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Displacement of Valine from Intact Sea-Urchin Eggs by Exogenous Amino Acids

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Displacement of Valine from Intact Sea-Urchin Eggs by Exogenous Amino Acids 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 /<c/ml in 5 ml of artificial sea water at 20 °C for 1 h (unfertilized eggs, upper graph) or | h (fertilized eggs, 30 min after fertilization, lower graph) followed by four washings (by hand-centri- fugation) with artificial sea water. The preloaded unfertilized (10160 eggs/tube) and fertilized (17780 eggs/tube) eggs were then incubated for 1 h at 20 °C in a total volume of 0-5 ml, in duplicate tubes, of artificial sea water containing the individual [lrC]- amino acids each at 0-028 M, except tyrosine which was 0-00032 M. The suspension was then hand-centrifuged and the supernatant fraction assayed for radioactivity as given under Materials and Methods. The individual results of the duplicate deter- minations are represented by each member of the pairs of bars, and the values are given as counts per minute (cpm). 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.
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