A SURVEY OF TRANSAMINASES IN PLANTS* BY MARY JANE KREKO LEONARD AND R. H. BURRIS

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1 A SURVEY OF TRANSAMINASES IN PLANTS* BY MARY JANE KREKO LEONARD AND R. H. BURRIS (From the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison) (Received for publication, July 14, 1947) The transamination reaction of Braunstein and Kritzmann (1) has been studied in green plant tissue by Virtanen and Laine (2), Cedrangolo and Carandante (3), Albaum and Cohen (4), and Rautanen (). Since few data have been presented on the distribution of the transaminase enzymes in various plant tissues and kinds of plants, more data are necessary in order to evaluate the role of the transamination react.ion in protein metabolism. The occurrence of the asparticglutamic transaminase was investigated in t,he parts of twentytwo plant species at various ages. Methods Preparation of Enzyme ExtractExtracts were prepared by comminution of plant tissuewith a Nixtamal mill, by hand grinding with sand, or with a glass homogenizer (6). Samples were icecooled either before or immediately after grinding. Extracts were adjusted to ph 8.0 with the aid of a glass electrode. All preparations were centrifuged in a Beams airdriven, spinning top centrifuge (7) and the supernatant was employed. Total nitrogen of homogenates and saps was determined by the semimicro Kjeldahl method (8). The embryos used in the studies were prepared by sterilizing seeds Ivith 1: 00 HgC12. The seeds were soaked in sterile distilled water on blotting paper in Petri dishes and then germinated by incubation in a moist chamber in the dark at 30. Barley was grown under cont.rolled condit,ions of moisture and temperature in a malting chamber. Embryos were carefully dissected from the seeds and homogenized in icecold 0.1 M phosphate buffer (ph 8.0). Unheated wheat germ (2 per cent by weight in buffer solution) was likewise homogenized. The 2,4, and 6 weekold plants used in the experiments were grown in a greenhouse in sand supplied with nutrient solution. In sampling, petioles were treated as stem tissue. tissue included only the leaf blade. *Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. These studies were supported in part by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. 701

2 702 TRANSAMINASES IN PLa4NTS After grinding, the sap was expressed through cheesecloth and centrifuged. In almost all experiments the undiluted sap was used. Analytical MethodsGlutamic acid was determined with a bacterial decarboxylase as described by Umbreit and Gunsalus (9). Oxalacetic acid was determined by the aniline citrate method (). SubstratesOxalacetic acid was prepared from sodium diethyl oxalacetate (lo), and the aketoglutaric acid was kindly supplied by Dr. F. H. Stodola (11). Merck amino acids were used. Procedure The following transamination reaction was studied : (4 (1) LGlutamic acid + oxalacetic acid &I? cyketoglutaric acid + Laspartic acid (6) The forward reaction, Reaction 1, a, was carried out in 18 X 10 mm. testtubes which were attached to a mechanical shaker and immersed in a water bath maintained at 30. No attempt was made to provide anaerobic conditions. A 4 to ml. sample of the transaminase preparation (ph 8.0) was incubated with 2.0 ml. of 0.06 M oxalacet,ic acid for minutes. Then 2 ml. of 0.06 M glutamic acid (ph 8.0) were added and the reaction allowed to proceed for various incubation periods. The reaction was stopped by the addition of 1.0 ml. of per cent sulfuric acid. Controls containing oxalacetic acid and enzyme and controls containing glutamic acid and enzyme to test recovery were included. In order to decompose any remaining oxalacetic acid, the contents of the tubes were adjusted to ph 2.0 and were immersed in a boiling water bath for 1 hour. After cooling, the samples were carefully adjusted to ph.0, transferred to ml. volumetric flasks, and made up to volume. Each sample was then centrifuged and a 2.0 ml. aliquot was placed in a Warburg flask for analysis. After a minute equilibration period at 30, 0. ml. ( mg.) of glutamic acid decarboxylase preparation (9), suspended in potassium acid phthalate buffer (ph.0), was added from the side arm and evolution of CO2 was measured. For measuring the reverse reaction, Reaction 1, b, the enzyme preparation and 0. ml. of 0.03 M nnaspartic acid (ph 8.0) were placed in the main compartment of the Warburg flask. In one arm 0. ml. of 0.03 M aketoglutaric acid (ph 8.0) was placed, and in the other side arm 0. ml. of aniline citrate (equal volumes of 0 per cent citric acid and freshly distilled aniline). After minutes equilibration at 30, the aketoglutaric acid was mixed with the sample and the transamination reaction allowed to proceed. The reaction was stopped and analyzed by adding the aniline citrate. The decomposition of the oxalacetic acid formed in the transami

3 M. J. K. LEONARD AND R. H. BURRW 703 nation reaction was essentially complete in minutes. Controls with aketoglutaric acid omitted were included. Results Germinating Seed EmbryosTable I presents data on the ability of germinating seed embryo homogenates to catalyze Reactions 1, a and 1, b. Green Sweet Garden Radish Cucumber Clover Germinating beans corn peas Soy beans Vicland oats TABLE Transamination in 48 HourOld Germinating i Seed Embryos seed Transamination period min. 1: T Reaction 1, 0 fer cent I per cent ransam 1 inationt QT PUt T, Oxalacetic a cid formed Reaction rransamination$ _ micromolcs per cent b QT (N)t * The percentage of initial glutamic acid (24 micromoles in the aliquot assayed, equivalent to 38 microliters of CO2 on decarboxylation) added to the controls which remained at the end of the transamination period. t The percentage of initial glutamic acid which was consumed in Reaction 1, a. $ QT (N) = (micromoles of amino acid transaminated)/(mg. of N X hours). 0 Percentage of initial added aspartic acid which was transaminated, as measured by oxalacetic acid formation. In the controls for Reaction 1, a, in which glutamic acid and the enzyme preparation were incubated (no oxalacetic acid added) and the amount of glutamic acid then determined, a small amount of the initial glutamic acid disappeared. Since this amount may easily have been consumed by means of the transamination reaction, it was decided to base percentage transamination on the initial quantity of glutamic acid present. The percentages of transamination in minutes for Reaction 1, b are onehalf to onethird those for Reaction 1, a. This ratio agrees with that recorded in the literature for oat seedlings (4).

4 704 TRANSAMINASES IN PLANTS Reno Malting temperature, 116. Manchuria Korsbyg Kindred Reno Manchuria Korsbyg Kindred Reno Manchuria Korsbyg Kindred Variety Reno Manchuria Korsbyg Kindred TABLE Transamination in Barley; Reaction 1, b Age hrs II I Transamination period min Transamination QT 09 per cent Moisture Nitrogen (dry weight basis) ger cent per cent Reaction 1, b was studied in several varieties of barley at various stages of malting. The values obtained are recorded in Table II. Two varieties,

5 M. J. K. LEONARD AND R. H. BURRIS 70 Reno and Manchuria, have a high nitrogen content and germinate rapidly. Both germinated while being steeped and were quite advanced at the 24 hour stage. Korsbyg and Kindred, on the other hand, have a lower nitrogen content and germinate slowly. The faster germinating barley had the higher transaminase activity; QT (N) values at all st.ages were much higher than those for the more slowly germinating barley. It is of interest to note the sharp fall of transaminase activity of 72 hourold embryos of the Korsbyg and Kindred varieties. QT (N) values were high at 24 hours, decreased at 72 hours, and then increased again at 120 hours. These temporal changes did not occur with the Reno and Manchuria varieties. The competition of other enzymatic systems is probably more in evidence in the slower germinating barley, Korsbyg and Kindred, than in the faster germinating barley. TABLE III Transamination in Wheat Germ Reaction 1, (I Reaction 1, b Transamination period Glutamic acid Transami O.&acetic Transamitransaminated nation QT (W acid formed nation QT W) min. micromoles per cent micromoles per cent Wheat GermThe transaminase activity of commercial unheated wheat germ was investigated; its use was suggested to us by Dr. P. P. Cohen who had found it a convenient material with high transaminase activity. Table III presents dat,a obtained for Reactions 1, a and 1, b. Examination of the percentages of transamination for the different periods of time reveals that Reaction 1, a is about 3 times as fast as Reaction 1, b. This ratio again agrees with that recorded in the literature (4). Wheat germ preparations were also tested for their ability to transfer the amino group from alanine to arketoglutarate. This transamination, as measured by pyruvate formation, occurred at a rat.e comparable to,that of Reaction 1 and gave a QT (N) of 40.6 over a minute period. Young and Mature Plant TissueThe distribution of glutamicaspartic transaminase was studied in a number of young and mature plants. Values lqt(n) = (micromoles of amino acid transaminated)/(mg. of N X hours). This expression of transamination on a micromolar basis when multiplied by is equivalent to the &TN on an equivalent CO, basis as used by Lichstein and Cohen (12).

6 706 TRANSAMINASES IN PLANTS TABLE IV Transamination in Plant Tissue: Reaction 1. 6 AS Tissue mwamination period lx$c$ic formed Ikansamination IT W) Soy bean Potato Tomato Beet Lettuce Cabbage Barley, Manchuria Korsbyg Carrot Squash Green tomatoes I apples Garden peas wks. 2 6 Nodules St,em + stem 2 + stem stem 2 + Fruit I 12 Pods ntin. nicromoles ger cent ii

7 M. J. K. LEONARD AND R. H. BURRIS 707 Plant Age TABLE IVConcluded Tissue Ihnsamination period Ox;$etic formed Transmination Kohlrabi Green Maple Pine Soy bean Potato beans Plant wks Nodules Needles TABLE V micromoles per cent Transamination in Plant Tissue; Reaction 1, a, Minutes Tomato 6 Garden peas 12 Squash Barley, Korsbyg 2 Age ruks. 2 _ Nodules Pods Control recovery glutamic acid + :nzyme) Gl;zCc transaminated Transamination per cent nicromoles per cent QT(W for Reaction 1, b are presented in Table IV. Soy beans, potatoes, tomatoes, and beets were investigated at the 2 week stage and again at the 6 or week stage. Transamination ability tended to decrease with age in soy beans, tomatoes, and beets, but 6 weekold potato plants showed a slightly greater activity than the 2 weekold plants. The greatest activity, expressed on a nitrogen basis, was always found in the roots (except beet and cabbage). In all examples cited greater activity was found in the leaves of the 2 weekold plants than in the leaves of mature plants (except potato). Carrot root had an abnormally high QT (N). s and nodules of green beans were more active than the leaves or stems. The active nitrogen metabolism in green pea pods probably accounted for the high values in the pods, in contrast to the lower values found in the other tissues of the pea. The fleshy stem of kohlrabi had high activity and this was also true of

8 708 TRANSAMINASES IN PLANTS weekold cabbage stems. The presence of transaminase in squash fruit was of interest. The enzyme could not be detected in any appreciable amounts in such fruits as green apples or green tomatoes. Reaction 1, a was also studied in various plant tissues. The results are presented in Table V. Greatest activity, on a nitrogen basis, is again exhibited by the roots. DISCUSSION Transaminase activity was demonstrated in various tissues from many types of plants. Activity was always found in tissues containing significant quantit,ies of crude protein, but in tissues low in protein, such as green apples, green tomatoes, maple leaves, and pine needles, marked activity could not be demonstrated. Although mature plants generally have a less intense protein metabolism than germinating seeds, their tissues showed high transaminase activity. The QT (N) values for many mature tissues were of the same order of magnitude as for germinating seeds. Leaves had active systems, showing a higher percentage transamination of substrate in a given length of time per ml. of sap than did either roots or stems. However, a more valid comparison can be made from the QT (N) values which are based on the nitrogen content of the enzyme preparations; roots, with a low nitrogen content, showed higher QT (N) values than leaves. In view of the general occurrence and high activity of transaminase in plant tissues, it seems probable that transamination functions actively in the protein metabolism of plants. SUMMARY 1. Embryos of germinating seeds were tested for their ability to catalyze the forward and reverse glut amicaspartic transamination reaction. In the case of malt sprouts the transamination quotient tended to increase with age and leveled off at the 120 hour stage. 2. Very active transaminases, catalyzing both the glutamicaspartic and glutamicalanine transaminations, mere found in unheated wheat germ. 3. The presence and distribution of asparticglutamic transaminase in mature higher plants was demonstrated., stem, root, fruit, and nodular tissue all catalyzed the reaction. tissue was found to be very active per unit volume of sap, but roots and nodules had higher QT(N) values. 4. The transamination rate per unit of tissue decreased with age in the growing plant. At mat.urity the QT (N) values for the various tissues were markedly lower than for the 2 weekold plant tissues. BIBLIOGRAPHY 1. Braunstein, A. E., and Kritzmann, M. G., Enzymologia, 2, 129 (1937). 2. Virtanen, A. I., and Laine, T., Nature, 141, 748 (1938).

9 M. J. K. LEONARD AND R. H. BURRIS Cedrangolo, F., and Carandante, G., Boll. Sot. ital. biol. sper., 16, 482 (1940). 4. Albaum, H. G., and Cohen, P. P., J. Biol. Chem., 149, 19 (1943).. Rautanen, N., J. Biol. Chem., 163,687 (1946). 6. Potter, V. R.: and Elvehjem, C. A., J. Biol. Chem., 114,49 (1936). 7. Beams, J. W., Weed, A. J., and Pickels, E. G., Science, 78,338 (1933). 8. Umbreit, W. W., and Bond, V. S., Ind. and Eng. Chem., Anal. Ed., 8,276 (1936). 9. Umbreit, W. W., and Gunsalus, I. C., J. Biol. Chem., 169, 333 (194).. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and related methods for the study of tissue metabolism, Minneapolis (194). 11. Lockwood, L. B., and Stodola, F. H., J. Biol. Chem., 164, 81 (1946). 12. Lichstein, H. C., and Cohen, P. P., J. Biol. Chem., 167,8 (194).

10 A SURVEY OF TRANSAMINASES IN PLANTS Mary Jane Kreko Leonard and R. H. Burris J. Biol. Chem. 1947, 170: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#reflist1