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

Woo, K. C.; Atkins, Craig A.; Pate, John S.

doi:10.1104/pp.67.6.1156

Cited by 18 publications

(4 citation statements)

References 14 publications

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“…Studies with cell-free extracts of cowpea and soybean have demonstrated that these compounds are formed from the oxidation of purine bases and nucleotides (1,4,17,19,20) derived by a pathway of de novo purine synthesis which utilizes products of N2 fixation (5). Although IMP has been detected as the initial nucleotide product of de novo purine synthesis in cowpea nodules (5), it is not clear whether IMP is metabolized principally through inosine and hypoxanthine or through oxidation to XMP2 (Fig.…”

Section: Methodsmentioning

confidence: 99%

Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Shelp¹,

Atkins²

1983

Plant Physiol.

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Cell-free extracts from nodules of cowpea ( Vigna unguiculata L. (Walp.) cv Caloona:Rhizobium strain CB756) prepared in the presence of 15% (v/ v) glycerol showed high rates (30 to 60 nanomoles NAD reduced per minute per gram fresh weight nodule) of inosine monophosphate oxidoreductase (EC 1.2.1.14) activity. The enzyme was labile (half-life of activity less than 3 hours) but could be stabilized for up to 18 hours by inclusion of the substrates NAD and inosine monophosphate in the breaking media. Activity showed a broad pH optimum between 8.5 and 9.5, had an apparent K. (inosine monophosphate) of 4 and 12 micromolar at pH 7.5 and 9.0, respectively, and was largely (96%) associated with the plant ceil cytosol fraction of the nodule.Metabolism of 18-14Clinosine monophosphate and il-'4Ciglycine by the cell-free system showed two pathways for purine base production from inosine monophosphate, one via xanthosine monophosphate, xanthosine, and xanthine, the other via inosine and hypoxanthine. The proportion of inosine monophosphate utilized by inosine monophosphate oxidoreductase and the xanthine-based pathway was increased from 30% at 0.5 millimolar to 80% at 0.01 millimolar inosine monophosphate. The data are interpreted to indicate that in vivo inosine monophosphate oxidation rather than dephosphorylation is the predominant metabolic route leading to ureide synthesis and that inosine monophosphate provides the link between de novo purine nucleotide synthesis in the plastid and ureide production in the plant cell cytosol. doreductase (EC 1.2.1.37) in crude (4) or highly purified preparations (6) of nodules. Application of the inhibitor to intact plants results in xanthine accumulation in the nodules (4,8,10) consistent with metabolism through IMP oxidoreductase (EC 1.2.1.14) in vivo. In cell-free extracts, labeled hypoxanthine rather than xanthine accumulates from (1) Freshly harvested nodules from 4-to 6-week-old plants were washed with deionized H20 and crushed in a chilled mortar and pestle with I to 2 volumes of 50 mm Hepes-KOH buffer (pH 7.5), containing 25 mm KCI, 5 mM MgCl2, 1% (w/v) PVP, 15% (w/v) glycerol, 10 mm ME, 5 nms DTT, 2 mm GSH, and 0.25 M sucrose.

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Section: Methodsmentioning

confidence: 99%

Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Shelp¹,

Atkins²

1983

Plant Physiol.

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“…Since the enzymes of de novo purine synthesis and uricase were found in both cell types, Shelp et al (21) concluded that both cell types were capable of ureide synthesis. However, Shelp et al (21) proposed that uricase may be active only in the uninfected cells in vivo owing to the 02 requirement for the uricase reaction (30) and the presence of the 02-binding protein, Lb2, in the infected cells (22).…”

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confidence: 99%

Intercellular Nodule Localization and Nodule Specificity of Xanthine Dehydrogenase in Soybean

Triplett¹

1985

Plant Physiol.

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ABSTIRACIThe distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellulr localizaton of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies againt purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slihtly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test. Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by aflopurinol and was observed only in the infected nodule cells ofthese species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.The ureides, allantoin and allantoic acid, are the predominant forms of nitrogen transported from nitrogen-fixing soybean nodules to other plant parts (15,23). Synthesis of ureides occurs in the plant cells of nodules via de novo purine synthesis followed by purine oxidation (19). The intracellular and intercellular localizations of the enzymes responsible for ureide synthesis has received significant attention recently (6,10,21 with a Beckman DU-7U spectrophotometer. Purification of XDH from field nodules proved to be more difficult than the procedure used for greenhouse nodules (25). All steps were performed at 4°C. The buffer used throughout the purification was 10 mm K-phosphate (pH 7.8) with I mM DTE. Field nodules (200 g) were ground in a Waring Blendor with 500 ml buffer and 50 g insoluble PVP. The extract was then centrifuged at l0,OOOg for 20 min to remove debris, bacteroids, and intact organelles. The supernatant, which is referred to as the crude extract, was brought to 30% saturation with solid (NH4)2SO4. After stirring for 20 min, the sample was centrifuged at 10,OOOg for 20 min. The supernatant was brought to 45% saturation with solid (NH4)2SO4. After stirring and centrifugation, the pellet was resuspended in 30 ml of 20% (NH4)2SO4. The resuspended pellet was then applied to a 50-ml octyl-Sepharose column equilibrated with 20% (NH4)2SO4 in buffer. A 400-ml gradient of 20 to 0% (NH4)2SO4 in buffer was applied to the column. XDH was eluted with 100 ml of the above buffer without (NH4)2SO4. Active fractions were pooled and applied to a 50-ml DEAE-Sephadex column equilibrated with buffer. A 400-ml gradient of 0 to 400 mM...

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“…The catabolic route to ureide biogenesis is supported by the following observations: 1. high levels of xanthine (XAN) dehydrogenase, uricase, and allantoinase activity are present in nodules of cowpea (2, 5, 7), soybean (40, 41, 115, 135), and garden bean (102,105,136,137); 2. the levels of these enzymes are normally low in nodules of amide-exporting legumes such as lupin (102,105) and pea (28); 3. the levels of these enzymes increase in response to the onset of N2 fixation, NH4 + assimilatory activity, and ureide export within the nodule (2,5,7,105,115); and 4. the addition of allopurinol, an irreversible inhibitor of XAN dehydrogenase, results in a decrease in the levels of ureides in nodules and ureide export in the xylem and an increase in XAN in the nodule (2,5,40,142). The ability of cell-free extracts of nodules to convert intermediates such as IMP, XMP, XAN, and hypoxanthine into ureides and for allopurinol to block this conversion (142,152,153) substantiate the competency of nodules to form ureides via purine oxidation.…”

Section: Biogenesis Of Allantoin and Allantoic Acidmentioning

confidence: 84%

“…Label from e4C]GL Y, a purine precursor, was incorporated into ureides (152,153) in cell-free extracts of cowpea nodules. These results are consistent with the role of de novo purine synthesis but do not discount a condensation mechanism of synthesis.…”

Section: Biogenesis Of Allantoin and Allantoic Acidmentioning

confidence: 99%

Products of Biological Nitrogen Fixation in Higher Plants: Synthesis, Transport, and Metabolism

Schubert¹

1986

Annu. Rev. Plant. Physiol.

355

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

Cited by 18 publications

References 14 publications

Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Intercellular Nodule Localization and Nodule Specificity of Xanthine Dehydrogenase in Soybean

Products of Biological Nitrogen Fixation in Higher Plants: Synthesis, Transport, and Metabolism

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