Nucleoside Phosphorylase Activity Research Articles

Effects of Orally Administered 5-Fluorouracil and Its Derivative 5′-Deoxy-5-fluorouridine on 7,12-Dimethylbenz[β]Anthracene-Induced Breast Carcinomas in Rats

Antitumour activity of 20 mg/kg 5-fluorouracil (5-FU) and 100 or 200 mg/kg 5'-deoxy-5-fluorouridine (5'-DFUR) was assessed by tumour regression, light and electron microscopic changes, and immunohistochemical variations in broxuridine labelling in 7,12-dimethyl-benz[a]anthracene- (DMBA-) induced breast carcinomas in rats. The relative regression of tumours was significantly (P less than 0.05) greater in animals receiving 5-FU or 5'-DFUR than in control animals. Light microscopic changes became apparent after 3 or 4 days' treatment and were more evident after 4-21 days. Electron microscopic degeneration of tumours was observed after 1 day. The broxuridine labelling index decreased significantly (P less than 0.05) in rats receiving 5-FU and especially in those receiving 5'-DFUR. Concentrations of 5-FU in DMBA-induced breast carcinomas were significantly (P less than 0.05) higher in rats treated with 5'-DFUR than in those treated with 5-FU, and the histological degeneration was significantly (P less than 0.05) more advanced in 5'-DFUR- than in 5-FU-treated animals, possibly due to the higher pyrimidine nucleoside phosphorylase activity in the former treatment group.

Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio

Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect. We therefore measured the activity of adenosine kinase, adenosine deaminase, nucleoside phosphorylase and xanthine oxidoreductase in homogenates of hearts and myocytes from neonatal and adult rats. In hearts the activity of adenosine deaminase and nucleoside phosphorylase (10–20 U/g protein) changed relatively little. However, adenosine kinase activity decreased from 1.3 to 0.6 U/g ( P < 0.025), and xanthine oxidoreductase activity increased from 0.02 to 0.85 U/g ( P < 0.005). Thus the ratio in activity of these rate-limiting enzymes for anabolism and catabolism dropped from 68 to 0.68 during cardiac development. In contrast, the ratio in myocytes remained unchanged (about 23). The large difference in adenosine anabolism/catabolism ratio, observed in heart homogenates, could explain why ATP breakdown due to hypoxia is lower in neonatal than in adult heart. Because this change is absent in myocytes, we speculate that mainly endothelial activities of adenosine kinase and xanthine oxidoreductase are responsible for this shift in purine metabolism during development.

A pleitropic mutation affecting purine metabolism in Bacillus subtilis

A pleiotropic mutation ( cpm) which is localized in the vicinity of the spoOA gene is described in Bacillus subtilis. The mutation inhibits spore formation, rendering bacteria auxotrophic for adenine and tyrosine, enhances sensitivity to antibodiotics, decreases cell motility, inhibits the ability to grow on pentoses and to maintain bacteriophage multiplication, induces severalfold the activities of alkaline proteinase and α-amylase. At the same time, the cpm mutant starts to excrete inosine into the growth medium. This excretion most probably is explained by 1 50-fold increase in the activity of inosine monophosphate: 5′-nucleotidase and a 10-fold decrease in the activity of purine nucleoside phosphorylase. The inosine production and Ade − phenotype of the cpm mutant is not accompanied by the change in the activity of succinyl adenosine monophosphate synthetase. The nature of the mutation is discussed.

A pleitropic mutation affecting purine metabolism inBacillus subtilis

A pleiotropic mutation (cpm) which is localized in the vicinity of the spoOA gene is described in Bacillus subtilis. The mutation inhibits spore formation, rendering bacteria auxotrophic for adenine and tyrosine, enhances sensitivity to antibodiotics, decreases cell motility, inhibits the ability to grow on pentoses and to maintain bacteriophage multiplication, induces severalfold the activities of alkaline proteinase and α-amylase. At the same time, the cpm mutant starts to excrete inosine into the growth medium. This excretion most probably is explained by 1 50-fold increase in the activity of inosine monophosphate: 5′-nucleotidase and a 10-fold decrease in the activity of purine nucleoside phosphorylase. The inosine production and Ade− phenotype of the cpm mutant is not accompanied by the change in the activity of succinyl adenosine monophosphate synthetase. The nature of the mutation is discussed.

Genetic deficiency of purine nucleoside phosphorylase in the mouse. Characterization of partially and severely enzyme deficient mutants

Two independent mutations of purine nucleoside phosphorylase were identified in the first-generation progeny of male mice that had been treated with the mutagen N-ethylnitrosourea and mated to untreated females. The common allele in inbred strains is Np-1a and the mutants are assigned the gene symbols Np-1e and Np-1f. Heterozygotes had approximately half normal purine nucleoside phosphorylase activity in erythrocytes and activity of homozygotes was 17 and 5% of NP-1A for NP-1E and NP-1F, respectively. The following properties are consistent with both Np-1e and Np-1f being point mutations: the expression of residual but markedly reduced activity with normal Michaelis constants for inosine and phosphate, altered isoelectric points, and increased thermal lability. The reduction in erythrocyte activity was also evident in other tissues. A metabolic consequence of the mutations was increased purine nucleoside excretion. Inosine and guanosine, total 150 +/- 84 microM, and inosine, deoxyinosine, guanosine, and deoxyguanosine, total 1490 +/- 190 microM, were present in urine of Np-1e/Np-1e and Np-1f/Np-1f mice, respectively, but not in normal urine, less than 10 microM.

A new colorimetric assay for purine nucleoside phosphorylase

A new colorimetric procedure for the determination of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activity is described. In this procedure, the hydrogen peroxide formed in the PNP-xanthine oxidase reaction is used to oxidize the chromogenic reagents--3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone using the enzyme peroxidase. The rate of enzyme reaction is followed at 520 nm. This procedure correlated well with the UV method (290 nm), with a correlation coefficient of 0.98 (P less than 0.005). Within-run and between-run precision (CV) were less than 2.8% and 3.7%, respectively. Here we also describe an optimized NAD-dependent method (340 nm) for PNP determination. The colorimetric method is superior to both the 340 nm and the UV methods in terms of both sensitivity and precision. The mean erythrocyte PNP activity for 17 healthy subjects was 11.88 U/mL packed cells for the NAD-dependent method and 13.22 U/mL packed cells for the colorimetric method.

T-lymphocyte dysfunction in the elderly associated with zinc deficiency and subnormal nucleoside phosphorylase activity: Effect of zinc supplementation

The present study was conducted in an attempt to explore a possible mechanism for zinc-deficiency-induced T-lymphocyte dysfunction in the elderly. Eight elderly subjects aged 65–78 years served in this study. All subjects were anergic, zinc-deficient and had low activity of erythrocyte nucleoside phosphorylase. Baseline values for zinc status and immunological indices were established after which subject were treated with zinc acetate ( 60 mg elemental zinc per day given orally) for a period of 4 1 2 months. Zinc supplementation resulted in a significant increase in the concentrations of zinc in plasma ( P < 0.001), erythrocytes ( P <0.05), lymphocytes ( P < 0.001) and neutrophils ( P < 0.005). The activity of nucleoside phosphorylase in erythrocytes increased significantly ( P < 0.001) as a result of zinc treatment. This was associated with significant improvement in the delayed cutaneous hypersensitivity reactions.

Purine nucleoside phosphorylase in erythrocytes: Determination of optimum reaction conditions

Reaction conditions for determining the activity of purine nucleoside phosphorylase (PNP; E.C. 2.4.2.1) were investigated. We examined the kinetic parameters for the enzymatic reaction with respect to the substrates inosine and phosphate. We confirmed the pH optimum, established the optimal concentration of xanthine oxidase and that of calcium and magnesium. The Km values for inosine and phosphate were found to be 60 uM and 667 uM, respectively. Optimum assay conditions for PNP activity were established. This optimized method has been compared with other procedures and found to be more sensitive and to yield significantly higher activities. The experimental variation of a manual procedure using these optimum reaction conditions was less than 4.5%. The mean erythrocyte PNP activity of 28 healthy subjects was estimated to be 9.71 U/mL packed cells at 25 degrees C and 18.60 U/mL packed cells at 37 degrees C.

Determination of adenosine deaminase and purine nucleoside phosphorylase activities using high-performance liquid chromatographic and radiochromatographic methods

Two methods for the determination of adenosine deaminase and purine nucleoside phosphorylase activities were compared. The high-performance liquid chromatographic (HPLC) technique used separation on a reversed-phase silica column and exhibited adequate sensitivity and a markedly higher rate of analysis compared with that of the paper radiochromatographic method. Correlation analysis of the results obtained by the two methods on a set of lymphoid cells from 25 patients with lympho-proliferative disorders confirmed the utility of the HPLC technique in clinical investigations.

Differential Expression of Adenosine Deaminase Isozymes in Acute Leukemia

Total adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities were measured in cell samples from 13 cases of de novo acute leukemia and from three cases of chronic myeloid leukemia in blast crisis (CMLBC). These cases could be separated into lymphoid and nonlymphoid types on the basis of enzyme activity, with two misclassifications. However, PNP activity added little or no discriminatory information. Analysis for expression of the various molecular weight (mol wt) ADA isozymes, ADA1 (40 Kd) and ADA2 (110 Kd), revealed that ADA2 was expressed exclusively in nonlymphoid cells whereas ADA1 was found in both lymphoid and nonlymphoid cell types. Identification of ADA2 divided these leukemia cases into lymphoid and nonlymphoid types with no misclassifications (P = .0002; Fisher's exact test). Acute nonlymphoblastic leukemia (ANLL) with a monocytic component tended to have a greater percentage of ADA2 than ANLL without a monocytic component. These studies suggest that ADA2 may be a novel biochemical marker for an immature nonlymphoid cell.

Differential expression of adenosine deaminase isozymes in acute leukemia

Total adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities were measured in cell samples from 13 cases of de novo acute leukemia and from three cases of chronic myeloid leukemia in blast crisis (CMLBC). These cases could be separated into lymphoid and nonlymphoid types on the basis of enzyme activity, with two misclassifications. However, PNP activity added little or no discriminatory information. Analysis for expression of the various molecular weight (mol wt) ADA isozymes, ADA1 (40 Kd) and ADA2 (110 Kd), revealed that ADA2 was expressed exclusively in nonlymphoid cells whereas ADA1 was found in both lymphoid and nonlymphoid cell types. Identification of ADA2 divided these leukemia cases into lymphoid and nonlymphoid types with no misclassifications (P = .0002; Fisher's exact test). Acute nonlymphoblastic leukemia (ANLL) with a monocytic component tended to have a greater percentage of ADA2 than ANLL without a monocytic component. These studies suggest that ADA2 may be a novel biochemical marker for an immature nonlymphoid cell.

Purine and Pyrimidine Metabolism in Mollicutes Species

We studied the purine enzyme activities in dialyzed cytoplasmic extracts from the following eight species, representing four genera, of Mollicutes: Mycoplasma pneumoniae FHT (T = type strain) and M129, Mycoplasma bovigenitalium PG-11T, Mycoplasma hominis PG-21T and 1620, Mycoplasma genitalium G-37T, Mycoplasma hyopneumoniae JT, Ureaplasma urealyticum T960T, Spiroplasma citri Maroc-R8A2T, and Anaeroplasma intermedium 5LA. In an investigation of purine nucleoside kinase activity we also included M. hominis 13408, 10144, 13428, 1612, 1184, and Botte. All of these Mollicutes species except U. urealyticum had purine phosphoribosyltransferase activity for adenine, hypoxanthine, and guanine; U. urealyticum had only adenine phosphoribosyltransferase activity. All of the organisms had nucleoside phosphorylase activity which used either ribose 1-phosphate or deoxyribose 1-phosphate and adenine, hypoxanthine, or guanine for the synthesis of nucleosides and adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, or deoxyinosine in the reverse direction. All had 5′-nucleotidase activity for adenosine monophosphate, deoxyadenosine monophosphate, inosine monophosphate, or guanosine monophosphate. Only M. hominis 1620, 13408, 10144, and 13428, A. intermedium, and S. citri had pyrophosphate-dependent nucleoside kinase activity. Only S. citri had nucleoside kinase activity with adenosine triphosphate and deoxyguanosine. We studied pyrimidine enzyme activities in all of the Mollicutes species except M. hominis and M. bovigenitalium. All of the Mollicutes species assayed had thymidine, thymidylate, and deoxycytidine kinase and thymidine and uridine phosphorylase activities. All of the Mycoplasma spp. had deoxycytotidine monophosphate and cytidine-deoxycytidine deaminase activities. All of the Mycoplasma spp. and U. urealyticum lacked deoxyuridine triphosphatase activity. U. urealyticum lacked deoxycytidine monophosphate deaminase activity, but otherwise it resembled all of the Mycoplasma spp. A. intermedium and S. citri differed from each other and from Mycoplasma spp. and U. urealyticum in the patterns of pyrimidine enzyme activities. Pyrophosphate-dependent nucleoside kinase activity was the most variably detected activity. None of the Mycoplasma spp. except four of eight strains of M. hominis had this kinase activity. Likewise, U. urealyticum did not have the pyrophosphate-dependent nucleoside kinase activity; however, A. intermedium and S. citri did have this enzyme activity. The absence of deoxyuridine triphosphatase activity in all Mycoplasma spp. may be related to their proposed rapid evolution and the relative lack of conserved sequences in their 5S ribosomal ribonucleic acids.

Purine nucleoside phosphorylase: a histochemical marker for glial cells

The distribution of purine nucleoside phosphorylase activity has been investigated histochemically in rat and guinea-pig brain. At the light microscopical level, enzyme activity was most pronounced in glial cells in various anatomical regions of the rat brain. In contrast, the guinea-pig brain presented only a weak activity. Endothelial cells of both species were also reactive. These findings were confirmed by electron microscopy. Based upon anatomical position and morphologic characteistics, positive glial cells were identified as astrocytes. Precipitate-rich astrocytic processes could be easily demonstrated in between barely reactive neuronal fibers and around microvessels. A minority of astrocytes was devoid of reaction product. The present method may offer a valuable tool for the histopathological study of several types of disorders in which glial cells play a functional role.

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep.

Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (NuI) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nui). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NPH) is incompletely dominant to that for low activity (NPL). Inheritance data indicate that the Nu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labile NPL gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep

Cellular zinc in patients with diabetes mellitus

Although hyperzincuria appears to be a common finding in most diabetic subjects, plasma zinc concentration has been reported to be normal, increased, or decreased in these patients. In this study we assayed cellular zinc levels of diabetic subjects in order to assess zinc status. Zinc levels in plasma, lymphocytes, granulocytes, and platelets were lower than in controls. We also observed hyperzincuria in our subjects. The activity of nucleoside phosphorylase a zinc dependent enzyme, was decreased in the lymphocytes. Based on these data we conclude that our diabetic subjects were zinc deficient.

Decreased B lymphocyte ecto-5'nucleotidase and increased adenosine deaminase in mononuclear cells from patients infected with human immunodeficiency virus.

The levels of purine enzyme activities were studied in 10 patients with acquired immunodeficiency syndrome (AIDS) or AIDS related complex (ARC) and in 6 healthy individuals with antibodies against human immunodeficiency virus (HIV). All AIDS/ARC patients studied had ecto-5'nucleotidase (ecto-5'NUC) activity in B lymphocytes below the normal range and 4 out of 6 clinically healthy HIV-positive likewise had reduced activity. Increased numbers of activated B lymphocytes were found both in the group of healthy HIV positive individuals and in AIDS/ARC patients. Further studies are needed to define whether the decrease in ecto-5'NUC activity on the B lymphocytes is a result of increased activation of the cells or of a B cell defect. No significant changes were found in ecto-5'NUC levels in T lymphocytes or mononuclear cells (MNC), neither in the group of AIDS/ARC patients nor in the healthy HIV-positive group. Both AIDS/ARC patients and healthy individuals with antibodies against HIV had increased levels of adenosine deaminase (ADA) activity in mononuclear cells, but only in the group of AIDS/ARC patients was the increase significant. No changes were found in purine nucleoside phosphorylase (PNP) activity in the two groups tested. From these investigations of purine enzyme levels and other markers of immune function in both sick and healthy HIV infected individuals we conclude that the observed changes in ecto-5'NUC and ADA activities in HIV infected patients are not a direct result of the HIV infection but develop early in the course of the disease.

96 Enzyme therapies for hyperuricemia

The relationship between dietary factors and the prevalence of gout is well known. Many gouty patients eat food rich in purines and excrete increased urinary urate. The dietary purines may contribute to hyperuricemia. When enzyme activity related to purine metabolism in the human digestive juice was measured, nucleotidase, purine nucleoside phosphorylase and xanthine oxidase activity was shown in bile and pancreatic juice, although they were not so high. Therefore, the effect in chickens of oral administration of enzymes related to purine catabolism on plasma uric acid concentrations was studied. After oral administration of inosine to chickens, plasma uric acid concentrations increased markedly. Prior oral administration of the enzymes purine nucleoside phosphorylase, xanthine oxidase and uricase prevented tiie rise in plasma uric acid due to inosine intake. It seems that these enzymes administered orally degrade the dietary purines during gastrointestinal passage. Secondly, liposomal-entrapped methoxypolyethyleneglycol(PEG) modified uricase was used to study its plasma urate lowering effect after oral administration to chickens. Plasma uric acid concentrations fell gradually and were accompanied by a rise in plasma uricolytic activity. Oral administration of purine enzymes and/or PEG-modified uricase liposomes may have a useful effect as a new enzyme therapy for hyperuricemia and gout.

Pathways of adenine nucleotide catabolism in primary rat cardiomyocyte cultures

The pathways of adenine nucleotide catabolism were investigated in cultured beating cardiomyocytes. The activity of the enzymes involved in AMP degradation was assayed in cell extracts. Fluxes of label from ATP to the various purine derivatives were measured in intact cells. Under physiological conditions, cells degraded AMP through deamination to IMP. IMP was rapidly degraded to inosine, hypoxanthine, xanthine and uric acid, which were effluxed from the cells. This is in accord with the fact that the activity of AMP deaminase (EC 3.5.4.6) was 7-fold that of AMP 5'-Nucleotidase (EC 3.1.3.5). Mild ATP-degradation, induced by inhibition of glycolysis by iodoacetate, caused no alterations in the degradation pathways (more than 85% through deamination to IMP). However, fast ATP-degradation (83% of adenine nucleotides/10 min), induced by simultaneous inhibition of glycolysis and electron transport (by antimycin A), caused increased dephosphorylation of AMP to adenosine (50% of total AMP-degradation). The cardiomyocyte extracts were found to contain a significant activity of purine nucleoside phosphorylase (EC 2.4.2.1). Despite the presence of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), salvage of hypoxanthine to IMP, both at physiological as well as at conditions associated with ATP degradation, was slow. The salvage of adenosine appeared to be efficient at physiological conditions, but not at fast rates of ATP degradation.

Comparison of the pyrimidine nucleoside phosphorylase activity in human tumors and normal tissues

The novel fluoropyrimidine, 5'-deoxy-5-fluorouridine (5'-dFUDR) has strong tumor-inhibiting effect without severe cytotoxic effects on the normal cells. This pro-drug is metabolized by the enzyme pyrimidine nucleoside phosphorylase (PN'ase) to the active form 5-fluorouracil. Comparative determinations of pyrimidine nucleoside phosphorylase activity in tissue extracts revealed a significantly higher activity in the human gastrointestinal cancer tissues (6.84 +/- 0.70 nmol/mg protein) than in normal tissues from the same organ (2.37 +/- 0.21 nmol/min/mg protein).