Nucleotidase Enzyme Research Articles

Development of Antibody‐Coated Magnetite Nanoparticles for Biomarker Immobilization

Magnetic nanoparticles (MNPs) have great potential in biomedical applications because of their magnetic response offers the possibility to direct them to specific areas and target biological entities. Magnetic separation of biomolecules is one of the most important applications of MNPs because their versatility in detecting cancer biomarkers. However, the effectiveness of this method depends on many factors, including the type of functionalization onto MNPs. Therefore, in this study, magnetite nanoparticles have been developed in order to separate the 5′‐nucleotidase enzyme (5eNT). The 5eNT is used as a bio‐indicator for diagnosing diseases such as hepatic ischaemia, liver tumor, and hepatotoxic drugs damage. Magnetic nanoparticles were covered in a core/shell type with silica, aminosilane, and a double shell of silica‐aminosilane. A ScFv (fragment antibody) and anti‐CD73 antibody were attached to the coated nanoparticles in order to separate the enzyme. The magnetic separation of this enzyme with fragment antibody was found to be 28% higher than anti‐CD73 antibody and the enzyme adsorption was improved with the double shell due to the increased length of the polymeric chain. Magnetite nanoparticles with a double shell (silica‐aminosilane) were also found to be more sensitive than magnetite with a single shell in the detection of biomarkers.

Homocysteine modifies extracellular ATP availability in macrophages

Increased levels of plasma homocysteine (hyperhomocysteinemia-HHcy) are associated to the development of coronary artery disease (CAD), peripheral vascular disease and thrombosis. In addition, recent studies have shown that inflammation, probably mediated by macrophages, mediates the pathogenesis associated to high levels of homocysteine (Hcy). In the present study, we evaluated the Hcy effects in the ATP hydrolysis and its breakdown products in murine macrophages. The results showed that micromolar concentrations of Hcy increased the ATP, ADP and AMP hydrolysis. Additionally, our results show decreased inosine levels in the extracellular milieu of Hcy-exposed macrophages. The increasing in ATP, ADP and AMP hydrolysis are not explained by increased transcription or protein expression of NTPDases and ecto-5′-nucleotidase (ecto-5′-NT/CD73) enzymes. Moreover, the formation of reactive oxygen species did not interfere in the Hcy effects, which suggest that Hcy or Hcy metabolites act directly on the modulation of NTPDases and ecto-5′-NT/CD73 activities. In conclusion, Hcy induces the rapid breakdown of ATP, ADP and AMP to adenosine (ADO), which is classically known as an anti-inflammatory response in immune cells. However, by the action of these enzymes, the extracellular adenosine generated during Hcy treatment probably is uptaken into the cells, as evidenced by the decreased in inosine formation, and thus collaborating to the inflammatory complications associates to HHcy

Estradiol Regulation of Nucleotidases in Female Reproductive Tract Epithelial Cells and Fibroblasts

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5′-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5′-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.

Identification of novel chromone based sulfonamides as highly potent and selective inhibitors of alkaline phosphatases

A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (Ki, up to 0.01 ± 0.001 μM) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5′-nucleotidase enzyme.

Abstract 4595: Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

Abstract Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease. Using whole exome sequencing, here we identify mutations in NT5C2, a 5’-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL. Citation Format: Gannie Tzoneva, Arianne Perez-Garcia, Zachary Carpenter, Hossein Khiabanian, Valeria Tosello, Maddalena Allegretta, Elisabeth Paietta, Janis Racevkis, Jakob M. Rowe, Martin S. Tallman, Maddalena Paganin, Jana Hoff, Renate Kirschner-Schwabe, Teresa Palomero, Raul Rabadan, Adolfo Ferrando. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2013-4595

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease1,2. Using whole exome sequencing, here we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL.

A rapid method to assess the stage differentiation in Leishmania donovani by flow cytometry

In this study we describe a rapid and novel method to assess the morphological stage differentiation in Leishmania donovani by flow cytometry (FCM). FCM is fast, accurate, and inexpensive to study the stage differentiation of promastigote into L. donovani axenic amastigote (LdAxAm). The non-flourimetric FCM method is easy to perform; with requirement of little expertise, and provides unambiguous results. It is an advanced tool, requires minimal time, and no fluorescent dyes. The gradual increase of differentiation and reduction in size from promastigote stage to LdAxAm leads to peak shifting from right to left on histogram. Earlier reports assessed the stage differentiation of Leishmania by studying the expression of stage specific markers like surface or secretory proteins and genes. For validation, conventional methods like microscopic analysis are used. These methods are quite expensive, laborious and time consuming. Non-flourimetric morphological parameters were further validated by conventional methods like optical and scanning electron microscopy. Additionally, differential expression of stage specific genes (e.g. upregulation of amastin and ATP binding cassette A3 (ABCA3) transporter gene transcripts) and differential activity of enzymes (down regulation of secretory acid phosphatase (SAcP) and 3'-nucleotidase enzyme activity) in LdAxAm suggest stage differentiation. Therefore, we believe that our method is an alternative tool for high reproducibility and reliability in assessment of stage differentiation.

CD73‐generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?

Nucleosides fromPhlebotomus papatasiSalivary Gland Ameliorate Murine Collagen-Induced Arthritis by Impairing Dendritic Cell Functions

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4(+) Th17 cell response. We identified adenosine (ADO) and 5'AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2(A) receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5'-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5'AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5'AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Suppression of 5′-Nucleotidase Enzymes Promotes AMP-activated Protein Kinase (AMPK) Phosphorylation and Metabolism in Human and Mouse Skeletal Muscle

The 5'-nucleotidase (NT5) family of enzyme dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. We hypothesized that gene silencing of NT5 enzymes to increase the intracellular availability of AMP would increase AMP-activated protein kinase (AMPK) activity and metabolism. We determined the role of cytosolic NT5 in metabolic responses linked to the development of insulin resistance in obesity and type 2 diabetes. Using siRNA to silence NT5C2 expression in cultured human myotubes, we observed a 2-fold increase in the AMP/ATP ratio, a 2.4-fold increase in AMPK phosphorylation (Thr(172)), and a 2.8-fold increase in acetyl-CoA carboxylase phosphorylation (Ser(79)) (p < 0.05). siRNA silencing of NT5C2 expression increased palmitate oxidation by 2-fold in the absence and by 8-fold in the presence of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside. This was paralleled by an increase in glucose transport and a decrease in glucose oxidation, incorporation into glycogen, and lactate release from NT5C2-depleted myotubes. Gene silencing of NT5C1A by shRNA injection and electroporation in mouse tibialis anterior muscle reduced protein content (60%; p < 0.05) and increased phosphorylation of AMPK (60%; p < 0.05) and acetyl-CoA carboxylase (50%; p < 0.05) and glucose uptake (20%; p < 0.05). Endogenous expression of NT5C enzymes inhibited basal lipid oxidation and glucose transport in skeletal muscle. Reduction of 5'-nucleotidase expression or activity may promote metabolic flexibility in type 2 diabetes.

Spermicidal action of a protein isolated from ethanolic root extracts of Achyranthes aspera: An in vitro study

A previous study conducted in our department, showed that 50% ethanolic extract of the roots of Achyranthes aspera possess spermatotoxic effects. Preliminary studies also revealed that the active principle may be a protein. In this study a 58 kDa Achyranthes protein (Ap) was isolated from Achyranthes aspera using standard protocols and their effects on the rat sperm was studied in vitro in comparison with nonoxynol-9 (N-9). The sperm immobilization studies showed that about 150 μg of Ap was able to immobilize sperms completely within seconds at a lower concentration than N-9 (250 μg). The sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in the Ap-treated and N-9 treated groups in comparison to the control. In the Ap and N-9 treated groups the number of acrosome reacted cells were found to be high and it also caused agglutination of the sperms indicating the loss of intactness of the plasma membrane which was further supported by the significant reduction in the activity of membrane bound 5' nucleotidase and acrosin enzyme. Hence this study showed that the protein isolated from the roots of Achyranthes aspera possess spermicidal activity in vitro and can act as a spermicide similar to that of nonoxynol 9. Ap also possessed spermicidal activity against human sperms in vitro.

ChemInform Abstract: 5′‐Nucleotidases and Their New Roles in NAD+ and Phosphate Metabolism

AbstractReview: 103 refs.

5′-Nucleotidases and their new roles in NAD+ and phosphate metabolism

5′-Nucleotidase (EC 3.1.3.5) designates a set of enzymes, which catalyze the hydrolysis of ribonucleoside and deoxyribonucleoside monophosphates into the corresponding nucleosides plus orthophosphate. 5′-Nucleotidases are classified according to subcellular localization, nucleobase specificity and their ability to hydrolyze deoxynucleoside monophosphate substrates. Membrane-bound 5′-nucleotidases are ectoenzymes principally involved in salvage of extracellular nucleosides, and often display a preference toward adenosine monophosphate, thereby modulating signal transduction cascades involving purinergic receptors. Cytosolic 5′-nucleotidases are members of the haloacid dehalogenase superfamily of enzymes, which are two-domain proteins containing a modified Rossman fold as the core and a variable cap structure. Extracellular and intracellular 5′-nucleotidase activities participate in purine and pyrimidine salvage to support balanced synthesis of nucleotides, which is critical for maintaining high fidelity DNA replication. While the production of ribonucleosides from ribonucleotides by 5′-nucleotidases remains the most well studied function, it appears that the physiological functions of these activities are more broad. Indeed, Sdt1, previously termed a pyrimidine-specific 5′-nucleotidase, and Isn1, previously termed an inosine monophosphate (IMP)-specific 5′-nucleotidase, have recently been implicated in catabolic processes in nicotinamide adenine dinucleotide (NAD+) metabolism, and are regulated by the NAD+ precursor vitamin nicotinic acid, glucose and phosphate availability in the medium. In addition, Usha, Pho5, Sdt1 and Phm8 are phosphate starvation-induced 5′-nucleotidases with diverse substrate specificities that liberate phosphate under phosphate starvation conditions. Here we review 5′-nucleotidase enzyme structure, catalytic mechanism and substrate specificity and focus on new biological roles for these enzymes in nucleotide, NAD+ and phosphate metabolism.

Preface: special issue on medicinal chemistry of purines

The concept of preparing a special issue of Purinergic Signalling to highlight progress in the medicinal chemistry of purines arose during the 8th International Symposium on Adenosine and Adenine Nucleotides, which was held in Ferrara, Italy, from 24 May to 28 May 2006. This meeting emphasized the accelerated pace of introduction of new compounds, including preclinical and clinical candidates, that act through purine-related mechanisms. The suggestion to broaden the scope to include the intracellular actions of purines, notably through protein kinases, was made by Dr. Michael Williams, as an Associate Editor of this journal. Consequently, this volume reveals new ligands for modulation of the function of proteins that are involved in purine action, encompassing both receptors and enzymes. This purview could be described under the umbrella of the “purinome,” which represents numerous critical interactions in cellular function. Given the limited space available, we are unable to effectively showcase all major aspects of the purinome, but rather have provided representative segments. The issue is written from the perspective of the medicinal chemist but in a manner comprehensible to those in the biological sciences. Both review chapters and original chemical research reports are included in this volume. We commend Prof. Geoff Burnstock, Editor in Chief of the journal, for enthusiastically supporting the inclusion of a dedicated issue on medicinal chemistry of purines and for recognizing a central role played by medicinal chemistry in the recent exponential growth of this field. The protein targets of compounds in this volume include twelve G protein-coupled receptors that respond to nucleosides and nucleotides, i.e., both adenosine (four subtypes) and P2Y receptors (eight subtypes), and seven nucleotide-gated ion channels, i.e., P2X receptors. A chapter by Carroll et al. reviews the P2X7 receptor. In addition, agonists (Baraldi et al., Gao et al., Kim et al.), antagonists (Kalla and Zablocki), and allosteric modulators (Goblyos and IJzerman) of the P1 family of adenosine receptors are described including compounds currently under active development. It is to be noted that at least four subtypes of the P2Y receptors are activated by pyrimidine nucleotides (Jacobson et al.). Thus, this issue is not exclusively limited to purines. These purine and pyrimidine receptors are widespread in the body and govern critical physiological processes, notably in the immune, cardiovascular, skeletal-muscular, and nervous systems. The volume also covers inhibition of the superfamily of >500 kinases, which are involved in intracellular signaling and are relevant to cancer (Garcia-Echeverria) and chronic inflammatory diseases (Bhagwat). The search for novel kinase inhibitors, which has become the focus of recent drug discovery efforts, has yielded eight small molecule drugs and a handful of protein therapeutics approved for human use to date. The number of kinase inhibitor drugs for the treatment of human disease is likely to increase in the near future. Also, the volume describes novel inhibitors of nucleotidase enzymes that interconvert extracellular nucleotides (Baki et al.), leading to a spectrum of biological effects. In conclusion, these fields deriving from the purinome have become a major focus in the pharmaceutical industry, and the promise of new drugs emerging from them is enormous. We hope that this special issue will be a good source of information and reference for basic and applied researchers in this field. 14 May 2008

NTPDase and 5′ ecto-nucleotidase expression profiles and the pattern of extracellular ATP metabolism in the Walker 256 tumor

In this study, we evaluated the NTPDases and ecto-5′-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5′-nucleotidase. By quantitative real-time PCR, Entpd1 ( Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5′-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.

Previous treatment with ebselen and vitamin E alters adenine nucleotide hydrolysis in platelets from adult rats experimentally demyelinated with ethidium bromide

Many aspects of the relationship between the demyelinating pathology and platelet function need to be elucidated. Thus, the activity of NTPDase and 5′-nucleotidase enzymes was analyzed in platelets from rats demyelinated with ethidium bromide (EB) and previously treated with ebselen (Ebs) and vitamin E (Vit. E). The animals were divided into four groups: for ebselen, the groups were: I — control (saline), II — (saline and Ebs), III — (EB) and IV — (EB and Ebs); and for vitamin E, the groups were: I — control (saline), II — (saline and Vit. E), III — (EB) and IV — (EB and Vit. E). After 3 and 21 days, the blood was collected and the platelets were separated for enzymatic assays. For the treatment with Ebs, the NTPDase activity for ATP substrate was significantly lower in groups II, III and IV ( p < 0.05) after 3 days, while after 21 days, a reduction was observed in group III ( p < 0.05). ADP hydrolysis was reduced in group II ( p < 0.05) and increased in group IV ( p < 0.05) after 3 days, while after 21 days there was an increase in group IV ( p < 0.05). In the treatment with Vit. E, ATP hydrolysis was lower in groups II, III and IV ( p < 0.05) after 3 and 21 days. ADP hydrolysis was increased in group II ( p < 0.05) after 3 days, and in group IV ( p < 0.05) after 21 days. However, 5′-nucleotidase activity was not altered by the treatments. These findings demonstrate that NTPDase activity in platelets is diminished in demyelinating events and the treatments with Ebs and Vit. E modulated adenine nucleotide hydrolysis.

Detection of a 3′, 5′-cyclic-AMP phosphodiesterase activity in the lichenized fungus Evernia prunastri

A 3′, 5′-cyclic-AMP phosphodiesterase (PDE) was detected and measured in the lichen Evernia prunastri. The percentage of hydrolysis of tritiated 3′, 5′-cyclic-adenosine monophosphate ([3H]-cAMP) and 3′, 5′-cyclic-guanosine monophosphate ([3H]-cGMP) by the PDE enzyme into tritiated 5′-adenosine-monophospahte ([3H]-AMP) and tritiated 5′-guanosine-monophospahte ([3H]-GMP) was measured by treating the PDE products with a 5′-nucleotidase enzyme present in snake venom. The lysate fraction (L) (plasma membranes and cell walls) and the supernatant (S) (soluble fraction of the cells) were tested. In both fractions, competition of unlabelled cAMP, but not unlabelled cGMP, was revealed. Specific competitive PDE inhibitors such as IBMX inhibited enzymatic activity. Although it is thought that in this species cAMP is regulated by red/far red light through PDE activity, this is the first report that seems to suggest the presence of a PDE activity specific for cAMP in lichenized fungi. However, this work is at a preliminary stage and despite the high levels of enzymatic activity with cAMP found in both fractions, data are still insufficient to state the absolute specificity for this nucleotide.

Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: Molecular identification, kinetic characterization and biochemical properties

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5′-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5–8.0, and absolute requirement for divalent cations (Mg 2+ > Ca 2+). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K m values were 464.2 ± 86.6 μM (mean ± SEM, n = 4), 137.0 ± 31 μM (mean ± SEM, n = 5) and 44.8 ± 10.2 μM (mean ± SEM, n = 4), and V max values were 655.0 ± 94.6 (mean ± SEM, n = 4), 236.3 ± 27.2 (mean ± SEM, n = 5) and 177.6 ± 13.8 (mean ± SEM, n = 5) nmol of inorganic phosphate min − 1 mg of protein − 1 for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5′-nucleotidase. The presence of NTPDases and 5′-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.

The anti‐snake venom properties of Tamarindus indica (leguminosae) seed extract

In Indian traditional medicine, various plants have been used widely as a remedy for treating snake bites. The aim of this study was to evaluate the effect of Tamarindus indica seed extract on the pharmacological as well as the enzymatic effects induced by V. russelli venom. Tamarind seed extract inhibited the PLA(2), protease, hyaluronidase, l-amino acid oxidase and 5'-nucleotidase enzyme activities of venom in a dose-dependent manner. These are the major hydrolytic enzymes responsible for the early effects of envenomation, such as local tissue damage, inflammation and hypotension. Furthermore, the extract neutralized the degradation of the Bbeta chain of human fibrinogen and indirect hemolysis caused by venom. It was also observed that the extract exerted a moderate effect on the clotting time, prolonging it only to a small extent. Edema, hemorrhage and myotoxic effects including lethality, induced by venom were neutralized significantly when different doses of the extract were preincubated with venom before the assays. On the other hand, animals that received extract 10 min after the injection of venom were protected from venom induced toxicity. Since it inhibits hydrolytic enzymes and pharmacological effects, it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of PLA(2), metalloproteinases, serine proteases, hyaluronidases and 5 cent-nucleotidases, the enzymes involved in several physiopathological human and animal diseases.