Zn2+-Dependent Acid Inorganic Pyrophosphatase Activity as Related to Other Pyrophosphatases in Oryza sativa

Acid inorganic pyrophosphatase on the one hand, and Mg²⁺-dependent alkaline inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase on the other hand showed opposite trends in their activities in rice (Oryza sativa L. cv. Ratna) seedlings grown in dark and sun. The opposite trends in their activities were also noted in rice seedlings grown from gamma-irradiated seeds and in detached rice leaves floated on water in dark. The ratios of Mg²⁺ dependent alkaline inorganic pyrophosphatase/acid inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase/acid inorganic pyrophosphatase changed significantly in response to the above physical treatments, but the ratio of Mg²⁺ dependent alkaline inorganic pyrophosphatase/Zn²⁺ dependent acid inorganic pyrophosphatase remained relatively stable. The conclusion is that Zn²⁺-dependent acid inorganic pyrophosphatase activity is the same as that of Mg²⁺-dependent alkaline inorganic pyrophosphatase and is different from that of acid inorganic pyrophosphatase, which requires no metal ion for activity. The acid and alkaline inorganic pyrophosphatase activities are due to separate enzyme proteins.     

Inhibition of Inorganic Pyrophosphatase from Escherichia coli with Inorganic Phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (P _i) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with P _i, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when P _i is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Antioxidant Effect of Gly-Gly-His on Cu(II)-catalyzed Autoxidation and Photosensitized Oxidation of Lipids

The level of alkaline inorganic pyrophosphatase (EC 3.6.1.1) in different plant leaves varies to a great extent. Out of twenty two families comprising of fifty one species of plant leaves studied, a very high level of activity was found in leaves of Amarantus blitum and Amarantus gangeticus, which belong to the Amarantaceae family. A possible role of this enzyme in the C₄ pathway of photosynthesis has been discussed. The purified enzyme from the leaf of Amarantus blitum was found to have optimum pH at 9.0, with magnesium being an absolute requirement. Excess substrate inhibits the enzyme activity.     

Alkaline inorganic pyrophosphatase and starch synthesis in amyloplasts

The aim of this work was to see if amyloplasts contained inorganic pyrophosphatase. Alkaline pyrophosphatase activity, largely dependant upon MgCl₂ but not affected by 100 M ammonium molybdate or 60–100 mM KCl, was demonstrated in exracts of developing and mature clubs of the spadix of Arum maculatum L. and of suspension cultures of Glycine max L., but not in extracts of the developing bulb of Allium cepa L. The maximum catalytic activity of alkaline pyrophosphatase in the above tissues showed a positive correlation with starch synthesis, and in the first two tissues was shown to exceed the activity of ADPglucose pyrophosphorylase. Of the alkaline pyrophosphatase activity in lysates of protoplasts of suspension cultures of Glycine max, 57% was latent. Density-gradient centrifugation of these lysates showed a close correlation between the distribution of alkaline pyrophosphatase and the plastid marker, nitrite reductase. It is suggested that much, if not all, of the alkaline pyrophosphatase in suspension cultures of Glycine max is located in the plastids.Abbreviations PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate     

A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

An alkaline inorganic pyrophosphatase is found in association with isolated spinach chloroplast membranes. The enzyme is not removed from chloroplasts by repeated washings in an iso-osmotic medium. Suspension of the chloroplasts in hyper- or hypo-osmotic medium, however, results in the loss of pyrophosphatase activity in the chloroplasts. Fractionation of an isolated chloroplast suspension by differential centrifugation yields chloroplast fractions possessing high levels of alkaline pyrophosphatase activity but practically devoid of cytoplasmic acid pyrophosphatase.The alkaline pyrophosphatase exhibits a pH optimum of 8.2–8.5. In addition, there is an absolute requirement for Mg²⁺, with maximal rates of pyrophosphate hydrolysis occurring at $\text{Mg}{}^{\mathrm{2+}}\text{PP}{}_{\mathrm{i}}$ ratios greater than 2. From these findings the actual substrate for the enzyme is evidently Mg₂P₂O₇⁰ with pyrophosphate (P₂O₇^4?) acting as a potent inhibitor.The enzyme is inhibited by high concentrations of ATP (>3 mm), but increasing the concentration of Mg²⁺ effectively relieves this inhibition. At lower ATP concentrations, however, there is a stimulation of pyrophosphatase activity.The rate of hydrolysis of pyrophosphate by isolated chloroplasts is not affected by methylamine, 4′-deoxyphlorizin, and light. The possible role of this enzyme in photophosphorylation is discussed.     

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg² was required foractivity, the activity with Mn², Co² and Zn² was 48, 48 and 182% of the activity observed with Mg², respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent K_m for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.     

A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an _{2 2} oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg²⁺ was required for activity. The pH optimum was 8 at 1 mM PP _i and 5 mM Mg²⁺. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent K _m for PP _i of 0.1 mM in the presence of 5 mM Mg²⁺. The V _max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K _cat of 1400 per second.The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedyre were applied.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Distribution and properties of a ‘lectin- like’ glycoprotein from leaves and stems of<Emphasis Type="Italic">Dolichos biflorus</Emphasis>: Implications on the role of lectins in plants

Cardiotoxin II of the Indian cobra(Naja naja) contains approximately four Mg²⁺ per mol. Complete demetallation of the toxin is achieved by three cycles of treatment with ethylenediamine tetraacetate and gel filtration. Reconstitution of toxin by treatment of the apo-protein with Mg²⁺ restores metal content and inorganic pyrophosphatase activity only to the extent of two atoms/mol and 65%, respectively. Use of Mg (II)-EDTA in the reconstitution experiment yields restoration of half the original enzyme activity. Mg²⁺ is required for the inorganic pyrophosphatase action of the toxin. A definitive statement on the non-essentiality of Mg²⁺ for the lethal toxicity of the toxin is not possible at present, although experimental observations indicate that demetallated toxin is as toxic as the native toxin. Based on this and the differing sensitivities of the enzyme and toxic activities of the toxin to heat, it is suggested that the reaction centres in the toxin for the two activities are different and that the pyrophosphatase activity is not causally connected with the lethal toxicity of the toxin     

The Effect of Pyrophosphate Analogues on the Activity of the Inorganic Pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate.     

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4: Ligand-induced molecular asymmetry

The three-dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Patterson's search technique and refined to an R-factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α₃)₂ into α₃′α₃″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re-arrangements for pyrophosphatase function is discussed.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998     

INORGANIC PYROPHOSPHATASE OF RAT LIVER MITOCHONDRIA : Correlation of Latency with Catalytic Properties and Intramitochondrial Location

Stimulation of Mg²⁺-dependent inorganic pyrophosphatase activity several fold by disruption of mitochondrial membranes does not appreciably alter the catalytic properties of the enzyme. Stimulation is due to increased accessibility of substrate to the enzyme, which is not solublized on activation. The enzyme is attached to the inside of the inner membrane, and under physiological conditions probably hydrolyzes only intramitochondrially-produced PP_i.     

Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.     

32P-labeling of bovine tryptophanyl-tRNA synthetase with [32P]pyrophosphate

The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of³²PP_i contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PP_i.Enzymes tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1)     

The Effect of Benzimidazole and Red Light on the Senescence of Detached Leaves of Oryza sativa cv. Ratna

Excised rice leaves (Oryza sativa L. cv. Ratna) werefloated on a 10^–3M solution of benzirnidazole under dark or continuous red light. Compared to the water control a degradation of chlorophyll, protein, RNA, DNA and a decrease in the activity of alkaline inorganic pyrophosphatase was delayed at the same time as an increase of α-amino nitrogen and the activity of acid inorganic pyrophosphatase occurred, Benzimidazole was more effective under red light than in the dark in retarding senescence. The possible role of inorganic pyrophosphatases is discussed with respect to biosynthesis during leaf senescence.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii

An inorganic pyrophosphatase (PPases) was cloned from the hyperthermophilic archaeon Pyrococcus horikoshii and was expressed in and purified from Escherichia coli. The recombinant inorganic pyrophosphatase (PhPPase) exhibited robust catalytic activity of the hydrolysis of pyrophosphate into two orthophosphates at high temperatures (70°C to 95°C). Thermostable pyrophosphatase activity was applied into polymerase chain reaction (PCR) due to its ability to push chemical equilibrium toward the synthesis of DNA by removing pyrophosphate from the reaction. A colorimetric method using molybdate and reducing agents was used to measure PCR progress by detecting and quantifying inorganic phosphate in the PhPPase-coupled PCR mixture. Compared to PCR mixtures without PhPPase, the thermostable PhPPase enhanced the amount of PCR product in the same number of cycles. Thus, thermostable PPase may overcome the limitations of thermodynamically unfavorable DNA polymerization in PCR by yielding more products.