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PubMed1981

Purification and some properties of inorganic pyrophosphatase from Streptococcus faecalis.

Inorganic pyrophosphatase [EC 3.6.1.1] from Streptococcus faecalis ATCC 8043 was purified to homogeneity as judged by slab gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The purification consisted of the following steps: streptomycin sulfate precipitation, (NH4)2SO4 treatment in a Sepharose CL-4B column, DEAE-Sepharose CL-6B chromatography, gel filtration on Ultrogel AcA 34, and preparative slab gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight and slab gel electrophoresis in the presence of sodium dodecyl sulfate to study the subunit molecular weight. The enzyme appeared to be composed of four subunits with molecular weights of approximately 32,500. The molecular weight of the native enzyme was about 128,000. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Ca2+ supported 24% and 5.6% of the activity observed with Mg2+, respectively. 2,4,6-Trinitrobenzene sulfonic acid inhibited the reductants containing SH-groups activated the enzyme suggesting that lysine and cysteine have essential roles in the enzyme.

Cations, Divalent↗
PubMed1981

Sulfohydrolytic degradation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and adenosine 5'-phosphosulfate (APS) by enzymes of a nucleotide pyrophosphatase nature.

The sulfohydrolytic activity to degrade active sulfate (3'-phosphoadenosine 5'-phosphosulfate, PAPS) and its precursor, APS (adenosine 5'-phosphosulfate), with a pH optimum at 9.5 was found to be widely distributed in various tissues of rats. In the liver, the activity was located in plasma membranes and endoplasmic reticula. Triton X-100 solubilized rough and smooth endoplasmic reticula gave two peaks of the activity on gel filtration, both of which had nucleotide pyrophosphatase activities, hydrolyzing the pyrophosphate linkages of ATP, NAD, and UDP-Glc, and the phosphodiester linkage of PNTP (p-nitrophenyl-thymidine 5'-monophosphate) besides PAPS and APS.

Adenine Nucleotides↗
PubMed1991

Inorganic pyrophosphate, nucleoside triphosphate pyrophosphatase, and cartilage fragments in normal human synovial fluid.

The association between calcium pyrophosphate dihydrate (CPPD) crystal deposition and ageing is unexplained. Levels of inorganic pyrophosphate (PPi) and activity of nucleoside triphosphate pyrophosphatase (NTPP) were estimated in synovial fluid from 91 asymptomatic normal knees, including five with isolated CPPD crystal deposition (70 subjects; median age 47, range 22-83 years). In non-crystal fluids, PPi levels and NTPP activities were low (median, interquartile range: 9.5, 7.6-12.3 microM, and 1, 0.4-2 microM/30 min/mg protein, respectively: n = 50) and a generalized increase with age was not demonstrated. Higher values for PPi and NTPP in all five subjects with isolated CPPD deposition supports involvement of altered PPi metabolism, unrelated to ageing per se, in crystal formation. Presumed cartilage fragments were identified by morphological characteristics in 70% of normal fluids: metachromatic staining and electron microscopy, undertaken in a limited number, supported identification of such particles as cartilage. This finding supports dynamic turnover of healthy matrix, and questions the usefulness of identification, without quantification, cartilage fragments as a measure of cartilage damage.

Adult↗
PubMed2003

Decrease in serum nucleotide pyrophosphatase activity in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a prototype of a group of rheumatic diseases referred to as spondyloarthropathy. AS patients show marked ectopic ossification in the spine, occasionally resulting in so-called bamboo spine. Although a strong association with HLA-B27 has been reported, its aetiology remains undetermined. Another rheumatic disease, ossification of the posterior longitudinal ligament of the spine (OPLL), demonstrates ectopic ossification of the spinal ligaments very similar to that of AS. Recently, nucleotide pyrophosphatase (NPPS) was implicated in the aetiology of OPLL: an Npps mutation was found to cause OPLL in mice, and an association between a polymorphism of the human NPPS gene and OPLL was identified. The clinical similarities between AS and OPLL led us to hypothesize that NPPS may also be implicated in the aetiology of AS. To elucidate the role of NPPS in the pathogenesis of AS, we examined serum NPPS activity and the possible association of the NPPS gene with AS. METHODS: Forty-four Japanese patients with AS, 43 patients with OPLL, and age- and sex-matched normal volunteers took part in this study. We determined serum NPPS activity using high-performance liquid chromatography and examined the association between AS and NPPS using single nucleotide polymorphisms (SNPs) of the NPPS gene. RESULTS: Serum NPPS activity in AS patients was significantly decreased compared with the controls (P < 0.0001). However, there was no association between AS and NPPS gene SNPs. CONCLUSION: NPPS is implicated in the pathogenesis of AS.

Adult↗
PubMed2003

Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions.

Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H-labeled and unlabeled nucleotides and adenosine, direct transfer of gamma-terminal phosphate from [gamma-32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate-specificity and competitive studies, we identified three purine-inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5'-nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP-generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40-70 micro mol/l. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.

Adenosine Deaminase↗
PubMed2004

Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase).

Adverse drug reactions to azathioprine (AZA), the pro-drug of 6-mercaptopurine (6-MP), occur in 15% to 28% of patients and the majority are not explained by thiopurine methyltransferase (TPMT) deficiency. Inosine triphosphate pyrophosphatase (ITPase) deficiency results in the benign accumulation of the inosine nucleotide ITP. 6-MP is activated through a 6-thio-IMP intermediate and, in ITPase deficient patients, potentially toxic 6-thio-ITP is predicted to accumulate. The association between polymorphism in the ITPA gene and adverse drug reactions to AZA therapy was studied in patients treated for inflammatory bowel disease. Sixty-two patients with inflammatory bowel disease suffering adverse drug reactions to AZA therapy were genotyped for ITPA 94C>A and IVS2 + 21A>C polymorphisms, and TPMT*3A, *3C, *2 polymorphisms. Genotype frequencies were compared to a consecutive series of 68 controls treated with AZA for a minimum of 3 months without adverse effect. The ITPA 94C>A deficiency-associated allele was significantly associated with adverse drug reactions [odds ratio (OR) 4.2, 95% confidence interval (CI) 1.6-11.5, P = 0.0034]. Significant associations were found for flu-like symptoms (OR 4.7, 95% CI 1.2-18.1, P = 0.0308), rash (OR 10.3, 95% CI 4.7-62.9, P = 0.0213) and pancreatitis (OR 6.2,CI 1.1-32.6, P = 0.0485). Overall, heterozygous TPMT genotypes did not predict adverse drug reactions but were significantly associated with a subgroup of patients experiencing nausea and vomiting as the predominant adverse reaction to AZA therapy (OR 5.5, 95% CI 1.4-21.3, P = 0.0206). Polymorphism in the ITPA gene predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6-thioguanine, should be considered for AZA-intolerant patients with ITPase deficiency.

Adolescent↗
PubMed2005

The extent of ossification of posterior longitudinal ligament of the spine associated with nucleotide pyrophosphatase gene and leptin receptor gene polymorphisms.

STUDY DESIGN: A case-control study using radiograph findings and the PCR assay with regard to the susceptibility and the severity of ossification of posterior longitudinal ligament of the spine (OPLL). OBJECTIVE: To analyze whether polymorphisms of the nucleotide pyrophosphatase (NPPS) gene and the leptin receptor gene predispose to an increased frequency and severity of OPLL. SUMMARY OF BACKGROUND DATA: The NPPS gene is responsible for ectopic ossification in the ttw mouse, an animal model for OPLL. The Zucker fatty rat, another animal model for OPLL, has a missense mutation in the leptin receptor gene. METHODS: Analysis of 172 OPLL patients and 93 non-OPLL controls was performed. Radiographs of the cervical, thoracic and lumber spine were analyzed to determine whether OPLL was present and to what degree. Genomic DNA was extracted from all participants. Polymorphisms of the NPPS gene and the leptin receptor gene were analyzed using the PCR assay. The association of the polymorphisms with the development and extent of OPLL were statistically evaluated. RESULTS: No significant association was found between the polymorphisms and the existence of OPLL in both the NPPS and the leptin receptor genes. However, the IVS20-11delT variant in the NPPS gene and the A861G variant in the leptin receptor gene were more frequent in patients with OPLL in the thoracic spine compared with patients whose OPLL was restricted to the cervical spine. CONCLUSION: The present results suggest that the IVS20-11delT variant of the NPPS gene and the A861G variant of the leptin receptor gene are associated with more extensive OPLL, but not with the frequency with which it occurs.

Adenine↗
PubMed1987

Inorganic pyrophosphatase activity in cell-free extracts of Ureaplasma urealyticum.

Cell-free extracts of Ureaplasma urealyticum strains Pi and T960 (CX8) (serovars 6 and 8, respectively) metabolized inorganic pyrophosphate (PPi). The inorganic pyrophosphatase (PPase) activity was greatest with Mg2+ as cofactor, but Mn2+ acted as a poor substitute. The PPases of the two serovars differed electrophoretically. Although the highest PPase activity was obtained using PPi as substrate, the enzyme could also utilize to a lesser degree both tripolyphosphate and trimetaphosphate. No activity was observed against beta-glycerophosphate, naphthyl phosphates, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, thiamin pyrophosphate, phosphoribosylpyrophosphate, ADP or ATP. Acid- and alkaline-phosphatase activities were observed with naphthyl phosphates as substrates, but they did not have the same electrophoretic mobility on gels as the PPase activity. U. urealyticum PPase was inhibited by oxidized glutathione, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, p-chloromercuribenzoic acid, Mn2+, Zn2+ and Ca2+. Neither reduced glutathione, L-cysteine nor Co2+ enhanced activity. PPi can act as a substrate or regulator of certain metabolic reactions, and PPi metabolism can function in bacterial bioenergetics; its role in ureaplasmas is presently unclear.

Cations, Divalent↗
PubMed2000

AVP2, a sequence-divergent, K(+)-insensitive H(+)-translocating inorganic pyrophosphatase from Arabidopsis.

Plant vacuolar H(+)-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775-1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K(+)-stimulated H(+) translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K(+)-insensitive, Ca(2+)-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H(+) translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.

Amino Acid Sequence↗
PubMed2000

Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms.

dUTP pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40 A resolution in a hexagonal crystal form and at 2.3 A resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5 A resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the alpha-, beta- and gamma-phosphates. The enzyme utilizes adaptive recognition to bind the alpha- and beta-phosphates. In particular, the alpha-beta phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.

Animals↗
PubMed2003

Overproduction, crystallization and preliminary diffraction data of ADP-ribose pyrophosphatase from Thermus thermophilus HB8.

An ADP-ribose pyrophosphatase from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography showed the protein to be in a dimeric state. This protein catalyses the Mg(2+)- or Zn(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose-5'-phosphate. It was crystallized in the absence and the presence of ADP-ribose by the hanging-drop vapour-diffusion method. Complete data sets were collected to 1.50 A resolution from the apo form using synchrotron radiation and to 2.0 A resolution from the complexed form. Both crystals belong to space group P3(1)21 or P3(2)21 and contain one molecule in the asymmetric unit.

Adenosine Diphosphate Ribose↗
PubMed2004

Production and preliminary analysis of perdeuterated yeast inorganic pyrophosphatase crystals suitable for neutron diffraction.

Yeast inorganic pyrophosphatase (Y-PPase) is a model system for studying phosphoryl-transfer reactions catalysed by multiple metal ions. To understand the process requires knowledge of the positions of the protons in the active site, which can be best achieved by neutron diffraction analysis. In order to reduce the hydrogen incoherent-scattering background and to improve the signal-to-noise ratio of the neutron reflections, deuterated protein was produced. Deuterated protein 96% enriched with deuterium was produced in high yield and crystals as large as 2 mm on one side were obtained. These crystals have unit-cell parameters a = 58.9, b = 103.9, c = 117.0 A, alpha = beta = gamma = 90 degrees at 273 K and diffract neutrons to resolutions of 2.5-3 A. The X-ray structure of the perdeuterated protein has also been refined at 273 K to 1.9 A resolution.

Crystallization↗
PubMed2004

Crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 71.7, b = 86.5, c = 92.5 A, alpha = beta = gamma = 90 degrees. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A resolution in-house.

Crystallization↗
PubMed1995

Inhibition of streptococcal growth, F-ATPase and pyrophosphatase by diphosphonates.

1-Hydroxyethane-1,1-diphosphonate (EHDP) and a variety of other diphosphonates, and also pyrophosphate, at millimolar levels were found to inhibit the growth of Streptococcus mutans GS-5. Inhibition appeared to be due mainly to chelation of Mg2+ and could be readily reversed through addition of Mg2+, or less effectively, by other divalent cations. The trianionic forms of the diphosphonates or pyrophosphate were more effective inhibitors than the dianionic forms. Diphosphonates and pyrophosphate did not inhibit glycolysis by S. mutans, assayed in terms of glucose utilization, or arginolysis by Streptococcus rattus FA-1, assayed in terms of ammonia production. However, they did act as buffers to moderate pH changes. Diphosphonates also were inhibitors of the F-ATPase of S. mutans by complex mechanisms only partly reversible with divalent cations. They also were inhibitors of the pyrophosphatase of the organism. However, intact cells were impermeable to the compounds, and inhibition of cytoplasmic or membrane enzymes did not appear to be involved in growth inhibition.

Cell Membrane Permeability↗
PubMed1979

Characterization of the membrane-bound inorganic pyrophosphatase in Rhodospirillum rubrum.

The membrane-bound inorganic pyrophosphatase (EC 3.6.1.1) from Rhodospirillum rubrum has been investigated with the tools of enzyme kinetics, and with two amino acid reagents, N-ethyl-maleimide (MalNET) and 4-chloro-7-nitrobenzofurazan (Nbf-Cl). 1. The concentration of the true substrate, MgPPi, was varied with constant concentrations of free Mg2+ or PPi. It was observed that Mg2+ acted as an activator. 2. Heat inactivation of the enzyme at 62 degrees C was slowed down in the presence of Mg2+. 3. MalNET and Nbf-Cl bind to the enzyme, and inhibit its activity. The effect of both reagents is dependent on the temperature. 4. A model is proposed where the 1:1 complex of Mg2+:PPi acts as substrate and Mg2+ interacts directly with the enzyme as an activator. PPi can bind to the enzyme, but is not hydrolyzed in the uncomplexed form.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Nucleotide pyrophosphatase from potato tubers. Purification and properties.

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800-fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr for the subunit of 74000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3-8.7. The enzyme is a glycoprotein. The purified enzyme exhibits the previously reported activities versus pyrophosphate linkages located at either the 5'-OH or 3'-OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3'- and 5'-phosphates, p-nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2',3'-phosphates. However, the relative rates of activity towards these substrates, and the corresponding V values, differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5')p2(5')A-A(5')p5(5')A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3',5'-phosphates. The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5'-terminal m7G(5')p3(5')Gm of intact reovirus mRNA. NAD+ was found to be the most effective inhibitor of enzyme activity versus thymidine 5'-p-nitrophenylphosphate, with a Ki = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2',3'-cAMP and thymidine 3'-p-nitrophenylphosphate inhibit hydrolysis of NAD+ noncompetitively and vice-versa.

Chromatography, Thin Layer↗
PubMed1990

The quaternary structure of Escherichia coli inorganic pyrophosphatase is essential for phosphorylation.

The hexameric inorganic pyrophosphatase (PPase) is irreversibly inactivated by phosphoric acid monoesters. The inactivation kinetics are consistent with the formation of a dissociable complex of the phosphoric acid monoester with the enzyme, followed by phosphorylation of the dicarboxylic amino acid of its active site. PPi and its analogues, binding at the regulatory site, release the inhibitor from the active site and thus restore PPase activity. Chemically identical subunits in the hexameric PPase interact, promoting their cooperativity in a reaction with phosphoric acid monoesters. The trimeric and monomeric PPase, exhibiting full catalytic activity, form a dissociable complex with the phosphoric acid monoesters but, in contrast to the hexameric PPase, do not form a covalent bond with them. This indicates that the native hexameric structure is essential for the irreversible inactivation of Escherichia coli PPase by phosphoric acid monoesters. Possible nontraditional pathways for activity regulation of PPase are discussed.

Binding Sites↗