Metabolism of purine nucleosides and phosphoribosylpyrophosphate in thymocytes and splenocytes of various mammalian species

1. Activities of ADA, PNP and AK were measured in splenocytes and thymocytes of newborn children, young horses, pigs, sheep, rats and mice and compared with the activities previously found in peripheral lymphocytes. 2. With all species, except horse, the activity of ADA (per 10(6) cells) was higher in thymocytes than in lymphocytes. Activity of ADA was highest in splenocytes of pig and sheep. Activity of ADA was lowest in all lymphoid cells of the horse and only about 10% of the activity in human splenocytes and lymphocytes. 3. With all species, except horse, the activity of PNP was lower in thymocytes than in lymphocytes. Activity of PNP was highest in human lymphocytes and lowest in ovine thymocytes. 4. Activity of AK is comparable in thymocytes of all species and always lower than the ADA activity. In splenocytes of man, horse and pig the activity of AK is comparable to that in thymocytes. 5. Activity of deoxyguanosine kinase was lowest in rat thymocytes and highest in those of man. 6. When enzyme activities are expressed per milligram of protein, the differences between thymocytes and lymphocytes are less pronounced. 7. Activity of PRPP synthetase per 10(6) cells was comparable in thymocytes, splenocytes and lymphocytes of the same species and between the various species. 8. The concentration of PRPP was lowest in ovine thymocytes and higher in splenocytes than in thymocytes of the same species, except man.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Long ultradian rhythms in red blood cells and ghost suspensions: Possible involvement of cell membrane

Summary Oscillations in glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD) activities were recorded in suspensions of intact human red blood cells (RBCs) exposed to various light regimens. The periods of these oscillations, defined as “long ultradian,” ranged between 13 and 18 h regardless of light regimen. The patterns of enzymatic activities were the same when assayed at each time point, in full hypotonic hemolysates, and membrane-free hemolysates. However, if hemolysates were prepared by sonication the activity pattern did not exhibit significant oscillations and the activity was higher than that recorded in hypotonic hemolysates. The observed rhythms may reflect a time-dependent attachment and detachment of enzyme molecules from cell membrane, suggesting that at the bound state the enzyme molecules are (temporarily) inactive. Oscillations with similar long ultradian periods were also observed in Ca⁺⁺ concentration of suspended RBCs and in the binding of Ca⁺⁺⁴⁵ to human RBC ghosts. Treatment of the RBCs with A₂C or Diamide before the preparation of the ghosts changed or distorted the rhythmic pattern of Ca⁺⁺⁴⁵ binding. These results point to the role of the membrane in processing the long ultradian oscillations. The relation between this type of oscillations to circadian rhythm is discussed.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Purine salvage pathway enzyme activities in human T-, B-, and null lymphocyte populations

Deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) result in distinctly different immunodeficient states. In order to better understand the roles of these two purine salvage pathway enzymes in normal immunity we have characterized their activities in peripheral blood T-, B-, and null (non-T, non-B) cell populations. We have found that T lymphocytes have significantly higher ADA activity than B or null cells (P < 0.001) only when expressed as a function of cell protein. Contrary to other reports B lymphocytes cannot be distinguished from T lymphocytes on the basis of PNP activity. When the enzyme activities are expressed per cell number null cells have a much higher PNP activity (mean ± SD = 461 ± 174 ng/hr/10⁶ cells) than either T (57 ± 10) or B (93 ± 51) lymphocytes (P < 0.001 and 0.002, respectively). Null cells also have a significantly greater protein content per cell (55.0 ± 9.6 μg/10⁶ cells) than T (13.3 ± 4.6) or B (18.7 ± 13.8) lymphocytes. When expressed as a function of cell number null cells have a higher mean, though not statistically significant, ADA activity than T and B lymphocytes. These results indicate that normal human peripheral blood T, B, and null cells can be distinguished on the basis of their ADA and PNP activities and that the method of expression of the activities is critical. The significantly higher PNP activity and protein content per cell in null lymphocytes is compatible with a less differentiated cell population which may include B- as well as T-cell precursors.     

利用复感儿T淋巴细胞作为外源性ADA基因表达效应细胞的实验探讨

对４１例健康儿童和１７例反复呼吸道感染患儿（复咸儿）的外周血淋巴细胞ＡＤＡ活性进行了检测．两例ＡＤＡ活性明显低下的复感儿的外周血Ｔ淋巴细胞被选作实验样品,经体外培养后,采用ｌｉｐｏｆｅｃｔｉｎ介导的的方法导入外源性ＡＤＡ基因．结果表明：体外培养的两例复感儿淋巴细胞中ＡＤＡ活性较基因转移前升高;细胞免疫学特性也在一定程度上获得改善     

Localization and identity of adenosine deaminase-positive cells in tissues of the young rat and calf

Rabbit antibody to calf adenosine deaminase (ADA) was used to localize this enzyme in tissues of the young rat and calf by the immunoperoxidase method. The distribution patterns of ADA in most tissues were similar for both species. Within the thymus gland, the enzyme was strongly expressed predominantly in cortical lymphocytes. In the spleen and lymph nodes, most lymphocytes of T-cell areas stained weakly for ADA, whereas only a small number of ADA-positive cells were found in B-cell areas. Clumps of strongly ADA-positive mononuclear blastoid and plasma cells were observed in the medullary regions of lymph nodes, around peri-arteriolar lymphocyte sheaths and in the red pulp of the spleen, and in the lamina propria of the intestine. Double immunofluorescence staining studies in the rat showed that some of these blastoid cells contained both ADA and immunoglobulins and appeared to be plasmablasts. Strong staining for ADA was also found, in both the rat and calf, in as yet unidentified mononuclear blastoid cells in the interstitium of non-lymphoid organs (kidney, heart, lung), in endothelial cells of some arterioles and capillaries, and in Kupffer cells of the liver. In addition, ADA was strongly expressed in calf bile canaliculi. These studies define areas in rat and calf tissues which contain ADA-positive cells and provide a model system for investigations of the relationship between ADA and the function and development of these cells.     

Adenosine deaminase activity in leukemia patients

The activities of adenosine deaminase (ADA) were measured in the blood plasma, erythrocytes, and lymphocytes of healthy reference persons and in patients affected with leukaemia. ADA is increased in patients with acute immature cell leukaemia, in patients with chronic lymphatic leukaemia it is comparatively low in lymphocytes. In chronic myeloic leukaemia ADA activities are different depending on the activity of the disease. ADA-activities in the blood plasma, erythrocytes, and lymphocytes do not correlate with each other. ADA-activities in leukaemias may be regarded as an indicator of increased purin metabolism rather a as parameter of disturbed cellular immunofunction.     

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.     

Membrane-associated purine metabolizing enzyme activities of human peripheral blood cells

Sealed and unsealed plasma membrane vesicles were prepared from human erythrocytes and lymphocytes. Phosphoribosylpyrophosphate synthetase (PRibPP synthetase), hypoxanthine phosphoribosyltransferase (HPRTase), and adenine phosphoribosyltransferase (APRTase) activities are detectable on both inside-out and right-side-out sealed vesicles. Ghost preparations were about 0.2%, 1%, and 1.2% of the total erythrocyte and 0.5%, 5.3%, and 9.7% of the lymphocyte APRTase, HPRTase, and PRibPP synthetase activities. The rapid decrease in these enzyme activities, upon further purification of the membranes, seemed to suggest that they might be loosely bound extrinsic proteins. Evidence confirming the localization of these enzymes on the cell surface was obtained by measuring production of [14C]AMP by intact cells in medium containing [14C]adenine, ribose 5-phosphate, and Mg2+ATP. The formation of AMP was linear with time and number of cells present. Magnesium and phosphate exerted different effects on the production of extracellular AMP than on intracellular, which involves transport as well as phosphoribosylation. Cytosoluble and membrane-bound APRTase and PRibPP synthetase exhibited different catalytic properties and sensitivities to effectors. Membranes of erythrocytes of HPRTase-deficient patients contain little or no HPRTase activity when assayed in the absence of Triton. Reisolation of these membranes from admixture with normal hemolysates did not result in any bound activity; thus, the membrane-bound activity is not an artifact of the isolation procedure. Lysis with Triton released activity equal to about half that of control membranes. This is further evidence that the enzyme is firmly bound to the membrane.     

Changes in adenosine deaminase and antioxidative enzyme activity in patients with lung diseases of dust etiology]

Activity of purine metabolism enzyme, adenosine deaminase (ADA) in lymphocytes and erythrocytes from patients with pneumoconiosis and chronic bronchitis was determined. ADA activity decrease was found in lymphocytes and erythrocytes for both decreases. The observed decrease of ADA activity indicates that after the long term work in mineral dust industry damage of purine metabolism takes place, that is similar to the disturbance observed under secondary immunodeficiency. Changes in activity of superoxide dismutase and catalase show some decrease of functional activity of anti-radical protection. Therefore the results obtained can be used for the selection of a group with highest risk of predisposition to these diseases.     

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide (CY, 200 mg/kg) and after whole-body gamma irradiation (5.5 Gy), applied alone or three days after CY application. In the first days after the treatment the enzyme activities were significantly depressed (p less than 0.01) with the exception of ADA in the spleen, where a high elevation (220-380%) in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation (270%). In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs.     

Adenosine Deaminase Activity in Serum and Lymphocytes of Rats Infected with Sporothrix schenckii

Sporotrichosis is a fungal infection of subcutaneous or chronic evolution, inflammatory lesions characterized by their pyogranulomatous aspect, caused by the dimorphic fungus Sporothrix schenckii. Adenosine deaminase (ADA) is a "key" enzyme in the purine metabolism, promoting the deamination of adenosine, an important anti-inflammatory molecule. The increase in ADA activity has been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this fungal infection. The objective of this study was to evaluate the activity of serum ADA (S-ADA) and lymphocytes (L-ADA) of rats infected with S. schenckii. We used seventy-eight rats divided into two groups. In the first experiment, rats were infected subcutaneously and in the second experiment, infected intraperitoneally. Blood samples for hematologic evaluation and activities of S-ADA and L-ADA were performed at days 15, 30, and 40 post-infection (PI) to assess disease progression. In the second experiment, it was observed an acute decrease in activity of S-ADA and L-ADA (P?相似文献   

Adenosine deaminase, 5′nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human bladder tissues

Activities of adenosine deaminase (ADA), 5′nucleotidase (5NT), xanthine oxidase (XO), superoxide dismutase (SOD), and catalase (CAT) enzymes were measured in cancerous and cancer-free adjacent bladder tissues from 36 patients.with bladder cancer and in control bladder tissues from 9 noncancer patients. Increased ADA and decreased XO, SOD, and CAT activities were found in cancerous bladder tissues compared with those of cancer-free adjacent tissues and of control bladder tissues. Differences were also found between enzyme activities in the bladder of different disease stages and grades. In the cancerous tissues, only positive intracorrelations were found, but in the cancer-free adjacent tissues and control tissues, both positive and negative correlations were established between enzyme activities. Results suggested that purine metabolism and salvage pathway activity of purine nucleotides were accelerated in the cancerous human bladder tissues via increased ADA and decreased XO activities, probably together with changes in some other related enzyme activities and, free radical metabolising-enzyme activities were depressed in cancerous bladder tissues, which indicated exposure of cancerous tissues to more radicalic stress.     

Uroporphyrinogen III cosynthetase in asymptomatic carriers of congenital erythropoietic porphyria

Mean activity of the enzyme uroporphyrinogen III cosynthetase in hemolysates from five asymptomatic carriers of bovine erythropoietic porphyria was intermediate to the means of the activities in normal controls and affected animals. Similarly, mean cosynthetase activity in hemolysates from eight presumed carriers of human congenital erythropoietic porphyria was lower than the mean for 38 nonporphyric individuals and higher than the activity in hemolysates from patients. These results are consistent with the known autosomal recessive mode of inheritance of the disorder in cattle and the presumed autosomal recessive inheritance in man. The activity of cosynthetase is higher in young red cells than in older ones, which explains the relatively high enzyme activities in blood from human patients with nonporphyric hemolytic disorders accompanied by reticulocytosis. It also explains why cosynthetase activity is higher in newborn porphyric calves than in older porphyric animals, because the former have a period of accelerated erythropoiesis and a high percentage of very young red cells in the circulation.Supported in part by U.S. Public Health Service Grants NB-05367 and AM-10858.     

Activities of enzymes that hydrolyze adenine nucleotides in lymphocytes from patients with rheumatoid arthritis

The purpose of this study was to investigate the activities of ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5; CD39) and adenosine deaminase (E‐ADA; EC 3.5.4.4) in lymphocytes from patients with rheumatoid arthritis (RA). Thirty patients diagnosed with RA through American College of Rheumatology criteria as well as 30 healthy patients were selected. Peripheral blood lymphocytes were isolated, and E‐NTPDase and E‐ADA activities were assayed. The results demonstrated an increased E‐NTPDase activity (both ATP and ADP as substrates) and a decreased E‐ADA activity in RA patients. These data suggest an organic effort to preserve the adenosine level, which is known to have anti‐inflammatory and analgesic properties, working as a potent suppressor of immune response. Copyright © 2012 John Wiley & Sons, Ltd.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced reduction of adenosine deaminase activity in vivo and in vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunosuppressant in several animal species. The purpose of this study was to determine if TCDD affected the activity of adenosine deaminase (ADA), a purine metabolizing enzyme that is vital to the proper functioning of the immune system. The effect of TCDD on ADA ctivity was studied in various tissues of male Balb/c mice (a TCDD-responsive strain) and DBA/2 mice (a less-responsive strain). Of the tissues examined after administration of TCDD in vivo (115 μ/kg, i.p.), ADA activity was found to be significantly reduced in thymic and splenic tissues of Balb/c mice at 24 hours postadministration. The enzyme activity in these affected tissues remained consistently low through 10 days postadministration. Such an effect of TCDD was both dose and time related in the thymic tissue of Balb/c mice. In contrast, no appreciable alterations in ADA activity were evident in any of the tissues of DBA/2 mice at any of the sampling intervals, indicating that such an effect of TCDD is likely to be mediated through the Ah receptor. This in vivo effect of TCDD on thymic ADA activity was also reproducible in situ where isolated whole thymuses were directly incubated with 10 nM TCDD. In this model, TCDD's effects on ADA activity were antagonized by known protein kinase or phosphorylation inhibitors such as quercetin, genistein, tyrphostin, and neomycin. These results indicate that the effect of TCDD on ADA activity in the thymus may be related to its property to elevate protein kinase activities in this tissue. ADA activity was also reduced in 3T3 cells that were treated with 10 nM TCDD in a low (1%) serum media. In contrast, 25 ng/mL epidermal growth factor (EGF) under such conditions consistently stimulated ADA activity. Interestingly, EGF at a similar concentration failed to elicit a stimulatory effect on ADA activity when cells were pretreated with TCDD. The property of TCDD to lower ADA activity under in vivo, in situ, as well as in vitro conditions appears to be largely related to its action to modulate protein phosphorylation activities.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37 degrees C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H2O2 and HbO2, may be related to the pathogenesis of hemolysis in favism.     

Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.