Activity of adenosine metabolism enzymes and spontaneous chemiluminescence in macrophages in the tumor growth process

Adenosine deaminase and 5'-nucleotidase activities as well as chemiluminescence emission were measured in peritoneal macrophages of Syrian hamsters in the growth process of tumours with different grade of malignancy. The adenosine deaminase activity was established to decrease, while the 5'nucleotidase activity--to increase in macrophages after the subcutaneous injection of tumour cells with high level of malignancy as compared with these values in normal cells. This is accompanied by a decrease of the macrophage chemiluminescence during the whole experimental period. At the same time adenosine deaminase and 5'-nucleotidase activities as well as chemiluminescence emission in peritoneal macrophages of hamsters treated with low-malignancy cells do not differ from these values in the control group.     

5'-Nucleotidase and adenosine deaminase activity in the rat brain upon prolonged effect of low radiation doses

The effect of combined low radiation doses (0.2-50.8 cGy) on the 5'-nucleotidase and adenosine deaminase activities in the rat hypothalamus, hippocamp and cerebral cortex during 45, 120 and 365 days was examined. It has been shown that the changes in the 5'-nucleotidase activity of the hypothalamus and hippocamp have a phase character. The direction of the changes in enzyme activity of the hypothalamus and hippocamp adenosine forming was dependent on the zone stay period and had the exactly opposite character depending on the early and prolonged stay period in the zone. 5'-nucleotidase activity was changed under the influence of mean and lesser doses with an increase of the zone stay period. No changes in the 5'-nucleotidase activity of the cerebral cortex were noted. No changes in the hypothalamic adenosine deaminase activity of rats that stayed in a zone during 45 days were revealed; under the effect of mean dose during 120 days the activity decreased and also in case of a higher dosage during one year. The adenosine deaminase activity in animal hippocamp decreased in rats only under the influence of the lesser dose, for 45-day period. The decrease in adenosine deaminase activity of the cerebral cortex that was noted under the effect of all the three doses during 45 days, the higher and mean doses during 120 days disappeared in a year.     

The action of pertussis vaccines and GMDP on the change in the enzymatic activity of macrophages from peritoneal exudate

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.     

Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensitivity

The maximal activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in quadriceps or soleus muscle from animals in which the sensitivity to insulin was changed. Most conditions caused no effect on the activities but exercise-training increased the activity of adenosine deaminase and cold exposure increased the activity of 5'-nucleotidase in soleus muscle: in addition, ageing decreased markedly the activities of all three enzymes in both muscles. When the activities are based on mg protein they are much higher in both white and brown adipose tissue than in muscle, suggesting that changes in adenosine concentration may be important in changing insulin sensitivity in adipose tissue whereas changes in adenosine receptor number may be more important in muscle.     

Subcellular distribution of adenosine system enzymes in the hypothalamus and hippocampus of the rat brain

The subcellular distribution of 5'-nucleotidase and adenosine deaminase in rat brain hypothalamus and hippocampus was studied. In the hippocampus the 5'-nucleotidase activity was shown to be much higher than in the hypothalamus, while the adenosine deaminase activity, contrariwise, is nearly two times as high as that in the hypothalamus. During the analysis of subcellular distribution 5'-nucleotidase and adenosine deaminase were detected in all fractions under study, i. e., in nuclear, soluble, myelin fractions as well as in synaptic membranes, synaptosomes and "pure" mitochondria. The highest 5'-nucleotidase activity was found in the myelinic and synaptic fractions both in the hypothalamus and in the hippocampus. The highest adenosine deaminase activity was detected in the soluble fraction of the above structures. The enzyme activity in synaptic membranes and synaptosomes was nearly two times as low.     

Activity of purine enzyme metabolism in rat thymocytes after irradiation and after administration of riboxine

It has been established, that the total X-ray irradiation of animals takes influence upon the functional activity of key enzymes of adenine nucleotides metabolism: adenylatekinase, AMP deaminase, 5'-nucleotidase, adenosine deaminase and purine nucleoside phosphorylase in rat's thymocytes. The increase of activity of the investigated enzymes is observed in our experiment, except 5'-nucleotidase, which activity is authentically decreasing after irradiation (1.0 and 7.78 Gy). The injection of the preparation riboxine to experimental animals 15 min prior to the exposure has normalized the purine exchange enzymes activity.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Enzymes of adenosine metabolism in mouse sperm suspensions

The enzymes of adenosine metabolism were investigated in suspensions of epididymal mouse spermatozoa incubated under conditions which support capacitation in vitro. High levels of adenosine deaminase activity were found in sperm suspensions, but the enzyme was located in the surrounding medium and was not intrinsic to spermatozoa. 5'-Nucleotidase was also present in the surrounding medium while in sperm cells it existed as an ecto-enzyme. Adenosine was not metabolized by washed spermatozoa under conditions used for the assay of adenosine deaminase or adenosine kinase, but it was metabolized rapidly by unwashed sperm suspensions. Incubation of sperm suspensions in conditions which modulate fertilizing ability resulted in small alterations in intrinsic 5'-nucleotidase activity of spermatozoa. In contrast, the activity of adenosine deaminase was not consistently modulated by such manipulations. Adenosine deaminase and 5'-nucleotidase exhibited similar kinetic parameters to enzymes from other sources and their activities were inhibited by coformycin and alpha, beta-methylene adenosine 5'-diphosphate, respectively. These studies highlight the low adenosine-metabolizing ability of spermatozoa coupled with the extensive metabolism in the medium which surrounds them. Extracellular adenosine metabolism can therefore occur and may modulate capacitation in vitro.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.     

Selective adenosine release from human B but not T lymphoid cell line

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.     

Intranephron distribution of purine-metabolizing enzymes in rats

The activities of purine-metabolizing enzymes, 5'-nucleotidase, adenosine deaminase, and purine nucleoside phosphorylase in microdissected rat nephron segments were measured. The specific activity of 5'-nucleotidase was highest in the proximal tubules and the cortical collecting duct, but low in the glomerulus. In contrast, the highest activity of adenosine deaminase was found in the glomerulus. The distribution pattern of purine nucleoside phosphorylase was similar to that of adenosine deaminase. These results suggest that various nephron segments can form adenosine and that the glomerulus exhibits highest capacities to metabolize this nucleoside.     

Adenosine deaminase activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages

Adenosine deaminase (ADA) undergoes changes in specific activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages. Monocyte adenosine deaminase activity increases during the first 3 days of culture; after 3 days the specific activity decreases below the levels observed for freshly isolated cells. In contrast, ADA activity of pulmonary alveolar macrophages increases throughout the 14-day culture period studied. Based on pH optima, starch gel electrophoresis and gel filtration column chromatography, the changes in adenosine deaminase activity in monocyte-macrophage cultures are related to changes in the molecular form of the enzyme. Freshly isolated monocytes contain mainly ES, while at day 14, starch gel and gel filtration experiments demonstrate the appearance of EI. Human pulmonary macrophages contain primarily EI or EL; following several days in culture, there is an increase in the amount of ES present.     

Activities and some properties of 5''-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine.

1. The maximal activities of 5'-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues. 2. Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K.,from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5. This evidence includes the effects of pH and temperature on the activities of the enzymes. 3. In many tissues, the activities of 5'-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate. In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo. 4. In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase. It is suggested that 5'-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration. 5. The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles. Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles.     

Transmural distribution of 5'-nucleotidase and of adenosine deaminase enzyme activities in the wall of the normal rat left ventricle

The transmural distributions of adenosine metabolizing enzymes (5'-nucleotidase and adenosine deaminase) were examined in normal rat hearts. It was found that the total activities of both enzymes vary in a biphasic manner across the left ventricular wall, such that the ratio of 5'-nucleotidase to adenosine deaminase is at a minimum near the midmyocardium.     

Antimetastatic effect of L-lysine-alpha oxidase

The influence of a new antitumor enzyme L-lysine alpha-oxidase on Lewis lung carcinoma spreading was studied in mice in which primary tumor had been removed. The enzyme was found to significantly decrease the extent and number of lung metastases as compared to mice which hadn't received L-lysine alpha-oxidase. This was matched by recovery of alveolar macrophages functional activity, as assessed by adenosine deaminase and 5' nucleotidase levels in these cells. Moreover, antimetastatic and cytostatic effect was confirmed by the measuring of polyamine concentration in mice erythrocytes.     

Activities of adenylate-degrading enzymes in muscles from vertebrates and invertebrates

Activities of adenylate-degrading enzymes in muscles of vertebrates and invertebrates were determined. Mammalian and fish muscles showed a markedly higher activity of AMP deaminase with a lower level of adenosine deaminase and 5'-nucleotidase. Cephalopods showed an active adenosine deaminase and a 5'-nucleotidase which preferred AMP as the substrate. Negligible deamination of AMP and adenosine and little phosphohydrolase activity toward AMP and IMP were observed in the shellfish muscles. Adenine nucleotides can be degraded to form IMP via the AMP deaminase reaction in vertebrate muscles, while dephosphorylation of AMP to adenosine, which is then converted to inosine, appears to proceed in cephalopods. Adenylates can be hardly degraded in shellfish muscles.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.