Metabolism of nicotinamide, adenine and inosine in developing microspore-derived canola (Brassica napus) embryos

The metabolic fate of [carbonyl-(14)C]nicotinamide, [8-(14)C]adenine and [8-(14)C]inosine was examined in microspore-derived canola (Brassica napus) embryos at different developmental stages: globular stage (day 10, stage 1), early cotyledonary stage (day 20, stage 2), late cotyledonary stage (day 25, stage 3), and fully developed stage (day 35, stage 4). Uptake of [8-(14)C]nicotinamide by the embryos was always rapid. A lower uptake rate was found for [8-(14)C]adenine and [8-(14)C]inosine, especially at stages 1 and 2. [Carbonyl-(14)C]nicotinamide was converted to nicotinic acid and further metabolized to pyridine nucleotides (NAD/NADP). Some radioactivity was also associated to nicotinic acid glucoside. [8-(14)C]adenine was efficiently utilized for the synthesis of adenine nucleotides and RNA. A small fraction of adenine was degraded to CO(2) via ureides. Up to 40% of [8-(14)C]inosine was salvaged to nucleotides and RNA, although degradation of [8-(14)C]inosine to CO(2) was pronounced. At stage 1, highest salvage activities of nicotinamide, adenine and inosine were observed. In contrast, the lowest purine salvage and highest purine catabolism were found in stage 3 embryos. These results suggest that both nicotinamide and purine salvage for NAD/NADP and purine nucleotides synthesis are extremely high in the globular stage (stage 1). These activities decrease gradually until the late cotyledonary stage (stage 3), before increasing again in the fully developed embryos (stage 4). Overall it appears that nicotinamide and purine salvage are required in support of active growth during the initial phases of embryogenesis and at the end of the maturation period, in preparation for post-embryonic growth.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

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     

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.     

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.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

Biosynthesis of coenzyme F420 and methanopterin in Methanobacterium thermoautotrophicum

Growing cultures of Methanobacterium thermoautotrophicum were supplemented with [U-¹⁴C]adenosine or [1-¹⁴C]adenosine. 7,8-Didemethyl-8-hydroxy-5-deazariboflavin (factor F₀) and 7-methylpterin were isolated from the culture medium. Hydrolysis of cellular RNA yielded purine and pyrimidine nucleotides. The ribose side chain of proffered adenosine is efficiently incorporated into cellular adenosine and guanosine nucleotide pools but not into pyrimidine nucleotides. Thus, M. thermoautotrophicum can utilize exogenous adenosine by direct phosphorylation without hydrolysis of the glycosidic bond, and AMP can be efficiently converted to GMP. Factor F₀ and 7-methylpterin had approximately the same specific activities as the purine nucleotides. It follows that the ribityl side chain of factor F₀ is derived from the ribose side chain of a nucleotide precursor by reduction. The pyrazine ring of methanopterin is formed by ring expansion involving the ribose side chain of the precursor, GTP.Abbreviations Factor F₀ 8-hydroxy-6,7-didemethyl-5-deazariboflavin - APRT adenine phosphoribosyltransferase - GPRT guanine phosphoribosyltransferase - PRPP phosphoribosylpyrophosphate - HPLC high performance liquid chromatography     

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.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

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.     

Ca2+ and pH responses to sequential additions of mitogens in single 3T3 fibroblasts: correlations with DNA synthesis

The progression of Swiss 3T3 fibroblasts from the quiescent state (G0) through G1 to DNA synthesis in S phase generally requires the synergistic action of two mitogens. The main aim of this study was to compare systematically the early Ca2+ and pH responses in quiescent cells to all of the pair combinations of eight mitogens (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha, epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate, insulin, 8-bromo-cAMP) with their subsequent effects on DNA synthesis. Each of the mitogens which caused inositol phosphate accumulation (bombesin, platelet-derived growth factor, vasopressin, prostaglandin F2 alpha) also activated Ca2+- and phospholipid-dependent protein kinase (protein kinase C) and generated both the Ca2+ and pH responses, although epidermal growth factor also generated the ionic responses without causing release of inositol phosphates or activation of protein kinase C. For sequential mitogen additions the ionic signals were measured in single cells as well as in cell populations to avoid ambiguities due to heterogeneity in the responses of the cells to the various mitogens. The modulating effects of the mitogens on the [Ca2+]i responses to subsequent mitogen additions varied widely, but detailed comparisons showed that the pattern of blocking effects could not be attributed solely to the effect of the first mitogen causing either maximal breakdown of phosphatidylinositol 4,5-bisphosphate or complete depletion of the intracellular Ca2+ pool or activation of protein kinase C. From these analyses it was concluded that the requirement for two mitogens for effective DNA synthesis could not be attributed to the summation to a critical threshold of either the ionic signals or phosphatidylinositol 4,5-bisphosphate breakdown, and that these responses are insufficient by themselves to cause the cells to progress to DNA synthesis in S phase.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

Inosine 5'-monophosphate vs inosine and hypoxanthine as substrates for purine salvage in human lymphoid cells

The ability of inosine 5'-monophosphate vs inosine or hypoxanthine to supply the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells was evaluated. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and make the cells dependent upon an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 25 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA at rates equal to that of untreated control cultures. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line, WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line No. 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, only inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells and suggest that this enzyme may have functional significance when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

Nucleotide and pentose synthesis after serum-stimulation of resting 3T6 fibroblasts.

We have investigated the de novo synthesis of intermediates of purine nucleotides in 3T6 fibroblasts and determined the manner by which the activity of this pathway is increased in resting cells by the addition of fresh serum. Within 30 minutes after stimulation, 3T6 cells began to synthesize increased amounts of purines by the de novo pathway as measured by increased amounts of formylglycinamide ribonucleotide, a representative intermediate of this pathway. Within 15 minutes after serum-stimulation 3T6 cells exhibited a substantial increase in their capacity to synthesize ribose compounds, particularly in the form of 5-phosphoribosylpyrophosphate (PRPP). The availability of PRPP appeared to be limiting for the synthesis of purine nucleotides in resting fibroblasts, but not necessarily in serum-stimulated cells. The amount of the enzyme PRPP synthetase as measured in vitro remained constant for at least the first four hours. Therefore, a study was made of various compounds known to activate PRPP synthetase in vitro. No evidence was found that suggested involvement of concentrations of cyclic nucleotides or phosphate. Experiments with methylene blue, an artificial electron acceptor that stimulates the production of ribose 5-phosphate by the hexose monophosphate shunt, indicated that one of the immediate consequences of the addition of serum is increased cycling of the pyridine nucleotide coenzymes, NADP⁺ and NADPH, and that the rapid increase in formation of ribose compounds and, consequently, purine nucleotides was caused as a result of modulation by this coenzyme. The relative ration of ATP:ADP:AMP as well as their concentrations remain constant in resting and serum-stimulated cells under normal assay conditions. However, there was a substantial decrease in ATP concentrations with a corresponding increase in AMP concentration with methylene blue in the assay buffer. The production of AMP from ATP was 5-fold greater in the serum-stimulated than in the resting fibroblasts. The increased production of AMP is thus serum-dependent and may reflect a basic enzymatic function of proliferative as compared to resting cells.     

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.     

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

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 sttages. This indicated that purine slavage was more importnt 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 theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells 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.     

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study.

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.