Diguanosine Tetraphosphate Research Articles

Dinucleoside polyphosphates: newly detected uraemic compounds with an impact on leucocyte oxidative burst

Dinucleoside polyphosphates (Np(n)N) have pathophysiologic roles in cardiovascular disease and are newly detected uraemic retention solutes. They were retrieved in human plasma, tissues and cells. Although their impact on several cell systems involved in vascular damage (endothelium, smooth muscle cells and thrombocytes) has been evaluated, their effect on different types of leucocytes has never been studied. This study evaluates, for the first time, the impact of Np(n)N on monocyte, granulocyte and lymphocyte oxidative burst activity at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) in whole blood. Diadenosine triphosphate (Ap(3)A) to diadenosine hexaphosphate (Ap(6)A) were tested to investigate the effect of the number of phosphate groups on reactive oxygen species (ROS) production. The effect of the type of nucleoside was evaluated by comparing adenosine guanosine tetraphosphate, diguanosine tetraphosphate, uridine adenosine tetraphosphate (Up(4)A) and diadenosine tetraphosphate (Ap(4)A). This study demonstrated that lymphocytes are especially susceptible to intracellular diadenosine polyphosphates. Depending on the phosphate chain length, different effects were observed. At baseline and with fMLP, Ap(4)A, Ap(5)A and Ap(6)A enhanced lymphocyted-free radical production. In addition, Ap(3)A, Ap(4)A and Ap(5)A increased PMA-stimulated ROS production in lymphocytes. Monocytes and granulocytes parallel the lymphocyte response albeit with an inhibition of Ap(6)A on granulocytes. Considering Np(n)N with four phosphate groups, Up(4)A showed the most important stimulatory effects on monocytes and Ap(4)A on lymphocytes. Np(n)N mainly have a leucocyte-activating impact, most significant for Ap(4)A, considering phosphate chain length, and for Up(4)A, considering the type of nucleosides. These results suggest that the pro-inflammatory effects of Np(n)N can contribute to the development of atherosclerosis, probably in the early stages of chronic kidney disease, but their chemical composition affects their activity.

Critical Role of the Heme Axial Ligand, Met95, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.

Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3′,5′-Cyclic Diguanylic Acid

Cyclic diguanylic acid (c-di-GMP; cGpGp) is a global second messenger controlling motility and adhesion in bacterial cells. Intracellular concentrations of c-di-GMP depend on two opposite activities: diguanylate cyclase, recently assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase, associated with proteins harboring the EAL domain. To date, little is known about the targets of c-di-GMP in the cell or if it affects transcriptional regulation of certain genes. In order to expand our knowledge of the effect of this molecule on the bacterial metabolism, here we report on the Escherichia coli transcriptional profile under high levels of c-di-GMP. We show that an important number of genes encoding cell surface and membrane-bound proteins are altered in their transcriptional activity. On the other hand, genes encoding several transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of motility and cell division genes were altered, and consistent with this was the physiological analysis of cells overexpressing yddV, a diguanylate cyclase; these cells displayed an abnormal cell division process when high levels of c-di-GMP were present. We also show evidence that the diguanylate cyclase gene yddV is co-transcribed with dos, a heme base oxygen sensor with c-di-GMP-specific phosphodiesterase activity. A delta dos::kan mutation rendered the cells unable to divide properly, suggesting that dos and yddV may be part of a fine-tuning mechanism for regulating the intracellular levels of c-di-GMP.

GTP:GTP guanylyltransferase: Trapping procedures for detecting and characterizing chemical nature of enzyme-nucleotide phosphoramidate reaction intermediate

This chapter focuses on trapping procedures for detecting and characterizing the chemical nature of enzyme-nucleotide phosphoramidate reaction intermediate. The chemical nature of the enzyme-nucleotide phosphoramidate intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. The chapter describes the formation and trapping of the enzyme-guanylate reaction intermediate along with experiments to establish the identity of the bound nucleotide and to determine that it represents a genuine reaction intermediate. The chemical stability of the intermediate yields information about the nature of the covalent linkage and may even suggest the identity of the amino acid involved. The intermediate in the ligases and capping enzymes involves an enzyme lysyl residue, while the others employ a histidyl residue. In the absence of sequence and structural information, as is the case with the Artemia Gp4G synthetase, characterization of the reaction intermediate can help in the tentative allocation of an enzyme to a particular protein family.

Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction.

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).

Formation of a CovalentNϵ2-Guanylylhistidyl Reaction Intermediate by the GTP:GTP Guanylyltransferase from the Brine ShrimpArtemia

The chemical nature of the enzyme–nucleotide phosphoramidate reaction intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets ofArtemia franciscanacysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. Labeling of the enzyme with [α-32P]GTP followed by isolation of the labeled phosphoamino acid by periodate treatment and alkaline hydrolysis and comparison of the product with phosphoamino acid standards by thin-layer and ion-exchange chromatography showed that the linkage involves the Nϵ2 ring nitrogen of an enzyme histidyl residue. Thus, this enzyme is distinct from the mRNA capping enzymes which can also synthesize Gp4G but which employ a lysyl–nucleotide intermediate. Based on its reaction mechanism and substrate specificity, GTP:GTP guanylyltransferase may belong to the GAFH superfamily which includes the histidine triad proteins, Ap4A phosphorylases, and galactose-1-phosphate uridylyltransferase.

Occurrence of millimolar concentrations of guanosine (5′)tetraphospho(5′)guanosine (Gp 4G) in encysted embryos of Thamnocephalus platyurus

One gram of encysted embryo of Thamnocephalus platyurus (an anostracan crustacea) contains around 17 μmoles of diguanosine tetraphosphate (Gp 4G) and 390 mg of protein, and traces of diguanosine triphosphate (Gp 3G). The identity of Gp 4G was established by its ultraviolet spectrum, insensitivity to alkaline phosphatase, analysis of its phosphodiesterase degradation products (GTP and GMP) by HPLC and, finally, by its 1H NMR characteristics.

Effect of diets containing adenosine, guanosine, inosine or xanthosine on the nucleotide content of Artemia. Influence of mycophenolic acid

Artemia uses the stored diguanosine tetraphosphate as a source of adenine and guanine nucleotides during development from the encysted gastrula to the free swimming larva. Further development of the larvae depends on a dietary source of purine rings. We have investigated the growth of Artemia in axenic cultures supplemented with 0.6 mg ml-1 of adenosine, guanosine, inosine or xanthosine. The total protein and soluble nucleotide content of Artemia grown in the presence of adenosine, guanosine or inosine was very similar, around (2 A260 units and 500 mg protein) and (4 A260 units and 1000 mg protein) after 4 and 6 days of postlarval development, respectively. The nucleotide pattern of those extracts subjected to HPLC were almost identical, the major peaks corresponding to ATP, ADP and AMP. Other nucleotides, not well characterized, were also present in those extracts. Mycophenolic acid (10 micrograms ml-1) inhibited the growth of Artemia (as measured by their protein and soluble nucleotide content) in the presence of adenosine and inosine as the purine source, and had no appreciable effect in the presence of guanosine. A quantitative analysis of the chromatographic peaks obtained from Artemia grown in the presence of any of the three nucleosides +/- mycophenolic acid showed that the effect of the antibiotic on each one of the chromatographic peaks was very similar, suggesting that Artemia, and probably other organisms as well, tend to maintain a balance between all nucleotides and to adjust the overall level to the limiting step(s) in their rates of synthesis/interconversion. Xanthosine was not able to support the development of Artemia.

Metabolic Heat Production by Artemia Embryos Under Anoxic Conditions

ABSTRACT The metabolic status of encysted Artemia embryos under anoxic conditions has been studied by calorimetry. Previous work demonstrated a very low, but still measurable, source of metabolic heat (31nWmg-1 drymass) after 6 days of anoxia, attributed to the catabolism of small amounts of carbohydrate storage compounds and the unusual nucleotide diguanosine tetraphosphate. Our results indicate that the heat measured drops below the detection limits of our calorimeter (11nWmg-1 drymass) after 1.5h of anoxia, in sharp contrast with previously published results. The possible sources of error that could explain this difference, and a re-evaluation of the metabolic status of cysts during anoxia, are discussed. We conclude that if the cysts are carefully protected from oxygen they become essentially ametabolic.

Human Placental ( Asymmetrical) Diadenosine 5′,5‴- P 1, P4-Tetraphosphate Hydrolase: Purification to Homogeneity and Some Properties

The diadenosine 5′,5‴- P 1, P 4-tetraphosphate ( asymmetrical) hydrolase (EC 3.6.1.17) from human placenta has been purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephecel, gel filtration on Sephadex G-100, and affinity elution from red Sepharose. The enzyme is a single polypeptide of M r 19,200. It exhibits maximum (100%) activity at pH 7.3 in the presence of 3mM MgCl 2 and 60, 50, and 40% of the activity in 1 mM CoCl 2, 0.1 mM ZnCl 2, and 0.5 mM MnCl 2, respectively. The K m value calculated for diadenosine tetraphosphate in the presence of Mg 2+ is 10 μM and in the presence of Zn 2+ 40 μM. Adenosine 5′-tetraphosphate, guanosine 5′-tetra-phosphate, and fluoride proved to he inhibitors of the diadenosine tetraphosphate hydrolase; the I 50 values were 6, 10, and 20 μM, respectively. Diguanosine tetraphosphate, bis-2,6-diaminopurine β-D-ribofuranoside tetraphosphate, and diadenosine pentaphosphate were substrates for the hydrolase; relative velocities of hydrolysis estimated for 0.5 mM diadenosine tetraphosphate and these other substrates were 1:0.51:0.44:0.20, respectively. Diadenosine tetraphosphate analogues with P 2 P 3 bridges such as CF 2, CCI 2, and CH 2 were hydrolyzed to adenosine 5′-phosphate and the corresponding adenosine 5′-triphosphate analogue. Interaction of the human placental diadenosine tetraphosphate hydrolase with diadenosine triphosphate and its phosphonate analogues was also tested. These compounds appeared to be competitive inhibitors with K i values lower than the K m for diadenosine tetraphosphate.

Heat dissipation during long-term anoxia inArtemia franciscana embryos: identification and fate of metabolic fuels

Microcalorimetric measurements of brine shrimp embryos during 6 days of anoxia indicated that heat dissipation was rapidly suppressed to 2.7% of control (aerobic) values over the first 9 h. Energy flow continued to decline slowly to 31 microW.g dry mass-1 (0.4% of control) during the subsequent 5.5 days. Within 2 h after returning anoxic embryos to aerobic conditions, heat dissipation rose to 77% of control rates. The calorimetric/respirometric (CR) ratio across this 2-h recovery period increased steadily from -226 to -346 kJ.mol O2-1). Prior to the anoxic exposures, hydrated embryos were incubated aerobically for 10 h to insure full initiation of carbohydrate metabolism (CR ratio = -484 kJ.mol O2-1). During the 6-day asymptotic approach to a nearly ametabolic state, trehalose and glycogen levels declined 18% and 13%, respectively. The majority of this utilization occurred within the first three days. Thermochemical calculations showed that carbohydrate catabolism accounted for 84% of the total heat dissipation measured over the 6-day anoxic bout; only 3% of the heat could be explained by the catabolism of diguanosine tetraphosphate (Gp4G). Analyses of embryo extracts by high performance liquid chromatography indicated that multiple acid end products were accumulated. Lactate and propionate reached 4.5 mM and 1.0 mM, respectively, but these compounds did not account quantitatively for the amount of carbohydrate utilized. However, the largest chromatographic peak that accumulated under anoxia has not been successfully identified. Fumarate and pyruvate levels decreased as anoxia proceeded. Thus, a perceptible energy flow in Artemia franciscana embryos still remained after 6 days of anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of guanosine nucleotide levels in thirteen populations of Artemia cysts

1. 1. Among 13 populations of Artemia cysts analyzed, the guanosine nucleotide content varied from 44 pmol/cyst (Thailand) to 125 pmol/cyst (Yugoslavia). 2. 2. The diguanosine tetraphosphate content of the Artemia cysts varied from 27 pmol/cyst (Thailand) to 78 pmol/cyst (Yugoslavia). 3. 3. The guanosine nucleotide content of Artemia cysts was significantly correlated ( r = 0.842) with the average cyst size (mass) among all populations analyzed irrespective of their mode of reproduction. 4. 4. A significant positive relationship was observed between the guanylate energy charge and cyst viability ( r = 0.623). 5. 5. No significant correlation ( r = 0.251) was found between guanylate energy charge and cyst size.

Short Communication Long-Term Anoxia in Artemia Cysts

ABSTRACT Among the many adaptations associated with the rigorous life history of the brine shrimp, Artemia, is the striking resistance of the encysted embryo (cyst) to oxygen lack (anoxia). Dutrieu and Chrestia-Blanchine (1966) reported that these cysts could be incubated in sea water under anoxic conditions for over 5 months without a decrease in viability. Subsequent work showed that anoxic cysts did not carry out a conventional lactate-producing metabolism and that the breakdown of trehalose, a disaccharide required for the energy metabolism of aerobic cysts, was not detected over an g-h period of anoxia (Ewing and Clegg, 1969). Comprehensive studies of the nucleotide pool by Stocco et al. (1972) suggested that utilization of the unusual guanine nucleotide diguanosine tetraphosphate (Gp4G) might provide the free energy presumably required to support the maintenance of anoxic cysts. Most recently, Hand and Gnaiger (1988) used calorimetric methods to show that anoxic energy metabolism, as reflected by heat production, was reduced to less than 2 % of aerobic values during short-term anoxia (10 h). Those authors also calculated that the utilization of Gp4G could account for only about 2 % of anoxic heat production and suggested that the very slow catabolism of trehalose might be a more likely explanation for their results. We examined that suggestion by measuring the concentrations of trehalose and other carbohydrates in cysts incubated under anoxic conditions for 3 months (Clegg and Jackson, 1989). We found no measurable change in trehalose and glycerol content under these conditions, but did observe a statistically significant decrease in the glycogen content of anoxic cysts. In the present paper we report comparable data from additional studies on anoxic cysts incubated for 3 and 6 months, and we measure the free amino acid pool, cyst dry mass and hydration.

Diguanosine 5′ ,5‴- P1 , P4-tetraphosphate and other purine nucleotides inhibit endoribonuclease VI from Artemia

The activity of the endoribonuclease VI from Artemia is sensitive to several purine nucleotides. The enzyme is non-competitively inhibited by diguanosine tetraphosphate ( K i = 75 μM), a nucleotide abundant in Artemia encysted gastrulae and located in the same particulate fraction as the gastrular ribonuclease. Diguanosine triphosphate and diadenosine tetraphosphate are less efficient inhibitors ( K i⋍200 μM). The ribonuclease is non-competitively inhibited by 5′-AMP ( K i = 10 μM) and 5′-GMP ( K i = 50 μM) but is insensitive to the corresponding 5′-phosphates of cytosine and uridine. Other purine mononucleotides inhibit the enzyme activity less efficiently. The modulation of the enzyme activity by these nucleotides is discussed in relation with the changes in ribonuclease activity during early development of Artemia.

Anaerobic Dormancy Quantified in Artemia Embryos: A Calorimetric Test of the Control Mechanism

Continuous measurement of heat dissipation from brine shrimp embryos during reversible transitions from aerobic development to anaerobic dormancy demonstrates a primary role for intracellular pH(pH(i))in this metabolic switching. Artificially elevating the depressed pH(i) during anoxia by adding ammonia markedly reactivates metabolism, as judged by increases in heat dissipation, trehalose catabolism, and the ratio of adenosine triphosphate to adenosine diphosphate. Energy flow during anaerobic dormancy is suppressed to 2.4 percent of aerobic values, which is the lowest percentage thus far reported for euryoxic animals. Use of diguanosine tetraphosphate stores cannot account for this observed heat dissipation. Thus, mobilizing trace amounts of trehalose may explain the energy metabolism during quiescence.

Diguanosine 5′,5‴ - P1, P4-tetraphosphate causes specific inhibition and desensitization of Artemia trypsin-like proteinase in the hydrolysis of a high-affinity, arginine-rich substrate

Diguanosine tetraphosphate (Gp 4G) inhibits Artemia trypsin-like proteinase. The inhibition is quite specific and presents a K i about 1 μM. Gp 4G modifies both maximum velocity and affinity of the proteinase. The proteinase, without losing activity, can be desentsitized to the inhibition by Gp 4G at low concentrations (1–10 μM). Gp 4G inhibits the hydrolysis of the high-affinity, arginine-rich substrate, protamine, to a lesser extent that of polylysine and does not affect the catalysis of other basic proteins. The results may suggest the involvement of Gp 4G in the regulation of the hydrolysis of polypeptides containing clusters of arginine.

IMP dehydrogenase from Artemia embryos: Molecular forms, purification and properties

1. 1. IMP dehydrogenase (EC 1.2.1.14) has been purified near homogeneity from Artemia embryos. 2. 2. The K m values for IMP and NAD + were 15 and 200 μM, respectively. 3. 3. GMP, XMP, GTP, guanosine 5′-tetraphosphate and diguanosine tetraphosphate (Gp 4G) were competitive inhibitors of the reaction towards IMP with K i values of 140, 180, 175, 120 and 87 μM, respectively. 4. 4. The enzyme from the 27,000 g supernatant can occur in a number of oligomeric forms (450, 375, 260 or 220 kDa) depending on the ionic strength of the medium. 5. 5. Upon precipitation with ammonium sulphate (0.3–0.4 saturation) the enzyme aggregates forming complexes of more than 1000 kDa.

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap 4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap 4A appeared to induce Michaelian kinetic behavior. The activation by Ap 4A was not dependent on the presence of either MgCl 2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.

Nucleoside diphosphate kinase from Artemia embryos

1.1. Nucleoside diphosphate kinase (EC. 2.7.4.6) has been purified 1900-fold with 17% yield from Artemia dormant embryos.2.2. The purified enzyme exhibits a ping-pong mechanism of reaction. The calculated Km values for ADP, GTP, ITP, UTP and CTP are 0.04, 0.17, 0.25, 0.27 and 0.62 mM, respectively.3.3. The role of this enzyme in the conversion of diguanosine tetraphosphate into ATP is also discussed. Maximum velocity of nucleoside diphosphate kinase (50 U/g dry cysts) is well in excess (around thousand-fold) over the enzymes which transform diguanosine tetraphosphate into AMP.

Binding of P1,P4-bis(5'-guanosyl)tetraphosphate to brain.

A brain preparation, consisting of nuclei and perikarya, was able to bind tritium-labeled diguanosine tetraphosphate ([3H]Gp4G). The binding was linear with both time and amount of extract and apparently presented two dissociation constant values (KD) of 0.16 and 0.6 microM, respectively, as determined by equilibrium binding experiments. Inhibition of [3H]Gp4G specific binding by 50% at equilibrium was accomplished by cold Gp4G, guanosine 5'-tetraphosphate, diguanosine triphosphate, and GTP at 0.7, 2.8, 3.1, and 10 microM concentrations, respectively. Diadenosine tetraphosphate (Ap4A) at concentrations up to 100 microM did not affect the observed binding of [3H]Gp4G to brain. These results suggest that this binding is specific and requires the existence of 4 phosphates plus at least 1 guanosine residue in the molecule. The binding of Gp4G to brain increased with time of development reaching a plateau at about 20 days after birth. The data are discussed in relation to previous results on the binding of Ap4A to brain and to DNA polymerase-alpha (Grummt, F., Waltl, G., Jantzen, H-M., Hamprecht, K., Huebscher, U., and Kuenzle, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6081-6085; Rapaport, E., Zamecnik, P. C., and Baril, E. F. (1981) Proct. Natl. Acad. Sci. U. S. A. 78, 838-842).