A simple assay of cyclic-AMP phosphodiesterase in the presence of interfering enzymes in Vibrio harveyi

PEI-cellulose thin layer chromatography is used to separate the reaction products formed from cyclic-AMP initiated by phosphodiesterase in crude extracts of bacteria.     

Rapid assay for cyclic AMP and cyclic GMP phosphodiesterases.

A rapid highly sensitive assay for cyclic AMP phosphodiesterase has been devised. After a 5-min incubation, cyclic AMP is readily resolved from 5′-AMP, adenosine, and inosine by ion-exchange thin-layer chromatography on 1.3 × 6.5-cm strips of PEI-cellulose for 7 to 8 min. This procedure combines the accuracy of the standard paper chromatography assay (1) with the speed of ion-exchange resin techniques (2), while surmounting some of the major drawbacks of the other two methods (3). Since chromatography on PEI-cellulose efficiently resolves cyclic GMP, 5′-GMP, and guanosine, this methodology has also been adapted to the measurement of cyclic GMP hydrolysis.     

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.     

Purine metabolism in Trypanosoma brucei gambiense

The procyclic forms of Trypanosoma brucei gambiense do not incorporate glycine or serine into ribonucleotides. Although de novo purine synthesis does not occur, all purine bases and ribonucleotides are interconverted, indicating the presence of active salvage pathways. Guanine is actively deaminated to xanthine by guanase activity. Purine ribonucleosides are cleaved to their respective free bases. The order of salvage efficiency for purine bases and their respective ribonucleotides is: adenine > hypoxanthine > guanine > xanthine.     

Chromatographic separation and identification of nucleotides from animal tissues and yeast cells on a polyethyleneimine cellulose column.

A procedure has been developed for the separation and identification of nucleotides by anion-exchange chromatography. The method includes column chromatography on PEI-cellulose with an unbuffered exponential salt gradient. This easily handled procedure gives a high degree of resolution and reproducibility and demands no personal attendance during elution. Application of the method is demonstrated with a mouse liver extract and extracts of yeast cells (Saccharomyces cerevisiae).     

Catabolic pathways of purine ribonucleotides and deoxyribonucleotides in lymphocytes

Deficiency of either one of the subsequent purine catabolic enzymes adenosine deaminase or purine nucleoside phosphorylase results in immunodeficiency disease in humans. However, the mechanism by which impairment of purine metabolism may cause immunodeficiency is unclear. In the present work we have studied the catabolism of purine ribonucleotides and deoxyribonucleotides in T lymphocytes to better understand the role of purine nucleoside phosphorylase and adenosine deaminase in the immune function. It was found that purine deoxyribonucleotides are degraded via catabolic pathways distinctly different from those used for purine ribonucleotide degradation. Thus both adenine and guanine ribonucleotides are deaminated to IMP whereas purine deoxyribonucleotides are exclusively dephosphorylated to the corresponding deoxyribonucleosides. These findings may explain the relatively higher degradation rates of purine deoxyribonucleotides in mammalian cells as compared to purine ribonucleotides. The catabolism of purine nucleotides is tightly linked to the active purine nucleoside cycles which consist of the phosphorolysis of purine nucleosides and deoxyribonucleosides to their corresponding bases, their salvage to monophosphates and back to the corresponding ribonucleosides. The above observations also imply that a possible role of the purine nucleoside cycles is to convert purine deoxyribonucleotides into their corresponding ribonucleotide derivatives. Deficiencies of purine nucleoside phosphorylase or of adenosine deaminase activities, enzymes which participate or lead to the purine nucleoside cycles, thus result in a selective impaired deoxyribonucleotide catabolism and immunodeficiency.     

Studies on the incorporation of precursors into purine and pyrimidine nucleotides via 'de novo' and 'salvage' pathways in normal lymphocytes and lymphoblastic cell-line cells

The incorporation of radioactive precursors into purine and pyrimidine nucleotides via 'de novo' and 'salvage' pathways was measured in normal lymphocytes, resting as well as proliferating, and lymphoblastic cell-line cells (MOLT-3). Lymphocytes stimulated with anti-CD3 were taken as actively proliferating lymphocytes (35% in the S-phase, 40 h after stimulation). The incorporation of the precursors in the purine and pyrimidine ribonucleotides was measured by a combination of anion-exchange high-performance liquid chromatography (HPLC) and on-line radioactivity measurement. The actively proliferating normal lymphocytes and MOLT-3 cells incorporated 30-500 times more of the various precursors in the ribonucleotides compared to normal resting lymphocytes. The imbalance in the nucleotide pool found in proliferating normal and lymphoblastic cells was reflected in the incorporation pattern of the various precursors. The activities of the branch-point enzymes IMP dehydrogenase and CTP synthetase most likely determine the differences in the composition of the nucleotide pools between resting and proliferating cells.     

The specificity of the interaction of bovine pancreatic ribonuclease A with natural and halogenated purine nucleotides.

The interaction between bovine pancreatic ribonuclease A (EC 3.1.4.22) and the purine nucleotides AMP, GMP, 6-chloropurine 5'-ribonucleotide and 8-bromoadenosine 5'-monophosphate was studied by u.v. difference spectroscopy. The stoicheiometry of the binding of the halogenated nucleotides to the enzyme shows a 1:1 ratio, as for the natural ones. The binding constants, Ka, for all four nucleotides at pH 5.5 were determined. They are within the same order of magnitude, though the nucleotides with a 6-amino group show a stronger interaction. The magnitude of the binding shows a reciprocal dependence on the ionic strength, which indicates an electrostatic interaction between ligand and enzyme. Finally, solvent-perturbation experiments show that all four nucleotides bind to the enzyme in a partially hydrophobic region. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule. The special synthesis and identification of 6-chloropurine 5'-ribonucleotide are discussed extensively. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule and thus the halogenated analogues are potential reagents for the affinity labelling of the purine-binding site.     

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucie gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine>inosine>guanosine>xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine>adenine>guanine>xanthine.     

Xanthine phosphoribosyltransferase from Streptococcus faecalis. Properties and specificity

Streptococcus faecalis (ATCC 8043) was shown to have a purine phosphoribosyltransferase specific for xanthine. This enzyme was separated from interfering activities by heat treatment, ammonium sulfate fractionation, hydroxylapatite chromatography, and affinity chromatography. The xanthine phosphoribosyltransfer activity of this preparation was stable between pH 5.6 and 10, had a pH optimum between pH 7.4 and 8.8, and had a particle weight of 42,000 as determined by G-100 Sephadex chromatography. An initial velocity analysis when plotted in double-reciprocal form resulted in a family of parallel lines which when extrapolated to infinite concentration gave K_m values for xanthine and PP-ribose-P of 20 and 53 μm, respectively. Inhibition studies with 42 purine and purine analogs indicated that oxo groups at positions 2 and 6 of the purine ring were required for optimal binding. The substitution of thio for oxo reduced binding to the enzyme ca. 20-fold. In contrast to its rigid specificity with respect to the 2,6-dioxo substituents, the enzyme bound a variety of 4,5-condensed pyrimidine systems containing a nitrogen at the position corresponding to the N-7 of xanthine. At concentrations of 1 mm, hypoxanthine, adenine, and 4,6-dihydroxypyrazolo[3,4-d]pyrimidine were converted to their corresponding ribonucleotides at rates approximately 0.1% of the rate for xanthine. Guanine was not detected as a substrate (rate <0.007% that of xanthine). The enzyme was inhibited by the ribonucleoside mono-, di-, and triphosphates of xanthine and guanine but not by those of adenine.     

Purine metabolism in Saccharomyces cerevisiae.

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) in sea urchin embryos

Sea urchin embryos were labeled with [3H]adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP4A fraction. The present result suggests that AP4A is actively synthesized in the sea urchin embryos.     

De novo purine ribonucleotide biosynthesis in adult Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

1. Radioactivity was found to be associated with purine ribonucleotides in extracts of gravid adult Angiostrongylus cantonensis from the lungs of rats when the parasite was incubated in a medium containing [14C]glycine. 2. The rate of incorporation was at least 0.2 pmole/min per mg of parasite and approximately equal amounts of adenine and guanine ribonucleotides were synthesized. 3. This is the first demonstration of the capacity for a mammalian parasitic helminth to synthesize purines de novo.     

Protein sequencing: thermal conversion of thiazolinone derivatives of amino acids to thiohydantoins.

A tlc method is presented for the separation on PEI-cellulose of adenine derivatives from the acid-soluble fraction of brain tissue. The procedure permits the quantitative estimation of radioactivity distributed among adenine nucleotides and the determination of ATP specific activity following labeling with [¹⁴C] adenosine. Two rapid one-dimensional separations produce excellent resolution of AMP, ADP, ATP, adenine, and adenosine from each other and from the corresponding guanosine derivatives. A two-dimensional procedure gives excellent separation of ATP from all other deoxy- and ribonucleotides. The recovery of the separated components by elution is better than 94% and requires few technical manipulations. The technique possesses the advantages of simplicity and sensitivity and is applicable to small amounts of biological material.     

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.     

Comparison of free ribonucleotide pool in the spleens of C57BL and DBA/2 mice and in leukemia cells sensitive and resistant to 5-fluorouracil

The amount of free purine and pyrimidine ribonucleotides in the spleens of mice (C57Bl and DBA/2) and in lympholeukemia cells (La and L1210), sensitive and with induced resistance to 5-fluorouracil, was determined by chromatography on a column with DEAE-cellulose. It was found that the cytidine ribonucleotide pool in the spleens of DBA/2 mice is 2 times lower as compared to C57Bl mice. The lympholeukemia cells (La and L1210) isolated from the animals also differed in their uridine nucleotide pools. The development of leukemia was accompanied by a decrease in ATP and GTP. No significant changes in the total amount of pyrimidine nucleotides under developing resistance to 5-fluororuacil were observed.     

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides. 3rd communication. Behaviour of 5-amino-1H-imidazole-4-carbonitrile derivatives

The potentially prebiotic synthesis of pyrimidine ribonucleotides by stepwise nucleobase assembly on arabinose-3-phosphate derivatives has been demonstrated in previous work. The generation of xylose-2-phosphate derivatives by aldolisation, and the behaviour of these compounds under the conditions of pyrimidine nucleobase assembly have also been described. In this paper, the scope for generation of purine nucleotides via 3,3'-anhydro-xylo-nucleotides is investigated. In neutral D2O solution, the potential intermediate 47 (Schemes 6 and 8) undergoes H-C2 --> D-C2 exchange, but no appreciable reaction with cyanide or cyanamide occurs. The exchange chemistry expands options for purine nucleobase assembly on sugar phosphate scaffolds.     

Effects of copper on mammalian cell components

Both deficiency and excess of copper induce toxic effects on mammalian cell systems in vivo and in vitro. The effects can be related to the affinities of Cu(II) ions for specific cell components. The nucleus is a potential site for temporary Cu storage while primary targets for free Cu(II) ions are the thiol groups which reduce the ions to Cu(I). Cu(II) ions show a high affinity for nucleic acids, binding with DNA both at intrastrand and interstrand levels, possibly through intercalation between GC pairs. The ability to chelate Cu(II) ions is seen to be of the order: purine greater than purine ribonucleotides greater than purine ribonucleoside greater than pyrimidine ribonucleotides. Copper is an integral part of enzyme activation and enters into the molecular structure of several proteins, like ceruloplasmin. Cu(II) ion is a potential mutagenic agent as seen by its property of inducing infidelity in DNA synthesis in vitro. Teratogenic activities of copper have been reported but carcinogenicity is not yet confirmed. Copper is an essential component of chromatin and is known to accumulate preferentially in the heterochromatic regions. External application of higher doses, however, induces both clastogenic effects and spindle disturbances. In certain forms, inorganic copper enhances the clastogenic activity of other agents. The most widely studied human genetic maladies linked with copper metabolism are Menkes' and Wilson's diseases. Several mutations are known which influence Cu homeostasis in mammals. Such mutations in mice have been used extensively for biochemical studies.