Planta 9 Springer-Verlag 1981
Planta I51, 289 292 (1981)
Two Routes for Asparagine Metabolism in Pisum sativum L. Robert J. Ireland and Kenneth W. Joy Department of Biology and Institute of Biochemistry, Carleton University, Ottawa, Ont. K1S 5B6,
Abstract. Asparagine, a major transport compound, is metabolized in P i s u m s a t i v u m by two enzymes, asparaginase (EC 220.127.116.11) 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 Km for asparagine (4.5raM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower Km for asparagine (3 mM) than the leaf asparaginase. Key words: Aminotransferase - Asparaginase paragine Nitrogen metabolism - Pisum.
In legumes, asparagine is the predominant nitrogen transport compound (see reviews by Pate 1973 ; Miflin and Lea 1977). The storage proteins of lupin seeds contain only 7 10% asparagine, and 15N-labelling studies have shown that most of the asparagine is rapidly metabolized to other amino acids prior to protein synthesis (Atkins et al. 1975). In spite of the common occurrence of asparagine, its path of utilization is not yet clear. Two main routes for asparagine metabolism have been suggested (see review by Lea and Fowden 1975). The enzyme which has been most
studied is asparaginase (EC 18.104.22.168) which yields aspartate and ammonia. The second route involves transamination of the a-amino nitrogen of asparagine by an aminotransferase. Another mechanism has also been suggested, involving asparagine in a reaction similar to the G O G A T system, but a number of workers have been unable to demonstrate the presence of this enzyme (see Miflin and Lea 1977). Examination of developing pea seeds showed the presence of a potassium-dependent asparaginase, but no asparagine aminotransferase was detected (Sodek et al. 1980). Asparagine aminotransferase activity is present in soybean leaves (Streeter 1977). The conversion of 2-ketosuccinamate (the product of transamination of asparagine) to 2-hydroxysuccinamate, and the accumulation of the latter compound have been demonstrated in peas and soybeans (Lloyd and Joy 1978); Bauer et al. (1977 a) showed that alanine nitrogen could be derived directly from asparagine, presumably by transamination. These results indicate that further studies of asparagine metabolism are necessary to clarify the roles played by various enzymes in asparagine utilization, since there are no reports surveying the various pathways in all regions of the same plant. In particular it is of interest to compare the two main sites of nitrogen utilisation, the growing leaves and the developing seeds. The results presented in this paper show that P i s u m s a t i v u m contains both asparaginase and asparagine-pyruvate aminotransferase, but that the contribution made by each enzyme varies in different parts of the plant. Material and Methods Plant Material. Pisum sativum L. (cv. Little Marvel; McKenzie,
Steele, Briggs Seeds, Brandon, Man., Canada) were surface-sterilized, germinatedin vermiculiteand grown in containers with Hoagland-type nutrient solution as described previously (Bauer et al. 1977b). Three-week-oldplants, with five leaves, were used for de-
290 termination of leaf enzymes. Pod development occurred when plants were 4-7 weeks old.
To extract aminotransferase, pea tissue (leaves, seeds or pods) was homogenized for 30 s in four volumes 5 0 m M tris-(hydroxymethyl)-aminomethane (Tris)-HCi buffer (pH 8.2) containing 0.1 m M ethylenediaminetetraacetic acid (EDTA) and 2 m M dithiothreitol, using a Polytron homogenizer (Brinkmann, Rexdale, Ont., Canada), setting 7. Pea roots were ground in the same medium using a mortar and pestle. The extracts were filtered through four layers of cheesecloth and centrifuged at 20,000 g for 20 rain. Polyethylene glycol 6,000 (BDH, Toronto, Canada) (50%, w/w, in 50 mM Tris-HC1, pH 8.2) was added dropwise to the supernatant to give a final concentration of 18% (w/w). After centrifugation at 20,000 g for 20 min, the pellets were resuspended in homogenisation buffer and assayed. For asparaginase extraction, the same procedure was used, except that 50 mM KC1 was included in the buffers, and 55% ammonium sulfate instead of polyethylene glycol was used for precipitation.
Enzyme Assays. Assay mixtures for asparagine-pyruvate aminotransferase contained enzyme, 20 m M L-asparagine, 20 m M sodium pyruvate and 50 m M Tris-HC1 buffer, pH 8.2, in a total volume of 1.5 ml. Asparaginase assay mixtures contained 30 m M KC1, but no pyruvate. Asparagine solutions were passed through ionexchange resin Dowex 1 (Sigma Chemical Corp., St. Louis, Mo., USA), formate form, at pH 6.5 to remove contaminating aspartic acid. The mixtures were incubated at 30 ~ C; 0.5-ml samples were removed at 0 and 20 rain and mixed with 25 mg 5-sulfosalicyclic acid, which precipitated proteins and stopped the reaction. The samples were centrifuged at 12,000 g for 4 min and the pH of the supernatant adjusted to 2 with NaOH. The production of alanine or aspartate in the samples was analysed with a Beckman 119BL amino-acid analyzer (Beckman Instruments, Palo Alto, Cal., USA) using the "hydrolysate" mode (sodium citrate buffers). Asparagine aminotransferase activity is expressed as nmol alanine produced.min -1 at 30 ~ C. Asparaginase activity is expressed as nmol aspartate produced, min- 1 at 30 ~ C. Asparagine-pyruvate aminotransferase activity was also determined in partially purified preparations by measuring production of 2-ketosuccinamic acid. The assay mixture was as above, but reactions were stopped by removing samples into 0.1 M NaOH. At alkaline pH, 2-ketosuccinamic acid has an absorption maximum at 289 nm (Cooper 1977); increase in A289 was linear for at least 1 h and the rate was the same as that for alanine production. This method was inaccurate when used with crude preparations.
Partial Purification of Asparagine-Pyruvate Aminotransferase. Pea leaf tissue was homogenized, filtered and centrifuged as described above. The pH of the supernatant was adjusted to 5.4 using 0.1 M HC1. After centrifugation at 20,000 g for 10 rain, the pH of the supernatant was adjusted to 8.2 using 0.1 M NaOH and the supernatant was brought to 30% saturation with ammonium sulfate. After centrifugation at 20,000 g for 10 rain, the supernatant was brought to 55% saturation ammonium sulfate and centrifuged again. The pellet was resuspended in 20 ml homogenisation buffer, then adjusted to 9 % (w/w) polyethylene glycol 6,000. After centrifugation, the polyethylene-glycol concentration of the supernatant was increased to 18% and the suspension centrifuged again. The pellet was resuspended in 5 ml homogenisation buffer and subjected to ion-filtration chromatography on a column (25 cm long, 2.3 cm diameter) of DEAE-Sephadex (Pharmacia, Montreal, Canada). The enzyme .was eluted with homogenisation buffer containing 0.5 M KC1, and active fractions were detected by assaying for production of 2-ketosuccinamic acid.
R.J. Ireland and K.W. Joy: Asparagine Metabolism in Pisum sativum Results
As reported by Sodek et al. (1980), pea asparaginase required the presence of K + ions for stability during isolation and for maximal activity in the assay. In the absence of K +, asparaginase activity was virtually undetectable in polyethylene glycol precipitates from extracts of leaves and pods; traces of activity were still detectable in similar extracts from whole seeds, which contain extremely high levels of the enzyme. It was found that ammonium ions could partially substitute for K § in activating the asparaginase. Thus an enzyme, prepared with no added K § and redissolved after precipitation by 55% saturated ammonium sulfate (approx. 2.5 M) still had about 40% of the activity of a similar sample prepared in the presence of K § Low concentrations of ammonium ions appear to be ineffective (Sodek et al. 1980). As aspartate can transaminate with pyruvate to produce alanine it was necessary to remove contaminating aspartate from stock solutions of asparagine used in the assay of transaminase, and also to eliminate asparaginase from these assays. For this reason polyethylene-glycol precipitation was used for the aminotransferase, since ammonium-sulfate precipitation effected partial recovery of asparaginase, even in the absence of K § The high level of asparaginase present in developing pea seeds (Table 1) is similar to that reported previously. The value presented in Table 1 for 14-dayold seeds is equivalent to 5 . 8 g m o l . h - l . s e e d -1, about 60% higher than that reported by Sodek et al. (1980) for seeds at a similar stage of development. We also confirm that the seed has hardly any asparagine-pyruvate aminotransferase activity (Table 1), although this enzyme is present in the pod tissue which Table 1. Distribution of asparaginase and asparagine-pyruvate aminotransferase in different tissues of Pisum sativum. Trace (tr) indicates less than 1 nmol. rain- 1. g i fresh weight. The asparagine-dependent production of alanine (from pyruvate) was used to measure aminotransferase Activity (nmol.min i . g lfr.wt. ) AsparAsparagineaginase pyruvate aminotransferase Leaf a (fully-expanded) Pod (i0 d after flowering) Whole 6d I0 d 14 d
seed (cotyledon + testa) after flowering after flowering after flowering
Roots See also Fig. I
32 139 494 2.2
tr tr tr tr
R.J. Ireland and K.W. Joy: Asparagine Metabolism in Pisum satirum
k , ASNase "~ 80
Fr, Wt . . . . . . . . . . .
291 Table 2. Effect of asparagine and inhibitors on levelsof some amino acids in growing pea leaves. Detached, 3-week-oldpea shoots were supplied through the xylem with combinations of asparagine (8 mM), methionine sulfoximine (0.8 mM) and sodium aminooxyacetate (4 mM). Solutions were adjusted to pH 6.4. After 2 h in light, young (half-expanded) leaves were ground in sulfosalicylicacid, 50 mg/ml. Amino-acid and ammonia levels were determined using a Beckman 119BL amino acid analyser in the "physiological" mode, using lithium-citrate buffers. "Untreated" represents levels at the beginning of the experiment Asp
Jamol.g 1 fresh weight Asn Glu Gln NH3
Leaf age (days after emergence From stipule)
Fig. 1. Activities of asparaginase () and asparagine-pyruvate aminotransferase (e) during growth of the fifth leaf of pea seedlings. Arrow indicates approximate time of full expansion. The asparagine-dependent production of alanine (from pyruvate) was used to measure aminotransferase
has lower levels of asparaginase. Substantial amounts of the aminotransferase were detected in leaves (Fig. 1), with similar levels in growing and mature leaves. In contrast, asparaginase showed a considerable change during leaf development; activity was high in the apical bud, but decreased rapidly in the expanding leaf (Fig. 1). R o o t tissue contains low levels of asparaginase activity and only trace amounts of the aminotransferase. Asparaginase prepared from seed tissue by ammonium-sulfate precipitation showed Michaelis-Menten type kinetics and had a K,, for asparagine of 3.0 mM, similar to the values of 3.2 m M for pea cotyledon and 3.7 m M for pea testa reported previously (Sodek et al. 1980). Asparaginase extracted from leaf tissue under the same conditions had a higher Km for asparagine, 8.0 raM. The partial purification of asparaginase-pyruvate aminotransferase achieved by precipitation and ionfiltration c h r o m a t o g r a p h y resulted in preparations containing no detectable asparaginase or aspartatepyruvate aminotransferase. The asparagine aminotransferase prepared from leaves by this procedure also showed Michaelis-Menten type kinetics, with a Km of 4.5 m M for asparagine, and 2.5 m M for pyruvate. Excised pea shoots were supplied with asparagine (8 raM) which was taken up through the xylem. The effect of asparagine and various inhibitors on the amino-acid composition of young leaves was determined (Table 2). When detached shoots were supplied for 2 h with water only, the anaino-acid composition of the leaves was similar to that of the untreated
Untreated +Ash + Asn+ methionine sulfoximine + Asn+ amino-oxyacetate +Asn+methionine sulfoximine + aminooxyacetate
5.04 4.83 2.80
8.12 6.93 2.24 1.54 12.32 4.41 5.25 1.47 13.37 3.08 0.28 19.74
12.53 8.68 3.36 10.85 11.41 0.91
tissue, with only slight decreases in levels of the amides (data not shown). When asparagine was provided, glutamine levels more than doubled over the 2-h period, and continued to increase to give four to five times higher levels after 4 h (not shown) ; there was an accompanying decrease in the level of glutamate. Glutamine synthesis could result from a direct transfer of nitrogen from asparagine, or by assimilation of a m m o n i a produced by deamidation. The latter interpretation is borne out by the data obtained in the presence of methionine sulfoximine, an inhibitor of glutamine synthetase: glutamine synthesis was sharply reduced, and a m m o n i a accumulated. Addition of aminooxyacetate, an aminotransferase inhibitor, decreased the glutamine accumulation and also inhibited the methionine sulfoximine-dependent ammonia accumulation (Table 2). In-vivo experiments show that aminooxyacetate has only a slight inhibitory effect on asparaginase, thus much of the supply of a m m o n i a must occur through an aminotransferasedependent deamidation step, rather than from asparaginase activity.
Discussion It is likely that asparagine metabolism in P. s a t i v u m can proceed by two distinct routes, and the enzyme distribution data indicate, that the predominant route may differ in different tissues. This explains why conflicting emphasis on alternate pathways has been given by different investigators. The very high levels of asparaginase in the seed indicate that this enzyme will be responsible for utilisation of virtually all o f
292 the asparagine, since the transaminase is present only in trace amounts. The products will be aspartate and ammonia. Aspartate will transaminate readily, but ammonia will require reassimilation, possibly by glutamine synthetase, although the level of this enzyme in pea cotyledon is low (Storey and Beevers 1978). A highly active glutamate dehydrogenase, effective at low ammonia concentrations, has been reported for pea seed (Pahlich and Gerlitz 1980) and may therefore be involved in a m m o n i a assimilation. The pea pod also plays an important role in processing nitrogen which ultimately enters the Seed (Storey and Beevers 1978) but asparaginase appears to be much less important in this tissue. Although substantial amounts of asparaginase are present in the youngest leaves, several lines of evidence indicate that the enzyme plays a minor role in this tissue. As shown here, the enzyme activity is falling rapidly when the leaf is entering its most rapid phase of growth and protein synthesis. Labelling studies with 15N (Bauer et al. 1977a) show that in the young pea leaf asparagine nitrogen appears most rapidly in alanine, presumably by transamination, and much smaller amounts are found in aspartate and ammonia, the products of asparaginase activity. Feeding experiments with shoots (Table 2) show that although supply of higher levels of asparagine results in ammonia production (immediately assimilated by glutamine synthetase), much of this ammonia results from aminotransferase-linked deamidation and not from asparaginase activity. This is also consistent with lr studies (Lloyd and Joy 1978) in which labelled carbon from asparagine accumulated predominantly in 2-hydroxysuccinamic acid, the reduced form of the transamination product 2-ketosuccinamic acid. It is not clear whether the keto- or the hydroxysuccinamate is the prime deamidation substrate. Streeter (1977) has shown the presence of a ketosuccinamate deamidase in soybean. We have detected both keto- and hydroxysuccinamate deamidase activities in crude extracts of pea leaves. Deamidation does not appear to be the major route for ketosuccinamate metabolism, since feeding experiments show that a high proportion is rapidly converted to hydroxysuccinamate (Lloyd and Joy 1978). Little is known of the subsequent pathways for metabolism of hydroxysuccinamate. In the light it accumulates wlth relatively little conversion in feeding experiments, but in the dark there must be some cleavage of the nitrogen group since much of the metabolised carbon appears in car-
R.J. Ireland and K.W. Joy: Asparagine Metabolism in Pisum sativurn bon dioxide. Nothing is known of turnover or diurnal fluctuations of this compound. It is clear that factors other than total measurable enzyme activity play a role in determining metabolic flow of asparagine through competing paths. Indeed, for mature leaves, although both potential enzyme systems are present, access of asparagine to either system is quite limited since a large proportion of the asparagine entering the leaf in the transpiration stream is re-exported without metabolism (Urquhart 1980). Factors such as kinetic properties of the enzymes, regulatory mechanisms and compartmentation may all play a role in controlling accessability of asparagine to the appropriate enzymes. The work was supported by a Natural Science and Engineering Research Council (Canada) grant. We are grateful to C. Shay for help with amino acid analysis.
References Atkins, C.A., Pate, J.S., Sharkey, P.J. (1975) Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds. Plant Physiol. 56, 807-812 Bauer, A., Joy, K.W., Urquhart, A.A. (1977a) Amino acid metabolism of pea leaves. Labeling studies on utilization of amides. Plant Physiol. 59, 920-924 Bauer, A., Urquhart, A.A., Joy, K.W. (1977b) Amino acid metabolism of pea 1eaves. Diurnal changes and amino acid synthesis from lSN-nitrate. Plant Physiol. 59, 915 919 Cooper, A.J.L. (1977) Asparagine transaminase from rat liver. J. Biol. Chem. 252, 2032-2038 Lea, P.J., Fowden, L. (1975) Asparagine metabolism in higher plants. Biochem. Physiol. Pflanzen 168, 3 14 Lloyd, N.D.H., Joy, K.W. (I978) 2-Hydroxysuccinamicacid: a product of asparagine metabolism in plants. Biochem. Biophys. Res. Commun. 81, 186-192 Miflin, B.J., Pea, P.J. (1977) Amino acid metabolism. Annu. Rev. Plant Physiol. 28, 299-329 Pahlich, E., Gerlitz, C. (1980) Derivations from Michaelis-Menten behaviour of plant glutamate dehydrogenase with ammonium as variable substrate. Phytochemistry 19, 11-13 Pate, J.S. (1973) Uptake, assimilation and transport of nitrogen compounds by plants. Soil Biol. Biochem. 5, 109-119 Sodek, L., Lea, P.J., Miflin, B.J. (1980) Distribution and properties of a potassium-dependent asparaginase isolated from developing seeds of Pisum sativum and other plants. Plant Physiol. 65, 2~26 Storey, R., Beevers, L. (1978) Enzymology of glutamine metabolism related to senescence and seed development in the pea (Pisum sativum L.). Plant Physiol. 61, 494-500 StreeteL J.G. (1977) Asparaginase and asparagine transaminase in soybean leaves and root nodules. Plant Physiol. 60, 235-239 Urquhart, A.A. (1980) Transport, metabolism and redistribution of xylem-borneamino acids in pea seedlings. Ph.D. thesis, Carleton University, Ottawa, Canada Received 21 September; accepted 19 November 1980