Effect of methionine sulfoximine on asparaginase activity and ammonium levels in pea leaves.

In developing leaves of Pisum sativum the levels of ammonium did not change during the light-dark photoperiod even though asparaginase (EC 3.5.1.1) did; asparaginase activity in detached leaves doubled during the first 2.5 hours in the light. When these leaves were supplied with 1 millimolar methionine sulfoximine (MSX, an inhibitor of glutamine synthetase, GS, activity) at the beginning of the photoperiod, levels of ammonium increased 8-to 10-fold, GS activity was inhibited 95%, and the light-stimulated increase in asparaginase activity was completely prevented, and declined to less than initial levels. When high concentrations of ammonium were supplied to leaves, the light-stimulated increase of asparaginase was partially prevented. However, it was also possible to prevent asparaginase increase, in the absence of ammonium accumulation, by the addition of MSX together with aminooxyacetate (AOA, which inhibits transamination and some other reactions of photorespiratory nitrogen cycling). AOA alone did not prevent light-stimulated asparaginase increase; neither MSX, AOA, or elevated ammonium levels inhibited the activity of asparaginase in vitro. These results suggest that the effect of MSX on asparaginase increase is not due solely to interference with photorespiratory cycling (since AOA also prevents cycling, but has no effect alone), nor to the production of high ammonium concentration or its subsequent effect on photosynthetic mechanisms. MSX must have further inhibitory effects on metabolism. It is concluded that accumulation of ammonium in the presence of MSX may underestimate rates of ammonium turnover, since liberation of ammonium from systems such as asparaginase is reduced by the effects of MSX.

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 (15)N 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 alpha-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.

Ureide metabolism in leaves of nitrogen-fixing soybean plants.

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [(14)C]urea-dependent NH(3) and (14)CO(2) production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH(3) per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH(3) and CO(2) production from the utilization of [2-(14)C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH(3) production. The almost complete sensitivity of NH(3) and CO(2) production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH(3) and two molecules of CO(2).

Diurnal variation of asparaginase in developing pea leaves.

Levels of asparaginase activity from developing pea leaves (Pisum sativum) were found to change on a daily basis, increasing during the light period and decreasing in the dark. During extended periods of light, high levels of activity were maintained, while prolonged dark reduced activity to a low value. Half-expanded leaves exhibited the greatest change in activity over the photoperiod. Very young or mature leaves displayed little or no diurnal variation in asparaginase activity.

Utilization of the amide groups of asparagine and 2-hydroxysuccinamic Acid by young pea leaves.

The fate of nitrogen originating from the amide group of asparagine in young pea leaves (Pisum sativum) has been studied by supplying [(15)N-amide]asparagine and its metabolic product, 2-hydroxysuccinamate (HSA) via the transpiration stream. Amide nitrogen from asparagine accumulated predominantly in the amide group of glutamine and HSA, and to a lesser extent in glutamate and a range of other amino acids. Treatment with 5-diazo,4-oxo-L-norvaline (DONV) a deamidase inhibitor, caused a decrease in transfer of label to glutamine-amide. Virtually no (15)N was detected in HSA of leaves supplied with asparagine and the transaminase inhibitor aminooxyacetate. When [(15)N]HSA was supplied to pea leaves, most of the label was also found in the amide group of glutamine and this transfer was blocked by the addition of methionine sulfoximine, which caused a large increase in NH(3) accumulation. DONV was not specific for asparaginase, and inhibited the deamidation of HSA, causing a decrease in transfer of (15)N into glutamine-amide, NH(3), and other amino acids. It is concluded from these results that use of the amide group of asparagine as a nitrogen source for young pea leaves involves deamidation of both asparagine and its transamination product HSA (possibly also oxosuccinamate). The amide group, released as ammonia, is then reassimilated via the glutamine synthetase/glutamate synthase system.

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 (15)N-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.

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor.

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [(14)C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [(14)C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

Subcellular Localization of Asparaginase and Asparagine Aminotransferase in Pisum sativum Leaves.

Protoplasts isolated from young and mature pea leaves (Pisum sativum L.) were broken and their contents fractionated by differential centrifugation or on sucrose-density gradients. Asparaginase was found only in the cytosol of young leaves. Asparagine aminotransferase was found in both young and mature leaves and was localized exclusively in the peroxisome. This corroborates the observation that asparagine transamination is catalyzed by the serine:glyoxylate aminotransferase.

Purification and properties of an asparagine aminotransferase from Pisum sativum leaves.

The enzyme responsible for the transamination of L-asparagine in pea leaves has been partially purified. It appears to be the same protein as the serine-glyoxylate aminotransferase. It is able to use serine or asparagine as amino donors and pyruvate or glyoxylate as amino acceptors. The reaction is reversible but the equilibrium is toward glycine or alanine production. The favored substrates are serine and glyoxylate: serine shows competitive inhibition toward asparagine, as does pyruvate toward glyoxylate. Substrate interaction and product inhibition patterns are consistent with a ping-pong mechanism. The enzyme has a pH optimum at 8.1. Gel filtration indicates a Mr of 105,000. Inhibition was caused by aminoxyacetate and hydroxylamine, but the enzyme was unaffected by isonicotinic acid hydrazide. The apoenzyme was resolved and was inactive: addition of pyridoxal 5'-phosphate restored 85% of the original activity.

Two routes for asparagine metabolism in Pisum sativum L.

Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K m for asparagine (3 mM) than the leaf asparaginase.

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm.

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK(c), is localized in the cytosol, and the other, PK(p), is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K(+) and Mg(2+). Both isozymes are inhibited by citrate, pyruvate, and ATP. PK(c) has a much broader pH profile than PK(p) and is also more stable. Both have the same K(m) (0.05 millimolar) for PEP, but PK(p) has a 10-fold higher K(m) (0.3 millimolar) for ADP than PK(c) (0.03 millimolar). PK(c) also has a higher affinity for alternate nucleotide substrates than PK(p). The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.

Isozymes of the glycolytic and pentose-phosphate pathways in storage tissues of different oilseeds.

Isozymes of hexose-phosphate isomerase (HPI; EC 5.3.1.9), pyruvate kinase (PK; EC 2.7.1.40) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) have been detected in the developing cotyledons of soybean (Glycine max (L.) Merr.), safflower (Carthamnus tinctorius L.) and sunflower (Helianthus annuus L.). In each seed there are two isozymes each of PK and HPI. The isozyme patterns of 6PGDH are more complex: soybean has two forms of the enzyme, safflower three, and sunflower six. In each tissue, at least 25% of the activity of each of the three enzymes is in the plastids. This supports the proposal that the glycolytic and pentose-phosphate pathways are operating in the plastids and that the plastids are the site of long-chain fatty-acid biosynthesis in developing oilseeds.

Isoenzymes of pyruvate kinase in etioplasts and chloroplasts.

Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity.