Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nucleotidases”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1991

The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase.

A soluble 'low-Km' 5'-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5'-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble 'low-Km' 5'-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5'-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble 'low-Km' 5'-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5'-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5'-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5'-nucleotidase. The soluble 'low-Km' 5'-nucleotidase, like the ecto-5'-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble 'low-Km' 5'-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5'-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.

5'-Nucleotidase↗
PubMed1983

Ecto-5'-nucleotidase activity in human T cell subsets. Decreased numbers of ecto-5'-nucleotidase positive cells from both OKT4+ and OKT8+ cells in patients with hypogammaglobulinemia.

T lymphocytes from control subjects were separated into subsets using monoclonal antibodies of the OKT series and complement lysis and analyzed for ecto-5'-nucleotidase activity both by quantitative radiochemical assay and a histochemical stain. T cells from 15 control subjects contained 54+/-4% OKT4(+) (helper/inducer) cells and 32+/-3% OKT8(+) (cytotoxic/suppressor) cells. Total T cell ecto-5'-nucleotidase activity was 10.9+/-2.1 nmol/h per 10(6) cells with 25+/-7% positive by histochemical stain. Ecto-5'-nucleotidase activity in OKT4-enriched populations was 5.43+/-1.8 nmol/h per 10(6) cells with 14+/-2% positive by histochemical stain; that in OKT8-enriched populations was 17.1+/-5.9 nmol/h per 10(6) cells with 35+/-8% positive by histochemical stain. Two of four patients with congenital agammaglobulinemia and four of seven patients with common variable immunodeficiency had decreased proportions of OKT4(+) T cells with corresponding increases in the proportions of OKT8(+) T cells (OKT4/OKT8 = 0.60 to 1.0 as compared with 1.7+/-0.2 for control subjects). All four patients with congenital agammaglobulinemia, and three of seven patients with common variable immunodeficiency also had low T cell ecto-5'-nucleotidase activity (<5.5 nmol/h per 10(6) cells). Ecto-5'-nucleotidase activity in OKT4- enriched populations isolated from four patients with low total T cell activity was 2.85+/-0.90 nmol/h per 10(6) cells with 10+/-4% positive by histochemical stain; that in OKT8-enriched populations was 6.82+/-1.7 nmol/h per 10(6) cells with 7.5+/-3% positive by histochemical stain. Thus, the number of ecto-5'-nucleotidase positive cells is decreased, especially in the OKT8(+) subpopulation, and the low total T cell ecto-5'-nucleotidase activity seen in these patients is due to fewer positive cells rather than to substantially less activity per cell. Our data indicate that ecto-5'-nucleotidase activity defines two subpopulations of T lymphocytes (ecto-5'-nucleotidase positive and negative), the proportions of which are markedly altered in many patients with hypogammaglobulinemia. In preliminary studies with seven patients, increased numbers of ecto-5'-nucleotidase negative T cells appeared to correlate with increased suppressor T cell activity toward in vitro immunoglobulin synthesis. Therefore, ecto-5'-nucleotidase may be a useful cell surface marker in the study of imbalances of regulatory T cell subsets in patients with antibody synthesis disorders.

Adenosine Deaminase↗
PubMed1973

Mutants of Escherichia coli K-12 "cryptic," or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (cyclic phosphodiesterase) activity.

Mutants of Escherichia coli have been selected for the absence of 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (2',3'-cyclic phophodiesterase). Mutants selected for the absence of 5'-nucleotidase are of two kinds: those that lack detectable activity for the enzyme (Ush(-)), and those that possess activity when cell extracts are assayed, but not when intact cells are assayed (cryptic; Crp(-)). The latter class is probably identical to a type of mutant previously reported by Ward and Glaser. When mutants are selected for the absence of 3'-nucleotidase, Crp(-)mutants are also obtained. Thus far, however, mutants totally lacking this enzyme have not been found. The location on the genetic map of one ush mutation is at position 11 min and that of one crp mutation at approximately 67 min. In the crp mutant, 5'-nucleotidase and 3'-nucleotidase remain located in the periplasm. This mutant is also cryptic for alkaline phosphatase but not for acid hexose phosphatase. Treatment of cells with ethylenediamine-tetraacetate substantially alleviated crypticity. These data are discussed in terms of the organization of periplasmic enzymes and of the outer membrane as a permeability barrier.

Acid Phosphatase↗
PubMed1968

The 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) of the Enterobacteriaceae.

All members of the Enterobacteriaceae possess distinct 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) that can be differentiated from the acid and alkaline phosphatases and the acid sugar hydrolases. The nucleotidases and cyclic phosphodiesterases of the various Enterobacteriaceae are remarkably similar in properties. All of the 5'-nucleotidases hydrolyze 5'-nucleotides, adenosine triphosphate, and uridine diphosphoglucose. Their pH optimum is from 5.7 to 6.1. The cyclic phosphodiesterases hydrolyze 3'-nucleotides, cyclic phosphonucleotides, bis-(p-nitrophenyl)phosphate, and p-nitrophenylphosphate. Their pH optimum is from 7.2 to 7.8. For both enzymes, cobalt showed optimal metal stimulation. An intracellular protein inhibitor for the 5'-nucleotidase is present in all of the Enterobacteriaceae. No inhibitor of cyclic phosphodiesterase activity exists, although hydrolysis of both cyclic phosphonucleotides and 3'-nucleotides is inhibited by ribonucleic acid. Neither of the enzymes is subject to control by phosphate level or by catabolite repression. Of the other bacteria studied, only Haemophilus and Bacillus subtilis contained significant 3'- or 5'-nucleotidase activity.

Bacillus subtilis↗
PubMed1986

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase.

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.

5'-Nucleotidase↗
PubMed1988

5'-Nucleotidase activities in human erythrocytes. Identification of a purine 5'-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate.

A purine 5'-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5'-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5'-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5'-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.

2,3-Diphosphoglycerate↗
PubMed1975

Biogenesis of plasmalemmal glycoproteins. Intracellular site of synthesis of mouse liver plasmalemmal 5'-nucleotidase as determined by the sub-cellular location of messenger RNA coding for 5'-nucleotidase.

1. Free and membrane-bound mouse liver polyribosomes were separated by prolonged density-gradient centrifugation of the post-mitochondrial supernatant. RNA was extracted from free and membrane-bound polyribosomes and mRNA purified by oligo(dT)-cellulose column chromatography. 2. Antisera against purified mouse liver plasma membrane 5'-nucleotidase and moust albumin were prepared and characterized. 3. Microinjection of equivalent amounts of mRNA from free and membrane-bound liver polyribosomes into Xenopus laevis oocytes indicated by immuno precipitation and sodium dodecylsulphate gel electrophoresis a higher proportion of mRNA coding for 5'-nucleotidase and serum albumin in membrane-bound polyribosomes than free polyribosomes. 4. Although small, significant amounts of serum albumin and 5'-nucleotidase were also coded for by mRNA purified from free polyribosomes. The results suggest that in vivo, mRNA in mouse liver membrane-bound polyribosomes codes for the synthesis of 17 times more 5'-nucleotidase than does the mRNA in free polyribosomes.

Animals↗
PubMed1992

Purification and characterization of cytoplasmic 5'(3')-nucleotidase from rabbit spleen: characteristic differences of the enzyme from the rat spleen nucleotidase.

1. A 5'(3')-nucleotidase, which preferably hydrolyzed 3'-dTMP and 3'-dUMP, was highly purified from rabbit spleen cytosol. 2. The enzyme also hydrolyzes 3'-UMP, 5'-dUMP and guanine nucleotides, but does not hydrolyze any adenine nucleotides or cytosine nucleotides. 3. The activity is dependent upon Mg2+, Co2+ or Mn2+; the addition of deoxyinosine stimulates the activity, and the pH optimum for the hydrolysis of 3'-dTMP is 7.0. 4. Although the catalytic properties of the enzyme are similar to the 5'(3')-nucleotidase from rat spleen, these nucleotidases differ in their molecular disposition. 5. The charge state of the rabbit enzyme is slightly basic, and the subunit M(r) is about 27 kDa, while the value of the rat enzyme is 26 kDa. 6. Immunochemical experiments with the use of antibodies against the purified nucleotidase indicate that enzymes from rabbit spleen and from rat spleen are composed of different polypeptides.

Animals↗
PubMed1984

Identification of thymidine nucleotidase and deoxyribonucleotidase activities among normal isozymes of 5'-nucleotidase in human erythrocytes.

The persistence of normal thymidine nucleotidase (ThyNase) activity in subjects with pyrimidine nucleotidase (PyrNase) deficiency suggested the possible existence of separate isozymes in normal human erythrocytes. This hypothesis was confirmed by studies of PyrNase-deficient individuals from five unrelated families. Erythrocytes deficient in PyrNase retained normal activity of an enzyme system preferentially active at pH 6.2 with a variety of 2'-deoxyribonucleoside 5'-monophosphate substrates, including those of uridine, thymidine, and cytidine. Lesser activities were observed with the corresponding ribonucleotides. Normal control hemolysates were also found capable of effectively dephosphorylating purine nucleotides (dAMP greater than AMP) when pH was lowered sufficiently from the pH 7.4-8.0 region commonly used in conventional assays. Variations in substrate specificity, pH optima, kinetics, and sensitivity to inactivation by Pb2+ indicated the existence of multiple 5'-nucleotidase isozymes in normal erythrocytes: PyrNase and deoxyribonucleotidase(s) that might function physiologically in the conversion of DNA-derived nucleotides to diffusible nucleosides. Evolution of such a unique 5'-nucleotidase suggests that normal erythroblast maturation and nuclear extrusion is accompanied by a degree of karyolysis sufficient to require dephosphorylation and clearance of DNA degradation products.

Deoxyribonucleotides↗
PubMed1972

Cytochemical localization of 5'-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5'-nucleotidase activity in microsomes.

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO(3))(2) and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb(2+), or Pb(2+) and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb(2+), and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb(2+) and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb(2+) and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.

Adenosine Monophosphate↗
PubMed1983

Analysis of human erythrocyte 5'-nucleotidases in healthy individuals and a patient deficient in pyrimidine 5'-nucleotidase.

Electrophoretic and quantitative analysis of pyrimidine 5'-nucleotidase in human erythrocytes from healthy individuals and a patient deficient in pyrimidine 5'-nucleotidase, using a range of substrates, has shown that the patient has a marked deficiency with UMP, CMP and dCMP as substrates but near normal levels of activity with dUMP and dTMP as substrates. The observations suggest that two separate structural gene loci coding for distinct 5'-nucleotidases with similar electrophoretic mobility exist in man. The genetic determination of these enzymes seems therefore to be homologous with that found in rodents.

5'-Nucleotidase↗
PubMed1975

Effects of low-level lead exposure on pyrimidine 5'-nucleotidase and other erythrocyte enzymes. Possible role of pyrimidine 5'-nucleotidase in the pathogenesis of lead-induced anemia.

Similarities between lead-induced anemia and a new hereditary erythorenzymopathy involving pyrimidine-specific 5'-nucleotidase prompted studies of the effects of lead on this and other erythrocyte enzymes. In vitro incubations of normal mature erythrocytes demonstrated that significant inhibition of pyrimidine 5'-nucleotidase occurred in the presence of lead at concentrations that had minimal effects on many other erythrocyte enzymes assayed simultaneously. Similarly, subjects with chronic lead intoxication secondary to industrial exposure exhibited substantial and consistent impairment of erythrocyte pyrimidine-5'-nucleotidase activity. Results suggest that lead-induced deficiency of this enzyme in maturing erythroid elements could, if sufficiently severe, result in induction of basophilic stippling and premature erythrocyte hemolysis analogous to that encountered in the genetically induced enzyme-deficiency syndrome.

Erythrocytes↗
PubMed1988

Red cell pyrimidine 5' nucleotidase deficiency: determination of nucleotidase activity and nucleotide content using HPLC.

The hereditary deficiency of erythrocyte pyrimidine 5' nucleotidase has been investigated using an HPLC anion-exchange procedure designed to measure both red cell nucleotide content and enzymic activity. Red cell nucleotide profiles were determined using a low pH phosphate buffer salt-gradient system in 20 min, whereas a low pH buffer alone permitted the determination of enzymic activity with each of six different nucleotide substrates in less than 4 min. Both the red cell nucleotide profiles and the enzymic activity of haemolysates from two affected brothers and their children agreed well with previously published values. This unified approach should prove useful for detailed studies of deficiencies involving isoenzymes of pyrimidine nucleotidase.

5'-Nucleotidase↗
PubMed1987

Chromatographic analysis of human erythrocyte pyrimidine 5'-nucleotidase from five patients with pyrimidine 5'-nucleotidase deficiency.

Human erythrocyte pyrimidine 5'-nucleotidase (P5N) was separated into two subclasses. P5N-I and P5N-II, by DEAE Bio-Gel A column chromatography. Their enzymological properties were studied using five normal subjects and five patients with different P5N deficiencies. Study of the normal subjects showed that P5N-I and P5N-II have distinctive properties, and P5N-II is similar to the 5'-nucleotidase in rat liver cytosol. The P5N-II from the five subjects with this deficiency had normal activity and other normal enzymological properties. However, the P5N-I from these patients had abnormal properties, including reduced activity. These variant enzymes had a high Michaelis constant for substrate cytidine 5'-monophosphate and were heat stable. The optimum pH was shifted towards the acidic side in two patients, towards the basic side in one, and was unchanged in the other two. These results strongly suggest that the main cause of P5N deficiency is an abnormality of P5N-I, probably arising from a structural gene mutation.

5'-Nucleotidase↗
PubMed1986

Erythrocyte arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N) as indices of lead exposure.

The activities of three erythrocyte (rbc) enzymes, arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N), were compared in 16 lead workers and 14 age matched controls as correlates of blood lead (PbB) and unextracted zinc protoporphyrin (EP) concentrations. Subjects with PbB of 0.9-2.5 microM (19-52 micrograms/dl) had 6.5 +/- 0.6 IU of P5N activity with uridine monophosphate (UMP) as substrate, significantly less (p less than 0.001) than the 12.0 +/- 0.7 IU activity of controls with PbB 0.3-0.6 microM (6-12 micrograms/dl). The mean activity of rbc dP5N with either deoxyuridine monophosphate (dUMP) or thymidine monophosphate as substrate, and of rbc arginase, did not differentiate the two groups. The correlation coefficients of ln PbB with the selected substrates for P5N and dP5N were: UMP, r = -0.75; dUMP, r = -0.61; TMP, r = -0.23. The correlation coefficient of ln PbB and arginase activity was -0.03. Rbc P5N (UMPase) is a significant correlate of PbB, equivalent to rbc protoporphyrin. HPLC assay of rbc UMPase activity is a sensitive and rapid assay that appears to meet criteria for a reliable clinical laboratory index of blood lead concentrations.

5'-Nucleotidase↗
PubMed1983

Pyrimidine nucleotidase deficiency with active dephosphorylation of dTMP: evidence for existence of thymidine nucleotidase in human erythrocytes.

Erythrocytes from a patient with classical pyrimidine nucleotidase (PyN) deficiency had less than 10% residual PyN activity with uridine 5'-monophosphate (UMP) or cytidine 5'-monophosphate (CMP) as substrate, but exhibited brisk nucleotidase activity with thymidine 5'-monophosphate (dTMP). This strongly suggests the existence of separate enzymes or isozymes of PyN in normal human erythrocytes--an hypothesis that should be tested by similar studies in other cases of severe PyN deficiency, whether induced by genetic defects or lead toxicity.

5'-Nucleotidase↗
PubMed1994

The bifunctional cytosolic 5'-nucleotidase: regulation of the phosphotransferase and nucleotidase activities.

The cytosolic 5'-nucleotidase specific for IMP, GMP, and their deoxyderivatives has been purified approximately 1000 times from calf thymus. The enzyme, in the presence of a suitable nucleoside, can act as a phosphotransferase, catalyzing the transfer of the phosphate moiety from a nucleoside monophosphate donor to a nucleoside acceptor, thus operating as an interconverting activity. This phosphorylating activity has drawn the attention of several research groups because the cytosolic 5'-nucleotidase represents the only cellular enzyme able to phosphorylate inosine and guanosine analogs, which are not substrates of known cellular nucleoside kinases. In this paper, we report the kinetic parameters of the bifunctional enzyme and its response to variations in adenylate energy charge. The results seem to indicate that in the presence of physiological concentrations of ATP and phosphate, the enzyme behaves mainly as a phosphotransferase, its activity being dependent only on the availability of a suitable nucleoside.

5'-Nucleotidase↗
PubMed1980

Cooperativity of the concanavalin A inhibition of bovine milk fat globule membrane 5'-nucleotidase. Response to extraction of nucleotidase and of putative cytoplasmic surface coat components.

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) of bovine milk fat globules can be solubilized by deoxycholate from either isolated globule membranes or washed cream. The solubilized and membrane-bound enzymes exhibit similar Km values and are inhibited by concanavalin A by an apparent noncompetitive process. The soluble enzyme shows positive cooperativity for the inhibition (Hill coefficient of 2) at 37 degrees C, but the membrane enzyme exhibits essentially no cooperation effect. At lower temperatures (5 or 20 degrees C) the cooperative effect in the inhibition of the soluble enzyme is lost. Colchicine and cytochalasin D failed to induce cooperativity of the concanavalin A inhibition of the membrane enzyme, but induction cooperativity occurred when membranes were extracted with glycine/EDTA/mercaptoethanol, releasing a major protein component with a polypeptide molecular weight of 155 000. We suggest that the interaction of this component with the membrane imposes restraints on the behavior of the nucleotidase which are reflected in the cooperativity of the inhibition of the enzyme by concanavalin A.

Animals↗