Purine-nucleoside 5’-monophosphates

In the assay described by Amici et al. (1994), a wide variety of pyrimidine and purine nucleoside 5 -monophosphates were separated from their nucleosides by chromatography on a Supelco LClg guard column (4.6 mm x 20 mm, 5 fim). The short column allows separations in less than a minute. The mobile phase was 0.1 M potassium phosphate buffer (pH 6.0) except in the case of adenosine and deoxyadenosine, when 5% methanol was also included. The flow rate was 2 mL/min. Compounds were detected by monitoring the effluent at 254 nm, although sensitivity could be improved in some cases by using a different wavelength. 

Chemical Synthesis.—Purine nucleoside 5 -monophosphates enriched with or 0 on the phosphate group are conveniently prepared by treating phosphorus pentachloride in dry triethyl phosphate with one equivalent of the appropriately labelled water to give PO]- or P 0]P0Cl3, which is not isolated but mixed with adenosine or guanosine in the same solvent. Work-up of the resulting 5 -phos-phorodichloridate in similarly labelled water permits the formation of pO]-or P 0]AMP (or GMP) in fair yield with good enrichment. The 5 -monophos-phates of the 5 -C-methyl uridines derived from 6-deoxy-D-allose and 6-deoxy-L-talose have been prepared via phosphorylation of the 2, 3 -0,0-ethoxymethyl-idene derivative of the nucleosides (1) with -cyanoethyl phosphate and DCC or TPS-Cl. The same method has been used to phosphorylate iV -benzoylated 2, 3 -0,C -isopropylidene derivatives of various 5 -C-alkyladenosine species (2) and also 4 -allyladenosine (3) as part of a study in which derivatives of AMP... 

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine nucleoside monophosphates, in a reaction analogous to the formation of orotidylate. Two salvage enzymes with different specificities recover purine bases. Adenine phosphorihosyltransferase catalyzes the formation of adenylate... 

Cydic purine nucleoside monophosphates (cAMP and cGMP) are important second messengers in metabolic regulation (i.e. they carry messages within the cell, triggered by extracellular hormones). 

Subsequent phosphorylation reactions produce purine nucleoside diphosphates (ADP and GDP) and triphosphates (ATP and GTP). The purine nucleoside monophosphates, diphosphates, and triphosphates are all feedback inhibitors of the first stages of their own biosynthesis. Also, AMP, ADP, and ATP inhibit the conversion of IMP to adenine nucleotides, and GMP, GDP, and GTP inhibit the conversion of IMP to xanthylate and to guanine nucleotides (Figure 23.22). 

It is apparent from the above considerations that an accelerated rate of purine biosynthesis de novo can occur, therefore, as a result of either elevated levels of PRPP in the cell or decreased levels of purine nucleotides especially the purine nucleoside monophosphates and ADP. Indeed, it seems likely that in many, if not all, clinical conditions to be described, alterations in the levels of both PRPP and purine nucleotides contribute to the accelerated rate of purine biosynthesis. Despite this prediction, the data currently available would suggest that the intracellular concentration of PRPP ordinarily plays a more... 

A more careful comparison of the inhibitory effects of AMP and GMP on the E. coli enzyme has shown that AMP is a slightly more potent inhibitor than GMP (35). The AMP concentration in . coli is approximately 0.03 mM, or about one-third itsifi (35). GMP levels are likely about 0.01 mM (35), although values as high as 0.15 mM have been reported (79). Thus, the case for specific feedback inhibition of the enzyme by AMP is not clear-cut, although the enzyme is probably sensitive to increases in purine nucleoside monophosphate concentrations. 

Since the discussion livened up again about the formation of nucleotides for DNA-replication and DNA-repair by the contested theory of Werner (1971) it seems important to study the problems of nucleotide formation during repair processes. Our own studies performed with the radiosensitive organism Byssochlamys fulva and the radioresistant Pullularia pullulans led us to suppose that the synthesis of purine nucleoside monophosphates via purinephosphoribosyltransferase (purine-PRT) is not as sensitive to radiation as the de novo synthesis. Therefore a relation between the salvage pathway and nucleotide formation during repair processes does not seem impossible. 

Excess purine nucleotides or those released from DNA and RNA by nucleases are catabolized first to nucleosides (loss of P.) and then to free purine bases (release of ribose or deoxyribose). Excess nucleoside monophosphates may accumulate when ... 

Most biologically active purines or purine nucleosides must be anabolized to 5 -nucleotides to exert their effects. "The 5 -nucleotides are in some cases further phosphorylated to the di- and triphosphates and the number of potential sites of action of these compounds is obviously greater than the number of potential sites of action of compounds metabolized only to the monophosphate level. But even... 

The nucleotides are among the most complex metabolites. Nucleotide biosynthesis is elaborate and requires a high energy input (see p. 188). Understandably, therefore, bases and nucleotides are not completely degraded, but instead mostly recycled. This is particularly true of the purine bases adenine and guanine. In the animal organism, some 90% of these bases are converted back into nucleoside monophosphates by linkage with phosphori-bosyl diphosphate (PRPP) (enzymes [1] and [2]). The proportion of pyrimidine bases that are recycled is much smaller. 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

Table 7.1.4 Concentration range of purine and pyrimidine metabolites in urine (pmol/mmol creatinine) from patients. ADA Adenosine deaminase, APRT adenine phosphoribosyltransferase, ASA adenylosuccinate lyase, DHP dihydropyrimidinase, DPD dihydropyrimidine dehydrogenase, HGPRT hypoxanthine-guanine phosphoribosyltransferase, PNP purine nucleoside phosphorylase, TP thymidine phosphorylase, UMPS uridine monophosphate synthase, / -UP fi-ureidopropionase... 

All biosynthetic pathways are under regulatory control by key allosteric enzymes that are influenced by the end products of the pathways. For example, the first step in the pathway for purine biosynthesis is inhibited in a concerted fashion by nucleotides of either adenine or guanine. In addition, the nucleoside monophosphate of each of these bases inhibits its own formation from inosine monophosphate (IMP). On the other hand, adenine nucleotides stimulate the conversion of IMP into GMP, and GTP is needed for AMP formation. 

Didanosine is a synthetic purine nucleoside analog that inhibits the activity of reverse transcriptase in HIV-1, HIV-2, other retroviruses and zidovudine-resistant strains. A nucleobase carrier helps transport it into the cell where it needs to be phosphorylated by 5 -nucleoiidase and inosine 5 -monophosphate phosphotransferase to didanosine S -monophosphate. Adenylosuccinate synthetase and adenylosuccinate lyase then convert didanosine 5 -monophosphate to dideoxyadenosine S -monophosphate, followed by its conversion to diphosphate by adenylate kinase and phosphoribosyl pyrophosphate synthetase, which is then phosphorylated by creatine kinase and phosphoribosyl pyrophosphate synthetase to dideoxyadenosine S -triphosphate, the active reverse transcriptase inhibitor. Dideoxyadenosine triphosphate inhibits the activity of HIV reverse transcriptase by competing with the natural substrate, deoxyadenosine triphosphate, and its incorporation into viral DNA causes termination of viral DNA chain elongation. It is 10-100-fold less potent than zidovudine in its antiviral activity, but is more active than zidovudine in nondividing and quiescent cells. At clinically relevant doses, it is not toxic to hematopoietic precursor cells or lymphocytes, and the resistance to the drug results from site-directed mutagenesis at codons 65 and 74 of viral reverse transcriptase. 

Again we allow for competition between the proton and metal ion for the basic N(l) sites on purine and N(3) sites on pyrimidine 5 -nucleoside monophosphates to find for neutral solutions at pH 7.4 the sequence... 

The conversion of IMP to AMP and GMP is shown in Figure 10.8. Note that GTP is required for the biosynthesis of AMP and ATP for GMP biosynthesis, and high-ATP levels channel the conversion of IMP to GMP. In addition, IMP dehydrogenase is inhibited by GMP. We therefore have additional loci for controlling cellular concentrations of purine nucleotides by regulating the fate of IMP. The conversion of nucleoside monophosphates to di- and triphosphates is discussed later. 

The activity is found in erythrocytes, platelets, and lymphocytes, and determination of its value aids in diagnosis of some blood disorders. In this assay, which can readily be used for purine and pyrimidine 5 - and 3 -nucleotidase activities, the nucleoside monophosphate (the substrate) was separated from the nucleoside (the product) using ion-pair reversed-phase HPLC with a mobile phase of 5% methanol-5 mAf potassium dihydrogen phosphate 0.25 mAf 1-decanesulfonic acid was also added to the mobile phase. The elution was carried out at room temperature and the eluent monitored at 254 nm. 

Modified Celluloses AC-10 CM DEAE ECTEOLA PEI Antioxidants Inorganics, metal ions Nucleoside mono-, di-, and tri-phosphates Purines, pyrimidines, nucleosides Monophosphate nucleosides... 

Tenofovir is not metabolized to a significant extent by CYPs and is not known to inhibit or induce these enzymes. However, tenofovir has been associated with a few potentially important pharmacokinetic drug interactions. A 300-mg dose of tenofovir increased the didanosine AUC by 44 to 60% probably as a consequence of inhibition of the enzyme purine nucleoside phosphorylase by both tenofovir and tenofovir monophosphate. These two drugs probably should not be used together, or if this is essential, the dose of didanosine should be reduced from 400 to 250 mg/day. 

AMP and GMP can be phosphorylated to the di- and triphosphate levels. The production of nucleoside diphosphates requires specific nucleoside monophosphate kinases, whereas the production of nucleoside triphosphates requires nucleoside diphosphate kinases, which are active with a wide range of nucleoside diphosphates. The purine nucleoside triphosphates are also used for energy-requiring processes in the cell and also as precursors for RNA synthesis (see Fig. 41.2). 

Fig. 41.10. Salvage of bases. The purine bases hypoxanthine and gnanine react with PRPP to form the nucleotides inosine and gnanosine monophosphate, respectively. The enzyme that catalyzes the reaction is hypoxanthine-gnanine phosphoribosyltransferase (HGPRT). Adenine forms AMP in a reaction catalyzed by adenine phosphoribosyltransferase (APRT). Nucleotides are converted to nucleosides by 5 -nucleotidase. Free bases are generated from nncleosides by purine nucleoside phosphorylase. Deamination of the base adenine occurs with AMP and adenosine deaminase. Of the purines, only adenosine can be directly phosphorylated back to a nucleotide, by adenosine kinase.

Pyrimidine bases are normally salvaged by a two-step route. First, a relatively nonspecific pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their respective nucleosides (Fig. 41.17). Notice that the preferred direction for this reaction is the reverse phosphorylase reaction, in which phosphate is being released and is not being used as a nucleophile to release the pyrimidine base from the nucleoside. The more specific nucleoside kinases then react with the nucleosides, forming nucleotides (Table 41.2). As with purines, further phosphorylation is carried out by increasingly more specific kinases. The nucleoside phosphorylase-nucleoside kinase route for synthesis of pyrimidine nucleoside monophosphates is relatively inefficient for salvage of pyrimidine bases because of the very low concentration of the bases in plasma and tissues. 

S ATP + deoxyadenosine <1, 3, 5, 6> (<1> involved in biosynthesis of nucleoside monophosphates from preformed deoxyribonucleosides [4] <3> key anaboHc enzyme for activation of purine and pyrimidine deoxyriho-nucleosides as weU as cytosine arabinoside and other anti-tumour drugs [9] <5> involved in nucleoside metabolism [11] <6> dGK/dAK plays an essential role in generating the dexyribonucleotide precursors, dGTP and dATP, for DNA metabolism [12]) (Reversibility <1, 3, 5, 6> [4, 9, 11, 12]) [4, 9, 11, 12]... 

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