific proteins and RNA polymerase activity in the immature chick oviduct. Biochim. Biophys. Acta, 145: 204-207. Rhodes, M. B., P. R. Azari and R. E. Feeney, 1958. Analysis, fractionation, and purification of egg white proteins with cellulose-cation exchanger. J. Biol. Chem. 230: 399-408. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons, Inc., New York.
NEWS AND NOTES (Continued from page 1093) was greatest for the modulus of elasticity and least for the shear values. There was no significant difference in modulus of toughness between raw and cooked muscle. Universal testing instrument measurements and the shear values all showed a significant difference between breast and thigh muscles for both raw and cooked turkeys. The shear values for raw and cooked muscle, and the modulus of toughness for raw also indicated a significant difference between the outer two sample depths of the breast muscle. It is thought
here that this is most likely the result of differences in structural characteristics between surface and deeper layers of the muscle. The second slice from the breast muscle had the lowest instrument values and the thigh the highest. Where sex exerted a significant effect, toms had the higher values with one exception. Where groups were significant, heavier birds were found to have larger shear and modulus of toughness values, but lighter birds had the larger values for maximum stress and modulus of elasticity. Age, where signif-
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A laying hen was injected with a total of one mCi. of a "C-labeled amino acid mixture over a ten hour period. Peak protein activities were observed in the second white, and the fifth and sixth yolks. Differences were observed in the specific activities of the individual white proteins from a single egg whereas the specific activities of two yolk fractions were similar and somewhat higher than that of a third fraction. A total of 11.7% of the administered radioactivity was located in the proteins of the whites of the first five eggs plus the yolk proteins of the first ten eggs with approximately one-hlaf of this total located in the white proteins of the second egg.
REFERENCES Bernardi, G., and W. H. Cook, 1960. An electrophoretic and ultracentrifugal study of the proteins of the high density fraction of egg yolk. Biochem. Biophys. Acta, 44: 86-96. Kritchevsky, D., C. R. Grau, B. M. Tobert and B. J. Kruecker, 1951. Distribution of radioactivity in the egg after feeding sodium acetate1-C". Proc. Soc. Exp. Biol. Med. 76: 741-743. Kritchevsky, D., and M. R. Kirk, 1951. Radioactive eggs. II. Distribution of radioactivity in the yolks. Proc. Soc. Exp. Biol. Med. 78: 200202.