Nitrogen Mobilization in Pea Seedlings. II. Free Amino Acids

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purfied from shoot tips of pea (Pisum sativum L. cv. Transaminases (aminotransferases, EC 2.6. 1), particularly those of plants, are quite incompletely described in regard to their amino acid and keto acid specificities. Some partially purified preparations are highly specific for particular amino acids (1, 2, 5, 30). Other preparations reportedly transaminate a variety of amino acids (7,8,12,15,25,29). Many workers have concentrated on transamination of aromatic amino acids. Two transaminase fractions have been isolated from rat brain, one of which showed highest activity toward tyrosine and phenylalanine and the other of which showed highest activity toward tryptophan and 5-hydroxytryptophan (4, 6, 26). Partially purified tryptophan transaminase from the bacterium Clostridium sporogenes catalyzed the transamination of tyrosine and phenylalanine, as well as tryptophan (23). A transaminase fraction from mung bean (Phaseolus aureus) also transaminated tryptophan, phenylalanine, and tyrosine, as well as several aliphatic amino acids (7,8).The purpose of these investigations was to purify partially and ascertain some properties of a transaminase fraction from pea shoots and to attempt to corroborate the evidence reported by Moore (20) and kept under a 16-hr photoperiod at 20 + 1 C with a light intensity of 900 to 1000 ft-c and an 8-hr dark period at 16 + 1 C. After 10 to 14 days, 50 to 70 g of shoot tips were removed immediately above the fifth node and placed in liquid nitrogen for overnight storage prior to use.Preparation of Enzyme Extract. Each sample of shoot tips was homogenized in a chilled mortar and pestle with 2 ml of 0.5 M borate buffer (pH 8.5), containing 150 ltM chloramphenicol and 1 mm dithiothreitol, per g fresh weight of tissue. The homogenate then was centrifuged at 40,000g for 10 min at 4 C, and the resulting supernatant was the crude enzyme extract. The extract was then brought to 25% (v/v) acetone with the addition of ice-cold acetone. After setting in an ice bath for 10 min, the 25% (v/v) acetone-crude extract was centrifuged at 10,000g for 10 min. The supernatant was then brought to 60% (v/v) acetone, allowed to set for 10 min in an ice bath and then was centrifuged at 10,000g for 10 min. The resultant supernatant was discarded, and the pellet was resuspended in 0.5 M borate buffer (pH 8.5) in a total of one-sixth of the original volume of crude extract. This suspension was then centrifuged at 40,000g for 10 min, and the resulting goldyellow colored supernatant was used as the acetone-precipitated enzyme fraction. In some studies, the acetone-precipitated enzyme was added to a column of hydroxylapatite (1.6 x 17 cm) which was previously equilibrated with 0.02 M KH,PO4-Na2HPO, buffer at pH 8.5. The column was then washed with 70 ml of 0.05 M sodium-potassium phosphate buffer (pH 8.5) and 50 ml of 0.08 M sodium-potassium phosphate buffer (pH 8.5). The enzyme was then eluted with 0.15 M sodium-potassium phosphate buffer...

Section: Resultsmentioning

confidence: 99%

Properties of an Aminotransferase of Pea (Pisum sativum L.)

Matheron¹,

Moore²

1973

“…In some plants such as the pea and Lathyrus (5), homoserine is present at quite high levels. It is particularly prominent during germination (4,6), and may have a role as a transport compound. Kinetic properties of enzymes of homoserine metabolism in the pea and nonaccumulating species have been compared, but results did not provide a convincing explanation for homoserine accumulation (17).…”

mentioning

confidence: 99%

The Role of Transamination in the Synthesis of Homoserine in Peas

Joy¹,

Prabha²

1986

ABSTRACIÌ ncubation of intact pea plants (Pisum sativum), or detached shoots, in continuous light caused a substantial increase (up to 4-fold in 2 days) in levels of homoserine. Amino acids supplied to leaves in the transpiration stream enhanced the accumulation, with glutamate, aspartate, and asparagine causing similar enhancement. Aminooxyacetate (AOA), a transamination inhibitor, at 1 millimolar prevented the accumulation. '4C-labeling experiments showed that succinate was a good source of carbon for homoserine synthesis; carbon from aspartate or asparagine was also incorporated into homoserine. For each precursor, the transfer of label was prevented by AOA. The keto acid analog of homoserine was rapidly transaminated in leaves to give homoserine. The results suggest that accumulating homoserine is synthesised by transamination rather than being derived from aspartate via the aspartate kinase/homoserine dehydrogenase pathway. The latter pathway was shown to be operating in the chloroplasts, and was sensitive to threonine (but was not inhibited by AOA), suggesting that this path has a role in synthesis of aspartatederived amino acids but is not involved in the accumulation of excess homoserine in the pea.Although homoserine is undetectable in most plants, it is thought to be an essential intermediate in the synthesis of threonine and methionine, and is considered to be derived from aspartate by the action of aspartate kinase, aspartyl semi-aldehyde dehydrogenase and homoserine dehydrogenase (9). In the leaf, this activity is located in the chloroplasts, including those of pea (1 1, 18). The pathway appears to be tightly regulated by threonine and lysine (9). In some plants such as the pea and Lathyrus (5), homoserine is present at quite high levels. It is particularly prominent during germination (4, 6), and may have a role as a transport compound. Kinetic properties of enzymes of homoserine metabolism in the pea and nonaccumulating species have been compared, but results did not provide a convincing explanation for homoserine accumulation (17).In our investigations of asparagine metabolism in the pea, several observations have suggested that homoserine was not directly derived from aspartate. There was a flow of nitrogen from the amino group of asparagine to homoserine, and this transfer was sensitive to the transamination inhibitor aminooxyacetate (15). In double labeling experiments, aspartate received considerable amounts of carbon and nitrogen from asparagine, whereas homoserine received mainly nitrogen (15). The deamidation inhibitor 5-diazo-4-oxo L-norvaline drastically inhibited production ofaspartate from asparagine, but had much less effect on transfer of nitrogen to homoserine (16). '5N-labeled alanine, asparagine, aspartate and glutamate were all effective as a source of nitrogen for homoserine synthesis (TC Ta, KW Joy, unpublished data). These results suggest a transfer of nitrogen to homoserine by transamination. Labeling experiments by other workers are also consistent with derivation of carbon,...

“…Enzyme studies (11) suggest that the amide group of glutamine is the preferred donor which is transferred to aspartic acid for asparagine synthesis. In pea plants, asparagine is important in transport of nitrogen in the xylem (5), and is also stored in leaves, having one of the largest pools of the amino acids (10). Homoserine is also present in large amounts in pea plants (9,10).…”

mentioning

confidence: 99%

Amino Acid Metabolism of Pea Leaves

Bauer¹,

Joy²,

Urquhart³

1977

Short term (2-hour) incorporation of nitrogen from nitrate, glutamine, or asparagine was studied by supplying them as unlabeled (14N) tracers to growing pea (Pisum sativum L.) leaves, which were previously labeled with 15N, and then following the elimination of 15N from various amino components of the tissue. Most components had active and inactive pools. Ammonia produced from nitrate was assimilated through the amide group of glutamine. When glutamine was supplied, its nitrogen was rapidly transferred to glutamic acid, asparagine, and other products, and there was some transfer to ammonia. Nitrogen from asparagine was widely distributed into ammonia and amino compounds. There was a rapid direct transfer to glutamine, which did not appear to involve free ammonia. Alanine nitrogen could be derived directly from aspargine, probably by transamination. Homoserine was synthesized in substantial amounts from all three nitrogen sources. Homoserine appears to derive nitrogen more readily from asparagine than from free aspartic acid. A large proportion of the pool of y-aminobutyric acid turned over, and was replenished with nitrogen from all three supplied sources.The central role of glutamine in primary nitrogen metabolism of higher plants has been demonstrated by several workers. The glutamine synthetase system is present in roots (8) and leaves (7,18,19), and a number of experiments with '5N have shown that glutamine is rapidly labeled in primary assimilation. In kinetic studies with 15N-nitrate (3) in young pea leaves, the amide group of glutamine had the highest rate of incorporation of ammonia, followed by glutamic acid and alanine; asparagine was extensively labeled, but at a slower rate which did not appear to be due to primary assimilation. Enzyme studies (11) suggest that the amide group of glutamine is the preferred donor which is transferred to aspartic acid for asparagine synthesis. In pea plants, asparagine is important in transport of nitrogen in the xylem (5), and is also stored in leaves, having one of the largest pools of the amino acids (10). Homoserine is also present in large amounts in pea plants (9, 10).There are relatively few data available on utilization or degradation of asparagine (11). Asparaginase has been suggested as providing the main pathway for asparagine utilization. In developing lupin seeds, asparagine was utilized as a nitrogen source, with the nitrogen appearing predominantly in ammonia, glutamine, and alanine, and asparaginase was very active in extracts of the tissue (2). 3 To whom reprint requests should be addressed.This paper describes experiments using l5N and '4N labeling of growing pea leaves, which allowed a comparison of the fate of nitrogen from nitrate, glutamine, and asparagine, during a period of 2 hr. Both amides supplied nitrogen to a range of compounds, including homoserine. Nitrogen from asparagine was extensively redistributed, and it was shown that asparagine could provide nitrogen for glutamine synthesis. MATERIALS AND METHODSThe objective was to fol...