Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Preservation of resuspended red cell concentrate. Analysis of purines, nucleosides and nucleotides in stored red cells

Purine nucleotides of red blood cells (RBC) during storage in two different media with addition of adenine/nicotinamide (NAP) or adenine/guanosine (CDS-AG) were estimated by HPLC. Synthesis of guanine nucleotides reached a maximum after 14 days in RBC stored with adenine/guanosine. The higher adenine concentration in the NAP solution (3 mmol/l) did not increase adenine consumption and the ATP-level of the erythrocytes. The adenylate energy charge (AEC) of RBC decreased from 0.91 to 0.63 during 42 days of storage in CDS-AG solution.     

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells was monitored using 8-14C-adenosine and 8-14C-adenine added to the incubation medium containing adenosine, pyruvate and inorganic phosphate (APP medium). Using 8-14C-adenosine it was shown that 21.7% of the isotope contained in the incubation medium penetrated red blood cells. Of that quantity about 50% becomes incorporated into nucleotides. Of the isotope 5.3% was found in hypoxanthine nucleotides (1.3% in ITP and 4.0% in IMP). During incubation of red blood cells in APP medium fortified with the 8-14C-adenine about 95% of isotope penetrated into cells and 60% of that quantity became incorporated into nucleotides. In hypoxanthine nucleotides only trace amounts of isotope were found (0.12% in IMP and 0.13% in ITP).     

Separation of cyclic AMP from adenine and hypoxanthine, their nucleosides and nucleotides by high voltage paper electrophoresis.

A simple and rapid technique which permits the separation of cyclic AMP, adenine, adenosine, hypoxanthine, inosine, 5′-AMP, IMP, ADP, and ATP by the use of unidirectional high-voltage paper electrophoresis has been described. The separation of these compounds based on their charge difference utilizes the following properties: (1) the protonation of the NH₂ group of the adenine, (2) the primary and secondary ionization of the phosphate group of the nucleotides, and (3) the formation of the chelated oxyderivative of boron with the two cis (OH) groups of the ribose moieties of nucleosides and nucleotides.     

Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34 degrees-36 degrees C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage-like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S-3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater-like structure (about 3-9 microns in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB-treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.     

The breakdown of adenine nucleotides in glucose-depleted human red cells.

1) The rate of 2,3-bisphosphoglycerate breakdown is independent of pH value. 2) The adenine nucleotide pattern at alkaline pH values with its characteristic lowering of ATP and the accompanying accumulation of fructose-1,6-bisphosphate is caused by a relative excess of the activity of the hexokinase-phosphofructokinase system as compared wity pyruvate kinase. 3) The breakdown of adenine nucleotides proceeds via AMP mainly through phosphatase and not via AMP deaminase. 4) The constancy of the sum of nucleotides as long as glucose is present is postulated to be due to resynthesis via adenosine kinase which competes successfully with adenosine deaminase. 5) A procedure is given to calculate ATPase activity of glucose-depleted red cells. The results indicate that the ATPase activity is less at lower pH values and declines with time. An ATPase with a high Km for ATP is postulated. 6) During glucose depletion ATP production is mostly derived from the breakdown of 2,3-bisphosphoglycerate and the supply from the pentose phosphate pool both of which proceed at a constant rate. The contribution of pentose phosphate from the breakdown of adenine nucleotides amounts to 40% of the lactate formed at pH 6.8 and is about twice the lactate at pH 8.1.     

Fluoride ion catalyzed alkylation of purines, pyrimidines, nucleosides and nucleotides using alky halides.

Alkyl halides react rapidly with purines and pyrimidines in the presence of fluoride ion. Alkylation of thymidine leads to novel dimeric nucleoside derivatives bridged through N3. Alkylation of thymidine mono and dinucleotides leads to alkylation at the base (N3) as well as diester and triester formation at the phosphate.     

The purine nucleotide cycle. Studies of ammonia production and interconversions of adenine and hypoxanthine nucleotides and nucleosides by rat brain in situ.

Electrical shock treatment produces a rapid loss of high energy phosphates in rat brain. The [ATP]/[ADP] ratio decreases to one-third of its control value within 10 s. The ammonia content increases 3-fold during the first minute after starting the stimulus. The total adenine nucleotide plus adenosine content of brain decreases an equivalent amount of hypoxanthine-containing compounds appears. Adenosine, inosine, and hypoxanthine accumulate, and there is a transitory accumulation of adenylosuccinate. The contents of ATP and creatine phosphate, and the [ATP]/[ADP] ratio, are rapidly restored to control values, but other metabolite contents are restored more slowly. The transient rise in adenylosuccinate and IMP provides evidence that the ammonia production is due in part, and possibly in whole, to the operation of the purine nucleotide cycle.     

Stimulation of DNA synthesis in Jurkat cells by synergistic action between adenine and guanine nucleotides.

P2-purinoceptor agonists stimulated the DNA synthesis of Jurkat cells via a pathway independent of cAMP and intracellular free calcium. The response was greatly enhanced by the synergistic action between adenine and guanine nucleotides, suggesting that binding sites of these nucleotides are different from each other, and the proliferation is stimulated by a novel interaction between adenine and guanine nucleotide receptors. The stimulatory effects of P2-agonists on proliferation was completely abolished by cholera toxin and attenuated by pertussis toxin, which suggests that substrates for cholera toxin and pertussis toxin are involved in the proliferative pathways associated with P2-purinoceptors.     

Synthesis of adenine and guanine nucleotides at the 'inosinic branch point' in lymphocytes of leukemia patients.

The synthesis of purine nucleotides has been studied in human peripheral blood lymphocytes from healthy subjects and patients affected by B-cell chronic lymphocytic leukemia (B-CLL). The rate of the synthesis was measured by following the incorporation of 14C-formate into the nucleotides of lymphocyte suspensions. The whole sequence AMP-->ADP-->ATP was found reduced in B-CLL lymphocytes; in the case of guanylates only the last step of the sequence GMP-->GDP-->GTP was significantly lower in the same cells. From the analysis of these results, combined with previous data, we conclude that purine metabolism undergoes an imbalancement during CLL, which is partially compensated, and point out the importance of studying concomitantly purine metabolism and nucleic acid synthesis in leukemia cells.     

The effects of adenine nucleotides and guanine nucleotides on urate synthesis de novo by isolated chick liver and kidney cells.

Isolated chick liver and kidney cells produce urate de novo from glycine, and this is partially inhibited by 1 mm-AMP and by 1 mm-GMP in liver cells but not in kidney cells. Azaserine fully inhibits this synthesis de novo, but attempts to isolate formylglycine amide ribonucleotide from azaserine-blocked cells were unsuccessful.     

Thermodynamic properties of enzyme-catalyzed reactions involving guanine, xanthine, and their nucleosides and nucleotides

The standard Gibbs energies of formation of species in the guanosine triphosphate and the xanthosine triphosphate series have been calculated on the basis of the convention that the standard Gibbs energy of formation for the neutral form of guanosine is equal to zero in aqueous solution at 298.15 K and zero ionic strength. This makes it possible to calculate apparent equilibrium constants for a number of enzyme-catalyzed reactions for which apparent equilibrium constants have not been measured or cannot be measured directly because they are too large. The eventual elimination of this convention is discussed. This adds ten reactants to the database BasicBiochemData3 that has 199 reactants. The standard transformed Gibbs energies of formation of these ten reactants are used to calculate apparent equilibrium constants at 298.15 K, 0.25 M ionic strength, and pHs 5, 6, 7, 8, and 9. The pKs, standard Gibbs energies of hydrolysis, and standard Gibbs energies of deamination are given for the reactants in the ATP, IMP, GTP, and XTP series.