Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza

We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots.     

Progression of programmed cell death in tobacco BY-2 cells is delineated by specific changes in de novo and salvage synthesis of purine nucleotides

Alterations in the pattern of purine nucleotide synthesis and degradation were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of purine nucleotides was estimated by following the metabolic fate of the [8-¹⁴C]5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR), the salvage synthesis was investigated using [8-¹⁴C]adenine and adenosine, and the degradation pathway was studied by following the incorporation of [8-¹⁴C]inosine. The results indicated that specific changes in purine metabolism occurred during the death programme of tobacco cells. During the early phases of PCD, increases in the salvage activity of adenine and adenosine were observed, and these were related to the high activity of the two major salvage enzymes: adenine phosphoribosyltransferase (APRT) and adenosine kinase (ARK). During the following stages, a large fraction of purine nucleotide was also produced through the de novo pathway, suggesting a tight regulation between salvage and de novo synthesis. These changes were strictly associated with PCD, as they did not occur if NO or hydrogen peroxide was increased individually, or if actinomycin, which inhibits the death programme, was added to the medium in the presence of NO and hydrogen peroxide. These changes in purine nucleotide synthesis represent an early metabolic switch which may be needed to ensure the proper execution of all the high-energy demand processes characteristic of the death programme.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Inhibition of caffeine biosynthesis in tea (Camellia sinensis) and coffee (Coffea arabica) plants by ribavirin

The effects of ribavirin, an inhibitor of inosine-5'-monophosphate (IMP) dehydrogenase, on [8-(14)C]inosine metabolism in tea leaves, coffee leaves and coffee fruits were investigated. Incorporation of radioactivity from [8-(14)C]inosine into purine alkaloids, such as theobromine and caffeine, guanine residues of RNA, and CO(2) was reduced by ribavirin, while incorporation into nucleotides, including IMP and adenine residues of RNA, was increased. The results indicate that inhibition of IMP dehydrogenase by ribavirin inhibits both caffeine and guanine nucleotide biosynthesis in caffeine-forming plants. The use of IMP dehydrogenase-deficient plants as a potential source of good quality caffeine-deficient tea and coffee plants is discussed.     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Effect of short-term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐¹⁴C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐¹⁴C]uridine, [2‐¹⁴C]uracil and more than 50% of [2‐¹⁴C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to ¹⁴CO₂ via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐¹⁴C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐¹⁴C]adenosine and [8‐¹⁴C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐¹⁴C]hypoxanthine was catabolised to ¹⁴CO₂, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [³H]Quinolinate, [carbonyl‐¹⁴C]nicotinamide and [carboxyl‐¹⁴C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

Characterization of purine nucleotide metabolism in primary rat cardiomyocyte cultures

Primary rat cardiomyocyte cultures were utilized as a model for the study of purine nucleotide metabolism in the heart muscle, especially in connection with the mechanisms operating for the conservation of adenine nucleotides. The cultures exhibited capacity to produce purine nucleotides from nonpurine molecules (de novo synthesis), as well as from preformed purines (salvage synthesis). The conversion of adenosine to AMP, catalyzed by adenosine kinase, appears to be the most important physiological salvage pathway of adenine nucleotide synthesis in the cardiomyocytes. The study of the metabolic fate of IMP formed from [14C]formate or [14C]hypoxanthine and that of AMP formed from [14C]adenine or [14C]adenosine revealed that in the cardiomyocyte the main flow in the nucleotide interconversion pathways is from IMP to AMP, whereas the flux from AMP to IMP appeared to be markedly slower. Following synthesis from labeled precursors by either de novo or salvage pathways, most of the radioactivity in purine nucleotides accumulated in adenine nucleotides, and only a small proportion of it resided in IMP. The results suggest that the main pathway of AMP degradation in the cardiomyocyte proceeds through adenosine rather than through IMP. About 90% of the total radioactivity in purines effluxed from the cells during de novo synthesis from [14C]formate or following prelabeling of adenine nucleotides with [14C]adenine were found to reside in hypoxanthine. The activities in cell extracts of AMP 5'-nucleotidase and IMP 5'-nucleotidase, which catalyze nucleotide degradation, and of AMP deaminase, a key enzyme in the purine nucleotide cycle, were low. The nucleotidase activity resembles, and that of the AMP deaminase contrasts the respective enzyme activities in extracts of cultured skeletal-muscle myotubes. The results indicate that in the cardiomyocyte, in contrast to the myotube, the main mechanism operating for conservation of nucleotides is prompt phosphorylation of AMP, rather than operation of the purine nucleotide cycle. The primary cardiomyocyte cultures are a plausible model for the study of purine nucleotide metabolism in the heart muscle.     

Brassinolide-improved development of <Emphasis Type="Italic">Brassica napus</Emphasis> microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of ¹⁴C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

The catabolism of endogenous adenine nucleotides in rat liver mitochondria

Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [¹⁴C]-adenosine yielded no [¹⁴C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.     

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Pyridine metabolism in tea plants: salvage, conjugate formation and catabolism

Pyridine compounds, including nicotinic acid and nicotinamide, are key metabolites of both the salvage pathway for NAD and the biosynthesis of related secondary compounds. We examined the in situ metabolic fate of [carbonyl-¹⁴C]nicotinamide, [2-¹⁴C]nicotinic acid and [carboxyl-¹⁴C]nicotinic acid riboside in tissue segments of tea (Camellia sinensis) plants, and determined the activity of enzymes involved in pyridine metabolism in protein extracts from young tea leaves. Exogenously supplied ¹⁴C-labelled nicotinamide was readily converted to nicotinic acid, and some nicotinic acid was salvaged to nicotinic acid mononucleotide and then utilized for the synthesis of NAD and NADP. The nicotinic acid riboside salvage pathway discovered recently in mungbean cotyledons is also operative in tea leaves. Nicotinic acid was converted to nicotinic acid N-glucoside, but not to trigonelline (N-methylnicotinic acid), in any part of tea seedlings. Active catabolism of nicotinic acid was observed in tea leaves. The fate of [2-¹⁴C]nicotinic acid indicates that glutaric acid is a major catabolite of nicotinic acid; it was further metabolised, and carbon atoms were finally released as CO₂. The catabolic pathway observed in tea leaves appears to start with the nicotinic acid N-glucoside formation; this pathway differs from catabolic pathways observed in microorganisms. Profiles of pyridine metabolism in tea plants are discussed.     

Purine metabolism in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) Seedlings

• 1.1. The metabolism of purine bases and nucleosides in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) was studied.
• 2.2. A large portion of absorbed [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]adenosine was salvaged in nucleotide and nucleic acid synthesis.
• 3.3. Most of the radioactivity of [8-¹⁴C]hypoxanthine and [8-¹⁴C]inosine was incorporated into allantoin and allantoic acid.
• 4.4. Activity of adenine phosphoribosyltransferase in enzyme extracts was much higher than that of hypoxanthme and guanine phosphoribosyltransferase(s).
• 5.5. Apparent activity of adenosine kinase was higher than that of inosine kinase. 6. NAD⁺-dependent xan thine dehydrogenase was detected in both cotyledons and embryonic axes of the seedlings.
• 6.7. The capacity of purine salvage was higher m 24 hr old cotyledons than 24 and 48 hr old embryonic axes. The reverse was observed concerning that of purine degradation.