Inhibition of photosynthesis in isolated spinach chloroplasts by inorganic phosphate or inorganic pyrophosphatase in the presence of pyrophosphate and magnesium ions

Inhibition of photosynthesis in isolated spinach chloroplasts by P_i is decreased by the presence of PP_i and increased with increasing Mg²⁺ concentration. Previously reported regulation of this photosynthesis by protein factors from spinach leaves appears to be due mostly to pyrophosphate phosphohydrolase (EC 3.6.1.1) activity which converts PP_i to P_i and to the effects of PP_i and Mg²⁺ on this pyrophosphatase activity.     

Thermodynamic analysis of F1‐ATPase rotary catalysis using high‐speed imaging

F₁-ATPase (F₁) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F₁ rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F₁ catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F₁ rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (P_i). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P_i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG^‡ = 53–56 kJ mol⁻¹, the contributions of the enthalpy (ΔH^‡), and entropy (ΔS^‡) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH^‡ and ΔS^‡. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.     

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.     

Constant Enthalpy Change Value during Pyrophosphate Hydrolysis within the Physiological Limits of NaCl

A decrease in water activity was thought to result in smaller enthalpy change values during PP_i hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 m NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 m NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of −35 kJ/mol was obtained for the halophile and non-halophiles at 1.5–4.0 and 0.1–2.0 m NaCl, respectively. These results show that solvation changes caused by up to 4.0 m NaCl (water activity of ∼0.84) do not affect the enthalpy change in PP_i hydrolysis. It has been postulated that PP_i is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.     

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)     

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Photosynthetic formation of inorganic pyrophosphate (PP_i) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP_i synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP_i synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP_i synthesis much less. Even at low rates of electron transport a significant rate of PP_i synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PP_i synthesis is inhibited. It is shown that PP_i synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.     

Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification

The onset of mineralization in embryonic chick femurs was studied as a model for the initiation of biological calcification. Electron microscopy confirmed the presence of calcifying matrix vesicles within newly formed bone, and showed that these vesicles were the initial site of crystal deposition. Matrix vesicles were first seen on day 6 of embryonic development, and already were present in considerable numbers on day 7, at which time mineral deposition was just beginning. As a reflection of initial mineralization the uptake of ⁴⁵Ca and ⁴⁰Ca into 7-day-old bones was studied during 2 days in organ culture. A control level of uptake was established using a defined culture medium, P-6. Addition of inorganic pyrophosphate (PP_i) to this medium caused a marked increase in calcium uptake into areas of matrix which normally calcify in vivo. The maximal ⁴⁵Ca uptake, greater than 4-fold, was achieved with 4 μg of P per milliliter of PP_i and was partially heat-inhibitable. Since the matrix vesicles are known to be rich in inorganic pyrophosphatase, it is proposed that mineralization is promoted in vesicles by the enzymatic hydrolysis of pyrophosphate. The membrane-bounded matrix vesicles appear to provide the necessary enzymes and environment to concentrate calcium and phosphate for initiating crystal formation.     

Proton Gradient Across the Tonoplast of Riccia fluitans as a Result of the Joint Action of Two Electroenzymes

Using pH-sensitive microelectrodes (in vitro) and acridine orange photometry (in vivo), the actions of the two tonoplast phosphatases, the tp-ATPase and the tp-PPase, were investigated with respect to how effectively they could generate a transtonoplast pH-gradient. Under standard conditions the vacuoles of the aquatic liverwort Riccia fluitans have an in vivo pH of 4.7 to 5.0. In isolated vacuoles a maximal vacuolar pH (pH_v) of 4.74 ± 0.1 is generated in the presence of 0.1 millimolar PP_i, but only 4.93 ± 0.13 in the presence of 2.5 millimolar ATP. Both substrates added together approximate the value for PP_i. Cl⁻-stimulates the H⁺-transport driven by the tp-ATPase, but has no effect on the tp-PPase. The transport activity of the tp-ATPase approximates saturation kinetics (K_½ ≈ 0.5 millimolar), whereas transport by the tp-PPase yields an optimum around 0.1 millimolar PP_i. The transtonoplast pH-gradient is dissipated slowly by weak bases, from which a vacuolar buffer capacity of roughly 300 to 400 millimolar/pH_v unit has been estimated. From the free energy (−11.42 kilojoules per mole) for the hydrolysis of PP_i under the given experimental conditions, we conclude that the PPase-stoichiometry (transported H⁺ per hydrolyzed substrate molecule) must be 1, and that in vivo this enzyme works as a H⁺-pump rather than as a pyrophosphate synthetase.     

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.     

Properties of xanthosine 5'-monophosphate-amidotransferase from Escherichia coli.

Xanthosine 5′-phosphate (XMP)-amidotransferase catalyzes the formation of guanosine 5′-phosphate (GMP) by aminating XMP with either the amide group of glutamine (amidotransferase) or ammonia (aminase). The glutamine-supported activity of the purified enzyme from Escherichia coli has been studied, and its properties have been compared with those of other amidotransferases. The following results have been obtained. (i) The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), irreversibly inhibits the amidotransferase activity. A maximal rate of inhibition by DON is achieved in the presence of XMP, ATP, and Mg²⁺ with a pseudo-first-order rate constant of 0.276 min^?1. (ii) The total number of sulfhydryl groups is approximately 22 per dimer (126,000 M_r). In the absence of substrates, about 8 sulfhydryl groups per dimer are titratable with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and in the presence of XMP, ATP, and Mg²⁺ an additional 6 cysteine residues per dimer become exposed. When the amidotransferase activity is inactivated by DON, only 8 sulfhydryl groups are titratable. DTNB, p-chloromercuribenzoate, and bromopyruvate all selectively inactivate the amidotransferase activity. These results are consistent with the hypothesis that cysteine residues which are exposed by the substrates are involved in the amidotransferase activity. (iii) The purified XMP amidotransferase contains a glutaminase activity which can be measured in the absence of GMP formation. The glutaminase activity requires XMP, Mg²⁺, and either psicofuranine, an analog of adenosine, or inorganic pyrophosphate (PP_i) and is inhibited by p-chloromercuribenzoate and DON. Maximal stimulation is observed with 100 μm psicofuranine or PP_i, and there is no further stimulation in the presence of both effectors. The apparent K_m is 31 μm with PP_i and 13 μm with psicofuranine; the V for glutamine hydrolysis is about 60% of the rate of the amidotransferase activity. The cooperative interactions between the binding of PP_i and psicofuranine have been confirmed. In the presence of 2.5 μm psicofuranine the K_m for PP_i is reduced 20-fold, but the maximal velocity is unchanged. Similarly, the apparent K_m for psicofuranine is reduced by low concentrations (10 μm) of PP_i. The “uncoupling” of the hydrolysis of glutamine from the amination of XMP is the basis for the reported inhibitory effects of psicofuranine and PP_i on the amidotransferase activity. (iv) Tris buffer selectively inhibits the XMP-amidotransferase activity by inhibiting the glutaminase activity. This inhibition is time dependent and reversible and may explain the previous reports on the inability of this enzyme to use glutamine as a substrate.     

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     

Yeast inorganic pyrophosphatase substrate recognition

Monodentate Co(NH₃)₅PP_i was determined not to be a substrate for yeast inorganic pyrophosphatase while P¹,P²-bidentate Co(NH₃)₄PP_i 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)₂PP_i, Cr(NH₃)₄PP_i, Cr(H₂O)(NH₃)₃PP_i, Cr(H₂O)₂(NH₃)₂PP_i, and Cr(H₂O)₄PP_i 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 K_m for Mg²⁺, the binding affinity of the complexes decreased with an increasing $\text{NH}{}_3\text{H}{}_2\text{O}$ ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)₂PP_i complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH₃)₄PP_i complex. The substrate activities of β,γ-bidentate Co(NH₃)₄PPP_i and α,β,γ-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.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Mg2+-Dependent,cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from storage roots of red beet (Beta vulgaris L.) posess a Mg²⁺-dependent, alkaline pyrophosphatase (PPase) activity which is further stimulated by salts of monovalent cations. The requirement for Mg²⁺ is specific. Mn²⁺ and Zn²⁺ permitted only 20% and 12%, respectively, of the PPase activity obtained in the presence of Mg²⁺ while Ca²⁺, Co²⁺ and Cu²⁺ were ineffective. Stimulation of Mg²⁺-PPase activity by salts of certain monovalent cations was due to the cation and the order of effectiveness of the cations tested was K⁺=Rb⁺=NH ₄ ⁺ >Cs⁺. Salts of Li⁺ and Na⁺ inhibited Mg²⁺-PPase activity by 44% and 24%, respectively. KCl-stimulation of Mg²⁺-PPase activity was maximal with 60–100 mM KCl. There was a sigmoidal relationship between PPase activity and Mg²⁺ concentrations which resulted in markedly non-linear Lineweaver-Burk plots. At pH 8.0, the optimal [Mg²⁺]:[PP_i] ratio for both Mg²⁺-PPase and (Mg²⁺+KCl)-PPase activities was approximately 1:1, which probably indicates MgP₂O₇ ^2- is the true substrate.Abbreviations BSA bovine serum albumen - EDTA ethylenediamine tetra-acetic acid, disodium salt - MES 2-(N-morpholino)ethanesulphonic acid - Mg _T ²⁺ total magnesium - P_i inorganic phosphate - PPase inorganic pyrophosphatase - PP_i inorganic pyrophosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

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.     

A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate

A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PP_i) was developed. PP_i was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl₂ and KF. The separated PP_i was dissolved by immersing the filter in 0.5 n H₂SO₄. Inorganic phosphate (P_i) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PP_i was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PP_i can be accurately assayed even when there is a 10⁴-fold excess of P_i. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PP_i. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PP_i in the same sample.     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.     

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P_i is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.     

A sensitive method for measuring pyrophosphate in the presence of a 10,000-fold excess of orthophosphate using inorganic pyrophosphatase

A simple method for measuring PP_i at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PP_i. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PP_i in urine is demonstrated.