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Permeability of chicken egg vitelline membrane to amino acids—Binding of amino acids to egg proteins
Camp. Biochem. Physiol. Vol. 82A, No. 2, pp. 289-292, 1985 Printed in Great Britain
0300-9629/85 $3.00+ 0.00 0 1985Pergamon Press Ltd
PERMEABILITY OF CHICKEN EGG VITELLINE MEMBRANE TO AMINO ACIDS-BINDING OF AMINO ACIDS TO EGG PROTEINS A. PONS, F. J. GARCIA, A. PALOU* and M. Departament de Bioquimica, Facultat de Ciencies, Universitat 07071 Mallorca, Balears, Spain and tFisiologia General, Universitat de Barcelona, 08028 Barcelona,
ALEMANY? de la Ciutat de Mallorca, Facultat de Biologia, Spain
(Received 3 January 1985)
Ahstraet-1. The fertile chicken egg shows higher amino acid concentrations in the yolk than in the albumen. A gradient of amino acid concentration across the vitelline membrane exists. 2. The vitelline membrane is freely permeable to amino acids: alanine, leucine and phenylalanine both from albumen to yolk and from yolk to albumen. 3. Most amino acids in the yolk are bound to protein, and are freed by alkaline treatment; a significant part of albumen amino acids are also bound to protein. Binding of amino acids to protein is observed both in vivo and in vitro systems. 4. Amino acid circulation and exchange across the vitelline membrane is controlled by its attachment to proteins at both sides of the membrane, thus maintaining the concentration gradients despite the permeability features.
INTRODUCMON There is a considerable variation in the values given for free amino acids in the unincubated avian egg (Emanuelsson, 1951, 1955; SBinz er al., 1983; Rota et al., 1984). Despite free amino acids being a very small
fraction of the total egg nitrogen (Schmidt et al., 1956; S&inz et al., 1983), their concentrations have been measured and considerable differences have been observed between albumen and yolk (Emanuelsson, 1951; Schmidt et al., 1956). It is also well known that amino acids constitute a significant source of energy for the developing chick embryo (Rupe and Farmer, 1955; Freeman and Vince, 1974), and that the production of ammonia from amino acids is significant in the early stages of its development (Clark and Fischer, 1957; Feeney and Allison, 1969). However, it is assumed that these amino acids come mainly from proteolytic processes observed even under conditions in which no incubation has taken place (Emanuelsson, 1951, 1955; Tausig, 1965). The existence of some proteolysis must be associated with increasing levels of free amino acids which might not be used by the embryo in unincubated eggs. Furthermore, the existing gradient of amino acid concentration between albumen and yolk (Emanuelsson, 1951, 1955; Sainz ef al., 1983) seems consistent with a lack of permeability of the vitelline membrane to amino acids, despite the yolk being the compartment that is used first for embryo sustainment and the necessity of interrelation between both compartments for energetic substrates and water transfer. In a previous work (Garcia et al., 1983) we found that glucose crosses the vitelline membrane easily despite steep gradients between albumen and yolk. This was shown to be due to differential affinity of the *To whom correspondence
should be addressed.
proteins in both compartments for binding free glucose. We have intended here to ascertain if there is a comparable process for amino acids that will help to explain the differences in the egg free amino acid concentrations, as well as the gradients existing between yolk and albumen. MATERL4L.SAND METHODS Unincubated fertile eggs of domestic fowl (Callus domesticus, white leghorn) were used. The eggs were injected in the white (Payne, 1976) with 0.1 MBq of uniformly labelled carrier free L-alanine-‘%, (specific radioactivity 6.3 GBq/mol) in 0.1 ml of 9 g/ml N&i. The injected eggs were incubated at 37.5”C during 24 hr in an automatically controlled incubator. After incubation, the eggs were opened and the fractions: white, yolk and vitelline membrane (the last washed with saline to prevent contamination with albumen and/or yolk) were separated and weighed. The vitelline membrane was homogenized in a motor-driven Tenbroek glass-Teflon homogenizer with 10 vol of distilled water. Aliquots of the homogenate, as well as samples of yolk and albumen, were used for total radioactivity determination through liquid scintillation counting without digestion. The three fractions were precipitated at 4°C with acetone (final concentration 55% by volume) (Arola et al., 1977). The supematant was used to determine the specific radioactivity associated to free amino acids (Pons et al., 1981). The acetone precipitate was delipidated with three washings with boiling absolute ethanol. The protein precipitate was then dissolved in 2N NaOH and boiled for 10 min to release bound amino acids. The excess alkali was neutralized with 6N HCl and the resulting solution used for protein determination (Reinhold, 1961) as well as for protein precipitation with trichloroacetic acid (final concentration loo/, by weight). The TCA precipitate and supematant were used to determine the radioactivity associated to protein and released free ammo acids, respectively (Pons et al., 1981). Total lipids were estimated by the Folch procedure (Folch et al., 1951, 1957). Glucose specific radioactivity was measured as previously described (Garcia et al., 1982). 289
A. POW et al.
290
To determine the permeability of the vitelline membrane to several amino acids, the double J-shaped tubes system previously described was used (Garcia et al., 1983). KrebsRinger avian phosphate buffer (Hale, 1966) containing different concentrations of uniformly labelled 14C-alanine, leucine and phenylalanine of high specific radioactivity. The osmolarity at both sides of the tube were corrected by changing the NaCl concentration of the buffer. Damaged or broken vitelline membrane preparations were discarded (Garcia et al., 1983). The appearance of radioactivity on the side that initially had no radioactive amino acid was determined by sampling at intervals up to 30 min and radioactive counting. The data were plotted and the mean rates of transfer were calculated for each concentration of the ammo acid used. Results showing a linear correlation between experimental points of less than 0.90 were discarded. The rates of transference obtained were plotted against the concentration of radioactive amino acid to determine the relationship between both parameters. Another group of eggs were put into a small plastic bag and hard-boiled in a water bath at 100°C for 15 min. After cooling and peeling off the shell, the egg was halved lengthwise and a strip was cut along the longest axis in the surface of one of the two halves with 2-3 mm of width and l-l .5 mm of thickness. This strip was rapidly positioned on graph paper and strips of about 0.5-l mm wide were cut from the strip, thus giving a sequence of bits that were weighed and minced. To prevent the de&cation of the samples this operation was carried out rapidly in a relatively humid atmosphere. The samples were left in the cold for 24 hr in tubes containing 0.5 ml of cold acetone-water mixtures (55% final concentration by volume). Then the samples were centrifuged and the supematants used for amino acid estimations (Allen et al., 1977). After correction of each sample for its weight (translated into width because of the uniform sixes of the strip form which they came) the data were plotted and superimposed on the actual structure of the other half of the egg used for measurements. Statistical comparisons between rates were done with the paired Student r-test.
Table 1 shows the distribution of the radioactive aianine injected in the egg after the first 24hr of incubation. There is a higher amount of radioactivity in the white--where the alanine was injected-than in the yolk, the vitelline membrane having an inter-
mediate amount of radioactivity. The proportion of amino acid present in the protein fraction is actually minimal in the albumen, and higher in the yolk and vitelline membrane. Most of the radioactivity is present in the form of free amino acids in the white, with a very low amount of transfer into the yolk. The amount of amino acids tightly bound to protein is considerable in the white (about la’/, of the free amino acids), but this proportion increased in the
Y
Fig. 1. Permeability of vitelline membrane of unincubated hen eggs to alanine, leucine and phenylalanine. The vertical axis corresponds to the net transfer of ammo acid in nmol see-’ cme2, and abscissae to amino acid concentration in mmoles. 1-l. W-r Y denotes that the study was done with the amino acid added to in the white compartment, thus measuring net transfer to the yolk. Y+ W indicated the reversed situation.
yolk, where this fraction was 55% that of free amino acids. The amount of radioactivity found both in glucose and in the lipid fraction was minimal. In Fig. 1 the plots of velocities of transfer of amino acid in both directions (yolk to white and white to yolk) measured in vitro against the concentration of the amino acid are presented. The range of concentrations tested in each direction were up to 34 fold those found under physiological conditions in the unincubated egg. The relationship between velocity of transfer and amino acid concentration was very similar for alanine and phenylalanine, and slightly smaller for leucine. No statistically sign&ant differences were found as to the direction of the transfer, i.e. the speed of transfer from yolk to white was comparable to that for white to yolk in different eggs tested. The correlations found were acceptable (P > 0.90) for most series of experiments despite not all points having being studied. In Fig. 2 the distribu-
Table 1. Distribution of the radioactivity in the main fractions of hen egg after the injection of 100 kBq of alanine in the albumen on day 0 after 24 hr of incubation Fraction Total radioactivity Protein Free amino acids Bound amino acids Free/bound amino acids ratio Lipid
Giuc0=
White
Vitellioe membrane
Yolk
1418 + 154 2.5 f 0.1 985 * 209 1ss*4 6.35 1.2kO.4 t2.5
411 &217 14.5 f 6.9 222 f 96 44.9 * 3.0 4.98 <2.s
162 * 30 13.7 f 1.7 75.3 * 30.0 41.S it 7.6 1.81 0.6 f 0.4 <2.5
The values presented are the mean * SEM of 5 different eggs and are expressed in Bq/g of white, yolk or vitelline membrane.
Chicken egg amino acids binding
DISTANCE FROM THE SHELL
( CM )
Fig. 2. Amino acid concentrations of the fractions cut lengthwise across a hard-boiled chicken egg. The fractions containing the vitelline membrane are shadowed. See the text for a description of the procedures.
tion of amino acid concentrations along the major axis of a hard-boiled egg are presented as described under Materials and Methods. The amino acid concentration in the albumen was considerably smaller than that of the yolk and much more uniform, despite the observed trend towards higher values near the vitelline membrane. The yolk amino acid concentrations were high and showed a lack of uniformity in adjoining samples, especially in the central parts of yolk. The content of amino acids in the samples that contained the vitelline membrane were comparable to those of the albumen. DISCUSSION
In a similar way to that found for carbohydrate (Garcia et al., 1983), the egg fractions have a significant capacity for binding amino acids. This ability is probably not associated with the synthesis of peptide bonds, as they are rather resistant to very short term alkaline treatment, which is known to release practically all radioactivity incorporated into protein by the albumen (in the yolk and vitelline membrane, a significant amount of radioactivity actually resists this treatment, although prolonged treatment with NaOH released them quantitatively). The amount of amino acids present in the yolk not in the form of protein is considerable when compared with that in the albumen. No reliable data on the actual non-protein amino acid in either structure could be presented, as all methods used in the literature (Folin and Wu, 1919; Neuberg et al., 1944; Somogyi, 1952; Bernt and Bergmeyer, 1967; Arola et al., 1977) measured only the amino acid solubilized from their protein binding site by their particular procedures for deproteinization, which are neither comparable nor uniformly reliable. Thus, the main problem of quantification of free amino acids remains unsolved. We have used two procedures, the relatively mild, cold-acetone precipitation and the strong combination of alkaline solubilization and protein denaturation. We have no absolute certainty however, that neither the “strong” procedure removed all amino acids nor that the “mild” method removed only the less tightly bound amino acids or only amino acids that were actually free. The complexity of the problem poses interesting questions such as the actual meaning of this ability to
291
bind amino acids, as this behavior has been only studied on very limited occasions (such as in spermatic fluid proteins: Busby et al., 1974). The yolk and vitelline membrane proteins showed considerable powerful ability to bind amino acids, as observed in the relative abundance of “free” and “bound” amino acid radioactivity upon injection of alanine. Very similar values have been obtained with other amino acids (unpublished results). The possibility that this incorporation was the result of protein synthesis from these amino acids (or else gluconeogenesis and their incorporation into glycoprotein) was studied in a cell-free suspension of yolk or white from unincubated eggs left in contact with radioactive amino acid solutions. There was a small incorporation of radioactivity into the acetone precipitates, which could be further released by alkaline treatment. This incorporation was very slow, partly because of the viscosity of protein solution and agreed with that of glucose incorporation (Garcia et al., 1983; Feeney et al., 1964). The pattern of “free” amino acid distribution along the axis of the egg, however, resulted in a reverse situation to that of glucose (Garcia et al., 1983), with higher attachment in the yolk than in the albumen. The vitelline membrane is freely permeable to the three amino acids tested: alanine, leucine and phenylalanine, very different in their metabolism and transport in the living cell (Christensen, 1973, 1975; Bender, 1975; Lemer, 1984). In addition, the ability to cross the vitelline membrane showed great similarity in both directions. Thus, it was assumed that the vitelline membrane constitutes no barrier to the diffusion of amino acids between both egg compartments. The presence of alanine radioactivity in the yolk after 24 hr of incubation agrees with this postulate despite the considerable concentration gradient of amino acids observed between yolk and white (Emanuelsson, 1951; Sainz et al., 1983) as has been found in this work. A higher affinity of yolk proteins for amino acids could help to explain this situation in a similar way to that found for glucose (Garcia et al., 1983). The considerable variability in amino acid concentration at neighbouring pieces of yolk could be a consequence of the concentric layered structure of this part of the egg (Bellairs, 1964; Freeman and Vince, 1974) with a significant variation of protein and lipid concentration along the lines of concentric layers (Bellairs, 1964; Freeman and Vince, 1974). The variability observed in amino acid levels could be a consequence of this particular structure associated to varying levels of protein. The actual meaning of the sequestering of free amino acids by proteins in the vitelline membrane and yolk could help concentrate and direct amino acids coming from proteolysis towards the embryo to allow for its development during its initial stages (Emanuelsson, 1951, 1955; Tausig, 1965). This concentrative effect must be complemented with a mechanism for easy release of amino acids bound to protein, although such processes are yet to be demonstrated. Acknowledgement-This work was supported by a research grant from the “Comisi6n Asesora de Investigacibn Cientlfica y Ttcnica” from the Government of Spain.
292
A. Pot i et al. REFERENCES
Allen P. C., Hill E. A. and Stokes A. M. (1977) Plusma Proteins, Analytical and Preparative Techniques, pp. 3-4. Blackwell, Oxford. Arola Ll., Herrera E. and Alemany M. (1977) A new method for deproteinization of small samplesof blood plasma for amino acid determination. Anal. Eiochern. 82, 236-239. Bellairs R. (1964) Biological aspects of the yolk of the hen’s egg. Ad& Morph. 4, 217-272. Bender D. A. (1975) Amino Acid Metaboltsm. Benjamin W. A., Clark H. and Fischer D. (1957) A reconsideration of nitrogen excretion by the chick embryo. J. exp. 2001. 136, 1-15. Bemt E. and Bergmeyer H. U. (1967) Glukose. Bestinnnungsmethoden. Arztl. Lab. 113, 472-475. Busby W. F. Jr.. Hele P. and Charm M. C. (1974) ADDared amino acid incorporation by ~jaculat& rabbit. spermatozoa. Biochim. biophys. Acta 330, 246-259. Christensen H. N. (1973) On the development of amino acid transport systems. Fed Proc. 32, 1%28. Christensen H. N. (1975) Biological Transport. Emanuelsson H. (1951) Proteolytic activity in the hen’s egg prior to incubation. Nature 168, 958-959. Emanuelsson H. (1955) Changes in the proteolytic enzymes of the yolk in the developing hen’s egg. Acta physiol. wand. 34, 124-134. Feeney R. E. and Allison R. G. (1969) Evolutionary Biochemistry of Proteins. Interscience, New York. Feeney R. E., Clary J. J. and Clark J. R. (1964) A reaction between glucose and egg white proteins in incubated eggs. Nature Ml, 192-193. Folch J., Ascoli I., Lees M., Meath J. A. and Le Baron N. (1951) Preparation of lipid extracts from brain tissue. 1. biol. Chem. 191, 833-841. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Folin 0. and Wu H. (1919) A system of blood analysis. J. biol. Chem. 38, 81-110. Freeman B. M. and Vince M. A. (1974) Development of the Avian Embryo. Chapman and Hall, London. Garcia F. J., Pons A., Alemany M. and Palou A. (1982)
Estimation of monossacharide radioactivity in biological samples through osazone derivatization. Analyl. Biochem. 120, 249-253. Garcia F. J., Pons A., Alemany M. and Palou A. (1983) Permeability of chicken egg vitelline membrane to glucose, gradients between albumen and yolk. Comp. Biothem. Physiol. 75B, 137-140. Hale L. J. (1966) Biological Laboratory Data. Chapman and Hall, London. Lemer J. (1984) Cell membrane amino acid transport processes in the domestic fowl (Gallus domesticus). Comp. Biochem. Physiol. 7EA, 2, 205-216. Neuberg C., Strauss E. and Lipkin L. E. (1944) Convenient method for deproteinization. Arch. Biochem. 4, 101-104. Payne L. N. (1976) The chick embryo. In The UFAW Handbook on the Care and Management of Laboratory Animals (Edited by UFAW), pp. 444-464. Churchill & Livingstone, Edinburgh. Pons A., Garcia F. J., Palou A. and Alemany M. (1981) A method for the estimation of amino acid radioactivity in biological samples. J. biochem. biophys. MethA 5, 153-156. Reinhold J. G. (1961) Proteinas totales, albuminas y globuhnas. In Mktodos Seleccionados de Amilisis Clinicos (Edited by Reines M.), Vol. 1, pp. 126-140. Aguilar, Madrid. Rota P., Sdinz F., Gonzalez M. and Alemany M. (1984) Structure and composition of the eggs from several avian species. Comp. Biochem. Physiol. VA, 307-310. Rupe C. 0. and Farmer Ch. J. (1955) Amino acid studies in the transformation of the hen’s egg to tissue proteins during incubation. J. biol. Chem. 213, 899-907. S&z F., Gonzalez M., Rota P. and Alemany M. (1983) Physical and chemical nature of eggs from six breeds of domestic fowl. Br. Poultry Sci. 24, 301-309. Schmidt G., Bessman M. J., Hickey M. D. and Thannhauser S. J. (1956) The concentrations of some constituents of egg yolk in its soluble phase. J. biol. Chem. 223, 1027-1031. Somogyi M. (1952) Notes on sugar determination. J. biol. Chem. 195, 19-23. Tausig M. P. (1965) Incorporation of amino acids into chick proteins during embryonic growth. Can. J. Biochem. Physiol. 43, 1099-l 110.
0300-9629/85 $3.00+ 0.00 0 1985Pergamon Press Ltd
PERMEABILITY OF CHICKEN EGG VITELLINE MEMBRANE TO AMINO ACIDS-BINDING OF AMINO ACIDS TO EGG PROTEINS A. PONS, F. J. GARCIA, A. PALOU* and M. Departament de Bioquimica, Facultat de Ciencies, Universitat 07071 Mallorca, Balears, Spain and tFisiologia General, Universitat de Barcelona, 08028 Barcelona,
ALEMANY? de la Ciutat de Mallorca, Facultat de Biologia, Spain
(Received 3 January 1985)
Ahstraet-1. The fertile chicken egg shows higher amino acid concentrations in the yolk than in the albumen. A gradient of amino acid concentration across the vitelline membrane exists. 2. The vitelline membrane is freely permeable to amino acids: alanine, leucine and phenylalanine both from albumen to yolk and from yolk to albumen. 3. Most amino acids in the yolk are bound to protein, and are freed by alkaline treatment; a significant part of albumen amino acids are also bound to protein. Binding of amino acids to protein is observed both in vivo and in vitro systems. 4. Amino acid circulation and exchange across the vitelline membrane is controlled by its attachment to proteins at both sides of the membrane, thus maintaining the concentration gradients despite the permeability features.
INTRODUCMON There is a considerable variation in the values given for free amino acids in the unincubated avian egg (Emanuelsson, 1951, 1955; SBinz er al., 1983; Rota et al., 1984). Despite free amino acids being a very small
fraction of the total egg nitrogen (Schmidt et al., 1956; S&inz et al., 1983), their concentrations have been measured and considerable differences have been observed between albumen and yolk (Emanuelsson, 1951; Schmidt et al., 1956). It is also well known that amino acids constitute a significant source of energy for the developing chick embryo (Rupe and Farmer, 1955; Freeman and Vince, 1974), and that the production of ammonia from amino acids is significant in the early stages of its development (Clark and Fischer, 1957; Feeney and Allison, 1969). However, it is assumed that these amino acids come mainly from proteolytic processes observed even under conditions in which no incubation has taken place (Emanuelsson, 1951, 1955; Tausig, 1965). The existence of some proteolysis must be associated with increasing levels of free amino acids which might not be used by the embryo in unincubated eggs. Furthermore, the existing gradient of amino acid concentration between albumen and yolk (Emanuelsson, 1951, 1955; Sainz ef al., 1983) seems consistent with a lack of permeability of the vitelline membrane to amino acids, despite the yolk being the compartment that is used first for embryo sustainment and the necessity of interrelation between both compartments for energetic substrates and water transfer. In a previous work (Garcia et al., 1983) we found that glucose crosses the vitelline membrane easily despite steep gradients between albumen and yolk. This was shown to be due to differential affinity of the *To whom correspondence
should be addressed.
proteins in both compartments for binding free glucose. We have intended here to ascertain if there is a comparable process for amino acids that will help to explain the differences in the egg free amino acid concentrations, as well as the gradients existing between yolk and albumen. MATERL4L.SAND METHODS Unincubated fertile eggs of domestic fowl (Callus domesticus, white leghorn) were used. The eggs were injected in the white (Payne, 1976) with 0.1 MBq of uniformly labelled carrier free L-alanine-‘%, (specific radioactivity 6.3 GBq/mol) in 0.1 ml of 9 g/ml N&i. The injected eggs were incubated at 37.5”C during 24 hr in an automatically controlled incubator. After incubation, the eggs were opened and the fractions: white, yolk and vitelline membrane (the last washed with saline to prevent contamination with albumen and/or yolk) were separated and weighed. The vitelline membrane was homogenized in a motor-driven Tenbroek glass-Teflon homogenizer with 10 vol of distilled water. Aliquots of the homogenate, as well as samples of yolk and albumen, were used for total radioactivity determination through liquid scintillation counting without digestion. The three fractions were precipitated at 4°C with acetone (final concentration 55% by volume) (Arola et al., 1977). The supematant was used to determine the specific radioactivity associated to free amino acids (Pons et al., 1981). The acetone precipitate was delipidated with three washings with boiling absolute ethanol. The protein precipitate was then dissolved in 2N NaOH and boiled for 10 min to release bound amino acids. The excess alkali was neutralized with 6N HCl and the resulting solution used for protein determination (Reinhold, 1961) as well as for protein precipitation with trichloroacetic acid (final concentration loo/, by weight). The TCA precipitate and supematant were used to determine the radioactivity associated to protein and released free ammo acids, respectively (Pons et al., 1981). Total lipids were estimated by the Folch procedure (Folch et al., 1951, 1957). Glucose specific radioactivity was measured as previously described (Garcia et al., 1982). 289
A. POW et al.
290
To determine the permeability of the vitelline membrane to several amino acids, the double J-shaped tubes system previously described was used (Garcia et al., 1983). KrebsRinger avian phosphate buffer (Hale, 1966) containing different concentrations of uniformly labelled 14C-alanine, leucine and phenylalanine of high specific radioactivity. The osmolarity at both sides of the tube were corrected by changing the NaCl concentration of the buffer. Damaged or broken vitelline membrane preparations were discarded (Garcia et al., 1983). The appearance of radioactivity on the side that initially had no radioactive amino acid was determined by sampling at intervals up to 30 min and radioactive counting. The data were plotted and the mean rates of transfer were calculated for each concentration of the ammo acid used. Results showing a linear correlation between experimental points of less than 0.90 were discarded. The rates of transference obtained were plotted against the concentration of radioactive amino acid to determine the relationship between both parameters. Another group of eggs were put into a small plastic bag and hard-boiled in a water bath at 100°C for 15 min. After cooling and peeling off the shell, the egg was halved lengthwise and a strip was cut along the longest axis in the surface of one of the two halves with 2-3 mm of width and l-l .5 mm of thickness. This strip was rapidly positioned on graph paper and strips of about 0.5-l mm wide were cut from the strip, thus giving a sequence of bits that were weighed and minced. To prevent the de&cation of the samples this operation was carried out rapidly in a relatively humid atmosphere. The samples were left in the cold for 24 hr in tubes containing 0.5 ml of cold acetone-water mixtures (55% final concentration by volume). Then the samples were centrifuged and the supematants used for amino acid estimations (Allen et al., 1977). After correction of each sample for its weight (translated into width because of the uniform sixes of the strip form which they came) the data were plotted and superimposed on the actual structure of the other half of the egg used for measurements. Statistical comparisons between rates were done with the paired Student r-test.
Table 1 shows the distribution of the radioactive aianine injected in the egg after the first 24hr of incubation. There is a higher amount of radioactivity in the white--where the alanine was injected-than in the yolk, the vitelline membrane having an inter-
mediate amount of radioactivity. The proportion of amino acid present in the protein fraction is actually minimal in the albumen, and higher in the yolk and vitelline membrane. Most of the radioactivity is present in the form of free amino acids in the white, with a very low amount of transfer into the yolk. The amount of amino acids tightly bound to protein is considerable in the white (about la’/, of the free amino acids), but this proportion increased in the
Y
Fig. 1. Permeability of vitelline membrane of unincubated hen eggs to alanine, leucine and phenylalanine. The vertical axis corresponds to the net transfer of ammo acid in nmol see-’ cme2, and abscissae to amino acid concentration in mmoles. 1-l. W-r Y denotes that the study was done with the amino acid added to in the white compartment, thus measuring net transfer to the yolk. Y+ W indicated the reversed situation.
yolk, where this fraction was 55% that of free amino acids. The amount of radioactivity found both in glucose and in the lipid fraction was minimal. In Fig. 1 the plots of velocities of transfer of amino acid in both directions (yolk to white and white to yolk) measured in vitro against the concentration of the amino acid are presented. The range of concentrations tested in each direction were up to 34 fold those found under physiological conditions in the unincubated egg. The relationship between velocity of transfer and amino acid concentration was very similar for alanine and phenylalanine, and slightly smaller for leucine. No statistically sign&ant differences were found as to the direction of the transfer, i.e. the speed of transfer from yolk to white was comparable to that for white to yolk in different eggs tested. The correlations found were acceptable (P > 0.90) for most series of experiments despite not all points having being studied. In Fig. 2 the distribu-
Table 1. Distribution of the radioactivity in the main fractions of hen egg after the injection of 100 kBq of alanine in the albumen on day 0 after 24 hr of incubation Fraction Total radioactivity Protein Free amino acids Bound amino acids Free/bound amino acids ratio Lipid
Giuc0=
White
Vitellioe membrane
Yolk
1418 + 154 2.5 f 0.1 985 * 209 1ss*4 6.35 1.2kO.4 t2.5
411 &217 14.5 f 6.9 222 f 96 44.9 * 3.0 4.98 <2.s
162 * 30 13.7 f 1.7 75.3 * 30.0 41.S it 7.6 1.81 0.6 f 0.4 <2.5
The values presented are the mean * SEM of 5 different eggs and are expressed in Bq/g of white, yolk or vitelline membrane.
Chicken egg amino acids binding
DISTANCE FROM THE SHELL
( CM )
Fig. 2. Amino acid concentrations of the fractions cut lengthwise across a hard-boiled chicken egg. The fractions containing the vitelline membrane are shadowed. See the text for a description of the procedures.
tion of amino acid concentrations along the major axis of a hard-boiled egg are presented as described under Materials and Methods. The amino acid concentration in the albumen was considerably smaller than that of the yolk and much more uniform, despite the observed trend towards higher values near the vitelline membrane. The yolk amino acid concentrations were high and showed a lack of uniformity in adjoining samples, especially in the central parts of yolk. The content of amino acids in the samples that contained the vitelline membrane were comparable to those of the albumen. DISCUSSION
In a similar way to that found for carbohydrate (Garcia et al., 1983), the egg fractions have a significant capacity for binding amino acids. This ability is probably not associated with the synthesis of peptide bonds, as they are rather resistant to very short term alkaline treatment, which is known to release practically all radioactivity incorporated into protein by the albumen (in the yolk and vitelline membrane, a significant amount of radioactivity actually resists this treatment, although prolonged treatment with NaOH released them quantitatively). The amount of amino acids present in the yolk not in the form of protein is considerable when compared with that in the albumen. No reliable data on the actual non-protein amino acid in either structure could be presented, as all methods used in the literature (Folin and Wu, 1919; Neuberg et al., 1944; Somogyi, 1952; Bernt and Bergmeyer, 1967; Arola et al., 1977) measured only the amino acid solubilized from their protein binding site by their particular procedures for deproteinization, which are neither comparable nor uniformly reliable. Thus, the main problem of quantification of free amino acids remains unsolved. We have used two procedures, the relatively mild, cold-acetone precipitation and the strong combination of alkaline solubilization and protein denaturation. We have no absolute certainty however, that neither the “strong” procedure removed all amino acids nor that the “mild” method removed only the less tightly bound amino acids or only amino acids that were actually free. The complexity of the problem poses interesting questions such as the actual meaning of this ability to
291
bind amino acids, as this behavior has been only studied on very limited occasions (such as in spermatic fluid proteins: Busby et al., 1974). The yolk and vitelline membrane proteins showed considerable powerful ability to bind amino acids, as observed in the relative abundance of “free” and “bound” amino acid radioactivity upon injection of alanine. Very similar values have been obtained with other amino acids (unpublished results). The possibility that this incorporation was the result of protein synthesis from these amino acids (or else gluconeogenesis and their incorporation into glycoprotein) was studied in a cell-free suspension of yolk or white from unincubated eggs left in contact with radioactive amino acid solutions. There was a small incorporation of radioactivity into the acetone precipitates, which could be further released by alkaline treatment. This incorporation was very slow, partly because of the viscosity of protein solution and agreed with that of glucose incorporation (Garcia et al., 1983; Feeney et al., 1964). The pattern of “free” amino acid distribution along the axis of the egg, however, resulted in a reverse situation to that of glucose (Garcia et al., 1983), with higher attachment in the yolk than in the albumen. The vitelline membrane is freely permeable to the three amino acids tested: alanine, leucine and phenylalanine, very different in their metabolism and transport in the living cell (Christensen, 1973, 1975; Bender, 1975; Lemer, 1984). In addition, the ability to cross the vitelline membrane showed great similarity in both directions. Thus, it was assumed that the vitelline membrane constitutes no barrier to the diffusion of amino acids between both egg compartments. The presence of alanine radioactivity in the yolk after 24 hr of incubation agrees with this postulate despite the considerable concentration gradient of amino acids observed between yolk and white (Emanuelsson, 1951; Sainz et al., 1983) as has been found in this work. A higher affinity of yolk proteins for amino acids could help to explain this situation in a similar way to that found for glucose (Garcia et al., 1983). The considerable variability in amino acid concentration at neighbouring pieces of yolk could be a consequence of the concentric layered structure of this part of the egg (Bellairs, 1964; Freeman and Vince, 1974) with a significant variation of protein and lipid concentration along the lines of concentric layers (Bellairs, 1964; Freeman and Vince, 1974). The variability observed in amino acid levels could be a consequence of this particular structure associated to varying levels of protein. The actual meaning of the sequestering of free amino acids by proteins in the vitelline membrane and yolk could help concentrate and direct amino acids coming from proteolysis towards the embryo to allow for its development during its initial stages (Emanuelsson, 1951, 1955; Tausig, 1965). This concentrative effect must be complemented with a mechanism for easy release of amino acids bound to protein, although such processes are yet to be demonstrated. Acknowledgement-This work was supported by a research grant from the “Comisi6n Asesora de Investigacibn Cientlfica y Ttcnica” from the Government of Spain.
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