Purine Nucleoside Phosphorylase Activity Research Articles

Purine nucleoside phosphorylase heterogeneity in the mouse as analyzed by isoelectric focusing and specific activity

1. 1. Isoelectric focusing in polyacrylamide gels identified three erythrocytic electrophoretic patterns of purine nucleoside phosphorylase in a survey of 16 mouse strains. 2. 2. Three strain-specific electrophoretic types were also evident in liver, kidney and spleen leukocytes. 3. 3. There are 3-fold differences in purine nucleoside phosphorylase activities between strains for several tissues; C57BL/6J and Mus spretus having the greatest and the least activity, respectively. 4. 4. Within strains there were up to 8-fold tissue-specific differences in activity with the order from greatest to least being: liver, kidney, spleen leukocyte, erythrocyte, heart.

First trimester diagnosis of adenosine deaminase deficiency.

Adenosine deaminase deficiency has been detected in the first trimester by direct analysis of enzyme activity in chorionic villi in a pregnancy at risk. Data for purine nucleoside phosphorylase activity in chorionic villi from healthy controls in the first trimester are also presented and should allow equally rapid diagnosis of this disorder.

Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi.

Cell extracts of Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, and Mycoplasma gallisepticum S6 were examined for 37 cytoplasmic enzyme activities involved in the salvage and biosynthesis of purines. All of these organisms had adenine phosphoribosyltransferase activity (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8). All of these organisms had purine-nucleoside phosphorylase activity (EC 2.4.2.1) in the synthetic direction using ribose-1-phosphate (R-1-P) or deoxyribose-1-phosphate (dR-1-P); this activity generated ribonucleosides or deoxyribonucleosides, respectively. The pyrimidine nucleobase uracil could also be ribosylated by using either R-1-P or dR-1-P as a donor. The synthesis of deoxyribonucleosides from nucleobases and dR-1-P has been reported from only one other procaryote, Escherichia coli (L. A. Mason and J. O. Lampen, J. Biol. Chem. 193:539-547, 1951). The reverse of this phosphorylase reaction is more widely known, and we found such activity in all mollicutes studied. Some Acholeplasma species but not the Mycoplasma species can phosphorylate deoxyribonucleosides to deoxyribomononucleotides by a PPi-dependent deoxyribonucleoside kinase activity, which was first reported in this group for the ribose analogs (V. V. Tryon and J. D. Pollack, Int. J. Syst. Bacteriol. 35:497-501, 1985). This is the first report of PPi-dependent purine deoxyribonucleoside kinase activity. An ATP-dependent purine deoxyribonucleoside kinase activity is known only in salmon milt extracts (H. L. A. Tarr, Can. J. Biochem. 42:1535-1545, 1964). Deoxyribomononucleotidase activity was also found in cytoplasmic extracts of these mollicutes. This is the first report of deoxyribomononucleotidase activity.

Activities of purine metabolising enzymes in lymphocytes of neonates and young children: correlates with immune function

Depressed activities of the following purine enzymes have been shown to result in immunodeficiencies: adenosine deaminase (ADA), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP). These enzymes and adenosine kinase (AK) were measured in cord blood lymphocytes of premature and small-for-gestational age infants since they have partial immunodeficiencies of unknown biochemical etiology which can persist for many years. We also measured these enzymes in 3 infants with various immunodeficiencies. Activities were compared with appropriate matched control groups. The results indicated normal ADA and PNP but significantly depressed AK ( P < 0.05) and HGPRT ( P < 0.001) activities in 10 premature/SGA infants when compared to 35 full-term normal infants. In the 3 immunodeficient children the results were as follows: Child 1 had a 2- to 3-fold decrease in ADA with normal PNP and AK activities; Child 2 had a 2- to 3-fold decrease in AK, 4-fold decrease in HGPRT with normal PNP and ADA activities; Child 3 had confirmed AIDS and a 4-fold decrease in ADA, 6-fold decrease in HGPRT with normal PNP activity. The possible role of these depressed purine enzyme activities found in lymphocytes is discussed in relation to the impaired immunity seen in these infants.

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

ChemInform Abstract: Synthesis of 8-Amino-3-deazaguanine via Imidazole Precursors. Antitumor Activity and Inhibition of Purine Nucleoside Phosphorylase.

8-Amino-3-deazaguanine (15), an analogue of both 3-deazaguanine (1) and 8-aminoguanine (6), an antitumor agent and a purine nucleoside phosphorylase (PNP) inhibitor, respectively, was synthesized from the ammonolysis of an imidazole precursor, methyl 2-(benzoylamino)-5-(cyanomethyl)-1H-imidazole-4-carboxylate (13). The requisite imidazole, methyl 2-(benzoylamino)-4-(methoxycarbonyl)-1H-imidazole-5-acetate (11), was prepared from the monoheterocyclic rearrangement of dimethyl 3-[(5-phenyl-1,2,4-oxadiazol-3-yl)amino]-2-pentenedioate (10) by NaH/DMF. Ammonolysis and subsequent dehydration of 11 provided the penultimate imidazole intermediate 13. Its deprotected (NaOMe/100 degrees C) product, methyl 2-amino-5-(cyanomethyl)-1H-imidazole-4-carboxylate (14), was also converted to 15. 8-Amino-3-deazaguanine, as its methanesulfonic acid (mesylate 7), exhibited an inhibition constant (IC50) of 9.9 microM against isolated mammalian PNP. It was a very weak inhibitor of T and B cell growth and did not enhance 2'-deoxyguanosine toxicity in the same cells. 8-Amino-3-deazaguanine mesylate was not significantly active in L1210 cells in vitro or L1210 leukemic mice. Thus, the amino group introduced in the 8-position of 3-deazaguanine enhances its PNP activity but diminishes its antitumor activity.

A human purine nucleoside phosphorylase deficiency caused by a single base change.

Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

1. 1. Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. 2. 2. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. 3. 3. In pigeon liver, only purine nucleoside phosphorylase activity was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. 4. 4. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.

Low cytidine deaminase levels in human hematopoietic cell lines

Purine and pyrimidine enzyme profiles of human cell lines have been investigated. A novel observation was the finding that most of the cell lines showed very low or undetectable levels of cytidine (deoxycytidine)deaminase, while they possessed pyrimidine 5′-nucleotidase, cytidine and deoxycytidine kinase activities. Most cell lines showed high levels of adenosine deaminase and purine nucleoside phosphorylase activities and low levels of purine 5′-nucleotidase. We propose that high adenosine deaminase and purine nucleoside phosphorylase activities and low cytidine deaminase activity may be of importance for immature hematopoietic cells in order to ensure a balanced synthesis of the DNA precursors.

Adenosine Deaminase and Purine Phosphorylase Activities in Lymphocytes and Red Blood Cells of Patients with Carcinoma of the Larynx

Lymphocytes from patients with cancer of the larynx have been characterized by the activity of two enzymes involved in purine metabolism--adenosine deaminase and purine nucleoside phosphorylase. It has been shown that purine nucleoside phosphorylase activity was 1.5-fold higher in lymphocytes of patients in comparison with a group of healthy individuals. Adenosine deaminase activity in lymphocytes was elevated three to four-fold. The authors suggest that especially the estimation of adenosine deaminase activity in lymphocytes would be a useful laboratory test in the study of immune responsiveness of patients with cancer of the larynx.

Chicken liver purine nucleoside phosphorylase activity dependency of the redox state of sulfhydryl groups

1. 1. Double reciprocal plots (1/v vs 1/S) for nucleoside substrates of chicken liver purine nucleoside phosphorylase were non linear at high inosine or deoxyinosine concentrations (>0.1 mM). The appearance of downward curvatures may be correlated with the oxidation of sulfhydryl groups of the enzyme. 2. 2. 5,5′-Dithiobis-(2-nitrobenzoic acid) reacts with four sulfhydryl groups in the native enzyme, but upon denaturation with sodium dodecilsulfate six sulfhydryl groups react with this reagent. 3. 3. Inosine, ribose-1-phosphate, hypoxanthine and orthophosphate partially protect sulfhydryl groups from the reaction with Ellman's reagent. 4. 4. Inhibition of purine nucleoside phosphorylase by p-chloromercuribenzoate and 5,5′-dithiobis-(2-nitrobenzoic acid) follows a second order reaction kinetics.

Assay of purine nucleoside phosphorylase in erythrocytes by flow-injection analysis with fluorescence detection.

A sensitive assay for purine nucleoside phosphorylase (PNP) in human erythrocytes is described. Erythrocyte lysate is incubated with the substrate inosine in the presence of phosphate for the enzyme reaction, and the resulting mixture is deproteinized with perchloric acid and neutralized. The supernatant containing the reaction product, hypoxanthine, is applied to a flow-injection system in which immobilized enzyme columns of xanthine oxidase, urate oxidase and horseradish peroxidase are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of hypoxanthine is measured fluorimetrically by reaction with 3- (p-hydroxyphenyl) propionic acid in the system. The lower limit of determination (signal-to-noise ratio=5) for hypoxanthine is 0.3 pmol in a 20-μl injection volume, which corresponds to a PNP activity of 0.117 μmol/min/ml erythrocytes. This method permits the assay of PNP in only 10 nl of human erythrocytes in 50 μl of erythrocyte lysate.

Decreased 5′nucleotidase activity in suppressor (OKT 8) T lymphocytes from homosexuals with AIDS-related complex: Nonassociation with enhanced deoxynucleoside toxicity

The purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′nucleotidase (5NT) have been shown to be important for normal lymphocyte maturation. Abnormalities of these enzymes have been associated with hereditary as well as acquired immunodeficiency states. Enzyme activity was measured in helper (OKT 4) and suppressor (OKT 8) lymphocyte subsets from 10 homosexuals with AIDS-related complex (ARC) and in 10 healthy controls. There were no significant differences in either mean ADA activity or mean PNP activity between ARC OKT 4 cells and control OKT 4 cells and between ARC OKT 8 cells and control OKT 8 cells. By contrast, mean 5NT activity was slightly decreased in OKT 4 cells from ARC patients compared with that of controls and more significantly diminished in ARC OKT 8 cells compared with that of controls. Both deoxyadenosine and deoxyguanosine, when incubated separately with OKT 4 and OKT 8 cells in the presence of EHNA, and ADA inhibitor, did not significantly inhibit lymphocyte blastogenesis to a greater extent in ARC patients than in controls. Hence, the decreases in 5NT activity most likely reflect lymphocyte immaturity and are not associated with biochemical abnormalities leading to increased deoxynucleoside toxicity.

Characterization of purine nucleoside phosphorylase in leukemia.

Purine nucleoside phosphorylase (PNP) activity was determined in mononuclear cells from 49 patients with various types of leukemia. A low level of PNP activity was found in mononuclear cells from patients with acute myeloid and lymphoblastic leukemia and with chronic lymphocytic leukemia. Enzymatic and immunological studies on PNP from leukemic cells of these patients revealed no differences in Michaelis constant for inosine, thermostability, electrophoretic mobility, immunological reactivity, or specific activity between the PNP of leukemic cells and that of normal mononuclear cells. These results suggest that the decrease in PNP activity of leukemic cells is due to a decreased rate of enzyme synthesis. Thus, the abnormality of PNP activity might be due to an alteration in the regulatory mechanism of enzyme synthesis in the purine metabolism in the leukemic clone.

Synthesis of 8-amino-3-deazaguanine via imidazole precursors. Antitumor activity and inhibition of purine nucleoside phosphorylase

8-Amino-3-deazaguanine (15), an analogue of both 3-deazaguanine (1) and 8-aminoguanine (6), an antitumor agent and a purine nucleoside phosphorylase (PNP) inhibitor, respectively, was synthesized from the ammonolysis of an imidazole precursor, methyl 2-(benzoylamino)-5-(cyanomethyl)-1H-imidazole-4-carboxylate (13). The requisite imidazole, methyl 2-(benzoylamino)-4-(methoxycarbonyl)-1H-imidazole-5-acetate (11), was prepared from the monoheterocyclic rearrangement of dimethyl 3-[(5-phenyl-1,2,4-oxadiazol-3-yl)amino]-2-pentenedioate (10) by NaH/DMF. Ammonolysis and subsequent dehydration of 11 provided the penultimate imidazole intermediate 13. Its deprotected (NaOMe/100 degrees C) product, methyl 2-amino-5-(cyanomethyl)-1H-imidazole-4-carboxylate (14), was also converted to 15. 8-Amino-3-deazaguanine, as its methanesulfonic acid (mesylate 7), exhibited an inhibition constant (IC50) of 9.9 microM against isolated mammalian PNP. It was a very weak inhibitor of T and B cell growth and did not enhance 2'-deoxyguanosine toxicity in the same cells. 8-Amino-3-deazaguanine mesylate was not significantly active in L1210 cells in vitro or L1210 leukemic mice. Thus, the amino group introduced in the 8-position of 3-deazaguanine enhances its PNP activity but diminishes its antitumor activity.

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors.

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.

Simplex Optimization for the Simultaneous HPLC Assay of the Activities of Purine-Nucleoside Phosphorylase and Hypoxanthine-Guanine Phosphoribosyl Transferase

Abstract The modified sequential simplex procedure is shown to be effective for maximizing a complex enzyme assay. The optlmum levels for two factors, pH and substrate concentration, for a coupled enzyme assay of hypoxanthine-quanlne phosphor Ibosy I-transferase (HGPRTase) and purine nucleoside phosphorylase (PNPase) were found by searching a factor space made up of these variables. The performance Index or response to be optimized was a function of the product of the two single activities. The maximum activity for this function was found at a pH of 7.9 and a concentration of Inosine in the reaction mix of 0.84 mM.

Levels of some purine metabolizing enzymes in lymphocytes from patients with Down's syndrome.

The activities of a number of purine metabolizing enzymes of erythrocytes and lymphocytes were determined in 18 subjects with Down's syndrome and in 18 age- and sex-matched control subjects. An increase of adenosine deaminase activity (adenosine or deoxyadenosine as substrates) was found in erythrocytes (P less than 0.001) as well as in lymphocytes (P less than 0.001) of Down's syndrome subjects compared to controls. The purine nucleoside phosphorylase activities in lymphocytes and plasma urate concentrations were also significantly higher in Down's syndrome subjects than in controls (P less than 0.001 and less than 0.02, respectively). Adenine phosphoribosyltransferase activities and hypoxanthine-guanine phosphoribosyltransferase activities in lymphocytes were identical in the two groups. In all subjects studied there were positive correlations between the erythrocyte adenosine deaminase activities, lymphocyte adenosine deaminase or deoxyadenosine activities, and plasma urate concentrations (P less than 0.05 in all cases), and between lymphocyte nucleoside phosphorylase and lymphocyte adenosine deaminase or deoxyadenosine deaminase activities (P less than 0.01 and less than 0.05, respectively). The results suggest that increased activities of some purine metabolizing enzymes found in both erythrocytes and lymphocytes may contribute to increased purine degradation and hyperuricemia in subjects with Down's syndrome. In addition, the increased adenosine deaminase and nucleoside phosphorylase activities may be related to the immunological dysfunction found in subjects with Down's syndrome.