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Metabolism of [carbonyl-14C]nicotinamide was surveyed in various plant materials including the model plants, Arabidopsis thaliana, Oryza sativa and Lotus japonicus. In all plants studied, nicotinamide was used for the pyridine (nicotinamide adenine) nucleotide synthesis, probably after conversion to nicotinic acid. Radioactivity from [carbonyl-14C]nicotinamide was incorporated into trigonelline (1- N-methylnicotinic acid) and/or into nicotinic acid 1 N-glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum, Medicago sativa and Raphanus sativus. Trigonelline synthesis from nicotinamide is generally greater in leaves than in roots. Na-Glc was formed as the major nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In seedlings of Chrysanthemum coronarium and Theobroma cacao, both trigonelline and Na-Glc were synthesized from [carbonyl-14C]nicotinamide. Trigonelline is accumulated in some seeds, mainly Leguminosae species. The pattern of formation of the nicotinic acid conjugates differs between species and organs. [ABSTRACT FROM AUTHOR]

To investigate the short-term (3h) 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. [ABSTRACT FROM AUTHOR]

Uridine and cytidine are major nucleosides and are produced as catabolites of pyrimidine nucleotides. To study the metabolic fates and role of these nucleosides in plants, we have performed pulse (2 h) and chase (12 h) experiments with [2-14C]uridine and [2-14C]cytidine and determined the activities of some related enzymes using tubers and fully expanded leaves from 10-week-old potato plants ( Solanum tuberosum L.). In tubers, more than 94% of exogenously supplied [2-14C]uridine and [2-14C]cytidine was converted to pyrimidine nucleotides and RNA during 2-h pulse, and radioactivity in these salvage products still remained at 12 h after the chase. Little degradation of pyrimidine was found. A similar pyrimidine salvage was operative in leaves, although more than 20% of the radioactivity from [2-14C]uridine and [2-14C]cytidine was released as 14CO2 during the chase. Enzyme profile data show that uridine/cytidine kinase (EC 2.7.1.48) activity is higher in tubers than in leaves, but uridine nucleosidase (EC 3.2.2.3) activity was higher in leaves. In leaves, radioactivity from [U-14C]uracil was incorporated into β-ureidopropionic acid, CO2, β-alanine, pantothenic acid and several common amino acids. Our results suggest two functions of uridine and cytidine metabolism in leaves; these nucleosides are not only substrates for the classical pyrimidine salvage pathways but also starting materials for the biosynthesis of β-alanine. Subsequently, some β-alanine units are utilized for the synthesis of pantothenic acid in potato leaves. [ABSTRACT FROM AUTHOR]

Suspension-cultured cells derived from seedlings of Bruguiera sexangulaare tolerant to NaCl. To examine the influence of long-term salt stress on glycolysis, we determined the effect of 100 mMNaCl on the activities of two key enzymes, phosphofructokinase (PFK, EC 2.7.1.11) and pyruvate kinase (PK, EC 2.7.1.40), and on the bypass enzymes, pyrophosphate: fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.49) and phosphoenolpyruvate phosphatase (PEPase, EC 3.1.3.60). From 10 days after NaCl treatment, increases were found in the activities of PFK, PK and PEPC. In contrast, there was little or no difference in the activities of PFP or PEPase. The short-term effect of salt stress was also investigated. NaCl (150 mM) caused a 1.4-fold increase in respiratory O2 uptake at 24 h after treatment. Alongside this respiratory rise, drastic changes in the levels of glycolytic metabolites were found: a decrease in the levels of glucose, glucose-6-phosphate and fructose-6-phosphate, and an increase in the levels of fructose-1, 6-bisphosphate and metabolites of the later steps of the glycolytic pathway. The crossover diagram of metabolites suggests that NaCl stimulates those steps catalysed by PFK and/or PFP. The in vitro activities of partially purified PFK and PFP were increased by the addition of 150 mMNaCl. The effect of salt on the kinetic properties of PFK and PFP was studied, and possible control mechanisms of glycolysis on salt stress are discussed. [ABSTRACT FROM AUTHOR]

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14 C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination. [ABSTRACT FROM AUTHOR]

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of 14C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO2 and β-ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphorihosyltransferase (EC 2.4.2.7), guanine phosphoribosyl transferase (EC 24.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the connversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 27.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonuclease(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]guanine, [18-14C]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. [ABSTRACT FROM AUTHOR]

Glucose‐6‐phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The Km value for glucose‐6‐phosphate is 1.6 × 10−4 and 6.3 × 10−4M at low (1.0–6.0 × 10−4M) and high (6.0–30.0 × 10−4M) concentrations of the substrate, respectively. The Km value for NADP+ is 1.4 × 10−5M. The enzyme is inhibited by NADPH, 5‐phosphoribosyl‐1‐pyrophosphate, and ATP, and it is activated by Mg2+, and Mn2+. In the presence of NADPH, the plot of activity vs. NADP+ concentration gave a sigmoidal curve. Inhibition of 5‐phosphoribosyl‐1‐pyrophosphate and ATP is reversed by Mg2+ or a high pH. It is suggested that black gram glucose‐6‐phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway. [ABSTRACT FROM AUTHOR]

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]jguanine. 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. [ABSTRACT FROM AUTHOR]

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot (Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [14C]-labelled adenosine into ATP and GTP were observed at the late growth stages. This indicated that purine salvage was more important at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of the enzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cell in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [32P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium. [ABSTRACT FROM AUTHOR]

Contents of purine alkaloids in different parts of tea (Camellia sinensis L. cv. Yabukita) seedlings, seeds and tissue cultures were determined with high‐performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4‐month‐old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds. Tracer experiments using [8‐14C]‐adenine indicate that (i) caffeine biosynthesis from [8‐14C]‐adenine occurs only in younger leaves,(ii) “salvage” of [8‐14C]‐adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8‐14C]‐adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue. The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the S-adenosylmethionine dependent N-methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent Mr of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute, to the production of caffeine. [ABSTRACT FROM AUTHOR]