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Results for “Pyrophosphatases”

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PubMed2002

Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase.

As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H(+) pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H(+)-ATPase, sensitive to concanamycin A and bafilomycin A(1). The other is a H(+)-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K(+). The operation of the H(+)-pyrophosphatase was dependent on the presence of a Mg(2+)-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H(+)-ATPase and a H(+)-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.

Animals↗

PubMed1997

Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies.

Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15 mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.

Amino Acid Sequence↗

PubMed1982

Role of glutathione in the regulation of inorganic pyrophosphatase activity in Streptococcus faecalis.

When the thiol-oxidizing agent diamide was added to a batch culture of Streptococcus faecalis during exponential growth, the specific activity of inorganic pyrophosphatase (EC 3.6.1.1) decreased to the level observed in the stationary phase. The effect of diamide was completely reversed by reduced glutathione. Furthermore, the ratio of reduced to oxidized glutathione and the specific activity of inorganic pyrophosphatase changed in a very similar fashion during batch culture. These findings, together with our earlier results, suggest that the activity of inorganic pyrophosphatase in S. faecalis is regulated by glutathione via the ratio of reduced to oxidized glutathione.

Cysteine↗

PubMed1980

Reconstitution of biological molecular generators of electric current. Inorganic pyrophosphatase.

Proteoliposomes have been reconstituted from soy-bean phospholipids (asolectin) and inorganic pyrophosphatase isolated from Rhodospirillum rubrum chromatophores. In the presence of Mg2+ ions, pyrophosphatase proteoliposomes were incorporated into a phospholipid-impregnated Teflon filter separating two solutions of an identical electrolyte content. Addition of inorganic pyrophosphate to the same compartment as proteoliposomes was found to induce generation of an electric potential difference between the two filter-separated compartments, the proteoliposomes-containing compartment being negatively charged. An electric potential difference of 15 mV and a current of 20 pA were observed. The electrogenic effect required Mg2+ and proved to be sensitive to fluoride, an inorganic pyrophosphatase inhibitor. Treatment with 10 microM N,N'-dicyclohexylcarbodiimide for several minutes was without influence upon pyrophosphate-induced membrane potential generation. Similar results were obtained in experiments with a proteoliposome suspension and a penetrating anion, tetraphenyl borate, which is a probe for membrane potential. The obtained data are discussed in connection with the results of studies on other enzymes as molecular generators of electric current.

Bacterial Chromatophores↗

PubMed1992

Cloning and sequencing of the gene for the cytoplasmic inorganic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. Two degenerate oligonucleotide probes, synthesized according to the N-terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries. The DNA-derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size. The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms.

Amino Acid Sequence↗

PubMed1993

Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae.

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.

Calcium↗

PubMed1963

REPRESSIBLE ACID PHOSPHOMONOESTERASE AND CONSTITUTIVE PYROPHOSPHATASE OF SACCHAROMYCES MELLIS.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Repressible acid phosphomonoesterase and constitutive pyrophosphatase of Saccharomyces mellis. J. Bacteriol. 86:805-813. 1963.-Saccharomyces mellis produces a nonspecific acid phosphomonoesterase (pH optimum of 5.5 to 6.0) when grown in a medium devoid of phosphate. Only minimal amounts of this enzyme are present in cells harvested from media containing phosphate. The enzyme requires no cofactors. It is inhibited by such anions as phosphate, arsenate, molybdate, and borate. S. mellis also contains an inorganic pyrophosphatase with a pH optimum of 7.5. The properties of this enzyme are distinctly different from those of the acid phosphomonoesterase. The pyrophosphatase requires Mg(++) for activity. This enzyme is constitutive, since it is present in cells regardless of the phosphate content of the growth medium.

Acid Phosphatase↗

PubMedDate not supplied

Protective role of trehalose in thermal denaturation of yeast pyrophosphatase.

Trehalose, a disaccharide of glucose, is accumulated in yeast cytosol when this organism is submitted to a stress condition. Recently it was shown that the level of trehalose increase up to 15 times when yeast cells are submitted to heat shock (De Virgilio et al., 1991). In this report we give evidence how trehalose may play an important role on the stress-survival of yeasts when submitted to a heat shock. We show that 1.5 M trehalose increases 13-fold the half-time for thermal inactivation (t0.5) of yeast cytosolic pyrophosphatase at 50 degrees C. This thermal protection conferred by trehalose is dose-dependent, after 10 min at 50 degrees C, a condition which inactivated pyrophosphatase, the presence of 2 M trehalose preserves 95% of total activity. Other carbohydrates were tested but were not so effective as trehalose. The presence of trehalose at high concentrations in the reaction medium at 35 degrees C inhibits pyrophosphatase activity. This inhibition is less effective at 50 degrees C suggesting that under this condition the enzyme is temperature-protected and active.

Disaccharides↗

PubMedDate not supplied

Trehalose protects yeast pyrophosphatase against structural and functional damage induced by guanidinium chloride.

Trehalose is accumulated at very high concentrations in yeasts when this organism is submitted to a stress condition. This report approaches the question on the protective effect of trehalose and its degradation product, glucose, against structural and functional damage promoted by guanidinium on yeast cytosolic pyrophosphatase. Here it is shown that both, 1 M trehalose or 2 M glucose, are able to attenuate at almost the same extent the conformational changes promoted by guanidinium chloride on the pyrophosphatase structure. On the other hand while 1 M trehalose increases 3.8 times the Ki (from 0.15 to 0.57 M) for guanidinium chloride inhibition of pyrophosphatase activity, 2 M glucose did not even duplicate this parameter (from 0.15 to 0.25 M). These data support evidences for a functional reason for the accumulation by yeasts of trehalose, and not other compound, during stress conditions.

Cytosol↗

PubMed2000

Active site interactions in oligomeric structures of inorganic pyrophosphatases.

Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers.

Binding Sites↗

PubMed1992

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.

Animals↗

PubMed1990

Purification of inorganic pyrophosphatase from rat salivary glands.

Inorganic pyrophosphatase [EC 3.6.1.1] isolated from rat sublingual and submandibular glands was purified 2300-fold and 2600-fold, respectively. The purified enzymatic preparations separated on electrophoresis into two protein bands, of which only one showed the pyrophosphatase activity. Inorganic pyrophosphatase from rat salivary glands is a monomeric anionic protein, its isoelectric point is 5.42 and 4.90 for the sublingual and submandibular glands, respectively.

Animals↗

PubMed1988

Kinetic characterization of a thermostable inorganic pyrophosphatase from Thermus thermophilus.

A kinetic characterization was performed for inorganic pyrophosphatase from Thermus thermophilus. The optimum activity with Mg2+ as the activating metal ion lies in a pH range between 8.3 and 9.5. The hydrolysis of inorganic pyrophosphate is also activated by Zn2+, Mn2+, and Co2+. Calcium ions are not activating at all. Tripolyphosphate is another substrate hydrolyzed by the enzyme but only with Zn2+ as the activating metal ion. Other potential substrates like ATP and cyclic metaphosphates are not hydrolyzed even at high enzyme concentrations. Computer modelling of kinetic data obtained from activity measurements with different total magnesium ion and pyrophosphate concentrations confirms a kinetic model which was shown to be valid also for inorganic pyrophosphatases from other microbial sources. The corresponding parameter values are given. The inorganic pyrophosphatase from T. thermophilus exhibits extremely high thermostability which is decreased by addition of EDTA indicating a stabilizing effect of divalent metal ions.

Enzyme Stability↗

PubMed1986

[Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. I. Phosphorylation and enzyme activation induced by ATP].

ATP phosphorylates the regulatory center of E. coli inorganic pyrophosphatase with the resultant 1,5-fold increase in the activity of the enzyme. The maximal incorporation of the ATP gamma-group into pyrophosphatase is 3 moles per mole of the protein. Pi likewise phosphorylates the enzyme regulatory center and lowers the pyrophosphatase activity by 10-15%. The ATP- and Pi-mediated phosphorylation processes are interrelated; ATP prevents phosphorylation by Pi and brings about rapid dephosphorylation of Pi-modified protein.

Adenosine Triphosphate↗

PubMed1986

[Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. II. Identification of the types of chemical bonds between phosphate and the enzyme].

A bond formed by phosphate with Pi-phosphorylated pyrophosphatase from E. coli was found to be labile in acidic and alkaline media and to be rapidly cleaved by hydroxylamine at neutral pH. N-Methylhydroxylamine modifies also activated carboxyl groups of the enzyme. Interaction of inorganic pyrophosphatase with ATP produces an alkali-resistant phosphoamide bond. A phosphorylated amino acid, identified as phosphohistidine, was isolated from the alkaline hydrolyzate of the ATP-phosphorylated pyrophosphatase.

Adenosine Triphosphate↗

PubMed1980

[Affinity labeling of inorganic pyrophosphatase from yeast by methylphosphate].

Yeast inorganic pyrophosphatase is specifically and irreversibly inactivated by methylphosphate. The high rate of inhibition, the protective effect of the substrate, the strict correlation between the degree of inhibition and the amount of the protein-bound reagent and the effect of saturation of the enzyme with methylphosphate provide evidence in favour of the reaction in the active center. Modification of two chemically identical enzyme subunits proceeds at different rates and results in a formation of phosphorylated subunits with different stability of the phosphate bond, which is indicative of the mutual effects of the pyrophosphatase subunits. The reaction between the modified enzyme and hydroxylamine suggests that the interaction between pyrophosphatase and methylphosphate entails modification of the carboxylic groups of two active centers, resulting in a formation of the acylphosphate bonds.

Affinity Labels↗

PubMed1981

[Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase].

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.

Binding Sites↗

PubMed1982

[Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast].

Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH. The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form. The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center. The enzyme binding to PPi is strongly stabilized by fluoride. The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule.

Hydrogen-Ion Concentration↗