Dendrostilbella sp. (85-25), a New Source of Trichodermin

A sensitive kinetic determination of serum 5′-nucleotidase activity is described. Nucleoside oxidase catalyzes the oxidative coupling reaction of phenolic compounds and 4-aminoantipyrine to form colored substances in proportion of the amount of nucleoside. The present method relies on this phenomenon. Human serum 5′-nucleotidase was assayed using 5′-AMP as a substrate. When adenosine liberated was oxidized by nucleoside oxidase in the presence of phenolic compounds and 4-aminoantipyrine, a colored substance was formed at the same time. The rate of colored substance formation was proportional to the 5′-nucleotidase activity. The proposed method shows a low variation and a good correlation with the reference method. 3′-Nucleotidase was also assayed in a similar manner.     

Human Homologue of Seta Binding Protein 1 Interacts with Cathepsin B and Participates in TNF-Induced Apoptosis in Ovarian Cancer Cells

Extracellular adenine nucleotide hydrolysis in the circulation is mediated by the action of an NTPDase (CD39, apyrase) and of a 5′-nucleotidase (CD73), presenting as a final product, adenosine. Among other properties described for adenine nucleotides, an anti-cancer activity is suggested, since ATP is considered a cytotoxic molecule in several tumour cell systems. Conversely, some studies demonstrate that adenosine presents a tumour-promoting activity. In this study, we evaluated the pattern of adenine nucleotide hydrolysis by serum and platelets from rats submitted to the Walker 256 tumour model. Extracellular adenine nucleotide hydrolysis by blood serum and platelets obtained from rats at, 6, 10 and 15 days after the subcutaneous Walker 256 tumour inoculation, was evaluated. Our results demonstrate a significant reduction in ATP, ADP and AMP hydrolysis in blood serum at 6, 10 and 15 days after tumour induction. In platelets, a significant reduction in ATP and AMP hydrolysis was observed at 10 and 15 days after tumour induction, while an inhibition of ADP hydrolysis was observed at all times studied. Based on these results, it is possible to suggest a physiologic protection mechanism against the tumoral process in circulation. The inhibition in nucleotide hydrolysis observed probably maintains ATP levels elevated (cytotoxic compound) and, at the same time, reduces the adenosine production (tumoor-promoting molecule) in the circulation.     

Inhibition of 5′-nucleotidase activity after growth of Dictyostelium discoideum

The specific activity of 5′-nucleotidase activity in cell-free extracts of Dictyostelium discoideum at both exponential and stationary growth phases was determined. The 5′-nucleotidase activity of both membrane and soluble fractions was determined. The results show that at exponential growth more activity is found in the soluble fraction. Furthermore, the results show that stationary phase cells contain about 10-fold less activity than cells at exponential growth. To determine if stationary phase cells contained an inhibitor of 5′-nucleotidase, purified membranes were incubated with a high speed supernatant (S-100) prepared from cells at this stage. The results showed not only a time and concentration dependent loss of membrane bound activity, but also that most of the lost activity could be recovered in a soluble form. This result suggested that the 5′-nucleotidase was being released by a factor in the S-100. Additional studies showed inactivation of the releasing factor by a protease and further, that this inactivation could be prevented by serine protease inhibitors. The specificity of releasing factor with respect to two other membrane bound activities was determined. The results indicated no loss of either 3′5′-cyclic phosphodiesterase or adenylate cyclase. In addition, the results of a comparison of the activity of the releasing factor at two stages of growth showed similar values at both exponential and stationary growth phase. This latter finding suggests that the loss of 5′-nucleotidase activity at stationary phase is not due to modulation of the releasing factor activity. An alternative mechanism is proposed.     

Activity of adenosine and thymidine metabolism enzymes in the blood of cancer patients of various ages

The activity of metabolic enzymes, adenosine and thymidine, has been studied in the blood serum and lymphocytes of healthy people and oncological patients aged 23-80. An increase in the activity of thymidine kinase (EC 2.7.1.2), an enzyme of thymidine biosynthesis, was observed in the blood serum of oncological patients against a background of a sharp decrease in the activity of thymidine phosphorylase (EC 2.4.2.4), a catabolic enzyme. The revealed enzymic shifts have been observed in breast cancer patients after 36, in patients with the stomach cancer--after 46. It is found that an increase in the activity of adenosine deaminase (EC 3.5.4.4) and 5-nucleotidase of AMP (EC 3.1.3.5) in the blood serum of oncological patients is accompanied by a sharp decrease in the activity of these enzymes in lymphocytes.     

Studies on the alkaline phosphatase and 5'-nucleotidase of Dictyostelium discoideum.

The alkaline phosphatase and 5′-nucleotidase activities of Dictyostelium discoideum are due to two distinct enzymes. Both enzymes are membrane bound, but over 90% of the 5′-nucleotidase activity is solubilized when the crude membrane fraction of the cell is treated with phospholipase C under conditions that release only 10% of the alkaline phosphatase.Part of the alkaline phosphatase activity can be detected in whole cells, suggesting that some of the enzyme molecules are located on the exterior surface of the plasma membrane. In contrast very low 5′-nucleotidase activity can be detected in whole cells. When membrane preparations, isolated from cells that had been surface labeled with ¹²⁵I, were subjected to sedimentation equilibrium on sucrose density gradients, the majority of the ¹²⁵I-radioactivity cosedimented with the alkaline phosphatase and 5′-nucleotidase activites, suggesting that both enzymes are plasma membrane components.The two enzymes have distinctly different pH optima, but otherwise their properties are remarkably similar. Both enzymes are inhibited by cyanide, sulfhydryl inhibitors and sulfhydryl reagents, although in each case the 5′-nucleotidase is slightly more susceptible. Both enzymes are inhibited by the levamisole analogue, R 8231, but the alkaline phosphatase is inhibited to a somewhat greater extent. Both enzymes are activated by incubation at 50 °C but inactivated by higher temperatures.The two enzymes increase in activity at identical times during differentiation, suggesting that they are under coordinate developmental control.     

5'-nucleotidase activity in developing chick pectoral muscle.

The activity of the plasma membrane enzyme 5′-nucleotidase varies dramatically during the embryonic development of chick pectoral muscle. The specific activity is greatest at early stages of differentiation (8-day embryos), falls to a minimum on days 12–14, then rises again in older embryos. In cultured muscle cells obtained from embryonic chick muscle the 5′-nucleotidase activity is essentially absent. Muscle cells grown in the presence of bromodeoxyuridine, an inhibitor of muscle differentiation, contain enhanced levels of 5′-nucleotidase activity. These results indicate that 5′-nucleotidase may be absent in muscle fibers, but present in other cells of muscle tissue.     

5′-Nucleotidase

Summary 5′-Nucleotidase and alkaline phosphatase activity was investigated in the developing kidney of the mouse by histochemical and electrophoretic methods. The growth of the kidneys was studied by determining the incorporation of radioactive thymidine by autoradiography. During development the isoenzyme patterns of 5′-nucleotidase and alkaline phosphatase behaved in a different way. In correlating the histochemical and electrophoretic changes, it has been found that the 5′-nucleotidase isoenzymes as well as the alkaline phosphatase isoenzymes are located in different parts of the kidney. In the convoluted part of the proximal tubule 5′-nucleotidase isoenzyme 3 and alkaline phosphatase isoenzyme 5 are present, while in the straight part of this tubule 5′-nucleotidase isoenzyme 5 and — upto three weeks — alkaline phosphatase isoenzyme 3 are located. So in tissue structures having different functional capacities, different isoenzymes of 5′-nucleotidase and alkaline phosphatase are found.     

Circadian Rhythms of Serum Concentrations of 12 Enzymes of Clinical Interest

A total of 25 apparently healthy adults (13 men and 12 women), 29.5 years (SD = 3.6 years) of age, served as subjects in a 24-h study conducted in Barcelona, Spain, in the spring of 1990. The group had a homogeneous pattern of meals, activity, and behavior. Six blood samples were collected at 4-h intervals over a single 24-h period beginning at 10:00 h. The oral temperature was measured at 2-h intervals to facilitate an independent biological time reference for the local population being studied. The serum concentration of 12 enzymes of clinical interest were measured in each sample: creatine kinase, creatine kinase 2, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, alkaline phosphatase, cholinesterase, lactate dehydrogenase, lactate dehydrogenase 1, 5′-nucleotidase, pancreatic α-amylase, and triacylglycerol lipase. We supposed that all experimental data obtained for a quantity came from a single “hypothetical subject” that represented the central tendency of the population and then these data were analyzed for circadian rhythm by single cosinor. A statistically significant circadian rhythm was detected in all quantities studied (p ≤ 0.05) except for serum concentrations of pancreatic α-amylase and triacylglycerol lipase. The maximum daily rhythmic variation was ～ 10% (interval, 6–14%) for all quantities studied except pancreatic α-amylase (2.6%). This rhythmic variation is greater than the analytical variation except for 5′-nucleotidase and pancreatic α-amylase. The acrophases for the quantities studied (except that of triacylglycerol lipase) coincide with times near those of the oral temperature acrophase (18:01 local time). The results of this study will doubtless contribute to further documentation of the structure of the human circadian timing system and to establishment of time-qualified reference intervals for a defined group of subjects.     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Interstrain differences in changes in 5-nucleotidase activity in mouse macrophages in response to immunostimulation

The effect of immunostimulants on the activity of 5-nucleotidase in the macrophages of peritoneal exudate (MPE) has been investigated in mice of various strains. It has been demonstrated that in case of subcutaneous introduction of immunostimulants interstrain differences might be observed in the changes of MPE 5-nucleotidase activity. The decrease in the enzymatic activity in MPE was found to be the most pronounced in C57Bl/6 mice, while in AKR mice it was the least marked. CBA and C3 mice revealed no changes in MPE 5-nucleotidase activity in response to immunostimulation.     

Ectonucleotidase and acetylcholinesterase activities in silver catfish (Rhamdia quelen) exposed to different salinities

The purpose of this study was to investigate whether salinity adaptation can alter the purinergic (ecto-nucleoside triphosphate diphosphohydrolase; NTPDase and, 5′-nucleotidase) and cholinergic (acetylcholinesterase; AChE) systems in whole brain and blood tissue of the silver catfish, Rhamdia quelen. Silver catfish were gradually adapted to salinities of 0, 4 or 8 ppt and maintained at the experimental salinity for 10 days before brain and blood samples were collected. Blood AChE activity decreased significantly at 8 ppt and significant decreases in AChE activity were observed in the brain with salinity increases. ATP hydrolysis did not change between the groups. In contrast, ADP and AMP hydrolysis in silver catfish maintained at salinities of 4 and 8 ppt were significantly higher than those kept at 0 ppt. In conclusion, this study showed that there is an enhancement in the NTPDase (ADP hydrolysis) and 5′-nucleotidase activities in the brains of silver catfish exposed to increased salinity. Therefore, the activities of these enzymes can act as markers of salinity changes.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence

Ecto-3′-nucleotidase/nuclease (3′NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3′mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3′-nucleotidase activity (La3′-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3′-nucleotidase inhibitor and approach the possible involvement of ecto-3′-nucleotidase in cellular adhesion. La3′-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3′-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu²⁺ ions. Interestingly, ecto-3′-nucleotidase activity is 60-fold higher than that of ecto-5′-nucleotidase in L. amazonensis. Additionally, ecto-3′-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage–parasite attachment/invasion was increased by 400% in the presence of adenosine 3′-monophosphate (3′AMP); however, this effect was reverted by TTM treatment. We believe that La3′-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.     

The effect of high protein diet on the regulatory properties of AMP deaminase from chicken liver

Feeding high protein diet for 5 days caused a 3,5-fold and 2-fold increase of the activity of xanthine dehydrogenase (EC 1.2.1.37) and 5-nucleotidase (EC 3.1.3.31) respectively, in chicken liver. Six hours after feeding the high protein diet there was no change in either enzyme activity although a 3-fold increase in the level of serum uric acid was observed. High protein diet considerably decreased the activity of AMP deaminase at low, but not at high substrate concentration. The activity ratio, measured at 10.0 and 0.16 mM AMP increased from 14:1 (low protein diet) to 23:1 and 24:1 after 6 h and 5 days of high protein diet, respectively. It has been suggested that feeding birds a high protein diet may cause transformation of liver AMP deaminase (EC 3.5.4.6) from a low Km form toward a high Km form.     

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5′-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K_m value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5′-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5′-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 ± 0.1°C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5′-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.     

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.     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

Effects of alpha-interferon on the level of adenosine metabolism enzymes and bactericidal activity of macrophages in staphylococcal infection]

In staphylococcal infection the changes in functional ability of macrophages occur: their oxygen-depending bactericidity and adenosine-desaminase activity are depressed 5-nucleotidase ability increases. Introduction of homologous alpha-IFN in the dose of 1 x 10(3) u/mouse leads to enhancing macrophage bactericidity of the animals infected, inhibits their 5-nucleotidase activity and enhances adenosine desaminase one. Influence of alpha-IFN on the activity of adenosine metabolism enzymes in macrophages can be considered one of the most important mechanisms of its modulating effect in bacterial infections.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Effects In Vitro of Guanidinoacetate on Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Tissues from Adult Rats

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. The aim of this work was to investigate the in vitro effect of guanidinoacetate in NTPDase, 5′-nucleotidase and acetylcholinesterase activities in the synaptosomes, platelets and blood of rats. The results showed that in synaptosomes the NTPDase and 5′-nucleotidase activities were inhibited significantly in the presence of GAA at concentrations of 50, 100, 150 and 200 μM (P < 0.05). However, in platelets GAA at the same concentrations caused a significant increase in the activities of these two enzymes (P < 0.05). In relation to the acetylcholinesterase activity, GAA caused a significant inhibition in the activity of this enzyme in blood at concentrations of 150 and 200 μM (P < 0.05), but did not alter the acetylcholinesterase activity in synaptosomes from the cerebral cortex. Our results suggest that alterations caused by GAA in the activities of these enzymes may contribute to the understanding of the neurological dysfunction of GAMT-deficient patients.