J. Cell Set. 3, 515-527(1968) 515 Printed in Great Britain
DISPLACEMENT OF VALINE FROM INTACT SEA-URCHIN EGGS BY EXOGENOUS AMINO ACIDS
J. PIATIGORSKY* AND A. TYLER Division of Biology, California Institute of Technology, Pasedena, California, U.S.A.
SUMMARY Unfertilized and fertilized eggs of the sea urchin Lytechinus pictus were preloaded with [14C]valine and exposed to individual solutions of each of the twenty 'coded' [llC]amino acids in artificial sea water. After 1 h incubation the amount of radioactivity in the medium was determined. The radioactivity was effectively displaced by most of the other neutral [11C]amino acids that are known to compete with valine for uptake. A chromatographic test with fertilized eggs showed the displaced radioactivity to be [14C]valine and not some metabolic product. Addition of acidic, basic or some neutral amino acids that are known to be poor inhibitors of valine uptake did not cause significant quantities of label to appear in the medium. For the unfertilized eggs, the concentration of acid-soluble label remained many hundreds of times greater in the egg fluid than in the sea water. Tests indicated that efflux of [14C]valine and subsequent competition for re-entry is a primary factor responsible for the displacement phenomenon. That this may not be the sole factor is suggested by the fact that some amino acids that are known to be powerful inhibitors of valine uptake were found to be only weak displacers of [14C]valine. Neither [14C]arginine nor [14C]glu- tamic acid were displaced in significant amounts from preloaded unfertilized or fertilized eggs by any of the tested [''CJamino acids. Attempts were made to utilize the displacement of ['"CJvaline to elevate the incorporation of [14C]valine and of other labelled amino acids into protein by intact eggs. Unfertilized and fertilized eggs were pretreated with related [11C]amino acids and then exposed to [14C]valine or a mixture of [14C]amino acids. The results varied in the different tests, ranging from no significant increase to 2-fold.
INTRODUCTION Experiments (Mitchison & Cummins, 1966; Tyler, Piatigorsky & Ozaki, 1966) with sea-urchin eggs have demonstrated a competition among amino acids for entrance into these cells. The competition occurs principally among those belonging to the same group. In the competition experiments, data (Table 2 of Tyler et al. 1966) were obtained which indicated also that the incorporation into protein of one particular labelled amino acid, namely [14C]valine, could be increased by pre-treatment of the eggs with a mixture of other non-radioactive amino acids. One possible interpretation of this result is that the pre-treatment partially displaces [12C]valine from the available amino acid pool. This would result in a higher endogenous specific radioactivity of [wC]valine in the pre-treated eggs than in the controls and consequent higher pro- portion of [14C]valine than of [12C]valine in the synthesized protein. • Present address: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. 33 Cell. Sci 3 516 J. Piatigorsky and A. Tyler The present investigation is a further study of the ability of one amino acid to dis- place another in unfertilized and in fertilized sea-urchin eggs. Exchanges among amino acids and among other substances have been demonstrated in other kinds of cells. In the first place, experiments (Lockart & Eagle, 1959; Eagle, 1963) with cultured mammalian cells have demonstrated that amino acids, as well as other material necessary for growth, readily leak out of cells cultured at a low population density. Experiments with rabbit erythrocytes (Park et al. 1956) and human erythrocytes (Rosenberg & Wilbrandt, 1958) have shown that labelled xylose and glucose, respectively, can be made to leave the cells against a concentration gradient simply by the addition of the same sugar or of closely related sugars to the medium of the cell suspension. Investigations with cultured Ehrlich ascites carcinoma cells (Heinz, 1954; Heinz & Walsh, 1958) have shown that preloading the cells with glycine or with other amino acids that competitively inhibit the uptake of glycine promotes increase in the rate of additional glycine uptake. Heinz & Walsh (1958) have interpreted the increase in glycine influx as an increase in the rate of glycine exchange after preloading the cells. From experiments of this sort with various kinds of cells (see Wilbrandt & Rosenberg, 1961; Christensen, 1962; Eagle, 1963, for reviews) it is evident that the distribution of a substance between the inside and outside of intact cells can be affected by the addition of another, related, material to the medium. The present study demonstrates that [14C]valine diffuses from preloaded unfertilized or fertilized eggs into the medium. Addition to the sea water of an excess of individual neutral amino acids that compete with valine for uptake causes the ["CJvaline to redistribute itself between the inside and outside of the eggs so that large amounts of radioactivity are lost from the eggs. The tests also show that addition of basic and acidic amino acids or of neutral amino acids that do not compete with valine for uptake does not cause significant repartitioning of p*C]valine between the eggs and medium. Further, the tests show that no appreciable amount of radioactivity is displaced from eggs preloaded with ["CJarginine or [uC]glutamic acid after addition of the various kinds of amino acids to the suspension.
MATERIALS AND METHODS Eggs of the sea urchin Lytechinus pictus were obtained by KCl-induced shedding, deprived of their gelatinous coat, and cultured in artificial sea water as described elsewhere (Tyler & Tyler, 1966). The tests were made with the 20 L-amino acids commonly found in proteins. The procedure involved preloading the eggs with the p4C]amino acid, washing by centri- fugation and subsequent incubation of the eggs in duplicate suspensions with the individual p2C]amino acids as reported in the legend to Fig. 1. Radioactivity was determined by scintillation counting (Tri-carb Spectrometer), with about 50% efficiency, by the filter paper method of Mans & Novelli (1961) as modified by Tyler (1966). The measurements were made on aliquot samples of the supernatant fluids, before and after incubation of the preloaded eggs with the [12C]amino acids or with Displacement of valine from sea-urchin eggs 517 artificial sea water. Also, the radioactivity of samples of the last wash-water, and of the washed preloaded eggs, after incubation in artificial sea water and lysis with distilled water, was similarly determined. The radioactivity of the egg material was
VAL MET THR ALA HIS ILU TYR LEU ASN PHE GLN TRY Unfertilized eggs SER GLY LYS GLU CYS PRO ASP ARG None 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 15 20 25 30
VAL THR MET ALA HIS LEU GLN ASN ILU TRY TYR Fertilized eggs SER PHE GLY CYS LYS PRO GLU ASP ARG None 1 I I 1 8 12 16 Counts/mm x10"3 Fig. 1. [14C]valine displacement from unfertilized and fertilized eggs of Lytechinus pictus in the presence of various individual [12C]amino acids. Eggs were preloaded with [14C]valine (sp.act. 208-5 c/mole) by exposure to 1 /
33-2 518 J. Piatigorsky and A. Tyler measured both before and after processing with 5 % trichloroacetic acid (TCA) so as to provide values both for total uptake and for incorporation into protein. Tests of the nature of the radioactivity in the supernatant fraction showed it to be completely soluble in TCA and identifiable as L-valine by paper chromatography as indicated below.
RESULTS AND COMMENTS Effect of \^2C~\amino acids on the displacement of [uC]valine from intact unfertilized and fertilized eggs The results of one set of tests of the ability of individual amino acids to displace radioactivity from eggs preloaded with ["CJvaline are illustrated in Fig. 1. The length of the horizontal bars represents the amount of radioactivity that was present in the sea water after the preloaded eggs were incubated for 1 h in sea water (lowermost bars), or in sea water containing one of the p2C]amino acids listed along the ordinate. The amino acids added to the medium were all at concentrations many times higher than that at which their rate of incorporation is saturated (Tyler, 1965; Tyler et al. 1966), with the exception of arginine, proline and tyrosine. Arginine was approximately at the half-saturating concentration and proline and tyrosine were just below the saturating concentration with respect to their incorporation into protein. It is clear that in the absence of added [^CJamino acid very little radioactivity appears in the surrounding medium. On the other hand, the presence of certain amino acids in the medium, notably neutral ones, results in appreciable accumulation of radioactivity in the sea water. One other complete set of tests and two other partial sets gave similar results. Values for the ratio of the radioactivity (cpm) in the sea water after 1 h incubation in the presence of a [^CJamino acid to that in the eggs plus the medium are given in Table 1. This ratio represents the fraction of the total radioactivity lost from the eggs. The percentage of radioactivity lost from the unfertilized eggs (Expts 1 and 2) after incubation with a non-radioactive amino acid is consistently higher than that lost from the fertilized eggs (Expt 3). This is explained by the fact that there is considerably less incorporation of the accumulated [14C]valine into protein by the unfertilized eggs than by the fertilized eggs. Only about 10% of the label is in- corporated into protein by the unfertilized eggs, while 90 %, or more, of the radio- activity is found in protein in the fertilized eggs. The relative amounts of radioactivity displaced from the eggs by each of the [12C]amino acids, however, are approximately the same in the experiments with the unfertilized eggs as in those with the fertilized eggs. Thus it is likely that the mechanism by which one amino acid displaces another is the same before and after fertilization. For the unfertilized eggs greater than 50 % displacement of the accumulated radioactivity was obtained by incubation with MET, THR and VAL. Less than 50% but greater than 10% displacement of radioactivity from the eggs resulted from addition to the medium of ALA, ILU, HIS, TYR, LEU, ASN, PHE, TRY, GLN and SER, listed in decreasing order of the average values of the two experiments given in Table 1. Incubation with GLY, LYS, Displacement of valine from sea-urchin eggs 519 GLU, ASP, PRO and ARG displaced only 10% or less of the accumulated radio- activity. A comparable result was obtained with the fertilized eggs except that VAL, MET and THR displaced somewhat less than half the radioactivity from the eggs and that PHE and SER displaced less than 10% of the 14C. This may be attributed to the relatively high incorporation of label into protein by the fertilized eggs as mentioned above. These experiments show that [14C]valine can be displaced from intact unfertilized and fertilized eggs by the addition of other neutral amino acids to the medium. On the other hand, addition of acidic and basic amino acids, such as ASP, GLU,
Table 1. Influence of individual [12C]amino acids on the redistribution of^Cjvaline between the inside and outside of unfertilized and fertilized eggs of Lytechinus pictus* Unfertilized eggs t it Expt 2t eggs, ExpAt 'Displacing' t s t Expt 3f [1JC]amino acid M [E] M [E] M at 0028 M M + E [M] M+E [M] M+E Alanine 0-49 49 0-41 97 0-28 Arginine 0-04 1748 o-oi 5875 o-oo Asparagine 0-31 128 O-2I 290 0-14 Aspartic acid 0-04 1617 0-02 4446 o-oo Cysteine 0-06 976 O-OS 1452 o-oi Glutamic acid 0-08 683 0-08 908 o-oi Glutamine O-2O 240 0-17 372 0-16 Glycine O-II 518 0-09 830 0-04 Histidine 0-39 84 040 i°5 O-2O Isoleucine 0-32 118 0-49 68 0-12 Leucine 032 120 0-28 191 0-17 Lysine o-io 563 0-09 842 O-O2 Methionine o-54 37 0-54 54 0-28 Phenylalanine 0-22 216 023 254 0-06 Proline 0-03 1791 003 2736 o-oi Serine O-I7 289 015 426 0-07 Threonine 0-51 44 o'57 43 0-30 Tryptophan O-I9 257 O-22 277 O-II TyrosineJ o-35 186 0-42 93 O-II Valine 0-53 37 °'54 52 0-47 None 0-04 1603 0-09 874 o-oi • These data (Expts 1 and 3) were obtained from the tests (average values of duplicate determinations) shown in Fig. 1. An additional test (Expt 2) was conducted under the same conditions as given in the legend to Fig. 1 except that incubation with the 12C-amino acids was at a cell density of 8540 eggs/tube. f M _ The ratio of the total radioactivity in the medium (M) to that in the medium M + E ~ Plu8 the egg8 (M + E). [E] —— = The ratio of the concentration (cpm/ml) of acid-soluble radioactivity in the egg-fluid [M] to that in the medium. The fluid volume of the eggs is assumed to be about 80 %. % At 0-00032 M. 520 J. Piatigorsky and A. Tyler ARG and LYS, does not result in the removal of appreciable amounts of [14C]valine from the eggs. Several other amino acids, namely PRO, GLY and CYS, are cate- gorized as neutral but lack the capacity to displace significant quantities of ["CJvaline from the eggs. The data in Table i also lists for the unfertilized eggs the ratios of the concen- trations (in cpm per unit volume) of the acid-soluble radioactivity inside the egg (assuming a fluid volume of 80% (cf. Leitch, 1934)) to that in the medium. The values show that the concentration of acid-soluble radioactive material (presumably [^Jvaline) is much higher inside the egg than in the surrounding medium. For the eggs in sea water, the average values are 1603 (Expt 1) and 874 (Expt 2). It is likely that the latter value for Expt 2 is somewhat low because of a spurious value for one of the duplicate determinations which showed a somewhat higher than usual radioactivity in the supernatant sea water. This is evidenced also by the fact that higher ratios were obtained in Expt 2 in the tests with the basic and acidic amino acids as well as those neutral amino acids that did not displace appreciable amounts of radioactivity from the eggs. For the eggs suspended in the 'displacing' p2C]amino acids the concentrations within the eggs are, of course, lowered but are still a great many times higher than in the medium. Thus the values range from about 40 to 50 for the neutral [12C]amino acids that effectively displaced label from the eggs, to several thousand for the [^C] basic, acidic and neutral amino acids that did not cause appreciable loss of radioactivity into the sea water. The corresponding analysis for the fertilized eggs cannot be done because of the high extent of incorporation into protein, which implies a rapid change in the con- centration of the [14C]valine in the pool.
Identification of displaced radioactive material as valine As a further check that the radioactive material that appears in the medium is principally valine, the material was examined by paper chromatography. The tests were done with fertilized eggs. A sample of fertilized eggs (30 min after fertilization) were preloaded with pHJvaline (sp.act. 870 c/mole) by incubating for £ h at 20 °C in artificial sea water containing 5 /ic/ml of 3H. The eggs were washed in ice-cold sea water and o-i ml of the suspension containing 19000 eggs added to 0-4 ml of sea water and to 0-4 ml of 0-035 M P2C]valine in sea water. A scintillation count on a sample of the last washing showed that about 400 cpm were transferred with the eggs. After incubation in sea water a total of 2600 cpm appeared in the medium. The supernatant of the suspension in ["CJvaline medium had a total of 16500 cpm. A 0-3 ml sample of this supernatant fluid was absorbed with a similar volume of Dowex-50 ion-exchange resin (HC1 and water-washed) and eluted with 0-4 N NH40H. After evaporation to dryness the residue was dissolved in 0-06 ml of water, spotted on Whatman no. 3 filter paper and subjected to ascending chromatography in a pyridine-acetic acid-water mixture (50-35-15). Under these conditions the RF value for valine is 0-73. The paper after drying was cut into thirteen sections covering the 12-5 in. from Displacement of vaUne from sea-urchin eggs 521 origin to solvent front, with papers nos. 1 and 13 being £ in. wide and the others 1 in. wide. Measurements of radioactivity gave the following values. Paper strip no. ... 12345678 9 10 11 12 13 Minus background cpm 12 8 6 11 10 15 8 29 531 4529 256 61 36 From our estimate of absorption loss and quenching the values represent about complete recovery of radioactivity. The peak of radioactivity appears in the expected paper strip corresponding to the RF value of 073 for valine.
Evidence concerning mechanism of displacement of [^C^aline by the added neutral amino acids The amino acids that displace ["CJvaline from intact eggs are, for the most part, the same as those that can competitively inhibit the uptake of ["CJvaline (Tyler et al. 1966); namely, most neutral amino acids compete with valine while acidic and basic amino acids have very little influence on the uptake of valine.
Table 2. Effect of dilution and of an individual neutral, acidic and basic amino acid on the displacement of [uC]valine from unfertilized and fertilized eggs of Lytechinus pictus*
Ratio of Cpm in cpm in supernatant supernatant sea water to total cpm in after 1 h at 20 °C supernatant and eggs A A V LJ1UI11C { i Incubation medium (ml) Unfertilized Fertilized Unfertilized Fertilized
Artificial sea water (s.w.) 0-5 153 76 0-003 o-ooi 170 20 Artificial s.w. io-o 742 283 0-015 0-008 832 284 0-028 M Arginine in s.w. o-5 in 62 0-004 0-002 342 67 0-028 M Glutamic acid in o-S I57O 124 0-03 0003 s.w. 1570 114 0-028 M Methionine in s.w. 0"5 15700 19000 0-30 0-51 1595° 18600 0-028 M Valine in s.w. o-5 11 800 21450 0-26 0-56 15400 19360 • Experimental procedure as given in the legend to Fig. 1 except that there were 31040 unfertilized and 37680 fertilized eggs per tube. Unfertilized and fertilized (1 h after fertili- zation) eggs were preloaded with [14C]valine (sp.act. 208-5 c/mole) for 1 h. Average total uptake (not treated with TCA): unfertilized eggs = 52900 cpm; fertilized eggs = 36200 cpm. Average total incorporation into protein (treated with TCA as given in Materials and Methods): unfertilized eggs = 4000 cpm; fertilized eggs = 25300 cpm.
Since it is not known to what extent the present systems may approach equilibrium conditions with respect to the distribution of amino acids inside and outside the cell, tests were made to determine the degree to which competition for re-entry may be a factor in the present results. In one experiment aliquots of eggs that had been preloaded with [14C]valine were incubated (20 °C) in io-o ml and in 0-5 ml of 522 J. Piatigorsky and A. Tyler artificial sea water. Also additional aliquots were incubated in 0-5 ml of individual solutions of [12C]arginine, [12C]glutamic acid and [^CJmethionine, as well as in 0-5 ml of a solution of [^CJvaline, all in artificial sea water. The suspensions were stirred continuously so as to avoid local accumulation of the [uC]valine in the vicinity of the eggs. After 1 h the eggs were centrifuged and a sample of the medium was assayed for its radioactivity. The results are given in Table 2. Both [12C]valine and [12C]methionine displaced large amounts of the ["CJvaline from the eggs, while P2C]arginine was ineffective. [12C]glutamic acid showed some effectiveness with un- fertilized eggs but not with the fertilized eggs. The immediately pertinent information comes from the tests of incubation in the two different volumes of sea water not containing added amino acid. The preloaded eggs suspended in io-o ml were 20 times more dilute than those in 0-5 ml. As the figures given in Table 2 show, roughly 5 times as much radioactivity appeared in the supernatant fluids of the dilute than of the more concentrated suspension. Thus the radioactivity was approximately 4 times more concentrated in the suspensions con- taining the smaller than in those with the larger volume. This contrasts with the difference in volume, which was 20-fold. From these figures it appears that radio- activity leaks from the preloaded eggs in artificial sea water but that progress towards equilibrium is relatively slow. This was examined further in a second type of experiment in which the sea water was removed and replaced with new media at successive short intervals and the sum of the radioactivities was compared with that from a single supernatant fraction covering the total time. Suspensions of unfertilized and fertilized eggs (80 min after fertilization) were preloaded with ["CJvaline, washed, and 2 ml aliquots, containing about 700000 eggs, placed in each of four tubes. The radioactivity in the eggs amounted to 12000 cpm for the unfertilized and 24000 cpm for the fertilized samples. In two tubes of each set the sea water was replaced 8 times (as indicated) during a period of 1 h while in the other two tubes it was unchanged. All were subjected to continuous gentle stirring. The values for the radioactivity found in the supernatant fractions are shown in Table 3. From these figures it appears that approximately as much ["CJvaline may be obtained in the medium in 5 min of incubation of the preloaded eggs as in 1 h. The lower over-all values for the fertilized eggs probably reflect, once again, the smaller intracellular pool of free ["CJvaline because of higher rate of incorporation into protein. Since incubation for 5 or 10 min can yield values as high as incubation continuously for 1 h the latter values would appear to be approximately those defined by the equilibrium conditions. It may then be concluded that influx, as well as efflux, of [^CJvaline is occurring at a significant rate during the incubation period. Therefore, in the experiments in which a [^CJamino acid is added to the medium, competition of the latter with re-entering p4C]valine is a significant factor contributing to the accumulation of radioactivity in the medium. The more rapid approach to equilibrium in the sequential-washing experiment than in the two-volume experiment may be explained by the different conditions in the two cases. The larger volume in the Displacement of valine from sea-urchin eggs 523 latter results in a lower concentration of radioactivity in the media, a condition which would be associated with a slower rate of re-entry. In the absence of further data on the kinetics of influx and efflux, the extent to which competition for re-entry serves to account for the displacement phenomenon cannot be accurately evaluated. If it were assumed that equilibrium values were reached in approximately 5 min then the amount of [14C]valine accumulating in the medium in 1 h in the presence of a large excess of a competing [12C]amino acid could be about 12 times that in absence of the [12C]amino acid. The data of Table 2 show that some 100 times more radioactivity can accumulate in the sea water when the preloaded unfertilized eggs are exposed to either [12C]valine or [12C]methionine than when they are incubated in the absence of these amino acids or in the presence of the 'non-competing' [12C]arginine. The corresponding values for the fertilized eggs are about 400 times. It may be concluded, then, either that equilibrium is approximated in much less than 5 min under these conditions or that other factors besides compe- tition or re-entry contribute significantly to the displacement phenomenon.
Table 3. Efflux of radioactivity from unfertilized and fertilized eggs preloaded urith [uC]valine*
Time Cpm in supernatant A licr vuiy 1 (min) Unfertilized Fertilized °-5 90 3 5-10 60 6 10-15 108 15 15-20 108 IS 20-30 90 12 30-40 126 3 40-50 IS 48 50—60 21 3 Sums 618 0-60 98 IS Experimental details given in the text.
Experiments with ^Qarginine and [uC]glutamic acid. In four sets of experiments that were performed with eggs preloaded with [14C]arginine and one set with ["C]- glutamic acid no appreciable displacement was obtained with any of the twenty [12C]amino acids. In these experiments preloading was of the same order of magnitude as in the experiments with [14C]valine. Incorporation of [uC]amino acids into protein by unfertilized and fertilized eggs after pre-treatment with [nC]amino acids. As mentioned in the Introduction, the lowering of the concentration of a particular amino acid inside the egg may be expected to increase the extent of labelling of protein obtained with a given amount of added labelled amino acid. The data listed in Table 4 show that pre-treatment of unfertilized or fertilized eggs with [^CJthreonine, one of the more effective 'displacing' amino acids, can result in elevated incorporation of labelled p4C]valine into protein. The amount of 524 J. Piatigorsky and A. Tyler additional incorporation of valine varied in different experiments, ranging from 1*03- (Expt 3) to i-40-fold (Expt 2) and from 1-18- (Expt 1) to i-3i-fold (Expt 2) for unfertilized and fertilized eggs, respectively. In a fourth experiment pre-treatment was done with a mixture of three 'non-competing' [12C]amino acids, namely methio- nine, lysine and glutamic acid, followed by incubation in [14C]valine, arginine and
Table 4. Incorporation of [uC]valine into protein by unfertilized and fertilized* eggs of Lytechinus pictus after pre-treatment with [l2C]amino acids
Unfertilized eggs Fertilized eggs A Cpm in protein Cpm in protein of io41 eggs Ratio: of io4 eggs Ratio: A Pre-treated A Pre-treated Experiment Control Pre-treated control Control Pre-treated control
it 1963 2294 116 22838 23 592 1-18 1817 2098 — 14189 20148 — 4 695 1 132 1-40 i35°i 15582 1-31 855 1044 — 11 208 16841 — 3§ 3197 2828 1 03 — — — 2897 3424 — — — — 4ll 5 552 11465 2-04 56766 66813 i-i5 5 74O II538 — 56139 63206 — • Experiment 1 pre-treated 30 min after fertilization; Expt 2 pre-treated 60 min after fertilization. f Pre-treatment with [llC]L-thxeonine at o-i M for i h at 20 °C followed by incubation for 30 min at 20 °C with [sH]L-valine (sp.act. 870 c/mole) at 5 /tc/ml in a total volume of i-o ml. J Pre-treatment and incubation for 20 min as in Expt 1. § Pre-treatment and incubation for 15 min as in Expt 1. || Pre-treatment with a mixture of ["CjL-methionine, [19C]L-lysine and ["CjL-glutamic acid, each at O-OI7M, for 1 h at 20 °C, followed by incubation for 30 min at 20 °C with a mix- ture of ["C]L-valine (sp.act. 208-5 c/mole), [14C]L-arginine (sp.act. 222 c/mole) and [14C]L- aspartic acid (sp.act 164 c/mole) each at 0-5 /tc/ml in a total volume of i-o ml. aspartic acid. This gave a 2-fold increase in incorporation of the [14C]amino acids for the unfertilized eggs and some i-15-fold for the fertilized eggs. From the afore- mentioned results of tests indicating lack of ready displaceability of glutamic acid and arginine it is uncertain that they contribute significantly to the high value obtained in this test with the unfertilized eggs. Preliminary tests of this type reported elsewhere (Tyler et al. 1966) also gave variable results ranging from no appreciable effect to a 2-19-fold increase of ["CJvaline incorporation into protein by unfertilized eggs after pre-treatment for 1 h with an amino acid mixture lacking valine. That the increase in incorporation of the P*C]valine into protein after pre-treatment is not attributable to an increase of uptake was shown in the cited experiments and confirmed in the present ones. These results, then, are interpreted to mean that for a given external concentration of [^Cjvaline the endogenous specific activity can be made higher by pre-treatment of the eggs with another [12C]amino acid capable of displacing valine. Displacement of valine from sea-urchin eggs 525
DISCUSSION The present results indicate that composition of the endogenous pool of free amino acids in unfertilized and fertilized sea-urchin eggs may be altered in response to addition of various amino acids to the medium. In particular, a labelled neutral amino acid, [14C]valine, was shown to be displaced from eggs in which it has been taken up, by the addition of certain other neutral, but not acidic or basic amino acids. The effectiveness of the various amino acids in displacing [aiC]vaUne correlates with the previously reported (Tyler et al. 1966) competition among the various amino acids for entry into the cell. That the displaced radioactivity is [14C]vaUne rather than some metabolic product was indicated by the facts that the label could be evaporated to dryness, was soluble in ice-cold 5 % TCA and migrated chromatographically as valine. The experiment (Table 2) in which the external volume of the egg suspensions was varied demonstrates that radioactivity leaks slowly from the preloaded eggs when they are suspended in artificial sea water. This observation was confirmed in the test (Table 3) involving sequential washing of the labelled eggs followed by determinations on the supernatant fractions. It appears likely, then, that in the presence of exogenous amino acids which inhibit valine uptake a competition for re-entry contributes to the accumulation of ["CJvaline outside of the eggs. This interpretation is supported by the good correlation between the competing and displacing ability (Tyler et al. 1966) of an amino acid with respect to valine. Thus methionine, threonine, alanine and histidine, which are highly effective in displacing valine, are among those previously shown to be strong competitive inhibitors of valine uptake. Also, aspartic acid, glutamic acid, arginine, lysine and proline, which are poor displacers of valine, are among those that were found to be weak inhibitors of valine uptake. There are, however, several important exceptions to the competitor-displacer relationship. Cysteine, phenylalanine, leucine, isoleucine and, for unfertilized eggs, tryptophan and tyrosine, displaced relatively small amounts of [14C]valine, although they are powerful inhibitors of valine uptake. These exceptions may be taken to mean that displacement may involve other factors besides competition for re-entry, such as, for example, a linked exchange flux. It is not possible to assess at this time the relative importance in the displacement phenomenon of competition for re-entry and of an exchange reaction between exogenous amino acids. In the present experiments eggs preloaded with ["CJarginine or ["CJglutamic acid retained almost all of their radioactivity regardless of the type of [12C]amino acid that was added to the medium. This may be due to the larger endogenous pools of the corresponding [12C]amino acids (Tyler, unpublished) or it may reflect a more tightly bound state for those amino acids or some other homeostatic mechanism. At present it is not possible to decide between these alternatives. Cells of marine invertebrates generally are efficient in the accumulation and retention of amino acids (Stephens & Schinske, 1961; Stephens & Virkar, 1966; Mitchison & Cummins, 1966; Tyler et al. 1966). This contrasts to the situation in cultured 526 J. Piatigorsky and A. Tyler mammalian cells where low population densities result in considerable leakage of non-essential amino acids, as well as other materials, at a faster rate than the cell can synthesize them (Lockart & Eagle, 1959; Eagle, Piez & Oyama, 1961; Eagle & Piez, 1962; see Levintow & Eagle, 1961; for reviews Eagle, 1963). The mammalian cells will not grow in dilute suspensions, then, unless the medium is enriched by the addition of the appropriate amino acids. This difference between cells of mammals and those of marine invertebrates is not altogether surprising in view of the fact that mammalian cells are adapted, in vivo, to very high population densities while those of marine invertebrates, in particular eggs of marine forms, must function when sur- rounded by large volumes of sea water deficient in amino acids. In the present experiments the possibility was examined that the redistribution phenomenon might be put to practical use so as to increase the effectiveness of radioactive labelling of proteins synthesized by the cells. Thus the lowering of the concentration of certain amino acids in the internal pool, as a result of a pre-treatment with other related ones could be expected to permit the attainment of higher specific radioactivity in the internal pool when the [14C] isotopes of the former are subsequently added. This is, of course, subject to the provision that the reductions in pool-size do not exceed the levels at which the concentration of the particular amino acid becomes rate-limiting. There would, presumably, be an optimum concentration at which the lowering of rate of protein synthesis by reduction of the endogenous pool- size is maximally compensated by the increase in specific radioactivity that is obtained. It is likely that the sea-urchin egg responds to the deficiency of amino acids by renewed synthesis, possibly by the release of feedback inhibition and repression of enzyme synthesis, as is well documented in micro-organisms (see Umbarger, 1961a, b; Vogel, 1961) and as may also occur in animal cells (Wilson & Pardee, 1964). None the less, labelling experiments performed before the eggs have completed restoring the amino acids to the partially depleted pool may result in an increased incorporation of the labelled amino acid into protein presumably without affecting the synthesis of protein. In the present experiments and in those reported previously (Tyler et al. 1966) the values have ranged from no significant effect to an approximately 2-fold increase for the incorporation of ["CJvaline into protein after preliminary treat- ment of the eggs with a related non-radioactive amino acid or mixture of amino acids. Since the pool of free amino acids is likely to differ from one batch of eggs to another, the effectiveness of the pre-treatment in optimally depleting the pool of a particular amino acid will correspondingly differ. For practical purposes the pre- treatment method would appear to be of rather limited use. It could be helpful in special cases involving the labelling of particular proteins known to be rich in a given amino acid. For studies involving bulk protein synthesis and synthesis of proteins of average amino acid composition, maximal labelling may be best achieved by using non-competing mixtures of amino acids having high specific activities, as previously noted (Tyler et al. 1966). We wish to acknowledge the effective technical assistance of Peter N. Redington and Edgar E. Vivanco. This work was supported by a grant from the National Institutes of Health (8 RO 1 HD-03476) and the National Science Foundation (GB 28). Displacement of valine from sea-urchin eggs 527
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