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

Abstract

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

A new caffeine biosynthetic pathway in tea leaves: utilisation of adenosine released from the S‐adenosyl‐L‐methionine cycle

“…Leaf segments were incubated with [8][9][10][11][12][13][14] C]SAH and 10 mM sucrose in 2.0 ml of 30 mM potassium phosphate bu¡er (pH 5.6) at 27³C for 18 h. Yokota (Teikyo University, Utsunomiya, Japan) for his skilled assistance in the preparation of Fig. 2.…”

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…The activity of UK is usually higher than that of UPRT and thus uridine is more efficiently salvaged to UMP than is uracil (e.g. Kanamori-Fukuda et al 1981;Ashihara et al, 2000). UK has been partially purified from higher plants and its enzymatic properties have been well characterized (Deng andIves, 1972 and1975).…”

Dual function of pyrimidine metabolism in potato (Solanum tuberosum) plants: pyrimidine salvage and supply of β‐alanine to pantothenic acid synthesis

“…The administration of 14 C precursors of labelled metabolites was performed aseptically according to the protocols described by Ashihara et al (2000). Briefly, samples (15 leaf disks or 12 tuber disks) were placed into 1.8 ml of 20 mM potassium phosphate buffer (pH 5.7) in the main compartment of a 30-ml Erlenmeyer flask.…”

“…Labelled metabolites were analysed with thin-layer chromatography by the methods of Ashihara et al (2000) and Katahira and Ashihara (2002). The spots of authentic standard of b-alanine and pantothenic acid were visualized with ninhydrine and I 2 vapour, respectively.…”

“…The homogenate was centrifuged at 20 000 g for 20 min at 4 C. Aliquots of the supernatant (2.5 ml) were desalted on a prepacked column of Sephadex G-25 (PD-10, Amersham Pharmacia Biotech, Uppsala, Sweden) and used for enzyme preparation. The activity of all enzymes in this study was determined radiochemically according to the method described by Ashihara et al (2000). The composition of the reaction mixture for determining the activities of enzymes, except for uridine nucleosidase, was as described in a previous article (Katahira and Ashihara 2002).…”