Purine Nucleoside Phosphorylase Activity Research Articles

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.

Characterization of the metabolic forms of 6-thioguanine responsible for cytotoxicity and induction of differentiation of HL-60 acute promyelocytic leukemia cells.

HL-60 human acute promyelocytic leukemia cells that lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6-thioguanine (TG) and, in contrast to parental HL-60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT- HL-60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6-Thioguanosine (TGuo) had limited activity, while beta-2'-deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT- HL-60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8-aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.

Enzymes of nucleic acid metabolism as biochemical markers of T cell development in mouse

The levels of three purine salvage enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5′-nucleotidase (5′-N), which are known to be associated with certain immunodeficiency disorders were determined in mouse T lymphocytes. These enzymes showed characteristic changes depending on the stage of T cell development. The activity of ADA was 5-fold higher in thymocytes compared to splenic T cells. On the other hand, the splenic T cells displayed a 2-fold and a 4–8 fold greater activity of PNP and 5′-N, respectively than those of thymocytes. The apparent K m and V max values have been determined for all the 3 enzymes in the immature and mature T cells. The data demonstrate that the absolute and relative activity of these enzymes may be used as biochemical markers to characterize the T lymphocytes during different stages of differentiation.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaroytic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C 18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing pairedion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formyin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent K m of 18.2 μ m and an apparent V max of 32.4 mmol min −1 mg −1 were observed for the activity. A purine nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent K m of 236 μ m and V max of 2.78 μmol min −1 mg −1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent K i value of 136 μ m was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a K m value for GTP, IMP, and aspartic acid of 23, 34, and 714 μ m, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have K m and V max values for hypoxanthine of 55.5 μ m and 34.3 nmol −1 min −1 mg 1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures. The methodology is extended to interpreting a series of enzyme reactions in which the product of the first was the substrate for the second.

Purine metabolizing enzymes in lymphocytes from patients with solid tumors.

The activities of five clinically important enzymes of purine metabolism have been determined in lymphocytes from 62 patients with various types of solid tumors. The activity of purine nucleoside phosphorylase was increased in all patient groups studied, i.e. small cell bronchogenic carcinoma (n = 30), carcinoma of the breast (n = 17) and other tumors (n = 15), compared to cells form normal donors. Activities of adenosine deaminase, adenine phosphoribosyltransferase (APRT), hypoxanthine (guanine) phosphoribosyltransferase (HGPRT), and 5'-nucleotidase (5'-NUC) vary little from control values, except for lower levels of APRT in lymphocytes from patients with carcinoma of the breast. In patients with small cell bronchogenic carcinoma, enzyme levels were also determined in granulocytes, where increased APRT activity was found. Following cytostatic treatment of these patients, significant decreases were seen in lymphocytic HGPRT and 5'-NUC activities.

Biochemical and functional characterization of lymphocytes from a horse with lymphosarcoma and IgM deficiency

Neoplastic lymphocytes from a horse with lymphosarcoma and IgM deficiency were analyzed for ability to grow in culture; surface and cytoplasmic IgM; functional activity in blastogenesis, cytoxicity, and suppressor assays; and activities of six enzymes involved in purine and pyrimidine metabolism. The cells lacked surface and cytoplasmic IgM. They had elevated activity of adenosine deaminase and reduced activity of purine nucleoside phosphorylase. Neoplastic cells were nonresponsive in blastogenesis assay and did not kill allogeneic lymphocyte target cells or YAC-1 targets in a lectin-dependent cytotoxicity assay, however, the cells were active in a suppressor assay. They were grown for 16 weeks in cultures supplemented with interleukin 2, during which time the cells retained suppressive activity. These results are consistent with a T cell lymphoma of suppressor cell origin, and may explain the deficiency of IgM observed in some horses with lymphoreticular neoplasms.

Adenosine deaminase, purine nucleoside phosphorylase and 5'-nucleotidase activities in infectious mononucleosis.

The present study was undertaken to study 3 selected purine pathway enzymes in infectious mononucleosis (IM). The enzymes concerned are adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5′-nucleotidase (5′-N). The enzymes were selected since the absence of these enzymes have been associated with clinically defective lymphoid function (1), and because abnormalities in the activities of ADA and PNP have been observed in leukemia (2,3) In 1974 Quagliata et al. (4) claimed that 5′-N may also be usefull in the biochemical characterization of subgroups of patients with lymphoid leukemia. In the early phase of IM they found a decrease of 5′-N.

Intralymphocytic adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNPase) activity in chronic lymphocytic leukemia (CLL): effect of thymostimulin.

ADA (E.C.3.5.4.4.) and PNPase (E.C.2.4.2.1.) are enzymes which have been extensively studied by imnunologists ever since it was noted that a deficiency of either one of these enzymes could be associated with severe congenital immunodeficiency. Though the catalytic actions of these enzymes are sequential, the absence or the alteration of one of the two enzymes causes different types of congenital immunodeficiencies. ADA insufficiency causes a severe combined immunodeficiency while PNPase insufficiency alters delayed immunity (1,2). JVbreover it has been demonstrated that these enzymes are involved in lymphocytic ontogeny both in animals (3) and in man (4).

Purine nucleoside metabolizing enzyme activities in mouse thymocytes at different stages of differentiation and maturation

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA AK and ADA PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.

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.

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.

Comparison of purine degradative enzymes and terminal deoxynucleotidyl transferase in T cell leukaemias and in normal thymic and post‐thymic T cells

Summary. Adenosine deaminase (ADA), ecto 5’ nucleotidase (5′NT), purine nucleoside phosphorylase (PNP) and terminal deoxynucleotidyl transferase (TdT) were measured in the cells of patients with acute or chronic T cell leukaemia and compared with normal putative prothymocytes (large, blast‐like cortical thymocytes), cortical and medullary thymocytes and peripheral blood T lymphocytes. Distinct patterns of enzyme activities were found in the individual types of T cell leukaemia. Mean ADA, TdT and 5′NT activities in thymic acute lymphoblastic leukaemia (Thy‐ALL) were 41·9 u/108 cells, 31·1 u/108 cells and 4·7 u/106 cells respectively; in chronic T cell leukaemia they were 7·1 u/108 cells, 0·6 u/108 cells and 18·1 u/106 cells respectively. Mean PNP activity was similar between these two groups of leukaemia (68·6 u/106 cells in Thy‐ALL and 77·9 u/106 cells in chronic T cell leukaemia). The activities of these four enzymes in OKT4+ chronic T cell leukaemia did not differ significantly from those in the OKT8+ chronic T cell leukaemia cases. The activities of TdT, ADA, PNP and 5′NT in Thy‐ALL closely resembled those in normal immature thymocytes, and in the chronic T cell leukaemias showed a similar pattern of enzyme activities to that of mature T lymphocytes. These findings are consistent with surface phenotypic studies of T cell malignancies which suggest that different T cell leukaemias represent malignant proliferation of T cell clones arrested at different stages of T cell differentiation. They also demonstrate the value of biochemical markers in defining the different subtypes of acute and chronic leukaemia.

Changes in purine nucleoside phosphorylase activity during thymosin-induced human null cell differentiation

Purine nucleoside phosphorylase (PNP) is a purine salvage pathway enzyme which we have found to be 8–10 times more active (per cell) in human peripheral blood null lymphocytes than in T lymphocytes. To test the hypothesis that null cells are, in part, pre-T lymphocytes we have defined an in vitro system for null cell differentiation into T cells and examined PNP activity during this differentiation process. We found that about 10% of human null cells could be driven to differentiate into T cells using thymosin fraction 5 (TF5) an extract of bovine thymus glands. The response to TF5 was dose related up to 250 μg/ml with a maximum response occurring by 42–46 hr incubation. Exposure to TF5 was necessary for more than 4 hr but no more than 8 hr in order to obtain a maximum response. Both OKT4 and OKT8 positive cells were present in the newly differentiated T cell population but OKT8 positive cells appeared to predominate ( OKT4 OKT8 = 0.69 ± 0.30 , mean ± 1 SD). The differentiation process did not involve DNA synthesis but was inhibited at 4 °C. In the newly differentiated T cells PNP activity per cell was 8- to 10-fold lower (36 ± 23 nm/hr/10 6 cells) than in null cells (311 ± 136), and was at a level similar to mature T cells (56 ± 7). Thus, human peripheral blood null cells can be induced to differentiate into T lymphocytes which can be characterized by both surface markers and biochemical parameters. Future studies will look at the function of TF5-induced T cells and the regulation of PNP activity during the differentiation process.

Effect of immunosuppressive agents on human T and B lymphoblasts

We have studied the effects of various immunosuppressive drugs on the growth of human-derived T (MOLT-4) and B (MGL-8) lymphoblasts. In addition, we have examined whether the lymphotoxic effect of any of these drugs could be attributed to inhibition of either adenosine deaminase (DDA) or purine nucleoside phosphorylase (PNP). Results indicated that 1-β- d-arabinofuranosylcytosine (Ara-C), methotrexate and chlorambucil were four to seven times more toxic for T than for B cells, while azathioprine, 6-thioguanine, 6-mercaptopurine, and 5-fluorouracil were highly toxic for both T and B cells. Cyclophosphamide and oxisuran were lymphotoxic only at concentrations exceeding 300 μM. Deoxyadenosine (50 μM), deoxyguanosine (10 μM) and deoxycoformycin (10 μM) failed to enhance T cell toxicity when individually combined with each drug. None of the drugs tested inhibited T or B lymphoblast ADA or PNP activity. With the exception of Ara-C, neither dATP nor dGTP accumulated in T lymphoblasts incubated in the presence of any of the drugs. We conclude that the cell culture system used in this investigation is useful for identifying lymphotoxic and T cell-specific immunosuppressive agents. However, none of the drugs studied appeared to function as an inhibitor of, or a competitive substrate for, either ADA or PNP.

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.

Analysis of purine nucleoside phosphorylase from human T and non-T lymphocytes of different maturation stages

In the last few years evidence has been growing that purine/pyrimidine metabolism changes during maturation of lymphocytes. In this view enzyme characteristics of lymphocytes may act as biochemical markers of lymphocyte maturation. Human purine nucleoside phosphorylase (PNP) has been studied in extracts of T lymphocytes isolated from thymic tissue, cord blood, and peripheral blood, as well as in extracts of non-T lymphocytes from cord blood, peripheral blood and tonsils. Using inosine or deoxyinosine as a substrate, complicated kinetics of PNP are observed which may be caused by the presence of multiple active enzyme species. The number of PNP-active molecular forms increases with the maturation stage of the T and B lymphocytes as shown by cellogel electrophoresis. PNP activity of thymocytes as compared to peripheral blood T cells is low; PNP activity of peripheral blood B cells as compared to neonatal B cells does not differ significantly. The difference found in PNP activity between thymocytes and peripheral blood T cells is caused by the difference in amount of PNP protein, as has been demonstrated by immunoprecipitation tests.

Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression.

Data are presented which indicate that the repression of pur gene expression seen after the addition of preformed purines to cultures of Salmonella typhimurium is the consequence of the presence or the formation of the purine bases, hypoxanthine and guanine. This conclusion is based on the following observations. First, it was impossible to find a correlation between the size of any individual purine nucleotide pool and the level of the first four enzymes in the de novo biosynthetic pathway. Second, adenine plus guanosine served as a perfect source of purine nucleotides, but their presence caused no repression of pur gene expression if the cells lacked purine nucleoside phosphorylase activity. This enzyme is needed to convert adenine and guanosine to hypoxanthine and guanine, but not for their conversion to nucleotides. Third, addition of guanine to a strain lacking guanine phosphoribosyltransferase (gpt) resulted in a repression of the level of the purine de novo biosynthetic enzymes, a reduction of the growth rate, and a fall in the pools of ATP and GTP. Addition of hypoxanthine to a strain lacking hypoxanthine phosphoribosyltransferase (hpt) had a similar, although weaker, effect. If the cells lacked both hypoxanthine and guanine phosphoribosyltransferases (hpt gpt), their basal level of the purine de novo biosynthetic enzymes was repressed in minimal medium. Such cells grow slower than wild-type cells and excrete purines, probably due to the inability to salvage endogenously formed hypoxanthine and guanine.

Metabolism of purine nucleosides in human and ovine lymphocytes and rat thymocytes and their influence on mitogenic stimulation

1. Phosphorolysis and phosphorylation rates of inosine, guanosine, and deoxyguanosine were determined in disrupted and intact human and ovine lymphocytes and rat thymocytes and related with their effect on mitogenic stimulation. 2. Activity of purine nucleoside phosphorylase (EC 2.4.2.1) was about 10 times higher in extracts of human lymphocytes than those of ovine lymphocytes and rat lymphocytes. Apparent K m values for inosine and guanosine were higher in human lymophocytes (about 100 μM) than in ovine lymphocytes (50 μM). Apparent K m values for deoxyguanosine were about 100 μM in the extracts of all three cell types. 3. In extracts of human and ovine lymphocytes the presence of guanosine kinase activity was established. Deoxyguanosine kinase activity was detected in all three cell types. 4. The rate of phosphorylation of deoxyguanosine was much lower than the rate of phosphorolysis both in extracts and in intact cells. 5. Deoxyguanosine, guanosine and inosine were incorporated by intact cells into nucleotides and nucleic acids. This incorporation of deoxyguanosine and guanosine was only partially due to phosphorolysis and subsequent conversion by hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The incorporation of inosine appeared to be due completely to this route. 6. Inosine (0.5 mM) did not inhibit thymidine incorporation of phytohemagglutinin-stimulated human and ovine lymphocytes. At the same concentration deoxyinosine caused 50% inhibition, but guanosine and deoxyguanosine inhibited almost completely. Thymidine incorporation of concanavalin A-stimulated rat thymocytes was hardly inhibited by 0.5 mM inosine, deoxyinosine and guanosine, but 50 μM and 0.5 mM deoxyguanosine caused 25% and complete inhibition, respectively.

Effect of thymostimulin on human lymphocyte adenosine deaminase and purine nucleoside phosphorylase activities: physiological and therapeutic effects.

Thymostimulin (TP1 — Serono), which has several immunobiological activities in vivo and in vitro, is a pool of thermostable polypeptides extracted from calf thymus and has been used in the treatment of primary and secondary immunodeficiencies (1, 2). However, the immunobiological properties and the mechanism of action of this cellular extract is still not completely understood. For this reason, we have studied the activity of thymostimulin on the intracellular levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) enzymes in peripheral blood lymphocytes of patients with hematological malignancies. We also hoped to reveal the effects of thymostimulin on lymphocytes from cord blood and from elderly patients. In fact, these enzymes have important roles in both lymphocyte ontogenesis (3, 4) and in the maintenance of immunological functions (5, 6).

Elevated erythrocyte phosphoribosylpyrophosphate and ATP concentrations in Japanese sumo wrestlers.

1. Japanese sumo wrestlers have a diet rich in energy, which results in marked obesity. Their plasma urate and triglyceride levels were significantly elevated. 2. Erythrocyte phosphoribosylpyrophosphate (PRPP) and ATP concentrations in sumo wrestlers were significantly elevated when compared to the levels in control subjects. 3. There were no significant differences in erythrocyte PRPP synthetase (EC 2.7.6.1), purine nucleoside phosphorylase (EC 2.4.2.1) and hypoxanthine guanine phosphoribosyl transferase (EC 2.4.2.8) activities between sumo wrestlers and control subjects. 4. Erythrocyte adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and adenine phosphoribosyl transferase (EC 2.4.2.7) activities in sumo wrestlers were significantly elevated. 5. It seems that sumo wrestlers have an increased turnover of adenine nucleotides which may contribute to hyperuricaemia.