Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinase(s)

Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).     

Purine metabolism in cultured human fibroblasts derived from patients deficient in hypoxanthine phosphoribosyltransferase, purine nucleoside phosphorylase, or adenosine deaminase.

Rates of purine synthesis de novo, as measured by the incorporation of [14C]formate into newly synthesized purines, have been determined in cultured human fibroblasts derived from normal individuals and from patients deficient in adenosine deaminase, purine nucleoside phosphorylase, or hypoxanthine phosphoribosyltransferase, three consecutive enzymes of the purine salvage pathway. All four types of cell lines are capable of incorporating [14C]formate into purines at approximately the same rate when the assays are conducted in purine-free medium. The purine overproduction that is characteristic of a deficiency in either the transferase or the phosphorylase and that results from a block in purine reutilization can be demonstrated by the resistance of [14C]formate incorporation into purines to inhibition by hypoxanthine in the case of hypoxanthine phosphoribosyltransferase-deficient fibroblasts and by resistance to inhibition by inosine in the case of purine nucleoside phosphorylase-deficient fibroblasts.     

Purine nucleoside phosphorylase deficiency: biochemical properties and heterogeneity in two families.

The biochemical features of two families with purine nucleoside phosphorylase deficiency are compared. Laboratory studies and an evaluation of kinetic and physical properties of erythrocyte purine nucleoside phosphorylase give evidence that a) the degree of abnormality in uric acid and nucleoside concentrations in plasma and urine reflect the severity of the enzymatic deficiency and b) structural alterations of the mutant enzymes result from structural gene mutations and demonstrate genetic heterogeneity in the disease purine nucleoside phosphorylase deficiency.     

Purine nucleotide metabolism in promastigotes of Leishmania tropica: inhibitory effect on allopurinol and analogues of purine nucleosides.

The catabolism of adenosine 5'-monophosphate in promastigotes of L. tropica suggests the presence of a parasite specific pathway. In continuing the investigation on enzymes of this pathway two purine nucleoside cleaving enzymes have been found, adenosine phosphorylase and inosine nucleosidase. Various purine nucleoside analogues inhibited the activity of both enzymes. A mode of action of the growth inhibition of L. tropica promastigotes by allopurinol has been suggested on the basis of Michaelis and inhibitor constants.     

Adenosine cycle in African trypanosomes

African trypanosomes can convert adenosine to adenosine monophosphate. However, in Trypanosoma brucei, as in T. vivax and T. congolense, most of the adenosine is broken down to adenine before conversion to the nucleotide by adenine phosphoribosyltransferase. Trypanosoma brucei and T. vivax use the purine nucleoside hydrolase for adenosine cleavage while T. congolense uses purine nucleoside phosphorylase for the nucleoside cleavage. Trypanosoma vivax also deaminates adenine to hypoxanthine before its conversion to adenosine monophosphate by way of inosine monophosphate. All African trypanosomes lack adenosine deaminase. This finding particularly demonstrates that the effectiveness of the therapy of African trypanosomiasis with adenosine analogue drugs will depend upon the strain of trypanosome which causes the infection.     

Purine Nucleoside Phosphorylase Deficiency

The biochemical features of two families with purine nucleoside phosphorylase deficiency are compared. Laboratory studies and an evaluation of kinetic and physical properties of erythrocyte purine nucleoside phosphorylase give evidence that a) the degree of abnormality in uric acid and nucleoside concentrations in plasma and urine reflect the severity of the enzymatic deficiency and b) structural alterations of the mutant enzymes result from structural gene mutations and demonstrate genetic heterogeneity in the disease purine nucleoside phosphorylase deficiency.     

Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase.

An inherited deficiency of adenosine deaminase (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes severe combined immunodeficiency disease in humans. A similar deficiency in purine nucleoside phosphorylase (Puo phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) engenders a selective cellular immune deficit. To elucidate the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting human lymphocytes of the Ado deaminase substrates deoxyadenosine and adenosine and the Puo phosphorylase substrate deoxyguanosine. When Ado deaminase was inhibited, micromolar concentrations of deoxyadenosine progressively killed nondividing helper and suppressor-cytotoxic T cells, but not B cells. The toxicity required phosphorylation, with subsequent dATP formation. The deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, and adenine arabinonucleoside also killed resting T cells. Cell death was unrelated to inhibition of adenosylhomocysteinase (EC 3.3.1.1) but was preceded by a gradual decline in ATP levels. As much as 1 mM deoxyguanosine did not impair resting lymphocyte viability, despite the synthesis of dGTP. The combination of 200 microM adenosine plus 500 microM homocysteine thiolactone killed dividing lymphocytes but had no discernible toxic effect toward resting T cells, which accumulated adenosylhomocysteine over a 4-hr period but thereafter excreted the nucleoside into the culture medium. The different clinical syndromes associated with genetic deficiencies of Ado deaminase and Puo phosphorylase may be explained by the ability of dATP to kill mature resting T lymphocytes by depleting ATP levels.     

Erythrocyte and Leucocyte Enzymes in a Case of Paroxysmal Nocturnal Haemoglobinuria

In a patient with paroxysmal nocturnal haemoglobinuria (PNH) enzymatic activities of erythrocytes and leucocytes were studied. Studies of autohaemolysis were also performed. The following erythrocytary enzymes were measured: Glucose-6-phosphate dehydro-genase (G-6-PD), pyruvate kinase (PK), glutathione reductase (GR), and acetyl-cholinesterase (AcChE). The following enzymes were measured in leucocytes: Adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and adenosine kinase. Normal activity of G-6-PD, GR and PK in erythrocytes was found. In leucocytes and lymphocytes activity of purine nucleoside phosphorylase was reduced. Autohaemolysis in vitro was increased, which could not be compensated by addition of glucose or ATP.     

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.     

Erythrocyte and leucocyte enzymes in a case of paroxysmal nocturnal haemoglobinuria.

In a patient with paroxysmal nocturnal haemoglobinuria (PNH) enzymatic activities of erythrocytes and leucocytes were studied. Studies of autohaemolysis were also performed. The following erythrocytary enzymes were measured: Glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK), glutathione reductase (GR), and acetylcholinesterase (AcChE). The following enzymes were measured in leucocytes: Adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and adenosine kinase. Normal activity of G-6-PD, GR and PK in erythrocytes was found. In leucocytes and lymphocytes activity of purine nucleoside phosphorylase was reduced. Auto-haemolysis in vitro was increased, which could not be compensated by addition of glucose or ATP.     

The differences in purine metabolism between T and B lymphocytes

In this study enzyme activities involved in purine metabolism were measured in T and B lymphocytes separated by E and EAC rosetting methods. Adenosine deaminase, purine nucleoside phosphorylase and HGPRTase activities were significantly elevated in T cells, compared to the activities in B cells. There were no significant differences in adenosine kinase and APRTase activities between T and B lymphocytes. In contrast, PRPPsynthetase activities were higher in B cells than in T cells. The uptake of various radiolabeled precursors by mitogen stimulated lymphocytes was studied. The uptake of 14C-formate by the mitogen stimulated lymphocytes was markedly lower, compared to that of 14C-adenosine and or 14C-purine bases. The uptake of 14C-adenosine by PHA stimulated lymphocytes was considerably higher than that of Con A or PWM stimulated lymphocytes. However, the uptake of 14C-hypoxanthine into lymphocytes stimulated with PWM was increased by comparison with unstimulated lymphocytes. From these results it seems that adenosine plays a central role in the metabolism of T cells, and that purine bases are preferentially utilized in B cells.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: Decreased purine nucleoside phosphorylase and adenosine deaminase activity

Summary Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.This work was supported in part by the Austrian Research Fund (Project No. 3796).     

Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocytes from patients with acquired immune deficiency syndrome

The purine metabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are important in lymphocyte differentiation, and genetic deficiencies of either enzyme have been associated with hereditary immunodeficiency states. Both ADA and PNP activity were measured in null cell-enriched and T cell-enriched peripheral blood lymphocytes from 16 patients with the acquired immune deficiency syndrome (AIDS), seven patients with the AIDS-related symptom complex (ARC), and seven asymptomatic homosexuals. ADA activity in nmol/10(6) lymphocytes/h was significantly elevated in null lymphocytes from AIDS (161 +/- 12) as compared with 23 healthy heterosexual controls (127 +/- 8;P less than .025). PNP activity was also significantly increased in null lymphocytes from AIDS patients (96 +/- 10;P less than .005) as well as those from ARC patients (84 +/- 11:P less than .025) relative to controls (61 +/- 5). No significant differences in enzyme activity were noted in T cell-enriched cells in any group. Along with elevated enzyme activity, AIDS patients had small yet significant increases in the percentages of HLA-DR (P less than .025), terminal deoxynucleotidyl transferase (TdT) (P less than .0001), and peanut agglutinin receptor (P less than .0001) positive lymphocytes in the null fraction compared with controls. TdT-positive cells appeared morphologically as large lymphoblasts with irregular nuclei. The data imply that the cellular immune deficiency in AIDS is not a result of deficiencies in lymphocyte ADA or PNP activity, but is more likely associated with an increase in an immature and/or activated lymphocyte subset.     

Metabolic fate of hypoxanthine and inosine in cultured cardiomyocytes.

The metabolic fate of labeled hypoxanthine and inosine, degradation products of adenine nucleotides, was studied in cultured beating cardiomyocytes, in order to assess the physiological significance of their contribution to salvage nucleotide synthesis in the heart. Inosine and hypoxanthine were found to be incorporated into nucleotides by a similar rate, but in the presence of 8-aminoguanosine, a potent inhibitor of purine nucleoside phosphorylase (EC 2.4.2.1), the rate of inosine incorporation into nucleotides was markedly reduced (by 75%), indicating that inosine incorporation to IMP (inosinic acid) occurs following its degradation to hypoxanthine. The proportion of hypoxanthine converted to IMP by hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) is markedly greater than that degraded to xanthine and uric acid by xanthine oxidase (EC 1.3.2.3). However, close to 50% of the IMP formed was degraded to inosine by IMP 5'-nucleotidase (EC 3.1.3.5). The results demonstrate the activity of the following futile cycle in the cardiomyocytes: hypoxanthine----IMP----inosine----hypoxanthine. The rational for the activity of this energy consuming cycle is yet unclear.     

Lymphoblast purine pathway enzymes in B-cell acute lymphoblastic leukemia

Activities of enzymes of the purine metabolic pathway, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'- nucleotidase (5'-N), were investigated in the lymphoblasts of a patient with B-cell acute lymphoblastic leukemia. These lymphoblasts exhibited increased ADA activity and diminished activities of both PNP and 5'N' as compared to normal lymphocytes as well as non-T, non-B leukemia cells. This enzymatic pattern is identical to that which has been described in T-cell leukemic lymphoblasts and differs from that which has been observed in the malignant cells of undifferentiated B-cell lymphomas. These data suggest that there is biochemical heterogeneity within the spectrum of B-cell malignancies. Furthermore, inhibitors of ADA may be of use in those B-cell lymphoid neoplasms that exhibit increased ADA activity.     

Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.

Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout.     

Enzyme analysis of lymphoproliferative diseases: a useful addition to cell surface phenotyping

Lymphocytes from patients with acute and chronic T-cell malignancy or chronic T gamma lymphocytosis were characterized by studying the activity of three enzymes involved in purine metabolism and by determining the isoenzyme pattern of lactate dehydrogenase (LDH) in addition to analysis of surface marker expression with monoclonal antibodies. Four clinically different types of disease were distinguished on the basis of the enzyme parameters. Lymphocytes from patients with acute lymphocytic leukemia (T-ALL) showed an enzyme profile similar to that of normal thymocytes, i.e., an elevated level of adenosine deaminase (ADA) activity as compared with normal T lymphocytes, reduced activities of purine 5'nucleotidase (5'NT) and purine nucleoside phosphorylase (PNP), and a binomial distribution of the LDH isoenzyme pattern. Cells from "null"-ALL patients had an ADA/PNP ratio that was intermediate between that of normal T cells and that of T-ALL cells or thymocytes, but their 5'NT activity and LDH isoenzyme pattern were thymocyte-like. Patients with chronic T-cell proliferation were subdivided into those with chronic T gamma lymphocytosis and those with proven chronic T malignancy. The lymphocytes from these patients had ADA and PNP activities within the ranges of those of normal T lymphocytes. However, the ADA activity and/or the ADA/PNP ratio were consistently higher in the cells from the patients with chronic T gamma lymphocytosis than in those with chronic T malignancy. The enzyme profile of the cells from the T gamma patients was similar to that of T gamma cells of normal individuals. The cells from patients with chronic T malignancies showed a heterogeneous enzyme pattern as compared with that of normal T lymphocytes. Analysis with monoclonal antibodies enabled us to distinguish null-ALL patients from the other leukemias studied, but a distinction between chronic and acute T-cell proliferation disease, for instance, was not possible with monoclonal antibodies alone. Our data demonstrate that the enzyme profiles studied provide supplementary information for classification and diagnosis of lymphoproliferative diseases to that obtained with cell surface markers alone.     

Approaches to gene therapy in disorders of purine metabolism

Model studies with gene transfer technology using retroviral vectors expressing the cloned human genes for adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine guanine phosphoribosyltransferase (HPRT) indicate that these disorders may eventually be treated by the introduction of the normal gene into defective cells. The autologous transplantation of bone marrow infected in vitro with retroviral vectors is the most developed approach at this time. The potential usefulness of this and other approaches for each of these three disorders of purine metabolism are discussed.     

The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum relies on the acquisition of host purines for its survival within human erythrocytes. Purine salvage by the parasite requires specialized transporters at the parasite plasma membrane (PPM), but the exact mechanism of purine entry into the infected erythrocyte, and the primary purine source used by the parasite, remain unknown. Here, we report that transgenic parasites lacking the PPM transporter PfNT1 (P. falciparum nucleoside transporter 1) are auxotrophic for hypoxanthine, inosine, and adenosine under physiological conditions and are viable only if these normally essential nutrients are provided at excess concentrations. Transport measurements across the PPM revealed a severe reduction in hypoxanthine uptake in the knockout, whereas adenosine and inosine transport were only partially affected. These data provide compelling evidence for a sequential pathway for exogenous purine conversion into hypoxanthine using host enzymes followed by PfNT1-mediated transport into the parasite. The phenotype of the conditionally lethal mutant establishes PfNT1 as a critical component of purine salvage in P. falciparum and validates PfNT1 as a potential therapeutic target.     

Normal thermostability of hypoxanthine guanine phosphoribosyltransferase in erythrocytes from Werner's syndrome patients

The thermostability of erythrocyte hypoxanthine guanine phosphoribosyltransferase of 2 Werner's syndrome patients was compared with that of normal subjects of different ages. No significant difference was observed regarding the thermal stability of the enzyme among normal subjects and Werner's syndrome patients. The activities of other erythrocyte enzymes, phosphoribosylpyrophosphate synthetase, adenine phosphoribosyltransferase, adenosine deaminase and purine nucleoside phosphorylase, were similar between Werner's syndrome and normal subjects.