Isolation and primary characterization of two forms of inorganic pyrophosphatase from ox heart muscle mitochondria

A method allowing to obtain two highly purified forms (membrane-bound and soluble ones) of inorganic pyrophosphatase from bovine heart mitochondria is described. Both forms have the isoelectric point of 5.8, pH optimum with Mg2+ at 7--9, are maximally stable at pH 5.8 and absolutely specific to PPi and require Mg2+ or Co2+ for their activity. The soluble pyrophosphatase is also activated by Zn2+. Besides, the two forms differ in their electrophoretic mobilities, affinity for DEAE cellulose and stability in acidic (pH less than 5.8) and alkaline media.     

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.     

Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase.

In this work the two interconvertible forms of inorganic pyrophosphatase (EC 3.6.1.1) of Streptococcus faecalis were shown to differ in kinetics. The highly active form of the enzyme was more sensitive to the changes in the Mg2+ concentration, and thus also more sensitive to the inhibition caused by ATP, which competes with PPi for the chelation of Mg2+ ions. We have previously described a kinetic model for the less-active form of S. faecalis inorganic pyrophosphatase [Lahti & Jokinen (1985) Biochemistry 24, 3526-3530]. The kinetic model of the highly active enzyme form is proposed to be a modification of the model of the less-active form in which enzyme activation by free Mg2+ is necessary for the reaction to occur. In this model the enzyme exists in two states, referred to as R- and T-states. In the absence of ligands the enzyme is in the T-state. R-state, i.e. the catalytically active state, exists only in the presence of free Mg2+. Mg1PPi2- is the primary substrate, and free pyrophosphate is a weak inhibitor that cannot serve as a substrate for the highly active form of S. faecalis inorganic pyrophosphatase. This model closely resembles that previously presented for yeast inorganic pyrophosphatase.     

Various physico-chemical properties of inorganic pyrophosphatase from the mouse spleen

Manganese, calcium and mercury ions, as well as p-chloromercury benzoate and dithiothreitol are studied for their effect on the activity of inorganic pyrophosphatase (EC 3.6.1.1) of mice spleen. It is shown that Ca2+ and Mn2+ are inhibitors of this enzyme, but Mn2+ in low concentrations may replace Mg2+ in the pyrophosphatase reaction. Hg2+ and p-chloromercury benzoate inhibit the pyrophosphatase activity essentially but not completely. Mice spleen pyrophosphatase is very labile: its preincubation without the substrate for 30 min at 37 degrees C leads to a complete loss of the activity. Neither glycerol, nor glutathione and cysteine but magnesium ions, dithiothreitol and 2-mercaptoethanol protect the enzyme from inactivation. The enzyme is purified by the sulphate ammonium salting-out, gel filtration on Sephadex G-100 as well as by isoelectrofocusing in 5% PAAG. Then pyrophosphatase is eluted from gel and subjected to electrophoresis in the plane layer of the linear gradient of 5-15% PAAG with SDS or 5-25% PAAG without denaturing conditions. One zone corresponding to the molecular mass of 70 kDalton is obtained. It is splitted into two zones in electrophoresis with SDS and 2-mercaptoethanol.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Investigations of the metal ion-binding sites of yeast inorganic pyrophosphatase

Yeast inorganic pyrophosphatase was found to bind two Mn2+ per subunit in the absence of phosphate and three Mn2+ per subunit in the presence of phosphate. Kinetic studies of the pyrophosphatase-catalyzed hydrolysis of Cr(NH3)4PP and Cr(H2O)4PP were carried out with Mn2+ and with Mg2+ as activators. The results from these studies suggest that three divalent cations per pyrophosphatase active site are required for catalysis. NMR and EPR studies were conducted to evaluate the relative location of the metal ion binding sites on the enzyme. The two Mn2+ ions bound to the free enzyme are in close enough proximity to magnetically interact. Analysis of the NMR and EPR data in terms of a dipolar relaxation mechanism between Mn2+ ions provides an estimate of the distance between them of 10-14 A. When the diamagnetic substrate analog [Co(NH3)4PNP]- or intermediate analog [Co(NH3)4 (P)2]- are bound to pyrophosphatase, two Mn2+ ions still bind to the enzyme and their magnetic interaction increases. In the presence of these Co3+ complexes, the Mn2+--Mn2+ separation decreases to 7-9 A. Several NMR and EPR experiments were conducted at low Mn2+ to pyrophosphatase ratios (approximately 0.3), where only one Mn2+ ion binds per subunit, in the presence of Cr3+ or Co3+ complexes of PNP or PP. Analysis of the Mn2+--Cr3+ dipolar relaxation evident in proton NMR and EPR data provided for the calculation of Mn2+--Cr3+ distances. When the substrate analog CrPNP was present, the Mn2+--Cr3+ distance was congruent to 7 A whereas, when Cr(P)2 was bound to pyrophosphatase, the Mn2+--Cr3+ distance was congruent to 5 A. These results strongly support a model for the catalytic site of pyrophosphatase that involves three metal ion cofactors.     

Reversible inactivation of rat liver inorganic pyrophosphatase by substrate and its analogs.

Dissociation of Mg2+ from one of the two metal-binding sites whose occupancy is absolutely required for catalysis by rat liver inorganic pyrophosphatase is a slow reaction (tau 1/2 = 3 h). Polycarboxylic Mg2+ complexons markedly accelerate this process due to their binding with Mg2+ on the enzyme. PPi, ATP and a number of diphosphonate analogs of PPi also bind with Mg2+ on the enzyme with concomitant decrease in enzyme activity by 75% but do not release the bound Mg2+. The resulting ternary complex rapidly (tau 1/2 of several seconds) dissociates upon dilution into substrate-free medium. PPi and imidodiphosphate, which are substrates for pyrophosphatase, decrease the rate of reactivation by at least two orders of magnitude. The results can be explained by existence of two interconvertible forms of the enzyme, of which one is inactive and is stabilized by substrate or its analogs.     

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.     

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.     

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

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Kinetic models for the action of cytosolic and mitochondrial inorganic pyrophosphatases of rat liver

Initial rates of PPi hydrolysis by cytosolic and mitochondrial inorganic pyrophosphatases of rat liver have been measured in the presence of 0.2-100 microM MgPPi and 0.01-50 mM Mg2+ at pH 7.2 to 9.3. The apparently simplest model consistent with the data for both enzymes implies that they bind substrate, in the form of MgPPi, and three Mg2+ ions, of which two are absolutely required for activity. The third metal ion facilitates substrate binding but decreases maximal velocity for the cytosolic enzyme, while substrate binding is only modulated for the mitochondrial enzyme. The model is also applicable to bovine heart mitochondrial pyrophosphatases. The active form of the substrate for the cytosolic pyrophosphatase is MgP2O7(-2); the catalytic and metal-binding steps require a protonated group with pKa = 9.2 and an unprotonated group with pKa = 8.8, respectively. The results indicate that the mitochondrial pyrophosphatase is more sensitive to variations of Mg2+ concentration in rat liver cells than is the cytosolic one.     

The formation of enzyme-bound and medium pyrophosphate and the molecular basis of the oxygen exchange reaction of yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase, with 10 mM 32Pi and 10 mM Mg2+ present at pH 7.3 TO 7.6, rapidly forms enzyme-bound pyrophosphate equivalent to about 5% of the total catalytic sties on the two enzyme subunits. The enzyme thus appears to bind PPi so as to favor thermodynamically its formation from Pi. The enzyme catalyzes a measurable equilibrium formation of free PPi at a much slower rate. Under similar conditions, the enzyme catalyzes a rapid exchange of oxygen atoms between Pi and water with the relative activation by metals being Mg2+ greater than Zn2+ greater than Co2+ greater than Mn2+. Millisecond mixing and quenching experiments demonstrate that the rate of formation and cleavage of the enzyme-bound PPi is rapid enough to explain most or all of the oxygen exchange reaction.     

Organic pyrophosphates as substrates for human alkaline phosphatases

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Crystalline inorganic pyrophosphatase isolated from baker's yeast

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

Chloroplast inorganic pyrophosphatase

An alkaline, Mg²⁺-dependent inorganic pyrophosphatase has been isolated from previously isolated spinach chloroplast. The activity of the enzyme was increased 100-fold, with a 42% yield, upon purification from the total soluble chloroplast enzymes. The pH optimum for the enzyme shifts from 9.0 at 5 mM Mg²⁺ to 7.0 at 40 mM Mg²⁺. The substrate for the reaction appears to be magnesium pyrophosphate, and anionic pyrophosphate is an effective inhibitor. There seems to be also an activating effect of Mg²⁺ on the enzyme at pH 7. No other cation substitutes for Mg²⁺ in activating the hydrolysis of pyrophosphate. Among anions tested, only F⁻ caused severe inhibition. The enzyme is inactive towards fructose 1,6-diphosphate, thiamine pyrophosphate, ATP, and ADP. The possibility that this enzyme is subject to metabolic regulation is discussed in relation to an indicated role of pyrophosphate in the regulation of photosynthetic carbon reduction.     

Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. I. Characterisation of chloroplast pyrophosphatase and its relation to the response to exogenous pyrophosphate

1. 1. A soluble, alkaline, Mg²⁺-dependent inorganic pyrophosphatase (EC 3.6.1.1) has been isolated from the stroma of intact spinach and pea chloroplasts and purified some 100-fold. The enzyme has a high specificity for inorganic pyrophosphate and Mg²⁺, and exhibits maximal activity at pH 8.2–8.6. The enzyme shows allosteric characteristics with Mg²⁺ as activator and optimal rates are obtained with a ratio of Mg²⁺ to PP_i of approximately 4 to 1. The enzyme is inhibited by anionic PP_i and by its own reaction product, orthophosphate.

2. 2. If Mg²⁺ is excluded from the medium in which isolated chloroplasts are assayed, active photosynthetic oxygen evolution can still be observed. The addition of P_i, but not PP_i, will then offset a phosphate deficiency. If external Mg²⁺ is present PP_i will also offset a phosphate deficiency and in these circumstances the rapidity and nature of the response is related to the external pyrophosphatase activity.

3. 3. Evidence is presented that the chloroplast envelope is relatively impermeable to PP_i and that the response to added PP_i is due to external hydrolysis followed by entry of P_i to the chloroplast. These results have significance concerning proposed mechanisms for control of photosynthesis.

Two pathways for phosphate/water oxygen exchange by yeast inorganic pyrophosphatase

A scheme of Mg2+ and Pi binding to yeast inorganic pyrophosphatase has been deduced from the concentration dependencies of the rate of oxygen exchange between Pi and water. The exchange reaction requires the binding of MgPi and free Pi (pathway I) or two MgPi (pathway II) in addition to two Mg2+ ions bound in the absence of Pi. Pathway II predominates above 0.16 mM Mg2+. The rate of formation of bound PPi from bound Pi for pathway II is three times as high as that for pathway I. The results suggest that the binding of the fourth Mg2+ ion to pyrophosphatase stimulates its synthetic vs its hydrolytic capability.     

H(+)-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases.

The effects of divalent cations, especially Ca2+ and Mg2+, on the proton-translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5 = 42 microM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 microM. CaCl2 decreased the enzyme activity to less than 60% at 40 microM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 microM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2(+)-PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme. The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immunoreactivity.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.