Dependent Inorganic Pyrophosphatase Research Articles

Properties and regulation of Mg 2+-dependent chloroplast inorganic pyrophosphatase from Sorghum vulgare leaves

A Mg 2+ dependent inorganic pyrophosphatase from chloroplasts of Sorghum vulgare has been purified 275-fold to electrophoretic purity with an overall recovery of about 25% activity. Estimations of native and monomeric relative molecular weights by size exclusion chromatography and denaturing electrophoresis suggest that the holoenzyme is a monomer of 42 ± 1.5 kDa. A high specificity for tetrasodium pyrophosphate ( PP i) as substrate has been observed, as the other phosphoesters tested were virtually unaffected. The Mg 2+: PP i ratio of 5:1 at pH 8.0 shifts to 2.5:1.0 at pH 9.0 and 10:1 at pH 7.0. None of the divalent cations tested could substitute for Mg 2+. Further, in the presence of Mg 2+, these divalent cations inhibit the catalytic hydrolysis of PP i. EDTA rapidly and irreversibly inactivates the purified enzyme in a biphasic manner. Of the metabolites tested, P i and l-malate significantly inhibited the catalytic activity of the enzyme. Malate inhibits the enzyme through an allosteric mechanism. A Hill plot of this inhibition shows that at least two molecules of malate bind to each molecule of the purified enzyme. The likely physiological significance of this result is discussed.

An alkaline magnesium dependent inorganic pyrophosphatase from the leaf of Amarantus blitum L.

The purified inorganic pyrophosphatase from the leaf of Amarantus blitum shows absolute magnesium requirement for its enzyme activity. For maximum enzyme activity, the Mg : PPi ratio was found to be 10:1 at the optimum pH of 9.0. This ratio varies with the shift of the pH. was found to be the true substrate for the enzyme in the presence of the free Mg2+ ion. Some divalent metal ions and the fluoride anion severely inhibit the enzyme activity in the presence of Mg2+. The Michaelis constant (Km) and molecular weight of the enzyme were found to be 4.95·−6 m and 32,860, respectively. This enzyme is strictly specific for inorganic pyrophosphate.

Purification of kinetic properties of human erythrocyte Mg 2+-dependent inorganic pyrophosphatase

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte henolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 μmol PP i hydrolysed · min −1 · mg −1 at pH 8 ad 37°C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg 2+ concentration in different ways. This ion constitutes part of the substrate (the Mg · PP i complex; K m = 1.4 · 10 −4 M) and probably acts as an allosteric activator (kinetic activation constant: K a Mg 2+ = 7.5 · 10 −4 M). Equilibrium binding studies performed in the absence of PP i showed 4 binding sites for Mg 2+, all having the same high affinity (dissociation constant: K d Mg 2+ = 4 · 10 t-6 M). Since the concentration of free Mg 2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.

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, Mg 2+-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 Mg 2+, and exhibits maximal activity at pH 8.2–8.6. The enzyme shows allosteric characteristics with Mg 2+ as activator and optimal rates are obtained with a ratio of Mg 2+ 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 Mg 2+ 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 Mg 2+ 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.

Chloroplast inorganic pyrophosphatase

An alkaline, Mg 2+-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 Mg 2+ to 7.0 at 40 mM Mg 2+. 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 Mg 2+ on the enzyme at pH 7. No other cation substitutes for Mg 2+ 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 of rat liver mitochondria. Correlation of latency with catalytic properties and intramitochondrial location.

Stimulation of Mg(2+)-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).

Allosteric properties of the Mg++-dependent inorganic pyrophosphatase in mouse liver cytoplasm.

The Mg++‐dependent inorganic pyrophosphatase of the mouse liver is localized primarily in the cytoplasm. Substrate is the complex ion MgPPi2−; free PPi2− and MgADP are strong inhibitors, while Mg3+‐ions behave as an activator for the enzyme. The [Mg2+]/[substrate] ratio for optimum activity decreases with increasing substrate concentration. At substrate saturation and at pH‐optimum of 8.0, the optimum ratio is 10:1. Under these conditions Vmax= 3.7 μmole × min−1× mg protein−1, and Km= 1.6 × 10−5 M. The enzyme shows allosteric properties of a K‐system with homotropic and heterotropic effects. Homotropic cooperative interactions with Hill coefficients of 2 have been shown for the substrate, the inhibitor MgADP, and the activator Mg2+. Under the influence of the substrate the cooperativity of the activator decreases, while that of the inhibitor increases, the cooperativity of the substrate itself decreases by means of the activator. If the enzyme is preincubated for 5 min at 40°, the allosteric cooperativity of the substrate is lost by desensitization. This effect is accompanied by a partial loss of activity. Pi4− > MgPP2− > Mg2+ > MgADP protect the pyrophosphatase against heat inactivation. The possibility that the pyrophosphatase reaction follows the kinetics of a substrate‐activated enzyme, which is inhibited by free pyrophosphate, could be excluded.