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Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3. [ABSTRACT FROM AUTHOR]

Abstract Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn2+-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (ki(ap)). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased ki(ap). The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.

We previously reported that knockout of the mazG (SA1292) gene decreases Staphylococcus aureus killing activity against silkworms. S. aureus MazG (SaMazG) has a nucleotide pyrophosphatase domain conserved among MazG family proteins, but its biochemical characteristics are unknown. In the present study, we purified recombinant N-terminal His-tagged SaMazG protein and examined its biochemical activity. SaMazG hydrolyzed GTP, UTP, dGTP, and TTP into nucleoside monophosphates. Hydrolytic activity of SaMazG against ATP, CTP, dATP, and dCTP was low or not detected. SaMazG exhibited high hydrolytic activity against 8-oxo-GTP and 8-oxo-dGTP, oxidized guanine nucleotides, with a V max / K m ratio more than 15-fold that of GTP. Furthermore, the S. aureus mazG knockout mutant was sensitive to hydrogen peroxide compared with the parent strain. These results suggest that SaMazG is a nucleotide pyrophosphatase hydrolyzing oxidized guanine nucleotides that contributes to the oxidative stress resistance of S. aureus. • SaMazG is a nucleotide pyrophosphatase against GTP, UTP, dGTP, and TTP. • SaMazG efficiently hydrolyzes 8-oxo-GTP and 8-oxo-dGTP. • Knockout of mazG sensitizes S. aureus to hydrogen peroxide. [ABSTRACT FROM AUTHOR]

Docking pose of compound 1x into the crystal structure of NPP3. A new series of sulfonate derivatives 1a – zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC 50 value of 0.387 ± 0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with an IC 50 value of 0.214 ± 0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC 50 value of 0.659 ± 0.007 µM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results. [ABSTRACT FROM AUTHOR]

Aims This study was performed to investigate the effect of PD-L1 polymorphisms on the susceptibility and prognosis of hepatocellular carcinoma (HCC) in a Chinese Han population. Methods Four single nucleotide polymorphisms (SNPs) of the PD-L1 gene, including rs2297136 (C > T), rs4143815 (C > G), rs2890658 (A > C) and rs17718883 (C > G) were examined in 225 HCC patients and 200 healthy controls using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Results Data revealed that the rs2297136 (C > T) SNP TT ( p  = 0.03) and rs4143815 (C > G) SNP GG genotypes ( p  < 0.001) were associated with significantly increased risks of HCC. No association was found between rs2890658 (A > C) SNP and HCC risk and this risk was significantly decreased in individuals with the rs17718883 SNP CG + GG genotype ( p  < 0.001). The rs2297136 (C > T) SNP CC + CT genotypes, the rs4143815 (C > G) CC genotype and the rs2890658 (A > C) AA genotype were associated with increased overall survival compared to their counterpart allelic genotypes ( p  < 0.001). The rs2890658 (A > C) SNP had no impact on the risk and prognosis of HCC ( p  > 0.05). Conclusions Our results indicated that three functional polymorphisms (rs2297136, rs4143815 and rs17718883) of the PD-L1 gene were associated with HCC risk and prognosis, suggesting that genetic variants of PD-L1 polymorphisms might be a possible prognostic marker for the prediction of HCC risk and development. Validation by a larger prospective study from a more diverse ethnic population is needed to confirm these findings. [ABSTRACT FROM AUTHOR]

A new methodology for the synthesis of fluorinated and non‐fluorinated 1H‐pyrazolo[3,4‐b]pyridin‐3‐ones was developed. The reactions proceed by cyclization of electron‐rich 3‐amino‐1H‐pyrazol‐5(4H)‐ones with 1,3‐diketones and the products were obtained in good to excellent yields and with excellent regioselectivity. The products, which can be regarded as deazapurine analogues, were tested for the alkaline phosphatase (h‐TNAP and h‐IAP) and nucleotide pyrophosphatase/phosphodiesterase (h‐NPP1 and h‐NPP3) inhibition profile. Most of the derivatives exhibited selective and potent inhibition on tissue non‐specific and intestinal alkaline phosphatase and nucleotide pyrophosphatase (NPP1 and NPP3) enzymes. Possible binding modes of the most potent inhibitors were studied by molecular docking analysis. [ABSTRACT FROM AUTHOR]

The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound. Of the 26 derivatives prepared, four compounds were found to have K i  < 105 nM against human NPP1. [ABSTRACT FROM AUTHOR]

A new approach to arylated 2-trifluoromethylquinolines based on novel regioselective Suzuki-Miyaura coupling reactions has been developed. Moreover, site-selective, chemo-selective amination reactions were performed. The new 2-trifluoromethylquinoline derivatives were tested as potential NPPs inhibitors and evaluated for their potential to inhibit two families of ecto-nucleotidases, i.e. NPPs and nucleoside triphosphate diphosphohydrolases (NTPDases). Several derivatives were active on a nanomolecular concentration. The results were validated based on docking studies to study the active binding site of the molecules. [ABSTRACT FROM AUTHOR]

Almost 25 years ago, an enzyme named zeatin cis-trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involved in the metabolism of plant hormones cytokinins was generally accepted by plant biologists, the corresponding protein and encoding gene have not been identified to date. Based on the original paper, we purified and identified an enzyme from maize, which shows the described zeatin cis-trans isomerase activity. The enzyme belongs to nucleotide pyrophosphatase/phosphodiesterase family, which is well characterized in mammals, but less known in plants. Further experiments with the recombinant maize enzyme obtained from yeast expression system showed that rather than the catalytic activity of the enzyme itself, a non-enzymatic flavin induced photoisomerization is responsible for the observed zeatin cis-trans interconversion in vitro. An overexpression of the maize nucleotide pyrophosphatase/phosphodiesterase gene led to decreased FAD and increased FMN and riboflavin contents in transgenic Arabidopsis plants. However, neither contents nor the ratio of zeatin isomers was altered suggesting that the enzyme is unlikely to catalyze the interconversion of zeatin isomers in vivo. Using enhanced expression of a homologous gene, functional nucleotide pyrophosphatase/phosphodiesterase was also identified in rice. [ABSTRACT FROM AUTHOR]

Ectonucleotide pyrophosphatase/phosphodiesterase 3 (Enpp3) is involved in multiple physiological processes, such as morphological changes and inflammatory processes. The present study investigated the spatiotemporal expression pattern and regulatory mechanisms controlling expression of Enpp3 in the rat ovary during the periovulatory period. Immature female rats were injected with pregnant mare serum gonadotropin to stimulate follicular development. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after human chorionic gonadotropin (hCG) administration. Real-time polymerase chain reaction analysis revealed that messenger RNA (mRNA) for Enpp3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. In situ hybridization analysis demonstrated that Enpp3 mRNA expression was induced in theca cells at 4 hours after hCG, and the expression remained elevated until 12 hours after hCG. The expression of Enpp3 mRNA was stimulated in granulosa cells at 8 hours and reached the highest expression at 12 hours. Localization of Enpp3 mRNA was observed in newly forming corpora lutea by in situ hybridization. The hCG-stimulated expression of Enpp3 mRNA was blocked by a protein kinase C inhibitor (GF109203) instead of the protein kinase A inhibitor (H89). Furthermore, Enpp3 induction is dependent on new protein synthesis. Inhibition of progesterone action did not alter Enpp3 mRNA expression, whereas inhibition of prostaglandin synthesis or the epidermal growth factor pathway diminished Enpp3 mRNA levels. In conclusion, our findings suggest that the induction of the Enpp3 mRNA may be important for the morphological changes and inflammatory response during ovulation and luteinization. [ABSTRACT FROM AUTHOR]

Background: We examined associations between single-nucleotide polymorphisms (SNPs) of IFITM3, TLR3, and CD55 genes and influenza clinical outcomes in Chinese.Methods: A multicenter study was conducted on 275 adult cases of avian (H7N9) and pandemic (H1N1pdm09) influenza. Host DNA was extracted from diagnostic respiratory samples; IFITM3 rs12252, TLR3 rs5743313, CD55 rs2564978, and TLR4 rs4986790/4986791 were targeted for genotyping (Sanger sequencing). The primary outcome analyzed was death.Results: IFITM3 and TLR3 SNPs were in Hardy-Weinberg equilibrium; their allele frequencies (IFITM3/C-allele 0.56, TLR3/C-allele 0.88) were comparable to 1000 Genomes Han Chinese data. We found over-representation of homozygous IFITM3 CC (54.5% vs 33.2%; P = .02) and TLR3 CC (93.3% vs 76.9%; P = .04) genotypes among fatal cases. Recessive genetic models showed their significant independent associations with higher death risks (adjusted hazard ratio [aHR] 2.78, 95% confidence interval [CI] 1.29-6.02, and aHR 4.85, 95% CI 1.11-21.06, respectively). Cumulative effects were found (aHR 3.53, 95% CI 1.64-7.59 per risk genotype; aHR 9.99, 95% CI 1.27-78.59 with both). Results were consistent for each influenza subtype and other severity indicators. The CD55 TT genotype was linked to severity. TLR4 was nonpolymorphic.Conclusions: Host genetic factors may influence clinical outcomes of avian and pandemic influenza infections. Such findings have important implications on disease burden and patient care in at-risk populations. [ABSTRACT FROM AUTHOR]

Absorption of dietary sphingomyelin (SM) requires its initial degradation into ceramide, a process catalyzed by the intestinal enzyme alkaline sphingomyelinase (alk-SMase, NPP7, ENPP7). alk-SMase belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, the members of which hydrolyze nucleoside phosphates, phospholipids, and other related molecules. NPP7 is the only paralog that can cleave SM, and its activity requires the presence of bile salts, a class of physiological anionic detergents. To elucidate the mechanism of substrate recognition, we determined the crystal structure of human alk- SMase in complex with phosphocholine, a reaction product. Although the overall fold and catalytic center are conserved relative to other NPPs, alk-SMase recognizes the choline moiety of its substrates via an NPP7-specific aromatic box composed of tyrosine residues. Mutational analysis and enzymatic activity assays identified features on the surface of the protein--a cationic patch and a unique hydrophobic loop--that are essential for accessing SM in bile salt micelles. These results shed new light on substrate specificity determinants within the NPP enzyme family. [ABSTRACT FROM AUTHOR]

Nucleotide pyrophosphatase/phosphodiesterase type 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its major substrate is ATP which is converted to AMP and diphosphate. NPP1 was proposed as a new therapeutic target in brain cancer and immuno-oncology. Several NPP1 inhibitors have been reported to date, most of which were evaluated vs. the artificial substrate p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP). Recently, we observed large discrepancies in inhibitory potencies for a class of competitive NPP1 inhibitors when tested vs. the artificial substrate p-Nph-5'-TMP as compared to the natural substrate ATP. Therefore, the goal of the present study was to investigate whether inhibitors of human NPP1 generally display substrate-dependent inhibitory potency. Systematic evaluation of nucleotidic as well as non-nucleotidic NPP1 inhibitors revealed significant differences in determined Ki values for competitive, but not for non- and un-competitive inhibitors when tested vs. the frequently used artificial substrate p-Nph-5'-TMP as compared to ATP. Allosteric modulation of NPP1 by p-Nph-5'-TMP may explain these discrepancies. Results obtained using the AMP derivative p-nitrophenyl 5'-adenosine monophosphate (p-Nph-5'-AMP) as an alternative artificial substrate correlated much better with those employing the natural substrate ATP. [ABSTRACT FROM AUTHOR]

There are multiple bioinformatics tools available for the detection of coding driver mutations in cancers. However, the prioritization of pathogenic non-coding variants remains a challenging and demanding task. The present study was performed to discriminate non-coding disease-causing mutations and prioritize potential cancer-implicated long non-coding RNAs (lncRNAs) in liver cancer using a logistic regression model. A logistic regression model was constructed by combining 19,153 disease-associated ClinVar and human gene mutation database pathogenic variants as the response variable and non-coding features as the predictor variable. Genome-wide association study (GWAS) disease or trait-associated variants and recurrent somatic mutations were used to validate the model. Non-coding gene features with the highest fractions of load were characterized and potential cancer-associated lncRNA candidates were prioritized by combining the fraction of high-scoring regions and average score predicted by the logistic regression model. H3K9me3 and conserved regions were the most negatively and positively informative for the model, respectively. The area under the receiver operating characteristic curve of the model was 0.92. The average score of GWAS disease-associated variants was significantly increased compared with neutral single nucleotide polymorphisms (5.8642 vs. 5.4707; P<0.001), the average score of recurrent somatic mutations of liver cancer was significantly increased compared with non-recurrent somatic mutations (5.4101 vs. 5.2768; P=0.0125). The present study found regions in lncRNAs and introns/untranslated regions of protein coding genes where mutations are most likely to be damaging. In total, 847 lncRNAs were filtered out from the background. Characterization of this subset of lncRNAs showed that these lncRNAs are more conservative, less mutated and more highly expressed compared with other control lncRNAs. In addition, 23 of these lncRNAs were differentially expressed between 12 pairs of liver cancer and adjacent normal specimens. The logistic regression model is a useful tool to prioritize non-coding pathogenic variants and lncRNAs, and paves the way for the detection of non-coding driver lncRNAs in liver cancer. [ABSTRACT FROM AUTHOR]

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1- NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/ mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs. [ABSTRACT FROM AUTHOR]

Background The effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5′- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide. Methods Rats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB + AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR). Results The results revealed that there was a significant ( P < 0.01) reduction in E-NPP activity in EB group (1.63 ± 0.10 nmol p -nitrophenol released/min/mg protein) when compared to CT group (2.33 ± 0.14 nmol p -nitrophenol released/min/mg protein). However, treatment with chlorogenic acid significantly ( P < 0.05) increased E-NPP activity in EB group. Furthermore, no significant ( P > 0.05) change was observed in the E-NPP activity of EB + AC group (2.19 ± 0.08 nmol p -nitrophenol released/min/mg protein) when compared to CT group (2.33 ± 0.14 nmol p -nitrophenol released/min/mg protein). In addition, there was a significant ( P < 0.05) increase in AMP hydrolysis in EB rat group when compared to CT group. No significant ( P > 0.05) difference was observed in AMP hydrolysis between AC, AC + EB and CT groups. Conversely, there were no significant ( P > 0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC + EB and CT groups). Likewise, there were no significant ( P > 0.05) changes in E-ADA activity and percentage platelet aggregation among all groups studied. Similarly, no significant ( P > 0.05) change was observed in the expression of E-NTPDase 1, 2 and 3 in all the groups tested. Conclusions Our study revealed that chlorogenic acid may modulate the hydrolysis of adenine nucleotides in platelets of rats demyelinated and treated with chlorogenic acid via alteration of E-NPP and ecto-5′-nucleotidase activities. [ABSTRACT FROM AUTHOR]

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methyl-CpG binding protein 2 ( MECP2 ) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50 kDa protein, i.e., the N -linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N -glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N -glycosylation of NPP-5. [ABSTRACT FROM AUTHOR]

BA1544 from Bacillus anthracis was previously annotated as a transcription factor for the gene cluster ba1554 - ba1558 , but has not been experimentally characterized. B. anthracis is an obligate pathogen causing fatal inhalational anthrax, and BA1544 is absolutely conserved in Bacillus species, including Bacillus cereus , Bacillus thuringiensis and Bacillus mycoides , with 100% sequence identity. To address the function of BA1544, we performed structural and biochemical studies, which revealed that BA1544 is a MazG protein. Thus, herein, the protein is defined as Bacillus -conserved MazG (BcMazG). Like other MazG structures, BcMazG assembles into a tetrameric architecture. Each monomer adopts a four-α-helix bundle that accommodates a metal ion using four acidic residues, and presents one putative substrate-binding site. Enzymatic characterization demonstrated that BcMazG is a nucleoside triphosphate (NTP) pyrophosphohydrolase and prefers adenosine triphosphate as a substrate among canonical NTPs. Moreover, structural comparison of BcMazG with its homologues revealed a potential regulation mechanism whereby the enzymatic activity of BcMazG is regulated by its C-terminal region. [ABSTRACT FROM AUTHOR]

The target tracer carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives, N -(3-[ 11 C]methoxy-4-methoxyphenyl)-2-((5-methoxy-3 H -imidazo[4,5- b ]pyridin-2-yl)thio)acetamide (3-[ 11 C] 4a ) and N -(4-[ 11 C]methoxy-3-methoxyphenyl)-2-((5-methoxy-3 H -imidazo[4,5- b ]pyridin-2-yl)thio)acetamide (4-[ 11 C] 4a ); 2-((6-amino-9 H -purin-8-yl)thio)- N -(3-[ 11 C]methoxy-4-methoxyphenyl)acetamide (3-[ 11 C] 8a ) and 2-((6-amino-9 H -purin-8-yl)thio)- N -(4-[ 11 C]methoxy-3-methoxyphenyl)acetamide (4-[ 11 C] 8a ), were prepared by O -[ 11 C]methylation of their corresponding precursors with [ 11 C]CH 3 OTf under basic condition (2 N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields based on [ 11 C]CO 2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555 GBq/μmol. [ABSTRACT FROM AUTHOR]

The bistably expressed K-state of B acillus subtilis is characterized by two distinct features; transformability and arrested growth when K-state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA+ protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA-dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)pp Gpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)pp Gpp in K-state cells, which are thus trapped in a non-growing state until ComGA is degraded. We show that some K-state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet-hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B . subtilis in the soil. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is a member of the nucleotide pyrophosphatase/phosphodiesterase family of ectoenzymes that hydrolyzes phosphodiester bonds of various nucleotides. It possesses lysophospholipase D activity, catalyzing the hydrolysis of lysophosphatidylcholine into lysophosphatidic acid (LPA), and it is considered the major LPA-producing enzyme in the circulation. LPA is a bioactive phospholipid with diverse functions in almost every mammalian cell type, which exerts its action through binding to specific G protein-coupled receptors and stimulates various cellular functions, including migration, proliferation and survival. As a consequence, both ATX and LPA have attracted the interest of researchers, in an effort to understand their roles in physiology and pathophysiology. The present review article aims to summarize the existing knowledge as to the implications of ATX in chronic inflammatory diseases and cancer and to highlight the low molecular weight compounds, which have been developed as leads for the discovery of novel medicines to treat inflammatory diseases and cancer. [ABSTRACT FROM AUTHOR]

YhdE, a Maf-like protein in Escherichia coli, exhibits nucleotide pyrophosphatase (PPase) activity, yet its cellular function remains unknown. Here, we characterized the PPase activity of YhdE on dTTP, UTP and TTP and determined two crystal structures of YhdE, revealing ‘closed’ and ‘open’ conformations of an adaptive active site. Our functional studies demonstrated that YhdE retards cell growth by prolonging the lag and log phases, particularly under stress conditions. Morphology studies showed that yhdE-knockout cells transformed the normal rod shape of wild-type cells to a more spherical form, and the cell wall appeared to become more flexible. In contrast, YhdE overexpression resulted in filamentous cells. This study reveals the previously unknown involvement of YhdE in cell growth inhibition under stress conditions, cell-division arrest and cell-shape maintenance, highlighting YhdE’s important role in E. coli cell-cycle checkpoints. [ABSTRACT FROM AUTHOR]

Nucleotidepyrophosphatase/phosphodiesterase 1 (NPP1) belongs tothe family of ecto-nucleotidases, which control extracellular nucleotide,nucleoside, and (di)phosphate levels. To study the (patho)physiologicalroles of NPP1 potent and selective inhibitors with drug-like propertiesare required. Therefore, a compound library was screened for NPP1inhibitors using a colorimetric assay with p-nitrophenyl5′-thymidine monophosphate (p-Nph-5′-TMP)as an artificial substrate. This led to the discovery of 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (5a) as ahit compound with a Kivalue of 217 nM.Subsequent structure–activity relationship studies led to thedevelopment of purine and imidazo[4,5-b]pyridineanalogues with high inhibitory potency (Kivalues of 5.00 nM and 29.6 nM, respectively) when assayed with p-Nph-5′-TMP as a substrate. Surprisingly, the compoundswere significantly less potent when tested versus ATP as a substrate,with Kivalues in the low micromolar range.A prototypic inhibitor was investigated for its mechanism of inhibitionand found to be competitive versus both substrates. [ABSTRACT FROM AUTHOR]

Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2′3′-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2′3′-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1−/− mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2′3′-cGAMP (2′3′-cGsAsMP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1−/− lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2′3′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic. [ABSTRACT FROM AUTHOR]

A series of Schiff base triazoles 1 – 25 was synthesized and evaluated for their nucleotide pyrophosphatase/phosphodiesterase-1 inhibitory activities. Among twenty-five compounds, three compounds 10 (IC 50 = 132.20 ± 2.89 μM), 13 (IC 50 = 152.83 ± 2.39 μM), and 22 (IC 50 = 251.0 ± 6.64 μM) were identified as potent inhibitors with superior activities than the standard EDTA (IC 50 = 277.69 ± 2.52 μM). The newly identified inhibitors may open a new avenue for the development of treatment of phosphodiesterase-I related disorders. These compounds were also evaluated for carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase inhibitory potential and were found to be inactive. The compounds showed non-toxic effect towards PC3 cell lines. [ABSTRACT FROM AUTHOR]

Abstract: Calcific aortic valve disease (CAVD) is a chronic disorder characterized by an abnormal mineralization of the leaflets, which is accelerated in bicuspid aortic valve (BAV). It is suspected that mechanical strain may promote/enhance mineralization of the aortic valve. However, the effect of mechanical strain and the involved pathways during mineralization of the aortic valve remains largely unknown. Valve interstitial cells (VICs) were isolated and studied under strain conditions. Human bicuspid aortic valves were examined as a model relevant to increase mechanical strain. Cyclic strain increased mineralization of VICs by several-fold. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analyses revealed that mechanical strain promoted the formation of mineralized spheroid microparticles, which coalesced into larger structure at the surface of apoptotic VICs. Apoptosis and mineralization were closely associated with expression of ENPP1. Inhibition of ENPP1 greatly reduced mineralization of VIC cultures. Through several lines of evidence we showed that mechanical strain promoted the export of ENPP1-containing vesicles to the plasma membrane through a RhoA/ROCK pathway. Studies conducted in human BAV revealed the presence of spheroid mineralized structures along with the expression of ENPP1 in areas of high mechanical strain. Mechanical strain promotes the production and accumulation of spheroid mineralized microparticles by VICs, which may represent one important underlying mechanism involved in aortic valve mineralization. RhoA/ROCK-mediated export of ENPP1 to the plasma membrane promotes strain-induced mineralization of VICs. [Copyright &y& Elsevier]

Summary: Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning. [Copyright &y& Elsevier]

Background and Purpose Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 ( CD39), ecto-5′-nucleotidase ( CD73), nucleotide pyrophosphatase phosphodiesterase 1 ( NPP-1) and tissue non-specific alkaline phosphatase ( TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors ( A1R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1R agonists neither affect basal osteoclastogenesis nor do they reverse A1R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Experimental Approach Osteoclast formation derived from wild-type, CD39 knockout ( KO), CD73 KO, NPP- 1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression ( Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Key Results Rolofylline inhibited osteoclast formation in a dose-dependent manner ( IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold. Conclusions and Implications Based on these findings, we hypothesize that the A1R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1R antagonists act as inverse agonists to release A1R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation. [ABSTRACT FROM AUTHOR]

Elevatednucleotide pyrophosphatase/phosphodiesterase-1 (NPP1)activity is implicated in health disorders including pathologicalcalcification. Specific NPP1 inhibitors would therefore be valuablefor studying this enzyme and as potential therapeutic agents. Herewe present a combined computational/experimental study characterizing13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors.All analogues at 100 μM inhibited (66–99%) the hydrolysisof pnp-TMP by both recombinant NPP1 and cell surface NPP1 activityof osteocarcinoma (HTB-85) cells. These analogues only slightly alteredthe activity of other ectonucleotidases, NPP3 and NTPDases. The Ki,appvalues of the seven most potent and selectiveinhibitors were in the range of 0.5–56 μM, all with mixedtype inhibition, predominantly competitive. Those molecules were dockedinto a newly developed homology model of human NPP1. All adopted ATP-likebinding modes, suggesting competitive inhibition with the endogenousligand. NPP1 selectivity versus NPP3 could be explained in terms ofthe electrostatic potential of the two proteins that of NPP1 favoringnegatively charged ligands. Inhibitor 2that had thelowest Ki,app(0.5 μM) was alsoinactive toward P2Y receptors. Overall, analogue 2isthe most potent and selective NPP1 inhibitor described so far. [ABSTRACT FROM AUTHOR]

Highlights: [•] ITPA cleanses nucleotide pools of naturally-occurring, dangerous base analogs. [•] Mutations in model organisms lead to genomic instability, heart defect and death. [•] Reduced ITPA activity/polymorphism are linked to adverse reactions to certain drugs. [•] Pro32Thr ITPA variant has incorrectly folded structure and limited action in cells. [•] Dominant negative effect of P32T variant in cells caused by mixed dimer degradation. [Copyright &y& Elsevier]

The tumor microenvironment is characterized by of high levels of extracellular nucleotides that are metabolized through the dynamic and sequential action of cell surface enzymes (ectoenzymes). These ectoenzymes operate according to their spatial arrangement, as part of (1) continuous (molecules on the same cell) or (2) discontinuous (molecules on different cells) pathways, the latter being facilitated by restricted cellular microenvironment. The outcome of this catabolic activity is an increase in the local concentration of adenosine, a nucleoside involved in the control of inflammation and immune responses. The aim of the work presented here was to demonstrate that a previously unexplored enzymatic pathway may be an alternate route to produce extracellular adenosine. Our data show that this new axis is driven by the nucleotide-metabolizing ectoenzymes CD38 (an NAD+ nucleosidase), the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, also known as CD203a or PC-1) and the 5' ectonucleotidase (5'-NT) CD73, while bypassing the canonical catabolic pathway mediated by the nucleoside tri- and diphosphohydrolase (NTPDase) CD39. To determine the relative contributions of these cell surface enzymes to the production of adenosine, we exploited a human T-cell model allowing for the modular expression of the individual components of this alternative pathway upon activation and transfection. The biochemical analysis of the products of these ectoenzymes by high-performance liquid chromatography (HPLC) fully substantiated our working hypothesis. This newly characterized pathway may facilitate the emergence of an adaptive immune response in selected cellular contexts. Considering the role for extracellular adenosine in the regulation of inflammation and immunogenicity, this pathway could constitute a novel strategy of tumor evasion, implying that these enzymes may represent ideal targets for antibody-mediated therapy. [ABSTRACT FROM AUTHOR]

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m7GMP) or diphosphate (m7GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m7GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m7GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity form7GMP)GMP. CMP is a slightly better substrate but again with a higherKm. The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not acceptm7GMP as a substrate. We suggest that the m7G-specific nucleotidases protect cells against undesired salvage of m7GMP and its incorporation into nucleic acids. [ABSTRACT FROM AUTHOR]

Abstract: A highly sensitive capillary electrophoresis method has been developed to monitor the activity of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and screen for NPP inhibitors. In this method, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) was used as an artificial substrate, and separation of reaction products was performed on a dynamically coated capillary. We found that the optimal capillary electrophoresis (CE) conditions were as follows: fused-silica capillary (20cm effective length×75.5μm (id)), electrokinetic injection for 60s, 70mM phosphate buffer containing polybrene 0.002%, pH 9.2, constant current of −80μA, constant capillary temperature of 15°C and detection at 400nm. To allow precise quantification, 2-methyl-4,6-dinitrophenol (dinitrocresol) was applied as an internal standard. The limit of detection (LOD) and the limit of quantification (LOQ) were 137 and 415nM, respectively. This new method was shown to be over 8-fold more sensitive than the conventional spectrophotometric assays and 16-fold more than the previously reported CE procedure, and the results (K m values for NPP1 and NPP3, K i values for standard inhibitors) obtained were in accordance with previous literature data. Therefore, this new method is an improvement of actual techniques and could be used as a quick and standard analytical technique for the identification and characterization of NPP inhibitors. [Copyright &y& Elsevier]

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosppiodiesterase4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADPmediated platelet aggregation at the site of vascular injury. [ABSTRACT FROM AUTHOR]

Several members of the Alkaline Phosphatase (AP) superfamily exhibit a high level of catalytic proffciency and promiscuity in structurally similar active sites. A thorough characterization of the nature of transition state for different substrates in these enzymes is crucial for understanding the molecular mechanisms that govern those remarkable catalytic properties. In this work, we study the hydrolysis of a phosphate diester, MpNPP-, in solution, two experimentally well-characterized variants of AP (R166S AP, R166S/E322Y AP) and wild type Nucleotide pyrophosphatase/phosphodiesterase (NPP) by QM/MM calculations in which the QM method is an approximate density functional theory previously parametrized for phosphate hydrolysis (SCC-DFTBPR). The general agreements found between these calculations and available experimental data for both solution and enzymes support the use of SCC-DFTBPR/MM for a semiquantitative analysis of the catalytic mechanism and nature of transition state in AP and NPP. Although phosphate diesters are cognate substrates for NPP but promiscuous substrates for AP, the calculations suggest that their hydrolysis reactions catalyzed by AP and NPP feature similar synchronous transition states that are slightly tighter in nature compared to that in solution, due in part to the geometry of the bimetallic zinc motif. Therefore, this study provides the first direct computational support to the hypothesis that enzymes in the AP superfamily catalyze cognate and promiscuous substrates via similar transition states to those in solution. Our calculations do not support the finding of recent QM/MM studies by López-Canut and co-workers, who suggested that the same diester substrate goes through a much looser transition state in NPP/AP than in solution, a result likely biased by the large structural distortion of the bimetallic zinc site in their simulations. Finally, our calculations for different phosphate diester orientations and phosphorothioate diesters highlight that the interpretation of thio-substitution experiments is not always straightforward. [ABSTRACT FROM AUTHOR]

The article offers brief information on nucleotide pyrophosphatase/ phosphodiesterase 7 (NPP7), only member of the mammalian NPP enzyme family which acts as a sphingomyelinase, hydrolyzing sphingomyelin (SM) for forming phosphocholine and ceramide. It mentions that it also hydrolizes lysophosphatidylcholine (LPC). It also mentions the two contributers of NPP7 to hydrolyze SM which includes second hydrophobic tail of SM occupying hydrophobic binding pocket and other is the leucine residue.

Autotaxin (ATX) is an ecto-enzyme responsible for lysophosphatidic acid (LPA) production in blood. ATX is present in various biological fluids such as cerebrospinal and seminal fluids and accounts for bulk LPA production in these fluids. ATX is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family and was originally isolated from conditioned medium of melanoma cells as an autocrine motility stimulating factor. LPA, a second-generation lipid mediator, binds to its cognate G protein-coupled receptors through which it exerts a number of biological functions including influencing cell motility and proliferation stimulating activity. Some of the biological roles of LPA can be mediated by ATX. However, there are other LPA-producing pathways independent of ATX. The accumulating evidences for physiological and pathological functions of ATX strongly support that ATX is an important therapeutic target. This review summarizes the historical aspects, structural basis, pathophysiological functions identified in mice studies and clinical relevance discovered by measuring the blood ATX level in human. The general features and functions of each NPP family member will be also briefly reviewed. The presence of the ATX gene in other model organisms and recently developed ATX inhibitors, both of which will be definitely useful for further functional analysis of ATX, will also be mentioned. [ABSTRACT FROM PUBLISHER]

The article offers information on the genome wide association and drug response biomarkers related to the gemcitabine and arabinosylcytosin pharmacogenomics. It offers information on the individual variation presented by cancer patients in their response to chemotherapeutic agents. It offers information on the data related to genome-wide single nucleotide polymorphisms (SNPs) and mRNA expression. It mentions that gemcitabine and AraC are widely used in the treatment of various cancers. It mentions that GWA approach using cell-based model system depicts the mechanism for variation in cytidine analogue response.

Membrane glycoproteins of neural cells play crucial roles in axon guidance, synaptogenesis, and neuronal transmission. We have here characterized membrane glycoproteins containing terminal α-mannose residues in rat brain membranes. Affinity purification using Galanthus nivalis agglutinin, that is highly specific for terminal α-mannose residues, revealed a 50-kDa protein as well as 80-kDa SHPS-1 and 45-kDa β2 subunit of Na,K-ATPase in rat brain membranes. Combination of N-terminal peptide sequencing and mass spectrometry indicated that the 50-kDa protein was rat nucleotide pyrophosphatase-5 (NPP-5). In contrast to other NPPs, NPP-5 was a type-I transmembrane protein. Northern blot analysis showed that NPP-5 was highly expressed in brain, but also expressed in other peripheral tissues. However, we could not detect either the NPP activity or the lysophospholipase D activity in the immunoprecipitates with antibodies to NPP-5 from rat brain membranes. These data, therefore, suggest that NPP-5 is a neural oligomannosidic glycoprotein that may participate in neural cell communications. [Copyright &y& Elsevier]

Diadenosine polyphosphates (Apn As) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from Apn A. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5′,5′′′-P1 ,P3 -triphosphate (Ap3 A), diadenosine 5′,5′′′-P1 ,P4 -tetraphosphate (Ap4 A), and diadenosine 5′,5′′′-P1 ,P5 -pentaphosphate, (Ap5 A), and the diguanosine polyphosphate, diguanosine 5′,5′′′-P1 ,P4 -tetraphosphate (Gp4 G). Each of the three enzymes hydrolyzed Ap3 A, Ap4 A, and Ap5 A at comparable rates. Gp4 G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4 A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the α,β-pyrophosphate bond. Apn A hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (K m ) values for Ap3 A were 5.1 µm, 8.0 µm, and 49.5 µm for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues. [ABSTRACT FROM AUTHOR]

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized.E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-β and glucocorticoids, but the signal transduction pathways that control expression are poorly documented.NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes.In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases. [Copyright &y& Elsevier]

The spinach (Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1–68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells. [ABSTRACT FROM AUTHOR]