Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

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.     

Purine deoxynucleoside salvage in Giardia lamblia

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines, including deoxythymidine. Dependence on deoxynucleoside salvage is extremely unusual among eucaryotic cells (Moore, E. C., and Hurlbert, R. B. (1985) Pharmacol & Ther. 27, 167-196). The present study investigates the possibility that giardia lacks ribonucleotide reductase and depends entirely on deoxynucleoside salvage. A ribonucleotide reductase inhibitor, hydroxyurea, at concentrations up to 2 mM had no effect on the growth of giardia. This is 15-20 times the ED50 of hydroxyurea for the protozoans Trypanosoma cruzi, Trypanosoma gambiense, and Leishmania donovani. A lysate of giardia had no detectable ribonucleotide reductase. Although radiolabeled adenine, adenosine, guanine, and guanosine were readily incorporated into RNA by cultured cells, no adenine or adenosine and only trace amounts of guanine and guanosine were detectable in DNA. This is in contrast to deoxynucleosides, where 58% of deoxyadenosine and 10% of deoxyguanosine incorporated into nucleic acid were found in DNA. Phosphorylation of both deoxyadenosine and deoxyguanosine was catalyzed by a cell lysate of giardia when nucleoside kinase co-substrates were included in the assay but not when phosphotransferase co-substrates were present. The absence of detectable ribonucleotide reductase, the failure to incorporate purine nucleobases and nucleosides into DNA to any significant extent, the ready incorporation of deoxynucleosides into DNA, and the demonstration of a purine deoxynucleoside kinase suggest that giardia are dependent on the salvage of exogenous deoxynucleosides.     

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.     

Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation

The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 microM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 microM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 microM deoxyguanosine was inhibited approximately 90% by 10 microM deoxycytidine or dCTP and was inhibited 70% by 200 microM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 microM, phosphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 microM, was partially inhibited by 200 microM adenosine, 34%; 200 microM deoxyguanosine, 10%; and 100 microM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 microM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.     

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

Purine metabolism in Toxoplasma gondii

We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.     

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.     

Pentatrichomonas hominis: purine salvage pathway

1. Pentatrichomonas hominis was found incapable of de novo synthesis of purines. 2. Pentatrichomonas hominis can salvage adenine, guanine, hypoxanthine, adenosine, guanosine and inosine, but not xanthine for the synthesis of nucleotides. 3. HPLC tracing of radiolabelled purines or purine nucleosides revealed that adenine, adenosine and hypoxanthine are incorporated into adenine nucleotides and IMP through a similar channel while guanine and guanosine are salvaged into guanine nucleotides via another route. There appears to be no direct interconversion between adenine and guanine nucleotides. Interconversion between AMP and IMP was observed. 4. Assays of purine salvage enzymes revealed that P. hominis possess adenosine kinase; adenosine, guanosine and inosine phosphotransferases; adenosine, guanosine and inosine phosphorylases and AMP deaminase.     

Purine metabolism in Acholeplasma laidlawii B: novel PPi-dependent nucleoside kinase activity

Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion. Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown. Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside. Adenosine, inosine, xanthosine, and guanosine were converted to their base. No ATP-dependent phosphorylation of nucleosides to mononucleotides was found. However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected. Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found. Interconversion of GMP to AMP was detected. Enzyme activities for the interconversion of AMP to GMP were not detected. Therefore, A. laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates. De novo synthesis of purines was not detected. This study demonstrates that A. laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp. mycoides. This is the first report of a PPi-dependent nucleoside kinase activity in any organism.     

Human placental deoxyadenosine and deoxyguanosine phosphorylating activity

We studied deoxyadenosine and deoxyguanosine phosphorylating activities in human placental cytosol. The specific activities of nucleoside kinase enzymes in nanomoles per h per mg +/- SD were as follows: adenosine kinase, 30 +/- 14; deoxyadenosine kinase, 12 +/- 2; deoxycytidine kinase, 0.30 +/- 0.04; and deoxyguanosine kinase, 27 +/- 16. Three major activities were resolved by ion exchange and affinity chromatography: deoxyguanosine-deoxycytidine kinase, deoxycytidine-deoxyadenosine kinase, and adenosine-deoxyadenosine kinase. Two other activities contained significant quantities of deoxyadenosine kinase. Deoxyguanosine-phosphorylating activity eluted as a single peak in association with deoxycytidine kinase. This deoxyguanosine-deoxycytidine kinase had an apparent molecular weight of 54,000, a Stokes radius of 31 A, and apparent Km values of 10, 130, and 14 microM for deoxyguanosine, deoxycytidine, and ATP, respectively. Four peaks of deoxyadenosine phosphorylating activity were resolved by affinity chromatography with AMP-Sepharose 4B. Adenosine-deoxyadenosine kinase had an apparent molecular weight of 38,000, a Stokes radius of 27.4 A, and apparent Km values of 0.4, 510, and 75 microM for adenosine, deoxyadenosine, and ATP, respectively. Attempts to distinguish whether adenosine-deoxyadenosine kinase was one enzyme with these two activities or two separate enzymes suggested that the former was the case. Deoxycytidine-deoxyadenosine kinase had apparent Km values of 0.7, 670, and 12 microM for deoxycytidine, deoxyadenosine, and ATP, respectively. Its apparent molecular weight was estimated to be 49,000 and its Stokes radius 30 A. Two other minor peaks of deoxyadenosine-phosphorylating activity had characteristics different from either deoxycytidine kinase or adenosine kinase-associated deoxyadenosine kinase. Our studies indicate that human placental cytosol contains a complex mixture of nucleoside kinase enzymes.     

Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.     

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.     

End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli

Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.     

Helicobacter pylori relies primarily on the purine salvage pathway for purine nucleotide biosynthesis

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori. Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an Δapt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway.     

Adenosine, deoxyadenosine, and deoxyguanosine induce DNA cleavage in mouse thymocytes

When thymocytes were cultured with adenosine, deoxyadenosine, or deoxyguanosine at 1 mM for 24 h, DNA cleavage at internucleosomal sites with multiples of approximately 180 bp was induced, followed by lactate dehydrogenase release into the medium. In the presence of coformycin, an adenosine deaminase inhibitor, or formycin B, a purine nucleoside phosphorylase inhibitor, DNA cleavage was induced by these nucleosides at concentrations of less than 50 microM. Other purine and pyrimidine ribo- and deoxyribonucleosides did not induce DNA cleavage or LDH release. Because thymocyte nuclei contain a Ca2+,Mg2+-dependent endonuclease, which preferentially cuts DNA in its linker regions, DNA fragmentation induced by the three purine nucleosides was suggested to occur through increased activity of the endonuclease. The DNA cleavage induced by the nucleosides required protein phosphorylation and synthesis, inasmuch as it was inhibited by an inhibitor of protein kinases, H-7, and by an inhibitor of protein synthesis, cycloheximide. The inhibition of DNA cleavage was accompanied by a reduction in lactate dehydrogenase release, suggesting a causal relationship between DNA cleavage and cell death. The DNA cleavage and subsequent cell lysis might be related to the selective thymocyte deletion observed in patients with adenosine deaminase or purine nucleoside phosphorylase deficiency.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Deoxyguanosine toxicity on lymphoid cells as a cause for immunosuppression in purine nucleoside phosphorylase deficiency.

To delineate the pathogenesis of the immunodeficiency disease associated with purine nucleoside phosphorylase deficiency, the effects of guanosine, inosine, deoxyguanosine and deoxyinosine on the growth of a mouse T cell lymphoma line in culture were studied. Of these four purine nucleosides, deoxyguanosine was the most toxic. At 5 x 10^?6 to 10^?5 M, deoxyguanosine inhibits growth of the lymphoma cells; higher concentrations result in complete killing. The cytotoxic effects of this deoxynucleoside can be prevented by simultaneous addition to culture medium of deoxycytidine and hypoxanthine. Determination of nucleotide pools in deoxyguanosine-treated cells shows a marked reduction of the deoxycytidine triphosphate and the adenine ribonucleotide pools, accompanied by a sharp rise in the guanosine deoxyribonucleotide and a smaller increase in the corresponding ribonucleotide pools.Deoxyguanosine as well as guanosine, inosine and deoxyinosine were known to accumulate to relatively high levels in the plasma of a patient with T cell immunodeficiency disease associated with purine nucleoside phosphorylase deficiency. The other three purine nucleosides are much less toxic than deoxyguanosine. Thus it is very probable that the patient's clinical manifestations of T lymphocytopenia are the consequence of deoxyguanosine inhibition of lymphoid cell proliferation, resulting from depletion of deoxycytidine triphosphate and adenine nucleotides.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.