TRANSAMINATION AND PROTEIN SYNTHESIS IN GERMINATING OAT SEEDLINGS*

TRANSAMINATION AND PROTEIN SYNTHESIS IN GERMINATING OAT SEEDLINGS* BY HARRY G. ALBAUMt AND PHILIP P. COHEN (From the Laboratory of Physiological Chemistry, University of Wisconsin, Madison) (Received for publication, April 12, 1943) The discovery of the transamination reaction by Braunstein and Kritzmann (1) has led to the suggestion that this reaction is of major importance in protein metabolism in plant and animal tissues. Thus Virtanen and Laine (2) have postulated that in the leguminous plants the first amino acid synthesized is aspartic acid, which in turn participates in the synthesis of the remaining amino acids by transamination with the corresponding crketo acids. Unfortunately, quantitative data for plant tissues in particular have been strikingly inadequate, not only for support of the more elaborate theories, but even for the mere demonstration that transamination occurs at an appreciable rate. On the whole the studies with plant tissues have suffered from a poor choice of material and experimental conditions, in addition to inadequate analytical methods. Thus Cedrangolo and Carandante (3) reported that aqueous extracts of different seeds and germinated seedlings were most active in transaminating the system, Lu-ketoglutaric acid plus aspartic acid. The rates observed, however, were extremely low and of questionable significance (4). The reverse reaction was not investigated. No transamination was observed to occur in Chlorella (4), but the experimental conditions employed were not optimum in view of the findings reported in this paper. Kritzmann (5) has reported the preparation of transaminating enzymes from animal and plant sources, and has found their properties to be similar. She has maintained the view that two transaminating systems exist, one concerned with glutamic (or a-ketoglutaric) acid, and the other with aspartic (or oxalacetic) acid. The latter system is reported by Kritzmann to be most readily prepared, free of the glutamic acid system, by extracting coarsely ground pea seedlings. Previous studies, however, by one of us (6-9) have led to the view that a single enzyme system is involved, the chief substrates for which are glutamic acid plus oxalacet,ic acid. In this paper experiments are reported in which the reaction (1) Z(+)-Glut.amic acid + oxalacetic acid <E b or-ketoglutaric acid + I(-)-aspartic acid * This investigation was aided by a grant from the Jane Coffin Childs?*Zemorial Fund for Medical Research. t National Research Council Fellow in the Natural Sciences, 1942.-43. 19

20 TRANSAMINATION AND PROTEIN SYNTHESIS was quantitatively investigated in embryos from oat seedlings at various stages of germination. Simultaneous estimations of different nitrogen fractions of the embryos are also reported for the purpose of correlating transamination with protein metabolism. Procedure Preparation of Oat Embryos--Oat embryos used in the studies were prepared in the following way. Grains of Avena sativa L. var. Vicland were hulled, soaked in distilled water for 2 hours in the light, and germinated in the dark at room temperature for 24 hours. After germination, the grains were planted in beakers in contact with moist filter paper, as previously described (lo), to which distilled water had been added. The plants were then allowed to continue their growth in the dark. Immediately after the initial soaking period and at 24 hour intervals thereafter groups of seedlings were removed from the beakers and the embryos were carefully dissected from the endosperms. Preparation of Homogenates and Incubation Mixtures-Groups of embryos varying in number between ten and 100, depending on the age, were homogenized in ice-cold 0.1 M phosphate buffer with a stainless steel homogenizer. In most of the experiments the ph was checked and when necessary adjusted to ph 8.0 with 1 M sodium bicarbonate. In other experiments the desired ph was attained by using a phosphate buffer of approximately the desired ph and then adjusting as before. 1 ml. samples of the homogenate were pipetted into test-tubes or 50 ml. flasks to which, in the case of the complete system (Reaction I, a), the following additions were made, 0.3 ml. of 0.1 M glutamate and 0.3 ml. of 0.1 M oxalacetate (or corresponding amounts of or-ketoglutarate and aspartate in the case of Reaction 1, b). The glutamate and oxalacetate were adjusted to the proper ph before use. The oxalacetate was added to the system after the homogenate plus glutamate was shaken for 10 minutes at 38 to allow for temperature equilibration. The substrate concentration was 0.0187 M. The reaction was stopped, at the end of varying periods of incubation with continuous shaking, by the addition of 1.0 ml. of 10 per cent sodium tungstate and 1.0 ml. of 10 per cent sulfuric acid. 2.4 ml. of water were added to make a final volume of 6.0 ml., and the mixture filtered. Analytical Procedures-Aspartic acid formation was determined by the chloramine-t method previously described (7). Determinations were usually carried out in duplicate. For the estimation of cu-ketoglutaric acid, the method of Krebs was employed (11). Total and soluble (non-protein) nitrogen was routinely determined on all homogenates and filtrates by a micro-kjeldahl method in the usual way.

H. G. ALBAUM AND I. P. COHEN 21 Results Comparison of Rate of Reaction 1, a by Aspartic Acid and a-ketoglutaric Acid Determinations-The rates of Reaction 1, a as measured both by aspartic acid and cu-ketoglutaric acid formation with the same incubation mixture are shown in Table I. While the rates of transamination are lower than those shown in Fig. 2, which were obtained at ph 8.0, which is closer to the optimum ph of 8.6, the values obtained by the two methods show good agreement. The significance of the data in Table I is that, first, the two independent analytical methods yield the same results, TABLE Rate of Transamination As Measured by Aspartic Acid and a-ketoglutaric Acid Formation in Same Incubation Mixture 2 hour-old embryos; ph 7.4. The per cent transamination was calculated on the basis of 672 microliters of added glutamic acid. IlKUbation time min. 15 30 60 120 - CO, evolved microliters 744 754 835 858 942 915 985 955 1020 Aspartic acid formation ~- - microliters microliters cot??zicroliters 749 615 134 847 615 232 929 615 314 986 615 371 ination per cent 19.9 34.5 46.7 55.2 I 02 uptake ndcroliters 116.5 a-ketoglutaric acid formation Blank, 02 uptake. nzdcroliters 44 AOz uptake nicroliters 72.5 x-keto- :lutaric acid 166.5 44 122.5 245 190.5 44 146.5 293 222 44 178 356 ransamination per cent 21.5 36.5 43.7 53.1 and second, that the course of Reaction 1, a is as depicted, and thus the measured rates in this system are not due to, nor influenced by, any secondary reactions which would serve either to utilize the substrates, glutamic and oxalacetic acids on the one hand, or the reaction products, aspartic and cy-ketoglutaric acids, on the other hand. E$ect of ph on Reaction 1, a-the effect of ph on Reaction 1, a (with embryos from grains soaked for 2 hours) is shown graphically in Fig. 1. It is seen that the optimum ph is at 8.6, with a relatively rapid decline in activity with both increasing and decreasing ph. This is somewhat in contrast to animal transaminase which shows an optimum activity at ph 7.5 (8), with a relatively slower decrease in activity with change in ph. Whether the optimum ph for these plant homogenates would obtain

22 TRAWSAMINATION rlnd PROTEIW SYNTHESIS for more purified preparations, it is not possible to predict. On the other hand, Kritzmann (5) has reported that the optimum ph for aqueous extracts of pea seedlings is the same as that for animal preparations; viz., 7.4. Preliminary experiments in this study were carried out at ph 7.4 because of Kritzmann s findings (5) and those reported for animal transaminase (8). However, as a result of the data shown in Fig. 1, all subsequent] experiments were conducted at ph 8.0. The optimum ph was not employed, because it seemed desirable to use a phosphate buffer. Rates of Reactions I, a and 1, b-the rates of Reactions 1, a and 1, b are shown graphically in Fig. 2. It is seen that Reaction 1, a procends at a relatively rapid rate initially and approaches more slowly an equilibrium PH FIG. 1. pii-activity curve (Reaction 1, a). 2 hour-old embryos level after about 60 per cent transamination. Reaction 1, b reveals a much slower initial rate, the equilibrium level being reached after about 20 per cent transamination. It is apparent that Reaction 1, a proceeds at a rate about 3 times as fast as Reaction 1, b. Values of this same order have been reported for purified transaminase from animal sources (8) and animal tissue homogenates (9). Changes in Transaminase Activity (Units) and Protein and Non-Protein Nitrogen at Di$erent Stages of Embryo Development-The units of transaminase activity present at different stages of embryo development are shown in Fig. 3. One transaminase unit is defined as that amount of enzyme which will produce 10 microliters of aspartic acid (or a-ketoglutaric acid) from a 0.0187 M concentration of glutamic acid plus oxalacetic acid in 15

H. G. ALRAUM AND P. P. COHEN 23 minutes at ph 8.0 and 38. In addition, protein nitrogen (total nitrogen minus soluble nitrogen) and soluble nitrogen (75 per cent of which is 80 a---------e- 60. l -e- i+! a Z 40. 20- / / 0 El00 0 0510. 20 40 60 TIME IN MINUTES PIG. 2. Rates of Reactions 1, a and 1, b. 2 hour-old embryos; ph 8 36 @ Z I- 24 5 0 TIME IN HOURS FIG. 3. Changes in transaminase activit,y and protein and non-protein nitrogen at different stages of embryo development. ph 8; Reaction 1, a. amino nitrogen by the chloramine-t method) levels at the same stages are presented.

24 TRANSAMINATION AND PROTEIN SYNTHESIS It can be seen from Fig. 3 that during the first 24 hours there is a slow and parallel increase in transaminase units, protein nitrogen, and soluble nitrogen levels. Between 24 and 48 hours, the soluble (or amino) nitrogen levels increase sharply. At about 48 hours, the transaminase units increase sharply. However, the protein nitrogen continues to increase at the same slow rate as during the first 24 hours up until the 72 hour stage. At this time the protein nitrogen levels rise sharply. Thus at 72 hours the transaminase units and soluble (or amino) nitrogen levels have reached relatively high values, subsequent to which time the protein nitrogen starts to increase rapidly. It would thus appear that the rate of protein synthesis is accelerated when the transaminasc activity and the soluble (or amino) nitrogen values reach a certain level. It should be emphasized that, between 24 and 72 hours, the rate of synthesis of transaminase is more rapid than the rate of synthesis of total protein. This in itself suggests that there is a preferential synthesis of a key protein, transaminase, which either directly or indirectly influences the rate of total protein synthesis. After 72 hours the rates of transaminase synthesis and protein synthesis are parallel, again emphasizing the intimate association of these two constituents. It is at this 72 hour stage that other enzyme systems start appearing or enzyme systems already present increase markedly in activity (12, 13). It is of interest to note that after 72 hours the soluble (or amino) nitrogen levels continue to rise along with the protein nitrogen level. It might be expected that if the protein were being synthesized from the amino acids (soluble nitrogen) present the latter values would decrease. During these early stages, however, the soluble (amino) nitrogen keeps increasing because of the rapid rate of transfer of soluble nitrogen from the endosperm which rapidly loses its protein nitrogen and supplies it to the embryo (12). Thus we have the conversion of the endosperm protein to the embryo protein through the medium of soluble (amino) nitrogen. The function of transaminase thus might appear to be that of rearranging or interconverting the soluble (amino) nitrogen provided by the endosperm into the components necessary for synthesis of embryo proteins. The broader implications of this will be cqnsidered under the discussion. Q tyansaminaiion Values at Different Developmental Stages-A comparative measure of transaminase activity is conveniently expressed in terms of the conventional Qcransamination values. However, since these values are usually expressed on the basis of the dry weight of the tissue, erroneous values are obtained with plants owing to the increasing amounts of supporting tissue, such as cellulose, with development. A more satisfactory basis for expressing Qtransamination values is on the basis of mg. of protein, since the latter more adequately reflects the metabolically active tissue of the plant. With values are presented in Table II both on a dry this in mind, Qtransamination

H. G. ALBAUM AND P. P. COHEN 25 weight basis (QT) and on a protein basis (Q Tcrr.)) at different stages of embryo development. It is apparent from Table II that QT values decrease with embryo development, while the QTcPr.) values increase. The latter values undoubtedly are a better reflection of the changes in transaminase activity than the former. The Qr(rr.) values are remarkably high and indicate that at least in this plant tissue transaminase activity is not only high initially but actually increases about 4-fold after 120 hours. During the first 24 hours the values change very little, but between 24 and 72 hours the values are tripled, while between 96 and 120 hours they are quadrupled. The QT(rr.) value for Reaction 1, b in 2 hour-old seedlings is of the order of 100. Calculation of the QT(rT.) values for the same reaction in extracts from pea and corn seedlings from the data of Cedrangolo and Carandante (3, 4) gives values of 1.1 and 3.8 respectively. TABLE Change in QT and QT(P~.) with Time in Developing microliters substrate transaminated QT = mg. dry weight X hrs.. microliters substrate transaminated QT(PL) = mg. protein X hrs. Time hrs. 2 24 48 72 96 QT II Oat Embryos @T(Pr.) 109 268 158 295 154 784 94 805 73 905 The QT(rr,) values for plant tissue are more or less comparable with QT values for animal tissue. In a comparison of the transaminase activity of animal and plant tissues it appears that in both cases there is a progressive increase with age (14). However, the values for the oat seedling are several times greater than those reported with the most active animal tissues. Thus pigeon breast muscle has a QT value of 450. On the other hand, purified transaminase preparations from pig heart muscle have values of the order of 1650 (8). DISCUSSION The exact manner in which transamination can function in synthetic reactions of protein is as yet not clear. Two possibilities merit consideration. In the first place, if one considers protein synthesis to proceed by the reversed action of proteolytic enzymes, then it is difficult to see how

2G TRANSAMINATION AND PROTEIN SYNTHESIS transamination can tie up directly with this process. On this basis one must assume that transamination serves the function of rearranging or interconverting the amino acid substrates in such a way as to insure the proper concentrations and kinds of amino acids. The two amino acids, glutamic and aspartic (and their corresponding amides and cy-keto acids), are known to be of special importance in intermediary protein metabolism in plants (15, 16). Unfortunately little is known about the influence of individual or combinations of different amino acids on the synthetic reactions of protein. In the second place, the possibility exists that protein synthesis is not a simple reversal of proteolysis. The synthesis of peptides by condensation of glyoxals with amino compounds, followed by dehydrogenation and transamination, has been suggested by Linderstrom-Lang (17). More recently Herbst and Shemin (18) have actually demonstrated the in vitro synthesis of dl-alanylalanine from pyruvylalanine by non-enzymatic transamination in aqueous solu- Con and have presented an attractive scheme for biological peptide synthesis from keto and amino acids. This scheme complements the classical concept of peptide synthesis and is consistent with the findings of Schoenheimer and his coworkers on the lability of the peptide link. The schemes of Linderstrom-Lang and of Herbst and Shemin possess features which are consistent with the facts presented in this paper, in addition to providing a direct rble for transamination in protein synthesis. Thus the fact that transaminase activity increases ahead of protein synthesis in the germinating oat seedling is in keeping with the above. Since an active carbohydrate metabolism makes its appearance in the germinating oat seedling at about the same time that the rate of protein synthesis increases rapidly (13), a ready source for ol-keto acids and possibly glyoxals is provided. While it is not certain that glyoxals are normal metabolic intermediates, it has been shown that germinating peas and beans are capable of converting hexose diphosphate to methylglyoxal (19), and that this activity increases with time of germination. The apparent metabolic relationship of protein synthesis, carbohydrate metabolism, and transamination in the germinating oat seedling is thus in keeping with the above schemes. In assessing the possible role of transamination in intermediary metabolism the schemes of Linderstrom-Lang and of Herbst and Shemin offer a more reasonable working hypothesis than the suggestion that tsansamination merely serves to rearrange or interconvert amino acids. 1. The transamination reaction SUMMARY (1) Z(+)-Glutamic acid + oxalacetic acid zl-( cu-ketoglutaric acid + I(-)-aspartic acid

H. G. ALBAUM AND P. P. COHEN 27 has been studied in homogenates of developing oat embryos. The optimum ph of the system is 8.6. Reaction 1, a proceeds at a rate 3 times as fast as Reaction 1, b. 2. Transaminase activity and non-protein nitrogen (75 per cent of which is amino nitrogen) increase more rapidly than total protein during the first 72 hours. After this time they increase at parallel rates up to 120 hours. 3. Qtransamination values of oat embryos, calculated on the basis of protein content, increase with time and reach values of the order of 900 after 9G hours development. 4. The relationship of transamination to protein synthesis is discussed. BIBLIOGRAPHY 1. Braunstein, A. E., and Kritzmann, M. G., Enzymologia, 2, 129 (1937). 2. Virtanen, A. I., and Laine, T., Nature, 141, 748 (1938). 3. Cedrangolo, F., and Carandante, G., Arch. SC. biol., 26, 363 (1940). 4. Cohen, P. P., Federation Proc., 1, 273 (1942). 5. Kritzmann, M. G., Nature, 143, 603 (1938). 6. Cohen, P. P., Rio&em. J., 33, 1478 (1939). 7. Cohen, P. P., J. Biol. Chem., 136,565 (1940). 8. Cohen, P. P., J. Biol. Chem., 136, 585 (1940). 9. Cohen, P. P., and Hekhuis, G. L., J. Biol. Chem., 140, 711 (1941). 10. Kaiser, S., and Albaum, H. G., Am. J. Bot., 26, 749 (1939). 11. Krebs, I-I. A., Biochem. J., 32, 108 (1938). 12. Albaum, H. G., Donnelly, J., and Korkes, S., Am. J. Bot., 29, 385 (1942). 13. Albaum, H. G., and Eichel, B., Am. J. Bot., 30, 18 (1943). 14. Cohen, P. P., and Heklmis, G. L., Cancer Research, 1, 620 (1941). 15. Chibnall, A. C., Protein metabolism in the plant, New Haven (1939). 16. Vickery, H. B., Pucher, G. W., Schoenheimer, R., and Rittenberg, D., 6. Biol. Chem., 136, 531 (1940). 17. Linderstrem-Lang, K., Annual review of biochemistry, Stanford University, 8, 37 (1939). 18. Herbst, R. M., and Shemin, D., J. Biol. Chem., 147, 541 (1943). 19. l\euberg, C., and Kobel, M., Biochem. Z., 229, 433 (1930).

TRANSAMINATION AND PROTEIN SYNTHESIS IN GERMINATING OAT SEEDLINGS Harry G. Albaum and Philip P. Cohen J. Biol. Chem. 1943, 149:19-2. Access the most updated version of this article at http://www.jbc.org/content/149/1/19.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/149/1/19.citation.full.ht ml#ref-list-1