Pentose phosphates in nucleoside interconversion and catabolism

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.     

Uptake and utilization of nucleosides for energy repletion

In this paper, we report that cells undergoing metabolic stress conditions may use the ribose moiety of nucleosides as energy source to slow down cellular damage. In fact, the phosphorolytic cleavage of the N-glycosidic bond of nucleosides generates, without energy expense, the phosphorylated pentose, which through pentose phosphate pathway and glycolysis, can be converted to energetic intermediates. In this respect, nucleosides may be considered as energy source, alternative or supplementary to glucose, which may become of primary importance especially in conditions of cellular stress. In accordance with the role of these compounds in energy repletion, we also show that the uptake of nucleosides is increased when the energetic demand of the cell is enhanced. As cell model, we have used a human colon carcinoma cell line, LoVo, and the depletion of ATP, with a concomitant fall in the cell energy charge, has been induced by exclusion of glucose from the medium and pre-incubation with oligomycin, an inhibitor of oxidative phosphorylation. In these conditions of energy starvation, we show that the uptake of 2'-deoxyadenosine in LoVo cells is significantly enhanced, and that the phosphorylated ribose moiety of inosine can be used for energy repletion through anaerobic glycolysis. Our data support previous reports indicating that the phosphorylated ribose stemming from the intracellular catabolism of nucleosides may be used in eukaryots as energy source, and advance our knowledge on the regulation of the uptake of nucleosides in eukaryotic cells.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Major determinants of purine excretion from human lymphoblasts

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.     

Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides.

The overall goal of this study was to determine the mechanisms by which nucleosides are transported in choroid plexus. Choroid plexus tissue slices obtained from rabbit brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturable with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhibited by naturally occurring ribo- and deoxyribonucleosides (adenosine, cytidine, and thymidine) but not by synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine arabinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, formycin B) and pyrimidine nucleosides (uridine and cytidine) were potent inhibitors of Na(+)-thymidine transport with IC50 values ranging between 5 and 23 microM. Formycin B competitively inhibited Na(+)-thymidine uptake and thymidine trans-stimulated formycin B uptake. These data suggest that both purine and pyrimidine nucleosides are substrates of the same system. The stoichiometric coupling ratios between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.     

Mononucleotide Metabolism in the Rat Brain After Transient Ischemia

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Depletion and recovery of ATP in V79 cells with exposure to inhibitors of glycolysis and oxidative phosphorylation

Summary Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP. Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal exhibited normal uridine uptake. This work was supported in part by Grant GM24271 from the National Institutes of Health, Bethesda, Maryland.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

AMP deamination delays muscle acidification during heavy exercise and hypoxia

In silico studies carried out by using a computer model of oxidative phosphorylation and anaerobic glycolysis in skeletal muscle demonstrated that deamination of AMP to IMP during heavy short term exercise and/or hypoxia lessens the acidification of myocytes. The concerted action of adenylate kinase and AMP deaminase, leading to a decrease in the total adenine nucleotide pool, constitutes an additional process consuming ADP and producing ATP. It diminishes the amount of ADP that must be converted to ATP by other processes in order to meet the rate of ADP production by ATPases (because the adenylate kinase + AMP deaminase system produces only 1 ATP per 2 ADPs used, ATP consumption is not matched by ATP production, and the reduction of the total adenine nucleotide pool occurs mostly at the cost of [ATP]). As a result, the rate of ADP consumption by other processes may be lowered. This effect concerns mostly ADP consumption by anaerobic glycolysis that is inhibited by AMP deamination-induced decrease in [ADP] and [AMP], and not oxidative phosphorylation, because during heavy exercise and/or hypoxia [ADP] is significantly greater than the Km value of this process for ADP. The resultant reduction of proton production by anaerobic glycolysis enables us to delay the termination of exercise because of fatigue and/or to diminish cell damage.     

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.     

Effect of phosphorylation on glycolysis in the cells of assimilating millet leaf tissues

The effect of phosphorylation on glycolysis reactions was studied in respect with the rate of 1-14C-glucose metabolization and the composition of synthesized labelled products in isolated cells of assimilating millet leave tissues incapable to reassimilation of respiratory CO2. Data on oxygen metabolism in mesophyll protoplasts and in cells of parenchymal facing of vascular bundle sheaths in the absence and in the presence of electron acceptors (3,5-dichlorophenolindophenol and methylviologen) show that they retain adenylate pool and energy charge characteristic of photosynthetizing tissues. The glycolysis rate decreased in illuminated cells, which did not remove carbon products from chloroplasts. Analysis of compounds produced from 1-14C-glucose exogenous ADP effect on their ration and the change of adenylate energy charge in the presence of methylviologen demonstrates that the acting factor is a decrease of Pi and ADP concentrations in cytoplasm because of their use chloroplast phosphorylation. It is suggested, that a short-cut chain of glycolysis reactions may take place in intact cells assimilating CO2 in photosynthesis, and the capacity of this chain is determined by the type of carbon metabolism and photorespiration mechanism.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

Blood uridine concentration may be an indicator of the degradation of pyrimidine nucleotides during physical exercise with increasing intensity

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside.

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.     

Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.