Formation of Allantoin and Allantoic Acid from Adenine in Leaves of Acer saccharinum L.

The synthesis of '4C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from 18-'4Ciguanine or 18-'4Clhypoxanthine, but not from 18-4Cladenine, was demonstrated in a cell-free extract from N2-fixing nodules of cowpea (Walp.). The 14C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the 14C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.Ureides are important nitrogenous solutes in the metabolism of a wide range of plant species (4, 8, 16, 22, 28). They are especially prominent in certain tropical legumes, in which they act as major products of N2 fixation (9,14,19), as the principal compounds in which fixed N is transported from nodules to shoots in the xylem (15,20) and as a primary source of N for protein synthesis in shoots of nodulated plants relying on N2 fixation (9).Pathways suggested for ureide synthesis in plants involve either the condensation of 2 molecules urea and I molecule glyoxylate (5,24) or the aerobic oxidation of purines (3,6,22). With the latter pathway ( Fig. 1), deamination of guanine and adenine to xanthine and hypoxanthine, respectively (reactions I and 2), and the subsequent oxidation of hypoxanthine and xanthine (reaction 3) are envisaged as donor reactions to the formation of allantoin and allantoic acid from uric acid. Circumstantial evidence for this pathway in legume nodules comes from the demonstration of activity of enzymes of purine oxidation, namely xanthine dehydrogenase (EC 1.2.1.37), xanthine oxidase (EC 1.2.3.2), uricase (EC 1.7.3.3), and allantoinase (EC 3.5.2.5) (25,26), and in vivo inhibition of ureide synthesis in N-fixing plants by allopurinol (2, 7), a potent inhibitor of both xanthine dehydrogenase and xanthine oxidase.This study reports the direct synthesis of allantoic acid, allantoin, uric acid, and urea from guanine and hypoxanthine in a cell- (29). In all cases, major peaks of radioactivity in the column eluates were found to co-chromatograph with the elution profiles of uric acid and allantoic acid (Fig. 2). Allantoin and urea were recovered in the neutral fraction. Allantoin was converted to allantoic acid by hydrolysis in base and recovered for '4C assay by column chromatography as above.['4C]Urea was converted to "4CO2 with urease (Sigma Type IX) and the '4Co2 was recovered in ethanolamine. The radioactivity in all the above compounds was determined by liquid scintillation counting. It was assumed that all labeled produ...

Section: Introductionmentioning

confidence: 99%

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.]

Woo

Atkins²,

Pate³

1980

“…In contrast to the animal systems which excrete allantoin as a waste material, these plants accumulate it within cells, transport it to various organs, and further reutilize its nitrogen (24,29). Several authors (5,10,17), by experiments using 'C-labeled purines or glycine, have demonstrated that allantoin or allantoic acid is formed through oxidative purine decomposition by a well defined reaction sequence in animals and microorganisms. Mothes (24) and Reinbothe (29) have, however, discussed in their reviews another plausible mode of allantoin synthesis, i.e.…”

mentioning

confidence: 99%

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

Fujihara

Yamaguchi²

1978

104

Some studies on the effects of xanthble oxiase inhibitor a_opurIo 14-hydroxypyrazolo(34-)pyri.idel on alato met of soybean plants (Glychae max cv. T _ ) are reported.Soyben 'as asepticaly germinated for 96 hours on agar containing I _ilhlmlar allopurinol, contained only slight amounts of aUantoin, allantoic acid, and urea as compared with controls. Analysis of puries and pyrimidines of the allopurinol-treated seedlings sbowed marked accumulation of xanthine both in the cotyedons and seedlin axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyedons had fallen. For nodulated plants, allopurinol caused a sigificant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a strking accumulatfon of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated slings. Allopurl at a concentration of 50 micromolar strongly inbibited xasthine oxidase prepared from soybean nodules.The results suggested that the main pathway of alantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.Allantoin is well known as one of the principal end products of nitrogen metabolism in animals and is produced by a biosynthetic sequence involving the formation of inosinic acid and its subsequent stepwise breakdown. Generally a variety of microorganisms, e.g. bacteria (2, 7), yeasts (30), fungi (3), and algae (1) have the ability to decompose oxypurines and consequently allantoin.Some higher plants such as maple, comfrey, and leguminous plants also produce and accumulate large amounts of allantoin, up to half the amount of their total N (24). In contrast to the animal systems which excrete allantoin as a waste material, these plants accumulate it within cells, transport it to various organs, and further reutilize its nitrogen (24,29). Several authors (5,10,17), by experiments using 'C-labeled purines or glycine, have demonstrated that allantoin or allantoic acid is formed through oxidative purine decomposition by a well defined reaction sequence in animals and microorganisms. Mothes (24) and Reinbothe (29) have, however, discussed in their reviews another plausible mode of allantoin synthesis, i.e. direct formation from allantoic acid via condensation of urea and glyoxylic acid. This simple reverse reaction may be supported by the finding that when ['4Clurea was fed to banana leaves allantoic acid was heavily labeled (12), and by a report of Brunel (9) suggesting an enzymic synthesis of allantoic acid from urea and glyoxylic acid in higher fungi. While it is likely that in higher plants most allantoin and allantoic acid are formed by the same pathway as that demonstrated in microorganisms and in animal tissues, the possibility that some might be made by the condensation of urea with glyoxylic acid has not been eliminated.Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidi...

“…Thereafter, the radioactivity accumulates in ureides (allantoin and allantoic acid). Such a degradation of adenine derivatives into ureides has also been reported in leaves of Acer saccharinum (1) and Symphytum officinale (19) and the conversion of hypoxanthine to ureides has been shown in leaves of Symphytum (3) and in shoot tips of tea (21).…”

mentioning

confidence: 88%

Catabolism of Adenine Derivatives in Leaves

Nguyen¹

1980

The in vivo activity of xanthine dehydrogenase (E.C. 1.2.137) was followed in leaf discs excised from illuminated or darkened plants. In cotyledons of Pharbitis nil, 24 hours of darkness enhanced the in vivo activity of xanthine dehydrogenase which increased between 2 to 5-fold depending on the concentration of hypoxanthine of the solution where cotyledon discs were incubated. The same effect occurred in leaves of several other species, in plants with both (2,6,7,23). The formation of ureides from adenylic compounds involves three main steps: first, the deamination of adenine derivatives to hypoxanthine derivatives; second, the oxidation of hypoxanthine to xanthine and uric acid; then, the opening of purine ring leading to the formation of allantoin and allantoic acid (10). Early studies have shown that in cotyledons of Pharbitis neither the deaminative rate of adenine derivatives nor the degradative rate of uric acid increased in darkness, whereas, the oxidation of hypoxanthine is sharply higher in darkness than in light (16). The oxidation of hypoxanthine represents the first irreversible reaction leading to the catabolic pathway of purine derivatives. In leaves, it is achieved by a soluble molybdoflavoprotein, a NAD(P)+-xanthine dehydrogenase (E.C. 1.2.1.37) (18). Time course for the changes of the in vivo activity of xanthine dehydrogenase was previously determined (14): in darkness, the increase of activity begins after an 8-h lag period and reaches a plateau after 30 h. At this time, the transfer of plants in light causes a rapid decrease in the in vivo activity of xanthine dehydrogenase within the first 8 h of light.This paper reports that light affects the in vivo activity of xanthine dehydrogenase in leaves of various species. Because the present results show that the effect of light on the in vivo activity of xanthine dehydrogenase did not occur either in roots or in Chldeficient leaves, the level of the in vivo activity of xanthine dehydrogenase was dependent on light energy, and the in vivo activity of xanthine dehydrogenase could be decreased by supplying carbohydrates to leaves, it is thought that the role of light on the in vivo activity of leaf xanthine dehydrogenase is mediated through photosynthetic activity. Among the ways by which photosynthesis can affect the in vivo activity of xanthine dehydrogenase, the present data suggest strongly that light acts mainly by changing the osmotic conditions in leaves. MATERIALS AND METHODSPlant Material. Pharbitis seeds were prepared as described (15). They were sown in Vermiculite, and grown in Phytotron at Gifsur-Yvette with 16 h light per day with combined fluorescent and incandescent light, the photon flux density being 250,uE m-2s-2 at a constant temperature of 27 C and 70%o humidity. The