Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

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ABSTRACT'5NH4+ and I'5N1(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 '5N content (micrograms excess 15N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. 1"NH4+ was also appreciably assimilated into alanine.When 115NI(amide)-glutamine was fed to detached nodules, 15N was almost entirely in the amide nitrogen of asparagine. The result indicates the presence of active glutamine-linked asparagine synthesizing systems in soybean root nodules.The results from numerous in vivo studies with metabolic inhibitors (14) and from in vitro enzymic analyses (9-11, 14) have established glutamine-linked asparagine synthesis in higher plants, although the pathway providing the carbon skeleton to asparagine remains open (I 1). In most legumes so far considered, asparagine is the principal assimilation product of symbiotic nitrogen fixation (7,8,10,16). Scott et al. (10) found glutamine-linked asparagine synthetase in the cytosol fraction of lupin nodules and proposed a sequence for asparagine formation from atmospheric nitrogen fixed by nodule bacteroids. Bleeding sap of soybean nodules also contains high concentrations of asparagine (16), indicating the presence of active asparagine synthesizing and transporting systems in soybean nodules. Extensive attempts by Streeter (12) to demonstrate asparagine biosynthesis from various radioactive precursors have, however, failed, possibly because of the presence of a very active asparaginase in soybean nodules (12). We observed that soybean nodule bacteroids had a high ability to transform asparagine to aspartate (unpublished data). The existence of such an active asparagine-decomposing system in nodules makes in vitro enzymic or tracer studies on asparagine synthesis difficult. In the present in vivo tracer study, asparagine formation in soybean nodules was examined especially in regard to the source of the amide nitrogen of asparagine.The data from isotopic analysis in this communication clearly Ar, and incubated at 30 C for I h. Reaction was terminated by the addition of 2 ml cold 0.4 N HC104 to each vial. The contents of the vials were immediately transferred into a cold mortar, ground with a pestle, washed into a centrifuge tube, and centrifuged at 15,000g for 30 min at 0 C. The acid precipitate was washed in cold 0.2 N HC104, centrifuged again, and the combined supernatant fluid was neutralized to pH 6.5. The cleared solution was used for the fractionation of each nitrogen constituent.Separation of Nitrogen Compounds and Conversion to Ammonia. The supernatant solution was shaken vigorously with I o (w/ v) of Permutit for several min and centrifuged at l0,OOOg for 10 min. The ammonia free supernata...

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confidence: 95%

Asparagine Formation in Soybean Nodules

Fujihara¹,

Yamaguchi²

1980

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“…Enzymic and inhibitor studies (2,13,26) provided circumstantial evidence that ureide synthesis in these nodules might involve a purine-based pathway (3,10). 02 is required in this pathway for the oxidation of uric acid to allantoin by uricase (EC 1.7.3.3), from either soybean nodules (26) or animal tissue (4,11), which is stimulated by 100% 02 compared to the control treatments in air.…”

Section: Methodsmentioning

confidence: 99%

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules

Woo¹,

Atkins²,

Pate³

1981

Tbe effect of°2 and pH on the in vitro synthesis of IC-labeled ureides from IS-Clhypoxanthine in a cell free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.733) activty, namely low 02 concentrations and low pH, ureide synthesis was inhibited and the 'C label incorporated into uric acid was increased. Conversely, conditos which increased uricase actinty, namelY high 02 concentrations and high pH, also stimulated ureide synthesis, and the 'C label was incorporated principaly into allantoin. The overal response of the system to 02 concentration and pH indicated that the per cent distribution of total 'C label incorporated into uric acid was inversely related to that into alantoi In the present study there was evidence that uricase (EC 1.733) controfled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.137) and as a consequence ureide synthesis from 18_14Clhypoxan-thine was also inhibited. MATERIALS AND METHODSAssays for ureide synthesis in the cytosolic fraction from cowpea nodules and the separation and identification of metabolic intermediates were carried out as previously described (33).Xanthine dehydrogenase (EC 1.2

“…Thus, azaserine is an inhibitor of de novo purine synthesis while allopurinol blocks purine degradation. Fujihara and Yamaguchi (15) found no difference in the amount of ureides in the roots of 3-d-old soybeans after treatment with azaserine. They concluded that de novo synthesis of purines is not involved in ureide production in young seedlings.…”

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confidence: 99%

“…Plants grown on N-free nutrient solution were harvested as described for azaserine treatment. Four seedlings were placed in flasks containing buffer A with or without 0.5 mm allopurinol (15). The base of the flasks were wrapped in aluminum foil.…”

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confidence: 99%

“…Plants grown on Nfree nutrient solution were harvested at 3, 8, and 12 d after imbibition. Seedlings were washed with dH20 and placed in 125-ml Erlenmeyer flasks (4 seedlings per flask) containing 10 mM K-phosphate buffer, pH 7.0 (buffer A) with or without 0.5 mM azaserine (15). The flasks were entirely covered with aluminum foil and placed on a rotary shaker (28C) in the dark for 24 h. After the incubation, seedlings were washed with dH20 and separated into shoots and leaves, roots, and cotyledons.…”

mentioning

confidence: 99%

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Purine Synthesis and Catabolism in Soybean Seedlings

Polayes¹,

Schubert²

1984

ABSTRACrThe ureides, allantoin and allantoic acid, are the major nitoge us substances transported within the xylem of Nrfixing soybeans (Glycine max L. Meff. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobiwm or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4day-old seedlings with 4qClserine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3-and 4day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with 1'4Ciserine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings. tration may result from (a) de novo synthesis of purines with subsequent metabolism to ureides, or (b) the degradation of preexisting nucleotides to form ureides. In either case the activity ofboth uricase and allantoinase, two enzymes involved in ureide synthesis via purine degradation, should be observed. The site of ureide synthesis can be determined by examining the patterns of activity of uricase and allantoinase.The pathway or ureide synthesis can be investigated by using the inhibitors azaserine and allopurinol. Azaserine blocks the transfer of the amide-N of glutamine to formylglycinamide ribonucleotide (30). Allopurinol inhibits the enzyme xanthine dehydrogenase (8). Thus, azaserine is an inhibitor of de novo purine synthesis while allopurinol blocks purine degradation. Fujihara and Yamaguchi (15) found no difference in the amount of ureides in the roots of 3-d-old soybeans after treatment with azaserine. They concluded that de novo synthesis of purines is not involved in ureide production in young seedlings. However, no data were presented for the effects of azaserine on the levels of ureides in the cotyledons.In this paper we examine the pattern of ureide accumulation in the developing soybean seedling. The role of de novo synthesis ofpurines in the production of ureides is addressed by examining the effect of inhibitors of purine metabolism on ureide synthesis and by measuring the incorporation of label from ['4C]serine into purines, allantoin and allantoic acid.The ureides, allantoin and allantoic acid, are the major xylary nitrogenous substances of soybeans grown in association with the bacterium, Rhizobium japonicum (17,20,25). The presence of ureides in the xylem stream has been linked to ...