Biosynthesis of purine nucleotides and methylated purines in higher plants.
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Antimetabolites↗
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Systematic variations in the content of the purine nucleotides in the steady-state perfused rat heart. Evidence for the existence of controlled storage and release of adenine nucleotides.
1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1mug/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3mumol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem.250, 6304-6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.
Adenine Nucleotides↗
Effect of histidine on purine nucleotide synthesis and utilization in Neurospora crassa.
Histidine affects de novo purine nucleotide synthesis and purine nucleotide pool utilization in Neurospora crassa. The former effect was assessed qualitatively by the presence or absence of purple pigment production in ad3B and ad3A mutants. Tryptophan also affected the de novo purine nucleotide synthesis. The effect of histidine on purine nucleotide pool utilization resulted in stimulated germination of ad8 and ad4 mutant conidia in adenine-deficient medium. Increased germination was correlated with increased net levels of nucleic acids in these strains. Possible mechanisms for the dual action of histidine are discussed.
Adenine↗
Purine nucleotide pyrophosphotransferase from Streptomyces morookaensis, capable of synthesizing pppApp and pppGpp.
Purine nucleotide pyrophosphotransferase was purified to apparent homogeneity from a culture filtrate of Streptomyces morookaensis. It is a monomeric protein with a molecular weight of 24 000-25 000, and its isoelectric point is 6.9. The enzyme synthesizes purine nucleoside 5'-phosphate (mono, di, or tri) 3'-diphosphates such as pppApp, ppApp, pApp, pppGpp, ppGpp and pppIpp by transferring a pyrophosphoryl group from the 5'-position of ATP, dATP and ppApp to the 3'-position of purine nucleotides. The purified enzyme catalysed the formation of 435 mumol of pppApp and 620 mumol of pppGpp from ATP and GTP per min mg protein under the standard conditions. The enzyme requires absolutely a divalent cation for activity, and optimum pH for the enzyme activity lay above 10 for Mg2+, for Co2+ and Zn2+ from 9 to 9.5, and for Fe2+ from 7.5 to 8. The following Michaelis constants were determined: AMP, 2.78 mM; ADP, 3.23 mM; GMP, 0.89 mM; GDP, 0.46 mM and GTP, 1.54 mM, in the case of ATP donor. The enzyme is inhibited by guanine, guanosine, dGDP, dGTP, N-bromosuccinimide, iodacetate, sodium borate and mercuric acetate.
Adenosine Triphosphate↗
Patterns of purine nucleotides in fish erythrocytes.
1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.
Animals↗
The purine nucleotide cycle in skeletal muscle.
The following evidence demonstrates that ammonia production in muscle occurs via the reactions of the purine nucleotide cycle: i) Extracts of cytosol which lack glutamine dehydrogenase produce ammonia under conditions that mimic muscle doing work. In such extracts a member of the purine nucleotide cycle (AMP, IMP, or adenylosuccinate) must be present in order that ammonia production can take place, ii) Perfused hindleg of rat or hindleg in situ produces ammonia during exercise, and there is a concomitant production of IMP and adenylosuccinate. Exercise causes a decrease in the contents of glutamate, aspartate, and glutamine and an increase in the content of alanine of perfused hindleg of rat. However, output of alanine does not change or is diminished during exercise. Glutamine output is diminished by exercise. Epinephrine increases the output and tissue content of ammonia and glutamine. It decreases the output and content of alanine, and it decreases the contents of aspartate and glutamate.
Adenosine Diphosphate↗
The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.
Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture.
AMP Deaminase↗
Synthesis and metabolic fate of purine nucleotides in cultured fibroblasts from normal subjects and from purine overproducing mutants.
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Adenine↗
Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.
A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.
Allosteric Regulation↗
The specificity of the interaction of bovine pancreatic ribonuclease A with natural and halogenated purine nucleotides.
The interaction between bovine pancreatic ribonuclease A (EC 3.1.4.22) and the purine nucleotides AMP, GMP, 6-chloropurine 5'-ribonucleotide and 8-bromoadenosine 5'-monophosphate was studied by u.v. difference spectroscopy. The stoicheiometry of the binding of the halogenated nucleotides to the enzyme shows a 1:1 ratio, as for the natural ones. The binding constants, Ka, for all four nucleotides at pH 5.5 were determined. They are within the same order of magnitude, though the nucleotides with a 6-amino group show a stronger interaction. The magnitude of the binding shows a reciprocal dependence on the ionic strength, which indicates an electrostatic interaction between ligand and enzyme. Finally, solvent-perturbation experiments show that all four nucleotides bind to the enzyme in a partially hydrophobic region. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule. The special synthesis and identification of 6-chloropurine 5'-ribonucleotide are discussed extensively. It is concluded that both halogenated and natural purine ribonucleotides interact in a similar manner with the enzyme molecule and thus the halogenated analogues are potential reagents for the affinity labelling of the purine-binding site.
Binding Sites↗
The purine nucleotide cycle. Control of phosphofructokinase and glycolytic oscillations in muscle extracts.
Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.
Adenosine Diphosphate↗
Increased purine nucleotide binding, altered polypeptide composition, and thermogenesis in brown adipose tissue mitochondria of cold-acclimated rats.
Rapid increases in atractyloside-insensitive binding of purine nucleotides (ADP or GDP) and in a polypeptide of 32 000 occur in brown adipose tissue mitochondria of the rat during acclimation to cold. The increased binding is apparent within 1 h and reaches a maximum after 3--7 days of exposure to 4 degrees C. The increase in the 32 000 peptide occurs more slowly and reaches a maximum after 2--3 weeks. There is a simultaneous decrease in a polypeptide of 96 000, apparent after 1 day and reaching a maximum after 1--2 weeks. Results are interpreted in terms of the appearance of an increased amount of the purine nucleotide-sensitive proton conductance pathway in association with the development of an enhanced thermogenic capacity of brown adipose tissue mitochondria during acclimation of the rat to cold.
Acclimatization↗
Effect of hepatocarcinogens on the adenine purine nucleotide cycle during the initiation phase of carcinogenesis.
Activities of the adenine purine nucleotide cycle enzymes, i.e., adenylosuccinate (SAMP) synthetase, SAMP lyase, and adenosine 5'-monophosphate deaminse, were determined in hepatic tissue of rats fed and/or given injections of 3'-methyl-4-dimethylaminoazobenzene, 4'-methyl-4-dimethylaminoazobenzene, thioacetamide, ethionine, or 2-acetylaminofluorene. SAMP lyase activity showed an early increase in all regimens containing hepatocarcinogens. Adenosine 5'-monophosphate deaminse showed increases with 3'-methyl-4-dimethylaminoazobenzene and thioacetamide but not with ethionine or 2-acetylaminofluorene. SAMP synthetase either was nonresponsive or else showed inhibition to the carcinogens. Increase in SAMP lyase activity was noted as early as 48 to 72 hr following i.p. injections of these carcinogens. The response of SAMP lyase was not duplicated by analogs of carcinogens such as 4'-methyl-4-dimethylaminoazobenzene or methionine. These data imply interaction of active carcinogens with SAMP lyase and to some extent adenosine 5'-monophosphate deaminse or to some mechanism responsible for their synthesis and/or release. This interaction may be a significant component of the initiation phase of carcinogenesis.
2-Acetylaminofluorene↗
Purine nucleotide metabolism in promastigotes of Leishmania tropica: inhibitory effect on allopurinol and analogues of purine nucleosides.
The catabolism of adenosine 5'-monophosphate in promastigotes of L. tropica suggests the presence of a parasite specific pathway. In continuing the investigation on enzymes of this pathway two purine nucleoside cleaving enzymes have been found, adenosine phosphorylase and inosine nucleosidase. Various purine nucleoside analogues inhibited the activity of both enzymes. A mode of action of the growth inhibition of L. tropica promastigotes by allopurinol has been suggested on the basis of Michaelis and inhibitor constants.
Allopurinol↗
Regulation of de novo purine synthesis in chick liver slices. Role of phosphoribosylpyrophosphate availability and of salvage purine nucleotide synthesis.
The differences between the uricotelic chick and the ureotelic rat, in the regulation of purine synthesis de novo, were studied in intact liver tissue. Chick liver, in comparison with rat liver, was found to contain a high activity of purine synthesis de novo, a high content and availability of 5-phosphoribosyl 1-pyrophosphate (PP-rib-P), comparable activity of PP-rib-P synthetase, and low activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and of adenine phosphoribosyltransferase (APRT). The results suggest that the intensive activity of the pathway of purine synthesis de novo in the chick liver is mediated by the high PP-rib-P concentration, which may be due at least in part to the relative partial deficiency of HGPRT.
Adenine↗
Effects of ethidium and isometamidium on adenosine triphosphate catabolism and purine nucleotide synthesis in Ehrlich ascites tumor cells in vitro.
Ethidium and isometamidium induce the breakdown of intracellular adenosine triphosphate in Ehrlich ascites tumor cells incubated in vitro. Ethidium induces appreciable adenosine triphosphate breakdown only when cells are incubated without glucose, whereas isometamidium produces this effect both in the presence and absence of glucose. In cells treated with isometamidium, purine nucleoside monophosphates accumulate, whereas these are mostly dephosphorylated when ethidium is used. Both ethidium and isometamidium inhibit purine nucleotide synthesis and incorporation of precursors into nucleic acids, although the magnitudes of these effects varied with the precursor used. Isometamidium inhibited the conversion of inosinate to adenine and guanine nucleotides, and both compounds partially inhibited the accumulation of phosphoribosyl pyrophosphate.
Adenine↗
The purine nucleotide cycle. Studies of ammonia production by skeletal muscle in situ and in perfused preparations.
Ammonia production by hind limbs of rat was studied in perfused preparations during rest, exercise, and hypoxia, and while the perfusate contained epinephrine or cyanide. Ammonia production by hind limbs was also studied in situ during rest and exercise. Strong correlations were observed between ammonia, IMP, and lactate formation. Adenylosuccinate was not present in detectable amounts in resting muscle (less than 1 nmol/g dry weight) but it appeared and rose sharply in exercising muscle, and then declined during recovery. The maximum found was about 18 nmol/g dry weight. When IMP accumulation exceeded 2.8 mumol/g dry weight, the formation of adenylosuccinate diminished, presumably because of an inhibition of adenylosuccinate synthetase by excess substrate. Exercise resulted in a greatly increased output of ammonia and a decreased output of alanine and glutamine. The tissue contents of aspartate and glutamate were decreased, while that of alanine was increased. The content of malate increased 3-fold during exercise. Epinephrine, hypoxia, and cyanide each caused an increased output of ammonia at the expense of the amino acid content of the tissue. These results provide evidence for the operation of the purine nucleotide cycle in skeletal muscle under various conditions that are associated with an increased rate of glycolysis.
Adenosine Monophosphate↗
Purine nucleotide cycle. Evidence for the occurrence of the cycle in brain.
Cell-free extracts of rat brain catalyze the reactions of the purine nucleotide cycle. Ammonia is formed during the deamination but not the amination phase of the cycle. The activity of adenylate deaminase in brain is sufficient to account for the maximum rates of ammonia production that have been reported. The activity of glutamate dehydrogenase is not sufficient to account for these rates of ammonia production. The activities of adenylosuccinate synthetase and adenylosuccinase are nearly sufficient to account for the steady state rates of ammonia production observed in brain. Demonstration of the cycle in extracts of brain is complicated by the occurrence of side reactions, in particular those catalyzed by phosphomonoesterase, nucleoside phosphorylase, and guanase.
4-Nitrophenylphosphatase↗