Expression of new human inorganic pyrophosphatase in thyroid diseases: its intimate association with hyperthyroidism

Inorganic pyrophosphatase (PPase) controls the level of inorganic pyrophosphate produced by biosynthesis of protein, RNA, and DNA. Thus, PPase is essential for life. PPase expression is unclear in the thyroid. We cloned a new human PPase, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase), and established a rabbit polyclonal anti-LHPPase antibody. This is the first study to determine the PPase expression by immunohistochemistry and Western blot. Intranuclear LHPPase expression of thyrocytes was enhanced most prominently in Graves' disease and autonomously functional thyroid nodule. To estimate a regulating factor of subcellular localization of LHPPase, we examined its expression of Graves' disease-derived thyrocytes in vitro with the disease-originated serum. Nuclear expression of LHPPase was lost in cultured thyrocytes even with the serum, while its cytoplasmic expression was retained. The data suggest that increased expression of LHPPase is associated with hyperthyroidism. Intranuclear expression of LHPPase may not be regulated by Graves' disease-derived serum factors.     

Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay

Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP_i. Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni²⁺-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI-TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.     

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     

Alkaline inorganic pyrophosphatase from orchid leaves

An alkaline inorganic pyrophosphatase (IP) from leaves of an orchid, Aranda Christine 130 (Arachnis hookerana var. luteola × Vanda Hilo Blue) was purified by acetone precipitation and chromatography on Sephadex G-75 and DEAE-cellulose. The IP gave a single band on non-denaturing gel electrophoresis at pH 8.3 and its M, determined by gel filtration, was 28 000. The pH optimum was 9 and the IP required Mg²⁺ for its activity and stability. The IP exhibited high specificity for PPi and attained a maximum activity at a Mg²⁺: PPi ratio of 10:1. Other cations tested could not replace Mg²⁺ and they were also found to be inhibitory. The IP was also inhibited by EDTA and F^? but not by iodoacetamide.     

Investigation of the role of the substrate metal ion in the yeast inorganic pyrophosphatase reaction

The substrate activities of a series of tripositive metal ion-pyrophosphate complexes with yeast inorganic pyrophosphatase were examined. While the Michaelis constants for these complexes were shown to be between one and two orders of magnitude greater than that of the natural substrate, [Mg(H2O)4PPi]2-, the turnover numbers were in general comparable to that of [Mg(H2O)4PPi]2-. These data suggest that the nature of the metal ion cofactor effects substrate binding but in most cases not catalysis. Thus, the role of the metal ion in catalysis is probably restricted to that of an electron sink.     

Kinetic and structural properties of inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi) and controls the level of PPi in cells. PPase plays an essential role in energy conservation and provides the energy for many biosynthetic pathways. The Helicobacter pylori pyrophosphatase (HpPPase) gene was cloned, expressed, purified, and found to have a molecular weight of 20 kDa. The K(m) and V (max) of HpPPase were determined as 214.4 microM and 594 micromol Pi min(-1) mg(-1), respectively. PPi binds Mg(2+) to form a true substrate that activates the enzyme. However, free PPi could be a potent inhibitor for HpPPase. The effects of the inhibitors NaF, ATP, iminodiphosphate, and N-ethylmaleimide on HpPPase activity were evaluated. NaF showed the highest inhibition of the enzyme. Crystal structures of HpPPase and the PPi-HpPPase complex were determined. HpPPase comprises three alpha-helices and nine beta-strands and folds as a barrel structure. HpPPase forms a hexamer in both the solution and crystal states, and each monomer has its own PPi-binding site. The PPi binding does not cause a significant conformational change in the PPi-HpPPase complex, which might represent an inhibition state for HpPPase in the absence of a divalent metal ion.     

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.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.     

Alkaline inorganic pyrophosphatase from immature wheat grains

Changes in the level of alkaline inorganic pyrophosphatase and ADPG-pyrophosphorylase were monitored in developing wheat grains at weekly intervals aft     

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.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.     

Alkaline inorganic pyrophosphatase from guar (Cyamopsis tetragonoloba) cotyledons

Alkaline inorganic pyrophosphatase from guar cotyledons was purified x 110 with about 34% recovery by (NH₄)₂SO₄ fractionation, acetone prec     

A novel calcium-dependent soluble inorganic pyrophosphatase from the trypanosomatid Leishmania major

A single-copy gene IPP encoding a putative soluble inorganic pyrophosphatase (LmsPPase, EC 3.6.1.1) was identified in the genome of the parasite protozoan Leishmania major. The full-length coding sequence (ca. 0.8 kb) was obtained from genomic DNA by polymerase chain reaction (PCR) and cloned into an Escherichia coli expression vector, and was overexpressed for functional protein purification and characterization. The recombinant LmsPPase, purified to electrophoretic homogeneity by a two-step chromatography procedure, exhibited a predicted molecular mass of ca. 30 kDa. The enzyme has an absolute requirement for divalent cations, exhibits a pH optimum of 7.5–8.0 and does not hydrolyze polyphosphates or adenosine triphosphate (ATP). LmsPPase differs from previously studied soluble pyrophosphatases with respect to cation selectivity, Ca²⁺ being far more effective than Mg²⁺. Comparisons to known sPPases show a short N-terminal extension predicted to be a mitochondrial transit peptide, and changes in active-site residues and the neighboring region. Subcellular fractionation of L. major promastigotes suggests a mitochondrial localization. Molecular phylogenetic analysis indicates that LmsPPase is a highly divergent eukaryotic Family I sPPase, perhaps an ancestral class of eukaryotic sPPases functionally adapted to a calcium-rich, probably mitochondrial, environment.     

Affinity labeling of inorganic pyrophosphatase from yeast by methylphosphate

Yeast inorganic pyrophosphatase is specifically and irreversibly inactivated by methylphosphate. The high rate of inhibition, the protective effect of the substrate, the strict correlation between the degree of inhibition and the amount of the protein-bound reagent and the effect of saturation of the enzyme with methylphosphate provide evidence in favour of the reaction in the active center. Modification of two chemically identical enzyme subunits proceeds at different rates and results in a formation of phosphorylated subunits with different stability of the phosphate bond, which is indicative of the mutual effects of the pyrophosphatase subunits. The reaction between the modified enzyme and hydroxylamine suggests that the interaction between pyrophosphatase and methylphosphate entails modification of the carboxylic groups of two active centers, resulting in a formation of the acylphosphate bonds.