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.     

Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.     

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.     

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     

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.     

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.     

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 and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.     

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.     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Metabolism of methylamine in the tea plant (Thea sinensis L.)

1. The metabolism of methylamine in excised shoot tips of tea was studied with micromolar amounts of [(14)C]methylamine. Of the [(14)C]methylamine supplied 57% was utilized by tea shoots during the 10h experimental period. 2. The main products of [(14)C]methylamine metabolism in tea shoots were serine, gamma-glutamylmethylamide, theobromine, caffeine and CO(2). There was also incorporation of the label into glutamate, aspartate, RNA purine nucleotides and S-adenosylmethionine. 3. The formation of methylamine from gamma-glutamylmethylamide was confirmed by feeding tea shoots with gamma-glutamyl[(14)C]methylamide. The products of gamma-glutamyl[(14)C]methylamide metabolism in tea plants were serine, theobromine, caffeine, glutamate and aspartate. 4. The results indicate that the oxidation of methylamine to formaldehyde is the first step of methylamine utilization. Labelled formaldehyde released by the metabolism of methylamine leads to the incorporation of the label into metabolites on the C(1) pathways of this compound. It is also suggested that formaldehyde is further oxidized via formate to CO(2). 5. The role of gamma-glutamylmethylamide in methylamine metabolism in tea plants is discussed. 6. Results support the view that theobromine is the immediate precursor of caffeine.     

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.     

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.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.     

Purine biosynthesis de novo in rat skeletal muscle.

Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.     

Hypoxanthine Transport and Metabolism in the Central Nervous System

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypoxanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [14C]hypoxanthine; however, 14C was readily released from choroid plexus principally as [14C]-hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid-soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [14C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C-nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

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.     

Leishmania mexicana: Purine metabolism in promastigotes,axenic amastigotes,and amastigotes derived from vero cells

Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [¹⁴C]formate and [¹⁴C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [¹⁴C]hypoxanthine, [¹⁴C]adenine, and [¹⁴C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (?60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.