20 research outputs found
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
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