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Bulletin of the World Health... 1977Studies using erythrocyte-free preparations of P. berghei were conducted with a view to improving knowledge of parasite metabolism, particularly nucleotide metabolism.... (Comparative Study)
Comparative Study
Studies using erythrocyte-free preparations of P. berghei were conducted with a view to improving knowledge of parasite metabolism, particularly nucleotide metabolism. The free parasites employed in these studies were prepared by saponin lysis of parasitized mouse erythrocytes in isotonic glucose solutions. A comparative study of post-lytic metabolic activity of free parasites prepared by saponin, ammonium chloride, or osmotic lysis indicated a significantly greater retention of metabolic activity in the saponin-lysis preparations. Separations of nucleoside mono-, di-, and triphosphates extracted from free parasites were performed by means of high pressure liquid chromatography (HPLC), and ATP was additionally measured by luciferin-luciferase assay. Studies designed to differentiate among uptake, phosphorylation, and subsequent incorporation of (3)H-adenosine into nucleic acids of the free parasite strongly suggested that adenosine is metabolized either outside or on the parasite membrane, being first deaminated to inosine and then deribosylated to hypoxanthine. Observations from HPLC and radioisotope precursor studies support a hypothesis in which hypoxanthine may be proposed as being a pivotal substrate for purine salvage by malarial parasites. Some of the key steps in purine salvage and pyrimidine biosynthesis were investigated, using radiolabel uptake studies and HPLC analysis of nucleotides of the free malarial parasite. These studies suggest that hypoxanthine uptake may constitute an important new basis for chemotherapeutic attack on the malarial parasite.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Erythrocytes; Hemolysis; Malaria; Methods; Mice; Nucleotides; Plasmodium berghei; Purine Nucleotides
PubMed: 338182
DOI: No ID Found -
Biochimie Mar 2017Uncoupling protein 1 (UCP1) is an integral membrane protein found in the mitochondrial inner membrane of brown adipose tissue, and facilitates the process of... (Review)
Review
Uncoupling protein 1 (UCP1) is an integral membrane protein found in the mitochondrial inner membrane of brown adipose tissue, and facilitates the process of non-shivering thermogenesis in mammals. Its activation by fatty acids, which overcomes its inhibition by purine nucleotides, leads to an increase in the proton conductance of the inner mitochondrial membrane, short-circuiting the mitochondrion to produce heat rather than ATP. Despite 40 years of intense research, the underlying molecular mechanism of UCP1 is still under debate. The protein belongs to the mitochondrial carrier family of transporters, which have recently been shown to utilise a domain-based alternating-access mechanism, cycling between a cytoplasmic and matrix state to transport metabolites across the inner membrane. Here, we review the protein properties of UCP1 and compare them to those of mitochondrial carriers. UCP1 has the same structural fold as other mitochondrial carriers and, in contrast to past claims, is a monomer, binding one purine nucleotide and three cardiolipin molecules tightly. The protein has a single substrate binding site, which is similar to those of the dicarboxylate and oxoglutarate carriers, but also contains a proton binding site and several hydrophobic residues. As found in other mitochondrial carriers, UCP1 has two conserved salt bridge networks on either side of the central cavity, which regulate access to the substrate binding site in an alternating way. The conserved domain structures and mobile inter-domain interfaces are consistent with an alternating access mechanism too. In conclusion, UCP1 has retained all of the key features of mitochondrial carriers, indicating that it operates by a conventional carrier-like mechanism.
Topics: Adipocytes, Brown; Adipose Tissue, Brown; Animals; Cardiolipins; Energy Metabolism; Fatty Acids; Gene Expression Regulation; Humans; Ion Transport; Mitochondria; Models, Molecular; Protons; Purine Nucleotides; Thermogenesis; Uncoupling Protein 1
PubMed: 28057583
DOI: 10.1016/j.biochi.2016.12.016 -
Journal of Bacteriology May 1982Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases....
Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.
Topics: Adenine; Bacteria; Biological Transport, Active; Cell Membrane; Guanine; Hypoxanthines; Kinetics; Purine Nucleosides; Purine Nucleotides; Purines; Seawater; Water Microbiology
PubMed: 7068527
DOI: 10.1128/jb.150.2.471-482.1982 -
Molecular Metabolism Feb 2018Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine...
OBJECTIVE
Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation.
METHODS
With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist.
RESULTS
Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP) and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration.
CONCLUSION
Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation.
Topics: Adipocytes, Brown; Adrenergic beta-Agonists; Animals; Cells, Cultured; Male; Mice; Mice, Inbred C57BL; Oxygen Consumption; Purine Nucleotides; Thermogenesis; Uncoupling Protein 1
PubMed: 29310935
DOI: 10.1016/j.molmet.2017.12.010 -
Kidney International. Supplement Feb 2001The prevalence of hyperhomocysteinemia in renal disease patients, its treatment by folate administration, and its aggravation by the 677 C-->T mutation of... (Review)
Review
The prevalence of hyperhomocysteinemia in renal disease patients, its treatment by folate administration, and its aggravation by the 677 C-->T mutation of methylene-tetrahydrofolate (methylene-THF) reductase has established the folate cycle as an important factor in the pathogenesis and management of renal disease. Proper function of the folate cycle depends on normal function of involved enzymes adequate of the vitamin and its correct disposition within the body. Vital processes in folate disposition include conversion of dietary folylpolyglutamates to monoglutamates, intestinal absorption, receptor and carrier-mediated transport across cell membranes, and cellular export. Folate coenzymes are responsible for the one-carbon unit transfer in intermediary metabolism and are required for several reactions in key metabolic processes, for example of purine, pyrimidine and methionine synthesis, and glycine and serine metabolism. Methionine synthase and its recently discovered reducing protein as well as methylene tetrahydrofolate reductase are key folate enzymes in homocysteine metabolism. Deficiencies of these enzymes are important causes of severe disease in the rare remethylation defects causing homocystinuria. Knowledge of their catalytic and molecular properties is important in understanding possible causes of moderate hyperhomocysteinemia, as for example, the well-known 677 C-->T transition of methylene tetrahydrofolate reductase.
Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Aminohydrolases; Biological Transport, Active; Folic Acid; Formate-Tetrahydrofolate Ligase; Histidine; Homeostasis; Homocysteine; Humans; Intestinal Mucosa; Kidney Diseases; Metabolism, Inborn Errors; Methylenetetrahydrofolate Dehydrogenase (NADP); Methylenetetrahydrofolate Reductase (NADPH2); Multienzyme Complexes; Oxidoreductases; Polyglutamic Acid; Purine Nucleotides; Serine; Tetrahydrofolates
PubMed: 11169015
DOI: 10.1046/j.1523-1755.2001.59780221.x -
The New Phytologist Mar 2023In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain...
In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.
Topics: Adenosine Triphosphate; DNA; Inosine Triphosphate; Nucleic Acids; Purine Nucleotides; Pyrophosphatases; RNA
PubMed: 36464781
DOI: 10.1111/nph.18656 -
The Journal of Biological Chemistry Mar 2015Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated...
Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.
Topics: Adenylosuccinate Synthase; Benzimidazoles; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; HeLa Cells; Humans; IMP Dehydrogenase; Magnetic Resonance Spectroscopy; Metabolomics; Multienzyme Complexes; Purine Nucleotides; Purines; Spectrometry, Mass, Electrospray Ionization
PubMed: 25605736
DOI: 10.1074/jbc.M114.628701 -
The Journal of Biological Chemistry Oct 1987Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the...
Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.
Topics: Azaserine; Cells, Cultured; Fibroblasts; Glutamine; Humans; Hypoxanthine Phosphoribosyltransferase; Pentosephosphates; Phosphoribosyl Pyrophosphate; Purine Nucleotides; Purines; Ribose-Phosphate Pyrophosphokinase
PubMed: 2444588
DOI: No ID Found -
Biochemical Pharmacology Aug 2011Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that... (Comparative Study)
Comparative Study
Structure-activity relationships for the interactions of 2'- and 3'-(O)-(N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases.
Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2',3'-(O)-(N-methylanthraniloyl) (MANT)-substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5. (M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases hypoxanthine>uracil>cytosine>adenine∼guanine≫xanthine. Omission of a hydroxyl group at the 2' or 3'-position reduced inhibitor potency as did introduction of a γ-thiophosphate group or omission of the γ-phosphate group. Substitution of the MANT-group by an ANT-group had little effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1 homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure-activity relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity with no preference for the "cognate" base adenine as verified by enzyme inhibition, fluorescence spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate development of non-nucleotide inhibitors.
Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Catalytic Domain; Cell Line; Guanosine Triphosphate; Mammals; Models, Molecular; Protein Binding; Purine Nucleotides; Pyrimidine Nucleotides; Spectrometry, Fluorescence; Spodoptera; Structure-Activity Relationship; ortho-Aminobenzoates
PubMed: 21620805
DOI: 10.1016/j.bcp.2011.05.010 -
Proceedings of the National Academy of... Dec 2018The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although...
The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products. Recently, a potentially prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been demonstrated, raising the question of the suitability of the 8-oxo-purines as substrates for prebiotic RNA replication. Here we show that the 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension, both as activated monomers and when present in the template strand; their presence at the end of a primer also strongly reduces the rate and fidelity of primer extension. To provide a proper comparison with 8-oxo-inosine, we also examined primer extension reactions with inosine, and found that inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. We propose that inosine, which can be derived from adenosine by deamination, could have acted as a surrogate for G in the earliest stages of the emergence of life.
Topics: Inosine; Models, Molecular; Nucleic Acid Conformation; Nucleotides; Origin of Life; Purine Nucleotides; Purines; RNA
PubMed: 30509978
DOI: 10.1073/pnas.1814367115