Protective effect of quercetin in ecto-enzymes,cholinesterases, and myeloperoxidase activities in the lymphocytes of rats exposed to cadmium

The ex vivo and in vitro effects of quercetin on NTPDase, adenosine deaminase (ADA), and acetycholinesterase (AChE) activities in lymphocytes, as well as the effects of quercetin on butyrylcholinesterase (BChE) activity in serum and myeloperoxidase (MPO) activity in plasma were determined in rats. For the ex vivo experiment, animals were orally exposed to Cadmium (Cd) for 45 days. Animals were divided into eight groups: saline/ethanol, saline/Querc 5 mg/kg, saline/Querc 25 mg/kg, saline/Querc 50 mg/kg, Cd/ethanol, Cd/Querc 5 mg/kg, Cd/Querc 25 mg/kg, and Cd/Querc 50 mg/kg. The ex vivo data showed an increase in the ATP and ADP hydrolysis and ADA activity in Cd-exposed rats when compared to the control group. The treatment with quercetin 25 and 50 mg/kg prevented this increase in the ATP and ADP hydrolysis, while the treatment with quercetin 5, 25, and 50 mg/kg prevented the increase in the ADA activity. AChE, BChE, and MPO activities ex vivo presented an increase in the Cd-exposed group when compared to the control group, and the treatment with quercetin 5, 25, and 50 mg/kg prevented this increase caused by Cd exposure. The in vitro experiment showed that quercetin 5, 10, 25, or 50 µM decreased the ADA activity proportionally to the increase of the concentrations of quercetin when compared to the control group. Thus, we can suggest that the quercetin is able to modulate NTPDase, ADA, AChE, and MPO activities and contribute to maintain the levels of ATP, adenosine, and acetylcholine normal, respectively, exhibiting potent pro-inflammatory and anti-inflammatory actions.     

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.     

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.     

Enzymes that hydrolyze adenine nucleotides in chronic renal failure: relationship between hemostatic defects and renal failure severity

The activities of the enzymes NTPDase (E.C.3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD 39) and 5'-nucleotidase (E.C.3.1.3.5, CD 73) were analyzed in platelets from patients with chronic renal failure (CRF), both undergoing hemodialysis treatment (HD) and not undergoing hemodialysis (ND), as well as from a control group. The results showed an increase in platelet NTPDase activity in CRF patients on HD treatment (52.88%) with ATP as substrate (P<0.0001). ADP hydrolysis was decreased (33.68% and 39.75%) in HD and ND patients, respectively. In addition, 5'-nucleotidase activity was elevated in the HD (160%) and ND (81.49%) groups when compared to the control (P<0.0001). Significant correlation was found among ATP, ADP and AMP hydrolysis and plasma creatinine and urea levels (P<0.0001). Patients were compared statistically according the time of hemodialysis treatment. We found enhanced NTPDase and 5'-nucleotidase activities between 49 and 72 months on HD patients. Our result suggests the existence of alterations in nucleotide hydrolysis in platelets of CRF patients. Possibly, this altered nucleotide hydrolysis could contribute to hemostasis abnormalities found in CRF.     

NTPDase activity in lymphocytes of rats infected by Trypanosoma evansi

Trypanosoma evansi is the aetiological agent of trypanosomosis in domestic animals. In this pathology, an inflammatory response can be observed and, as a consequence, the increase of extracellular adenine nucleotides such as ATP. These nucleotide concentrations are regulated by ectoenzymes such as NTPDase (EC 3.6.1.5, CD39), which catalyses the hydrolysis of ATP and ADP into AMP. In this study, the activity of NTPDase in lymphocytes of rats experimentally infected with T. evansi was evaluated. The animals were inoculated with the parasite and monitored by blood smear on a daily basis. The animals were then were divided into 4 groups according to the degree of parasitaemia and period of infection. The blood collections for enzyme analysis and lymphocyte count were performed on the 3rd (beginning of infection), 5th (acute infection) and 15th (chronic infection) days post-infection (p.i.). The control group was composed of non-infected animals. In the infected group a decrease in ATP hydrolysis (36%) was observed on the 3rd day p.i. and a decrease in ADP hydrolysis (62%) was observed on the 5th day p.i. when compared to the control. On the 15th day p.i., an increase in ATP (94%) and ADP (50%) hydrolysis was observed in the infected group. Considering these data it is suggested that NTPDase activity is altered on the surface of lymphocytes of rats infected with T. evansi at different time-points of infection.     

Enhanced NTPDase and 5′-nucleotidase activities in diabetes mellitus and iron-overload model

The activity of the enzymes NTPDase and 5′-nucleotidase was studied in both diabetes mellitus and an associated model of iron-overload. Rats were divided in five groups: citrate (CC), saline (S), diabetic (D), iron-overload (IO), and diabetic iron-overload (DIO). Diabetes was induced with alloxan (150 mg/kg), and iron-overload was induced with iron-dextran (10 intramuscular applications of ±80 mg/kg). The enzymatic activities were evaluated in the platelets. The results demonstrated an increase in the activity of NTPDase with substrates ATP and ADP (60% and 120%, respectively; P < 0.001), and 5′-nucleotidase (60%, P < 0.001). This increase was more intense in the IO and DIO groups. The results obtained in vitro showed an activation in ATP, ADP, and AMP hydrolysis between 1 μM and 1,000 μM ferric nitrate concentrations, being more pronounced at 100 μM and decreasing at 1,000 μM. We concluded that diabetes mellitus in association with iron-overload increased the hydrolysis of adenine nucleotides in platelets, contributing to the abnormalities found in these pathological conditions.     

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.     

Trypanosoma evansi: Activities of adenine nucleotide degradation enzymes in cerebral cortex of infected rats

This study aimed to evaluate the activities of the ectoenzymes NTPDase and 5′-nucleotidase in synaptosomes from cerebral cortex of rats experimentally infected with Trypanosoma evansi. The animals were divided in four groups (n = 10) according to the time and degree of parasitemia (groups A, B, C and D). The animals from group A were euthanized on day 3 (low parasitemia), group B on day 5 (high parasitemia) and group C on day 15 (low parasitemia). Group D consisted of healthy rats (not-infected, n = 15) and were divided in three periods (n = 5) in order to compare with the infected groups. After euthanasia, cerebral cortex was removed for the preparation of synaptosomes and enzymatic assays. Group A showed no changes in enzymatic activities compared with control. The hydrolysis of ATP, ADP and AMP by the enzymes NTPDase and 5′-nucleotidase were increased (P < 0.05) in group B (38%, 140% and 61%, respectively) when compared with control. In the group C it was observed a decreased (22%) hydrolysis of ATP when compared with control group. The activities of NTPDase and 5′-nucleotidase in synaptosomes alters the acute phase of the disease when the number of circulating parasites is high, thus the change observed is probably due to the parasitemia.     

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.     

Activities of enzymes that hydrolyze adenine nucleotides in platelets from rats experimentally demyelinated with ethidium bromide and treated with interferon-beta

The activities of the enzymes NTPDase (EC 3.6.1.5, apyrase, CD39) and 5'-nucleotidase (EC 3.1.3.5, CD73) were analyzed in platelets from rats submitted to demyelination by ethidium bromide (EB) and treated with interferon beta (IFN-beta). The following groups were studied: I - control (saline), II - (saline and IFN-beta), III - (EB) and IV - (EB and IFN-beta). After 7, 15 and 30 days, the animals (n=7) were sacrificed and the platelets were separated by the method of Lunkes et al. [Lunkes, G., Lunkes D., Morsch, V., Mazzanti, C., Morsch, A., Miron, V., Schetinger, M.R.C., 2004. NTPDase and 5'-nucleotidase in rats alloxan- induced diabetes. Diabetes Research and Clinical Practice 65, 1-6]. NTPDase activity for ATP and ADP substrates was significantly lower in groups II and III after seven days, when compared to control (p<0.001). At fifteen days, ATP hydrolysis was significantly lower in group III and IV and higher in group II (p<0.001), while there was an activation of ADP hydrolysis in group II (p<0.001), when compared with the control. 5'-nucleotidase activity was significantly higher in group IV (p<0.001) after seven days, and lower in the groups III and IV (p<0.001) after fifteen days in relation to the control. No significant differences were observed in NTPDase and 5'-nucleotidase activities after thirty days. In conclusion, our study demonstrated that the hydrolysis of adenine nucleotides is modified in platelets of rats demyelinated and treated with IFN-beta.     

Ethanol and acetaldehyde alter NTPDase and 5'-nucleotidase from zebrafish brain membranes

Alcohol abuse is an acute health problem throughout the world and alcohol consumption is linked to the occurrence of several pathological conditions. Here we tested the acute effects of ethanol on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5'-nucleotidase in zebrafish (Danio rerio) brain membranes. The results have shown a decrease on ATP (36.3 and 18.4%) and ADP (30 and 20%) hydrolysis after 0.5 and 1% (v/v) ethanol exposure during 60 min, respectively. In contrast, no changes on 5'-nucleotidase activity were observed in zebrafish brain membranes. Ethanol in vitro did not alter ATP and ADP hydrolysis, but AMP hydrolysis was inhibited at 0.5, and 1% (23 and 28%, respectively). Acetaldehyde in vitro, in the range 0.5-1%, inhibited ATP (40-85%) and ADP (28-65%) hydrolysis, whereas AMP hydrolysis was reduced (52, 58 and 64%) at 0.25, 0.5 and 1%, respectively. Acetate in vitro did not alter these enzyme activities. Semi-quantitative expression analysis of NTPDase and 5'-nucleotidase were performed. Ethanol treatment reduced NTPDase1 and three isoforms of NTPDase2 mRNA levels. These findings demonstrate that acute ethanol intoxication may influence the enzyme pathway involved in the degradation of ATP to adenosine, which could affect the responses mediated by adenine nucleotides and nucleosides in zebrafish central nervous system.     

Effect of subchronic treatment with mercury chloride on NTPDase, 5′-nucleotidase and acetylcholinesterase from cerebral cortex of rats

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl₂ on NTPDase (E.C. 3.6.1.5), 5′-nucleotidase (E.C. 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5′-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5′-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg²⁺, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.     

Hypothyroidism Enhanced Ectonucleotidases and Acetylcholinesterase Activities in Rat Synaptosomes can be Prevented by the Naturally Occurring Polyphenol Quercetin

Thyroid hormones have an influence on the functioning of the central nervous system. Furthermore, the cholinergic and purinergic systems also are extensively involved in brain function. In this context, quercetin is a polyphenol with antioxidant and neuroprotective properties. This study investigated the effects of (MMI)-induced hypothyroidism on the NTPDase, 5′-nucleotidase, adenosine deaminase (ADA), and acetylcholinesterase (AChE) activities in synaptosomes of rats and whether the quercetin can prevent it. MMI at a concentration of 20 mg/100 mL was administered for 90 days in the drinking water. The animals were divided into six groups: control/water (CT/W), control/quercetin 10 mg/kg, control/quercetin 25 mg/kg, methimazole/water (MMI/W), methimazole/quercetin 10 mg/kg (MMI/Q10), and methimazole/quercetin 25 mg/kg (MMI/Q25). On the 30th day, hormonal dosing was performed to confirm hypothyroidism, and the animals were subsequently treated with 10 or 25 mg/kg quercetin for 60 days. NTPDase activity was not altered in the MMI/W group. However, treatment with quercetin decreased ATP and ADP hydrolysis in the MMI/Q10 and MMI/Q25 groups. 5′-nucleotidase activity increased in the MMI/W group, but treatments with 10 or 25 mg/kg quercetin decreased 5′-nucleotidase activity. ADA activity decreased in the CT/25 and MMI/Q25 groups. Furthermore, AChE activity was reduced in all groups with hypothyroidism. In vitro tests also demonstrated that quercetin per se decreased NTPDase, 5′-nucleotidase, and AChE activities. This study demonstrated changes in the 5′-nucleotidase and AChE activities indicating that purinergic and cholinergic neurotransmission are altered in this condition. In addition, quercetin can alter these parameters and may be a promising natural compound with important neuroprotective actions in hypothyroidism.     

Moderate Red Wine and Grape Juice Consumption Modulates the Hydrolysis of the Adenine Nucleotides and Decreases Platelet Aggregation in Streptozotocin-Induced Diabetic Rats

This study investigated the ex vivo effects of the moderate red wine (RW) and grape juice (GJ) consumption, and the in vitro effects of the resveratrol, caffeic acid, gallic acid, quercetin, and rutin on NTPDase (nucleoside triphosphate diphosphohydrolase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), 5′-nucleotidase, and adenosine deaminase (ADA) activities in platelets and platelet aggregation from streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 10): control/saline, control/GJ, control/RW, diabetic/saline, diabetic/GJ, and diabetic/RW. RW and GJ were administered for 45 days; after this period, the blood was collected for experimental determinations. Results showed that NTPDase, E-NPP, 5′-nucleotidase, and ADA activities as well as platelet aggregation were increased in the diabetic/saline group compared to the control/saline group. Treatment with RW and GJ increased ectonucleotidases activities and prevented the increase in the ADA activity in the diabetic/GJ and diabetic/RW groups. Platelet aggregation was also decreased by the treatment with RW and GJ in the diabetic/GJ and diabetic/RW groups. In the in vitro tests, resveratrol, caffeic acid, and gallic acid increased ATP, ADP, and AMP hydrolysis, while quercetin and rutin decreased the hydrolysis of these nucleotides in platelets of diabetic rats. The ADA activity and platelet aggregation were reduced in platelets of diabetic rats in the presence of all polyphenols tested in vitro. These findings suggest that RW, GJ, and all polyphenols tested were able to modulate the ectoenzymes activities. Moreover, a decrease in the platelet aggregation was observed and it could contribute to the prevention of platelet abnormality, and consequently vascular complications in diabetic state.     

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.     

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.     

Histone deacetylases inhibitor trichostatin A increases the expression of Dleu2/miR-15a/16-1 via HDAC3 in non-small cell lung cancer

We aimed to examine the nucleoside triphosphate diphosphohydrolases (NTPDase) in lymphocytes; adenosine deaminase (ADA) and butyrylcholinesterase (BChE) in serum; and acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT) activity in whole blood; since these enzymes are involved in inflammation responses as well as in oxidative stress conditions. We also checked the levels of total thiols (T-SH), non-protein thiols (NPSH), and thiobarbituric acid reactive substances (TBARS) in serum of patients with lung cancer. We collected blood samples from patients (n = 31) previously treated for lung cancer with chemotherapy. Patients were classified as stage IIIb and IV according to the Union for International Cancer Control (UICC). The results showed a significant increase in the hydrolysis of ATP, ADP, and adenosine in patients when compared with the control group. The activity of AChE, SOD, and CAT as well as the T-SH and NPSH levels were higher in patients group and TBARS levels were lower in patients compared with the control group. These findings demonstrated that the enzymes activity involved in the control of inflammatory and immune processes as well as the oxidative stress parameters are altered in patients with lung cancer.     

The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'apyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.