Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

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     

Nucleotide pyrophosphatase/phosphodiesterase from Euphorbia characias latex: Purification and characterization

An authentic soluble metallo-protein nucleotide pyrophosphatase/phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 ± 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca²⁺ and 1 Mg²⁺ ion. Whereas Mg²⁺ was shown to be strongly bound to the enzyme, Ca²⁺ was easily removed by treatment with EDTA. Ca²⁺-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca²⁺ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 5′-monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.     

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.     

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.     

Hydrolysis of Inorganic Pyrophosphate in Dictyostelium discoideum

The hydrolysis of inorganic pyrophosphate has been studied in cell-free extracts prepared at different stages of development of Dictyostelium discoideum. Two enzyme reactions, pH optima 7.25 and 9.0, appear specific for inorganic pyrophosphate and have an absolute requirement for a divalent cation, preferably Mg²⁺. The enzyme specific activities do not change significantly during differentiation. Neither enzyme reaction is inhibited by orthophosphate and the presence of exogenous potassium phosphate does not affect the levels of pyrophosphalase at any stage. Exogenous glucose raises the pyrophosphatase levels in the sorocarps.     

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.     

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.     

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).     

Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca²⁺, the Michaelis constant (K_m) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg²⁺ the K_m of β-napthyl triphosphate hydrolysis was 9.0·10⁻⁶ M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.     

Purification and properties of a nucleotide pyrophosphatase from lentil seedlings

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 ± 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, whereas Cu²⁺, Zn²⁺, and Ni²⁺ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.     

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg²⁺ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent K _m values for Mg²⁺ and thiamine pyrophosphate were determined to be 24 M and 1.28 M. The apparent K _m value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.Abbreviations Tris-buffer 0,01 M tris-HCl buffer, containing 1 mM MgCl₂ 0.1 mM EDTA, 1.0 mM thiamine pyrophosphate, 2 mM mercaptopropanediol, pH 7.0     

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     

Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production

Fungi were isolated from natural soil samples and screened for extracellular dextranase synthesis. The strain F1002 was identified as Hypocrea lixii using a standard internal transcribed spacer ribosomal DNA analysis and was selected for extracellular dextranase synthesis. The enzyme was purified via ammonium sulfate precipitation and Sepharose 6B chromatography, which resulted in an 8.3-fold increase in the specific activity and a 10.73% recovery. This enzyme is a monomeric protein with a molecular mass of 62 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme, which was identified as an endodextranase, had an optimum pH of 5.0 and an optimum temperature of 25 °C. The dextranase activity was enhanced by Mg²⁺, Al³⁺, and especially Zn²⁺ at a low concentration, which improved its activity to 124.22%. The enzyme has a very high hydrolytic affinity toward high-molecular weight dextrans. Setting the concentrations of the H. lixii F1002 dextranase (2.31 U/mL) and dextrans (6%), as well as the reaction time (45 min), allowed the dextranase to hydrolyze dextrans of controlled molecular weights (20–70 kDa). Three types of oligodextrans with different molecular weights (namely, 69,376, 38,251, and 21,364 Da) were obtained, with a total yield of 80.32%.     

Enhancement of the rate of pyrophosphate hydrolysis by nonenzymatic catalysts and by inorganic pyrophosphatase

To estimate the proficiency of inorganic pyrophosphatase as a catalyst, ³¹P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PP_i) in the presence and absence of Mg²⁺ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPP_i²⁻ proceeds with a rate constant of 2.8 × 10⁻¹⁰ s⁻¹, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s⁻¹ under the same conditions. The resulting rate enhancement (2 × 10¹²-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH^‡ = −16.6 kcal/mol). The presence of Mg²⁺ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼10⁶-fold. Transfer to dimethyl sulfoxide accelerated PP_i hydrolysis by reducing the enthalpy of activation. Mg²⁺ increased the rate of PP_i hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation.     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Inorganic polyphosphates and phosphohydrolases inHalobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 μmol P_i/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content ofH. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities ofH. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase ofH. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective K_m of this enzyme with respect to pyrophosphate was 115 μM.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).