In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

Soluble NTPDase: An additional system of nucleotide hydrolysis in rat blood serum

The participation of a nucleoside triphosphate diphosphohydrolase in the nucleotide hydrolysis by rat blood serum was evaluated. Nucleoside triphosphate diphosphohydrolase and phosphodiesterase are enzymes possibly involved in ATP and ADP hydrolysis. The specific activity of the phosphodiesterase activity (using thymidine 5'-monophosphate p-nitrophenyl ester as substrate) was 4.92 +/- 0.73 (mean +/- SD, n = 10) nmol p-nitrophenol.min(-1).mg(-1) protein and the specific activities for ATP and ADP were 1.31 +/- 0.37 (mean +/- SD, n = 7) and 1.36 +/- 0.25 (mean +/- SD, n = 5) nmol Pi.min(-1).mg(-1) protein, respectively. A competition plot demonstrated that ATP and ADP hydrolysis occurs at the same active site. The effect of suramin and phenylalanine on ATP, ADP and thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was investigated. The results were opposite considering the hydrolysis of ATP and ADP and that of the substrate marker for the enzyme phosphodiesterase. These results are indicative of the presence of, at least, two enzymes participating in the serum nucleotide hydrolysis. The presence of cAMP did not affect the hydrolysis velocity of ATP and ADP, while thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was inhibited by cAMP by approximately 47%, suggesting that the hydrolysis of the ATP and ADP, under our assay conditions, occurs at a different site from the phosphodiesterase site. Both enzyme activities, in the rat blood serum, may be involved in the modulation of the nucleotide/nucleoside ratio in the circulation, serving an in vivo homeostatic and antithrombotic function. In addition, the phosphodiesterase may act on DNA or RNA liberated upon tissue injury and/or cell death.     

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.     

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5′-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5′-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 μL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1–1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Fluoxetine and nortriptyline affect NTPDase and 5'-nucleotidase activities in rat blood serum

Depression is a serious condition associated with considerable morbidity and mortality. Selective serotonin reuptake inhibitors and tricyclic antidepressants, such as fluoxetine and nortriptyline, respectively, were commonly used in treatment for depression. Selective serotonin reuptake inhibitors have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. ATP, ADP and adenosine are signaling molecules in the vascular system and nucleotidases activities are considered an important thromboregulatory system which functions in the maintenance of blood fluidity. Therefore, here we investigate the effect of in vivo (acute and chronic) and in vitro treatments with the antidepressant drugs on nucleotidases activities in rat blood serum. In acute treatment, nortriptyline decreased ATP hydrolysis (41%), but not altered ADP and AMP hydrolysis. In contrast, fluoxetine did not alter NTPDase and ecto-5'-nucleotidase activities. A significant inhibition of ATP, ADP, and AMP hydrolysis were observed in chronic treatment with fluoxetine (60%, 32%, and 42% for ATP, ADP, and AMP hydrolysis, respectively). Similar effects were shown in chronic treatment with nortriptyline (37%, 41%, and 30% for ATP, ADP, and AMP hydrolysis, respectively). In addition, there were no significant changes in NTPDase and ecto-5'-nucleotidase activities when fluoxetine and nortriptyline (100, 250, and 500 microM) were tested in vitro. Our results have shown that fluoxetine and nortriptyline changed the nucleotide catabolism, suggesting that homeostasis of vascular system can be altered by antidepressant treatments.     

E-NTPDases and ecto-5'-nucleotidase expression profile in rat heart left ventricle and the extracellular nucleotide hydrolysis by their nerve terminal endings

In this study, we have identified the E-NTPDase family members and ecto-5'-nucleotidase/CD73 in rat heart left ventricle. Moreover, we characterize the biochemical properties and enzyme activities from synaptosomes of the nerve terminal endings of heart left ventricle. We observe divalent cation-dependent enzymes that presented optimum pH of 8.0 for ATP and ADP hydrolysis, and 9.5 for AMP hydrolysis. The apparent K(M) values are 40 microM, 90 microM and 39 microM and apparent V(max) values are 537, 219 and 111 nmol Pi released/min/mg of protein for ATP, ADP and AMP hydrolysis, respectively. Ouabain, orthovanadate, NEM, lanthanum and levamisole do not affect ATP and ADP hydrolysis in rat cardiac synaptosomes. Oligomycin (2 microg/mL) and sodium azide (0.1 mM), both mitochondrial ATPase inhibitors, inhibit only the ATP hydrolysis. High concentrations of sodium azide and gadolinium chloride show an inhibition on both, ATP and ADP hydrolysis. Suramin inhibit more strongly ATP hydrolysis than ADP hydrolysis whereas Evans blue almost abolish both hydrolysis. AMP hydrolysis is not affected by levamisole and tetramisole, whereas 0.1 mM ammonium molybdate practically abolish the ecto-5'-nucleotidase activity. RT-PCR analysis from left ventricle tissue demonstrate different levels of expression of Entpd1 (Cd39), Entpd2 (Cd39L1), Entpd3 (Cd39L3), Entpd5 (Cd39L4) Entpd6, (Cd39L2) and 5'-NT/CD73. By quantitative real-time PCR we identify the Entpd2 as the enzyme with the highest expression in rat left ventricle. Our results contribute to the understanding about the control of the extracellular nucleotide levels in and cardiac system.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

Inhibition by ATP and activation by ADP in the regulation of flagellar movement in sea urchin sperm

ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.0-7.2 which is lower than the physiological pH of 8, about 90% of reactivated flagella were motionless at 1 mM ATP, while about 60% were motile at 0.02 mM ATP. The motionless flagella at 1 mM ATP maintained a single large bend or an S-shaped bend, indicating formation of dynein crossbridges in the axoneme. The ATP-dependent inhibition of flagellar movement was released by ADP, and was absent in outer arm-depleted flagella. Similar inhibition was also observed at 0.02 mM ATP when demembranated flagella were reactivated in the presence of Li+ or pretreated with protein phosphatase 1 (PP1). ADP also released this type of ATP-inhibition. In PP1-pretreated axonemes the binding of a fluorescent analogue of ADP to dynein decreased. Under elastase-treatment at pH 8.0, the beating of demembranated flagella at 1 mM ATP and 0.02 mM ATP lasted for approximately 100 and 45 s, respectively. The duration of beating at 0.02 mM ATP was prolonged by Li+, and that at 1 mM ATP was shortened by removal of outer arms. These results indicate that the regulation of on/off switching of dynein motile activity of flagella involves ATP-induced inhibition and ADP-induced activation, probably through phosphorylation/dephosphorylation of outer arm-linked protein(s).     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2+ and Ca2+. The inhibitors ouabain (0.01-1 mM), N-ethylmaleimide (0.01-4 mM), levamisole (0.10-0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03-30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50-300 microM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2+ and Ca2+ ATP/ADPase at 100 microM. Lanthanum decreased Mg2+ and Ca2+ ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach.     

The effect of ebselen on adenine nucleotide hydrolysis by platelets from adult rats

The extracellular hydrolysis of adenine nucleotides by intact rat blood platelets occurs by the action of a cascade of enzymes constituted by an NTPDase 3 (CD39, EC 3.6.1.5, apyrase) and a 5'-nucleotidase (CD73, EC 3.5.7.3), whose final product is adenosine. Ebselen is a seleno-organic compound that possesses low toxicity and exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and cytoprotective properties. The main objective of this study was to evaluate if the anti-inflammatory drug ebselen can modulate the extracellular adenine nucleotide hydrolysis by platelets from rats. Our results showed that ebselen, at final concentrations of 30 and 100 microM, inhibits in vitro ATP extracellular hydrolysis by 48 and 60%, respectively. Ebselen, at final concentrations of 100 and 130 microM, also inhibited the in vitro extracellular hydrolysis of ADP by 28 and 35%, respectively. However, this drug did not alter AMP hydrolysis by platelets in the appropriate assay conditions. Kinetic analysis showed that the inhibition of ADP and ATP hydrolysis by ebselen, in rat platelets, is of the uncompetitive type. The IC50 calculated from the results were 99 +/- 10 and 186 +/- 47 microM (mean +/- S.D., n = 3) for ATP and ADP hydrolysis, respectively.     

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.     

ATP and ADP hydrolysis in brain membranes of zebrafish (Danio rerio)

Nucleotides, e.g. ATP and ADP, are important signaling molecules, which elicit several biological responses. The degradation of nucleotides is catalyzed by a family of enzymes called NTPDases (nucleoside triphosphate diphosphohydrolases). The present study reports the enzymatic properties of a NTPDase (CD39, apyrase, ATP diphosphohydrolase) in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in a pH range of 7.5-8.0 in the presence of Ca(2+) (5 mM). The enzyme displayed a maximal activity for ATP and ADP hydrolysis at 37 degrees C. It was able to hydrolyze purine and pyrimidine nucleosides 5'-di and triphosphates, being insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (0.1 mM). A significant inhibition of ATP and ADP hydrolysis (68% and 34%, respectively) was observed in the presence of 20 mM sodium azide, used as a possible inhibitor of ATP diphosphohydrolase. Levamisole (1 mM) and tetramisole (1 mM), specific inhibitors of alkaline phosphatase and P1, P(5)-di (adenosine 5'-) pentaphosphate, an inhibitor of adenylate kinase did not alter the enzyme activity. The presence of a NTPDase in brain membranes of zebrafish may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in central nervous system of this specie.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis (P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis (P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate (P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5-di(adenosine-5')pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 microg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K(m) values of 77.6+/-10.2 and 106.8+/-21.0 microM, and V(max) values of 68.9+/-8.1 and 99.4+/-8.5 (mean+/-S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72+/-0.24% (mean+/-S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.     

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

ATP and ADP hydrolysis in the kidney and liver of fish, chickens and rats

We investigated NTPDase-like activity [ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases)] in liver and kidney membrane from silver catfish (Rhamdia quelen), chicken (Gallus gallus) and rat (Rattus norvegicus) under different conditions and in the presence of several inhibitors. The cation concentration required for maximal activity was 0.5, 1.5 and 2.0 mM for fish, chicken and rat liver, respectively (with ATP and ADP as substrates). The maximal activity in the kidney was observed at calcium concentrations of 0.5, 2.0, 1.5 mM (ATP) and 0.5, 1.5, 1.0 (ADP) for fish, chickens and rats, respectively. The results showed that the pH optimum for all animals and for the two tissues was close to 8.0. The temperature chosen was 25 °C for fish and 36 °C for chicken and rat preparations. Ouabain had no effect on the NTPDase-like activity of fish, chickens or rats. NTPDase activity was decreased in the presence of lanthanum in the chicken (ADP) and rat (ATP and ADP) liver. In the kidney, lanthanum inhibited fish ATP and rat ATP and ADP (0.2 mM) hydrolysis. N-ethylmaleimide (NEM) had an inhibitory effect on the kidney of all species at the concentration of 3.0 mM (ADP). Orthovanadate only inhibited fish membrane NTPDase; azide only inhibited the preparation at high concentrations (10 mM) and fluoride inhibited it at 10 mM (fish and chicken) and 5 mM (rat). Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. These results suggest that NTPDase-like activity shows a different behavior among the vertebrate species and tissues studied. Additionally, we propose that NTPDase1 is the main enzyme present in this preparation.