Enzyme Purine Nucleoside Phosphorylase Research Articles

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.

Central nervous system dysfunction and erythrocyte guanosine triphosphate depletion in purine nucleoside phosphorylase deficiency.

Developmental retardation was a prominent clinical feature in six infants from three kindreds deficient in the enzyme purine nucleoside phosphorylase (PNP) and was present before development of T cell immunodeficiency. Guanosine triphosphate (GTP) depletion was noted in the erythrocytes of all surviving homozygotes and was of equivalent magnitude to that found in the Lesch-Nyhan syndrome (complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency). The similarity between the neurological complications in both disorders indicates that the two major clinical consequences of complete PNP deficiency have differing aetiologies: neurological effects resulting from deficiency of the PNP enzyme products, which are the substrates for HGPRT, leading to functional deficiency of this enzyme. immunodeficiency caused by accumulation of the PNP enzyme substrates, one of which, deoxyguanosine, is toxic to T cells. These studies show the need to consider PNP deficiency (suggested by the finding of hypouricaemia) in patients with neurological dysfunction, as well as in T cell immunodeficiency. They suggest an important role for GTP in normal central nervous system function.

Characterization of arabinosylguanine resistance in a lymphoblastoid cell line.

In recent years, cancer chemotherapists have been searching for drugs that would selectively destroy malignant cells while sparing healthy host tissue. The discovery that dAdo and dGuo are more toxic to T compared to B lymphoblasts through the actions of their 5′-tri-phosphate derivatives (1) has spurred interest in developing analogs of these compounds that would exhibit a similar specificity of action, without being degraded by the purine catabolic enzymes adenosine deaminase (ADA) and purine nucleoside Phosphorylase (PNP). Using this rationale, 2-fluoroadenine arabinoside was developed as an ADA-insensitive analog of adenine arabinoside, and it is presently undergoing clinical trials as an antileukemic agent (2). A recent report (3) indicated that araG was resistant to cleavage by PNP, and exhibited selective toxicity for T relative to B lymphoblasts. We have previously compared the effects of araG in human lymphoblasts to those of araC, another arabinosyl antimetabolite that is in clinical use an an antileukemic agent (4). While araG was less potent than araC, it was far more selective in its cytotoxic effects. Based on these findings, araG shows promise as an antileukemic agent and merits greater study. In this paper, we report on the further characterization of the metabolism of araG in human cell lines exhibiting varying levels of sensitivity to araG.

The effects of PNP inhibition on rat lymphoid cell populations.

In humans an inherited deficiency of the enzyme purine nucleoside Phosphorylase (PNP; EC 2.4.2.1) is associated with a T cell deficiency and apparently normal B cells (1,2). Although a deficiency of PNP is present in all cells of deficient individuals, only the cells within the T lymphocyte lineage are affected. The immunodeficiency state associated with PNP deficiency is thought to arise from block(s) in T lymphocyte differentiation resulting from the accumulation of toxic metabolites of the enzyme’s substrate(s) (3-5).

A rapid, simple method for the micro-estimation of purine nucleoside phosphorylase activity in peripheral blood lymphocytes

Giblett and coworkers showed that a congenital deficiency of the enzyme purine nucleoside phosphorylase (PNP), which catalyses the reversible conversion of inosine to hypoxanthine, resulted in depressed T-cell but normal B-cell immunity [l]. This enzyme has since been shown to play a key role in the maturation of lymphocytes [2] and is used as a non-membrane biochemical marker in lymphoid malignancies [3]. Most PNP assays are compfex and involve the use of radioactive isotopes and chromatographic separation of product(s) and substrate. In this manuscript a rapid, simple spectrophotometric method for PNP estimation in lymphocytes using a Cobas-Bio centrifugal analyzer is described and compared with a radioisotopic assay. The spectrophotometric assay measures the PNP catalyzed conversion of inosine to hypoxanthine by estimating the rapid conversion of hypoxanthine to uric acid with xanthine oxidase.

Differential metabolism of guanine nucleosides by human lymphoid cell lines.

Deficiency of the enzyme purine nucleoside phosphorylase is associated with a specific depletion of T cells which is presumably mediated by its substrate, 2'-deoxyguanosine. Inhibitors of this enzyme are therefore being developed as potential immunosuppressive agents. We have compared the effects of 8-aminoguanosine, a competitive inhibitor of purine nucleoside phosphorylase, on the metabolism of 2'-deoxyguanosine by human T lymphoblasts, B lymphoblasts, and mature T-cell lines. 8-Aminoguanosine markedly potentiates the accumulation of dGTP in T lymphoblasts, but results in increased GTP levels in B lymphoblasts and mature T cells. GTP accumulation is associated with ATP depletion of a magnitude similar to that seen with an inhibitor of de novo purine biosynthesis, but does not result in inhibition of either DNA or RNA synthesis. In contrast, direct inhibition of de novo purine biosynthesis sharply decreased the incorporation of [3H]uridine into both DNA and RNA. We conclude that the mechanism of cell damage resulting from prolonged accumulation of GTP appears to involve more than inhibition of de novo purine biosynthesis and consequent ATP depletion. Perturbations in guanine nucleotide pools resulting from partial inhibition of purine nucleoside phosphorylase activity in vivo could result in cellular toxicity not limited to the target T cell population.

Lack of inhibition of purine nucleoside phosphorylase and adenosine deaminase in patients treated with azathioprine.

Deficiency of the purine salvage enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) are known causes of immunodeficiency. Evidence for inhibition of these enzymes was sought in 16 patients on azathioprine therapy by testing for deoxyguanosine (PNP deficiency) and deoxyadenosine (ADA deficiency) in urine using a novel phosphorescence method. These abnormal nucleosides were not found in urine of azathioprine treated patients or in 30 normal controls but were easily detected in urine from proven cases of PNP and ADA deficiency suggesting lack of in vivo inhibition of PNP and ADA by azathioprine.

2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation.

Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.

Evidence for a trans-dominant regulator of purine nucleoside phosphorylase expression in rat hepatoma cells.

Purine nucleoside phosphorylase (PNP) levels are modulated during the growth cycle of rat hepatoma cells and increase two- to three-fold as cells go from early exponential growth phase to stationary growth phase. A mutant of these hepatoma cells has been isolated which is deficient in PNP activity. Quantitative immunoprecipitation tests indicate that the decrease in enzyme activity is due to a decrease in the number of PNP molecules. The low level of PNP enzyme produced by the mutant, however, is indistinguishable from the wild-type enzyme, suggesting that the mutant may be defective in the ability to modulate PNP levels. Fusion of the mutant cells to wild-type parental cells results in hybrids that express the mutant phenotype. Segregants that arise from the hybrids show chromosome loss and reexpression of the wild-type parental phenotype, the mutant parental phenotype, and a 2S wild-type phenotype. These indicate the following about the defect in modulation in the mutant PNP-100: (1) it is trans dominant to the wild-type; (2) its effect is negative; (3) some genomic element is required for its continued effect; and (4) it does not act by obliterating its functioning counterpart in hybrid cells.

Inhibition of Purine Nucleoside Phosphorylase by 8-Aminoguanosine: Selective Toxicity for T Lymphoblasts

The guanosine analog 8-aminoguanosine is an effective inhibitor of the purine degradative enzyme purine nucleoside phosphorylase, both in vitro and in intact lymphoid cells. In a human lymphoblast tissue culture system, 8-aminoguanosine, in combination with low concentrations of 2'-deoxyguanosine, causes toxicity toward T cells but not B cells. The selective T cell toxicity correlates with increased accumulation of deoxyguanosine triphosphate in the treated T lymphoblasts.

Immunofluorescence: A sensitive and rapid method for the detection of purine nucleoside phosphorylase in single cells

Indirect immunofluorescence was used as a rapid, sensitive and specific method for the visualization of the enzyme purine nucleoside phosphorylase in single cells. The enzyme was localized throughout cytoplasm of human lymphoblasts and fibroblasts but not in cell nuclei. This method is valuable for the detection of mutant enzyme protein in cell-mediated immunodeficiencies caused by purine nucleoside phosphorylase deficiency since it does not rely on enzyme activity. It requires only a limited number of cells and can therefore be used for the rapid screening for the presence of cross-reactive protein in immunodeficiency diseases.

Deoxyguanosine toxicity in a mouse T lymphoma: Relationship to purine nucleoside phosphorylase-associated immune dysfunction

The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP. The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.

70] Purine nucleoside phosphorylase from Salmonella typhimurium and rat liver

Publisher Summary This chapter describes the purification procedure of purine nucleoside phosphorylase enzyme from Salmonella typhimurium and rat liver. When Salmonella typhimurium is grown in the presence of a purine ribonucleoside, purine nucleoside phosphorylase and phosphodeoxyribomutase are coordinately induced. But when cells are grown in the presence of deoxyribose or deoxyribonucleosides, four enzymes are induced. These include thymidine phosphorylase, purine nucleoside phosphorylase, phosphodeoxyribomutase, and deoxyribose-5-phosphate aldolase. The demonstration that purified purine nucleoside phosphorylase has specificity for both purine ribo- and deoxyribonucleosides is consistent with the hypothesis that a single gene codes for the enzyme but that the gene is regulated by more than one effector. The phosphorylase is present in a relatively high concentration in rat liver tissue accounting for about 0.2% of the soluble protein. The breakdown of inosine is followed by an increase in absorbance at 293 nm produced by uric acid in a coupled assay system with xanthine oxidase.

71] Purine nucleotide phosphorylase from rabbit liver

This chapter describes the purification procedure of purine nucleoside phosphorylase enzyme from rabbit liver. Isolation and purification procedures, and storage of the enzyme, are done between 4–10°. Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase) catalyzes the phosphorolysis and synthesis of nucleosides with almost equal efficiency. The enzyme from rabbit liver and bovine brain was purified to homogeneity and partially characterized. The assay measures the disappearance of guanosine, in the reverse reaction by the phosphorolysis of guanosine, which can be determined spectrophotometrically. The synthesis of the nucleoside by the ribosylation of purines is measured in the forward reaction. This is determined spectrophotometrically by recording the increase in the absorbance of the nucleoside product. The phosphorolysis is determined spectrophotometrically by measuring the increase in absorbance at 293 nm in a coupled system containing a phosphate buffer, nucleoside, xanthine oxidase, and purine nucleoside phosphorylase.

Metabolic studies of purine metabolism in the pig during the oral administration of guanine and allopurinol

(1) Allopurinol and guanine, both separately and together, were fed over varying periods to diiferent groups of large white/landrace cross pigs. Although allantoin and not uric acid represented the end point of purine metabolism in this species, total urinary purine excretion approximated that of a man of equivalent weight on a purine-free diet. No abnormality in the handling of guanine was found, up to 50 per cent of an exogenous load being absorbed, metabolized and rapidly excreted in the urine as allantoin. (2) Allopurinol alone, in increasing dosage, was capable of total saturation of the enzyme xanthine oxidase at high dosage levels, but produced only slight reduction in total purine excretion at any dosage. Allopurinol riboside was the principal urinary metabolite, increasing with increasing allopurinol dosage, even at dosages above enzyme saturation levels, when oxipurinol excretion had levelled off. Xanthine replaced allantoin as the principal urinary purine metabolite during allopurinol therapy but, despite saturation of the xanthine oxidase, allantoin excretion did not fall to zero and hypoxanthine excretion was not increased. Allopurinol also had an effect on pyrimidine excretion in the pig as indicated by increased urinary orotic acid and orotidine levels, an effect not eliminated when guanine was given together with allopurinol. (3) Combined therapy with allopurinol and guanine produced three additional effects to those when allopurinol was given alone, (a) Allopurinol reduced substantially the considerable increase in total purine excretion resulting from guanine alone, a finding difficult to explain on the basis of feedback inhibition of de novo purine synthesis. (b) Urinary hypoxanthine excretion increased and at the same time allopurinol riboside excretion decreased substantially, suggesting competitive inhibition of allopurinol riboside formation as mediated by the enzyme purine nucleoside phosphorylase. (c) Xanthine was again the principal urinary metabolite, but at levels in excess of its solubility, so that coprecipitation with oxipurinol occurred in the renal tubules causing a considerable degree of renal dysfunction. Crystals were not found in any other tissue, including muscle, so that no evidence of guanine gout was noted, nor any other abnormality of purine metabolism which could be related to leg weakness in pigs.

LINGCOD MUSCLE PURINE NUCLEOSIDE PHOSPHORYLASE

A purine nucleoside phosphorylase enzyme preparation, which catalyzed the general reaction ribose (deoxyribose) [Formula: see text] nucleoside (deoxynucleoside)+orthophosphate, was isolated from muscles of the lingcod (Ophiodon elongatus). The reaction was found to be about 85% in favor of nucleoside synthesis with 10 μM./ml. concentrations of reactants in the hypoxanthine–inosine system. With similar concentrations of reactants in the guanine–guanosine system the equilibrium was about 75% in favor of nucleoside synthesis, and with considerably higher concentrations of reactants in this system this percentage decreased markedly. Purified preparations were stable for over a year at −20 °C., contained two major protein components (zone electrophoresis and ultracentrifuge data), were rather heat labile, and possessed a maximum activity corresponding to liberation of 32 μM. orthophosphate/mg. protein N/hour. Evidence is given in support of the view that a single enzyme is responsible for the above reaction and that it possesses strict specificity for α-D-ribofuranose 1-phosphate and deoxyribose 1-phosphate. Of a large number of substituted purines investigated, only the following were active: hypoxanthine, xanthine, 6-mercaptopurine, 6-methylpurine, 8-azaguanine, guanine, adenine, and 2, 6-diaminopurine. Methods are given for the preparation, in good yield, of analytically pure dicyclohexylammonium salts of ribose 1-phosphate and deoxyribose 1-phosphate.

LINGCOD MUSCLE PURINE NUCLEOSIDE PHOSPHORYLASE

A purine nucleoside phosphorylase enzyme preparation, which catalyzed the general reaction ribose (deoxyribose) [Formula: see text] nucleoside (deoxynucleoside)+orthophosphate, was isolated from muscles of the lingcod (Ophiodon elongatus). The reaction was found to be about 85% in favor of nucleoside synthesis with 10 μM./ml. concentrations of reactants in the hypoxanthine–inosine system. With similar concentrations of reactants in the guanine–guanosine system the equilibrium was about 75% in favor of nucleoside synthesis, and with considerably higher concentrations of reactants in this system this percentage decreased markedly. Purified preparations were stable for over a year at −20 °C., contained two major protein components (zone electrophoresis and ultracentrifuge data), were rather heat labile, and possessed a maximum activity corresponding to liberation of 32 μM. orthophosphate/mg. protein N/hour. Evidence is given in support of the view that a single enzyme is responsible for the above reaction and that it possesses strict specificity for α-D-ribofuranose 1-phosphate and deoxyribose 1-phosphate. Of a large number of substituted purines investigated, only the following were active: hypoxanthine, xanthine, 6-mercaptopurine, 6-methylpurine, 8-azaguanine, guanine, adenine, and 2, 6-diaminopurine. Methods are given for the preparation, in good yield, of analytically pure dicyclohexylammonium salts of ribose 1-phosphate and deoxyribose 1-phosphate.