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PubMed2004

Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice.

The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.

5'-Nucleotidase↗
PubMed1981

5'-Nucleotidase from rat heart.

5'-Nucleotidase has been extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent molecular weight of the subunit is 74 000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5'-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS, AMP, isoAMP, GMP, and IMP. It shows no activity with nucleoside 2'- and 3'-monophosphates, sugar phosphates, and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.

5'-Nucleotidase↗
PubMed1982

Nucleoside exchange catalysed by the cytoplasmic 5'-nucleotidase.

The inhibition of the cytoplasmic 5'-nucleotidase (EC 3.1.3.5) by its product, inosine, was studied with a partially purified preparation of the enzyme from rat liver. Inhibition of Pi production was found to be due to exchange of the inosine moiety between inosine and IMP. Exchange was not catalysed by reversal of the hydrolytic reaction, suggesting, instead, the mediation of an enzyme-phosphate intermediate. Two models for the catalytic mechanism are proposed and rate equations for the dependence of Pi production on inosine concentration are derived. The experimentally determined dependence was consistent with a mechanism in which hydrolysis of the enzyme-phosphate intermediate occurred only when it was unoccupied by inosine. This conclusion suggests that inosine analogues that cannot participate in exchange should inhibit the enzyme. Such inhibitors might be useful in defining the enzyme's physiological role or as pharmacological agents to decrease breakdown of purine nucleotides. The possibility that nucleoside exchange provides an alternative route for the phosphorylation of mutagenic or cytotoxic nucleoside analogues should also be considered.

5'-Nucleotidase↗
PubMed1987

Purification, characterization and cellular localization of 5'-nucleotidase from Torpedo electric organ.

5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.

5'-Nucleotidase↗
PubMed1988

5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities.

Chromatography of soluble proteins from rat heart on phosphocellulose columns separates two 5'-nucleotidases. The first to emerge from the column shows a preference for AMP over IMP as substrate, whereas the second shows a preference for IMP over AMP. The properties of the IMP-preferring enzyme, including the conditions under which it is eluted from phosphocellulose columns, show it to be the enzyme studied by Itoh, Oka & Ozasa [Biochem. J. (1986) 235, 847-851]. The kinetic properties of the AMP-preferring enzyme indicate that it is likely to be the enzyme responsible for the production of adenosine under conditions of hypoxia and increased work load, and with metabolic stresses such as a high load of acetate.

5'-Nucleotidase↗
PubMed1982

Isolation and partial characterization of a 5'-nucleotidase specific for orotidine-5'-monophosphate.

A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This enzyme orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-beta-D-ribofuranosylbarbituric acid 5'-monophosphate (BMF), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate and beta-glycerophosphate, all of which--with the exception of the 2' or 3' monophosphates, ribose 5'-phosphate, and beta-glycerophosphate--are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SF reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase had a Mr 53,000, Km value of 1 mM for OMP, and Vmax value of 49 nmol/min . mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular "free" OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from aortic aciduria.

5'-Nucleotidase↗
PubMed1983

31P NMR study of erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency.

The composition of phosphate metabolites and the intracellular pH in erythrocytes from a patient with hereditary pyrimidine-5'-nucleotidase deficiency were examined using 31P NMR spectroscopy. Several resonances were identified in spectra from intact cells and from extracts. The 2,3-bisphosphoglycerate line intensities were normal but the NTP resonances were about twice normal due to the presence of millimolar quantities of pyrimidine phosphates. Several intense resonances were also observed in the diphosphodiester region of the spectrum. One compound contributing to these lines has been identified as cytidine diphosphocholine. The resonances of NTPs were in a position indicating that the additional triphosphates were also bound by Mg2+. Direct measurement shows that there is a nearly proportional increase in total cell Mg2+ in the patient's cells, in agreement with the interpretation of the spectra. The intracellular pH was about 0.2 unit lower in the patient's erythrocytes. This lower pH is due to the elevation in intracellular fixed negative charges and the shift in permeable anions consequent to the Donnan equilibrium. We suggest that the lower intracellular pH may explain the lower oxygen affinity of these cells in the presence of otherwise normal 2,3-bisphosphoglycerate levels and the increased Mg2+ triphosphates level, because the Mg2+ form of NTPs is known not to alter the oxygen affinity of hemoglobin under physiologic conditions. Furthermore, the lower intracellular pH can also explain the abnormalities in glycolytic intermediates observed for these cells.

5'-Nucleotidase↗
PubMed1997

Developmental disorder associated with increased cellular nucleotidase activity.

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5'-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

5'-Nucleotidase↗
PubMed2002

SDT1/SSM1, a multicopy suppressor of S-II null mutant, encodes a novel pyrimidine 5'-nucleotidase.

SDT1 (suppressor of disruption of TFIIS 1, YGL224c, also known as SSM1, suppressor of S-II null mutant 1) is Saccharomyces cerevisiae gene identified as a multicopy suppressor of 6-azauracil sensitivity in a null mutant of the transcription elongation factor S-II. We found that overproduction of SDT1 caused hyposensitivity to not only 6-azauracil but also 5-fluorouracil and 5-fluorocytosine. This hyposensitivity was limited to pyrimidine derivatives, and no effect was observed for non-pyrimidine drugs including such clinically used anti-fungal drugs as amphotericin B and fluconazole. Purified recombinant SDT1 protein specifically dephosphorylated 5'-UMP and 5'-CMP. These results suggested that SDT1 conferred pyrimidine-specific hyposensitivity by dephosphorylating active metabolites of 6- or 5-modified pyrimidines, i.e. 6- or 5-modified UMP. This is the first description of a highly specific pyrimidine 5'-nucleotidase in S. cerevisiae.

5'-Nucleotidase↗
PubMed1983

Association of plasma 5'-nucleotidase and alkaline phosphatase with production traits in chickens: effects of age and housing systems.

The housing system, age of pullets and their interaction had a significant effect on plasma 5'-nucleotidase and alkaline phosphatase activities. Activities were higher in birds in cages than in those on the floor, reflecting perhaps the stress of caging. Activities increased with age. Activities were higher in pullets selected for higher production. The type of housing had no effect on egg production, but age at first egg, egg weight and 40-week body weight were found to be affected significantly.

5'-Nucleotidase↗
PubMed1983

Association of plasma 5'-nucleotidase and alkaline phosphatase with production traits in chickens: genetic and phenotypic variability.

Heritability of plasma alkaline phosphatase and 5'-nucleotidase enzyme activities at 20, 35 and 50 weeks of age ranged from 0.047 to 0.275. Genetic and phenotypic correlations among enzyme activities at the three ages were positive and high. Genetic correlations of enzyme activities with production traits indicated that the bird having higher activities of these enzyme will grow faster, mature earlier and produce heavier but fewer eggs.

5'-Nucleotidase↗
PubMed1985

Serum 5'-nucleotidase in patients receiving anti-epileptic drugs.

Alkaline phosphatase (ALP), 5'-nucleotidase (5NT), and gamma-glutamyltransferase (GGT) were measured in sera from 335 adult and 93 pediatric inpatients receiving anti-epileptic drugs (AEDs). Among adults, 18 took only phenobarbital, 39 only carbamazepine, 159 only phenytoin, and 99 phenytoin with other AEDs. Of 93 children, 49 took phenytoin, and 44 took another AED. Among the 335 adults, only 118 had normal enzymes, but 316 had 5NT and ALP, each less than two times and GGT less than five times normal limits. Of 19 patients outside these ranges, 14 had either historic, physical, or biochemical evidence of liver or severe multisystem disease. The authors observed that the frequency of enzyme elevations in adults receiving AEDs was lower for 5NT (23%) than for GGT (54%) but similar to ALP (27%). The degree of elevation was also lower for 5NT and ALP than for GGT. Phenytoin was associated with the most frequent and highest enzyme elevations in both adult and pediatric patients. Consideration of these data is necessary in the evaluation of liver function in patients receiving AEDs.

5'-Nucleotidase↗
PubMedDate not supplied

Prediction of blood lead by HPLC assay of erythrocyte pyrimidine 5'-nucleotidase.

A simplified preparation of erythrocytes (rbc) for rbc pyrimidine 5'-nucleotidase (P5N) is described and correlated with blood lead. Washed but unfiltered rbc are incubated with uridine monophosphate (UMP) and the amount of uridine liberated is measured by high performance liquid chromatography (HPLC). UMPase activity of unfiltered rbc of 20 normal subjects was 12.4 +/- 2.4 units, comparable to reported data obtained under varying conditions of preparation and P5N assay. In 20 lead-exposed adults and 20 controls, the regression coefficient for blood lead (PbB) predicted by rbc UMPase was In PbB = 4.77 - 0.22 UMPase; r2 = .61. If UMPase less than or equal to 7.6 units (mean, 2.0 SD) is considered abnormal, sensitivity for prediction of a blood lead greater than or equal to 40 micrograms/dL is 80%. Specificity, the percent of subjects with a UMPase greater than 7.6 units who had a blood lead less than 40 micrograms/dL, was 96%. Wider utilization of P5N, measured as UMPase, is proposed as a biologic correlate of blood lead.

5'-Nucleotidase↗
PubMed1986

Nucleotide sequence and transcriptional analysis of the E. coli ushA gene, encoding periplasmic UDP-sugar hydrolase (5'-nucleotidase): regulation of the ushA gene, and the signal sequence of its encoded protein product.

The DNA sequence of the ushA gene, encoding UDP-sugar hydrolase (5'-nucleotidase), has been determined. The amino-terminal sequence encodes a signal peptide whose predicted processing site is confirmed by N-terminal amino acid analysis of purified mature UshA protein. The signal sequence contains a concentration of rare codons in comparison with the mature sequence. The origins of transcription from the ushA promoter have been determined, using primer extension. Three transcripts, originating within a 6 bp region, were identified and might be related to three overlapping potential -10 hexamers in the ushA promoter region. There was a discernable change in the relative proportion of these transcripts during growth-phase regulation of the ushA gene.

5'-Nucleotidase↗
PubMed1989

Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus.

Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.

5'-Nucleotidase↗
PubMed1984

Pyrimidine 5'nucleotidase and several other red cell enzyme activities in beta-thalassaemia trait.

The activity of 18 red blood cell (RBC) enzymes and reduced glutathione (GSH) content were measured in 70 normal subjects, in 50 heterozygous beta-thalassaemia carriers and in 50 non-thalassaemic patients with haemolytic anaemia and high reticulocyte counts. In addition, pyrimidine 5'nucleotidase (P5N) activity was also determined in 34 patients with hypochromic, microcytic, iron deficiency anaemia. beta-Thalassaemia trait was associated with an increase in almost all of the enzyme activities, except for 2,3-bisphosphoglycerate synthetase (BPGS) and glutathione reductase (GR) which were normal and for acetylcholinesterase (AChE) and P5N which were slightly and markedly decreased respectively. The increases in enzyme activities were similar to those observed in patients with non-thalassaemic reticulocytosis except for glyceraldehydephosphate dehydrogenase (GAPD), phosphoglyceratekinase (PGK), pyruvate kinase (PK), glutathione peroxidase (GPX) and adenylate kinase (AK) which were higher than in non-thalassaemic group of patients with increased number of reticulocytes. No correlation was found between the severity of P5N deficiency and the intensity of basophilic stippling which was present in 46 of 50 thalassaemic carriers here studied. In addition, GSH content and UV absorption spectra of deproteinized thalassaemic RBC extracts were also found to be normal. The present findings provide further information on the metabolic status of RBC in beta-thalassaemia trait and suggest a possible molecular explanation for the frequently observed basophilic stippling in this disease.

5'-Nucleotidase↗
PubMed1986

Red cell metabolism in hereditary pyrimidine 5'-nucleotidase deficiency: effect of magnesium.

Since pyrimidine nucleotides avidly bind magnesium, we tested the hypothesis that the haemolytic anaemia in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency is due to a state of functional magnesium depletion in the red cell (RBC). In haemolysates from normal subjects, cytidine triphosphate (CTP) inhibited the activity of pyruvate kinase in a competitive manner for magnesium. The CTP Ki was 0.4 mmol/l. CTP inhibited the activity of hexokinase in a competitive manner for ATP (Mg-ATP2-) with a Ki of 4 mmol/l. The inhibitory effect of CTP on both enzymes was overcome by increasing the magnesium content of the test system. Since CTP appeared to inhibit enzymes which required magnesium as a cofactor or Mg-ATP2- as a substrate, we tested the effect of exogenous magnesium on the metabolism of P5N deficient RBC. The autohaemolysis test, the incubated Heinz body assay and the rate of glucose oxidation by the pentose phosphate shunt were abnormal in the intact RBC from a patient with hereditary P5N deficiency. The addition of MgCl2 (6-10 mmol/l) did not improve these abnormal in vitro measures of metabolism in the P5N deficient RBC. This lack of effect of exogenous magnesium may be due to the slow uptake of magnesium by the human RBC.

5'-Nucleotidase↗