Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Simultaneous Determination of Purine and Pyrimidine Metabolites in Hprt-Deficient Cell Lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch–Nyhan syndrome and Kelley–Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5′-monophosphate (IMP) was different in Lesch–Nyhan syndrome, Kelley–Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.     

Circadian variations of adenosine level in blood and liver and its possible physiological significance

The role of adenosine as a possible physiological modulator was explored by measuring its concentration in different tissues during a 24-hour period. Initially the circadian variations of adenosine and other purine compounds such as inosine, hypoxanthine, uric acid and adenine nucleotides were studied in the rat blood. A daily cyclic response was observed, with low levels of adenosine from 08.00 - 20.00 h, followed by an increase from this time on. Inosine and hypoxanthine levels were elevated during the day and low at night. The uric acid changes observed indicate that the decrease in purine catabolism coincides with a decrease in inosine and hypoxanthine levels and an increase in adenosine. The blood adenine nucleotides, energy charge and phosphorylation potential remained constant during the day and showed oscillatory changes during the night. Similar studies were made in the liver, a primary source of circulating purines. Liver adenosine was high during the night while inosine and hypoxanthine remained low along the 24 hours. The results suggest that liver purine metabolism might participate in the maintenance and renewal of the blood purine pool and in the energy state of erythrocytes in vivo.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Heroin affects purine nucleotides catabolism in rats in vivo

To investigate the effect of heroin on purine nucleotides catabolism, a rat model of heroin administration and withdrawal was established. Concentrations of uric acid, creatinine, and urea nitrogen in plasma and ADA in plasma, brain, liver, and small intestine were tested. When two heroin administration groups were compared with the control group, the concentrations of plasma uric acid and ADA in plasma, brain, liver, and small intestine increased, whereas the plasma urea nitrogen concentrations in two heroin administration groups and the plasma creatinine concentration in the 3-day heroin administration group did not increase. It seemed that heroin exposure for a short time did not affect renal clearance rate notably. When two withdrawal groups were compared with two heroin administration groups, the concentrations of plasma uric acid and ADA in liver and small intestine decreased, but there was no significant reduction in ADA concentrations of the brain, while the plasma ADA concentrations in the two withdrawal groups were significantly higher than those of two heroin administration groups. When the two withdrawal groups were compared with the control group, there was no significant difference in the concentrations of plasma uric acid and ADA in liver and small intestine, while the concentrations of ADA in plasma and brain were still higher than those of the control group. The results imply that heroin administration may enhance the catabolism of purine nucleotides in the brain and other tissues by increased concentration of ADA and the effect may last for a long time in the brain.     

Nucleotides and inorganic phosphates as potential antioxidants

Highly reactive OH radicals, formed in an iron-ion catalyzed Fenton reaction, are implicated in many pathological conditions. The quest for Fenton reaction inhibitors, either radical scavenger or metal-ion chelator antioxidants, spans the previous decades. Purine nucleotides were previously studied as natural modulators of the Fenton reaction; however, the modulatory role of purine nucleotides remained in dispute. Here, we have resolved this long-standing dispute and demonstrated a concentration-dependent biphasic modulation of the Fenton reaction by nucleotides. By electron spin resonance measurements with 0.1 mM Fe(II), we observed an increase of •OH production at low purine nucleotide concentrations (up to 0.15 mM), while at higher nucleotide concentrations, an exponential decay of •OH concentration was observed. We found that the phosphate moiety, not the nucleoside, determines the pro/antioxidant properties of a nucleotide, suggesting a chelation-based modulation. Furthermore, the biphasic modulation mode is probably due to diverse nucleotide–Fe(II) complexes formed in a concentration-dependent manner. At ATP concentrations much greater than Fe(II) concentrations, multiligand chelates are formed which inhibit the Fenton reaction owing to a full Fe(II) coordination sphere. In addition to natural nucleotides, we investigated a series of base- or phosphate-modified nucleotides, dinucleotides, and inorganic phosphates, as potential biocompatible antioxidants. Ap₅A, inorganic thiophosphate and ATP-γ-S proved highly potent antioxidants with IC₅₀ values of 40, 30, and 10 μM, respectively. ATP-γ-S proved 100 and 20 times more active than ATP and the potent antioxidant Trolox, respectively. In the presence of 30 μM ATP-γ-S no •OH was detected after 5 min in the Fenton reaction mixture. The most potent antioxidants identified inhibit the Fenton reaction by forming full coordination sphere chelates.Patent pending.     

Radical Formation in the Rat Small Intestine During and Following Ischemia

Oxidative loading during the reperfusion of the proximal jejunum of rats following a one hour-period of complete ischemia was demonstrated in in vivo-experiments by the increases of the GSSG: total glutathione ratio and the concentration of TBA-RS. The pretreatment of the animals with the xanthine oxidoreductase inhibitor allopurinol diminished the accumulation of GSSG and of TBA-RS. It was concluded that the purine nucleotide degradation is an important source of oxygen reduction products in reoxygenated small intestine. The tissue concentrations of nucleotides, nucleosides and nucleobases were measured by an ion-pair reversed-phase HPLC separation. There occurred fast declines of ATP and GTP concentrations during ischaemia leading to temporary increases of nucleoside mono- and diphosphate pools. The hypoxanthine concentration is increased about twenty fold during oxygen deficiency. The ATP and GTP restoration during the reperfusion was accelerated in presence of allopurinol. The shares of the beneficial allopurinol effects are not yet clarified.     

Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3′-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg^{2 +} which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.     

Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi) in the rhesus monkey

Webster H. K., Haut M. J., Martin L. K. and Hildebrandt P. K. 1982. Purine and pyrimidine nucleotide profiles during synchronous malaria infection (Plasmodium knowlesi in the rhesus monkey. International Journal for Parasitology12: 75–79. Blood levels of purine and pyrimidine nucleotides were determined during synchronous infection by Plasmodium knowlesi in rhesus monkeys. Infected monkeys followed over 2–3 intraerythrocytic cycles showed variations in nucleotide pool levels characteristic of the predominant schizogonic growth stage. These changes in nucleotide levels as described for ‘ring’-stage, trophozoite growth and schizogony indicate a cyclically varying relationship between nucleotide concentrations and a specific stage of parasite development during the blood-phase of malaria infection.     

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.     

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively.     

Estimation of metabolic flux rates in liver purine catabolism of tumour-bearing mice by computer simulation of radioactive tracer experiments

Mouse hepatocytes from healthy control mice and from Ehrlich ascites tumour-bearing mice were used for tracer-kinetic studies of purine catabolism of liver cells during different periods of tumour growth. The dynamics of the radioactive tracers were modelled mathematically by a system of differential equations. Computer simulations, i.e. direct fitting of numerical solutions of these equations to the observed time-courses of metabolites and specific radioactivites, enables one to estimate unknown kinetic parameters of a simplified model of pathways of hepatic purine catabolism in tumour-bearing mice. There occurred great differences of metabolic flux rates between control hepatocytes, hepatocytes of mice during the proliferating period of tumour growth (6th day after inoculation of the tumour) and hepatocytes of mice during the resting period of tumour growth (12th day after inoculation of the tumour). The final purine degradation of hepatocytes prepared during the proliferating period was lower in comparison with that of control hepatocytes, but it was markedly higher in hepatocytes prepared during the resting period of tumour growth. The changes in hepatocyte purine catabolism during the proliferating period of tumour growth argue for transitions which aim at the maintenance of high purine nucleotide levels in the liver itself rather than for an increased nucleoside and nucleobase supply for the tumour. This suggestion is in accordance with the increased ATP level of the liver during the proliferating phase of tumour growth. The drastic acceleration of the final steps of hepatic purine catabolism forming uric acid and allantoin during the resting period of tumour growth was predominantly due to increased flux rate from xanthosine and guanine in accordance with increased catabolism of monophosphorylated nucleotides.     

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.     

Metabolic pool of purine compounds in erythrocytes of mice during growth of transplanted tumors

The pattern of purine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cells growth. Host erythrocytes purine nucleotides, nucleosides and bases were affected by the implanted tumors. The results indicated that the host erythrocytes markedly concentrated adenine and guanine nucleotides on the 5th and 11th-12th day of tumor growth. By contrast, content of nucleosides and bases were sharply decreased during the log growth phase (5th day) with the restoring of these precursors within the 11th-12th day (plateau phase). These observations indicate that aspects of the purine compounds metabolism in host erythrocytes are linked with tumor development.     

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.     

Acid-soluble purine and pyrimidine derivatives of growing, encysting and encystment-inhibited amoebae

The intracellular acid-soluble purine and pyrimidine derivatives of myxamoebae-swarm cells of Physarum flavicomum were investigated during growth, microcyst formation, and during adenine-inhibition of encystment, using high performance liquid chromatography (HPLC). We also studied the incorporation of exogenous radioactive adenine into the acid soluble purine derivatives and S-adenosyl-sulphur compounds separated by HPLC. The most abundant ribonucleoside monophosphate was AMP in the growing and 15 h encysting cells (NC), while it was UMP in the 15 h adenine-inhibited cells (AIC). ADP was the nucleoside diphosphate present in the greatest quantity in the growing and NC cells but it was CDP in the AIC. The nucleoside triphosphate in highest concentration was ATP, UTP, and GTP in growing, NC, and AIC, respectively. Guanosine was the most abundant nucleoside in all cells. The nucleobase occurring in greatest concentration was cytosine, cytosine and guanine, and adenine in the growing, NC, and AIC, respectively. The AMP content in the 15 h AIC was 2.1-fold higher than that of adenosine. The 15 h NC had the lowest adenylate energy charge, a value of 0.54 +/- 0.02, while the values for growing cells and the AIC were 0.62 +/- 0.02 and 0.76 +/- 0.01, respectively. [14C]-Adenine labelling studies (15 h) revealed the occurrence of purine nucleotide interconversion, as the label was detected not only in adenosine, AMP, ADP, ATP, but also in guanine, guanosine, GMP, GDP, GTP, as well as, in inosine monophosphate and xanthosine monophosphate. The percentage incorporation of the radiolabelled adenine into AMP was higher than into adenosine. An increased intracellular level of guanine nucleotides is associated with the inhibition of encystment. The extracellular adenine, rather than internal adenine sources, appears to be the primary precursor of nucleotide for S-adenosylmethionine synthesis during adenine-inhibition of encystment.     

Determination of metabolite and nucleotide concentrations in proliferating lymphocytes by 1H-NMR of acid extracts

Nuclear magnetic resonance (NMR) studies of extracts have proven to be a powerful window onto the intracellular machinery of cells and tissues. The major advantages of in vitro 1H-NMR, namely chemical preservation, simultaneous detection, identification, and quantitation of compounds, and sensitivity to a large variety of classes of compounds, are employed in this study to characterize the metabolic course of mitogen-stimulated proliferation of human peripheral lymphocytes. A reliable method to quantitate amino acids, metabolic intermediates, soluble membrane lipid precursors, and purine, pyridine and pyrimidine nucleotides is presented, using samples as small as 30 mg wet weight. A total of 53 substances were detected in lymphocytes and other blood cells. During the course of lymphocyte culture, changes in intracellular concentrations of lactate, taurine, inositol and nucleotides, including NAD, IMP and high-energy phosphates, were especially marked. 1H-NMR compares favorably to 31P-NMR and to HPLC, and is especially attractive in light of expectations for future in vivo application.     

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.     

Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTP[S] , GDP and ATP for maximal activation were 0.1mM, 5M,1 mM and 1 mM respectively. The activation caused by 1mM ADP was lower. The enzyme was not activated by 1mM AMP, but significant activation was observed by the addition of 1mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A ablished the activation. There were synergic effects between ATP and GTP, ATP and PIP₂, but not between ATP and GTP[S] , or PIP₂ and GTP[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP₂ scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP₂ is required for the activation induced by GTP. Protein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D, when the intracellular concentration of cAMP is increased.