Effect of D-galactosamine on nucleotides in the rat heart. Effects of cytidine and uridine administration

The treatment of rats by galactosamine (2 mmol/kg i.p.), which dramatically alters the metabolism of pyrimidine nucleotides in the liver, has been used to investigate the dynamics of pyrimidine nucleotides in the rat heart. Six hours after administration of the drug, the UTP and UDPG myocardial contents were decreased by respectively 40 and 52% while the sum of uracil nucleotides was increased by 66% and that of cytosine nucleotides by 15%. When administered 5 h after galactosamine treatment, cytidine (750 nmol/rat i.v.) induced a further increase in cytosine nucleotides (46% above control value 1 h later) without however effect on uracil nucleotides. On the other hand, the administration of uridine (250 nmol/rat, i.v. 5 h after galactosamine), while restoring UTP, UDPG and the pool of uracil nucleotides, provoked a decrease in cytosine nucleotide level (-17%). In the absence of galactosamine treatment, the administration of uridine and cytidine did not induce changes in nucleotide levels despite a rise in blood cytidine concentration. All these observations support the hypothesis that: 1. the pathway for cytosine nucleotide synthesis predominant in the heart is that utilizing preformed exogenous cytidine and 2. this pathway is mainly controlled by the intracellular concentration of UTP rather than that of CTP.     

Borrelia burgdorferi uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B. burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.     

Synthesis of phosphonate derivatives of uridine, cytidine, and cytosine arabinoside

The vinyl phosphonate derivatives of uridine, cytidine, and cytosine arabinoside (ara-C) have been prepared through oxidation of appropriately protected nucleosides to the 5' aldehydes and Wittig condensation with [(diethoxyphosphinyl)methylidine]triphenylphosphorane. Dihydroxylation of these vinyl phosphonates with an AD-mix reagent generated the new 5',6'-dihydroxy-6'-phosphonates. After hydrolysis of the phosphonate esters and the various protecting groups, the six phosphonic acids were tested for their ability to serve as substrates for the enzyme nucleotide monophosphate kinase and for their toxicity to K562 cells.     

Uridine-cytidine kinase III. Competition between uridine and cytidine for a single enzyme

Summary The combined phosphorylation of uridine and cytidine by a partially purified preparation of uridine-cytidine kinase has been studied with dual-substrate kinetics. The kinetic patterns obtained are consistent with the theoretical analysis for two competing, alternate substrates interacting with a single enzyme. Thus, despite feedback regulation of the kinase by both UTP and CTP, the results allow a clear conclusion that both nucleosides are phosphorylated by the same enzyme, and probably at a single site, rather than by two closely related isozymes, each specific for one pyrimidine.A preliminary report of these results has been presented (Proc. Amer. Assoc. Cancer Res.17 : 78, 1976).     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

Synthesis, salvage, and catabolism of uridine nucleotides in boron-deficient squash roots

Previous work has provided evidence that plants may require boron to maintain adequate levels of pyrimidine nucleotides, suggesting that the state of boron deficiency may actually be one of pyrimidine starvation. Since the availability of pyrimidine nucleotides is influenced by their rates of synthesis, salvage, and catabolism, we compared these activities in the terminal 3 centimeters of roots excised from boron-deficient and -sufficient squash plants (Cucurbita pepo L.). Transferring 5-day-old squash plants to a boron-deficient nutrient solution resulted in cessation of root elongation within 18 hours. However, withholding boron for up to 30 hours did not result in either impaired de novo pyrimidine biosynthesis or a change in the sensitivity of the de novo pathway to regulation by end product inhibition. Boron deprivation had no significant effect on pyrimidine salvage or catabolism. These results provide evidence that boron-deficient plants are not starved for uridine nucleotides collectively. Whether a particular pyrimidine nucleotide or derivative is limiting during boron deprivation remains to be examined.     

Determination of pyrimidine phosphoribosyltransferase and uridine kinase activities by an assay with DEAE-paper discs

Rapid (6 min washing step) and reliable methods for determining uridine kinase and pyrimidine phosphoribosyltransferase activities were devised. These procedures, consisting of a spotting technique on DEAE-discs followed by washing and elution, permitted the consistent recovery of about 90% of the nucleoside 5'-phosphate esters formed from radioactive precursors, either uridine or 5-fluorouracil, respectively. Of these precursors, less than 2% were retained on the discs. Direct counting of the discs (without elution), filtration by either gravity or suction, and the use of so-called activation of discs have not proved advantageous.     

Blood uridine concentration may be an indicator of the degradation of pyrimidine nucleotides during physical exercise with increasing intensity

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.     

Regulation of rat-liver glycoprotein: N-acetylneuraminic acid transferase activity by pyrimidine nucleotides.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N-acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N-Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide-sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.