Nucleoside Diphosphatase Activity Research Articles

Influence of cytomegalovirus infection on nucleoside diphosphatase activity in placenta

Introduction. Cytomegalovirus infection (CMVI) makes a significant contribution to the development of pregnancy pathologies, including dysfunction of the placenta. However, the mechanism of such an effect of CMV has not been precisely established. For the coordination of biochemical reactions in the placenta, an optimal concentration of substances, including nucleotides, is required. Nucleoside diphosphatase is an enzyme that catalyzes the hydrolysis of nucleotide diphosphates to nucleotide and phosphate.Aim. Determination of the activity of nucleoside diphosphatase in the placenta in physiological and complicated CMVI reactivation in the third trimester of pregnancy.Materials and methods. A study of 62 placentas obtained during childbirth at 38-40 weeks was carried out. The first group consisted of the placentas from women with CMVI reactivation in the third trimester of pregnancy and the second – the placenta of women without a history of infectious pathology. Diagnosis of CMVI was carried out by determining antibodies of class M and G by ELISA, as well as CMV DNA was detected by PCR. Histochemical analysis of nucleoside diphosphatase was performed according to the method of A.B.Novikoff and D.S.Goldfischer modified by Z.Lojda et al.Results. In the second group, the placenta nucleoside-diphosphatase had a pronounced activity in the syncytiotrophoblast and the connective tissue stroma of the villi. When CMVI was reactivated in the third trimester of pregnancy, a decrease in the intensity of the histochemical reaction to nucleoside-diphosphatase was found: the cytophotometric index in the first group significantly (p<0.001) decreased to 13.06±0.089 rel. units (in the second group it was 14.11±0.119 relative units).Conclusion. The decrease in the activity of nucleoside diphosphatase found during the reactivation of CMVI in the third trimester of pregnancy might indicate a decrease in nucleotide metabolism in the placenta. In our opinion, the study of enzymes involved in the exchange of nucleotides will help to reveal the mechanisms by which CMV causes disturbances in the functioning of the placenta, leading to the development of complications of pregnancy.

Нуклеозиддифосфатазы цестод Bothriocephalus scorpii (Cestoda: Bothriocephalidae)

Objective of research: To study the activities and properties of nucleoside- diphosphatase (NDPase) in cestode Bothriocephalus scorpii.
 Materials and methods: Cestodes were homogenized with 10 vol. of extraction medium. NDPase was detected in mitochondria and microsomes with substrates (IDP, GDP, UDP). Inorganic phosphorus was determined by the method of Kochetov (1980). The effects of 10 anthelmintic drugs on the activity of NDPase were studied. 
 Results and discussion: It was found that the mitochondrial and microsomal fractions of cestodes B. scorpii have nucleoside diphosphatase activity. The activity of nucleoside diphosphatase depends on substrates and Mg2+ ions. The impact of various effectors and ions (Ca2+, Mn2+, Zn2+) on enzyme activity was determined. Effects of 10 anthelmintic drugs on activity of nucleoside diphosphatase were studied. The anthelmintics Bitionol and Trichlorophen have been proved effective.

Novel Nucleoside Diphosphatase Contributes to Staphylococcus aureus Virulence

We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn(2+)- or Co(2+)-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic pathways and virulence gene expression and plays an important role in S. aureus virulence.

Gene set analysis of purine and pyrimidine antimetabolites cancer therapies

Responses to therapies, either with regard to toxicities or efficacy, are expected to involve complex relationships of gene products within the same molecular pathway or functional gene set. Therefore, pathways or gene sets, as opposed to single genes, may better reflect the true underlying biology and may be more appropriate units for analysis of pharmacogenomic studies. Application of such methods to pharmacogenomic studies may enable the detection of more subtle effects of multiple genes in the same pathway that may be missed by assessing each gene individually. A gene set analysis of 3821 gene sets is presented assessing the association between basal messenger RNA expression and drug cytotoxicity using ethnically defined human lymphoblastoid cell lines for two classes of drugs: pyrimidines [gemcitabine (dFdC) and arabinoside] and purines [6-thioguanine and 6-mercaptopurine]. The gene set nucleoside-diphosphatase activity was found to be significantly associated with both dFdC and arabinoside, whereas gene set γ-aminobutyric acid catabolic process was associated with dFdC and 6-thioguanine. These gene sets were significantly associated with the phenotype even after adjusting for multiple testing. In addition, five associated gene sets were found in common between the pyrimidines and two gene sets for the purines (3',5'-cyclic-AMP phosphodiesterase activity and γ-aminobutyric acid catabolic process) with a P value of less than 0.0001. Functional validation was attempted with four genes each in gene sets for thiopurine and pyrimidine antimetabolites. All four genes selected from the pyrimidine gene sets (PSME3, CANT1, ENTPD6, ADRM1) were validated, but only one (PDE4D) was validated for the thiopurine gene sets. In summary, results from the gene set analysis of pyrimidine and purine therapies, used often in the treatment of various cancers, provide novel insight into the relationship between genomic variation and drug response.

Absence of Nucleoside Diphosphatase Activities in the Yeast Secretory Pathway Does Not Abolish Nucleotide Sugar-dependent Protein Glycosylation

It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.

Ultrastructure of the cells forming amyloid fibers in Alzheimer disease and scrapie

Ultrastructural, three-dimensional reconstruction of cells surrounding the amyloid star in classical plaques in Alzheimer disease (AD) and histochemical studies of the cells associated with the deposits of amyloid fibers in scrapie were carried out. These studies showed that in both diseases, the fibers appear within the smooth endoplasmic reticulum (ER) and infoldings of cytoplasmic membranes of microglia/macrophages. Additional information about the site of formation of the amyloid fibers derives from histochemical studies of the localization of nucleoside diphosphatase (NDPase) activity. In normal microglia, this enzyme is associated with smooth ER and cell membranes. In the cells that form amyloid fibers, the NDPase activity is associated with the newly formed amyloid fibers within the distended cisternae of ER and the finger-like cytoplasmic projections. In the center of the amyloid star, the NDPase activity disappears. The presence of NDPase-positive amyloid fibers in the same location, where the enzyme is found in non-amyloid-forming cells, further supports our conclusion that the microglia/macrophages are the source of amyloid deposits. These studies also show that in spite of the differences in the proteins that produce the amyloid fibers in AD and scrapie, in both diseases, the microglia/macrophages play a key role in amyloid formation.

Nucleoside Diphosphatase and Glycosyltransferase Activities Can Localize to Different Subcellular Compartments in Schizosaccharomyces pombe

Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1+ did not affect Golgi N- or O-glycosylation, whereas disruption of Spynd1+ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.

Expression and Characterization of Soluble and Membrane-bound Human Nucleoside Triphosphate Diphosphohydrolase 6 (CD39L2)

Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP >> ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.

Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the γ-phosphate group of nucleoside triphosphates, and then attacks the β-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In constrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated kcat values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg2+ ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 °C, in the presence of millimolar Mg2+ concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.

Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the gamma-phosphate group of nucleoside triphosphates, and then attacks the beta-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In contrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated k(cat) values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg(2+) ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 degrees C, in the presence of millimolar Mg(2+) concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.

Polar tube formation and nucleoside diphosphatase activity in the microsporidian, Glugea stephani.

Polar tube formation and nucleoside diphosphatase activity in the microsporidian, Glugea stephani.

Microglia‐derived macrophages in early multiple sclerosis plaques

One of the characteristics of ongoing demyelination in multiple sclerosis (MS) is the accumulation of lipid-laden macrophages in active lesions. Little is known about the source of these macrophages in the early stages of plaque evolution as microglial-derived and haematogenous macrophages share morphological characteristics and most cell surface antigens. A key issue in understanding the pathogenesis of MS is the reliable identification of phagocytes capable of degrading myelin and presenting autoantigen to T cells at the onset of demyelination. Using a combination of histochemistry and immunocytochemistry, an average of 60% of EBM11+ phagocytes (EMBII is a pan-macrophage marker) in early active MS plaques, defined as lesions with myelin-containing phagocytes but no obvious parenchymal myelin loss around these cells, were judged to originate from microglia as they exhibited nucleoside diphosphatase activity, a microglial marker. Only 4-15% of EBM11+ phagocytes in these lesions exhibited non-specific esterase activity, an enzyme marker for monocytes and macrophages. In contrast, 30-80% of EBM11+ phagocytes in more advanced active plaques with partial or complete myelin loss in the parenchyma were non-specific esterase+. Lysosomal enzyme acid phosphatase activity was strongly exhibited by 90% of phagocytes in all active plaques and there was a significant correlation between numbers of acid phosphatase+ cells and oil red O+ foamy macrophages. The results indicate that microglia are the main population of phagocytes in the early stages of demyelination and may play an important role in the pathogenesis of MS.

Nucleoside diphosphatase (NDPase) activity associated with human ?-protein amyloid fibers

Nucleoside diphosphatase (NDPase) activity was studied by electron microscope cytochemistry in surgical specimens obtained from aged human cerebral cortices. The presence of NDPase activity on the surface of the microglial cells (MCs) and especially within the endoplasmic reticulum (ER) cisternae that are filled with amyloid fibers and that are in continuity with the extracellular amyloid deposits in plaques suggests a possible role of this enzyme in final elaboration of amyloid protein. The close structural relationship between MCs and amyloid plaques, suggesting the participation of these cells in the synthesis or final elaboration of amyloid fibers, was observed. The comparison of these observations with previously reported data on the distribution of NDPase in MCs and amyloid fibers in scrapie-infected mouse brain suggests that presumably similar mechanisms are acting in both cases. These observations, as compared with the results of other cytochemical and biochemical studies, also suggest that co-localization of NDPase activity with newly formed amyloid fibers in plaques can be associated with glycosyltransferase activities engaged in the amyloid or amyloid precursor protein glycosylation.

Enzyme Cytochemistry of the Alveolar Sacs and Golgian‐Like Cisternae in the Ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trophozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.

The endomembrane system of cholinergic and non-cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band of Broca: a cytochemical and immunocytochemical study.

Coronal vibratome sections of the rostral part of the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) nuclei were studied by an immunocytochemical technique using a monoclonal antibody against choline acetyltransferase (ChAT) and a double histochemical method for detection of acid phosphatase (AcPase) and nucleoside diphosphatase (NDPase) activity. The electron microscopic morphology of ChAT-immunoreactive and non-immunoreactive neurons was compared with similar neurons showing both AcPase and NDPase activity. ChAT-labeled and non-labeled neurons were well differentiated by the organization of the endomembrane system and especially by the structure of the rough endoplasmic reticulum (RER) and associated lamellar bodies. These results support the theory that the peculiar ultrastructure of the lamellar bodies in each neuron is related to the pattern of organization of the endomembrane system and its function. The significance of the lamellar bodies is discussed, and the data of the present work, together with findings described by other investigators. These data suggest that these bodies are predominant in efferent projection neurons in the basal forebrain nuclei.

A steady-state kinetic analysis of nucleoside diphosphatase activity of Golgi membranes

A steady-state kinetic analysis of nucleoside diphosphatase activity of Golgi membranes

Cytochemical localization of nicotinamide adenine dinucleotide phosphatase(NADPase) and nucleoside diphosphatase activities in the Golgi apparatus of the rat epididymis.

The ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) and thiamine pyrophosphatase (TPPase) or nucleoside diphosphatase (NDPase) activities was studied in the principal cells of the rat epididymis and compared with the pattern produced after monensin treatment.The NADPase activity was found positive in the intermediate cisternae near the cis side of the Golgi lamellae, to show a difference in compartment from that of TPPase on the trans side. After 60min treatment with monensin, most of the Golgi stacked lamellae exhibited dilation. The swelling occurred in all stacks without preferential effect on any particular Golgi subcompartment. With these enzymes, the characteristic staining patterns on the subcompartments in the Golgi apparatus were basically unchanged after treatment with monensin. However, some of the NADPase positive intermediate cisternae on the cis side of the Golgi apparatus were remarkably dilated.

Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii.

The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.

Electron cytochemical demonstration of phosphatase activity with microbody membranes of Basidiobolus haptosporus

Growth of cells of the potentially zoopathogenic fungus Basidiobolus haptosporus on a nutritionally defined medium with xanthine or urate as the nitrogen source results in greatly increased populations of microbodies. Modified Gomori procedures at the electron microscopic level suggested the single limiting membrane (and in some cases the granular matrix) of immature microbodies to be the exclusive subcellular locale(s) of alkaline phosphatase, 5'-nucleotidase and nucleoside diphosphatase activities. When grown in the presence of low inorganic phosphate, additional alkaline phosphatase activity was further identified cytochemically at and along profiles of endoplasmic reticulum and on inclusions previously described as "double-membraned vesicles". Cytochemical localization of acid phosphatase at microbody membranes was minimal if not ambiguous; Mg++-dependent adenosine triphosphatase and glucose-6-phosphatase were not identified at these locales. Quantitative biochemical estimates of alkaline phosphatase activity levels in particulate fractions initially increased with age of cells, perhaps as a function of the cultural induction and marked increase in immature microbody populations. We suggest that this enzyme may participate in some manner with protein translocation mechanisms associated with microbody biogenesis, ontogeny, and/or physiological function.

A rapid computer technique for analysing molecular interactions

A rapid method for analysing enzyme-substrate interactions using a discriminant analysis program is described. This technique identifies the structural features of substrate molecules which are important in determining metabolic activity. Two model systems, nucleoside diphosphatase activity of Golgi membranes and the interaction of yeast hexokinase with a range of D-sugars, are used as illustrations of the technique. The conclusions from both models are consistent with those previously obtained from analytical techniques.