Pyrimidine 5'-nucleotidase Research Articles

Regulation and localization of TOB and IFR1 in differentiating red cells

In differentiating red blood cells (RBCs) of the chick embryo, the synthesis of carbonic anhydrase (CAII) and pyrimidine 5′-nucleotidase (P5N-I) is triggered by the hypoxic mediators norepinephrine and adenosine via receptor-mediated cAMP formation. The process is accompanied by the induction of IFR1 and TOB which are putative regulators of transcription or translation in different cell types. The present investigation studied the erythroid TOB and IFR1 expression: mRNA and protein are up-regulated in post-mitotic RBCs from D11–19 treated with cAMP-elevating agonists. In contrast, immature RBCs of early embryos ( D5–7) fail to synthesize a significant amount of IFR1/TOB. In D11 RBCs, TOB and IFR1 are cytosolic proteins with different half-lives (TOB < 4 h, IFR1 > 12 h). Cytosolic fractionation characterized TOB as a free soluble protein while the abundant IFR1 ( c max∼3 μM) is completely associated with the ribosomal fraction. A putative function of both proteins as translational regulators is discussed.

Molecular basis of Japanese variants of pyrimidine 5'-nucleotidase deficiency.

The type-I isoform of pyrimidine 5'-nucleotidase (P5N-I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N-I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87-bp sequence. The sequence is located 6.2-kb downstream of the exon 2 and 2.7-kb upstream of the exon 3 sequence; consequently, the P5N-I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N-I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients were homozygous for each mutation. The mutant P5N-I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos-7. The degradation of P5N with 425C (L142P) was significantly faster than a wild-type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin-proteasome pathway.

Erythroid pyrimidine 5′-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia

AbstractA characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5′-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. Thep5n-I expression is induced (1) in definitive but not primitive RBCs by stimulation of β-adrenergic/adenosine receptors, and (2) in definitive RBCs by hypoxic incubation of the chicken embryo. Since embryonic RBCs increase their hemoglobin-oxygen affinity by degradation of nucleotides such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), the induction ofp5n-I expression can be seen as an adaptive response to hypoxia. (Blood. 2003;102:4198-4205)

Physiological significance and molecular genetics of red cell enzymes involved in the ribonucleotide metabolism

At the final maturation process red blood cells (RBC) are enucleated, becoming unable to synthesize nucleic acids as well as proteins. RBCs survive approximately 120 days in circulation using glucose as the sole energy source. Most crucial RBC functions depend on ATP to sustain physiological homeostasis. It is thus quite important that generation of ATP by glycolysis and replenishing of adenine nucleotide pools by the reaction, which is catalyzed by adenylate kinase (AK1). In turn, ribosomal RNA is degraded during remodeling of reticulocytes, and pyrimidine ribonucleotides become unnecessary for RBC viability. Thus they should be dephosphorylated by pyrimidine 5'-nucleotidase (P5N-I) and finally transported outside RBCs. There have been reported that hereditary deficiency of AK1 and P5N-I may cause shortened RBC life span, i.e. hemolytic anemia. In this review, we summarize physiological importance of these enzymes, which are involved in ribonucleotides metabolism during RBC maturation.

Expression of pyrimidine 5'-nucleotidase subclass I during erythrocyte maturation in rats.

The subclass I enzyme of rat pyrimidine 5'-nucleotidase (P5N-I), which preferentially hydrolyzes (deoxy)CMP and UMP, is distributed specifically in red blood cells (RBCs), and its activity increases approximately six-fold as compared to the control value after erythropoietic induction by phenylhydrazine administration. In this study, we detected rat P5N-I protein by using antibodies against the chicken P5N-I enzyme. The molecular mass of rat P5N-I was approximately 37 kDa, as estimated by gel filtration chromatography and Western blot analysis. The pI value of the enzyme was approximately 5.7. This protein band was detected only in RBC lysate extract, i.e., not in cytosol from the erythropoietic spleen. Protein mass of the P5N-I enzyme, estimated by immunoblot analysis, was increased in proportion to the enzyme activity after erythropoietic induction in rats. No phosphorylation of the enzyme protein was detected by immunoblot analysis with anti-phosphoserine or anti-phosphotyrosine antibody. In conclusion, these findings indicate that the rat P5N-I enzyme is expressed specifically in reticulocytes and may therefore be essential in the maturation process of rat erythrocytes.

Chicken erythrocyte pyrimidine 5'-nucleotidase: purification and characterization of the subclass I enzyme.

Nucleotidase activities resembling subclass I and subclass II of human pyrimidine 5'-nucleotidases (P5N) were detected in chicken red blood cells (RBCs). In chicken RBCs from untreated controls, the activity of the subclass II enzyme was about one third of that of subclass I enzyme, whereas that ratio was approximately 5:1 in rat or human RBCs. The subclass I activity in chicken RBCs was increased 5- to 6-fold upon erythropoietic induction by phenylhydrazine administration, but the subclass II activity did not increase under these conditions. The subclass I enzyme was purified to near homogeneity. Its molecular mass was about 35 kDa as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis. Its N-terminal 12 amino acids, PEFQKKTVHIKD, were also determined. The catalytic properties of the subclass I enzyme were very similar to those of the human enzyme with regard to substrate (preferential hydrolysis of CMP, dCMP, UMP), Km values, optimum pH, and metal ion requirements. Antibodies against chicken P5N subclass I were raised in rats. The chicken P5N-I as well as the rat P5N-I proteins could be detected by antibodies in Western blot analyses, but not the P5N-II proteins. These findings indicate that P5N subclass I may have an important function in chicken erythropoiesis.

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5′ nucleotidase deficiency: 25 years later

In 1972, Valentine et al described, under the name of ‘non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency’, an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden–Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of ‘haemolytic disorders associated with increased ATP’. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5′ nucleotidase (P5N). In conclusion, all these cases were formerly referred to as ‘high ATP syndromes’ because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

Characteristics of red cell pyruvate kinase (PK) and pyrimidine 5'nucleotidase (P5N) abnormalities in acute leukaemia and chronic lymphoid diseases with leukaemic expression.

Red cell pyruvate kinase (PK), pyrimidine 5'nucleotidase (P5N) and reduced glutathione content (GSH) were studied in 126 untreated patients with acute leukaemia (AL, 80 cases), chronic lymphocytic leukaemia (B-CLL, 38 cases) and B-cell lymphoma with leukaemic expression (LSCL, eight cases). Acute leukaemias were classified into lymphoblastic (ALL) and non-lymphoblastic (ANLL), the latter have been further sub-divided into four different variants according to FAB morphological criteria (1976). A significant decrease of PK activity was observed only in the ANLL group, leading to a clear-cut difference with the ALL group where a normal value was obtained. The decrease of P5N activity was similar in all the morphological variants of ANLL and no abnormalities in the low PEP assay system or after fructose 1,6-bisphosphate (Fru 1,6-P2) activation were observed. P5N activity was found to be significantly decreased in all groups of patients except in B-CLL, where it was normal. In regards to the different morphological groups of ANLL, a striking decrease of P5N activity was observed in the M3 variant. Although red cell GSH content was significantly increased in all groups of patients, no correlation was demonstrated between the raised GSH levels and the decreased P5N activities.

Pyrimidine 5′nucleotidase and several other red cell enzyme activities in β‐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.

New defects of pyrimidine metabolism.

The number of inherited defects of the pyrimidine metabolism described so far is small, compared to that of the purine metabolism. Combined deficiency of orotate phosphoribosyltransferase (OPRT) (EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (ODC) (EC 4.1.1.23), designated as type I hereditary orotic aciduria, presents with characteristic clinical features such as hypo-chromic anemia with a megaloblastic bone marrow and crystalluria. Only six patients have been described and, as far as we know, new cases have not been discovered recently. ODC deficiency with similar clinical phenomena and leading to increased urinary excretion of orotate and orotidine has been detected in only one patient (1). A third defect, a deficiency of pyrimidine 5′-nucleotidase (Py-5N). (EC 3.1.3.5.) in erythrocytes, is associated with chronic hemolytic anemia and prominent basophylic stippling of the erythrocytes due to accumulated pyrimidine nucleotides. An increasing number of patients have been reported, their detection being facilitated by the typical phenomena. We do not know whether the urinary pyrimidine profile in this condition is abnormal.

Hemolytic Anemia in Hereditary Pyrimidine 5′-Nucleotidase Deficiency: Nucleotide Inhibition of G6PD and the Pentose Phosphate Shunt

AbstractWe evaluated the erythrocytes of two patients with hereditary pyrimidine 5-nucleotidase deficiency. Significant findings included an increased reduced glutathione content, increased incubated Heinz body formation, a positive ascorbate cyanide test, and decreased intraerythrocytic pH. The pentose phosphate shunt activity of the patients’ red cells as measured by the release of 14CO2 from 14C-1-glucose was decreased compared to high reticulocyte controls. Glucose-6-phosphate dehydrogenase (G6PD) activity in hemolysates from control erythrocytes was inhibited 43% by 5.5 mM cytidine 5′-triphosphate (CTP) and 50% by 5.5 mM uridine 5′-triphosphate (UTP) at pH 7.1. CTP was a competitive inhibitor for G6P (Ki, = 1.7 mM) and a noncompetitive inhibitor for NADP+ (Ki = 7.8 mM). Glutathione peroxidase, glutathione reductase, and 6-phosphogluconate dehydrogenase were not affected by these compounds. Pentose phosphate shunt activity in control red cell hemolysate at pH 7.1 was inhibited to a similar degree by 5.5 mM CTP or UTP. Since the intracellular concentrations of G6P and NADP+ are below their Kms for G6PD, these data suggest that high concentrations of pyrimidine 5′-nucleotides depress pentose phosphate shunt activity in pyrimidine 5′-nucleotidase deficiency. Thus, this impairment of the pentose phosphate pathway appears to contribute to the pathogenesis of hemolysis in pyrimidine 5′-nucleotidase deficiency hemolytic anemia.

Relationship between lead concentration in blood and the activities of erythrocyte pyrimidine 5'-nucleotidase and delta-aminolevulinate dehydratase in the mice exposed to lead.

The relationship between the activities of both pyrimidine 5'-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) in erythrocytes and the concentration of lead in blood was investigated in the mice which were given ad libitum a drinking water containing lead of 10 to 500 ppm, for 30 days. From these results, it was demonstrated that the erythrocyte Py5N activity is inhibited by lead and its activity level is negatively correlated with the concentration of lead in blood (r = -0.78). In addition, it was suggested that the erythrocyte Py5N activity is a better indicator in the exposure to relatively high lead concentration while the ALA-D is a more sensitive indicator for evaluating the lead exposure of low and moderate levels.

Red-cell pyrimidine 5′-nucleotidase and lead poisoning

In an effort to determine its value as a biological index of lead poisoning, we investigated the red-cell pyrimidine 5'-nucleotidase (Py5N) activity, along with the classical parameters, in 22 subjects with varying degrees of lead intoxication. In all cases, the red-cell Py5N activity was found to be decreased, even when most of the other biological tests of lead intoxication remained negative. Below the level of 150 μg of lead per 100 ml of blood, the magnitude of the red-cell Py5N decrease was roughly proportional to the level of lead in peripheral blood. We conclude that red-cell Py5N is a convenient, reliable and sensitive index of lead exposure.