Dephosphorylation Of Nucleotides Research Articles

Effect of trimetazidine on the nucleotide profile in rat kidney with ischemia–reperfusion injury

Ischemia–reperfusion injury is often responsible for delayed graft function after transplantation. Trimetazidine (TMZ) is an antioxidant agent used to protect grafts from ischemia–reperfusion injury. The aim of the study was to examine the effect of TMZ on nucleotide profile in rat kidney with ischemia–reperfusion injury. The study was carried out on Wistar rats divided into two groups: animals treated with TMZ and control group receiving placebo. TMZ 10 mg/kg/day was administrated for 30 days. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (Ado), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), guanosine (Guo), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), uridine (Urd), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) were determined in kidney tissues after ischemia–reperfusion using HPLC. The total adenine nucleotide concentration (TAN) and adenylate energy charge (AEC) were also determined. Moreover the kidneys were evaluated histologically. Tissue concentrations of ATP, ADP, AMP, TAN and AEC were significantly increased in kidneys from rats treated with TMZ in comparison with rats receiving placebo. Concentrations of products of nucleotide degradation: inosine (Ino), guanosine (Guo) and uridine (Urd), as well as oxypurines: Hyp and Xan, were significantly decreased in rats treated with trimetazidine. Moreover, significantly less pronounced acute tubular necrosis was observed in kidneys of rats treated with TMZ. These results suggest that trimetazidine protects against dephosphorylation of nucleotides and ischemic damage.

Mammalian 5′(3′)-Deoxyribonucleotidase, cDNA Cloning, and Overexpression of the Enzyme in Escherichia coli and Mammalian Cells

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

Ascorbate Preserves Gastric Mucosal Metabolism and Microcirculation after Hemorrhagic Shock and Retransfusion in Rats

The gastric mucosal microcirculation and purine nucleotide metabolism were studied in rats after hemorrhagic shock and retransfusion. The mucosal surface density of perfused vessels (SDPV) and the mucosal levels of ATP, ADP, AMP, IMP, hypoxanthine and uric acid were measured following 15 min of hemorrhagic shock and 10 and 30 min after retransfusion, and the effects of pretreatment with allopurinol or ascorbate were studied. During shock there was a dephosphorylation of nucleotides and a decline in the SDPV. Retransfusion led to an additional reduction in the SDPV, but a complete restoration of preshock nucleotide levels 30 min after retransfusion. Allopurinol accelerated early rephosphorylation of nucleotides without effects upon SDPV while ascorbate completely preserved the mucosal level of energy-rich nucleotides 15 min after hemorrhagic shock and increased SDPV during early reperfusion. The results showed that there was a renewal of energy stores in gastric mucosa after hemorrhagic shock and retransfusion although parts of the vascular bed were not reperfused. The mucosal energy depletion after 15 min of hemorrhagic shock and part of the mucosal vessel injury after retransfusion were prevented by pretreatment with ascorbate.

Gastric mucosal microcirculation and purine nucleotide metabolism after retransfusion of rats in hemorrhagic shock.

Gastric mucosal microcirculation and purine nucleotide metabolism were studied after hemorrhagic shock and retransfusion in rats. The number of perfused microvessels and the concentration of adenosine triphosphate, adenosine monophosphate, inosine monophosphate, inosine, hypoxanthine, xanthine, and uric acid were investigated in mucosal biopsy specimens after 15 or 45 min of hemorrhagic shock and after 15 min of shock followed by 30 min of retransfusion. During shock a dephosphorylation of nucleotides and a decrease in the number of perfused microvessels occurred, the more the longer the duration of the shock period. Retransfusion led to an additional reduction in the number of perfused microvessels, but there was a partial restoration of high-energy phosphate metabolites in those areas of the mucosa which maintained a blood flow. The results indicate that there is a renewal of energy stores in gastric mucosa after hemorrhagic shock and reperfusion, although parts of the vascular bed are not reperfused.

Relation of 5′-nucleotidase and phosphatase activities with immunophenotype, drug resistance and clinical prognosis in childhood leukemia

Ecto-5′-nucleotidase (ecto-5′NT) catalyzes the extracellular dephosphorylation of nucleotides like IMP. Cytoplasmic 5′NT (cyto-5′NT) and non-specific (e.g. acid- and alkaline) phosphatases (AP) regulate the intracellular degradation of nucleotides. High NT and AP activities might cause a resistance to the thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We studied the relation between these enzymes and immunophenotype, drug resistance and prognosis in 77 children with acute lymphoblastic leukemia (ALL). Enzyme activities were assessed radiochemically; in vitro drug resistance was measured with the MTT assay. AP activities were higher in T-ALL and B-ALL than in precursor B-ALL. Cyto-5′NT activity was very low in all phenotypes and accounted for a significant proportion of total IMPase activity only in the very immature CD10 cμ − precursor B-ALL. CD10 + ALL cases with high ecto-5′NT activities showed a trend ( p = 0.065) for a lower probability of continuous complete remission than those with a low activity. Ecto-5′NT activity was not related to in vitro drug resistance to 6-TG. A weak correlation was found between in vitro 6-TG resistance and cyto-5′NT and AP activities. We conclude that high ecto-5′NT activities do not cause a resistance to 6-thiopurines in childhood ALL. Some patients have high cyto-5′NT and AP activities associated with 6-thiopurine resistance.

The role of 5′nucleotidase in therapy-resistance of childhood leukemia

Purine nucleotides become available for a cell by two routes, namely purine de novo synthesis and the purine salvage pathway. 5′Nucleotidase is a purine pathway enzyme and is present in two forms. Cytoplasmic 5′nucleotidase (cyto 5′NT) catalyzes the intracellular degradation of purine nucleotides into their corresponding nucleosides. The plasmamembrane bound form (ecto 5′NT) has its active site facing the external medium. Its function is the extracellular dephosphorylation of nucleotides, to which cells are generally impermeable, into nucleosides and the transport of these nucleosides through the cell membrane. Lymphoblastic 5′NT activity varies between different children with common-ALL. 5′NT positive cases have a higher relapse rate and thus a poorer prognosis than 5′NT negative cases (10). This can be explained by two hypotheses which are not mutually exclusive: 1. 1. Rescue hypothesis. When purine de novo synthesis is blocked by methotrexate (MTX) and/or 6-mercaptopurine (6-MP), the malignant cell has to rely on the purine salvage pathway. This pathway depends on the ecto-5′NT activity. So, leukemic cells might be resistant to MTX and/or 6-MP because of ecto-5′NT activity. 2. 2. Breakdown hypothesis. Leukemic cells are resistant to 6-MP because of the breakdown of the toxic nucleotide form of 6-MP into the nucleoside form by cyto-5′NT.

Effect of storage temperature on the dephosphorylation of nucleotides in fish muscle.

At a temperature range from -5°C to 20°C, the Arrhenius plot of the dephosphorylation of nucleotides in cod muscle exhibited one break point at around -2°C, the plot of carp muscle at about 5°C, but the plot of yellowtail exhibited two break points at around -2°C and 10°C. These break points at around -2°C disappeared on the addition of glycerin as an antifreeze. The Arrhenius plots of microsomal 5'-nucleotidase of carp and yellowtail exhibited one break point at 5°C and 10°C respectively, but the plot of cod showed no break point, in the presence of glycerin. The Km of microsomal 5'-nucleotidase of carp and yellowtail for IMP markedly increased with descending temperature below the break point. The break point of microsomal 5'-nucleotidase disappeared on the treatment with a surfactant. Therefore, the assumption made is, that the change in kinetics of dephosphorylation of nucleotides at around -2°C may be accounted for by the phase change of water in fish muscle, and the change at the temperature above freezing point may be accounted for by the lipid phase separation in membranes containing 5'-nucleotidase at the definite temperature which is specific to the species of fish.

Factors controlling the Pb(II) promoted dephosphorylation of nucleotides

The conditions under which Pb(II) promotes dephosphorylation of nucleotides have been studied with the Pb(II) complexes of several isomers of AMP, dAMP, GMP, and dGMP. A number of factors which together control the dephosphorylation reaction have been identified. These include (1) the tendency of Pb(II)-induced nucleotide base stacking, as evidenced by large enhancement in ultraviolet circular dichroism, to occur in the complexes and limit the reaction; (2) hydroxylation of the metal, either with weakening of the lead-nucleotide binding, or eventually with displacement of the nucleotide; and (3) the solubility of the complexes, which limits the reaction, but is increased by raising the temperature and by hydroxylation of the complexes. The pH range in which both base stacking and metal hydrolysis are minimized can define a “reaction window” for the complexes.

Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate.

The catabolism of GTP and ATP in intact human thymocytes was studied, and the effect of intracellular Pi concentrations on various catabolic reactions was evaluated. Induction of nucleotide catabolism was performed with either NaN3 or deoxyglucose. NaN3 induced an increase in intracellular Pi, whereas deoxyglucose induced a decrease in Pi in human thymocytes. At elevated Pi concentrations, an intracellular accumulation of GMP, IMP and AMP was observed, and the entry of these nucleotides into the nucleoside cycles was diminished. In contrast, at lowered Pi concentrations, there was no accumulation of nucleoside monophosphates, but the production of purine bases and nucleosides was increased as a result of enhanced nucleotide entry into the nucleoside cycles. The dephosphorylation of nucleotides thus seems to be regulated by intracellular Pi concentrations. ATP catabolism proceeded mainly via adenylate deamination at both elevated and lowered Pi concentrations. Guanylate formed during GTP catabolism was mainly dephosphorylated, but significant amounts of GMP were also deaminated to yield IMP.

Dephosphorylation of Nucleotides of 1-β-D-Arabinofuranosylcytosine in Relation to the Different Drug Sensitivity in Tumor Cells

Dephosphorylation of Nucleotides of 1-β-D-Arabinofuranosylcytosine in Relation to the Different Drug Sensitivity in Tumor Cells

On the effectiveness of EDTA in prolonging the shelf life of fresh fish

The purpose of this investigation was to ascertain whether EDTA might be useful in prolonging the keeping quality of fresh fish. The criteria of quality used were: the total bacterial count, the content of volatile basic nitrogen and trimethylamine, the degree of dephosphorylation of the nucleotides, the level of the hypoxanthine, and the organoleptic properties. The material consisted of rainbow trout (Salmo irideus) and Baltic herring (Clupea harengus var. membranus). The results showed that EDTA had a markedly favorable effect on the keeping quality of fish. Although the effect on the total bacterial count was only slight, a chemical and organoleptical comparison with control material revealed that spoilage in the EDTA-treated samples was definitely slower. In the case of fillets of rainbow trout, judged by the organoleptical method, the controls became unacceptable after storage for 18–20 days at +4°C, whereas after the same storage time the EDTA-treated samples were still free of off-odors and had a fair taste. Similarly, the values of volatile basic nitrogen, trimethylamine, and hypoxanthine increased at a slower rate, and the dephosphorylation of nucleotides proceeded less rapidly in the EDTA-treated fillets than in the controls. In the case of fillets of Baltic herring, sensory evaluation brought out the positive effect of the EDTA treatment. A corresponding difference was noted in the volatile basic nitrogen and trimethylamine values, and a slight difference in the hypoxanthine values. In this fish, the dephosphorylation of the nucleotides was rapid, and between the samples subjected to EDTA treatment and the control material there appeared only a small difference. With regard to the round Baltic herring, again, the differences between the two categories were less conspicuous; the chief difference was noted in the volatile basic nitrogen, but in the sensory evaluation the difference was not fully consistent.

Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

Fate of donor DNA in pneumococcal transformation

The molecular fate of homologous (pneumococcal) and heterologous ( Escherichia coli) [ 32P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 32P-containing products at the earliest time of observation was the same. About 50% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. coli as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and l-α-glycerophosphate and smaller amounts of the four 5′-deoxynucleotides. A plausible origin of labeled inorganic phosphate is the dephosphorylation of nucleotides. The labeled glycerophosphate, in turn, may be derived from inorganic phosphate, since artificially introduced [ 32P]phosphate rapidly equilibrated with a large pool of intracellular glycerophosphate. Thus, the initial products of entry are envisioned to be equal amounts of single strands and 5′-deoxynucleotides. This would be compatible with their origin in the degradation by a pneumococcal exonuclease of one strand of an incoming DNA duplex. On subsequent incubation, single strands of heterologous origin were slowly degraded; their breakdown products were used for de novo DNA synthesis. The disappearance of homologous single strands was much faster and corresponded to an increase of 32P in native DNA as well as to the recovery from eclipse of donor marker-transforming activity. It is concluded that single strands are precursors of genetically integrated DNA and that recombination involves the interaction of homologous single strands with host DNA. The ratio of genetically integrated DNA to total DNA incorporated suggests that only half, or somewhat more, of the single-stranded DNA was inserted as intact sequences. Inhibition of DNA synthesis by aminopterin gave results which were compatible with these conclusions.

The level of phosphorylation of nucleotides as related to the incorporation of [6- 14C]orotic acid into ribonucleic acid by rat-liver homogenates

1. 1. Evidence is presented to support the hypothesis that in rat-liver homogenates ATP inhibits the incorporation of radioactive uridine nucleotides into RNA. 2. 2. Increasing the concentration of MgCl 2 from 3 m M to 6 m M partially reverses inhibition by ATP. Higher concentrations of magnesium markedly inhibit incorporation regardless of the ATP concentration. 3. 3. Equilibrium between mono-, di-, and triphosphates is established at a level of 2.2-2.8 phosphates/adenosine by 20 min after the addition of ATP, ADP, or a mixture of AMP and ATP. It is only after rapid dephosphorylation of nucleotides has begun that significant incorporation of orotic acid into RNA is observed. 4. 4. When dephosphorylation of adenine nucleotides is almost complete, the specific activity of RNA reaches a maximum. Then the specific activity decreases, indicating that radioactive nucleotides are hydrolyzed from RNA faster than non-radioactive nucleotides.

DEPHOSPHORYLATION OF NUCLEOTIDES; THEIR ANALYSIS BY COUNTER-CURRENT DISTRIBUTION

DEPHOSPHORYLATION OF NUCLEOTIDES; THEIR ANALYSIS BY COUNTER-CURRENT DISTRIBUTION