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Results for “Pyrophosphatases”

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PubMed1991

[Catalytic properties of inorganic pyrophosphatase in rat liver mitochondria].

Intact rat liver mitochondria possess a very low hydrolytic activity, if any, towards exogenous pyrophosphate. This activity can be unmasked by making mitochondria permeable to PPi by toluene treatment or by disrupting them with detergents or ultrasound, thus indicating that the active site of pyrophosphatase is localized in the matrix. The initial rates of PPi hydrolysis of toluene-permeabilized mitochondria and purified pyrophosphatase were found to depend, in a similar manner, on PPi and Mg2+ concentrations. The simplest model consistent with these data in both cases implies that the reaction proceeds via two pathways and requires MgPPi as substrate and at least one Mg2+ ion as activator. In the presence of 0.4 mM Mg2+ (physiological concentration) the inhibition constant for Ca2+ is 12 microM and the enzyme activity is no less than 50% of the maximal one. The data obtained suggest that the activity of pyrophosphatase in mitochondria is high enough to keep free PPi concentration at a level close to the equilibrium one.

Animals↗

PubMed1990

[A comparison of pyrophosphatase and peroxidase as markers in the immunoenzyme analysis of the tick-borne encephalitis virus and antibodies to it].

The authors compare the sensitivities of pyrophosphatase and peroxidase conjugates with immunoglobulins in assays of tick-borne encephalitis (TBE) virus antigens and anti-TBE antibodies in human sera. Pyrophosphatase conjugates are similar to peroxidase ones in their enzymic and immunochemical characteristics and are not inferior to them in sensitivity in titration of antibodies and superior in assays with antigens. A number of advantages of pyrophosphatase conjugates, i.e. a simple preparation procedure, stable substrate and enzymic reaction product, color range convenient for visual recording, etc., make these conjugates preferable vs. the peroxidase ones and recommend them for wide use at diagnostic laboratories.

Antibodies, Viral↗

PubMed1991

Effects of pyrophosphatase on dissolution of calcium pyrophosphate dihydrate crystals.

Understanding the dissolution mechanisms involved in calcium pyrophosphate dihydrate (CPPD) crystals may prove important for the development of therapy for CPPD arthropathy. We demonstrate that yeast pyrophosphatase effectively dissolved CPPD crystals in solutions. Maximum enzymatic dissolution of CPPD crystals was achieved at neutral pH and when the enzyme had access to the crystal surface. The enzymatic dissolution of CPPD crystals was highly dependent on ambient [Mg++] and [Ca++]. The stimulating effects of Mg++ on crystal dissolution in the presence of the enzyme is due to stimulation of pyrophosphatase activity and to enhanced direct release of pyrophosphate ions from the crystal surface. The inhibiting effect of Ca++ on crystal dissolution by the enzyme is mainly due to the suppression of pyrophosphatase activity.

Calcium↗

PubMed1979

[Interrelationship between metabolic and genetic regulation of alkaline phosphatase and poly- and pyrophosphatases].

The effects of orthophosphate and mutations in the regulatory genes of alkaline phosphatase on the activities of pyrophosphatase and polyphosphatase of E. coli were studied. It was shown that orthophosphate represses the synthesis of alkaline phosphatase as well as that of polyphosphatase without having any effect on pyrophosphatase. The genes phoR and phoS are involved in the formation of a repressory complex both for alkaline phosphatase and polyphosphatase. The gene phoT is probably involved in a partial repression of pyrophosphatase synthesis.

Acid Anhydride Hydrolases↗

PubMed1978

[Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast].

A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done. It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator. Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate. The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase. The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.

Catalysis↗

PubMed1986

Characterization of Haemophilus influenzae nucleotide pyrophosphatase. An enzyme of critical importance for growth of the organism.

A nucleotide pyrophosphatase isolated from Haemophilus influenzae was purified to electrophoretic homogeneity and characterized with respect to molecular weight, substrate specificity, pH profile, thermal stability, functional group involvement, and effectiveness of selective inhibition. The enzyme catalyzes the hydrolysis of NAD to NMN and AMP and appears located appropriately to facilitate the internalization of NAD needed to satisfy the V-factor growth requirement of the organism. In the processing of NAD and structurally related substrates, the enzyme exhibited negative cooperativity. Structural alterations in the purine moiety of these dinucleotide substrates had pronounced effects on the negative cooperativity of the enzyme. AMP, ADP, and several related nucleotides were observed to be effective substrate-competitive inhibitors of the enzyme. Several of the dinucleotides serving as substrates for the nucleotide pyrophosphatase were evaluated with respect to substituting for NAD in supporting growth of the organism. AMP and ADP inhibited growth of the organism when NAD served as V-factor, and this inhibition correlated well with the inhibitory effects of these nucleotides on the purified nucleotide pyrophosphatase.

Haemophilus influenzae↗

PubMed1986

Inorganic pyrophosphatase activity in a plaque calcifying microorganism: Bacterionema matruchotii.

The presence and activity of an alcaline pyrophosphatase of Bacterionema matruchotii, a Gram positive filamentous organism present in the bacterial plaque and calculus was studied. A strong enzymatic activity was demonstrated. The optimal conditions for this activity (alcaline pH, magnesium cofactor) have to be related to the pyrophosphatase conditions found in other calcifiable structures. The activity was shown in intact bacteria as well as in the culture medium devoid of micro-organisms. The strongest activity having been demonstrated in broken cells, it can be postulated that the enzyme is active inside the cell. The hypothesis of a contribution of Bacterionema matruchotii to the mineral phase formation is proposed; it is possible that the pyrophosphatase produced by this microorganism contributes to the mineralization process involved in dental calculus formation.

Actinomycetaceae↗

PubMed1979

[Effect of magnesium ions on the thermostability of inorganic pyrophosphatase from baker's yeast].

The interaction of magnesium ions with inorganic pyrophosphatase from baker's yeast was studied by means of heat denaturation. The heat inactivation of this enzyme is a biphasic process. The velocities in the initial range and in the subsequent slower part of inactivation are diminished with rising Mg2+ concentration in the inactivation assay. A model is proposed which describes this behavior. It is assumed that two enzyme conformations exist in equilibrium whose conversion rates correspond to the inactivation rate in its order of magnitude. The equilibrium is shifted by Mg2+. The two enzyme species differ in their Mg2+ binding behavior as evidenced by differences in the half-saturation constants and the cooperativity of the binding. The same conclusions are drawn from the fluorimetric measurement of denaturation of inorganic pyrophosphatase. Besides, an additional Mg2+ binding site is demonstrable, the saturation of which obviously leads to stabilisation of part of the enzyme structure without protecting it against loss of enzymatic activity. With the same method the labilizing effect of Zn2+ on the structure of the inorganic pyrophosphatase from baker's yeast was studied.

Drug Stability↗

PubMedDate not supplied

[Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide].

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.

Ascomycota↗

PubMedDate not supplied

[Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide].

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).

Ascomycota↗

PubMed1967

Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations.

1. Inhibition of the pyrophosphatase and orthophosphatase activities of human liver and small-intestinal alkaline-phosphatase preparations by different classes of inhibitors has been studied. 2. Each type of substrate, pyrophosphate or orthophosphate, is a competitive inhibitor of hydrolysis of the other type. 3. l-Phenylalanine is a non-competitive inhibitor of both types of activity of the intestinal preparation, but inhibits neither activity of the liver enzyme. Arsenate is a competitive inhibitor of both activities of both preparations. For a given inhibitor, the values of K(i) are independent of the type of substrate used when measurements are made at the same pH. 4. Mg(2+) ions activate orthophosphatase but inhibit pyrophosphatase, except in very low concentrations. 5. These results are compatible with the presence in each tissue preparation of a single enzyme with one type of active centre, possessing both orthophosphatase and pyrophosphatase activities.

Journal Article↗

PubMed1980

Calcium Inhibition of Pyrophosphatase in Crude Plant Extracts: IMPLICATION OF SOLUBLE CALCIUM IN C(4) PHOTOSYNTHESIS.

Assays of alkaline pyrophosphatase activity in crude plant extracts are inhibited by soluble calcium coextracted with the enzyme from leaf tissues. Calcium concentrations in most extracts are high enough to interfere seriously with the assay.All C(4) plants examined kept their soluble calcium and interference with pyrophosphatase activity at very low levels, whereas the C(3) plants covered the whole range of soluble calcium concentration. The hypothesis is advanced that low soluble calcium might be a prerequisite for high activity of pyrophosphatase and the C(4) pathway.

Journal Article↗

PubMed1998

3-phosphohistidine and 6-phospholysine are substrates of a 56-kDa inorganic pyrophosphatase from bovine liver.

A 56-kDa inorganic pyrophosphatase isolated from bovine liver hydrolyzed PPi, imidodiphosphate, 3-phosphohistidine, and 6-phospholysine at rates of 0.11, 0.44, 1.09, and 1.22 mumol/min/mg protein, respectively, in a reaction mixture containing 1 mM MgCl2 at pH 8.2. The hydrolysis of imidodiphosphate was influenced by various treatments in a different manner from that of N-phosphorylated amino acids, indicating that the pyrophosphatase has two different catalytic sites for imidodiphosphate and N-phosphorylated amino acids, respectively. Evidence for separate catalytic sites consists of the following findings: the activity on hydrolysis of imidodiphosphate gave a bell-shaped pH curve with a peak at pH 6.5, while the activity on hydrolysis of N-phosphorylated amino acids maintained a high level in the pH range between 6.0 and 9.5. One hundred micromolar p-chloromercuriphenyl sulfonate inhibited the hydrolysis of imidodiphosphate by 35% and did not inhibit that of N-phosphorylated amino acids. Two millimolar magnesium chloride repressed the hydrolysis of imidodiphosphate and had no inhibitory effect on the hydrolysis of N-phosphorylated amino acids. Moreover, methylenediphosphonic acid, an analog of imidodiphosphate, stimulated the hydrolysis of imidodiphosphate in the presence of MgCl2, while it potentiated the substrate inhibition on hydrolysis of N-phosphorylated amino acids.

4-Chloromercuribenzenesulfonate↗

PubMed1998

Purification, properties, and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis.

The hydrolysis of magnesium pyrophosphate by inorganic pyrophosphatase from Bacillus subtilis required its specific, time-dependent, and prior activation by Mn2+ ions. This was reversed when Mn2+ ions were removed with EDTA. Free Mn2+ ions were not required for catalysis. Pyrophosphatase purified to near homogeneity gave a single main band of apparent M(r) 36,000 by SDS-PAGE, but of M(r) 34,000 by matrix-assisted laser desorption ionization-mass spectrometry. The native enzyme equilibrated at pH 7 between three distinct molecular forms. Exposure to Mn2+ generated a catalytically active trimer of specific activity about 5000 mumol pyrophosphate hydrolyzed/min/mg protein. Exposure to EDTA generated two catalytically inactive forms, a dimer at low ionic strength and a separate form, of uncharacterized multimeric nature, at molar concentrations of Na2SO4 or Li2SO4. The latter form was an intermediate in the dimer-trimer transition caused by addition or removal of manganese ions. Mn2+ reacted with this "intermediate" form, apparently by reversible association with two noninteracting binding sites of Kd approximately 0.005 and 0.35 microM, respectively. The properties of this enzyme may account in part for the unusual manganese requirements of B. subtilis and related species.

Bacillus subtilis↗

PubMed1994

Inorganic pyrophosphatase-based detection systems. I. Detection and enumeration of cells.

A novel technique, useful for detection and enumeration of both pro- and eukaryotic cells, has been developed. The method relies on the detection by a very sensitive assay of an enzyme, the inorganic pyrophosphatase, which is constitutively expressed in all cells. This technique has been used to demonstrate detection and enumeration of both Escherichia coli and Saccharomyces cerevisiae cells. Cells were incubated, for a chosen time period and at a specific temperature, in a specific lysis buffer which contained both lysing activity and inorganic pyrophosphate. The activity of the inorganic pyrophosphatase released from the cells after lysis was determined by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509). The amount of PPi hydrolyzed was proportional to the logarithm of the cell number. The sensitivity of the assay was dependent on several factors, such as cell type, incubation time, and incubation temperature. E. coli cells at concentrations of 1 x 10(4) cells/ml could be detected in an assay performed at 58 degrees C for 10 min and S. cerevisiae cells at concentrations of 2 x 10(4) cells/ml at 49 degrees C within 10 min. The assay could be designed both as a present/absent test, e.g., for determination of bacteriuria, or as a quantitative assay. Determination and enumeration of all types of cells is in principle possible by the appropriate design of the experimental parameters. Possible applications for the approach in a wide variety of areas, such as fundamental and medical sciences, clinical laboratories, food and dairy industry, pharmaceutical industry, water sanitary plants, and biomass determination in general, are discussed.

Escherichia coli↗

PubMed1995

Molecular cloning, characterization and expression analysis of isoforms encoding tonoplast-bound proton-translocating inorganic pyrophosphatase in tobacco.

The tonoplast-bound proton-translocating inorganic pyrophosphatase (V-type PPase) and the proton ATPase (V-type ATPase) are electrogenic proton pumps guaranteeing the energization of solute transport across the tonoplast. Using an Arabidopsis thaliana PCR cDNA fragment corresponding to clone ATAVP3 we have isolated 24 cDNA clones encoding tonoplast-bound inorganic pyrophosphatase of tobacco. Based on restriction analysis the cDNA clones could be grouped into three different classes. The complete nucleotide sequence of one member of each class (TVP5, TVP9 and TVP31) was determined. The cDNA clones contain an uninterrupted open reading frame of 2292 bp (TVP5), 2295 bp (TVP9) and 2298 bp (TVP31) coding for polypeptides of 764, 765 and 766 amino acids, respectively. The nucleotide sequence of the different clones is highly homologous within the coding region (79-89% identity) but differs strongly in the untranslated regions. The individual classes are encoded by single- or low-copy genes as judged from genomic gel blot experiments using 3'-specific probes. RNA analysis revealed that the accumulation of the specific transcripts is differentially regulated during leaf development.

Amino Acid Sequence↗

PubMedDate not supplied

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).

Amino Acid Sequence↗

PubMed1983

Yeast inorganic pyrophosphatase substrate recognition.

Monodentate Co(NH3)5PPi was determined not to be a substrate for yeast inorganic pyrophosphatase while P1,P2-bidentate Co(NH3)4PPi was turned over by the enzyme at a rate of 7.5 min-1. A kinetic analysis of the substrate activities of the P1,P2-bidentate complexes, Co(en)2PPi, Cr(NH3)4PPi, Cr(H2O)(NH3)3PPi, and Cr(H2O)2(NH3)2PPi, and Cr(H2O)4PPi was carried out in order to access the potential role of the metal-water ligands in productive binding. While substitution of the H2O ligands with NH3 ligands had a minimal affect on the Km for Mg2+, the binding affinity of the complexes decreased with an increasing NH3/H2O ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)2PPi complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH3)4PPi complex. The substrate activities of beta, gamma-bidentate Co(NH3)4PPPi and alpha, beta, gamma-tridentate Co(NH3)3PPP with pyrophosphatase were also tested. While both complexes were shown to bind tightly to the Mg2+-activated enzyme neither was hydrolyzed. On the other hand, in the presence of the Zn2+-activated enzyme the tridentate complex was turned over at a rate of 0.17 min-1 while the bidentate complex remained inert to hydrolysis.

Binding Sites↗