Modulation of nucleotide pyrophosphatase in plasmacytoma cells.

The effect of glucocorticoid hormones on the protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was examined in murine MOPC 315 plasmacytoma cells. Incubation of these cells with dexamethasone resulted in parallel increases in pyrophosphatase and phosphodiesterase specific activities. The incorporation of [3H]mannose into N-linked oligosaccharide precursors was also analyzed in cells following hormone modulation. In cells treated for 36 hours or cultured continuously with dexamethasone, the resulting increase in enzyme specific activities was accompanied by a decrease in [3H]mannose incorporation, consistent with the hypothesis that in some cell types, nucleotide pyrophosphatase activity is involved in the regulation of glycoprotein synthesis.     

Synthesis of triazole Schiff bases: Novel inhibitors of nucleotide pyrophosphatase/phosphodiesterase-1

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 (IC₅₀ = 132.20 ± 2.89 μM), 13 (IC₅₀ = 152.83 ± 2.39 μM), and 22 (IC₅₀ = 251.0 ± 6.64 μM) were identified as potent inhibitors with superior activities than the standard EDTA (IC₅₀ = 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.     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

Removal of 5''-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation.

The procedure for isolation of nucleotide pyrophosphatase (E.C. 3.6.1.9.) from potato has been modified to yield an endonuclease-free preparation purified 2300-fold. The enzyme was used for specific cleavage of pyrophosphate linkages in the 5'-terminal cap (m7GpppN) of several eukaryotic messenger RNAs. Enzymatic removal of 5'-terminal pm7G from reovirus, rabbit globin and Artemia salina mRNAs resulted in an almost complete loss (greater than 80%) of their template activities in a cell-free protein synthesizing system from wheat germ. Incubation with nucleotide pyrophosphatase did not decrease the translation of phage f2 RNA in an Escherichia coli cell-free system.     

Molecular cloning of cDNAs for human fibroblast nucleotide pyrophosphatase.

Nucleotide pyrophosphatase (NPPase) activity is markedly elevated in cultured skin fibroblasts from patients of Lowe's syndrome. cDNA clones encoding the NPPase were isolated using synthetic oligonucleotide probes designed on the basis of partial amino acid sequence of the enzyme purified from human placenta. The complete sequences of these clones yielded a merged sequence of 3508 bases. The polypeptide chain of the enzyme was deduced to comprise 873 amino acids with a calculated molecular weight of 99,703 and had the characteristics of a class II transmembrane protein. Ten potential N-glycosylation sites were detected in the protein. RNA blot analysis showed that human fibroblasts contain two minor mRNAs of 7.0 and 8.2 kb, respectively, in addition to a major 3.6-kb species that coincides with the merged cDNA in size. A computer search of a nucleotide sequence data-base revealed that plasma cell membrane glycoprotein PC-1, whose function was unknown at the time, is identical with the NPPase. Comparison of the amino acid sequence of the NPPase with the active site sequence of bovine 5'-nucleotide phosphodiesterase allowed the assignment of a putative active site domain to the central region of the COOH-terminal extracellular domain of the NPPase. The gene for human NPPase was localized to chromosome 6 at q22-q23 by in situ hybridization with a fragment of the NPPase cDNA.     

Elevation of nucleotide pyrophosphatase activity in skin fibroblasts from patients with Lowe's syndrome

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient[Fukui, S. etal. (1981) J. Biol. Chem. 256, 10313–10318] was found to be caused by elevated degradation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The enzyme involved in this degradation was then identified as an enzyme of nucleotide pyrophosphatase (EC 3.6.1.9) nature, cleaving the phosphosulfate linkage. The specific activities were 8 – 24 (mU/mg protein) in patients' fibroblasts, in contrast to 3 in normal and 5 – 14 in heterozygote cells. A possibility is discussed that the elevation of nucleotide pyrophosphatase activity is the primary genetic defect in Lowe's syndrome.     

The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5''-phosphate.

Synopsis A cytochemical method for the localization of nucleotide pyrophosphatase activity in plants employing naphthol AS-BI thymidine 5-monophosphate and -naphthyl thymidine 5-monophosphate as specific substrates is reported. Biochemical evidence for the validity of this method is presented and the synthesis of the naphthol AS-BI ester is described.The application of this cytochemical technique to shoots ofTriticum sp. and roots ofVicia faba has shown nucleotide pyrophosphatase to be ubiquitous in its distribution in these organs and to occur in a structurally-bound form in the cytoplasm. The highest activity was detected in developing fibres adjacent to the leaf vascular bundles, in the coleoptile epidermal and hypodermal cells and in the coleoptile and leaf xylem.     

Localization of nucleotide pyrophosphatase in the rat kidney

Summary Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freezedried sections. Nucleotide pyrophosphatase activity, expressed in mol·min^–1·mg protein^–1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichement of the activity during the purification of brush border vesicles was measured. A tenfold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.Supported by the Swiss National Foundation, grant nr. 3.813.084     

Localization of nucleotide pyrophosphatase in the rat kidney

Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freeze-dried sections. Nucleotide pyrophosphatase activity, expressed in mumol X min-1 X mg protein-1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichment of the activity during the purification of brush border vesicles was measured. A ten-fold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.     

Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.     

Novel nucleotide phosphonate analogues with potent antitumor activity

We have identified several nucleotide phosphonates demonstrating in vitro antiproliferative activity in several human cancer cell lines with IC(50) values in the microM range. The synthesis as well as structure-activity relationship are described.     

The role of nucleotide pyrophosphatase in Mullerian duct regression

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn²⁺ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from to rat fetuses (n = 77) were incubated with a-naphthyl thymidine-5′-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male (n = 16) Mullerian duct cells at days of gestation, but no hydrolysis was detected around female (n = 17) Mullerian duct cells at any stage. Controls (n = 24) incubated without substrate did not stain. Addition of exogenous ATP (n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro (n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.     

Purification and properties of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3-dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.     

Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast.

Use of Triton X-114 allowed us to develop a new method to separate hydrophilic oligosaccharidic material from hydrophobic oligosaccharide pyrophosphodolichols (oligosaccharide-PP-Dol). Taking advantage of this procedure we characterize, in yeast microsomal membranes, an enzymic activity that hydrolyses oligosaccharide-PP-Dol into oligosaccharidic material. H.p.l.c. analysis together with alkaline-phosphatase- and endo-N-acetyl-beta-D-glucosaminidase-susceptibility demonstrate that the oligosaccharidic released material is mainly composed of oligomannosides containing a chitobiose phosphate at the reducing end. The enzymic activity requires bivalent cations and is inhibited by pyrophosphate, NAD+ and bacitracin. As other, commercially available, pyrophosphatases have no action on lipid intermediates, the described pyrophosphatase activity appears to be the specific enzyme for oligosaccharide-PP-Dol. This enzymic splitting of the pyrophosphate bond might be the primary event in the catabolism of lipid intermediates.