Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Amino Acid metabolism of pea leaves: labeling studies on utilization of amides

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 asparagine, 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 γ-aminobutyric acid turned over, and was replenished with nitrogen from all three supplied sources.     

Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Asparagine formation in soybean nodules

¹⁵NH₄⁺ and [¹⁵N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of ¹⁵NH₄⁺ - feeding, the ¹⁵N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In ¹⁵N content (micrograms excess ¹⁵N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. ¹⁵NH₄⁺ was also appreciably assimilated into alanine.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy

The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective ¹⁵N-labeling of side-chain amides, however, can be a challenge due to enzyme-catalyzed exchange of amide groups during protein synthesis. In the present study, we developed an efficient way of selectively labeling the side chains of asparagine, or asparagine and glutamine residues with ¹⁵NH₂. Using the biosynthesis pathway of tryptophan, a protocol was also established for simultaneous selective ¹⁵N-labeling of the side-chain NH groups of asparagine, glutamine, and tryptophan. In combination with site-specific tagging of the target protein with a lanthanide ion, we show that selective detection of ¹⁵N-labeled side-chains of asparagine and glutamine allows determination of magnetic susceptibility anisotropy tensors based exclusively on pseudocontact shifts of amide side-chain protons.     

[15N] leucine as a source of [15N] glutamate in organotypic cerebellar explants

Approximately 26.0% of the [¹⁵N] glutamate and [alpha ¹⁵N] glutamine formed in organotypic cerebellar explants was derived from [¹⁵N] leucine. Approximately 14.0% of the ¹⁵NH₃ and [amide ¹⁵N] glutamine synthesized came from leucine nitrogen. Another 4.0% of the alpha nitrogen of both glutamate and glutamine was derived from [¹⁵N] valine. These results suggest that branched-chain amino acids, particularly leucine, may be important for the synthesis of glutamic acid by the brain.     

The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook

Analysis of soil solution from forest sites dominated by Eucalyptus grandis and Eucalyptus maculata indicates that soluble forms of organic nitrogen (amino acids and protein) are present in concentrations similar to those of mineral nitrogen (nitrate and ammonium). Experiments were conducted to determine the extent to which mycorrhizal associations might broaden nitrogen source utilization in Eucalyptus seedlings to include organic nitrogen. In isolation, species of ectomycorrhizal fungi from northern Australia show varying abilities to utilize mineral and organic forms of nitrogen as sole sources. Pisolithus sp. displayed strongest growth on NH₄⁺, glutamine and asparagine, but grew poorly on protein, while Amanita sp. grew well both on mineral sources and on a range of organic sources (e.g. arginine, asparagine, glutamine and protein). In sterile culture, non-mycorrhizal seedlings of Eucalyptus grandis and Eucalyptus maculata grew well on mineral sources of nitrogen, but showed no ability to grow on sources of organic nitrogen other than glutamine. In contrast, mycorrhizal seedlings grew well on a range of organic nitrogen sources. These observations indicate that mycorrhizal associations confer on species of Eucalyptus the ability to broaden their resource base substantially with respect to nitrogen. This ability to utilize organic nitrogen was not directly related to that of the fungal symbiont in isolation. Seedlings mycorrhizal with Pisolithus sp. were able to assimilate sources of nitrogen (in particular histidine and protein) on which the fungus in pure culture appeared to grow weakly. Experiments in which plants were fed ¹⁵N-labelled ammonium were undertaken in order to investigate the influence of mycorrhizal colonization on the pathway of nitrogen metabolism. In roots and shoots of all seedlings, ¹⁵N was incorporated into the amide group of glutamine, and label was also found in the amino groups of glutamine, glutamic acid, γ-aminobutyric acid and alanine. Mycorrhizal colonization appeared to have no effect on the assimilation pathway and metabolism of [¹⁵N]H₄⁺; labelling data were consistent with the operation of the glutamate synthase cycle in plants infected with either Pisolithus sp. (which in isolation assimilates via the glutamate synthase cycle) or Elaphomyces sp. (which assimilates via glutamate dehydrogenase). It is likely that the control of carbon supply to the mycorrhizal fungus from the host may have a profound effect on both the assimilatory pathway and the range of nitrogen sources that can be utilized by the association.     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L

Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO₂ and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, ¹⁵NH₄Cl was injected into the central pith of stem sections. The kinetics of ¹⁵N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of ¹⁵N derived from ¹⁵NH₄Cl was almost completely inhibited. Injections of amido-¹⁵N glutamine demonstrated a potential for ¹⁵N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.     

Amino Acid metabolism of pea leaves: diurnal changes and amino Acid synthesis from N-nitrate

In the young leaves of pea (Pisum sativum L.) plants, there was a diurnal variation in the levels of amino acids. In the light, total amino nitrogen increased for the first few hours, then stabilized; in the dark, there was a transient decrease followed by a gradual recovery. Asparagine, homoserine, alanine, and glutamine accounted for much of these changes. The incorporation of ¹⁵N into various components of the young leaves was followed after supply of ¹⁵N-nitrate. ¹⁵N appeared most rapidly in ammonia, due to reduction in the leaf, and this process took place predominantly in the light. A large proportion of the primary assimilation took place through the amide group of glutamine, which became labeled and turned over rapidly; labeling of glutamic acid and alanine was also rapid. Asparagine (amide group) soon became labeled and showed considerable turnover. Slower incorporation and turnover were found for aspartic acid, γ-aminobutyric acid, and homoserine. Synthesis and turnover of all of the amino acids continued at a low rate in the dark. γ-Aminobutyric acid was the only compound found to label more rapidly in the dark than in the light.     

GLUTAMYL, ASPARTYL AND AMIDE MOIETIES OF CEREBRAL PROTEINS: METABOLIC ASPECTS IN VITRO

Abstract— The total mixed proteins (excluding proteolipids) were isolated from cat cerebral cortex and subjected to acid and enzymic hydrolyses. Analyses on the hydrolysates were carried out by specific enzymic procedures to determine the glutamyl, glutaminyl, aspartyl and asparaginyl composition. The content of total glutamyl and total aspartyl residues was the same in all types of protein samples, with average values of 78 and 58 /miol/100 mg of protein, respectively. In biopsy samples approximately 45 per cent of each total was in the amide form. Preparation of slices of cerebral cortex for incubation was associated with deamidation in situ of 16 per cent of the protein-bound glutaminyl residues. The extent of deamidation was not increased by incubation or by prolonged hypoxia and was unaffected by prior anaesthesia or by incubation of slices with 10 mM-NH₄Cl or 40 mM-malonate. Slices prepared from animals intoxicated with methionine sulphoximine exhibited no deamidation. No deamidation was observed for slices of subcortical white matter, liver, kidney, testis or diaphragm of the cat. Cortical proteins from other species appeared to behave similarly to those of the cat. The 5-4 μmol of NH₃ released/g of fresh cortex could account for about 85 per cent of the endogenous free ammonia regularly encountered in such slices. Hence the labile fraction of protein-bound glutaminyl amide groups represents, as previously suspected, a major source of endogenous cerebral NH₃. Proteins isolated from cerebral cortical slices incubated with L-[U-¹⁴C]glutamic acid or L-[U-¹⁴C]glutamine contained 105 (±0.095) per cent of the total ¹⁴C metabolized. The ratios (x 100) of protein to free pool specific radioactivities (c.p.m.μmol) of glutamic acid and of glutamine were in the range 0-22 to 0-42, or of the same order as previously reported for other amino acids. Comparable results were obtained with proteins isolated from cerebral cortical slices incubated with 10 mM-¹⁵NH₄Cl or L-[amide-¹⁵N]glutamine or both. In the amide N of protein-bound glutaminyl residues the atoms per cent excess ¹⁵N ranged from 007 to 0-42. This degree of labelling could be accounted for completely by the turnover of the entire glutaminyl moiety, as indicated by the ¹⁴C studies. Simultaneous analyses of free pool NH₃ and glutamine suggested that transfer of glutamine from medium to slice involves deamidation as it is taken up and reamidation after entry.     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture

Solid-state [¹⁵N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium-assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen.     

Uptake, metabolism and distribution of nitrogen in crop plants traced by enriched and natural 15N: Progress over the last 30 years

The major findings of many years of research into plant N cycling are summarised in this review, firstly as revealed by ¹⁵N-enriched methods and secondly, in relation to natural ¹⁵N abundance (δ¹⁵N) in plants and their metabolites. This work has mainly been done in an agricultural context. As many groups especially attempt to relate δ¹⁵N to N cycling, atmospheric N deposition and the interactions of N with carbon budgets, we deem it useful to synthesize these major findings. Primary assimilation and distribution of N within plants were investigated from the ¹⁵N enrichment in individual plant organs and in individual amino acids after feeding them ¹⁵N-labelled compounds. In both roots and leaves, NH₄ ⁺ and NO₃ ^? were assimilated into amino acids, largely by a combination of glutamine synthetase (GS) and glutamate synthase (GOGAT). In the leaves, the transfer of glutamine (amide) N to glutamic acid was accelerated in the light, and amino N in some amino acids was deaminated to ammonia in the dark, followed by its incorporation into glutamine. The N in the growing parts such as growing leaves, filling grains and growing root parts were from two sources: re-allocation (phloem supply) of reserved N (amino acids), and currently-absorbed N. The metabolites from the mature parts may perform the roles of substrates for plant growth and signals for gene expression. δ¹⁵N values, measured for plants/soils and plant metabolites (inorganic N, amino acids, polyamines) were related with the acquisition, metabolism and distribution of N in plants. Small ¹⁵N/¹⁴N fractionation in the acquisition of N₂ and NO₃ ^? and large ¹⁵N/¹⁴N fractionation in NH₄ ⁺ uptake were found. The δ¹⁵N values of whole shoots or grains from field-grown crops were largely reflected major sources of N. In some legumes, ¹⁵N was enriched in their nodules and an extremely ¹⁵N-enriched compound was homospermidine. Nitrate reduction to ammonia (NR) and ammonia assimilation to glutamine (GS) showed large ¹⁵N/¹⁴N fractionations. Specific attention was paid to the δ¹⁵N values in xylem and phloem exudates compared to those of plant organs.     

N and C NMR determination of allantoin metabolism in developing soybean cotyledons

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state ¹³C and ¹⁵N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO₂. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO₂. The ¹³C-¹⁵N bonds of [5-¹³C;1-¹⁵N] and [2-¹³C;1,3-¹⁵N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH₄⁺ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH₄⁺ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine