Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released ¹⁴CO₂ from [ureido-¹⁴C]ureidoglycolate under conditions in which urease is not detectable. CO₂ and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K_m of 85 micromolar for ureidoglycolate. Glyoxylate and CO₂ are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO₂ and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2′-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [¹⁴C]urea production from [2,7-¹⁴C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of ¹⁴CO₂ release from [2,7-¹⁴C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of ¹⁴CO₂ is not dependent on the accumulation of free intermediates. That [2,7-¹⁴C]allantoate dependent ¹⁴CO₂ release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO₂ without a urea intermediate.     

Ureide metabolism in leaves of nitrogen-fixing soybean plants

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [¹⁴C]urea-dependent NH₃ and ¹⁴CO₂ production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH₃ per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH₃ and CO₂ production from the utilization of [2-¹⁴C] 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 NH₃ production. The almost complete sensitivity of NH₃ and CO₂ 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 NH₃ and two molecules of CO₂.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

Enzymic degradation of allantoate in developing soybeans

A Mn²⁺-dependent enzymic breakdown of allantoate has been detected in crude and partially purified extracts of developing soybeans. The products detected were CO₂, NH₃, glyoxylate, labile glyoxylate derivatives, and low levels of urea. Urea is initially produced at less than 10% the rate of urease-independent CO₂ release indicating that the activity is not allantoate amidinohydrolase (i.e. urea is not directly cleaved off allantoate). The urease-independent CO₂ releasing activity has an apparent K_m of 1.0 millimolar for allantoate. Ethylenediaminetetraacetate, borate, and acetohydroxamate (all at 10 millimolar) inhibit the enzymic production of NH₃, CO₂, and labile glyoxylate derivatives from allantoate. However, the potent urease inhibitor, phenyl phosphordiamidate does not inhibit CO₂ and NH₃ release indicating that the action of acetohydroxamate is not due to its inhibition of urease. That the allantoatedegrading activity was more than 5-fold greater in seed coats than in embryos is consistent with the data of Rainbird et al. (Plant Physiol 1984 74: 329-334) which indicate that available ureides are metabolized before reaching the embryo. 2-Ethanolthio, 2′ureido, acetic acid (NH₂COHNCHCO₂HSCH₂CH₂OH), the first allantoate-derived product detected by HPLC analysis, is an addition produced of mercaptoethanol with an unidentified enzymically produced ureido intermediate that is not derived from ureidoglycolate or oxalurate.     

Inhibition of glycine decarboxylation and serine formation in tobacco by glycine hydroxamate and its effect on photorespiratory carbon flow

Glycine hydroxamate is a competitive inhibitor of glycine decarboxylation and serine formation (referred to as glycine decarboxylase activity) in particulate preparations obtained from both callus and leaf tissue of tobacco. In preparations from tobacco callus tissues, the K_i for glycine hydroxamate was 0.24 ± 0.03 millimolar and the K_m for glycine was 5.0 ± 0.5 millimolar. The inhibitor was chemically stable during assays of glycine decarboxylase activity, but reacted strongly when incubated with glyoxylate. Glycine hydroxamate blocked the conversion of glycine to serine and CO₂in vivo when callus tissue incorporated and metabolized [1-¹⁴C]glycine, [1-¹⁴C]glycolate, or [1-¹⁴C]glyoxylate. The hydroxamate had no effect on glyoxylate aminotransferase activities in vivo, and the nonenzymic reaction between glycine hydroxamate and glyoxylate did not affect the flow of carbon in the glycolate pathway in vivo. Glycine hydroxamate is the first known reversible inhibitor of the photorespiratory conversion of glycine to serine and CO₂.     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

The effect of temperature on glycollate decarboxylation in leaf peroxisomes

[1-¹⁴C]glycollate was oxidised to¹⁴CO₂ by peroxisomes isolated from leaves of spinach beet about 3 times as rapidly at 35°C as at 25°C; the rate was further increased with rise in temperature to a maximum at 55°C. These increases are shown to be mainly due to the increased H₂O₂ available to oxidise glyoxylate non-enzymically as a result of the higher temperature coefficient of glycollate oxidase activity relative to that of catalase. These results are compared with similar increases in the rate of¹⁴CO₂ release between 25°C and 35°C when [1-¹⁴C]glycollate was supplied to leaf discs in light or darkness. The role of these reactions in accounting for the temperature effect on the release of photorespiratory CO₂ is discussed.Abbreviations PHMS Pyrid-2-yl--hydroxymethane sulphonate - FMN flavin mononucleotide     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Catabolism of porphobilinogen by etiolated barley leaves

When [2,4-¹⁴C]porphobilinogen (PBG) or [2 (aminomethyl),5-¹⁴C]PBG is administered to etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO₂, organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of ¹⁴CO₂ evolution from leaves fed [2,4-¹⁴C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of ¹⁴CO₂ evolution from [2(aminomethyl),5-¹⁴C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis.     

Mechanism of decarboxylation of glycine and glycolate by isolated soybean cells

Isolated soybean leaf mesophyll cells decarboxylated exogenously added [1-¹⁴C]glycolate and [1-¹⁴C]glycine in the dark. The rate of CO₂ release from glycine was inhibited over 90% by isonicotinic acid hydrazide and about 80% by KCN, two inhibitors of the glycine to serine plus CO₂ reaction. The release of CO₂ from glycolate was inhibited by less than 50% under the same conditions. This indicates that about 50% of the CO₂ released from glycolate occurred at a site other than the glycine to serine reaction. The sensitivity of this alternative site of CO₂ release to an inhibitor of glycolate oxidase (methyl-2-hydroxy-3-butynoate) but not an inhibitor of the glutamate:glyoxylate aminotransferase (2,3-epoxypropionate) indicates that this alternative (isonicotinic acid hydrazide insensitive) site of CO₂ release involved glyoxylate. Catalase inhibited this CO₂ release. Under the conditions used it is suggested that about half of the CO₂ released from glycolate occurred at the conversion of glycine to serine plus CO₂ while the remaining half of the CO₂ loss resulted from the direct oxidation of glyoxylate by H₂O₂.     

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO₂ concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with ¹⁴C showed that the barley mutants incorporated [¹⁴C]formate and [2-¹⁴C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway. Received: 14 August 1998 / Accepted: 30 September 1998     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves

The in vivo oxidation of the C₄ and C₅ of 5-aminolevulinic acid (ALA) to CO₂ has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of ¹⁴CO₂ evolution from leaves fed [4-¹⁴C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of ¹⁴CO₂ evolution from leaves fed [5-¹⁴C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C₄ is oxidized to CO₂ via the tricarboxylic acid cycle to a greater extent than is the C₅.     

Glyoxylate decarboxylation during photorespiration

At 25° C under aerobic conditions with or without gluamate 10% of the [1-¹⁴C]glycollate oxidised in spinach leaf peroxisomes was released as ¹⁴CO₂. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO₂ release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce ¹⁴CO₂ release from either [1-¹⁴C]glycollate or [1-¹⁴C]glyoxylate. Furthermore, the amount of available H₂O₂ (Grodzinski and Butt, 1976) was sufficient to account for all of the CO₂ release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure available H₂O₂ directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO₂ release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H₂O₂ at a known rate was used to replace the chloroplasts, similar rates of ¹⁴CO₂ release and [¹⁴C]glycine synthesis from [1-¹⁴C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO₂ arises during glyoxylate decarboxylation under the action of H₂O₂. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - INH isonicotinylhydrazide - PHMS pyridyl-2-yl--hydroxymethane sulphonic acid     

The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration

Large quantities of CO₂ are released within many photosynthesizing tissues in the light by the process of photorespiration. This CO₂ arises largely from the carboxylcarbon atom of glycolate, which is synthesized in chloroplasts during photosynthesis. Glyoxylate is then produced by the glycolate oxidase reaction. The glyoxylate may be directly decarboxylated to CO₂, but some investigators believe the glyoxylate must first be converted to glycine before CO₂ is released during photorespiration. Spinach chloroplasts with their envelope membranes removed in dilute buffer solution have now been shown to carry out the oxidative decarboxylation of [1-¹⁴C]glyoxylate, in the presence of light and manganous ions in an atmosphere containing oxygen, to yield 1 mole each of ¹⁴CO₂ and formate. Rates of enzymatic decarboxylation exceeding 50 μmoles of ¹⁴CO₂ mg chlorophyll⁻¹ hr⁻¹ were obtained at pH 7.6; hydrogen peroxide is probably the oxidant in the reaction. Heated chloroplasts are inactive under the standard conditions and there is an almost absolute requirement for each of the components listed above. Conditions for some other nonenzymatic decarboxylations of glyoxylate have also been described. [1-¹⁴C]Glycine is decarboxylated by the enzymatic system at only 1% of the rate of [1-¹⁴C]glyoxylate. Maize chloroplast preparations are much less active than spinach chloroplasts. The high rates of CO₂ produced by the spinach system directly from glyoxylate, as well as the need for light and oxygen, suggest that this reaction functions in photorespiration, and that CO₂ arises during photorespiration without glycine as a mandatory intermediate.     

Oxalate metabolism by tobacco leaf discs

The turnover rate of oxalate in leaf discs of Nicotiana tabacum, var Havana Seed, during photosynthesis was estimated to be 1 to 2 micromoles per gram fresh weight per hour. Radioactivity from the enzymic oxidation of [¹⁴C]oxalate rapidly appeared in neutral sugars (mainly sucrose), organic acids (mainly malate), and amino acids. Only 5% of the radioactivity was released to the atmosphere as ¹⁴CO₂, and no formate or formaldehyde could be detected. The metabolism of oxalate was not increased by raising the O₂ concentration from 1% to 21% to 60%, nor was the formation of [¹⁴C]oxalate from [2-¹⁴C]glyoxylate changed under the same conditions as was previously observed in vitro (Havir 1983 Plant Physiol 71: 874-878). While oxalate is not an inert end product of the glycolate pathway, it contributes little to the formation of photorespiratory CO₂.     

Further studies on o(2)-resistant photosynthesis and photorespiration in a tobacco mutant with enhanced catalase activity

The increase in net photosynthesis in M₄ progeny of an O₂-resistant tobacco (Nicotiana tabacum) mutant relative to wild-type plants at 21 and 42% O₂ has been confirmed and further investigated. Self-pollination of an M₃ mutant produced M₄ progeny segregating high catalase phenotypes (average 40% greater than wild type) at a frequency of about 60%. The high catalase phenotype cosegregated precisely with O₂-resistant photosynthesis. About 25% of the F₁ progeny of reciprocal crosses between the same M₃ mutant and wild type had high catalase activity, whether the mutant was used as the maternal or paternal parent, indicating nuclear inheritance. In high-catalase mutants the activity of NADH-hydroxypyruvate reductase, another peroxisomal enzyme, was the same as wild type. The mutants released 15% less photorespiratory CO₂ as a percent of net photosynthesis in CO₂-free 21% O₂ and 36% less in CO₂-free 42% O₂ compared with wild type. The mutant leaf tissue also released less ¹⁴CO₂ per [1-¹⁴C]glycolate metabolized than wild type in normal air, consistent with less photorespiration in the mutant. The O₂-resistant photosynthesis appears to be caused by a decrease in photorespiration especially under conditions of high O₂ where the stoichiometry of CO₂ release per glycolate metabolized is expected to be enhanced. The higher catalase activity in the mutant may decrease the nonenzymatic peroxidation of keto-acids such as hydroxypyruvate and glyoxylate by photorespiratory H₂O₂.     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

Catabolism of 2-methyloctanoic acid and 3beta-hydroxycholest-5-en-26-oic acid

1. 2-Methyl[1-¹⁴C]octanoic acid was synthesized from 2-bromo-octane and ¹⁴CO₂. 2. 2-Methyl[1-¹⁴C]octanoic acid was readily oxidized to propionic acid and carbon dioxide by mitochondrial preparations from liver, less readily oxidized by adrenal and kidney (mitochondria), and only poorly oxidized by heart, spleen and brown fat (mitochondria). 3. 3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was rapidly oxidized by mammalian-liver mitochondria to propionic acid and carbon dioxide. Caiman-liver and toad-liver mitochondria also oxidized this steroid acid. 4. The oxidation of propionic acid, octanoic acid and palmitic acid by mitochondrial preparations from these various tissues was also studied. 5. Added carnitine did not stimulate 2-methyloctanoic acid oxidation and feebly stimulated 3β-hydroxycholest-5-en-26-oic acid oxidation. 6. The significance of these results is discussed in relation to sterol catabolism in mammals and non-mammalian species.