The Regulation of Brain Nucleoside Utilization

The homeostatic regulation of intracellular purine and pyrimidine pools has long been studied at the level of de novo nucleotide synthesis. However, brain maintains the proper qualitative and quantitative nucleotide balance by salvaging preformed nucleosides, imported from blood stream, rather than by de novo synthesis from simple precursors. The main salvage enzymes are the nucleoside-kinases, catalyzing the ATP mediated phosphorylation of nucleosides in their 5’-position. Salvaged nucleoside-monophosphates are then either further phosphorylated, or converted back to nucleosides by a set of 5’-nucleotidases. This poses the following problem: why are nucleosides produced from nucleosidemonophosphates, to be converted back to the same compounds at the expense of ATP? As discussed in this article, the quantitative and qualitative intracellular balance of brain purine and pyrimidine compounds is maintained i) by the intracellular interplay between the rates of nucleoside-kinases and 5’-nucleotidases, ii) by the relative rates of the inward and outward nucleoside transport through equilibrative and concentrative transport systems, iii) by the metabolic cross-talk between extracellularly exported nucleoside-triphosphate breakdown and the intracellular process of nucleoside-triphosphate salvage synthesis

Purine and Pyrimidine Salvage in Whole Rat Brain. Utilization of ATP-derived Ribose-1-Phosphate and 5-Phosphoribosyl-1-pyrophosphate Generated in Experiments with Dialyzed Cell-free Extracts

The object of this work stems from our previous studies on the mechanisms responsible of ribose-1-phosphate- and 5-phosphoribosyl-1-pyrophosphate-mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L., Cappiello M., Cherri, S., and Ipata, P. L. (2000) Biochim. Biophys. Acta 1474, 70-74; Mascia, L., Cotrufo, T., Cappiello, M., and Ipata, P. L. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that when ATP at "physiological concentration" is added to dialyzed extracts of rat brain in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil-, and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nueleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase, and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1-pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L., and Ipata, P. L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our in vitro results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5-phosphoribosyl-1-pyrophosphate and ribose-1-phosphate pools, to be utilized for nucleotide resynthesis during reperfusion

Recent advances in structure and function of cytosolic IMP-GMP specific 5′nucleotidase II (cN-II)

Cytosolic 5′nucleotidase II (cN-II) catalyses both the hydrolysis of a number of nucleoside monophosphates (e.g., IMP + H2O→inosine + Pi), and the phosphate transfer from a nucleoside monophosphate donor to the 5′position of a nucleoside acceptor (e.g., IMP + guanosine →inosine + GMP). The enzyme protein functions through the formation of a covalent phosphoenzyme intermediate, followed by the phosphate transfer either to water (phosphatase activity) or to a nucleoside (phosphotransferase activity). It has been proposed that cN-II regulates the intracellular concentration of IMP and GMP and the production of uric acid. The enzyme might also have a potential therapeutic importance, since it can phosphorylate some anti-tumoral and antiviral nucleoside analogues that are not substrates of known kinases. In this review we summarise our recent studies on the structure, regulation and function of cN-II. Via a site-directed mutagenesis approach, we have identified the amino acids involved in the catalytic mechanism and proposed a structural model of the active site. A series of in vitro studies suggests that cN-II might contribute to the regulation of 5-phosphoribosyl-1-pyrophosphate (PRPP) level, through the so-called oxypurine cycle, and in the production of intracellular adenosine, formed by ATP degradation

What is the true nitrogenase reaction? A guided approach

Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2. In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) NBN triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H1 released by the hydrolysis of the 16 ATP molecules needed to fix the atmospheric N2 VC 2015 by the International Union of Biochemistry and Molecular Biology, 43(3):142–144, 2015

Metabolic regulation of uridine in the brain

The importance of nucleoside metabolism in brain followed the recognition that i) adult nervous system maintains its nucleotide pools in the proper qualitative and quantitative balance by salvaging preformed purine and pyrimidine rings, rather than by synthesizing nucleosides de novo from simple precursors, ii) adenosine, a purine nucleoside, acts as an extracellular signal, and exerts its protective effects by interacting with plasmamembrane bound purinergic G-protein coupled P2X receptors. More recently uridine, a pyrimidine nucleoside, has received considerable attention. Most of the uridine content of brain is supplied by its uptake from the plasma. An increasing body of evidence suggests that uridine exerts its function intracellularly in three distinct ways. It is phosphorylated to UTP, a pyrimidine nucleotide acting as a precursors for RNA and DNA synthesis, and as an extracellular neurotrophic signal. In combination with the -3 fatty acid decosahexaenoic acid and choline, uridine accelerates formation of synaptic membrane, being an obligatory precursor for CDP-choline synthesis. Finally, uridine can preserve the ATP pool via the conversion of its ribose-1-phosphate moiety into energetic intermediates of glycolysis. This article summarizes our present knowledge on uridine metabolism in the brain, with special emphasis on the mechanisms maintaining its intracellular homeostasis and on the cross talk between intracellular and extracellular uridine metabolism

Nucleoside recycling in the brain and the nucleosidome: a complex metabolic and molecular cross-talk between the extracellular nucleotide cascade system and the intracellular nucleoside salvage

The transports of nucleosides from blood into neurons and astrocytes are essential prerequisites to enter their metabolic utilization in brain. Adult brain does not possess the de novo nucleotide synthesis, and maintains its nucleotide pools by salvaging preformed nucleosides imported from liver. Once nucleosides enter the brain through the blood brain barrier and the nucleoside transporters, they become obligatory precursors for the synthesis of RNA and DNA and a plethora of other important functions. However, an aliquot of nucleotides are transferred into vesicular nucleotide transporters, and then in the extracellular space by exocytosis of the vesicles, where ATP and UTP interact with a vast heterogeneity of purine and pyrimidine receptors. Their signal actions are terminated by the ectonucleotidase cascade system, which degrades ATP and UTP into adenosine and uridine, respectively. The low specificity of the vesicular nucleotide transporters may explain the presence in the extracellular space of GTP and CTP, which are equally degraded to their respective nucleosides by the ectonucleotidases. The main four nucleosides are re-imported either into the same cell, or in adjacent cells, e.g. between two astrocytes, or between a neuron and an astrocyte, to regenerate nucleoside triphosphates. The molecular network of this metabolic cross-talk, involving the ectonucleotidases, the nucleoside transporters, the nucleotide salvage system, the nucleotide transport into the vesicular nucleotide transporters, and the exocytotic release of nucleotides, called by us the ‘‘nucleosidome’’, serves the nucleoside recycling in the brain, with a considerable spatial–temporal advantage