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Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrubEuphorbia characias(ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.

Over expression of nucleotide pyrophosphatase (NPPs) activity is associated with chondrocalcinosis, osteoarthritis, type 2 diabetes, neurodegenerative diseases, allergies and cancer metastasis. The potential of NPPs inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP inhibitors. Specifically, 2-bromo-7-methyl-5-oxo-5H-1,3,4-thiadiazolopyrimidine and its corresponding 6-fluoro derivatives were synthesized via a Suzuki–Miyaura reaction. The cross-coupling reaction with different arylboronic acids gave desired coupling products in good to excellent yields and showed wide functional group tolerance. Furthermore, all compounds were investigated for their potential to inhibit two families of ecto-nucleotidases, i.e. nucleoside triphosphate diphosphohydrolases (NTPDase) and NPPs. Interestingly, our compounds were identified as selective inhibitors of NPPs. Among derivatives 5a–5i, compound 5i (IC50 ± SEM = 0.39 ± 0.01 μM) was found to be the most potent inhibitor of h-NPP1 and compound 5h (IC50 ± SEM = 1.02 ± 0.05 μM) was found to be the most potent inhibitor of h-NPP3. Similarly, for fluorinated thiadiazolopyrimidones, derivative 6e (IC50 ± SEM = 0.31 ± 0.01 μM) exhibited the best inhibition of NPP1 and it was found that this compound exhibited ≈28 fold improvement in inhibitory potential as compared with the reference control i.e. Suramin (IC50 ± SEM = 8.67 ± 1.3 μM). Moreover, homology modelling and molecular docking studies of both inhibitors were carried out to suggest the putative binding mode of inhibitors with the respective enzyme i.e. h-NPP1 and h-NPP3.

Various N-fused isoquinoline derivatives were synthesized using a new one-pot reaction of 1-bromo-2-(2,2-difluorovinyl)benzenes with N–H group containing heterocycles followed by intramolecular palladium-catalyzed C–H arylation. The method described gives convenient access to diverse structures of N-fused polycyclic isoquinolines. Sixteen of the synthesized compounds were screened as potential human nucleotide pyrophosphatase/phosphodiesterase 1 and 3 (h-NPP-1 and h-NPP-3) inhibitors. The most effective h-NPP-1 inhibitor showed an IC50 value as high as 0.36 ± 0.06 μM, whereas the most potent h-NPP-3 inhibitor posessed an inhibitory value of 0.48 ± 0.01 μM. Kinetic and molecular docking studies of both most effective inhibitors were carried out.

NPP3 inhibitors are promising therapeutic agents due to their potential as anti-cancer, anti-metastatic and anti-neurodegenerative drugs. We have identified the first potent and selective inhibitors of human NPP3. We have also estimated the biochemical properties of the main NPP family members that can hydrolyse nucleotides using p-nitrophenyl-5'-thymidine monophosphate as substrate. Km values were found to be 20 ± 4 and 15 ± 6 � M for human NPP1 and NPP3, respectively. Maximum velocity (Vmax) values calculated for NPP1 and NPP3 were 12 ± 2 and 5 ± 1 nmol p-nitrophenol released/min/mg of crude protein, respectively. A series of benzothiazole derivatives and a series of 1,3,4-oxadiazole-2-thiones was tested on hNPP1 and hNPP3. Benzothiazoles were the most potent non competitive inhibi- tors of hNPP3 described to date. 1,3,4-oxadiazole-2-thiones were also identified as compounds which inhibited specifi- cally NPP3 over NPP1. The most potent compound was further characterized and found to exhibit a non competitive mechanism of inhibition.

A hydrolase activity directed against diadenosine 5′,5″′-P1,P4-tetraphosphate (Ap4A) has been solubilised and partially purified from the plasma membrane fraction of bovine adrenal medullary chromaffin tissue in order to determine its relationship to alkaline phosphodiesterase-I/nucleotide pyrophosphatase (PDase-I, EC 3.1.4.1). Activity with the specific dinucleoside tetraphosphatase (EC 3.6.1.17) substrate Ap4A and with the non-specific PDase-I substrate thymidine 5′-monophosphate p-nitrophenyl ester had Km and Vmax values of 2.0 μM and 600 pmol/min/mg protein and 0.2 mM and 26 nmol/min/mg protein respectively and co-chromatographed upon gel filtration and ion-exchange chromatography. Activity with the fluorescent substrates etheno-Ap4A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap4A. Immunoblotting of the most purified fraction with an antibody against mouse PC-1, one of the major PDase-I family members, detected bands of 240, 120 and 62 kDa corresponding to PC-1 dimer, monomer and proteolytic fragment. Therefore, the activity previously described as bovine adrenal chromaffin cell ecto(diadenosine polyphosphate hydrolase) (ecto-ApnAase) is a PDase-I, probably bovine PC-1.

The ectonucleoside pyrophosphatase phosphodiesterase 1 (NPP1/PC-1) is a member of the NPP enzyme family that is critical in regulating mineralization. In certain mineralizing sites of bone and cartilage, membrane-limited vesicles [matrix vesicles (MVs)] provide a sheltered internal environment for nucleation of calcium-containing crystals, including hydroxyapatite. MV formation occurs by budding of vesicles from the plasma membrane of mineralizing cells. The MVs are enriched in proteins that promote mineralization. Paradoxically, NPP1, the type II transmembrane protein that generates the potent hydroxyapatite crystal growth inhibitor inorganic pyrophosphate (PPi), is also enriched in MVs. Although osteoblasts express NPP1, NPP2, and NPP3, only NPP1 is enriched in MVs. Therefore, this study uses mineralizing human osteoblastic SaOS-2 cells, a panel of NPP1 mutants, and NPP1 chimeras with NPP3, which does not concentrate in MVs, to investigate how NPP1 preferentially targets to MVs. We demonstrated that a cytosolic dileucine motif (amino acids 49–50) was critical in localizing NPP1 to regions of the plasma membrane that budded off into MVs. Moreover, transposition of the NPP1 cytoplasmic dileucine motif and flanking region (AAASLLAP) to NPP3 conferred to NPP3 the ability to target to the plasma membrane and, subsequently, concentrate in MVs. Functionally, the cytosolic tail dileucine motif NPP1 mutants lost the ability to support MV PPiconcentrations and to suppress calcification. The results identify a specific targeting motif in the NPP1 cytosolic tail that delivers PPi-generating NPP activity to osteoblast MVs for control of calcification.

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.

We have identified in plasma membrane fractions isolated from rat hepatocarcinoma AS-30D ascites cells three glycoproteins of 125 kDa, 115 kDa and 105 kDa (gp125, gp115 and gp105) which become adenylylated using ATP as substrate, most readily in the presence of EDTA. The gp115 becomes also phosphorylated. The adenylylation of these tumor glycoproteins was much lower than that of a group of analogous adenylylatable glycoproteins (gp130, gp120-gp110 dimer and gp100) present in normal rat liver plasma membrane. The tumor glycoproteins were reversibly O-adenylylated at threonine residues, as was the case for their normal rat liver counterparts. The tumor gp115, and the gp120-gp110 dimer from normal rat liver were both isolated using either ATP-affinity chromatography and/or AMP-affinity chromatography. The gp120-gp110 dimer from normal rat liver was identified as the plasma cell differentiation antigen-1 (PC-1 protein), an ecto-5′ phosphodiesterase/nucleotide-pyrophosphatase (5′-PDE/NPPase). The gp115 from tumor cells also exhibited Zn2+-stimulated 5′-PDE and NPPase activities in alkaline conditions, although it appears to be distinct from the PC-1 protein. We have determined that the gp115 is an ecto-enzyme that catalyzes the hydrolysis of extracellular ATP, since its adenylylation and phosphorylation were detected in intact cells using extracellularly added [α-32P]ATP or [γ-32P]ATP, respectively, in the absence of any permeabilizing agent., This work was financed by grants to AV from the Comisión Interministerial de Ciencia y Tecnología (SAF99-0052), the Consejería de Educación y Cultura de la Comunidad de Madrid (08.5/0019/1997), and the Agencia Espanola de Cooperación Internacional (99CN0011).

The endogenous ADP-ribosylation of cytosolic proteins and the pattern of NAD degradation were analyzed in subcellular fractions of rat liver in order to investigate the modulation of these reactions by GTP-binding (G) proteins.

A simple and convenient procedure is described for the continuous fluorimetric assay of nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9) activity. It is based on the multifold increase in fluorescence emission intensity accompanying hydrolysis of a new fluorogenic substrate, ϵAp 2 ϵA (where ϵ = 1, N 6 -etheno), or of commercially available ϵNAD + and FAD, using potato nucleotide pyrophosphatase. The procedure is applicable to enzyme activity in tissue extracts, as well as to kinetic studies and screening of potential inhibitors. K m and V max / K m for the substrates, and K i values for various compounds, were evaluated. The most effective inhibitor of ϵAp 2 ϵA hydrolysis was the alternative substrate 8-bromo-NAD + ( K i = 7 μ M). The method is also useful for continuous assay of snake venom phosphodiesterase I-5′-exo-nuclease (EC 3.1.4.1). The preparation of ϵAp 2 ϵA is described, and spectral properties of the fluorogenic substrates listed. Both ϵAp 3 ϵA and ϵAp 4 ϵA were also shown to be fluorogenic substrates for nucleotide pyrophosphatase and phosphodiesterase I, and may be used for continuous fluorimetric assay of specific dinucleoside oligophosphatases.

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient [Fukui, S. et al . (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.

1. 1. Epiphyseal cartilage was separated into resting, ossifying and new bone regions. 2. 2. An acid phosphatase with sharp peaks at pH 3·0 and 6·0 was found in the soluble cartilage preparation. 3. 3. Activities of alkaline phosphatase, nucleotide pyrophosphatase, 5-nucleotidase and glycerophosphatase activities were higher in ossifying cartilage and new bone than in resting cartilage. 4. 4. It is suggested that phosphatases in the ossifying cartilage and new bone may play a very active role in preparing tissue for calcification.

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture-grade acidic fibroblast growth factor (FGF-1) correlates with a low-Mr contaminant. 1H-NMR analyses revealed EDTA in the total-volume fractions of a gel-filtration experiment, where all the inhibitory activity of the FGF-1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF-1 or the EDTA-containing fractions) was time-dependent, blocked by the substrate p-nitrophenyl-dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF-1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Publisher Summary This chapter highlights various experiments conducted to study biochemical characteristics of enzymes. Severo Ochoa was embarked on the isolation and purification of enzymes of the citric acid cycle in January 1946. By coupling aconitase to isocitric dehydrogenase, spectrophotometric assays, based on TPN reduction, took only a few minutes. Many ideas could be tested and discarded in the course of a day. After failing in a few attempts to crystallize the enzyme, Arthur Kornberg rebelled at continuing. Enzymes of astonishing specificity and catalytic potency linked the combustion of foodstuffs to the generation of ATP that made the cell grow and the muscle move. Severo Ochoa was embarked on the isolation and purification of enzymes of the citric acid cycle when Arthur came to his laboratory in January 1946. He suggested that Arthur work on aconitase, the enzyme that catalyzes the equilibrium. The purification of nucleotide pyrophosphatase from potatoes furnished a means for degrading TPN and distinguishing the location of its third phosphate.

When peripheral lymphocytes stimulated with phytohemagglutinin were permeabilized in vitro, (3H)dCTP acted as a precursor for DNA synthesis, but the formation of a compound soluble in organic solvents could also be demonstrated. The structure of the latter compound was studied analyzing the products formed after alkaline hydrolysis or an enzymatic treatment with nucleotide pyrophosphatase. Both treatments led to the formation of (3H)dCMP. When stimulated lymphocytes were labeled in vivo with (14C)glycerol before permeabilization and ulterior labeling with (3H)dCTP a double labeled compound was obtained. When this compound was submitted to alkaline hydrolysis, (3H)dCMP. and (l4C)glycerol-3-phosphate were obtained. It was concluded that the compound soluble in organic solvents was phosphatidyl-dCMP.

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2′-phosphate or 6-amino group, or both: 2′-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2′-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2′-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2′-O[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl)carbamoylethyl]-NAD (VI). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3′-phosphate isomer (Vb). The mixture was converted to Va via the 2′,3′-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3′-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.

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.