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At least 631 records · Page 35Linked to original sources
PubMed1985

Target size of 5'-nucleotidase in smooth muscle.

Target size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80,000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80,000 to 110,000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135,000 daltons in rat mesenteric artery and 210,000 daltons in rat myometrium.

5'-Nucleotidase↗
PubMed1981

Escherichia coli 5'-nucleotidase: purification, properties and its release by osmotic shock.

Escherichia coli 5'-nucleotidase was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. Its molecular weight was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, by gel exclusion chromatography, and by membrane filtration; values of 66 000, 48 500, and 15 000 to 30 000, respectively, were obtained. The enzyme was completely released from bacteria by osmotic shock treatment. The apparently anomalous behaviour of 5'-nucleotidase in terms of the molecular sieving hypothesis for the release of enzymes by osmotic shock proposed by Smith & Wyatt (1974) and extended by Broad & Smith (1979) is discussed.

5'-Nucleotidase↗
PubMed1981

Hereditary haemolytic anaemia due to red cell pyrimidine 5'-nucleotidase deficiency in two Irish families with a note on the benefit of splenectomy.

Hereditary haemolytic anaemia with basophilic stippling caused by pyrimidine 5'-nucleotidase deficiency is described in three members of two unrelated Irish families. In one family, the disease was moderately severe and the patient's condition was improved by splenectomy. In the other family the haemolytic anaemia was well compensated. In neither family was there a marked elevation of reduced glutathione. The implications are that pyrimidine 5'-nucleotidase deficiency is a heterogeneous disorder, both clinically and biochemically. In more severe forms splenectomy may be beneficial.

5'-Nucleotidase↗
PubMed1983

Purification and properties of bovine liver plasma membrane 5' nucleotidase.

5'-Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A-Ultrogel and 5' AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5'-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5-8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28 degrees C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.

5'-Nucleotidase↗
PubMed1982

The distribution of radioligand binding activity and 5'-nucleotidase activity in bovine caudate nucleus subcellular fractions.

The distribution of 5'-nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5'-nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.

5'-Nucleotidase↗
PubMed1982

Purification and characterization of bovine brain 5'-nucleotidase.

The 5'-nucleotidase located in the cytoplasmic fraction of bovine brain cortex was purified to electrophoretic homogeneity. The molecular weight was 134,000 daltons in the presence of sodium deoxycholate, whereas the enzyme formed high molecular weight aggregates in the absence of detergent. The purified enzyme showed the same kinetic and electrophoretic behaviour as the enzyme present in the original cytoplasmic fraction, and the presence of surfactants did not change the Km and Vm values. The nucleotidase from this source was a phosphohydrolase of 5'-mononucleotides acting on the deoxyribonucleotides and ribonucleotides of purines and pyrimidines. 5'-IMP was the preferred substrate; the optimum pH was 7.5. The study of the influence of the temperature on the initial reaction rates allowed calculation of the delta Ea and delta H degrees values. The variation of Vm and Km with a change in pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex.

5'-Nucleotidase↗
PubMed1987

The influence of theophylline and phenobarbital on rat brain 5'-nucleotidase.

Xanthines and barbiturates have opposing effects on cerebral function and metabolism. Since the xanthine theophylline is known to inhibit the enzyme 5'-nucleotidase, and thus endogenic adenosine release, an in vitro experiment was conducted on the assumption that theophylline and phenobarbital may have an opposing effect on 5'-nucleotidase activity. This assumption was confirmed. The hypothesis that barbiturates in general cause an increase in endogenic adenosine release seems compatible with the known functional and metabolic effects of barbiturates. Furthermore, accepting the hypothesis that cellular hypoxic-anoxic survival may ultimately depend on salvage of adenine nucleotides, this may even explain the dichotomy in the effect of barbiturates when used for protection of a hypoxic-anoxic insult.

5'-Nucleotidase↗
PubMed1985

Enzyme regulation in a trypanosomatid: effect of purine starvation on levels of 3'-nucleotidase activity.

Crithidia fasciculata, a nonpathogenic relative of the leishmanial and trypanosomal pathogens of humans and animals, showed a 3'-ribonucleotidase activity similar to that in Leishmania donovani. The level of 3'-nucleotidase activity in Crithidia was regulated by the availability of purines in the culture medium. Specifically, organisms obtained from culture medium depleted of purines contained elevated levels of enzyme activity compared to those grown in complete medium. The 3'-nucleotidase, located at the cell surface, may serve as a first step in purine salvage for these protozoa, which are unable to synthesize the purine ring de novo.

5'-Nucleotidase↗
PubMed1981

Measurement of 5'-nucleotidase in mouse peritoneal macrophages in listeriosis.

5'-Nucleotidase activity in peritoneal macrophages of mice was measured both before and after infection with Listeria monocytogenes in hosts which possessed high or low anti-listerial resistance either due to a genetically determined trait or as a result of splenectomy. Reduction in enzyme activity was directly related to the degree of infection that developed in the hosts and hence was inversely related to the level of anti-listerial resistance observed. Basal 5'-nucleotidase activity was significantly lower in noninfected hosts that possessed high anti-listerial resistance, possibly reflecting a higher level of native macrophage activation in these hosts.

5'-Nucleotidase↗
PubMed2002

Nucleotidase cascades are catalyzed by secreted proteins of the parasitic nematode Trichinella spiralis.

Extracellular nucleotides are signaling molecules whose receptor-mediated effects are involved in a variety of physiological responses in mammalian tissues. An overwhelming body of data indicate that inflammatory and other immune responses can be modulated by the availability and local concentrations of nucleotides via nucleotide receptor signaling, but this is only just beginning to be investigated in the context of infectious disease. Evidence is provided here that the parasitic nematode Trichinella spiralis can catalyze the conversion and thus modulate both the availability and concentration of extracellular nucleotides by means of the following secreted exoenzymes: apyrase, 5'-nucleotidase, and adenosine deaminase. These enzymes were characterized in terms of substrate specificity, kinetic behavior, pH, divalent cation preferences, and response to a series of compounds. The secreted 5'-nucleotidase was identified as a protein with an apparent molecular mass of 67 kDa after N-terminal amino acid sequencing of the purified protein. The presence of adenosine deaminase was confirmed in the secreted products by Western blotting with an antibody against a mammalian enzyme, as a protein with an apparent molecular mass of 38 kDa. These secreted proteins constitute an enzymatic cascade which catalyzes the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling.

5'-Nucleotidase↗
PubMed1986

Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.

5'-Nucleotidase↗
PubMed1988

Determination of pyrimidine 5'-nucleotidase (P5N) activity in whole blood as an index of lead exposure.

A simple method for determining pyrimidine 5'-nucleotidase (P5N) activity in whole blood has been developed, inhibiting the plasma activity for UMP-hydrolysis by concanavalin (Con A). Con A specifically inhibits the activity of plasma 5'-nucleotidase (5N) but does not affect erythrocyte P5N activity. The anticoagulant EDTA partially inhibits 5N activity but slightly activates P5N. P5N activity determined by the present method with heparinised blood and Con A was comparable with that by the method reported previously and correlated well with blood lead concentrations. The mean value and SD for P5N activity in normal subjects (n = 72) not exposed to lead are 16.2 and 2.5 mumole/h/g Hb, respectively. The present method can eliminate not only the isolation step of RBC but also Hb determination, the activity being expressed as mumole/h/l blood or RBC. Thus the procedures are so simplified that the assay may be used as a routine test for mass screening of lead exposure.

5'-Nucleotidase↗
PubMed1989

Protection of the kidney against ischemic injury by inhibition of 5'-nucleotidase.

We have evaluated the impact of inhibiting adenine nucleotide dephosphorylation on the metabolic and functional consequences of renal ischemia. Intramuscular injection of the ADP-analogue adenosine alpha, beta-methylene diphosphate (AMP-CP) achieved a 70% reduction in 5'-nucleotidase activity, as measured in crude extracts of rat kidney. AMPCP-treated animals had an increased residual nucleotide pool at the end of 45 min of ischemia compared with untreated rats. Assessment of renal ATP by 31P-nuclear magnetic resonance (31P-NMR) in vivo during reflow demonstrates the following: 1) higher rapid initial recovery of ATP (69.3 +/- 1.2 vs. 50.0 +/- 0.5% control value, P less than 0.005), 2) accelerated rate of ATP restoration (0.20 +/- 0.02 vs. 0.11 +/- 0.01% control/min, P less than 0.005), and 3) significantly enhanced renal ATP content after 120 min (93.6 +/- 2.0 vs. 63.1 +/- 0.7% control, P less than 0.005). Kidney function, as measured by the rate of inulin clearance 24 h after the insult, was also significantly improved in AMPCP-treated rats (725 +/- 50 vs. 313 +/- 28 microliters.min-1.100 g body wt-1). Thus inhibition of 5'-nucleotidase results in enhanced metabolic and functional recovery from a renal ischemic insult.

5'-Nucleotidase↗
PubMed1980

Additional data from two kindreds with genetically induced deficiencies of erythrocyte pyrimidine nucleotidase.

Two subjects, not previously reported in detail, had severe inherited deficiencies of erythrocyte pyrimidine nucleotidase. This was manifested hematologically by moderate hemolytic anemia with splenomegaly, morphologically by punctate basophilic stippling of Wright's stained erythrocytes, and biochemically by intraerythrocytic accumulation of pyrimidine nucleotides, elevated concentrations of reduced glutathione, and partial deficiencies of ribosephosphate pyrophosphokinase. All 5 of their children were asymptomatic and phenotypically normal except for intermediate reductions in activities of pyrimidine nucleotidase consistent with heterozygosity for an autosomal recessive defect.

5'-Nucleotidase↗
PubMed1986

5'-Nucleotidase activity in adult and fetal rabbit lungs.

5'-Nucleotidase was assayed in the membrane fractions isolated from rabbit lung homogenate. The enzyme activity was measured from the rate of hydrolysis of [U-14C]cytidine 5'-monophosphate (CMP). The optimal pH of the rate of hydrolysis is around 8.0 and the enzyme activity is stimulated by Mg2+. The apparent Km and Vmax of the enzyme in the microsomal fraction for CMP were 3.33 mM and 1.43 mumol/min/mg protein, respectively. During lung development the enzyme activity increased moderately at late gestational ages and reached its maximum level in adults (p less than 0.001). The developing profile of 5'-nucleotidase activity is similar to the developing pattern of the CMP content in rabbit lungs.

5'-Nucleotidase↗
PubMed1980

A distinctive activity of 5'-nucleotidase for dTMP in rat liver mitochondria.

The activities of 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5); adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4); AMP deaminase (AMP aminohydrolase, EC 3.5.3.6), and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver 5'-Nucleotidase (5'Nase) and ATP-(Mg2+)-ase activities were compared with similar enzyme activities in the plasma membrane (PM) fraction, obtained from the same biological material. In the regenerating liver, 5'Nase for dTMP diminished its activity by 56% (24 h after partial hepatectomy) and 35 +/- 4% for all substrates in the PM fraction (48 h after operation). In mitochondria, 5'Nase for dTMP manifests sigmoidal substrate activity curve (in contrast with all substrates in the PM fraction and remaining substrates in mitochondria). In vivo 5-azacytidine (a) administered 1 h after partial hepatectomy, prevented changes of 5'Nase activity: (b) administered 24 or 48 h after partial hepatectomy, stabilized low 5'Nase activity (in mitochondria for dTMP, in the PM fraction for all substrates) and decreased ATP-(Mg2+)-ase activity by 51 and 31% in mitochondria and the PM fraction respectively.

5'-Nucleotidase↗
PubMed1989

Purification and properties of a 5'-nucleotidase from rat renal membranes.

5'-Nucleotidase activity was solubilized from a particulate fraction of rat renal homogenates by Sulphobetaine 14. An 11,430-fold purification was achieved by a two-step chromatographic procedure using concanavalin-A Sepharose and ADP-agarose. SDS-PAGE of the purified material revealed a single polypeptide band with a Mr of 69,000. The enyzme exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine monophosphate (AMP) showed the highest value of V/Km. The Km for 5'-AMP is 5.1 mumol/l and V is 632 mumol/min/mg. The plot of activity versus pH shows a broad plateau between pH 6.8 and 8.0. The hydrolysis of 5'-AMP was competitively inhibited by adenosine 5'-triphosphate (ATP; Ki = 1.2 mumol/l), adenosine 5'-diphosphate (ADP; Ki = 0.032 mumol/l) and alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP; Ki = 0.005 mumol/l). All of the 5 detergents tested activated the enzyme. Sulphobetaine 14 was the most potent and resulted in a 4-fold stimulation by increasing V without change of Km. Addition of exogenous divalent cations was not required for activity. However, the enzyme was inhibited by EDTA. This inhibition was overcome by the addition of Co2+, Mn2+ and to a lesser extent of Mg2+. Hg2+, Zn2+, Cu2+ and Pb2+ inhibited in the low micromolar range. The properties of this enzyme from the rat kidney are similar to those reported in the literature for ecto 5'-nucleotidases from other sources.

5'-Nucleotidase↗
PubMed1980

A new spectrophotometric method for the determination of 5'-nucleotidase.

A spectrophotometric method is described for the determination of 5'-nucleotidase. In combination with the enzymes nucleoside phosphorylase and xanthine oxidase, inosine, formed by hydrolysis of 5'-IMP by 5'-nucleotidase, is cleaved phosphorolytically to hypoxanthine, which is oxidized to uric acid. In the presence of ethanol, the hydrogen peroxide formed is reduced by catalase and equivalent amounts of acetaldehyde are produced. The aldehyde is dehydrogenated (NADP-dependent) by aldehyde dehydrogenase and the production rate of NADPH is recorded at 334 nm. The inhibition of the unspecific cleavage of 5'-IMP by phosphatases is examined critically.

5'-Nucleotidase↗