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PubMed1988

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.

Amino Acid Sequence↗

PubMed1989

Intracellular PPi concentration is not directly dependent on amount of inorganic pyrophosphatase in Escherichia coli K-12 cells.

No correlation was observed between the level of inorganic pyrophosphatase (PPase) and the intracellular concentration of PPi in Escherichia coli cells. In exponentially growing cells the intracellular PPi concentration was in every case 1.5 nmol/mg (dry weight) or about 0.5 mM, even though the amount of PPase was varied from 15 to 2,600% of the control amount by mutation or by using a multicopy plasmid with an inserted gene (ppa) encoding PPase. The PPi concentration could, however, be increased or decreased from the control level under some stressful conditions.

Diphosphates↗

PubMed1999

Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes.

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H(+)-pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 microM) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM), oligomycin (1 microM), N-ethylmaleimide (100 microM), and KNO(3). AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H(+)-ATPase, H(+)-PPase, and (ADP-dependent) H(+)/Na(+) antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H(+)-PPase (both stages) and H(+)/Na(+) exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.

Adenosine Diphosphate↗

PubMed2002

Purification, physicochemical properties, and subcellular location of alkaline inorganic pyrophosphatase from sesame (Sesamum indicum L.) cotyledons.

Abstract: Changes in the levels of inorganic pyrophosphatases (PPases) were monitored in germinating sesame seeds at regular intervals. Activities of acid and alkaline PPases increased markedly in cotyledons up to day 4, remained at the peak level up to day 7, and then showed a considerable decline thereafter. An alkaline PPase was isolated and purified from 5-day-old sesame cotyledons following acetone precipitation, ammonium sulfate fractionation, and chromatography on DEAE-Sephadex. Current protocol yielded about 20% recovery of total activity with a 6.4-fold purification. The enzyme was a monomer with a molecular mass of 20.8 kDa. Some of the properties of alkaline PPase including stability, substrate specificity, ion requirement, and amino acid composition were studied. Alkaline PPase showed maximum activity at pH 8.6 in the presence of Mg2+ and at 50 degrees C. However, the metal ion could not protect the enzyme against thermal denaturation. Alkaline PPase was highly specific for inorganic pyrophoaphate (PP) as substrate and the Km value was 0.7677 +/- 0.0528 mM. Full activation of the enzyme was achieved with a Mg2+/PPi ratio of 2. Divalent metal ions such as Ca2+, Cu2+, and Zn2+ inhibited PPase activity. Mg2+, partially relieved the inhibition caused by adenosine 5'-triphosphate. Studies related to the localization of alkaline PPase in microbodies revealed that the enzyme was distributed between glyoxysomes and mitochondria, with the former containing more of it.

Adenine Nucleotides↗

PubMed1981

Localization of thiamine pyrophosphatase activity in motor end plates.

Thiamine pyrophosphatase (TPPase) activity was localized in the terminal arborization of the motor end plates both in rat and frog striated muscles. Electron microscopically the end product of the enzyme was seen in synaptic vesicles. That TPPase was axonally transported was indicated by its localization following experimental ligation of peripheral nerves. It is thus possible that TPPase may play a role in the synthesis of the synaptic mediator substance of the neuromuscular junction.

Animals↗

PubMed2001

Analysis of stress- and host cell-induced expression of the Mycobacterium tuberculosis inorganic pyrophosphatase.

BACKGROUND: The gene encoding the inorganic pyrophosphatase (PPase) of the intracellular pathogen Legionella pneumophila is induced during intracellular infection, but is constitutively expressed in Escherichia coli. The causative agent of tuberculosis, Mycobacterium tuberculosis, contains a well conserved copy of PPase. We sought to determine if expression of the M. tuberculosis PPase is regulated by the intracellular environment. RESULTS: A strain of Mycobacterium bovis bacille Calmette-Guérin (BCG) was constructed in which the Aequoria victoria green fluorescent protein (GFP) is controlled by the promoter of the M. tuberculosis ppa gene. After prolonged exposure of the recombinant BCG strain within murine bone-marrow-derived macrophages, there was no observed increased activity of the ppa promoter. Furthermore, there was no change in promoter activity after exposure to various stress stimuli such as reduced pH, osmotic shock, nutrient limitation or oxidative stress. CONCLUSIONS: These results suggest that macrophage induction of ppa is not a general phenomenon among intracellular pathogens.

Animals↗

PubMed1989

Dynamic distribution of the Golgi marker thiamine pyrophosphatase is modulated by brefeldin A in rat hepatoma cells.

Cytochemical electron microscopy of cultured rat hepatoma cells (AH-130) demonstrated that thiamine pyrophosphatase (TPPase) activity was localized in the Golgi complex. When the cells were treated with brefeldin A (BFA, 2.5 micrograms/ml) for 10 min, the characteristic structure of the Golgi stack was no longer observed, and TPPase was cytochemically stained in the vesicular and tubular structures scattered in the cytoplasm. A longer exposure of the cells to the drug (20 min to 1 h) resulted in the distribution of the TPPase activity in the endoplasmic reticulum (ER) and nuclear envelope. Such an unusual distribution of the enzyme activity, however, was reversible even in the presence of BFA. At 2 h after the exposure, the TPPase activity disappeared from the ER and was concentrated again in the vesicular and tubular structures. The enzyme activity was finally localized in the Golgi complex which was reassembled by 4 h after the exposure. The reversible effect of BFA may be due to a possible metabolism of the drug into an inert form during the incubation. Taken together, these results indicate that BFA causes a rapid disassembly of the Golgi complex and redistribution of the marker enzyme TPPase into the ER including the nuclear envelope. The spontaneous reversibility of the drug effect also favors a dynamic recycling of the Golgi marker between the ER and the Golgi complex under the conditions used.

Animals↗

PubMed1998

Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon.

The gene encoding an extremely stable inorganic pyrophosphatase from Sulfolobus sp. strain 7, a thermoacidophilic archaeon, was cloned and sequenced. An open reading frame consisted of 516 base pairs coding for a protein of 172-amino acid residues. The deduced sequence was supported by partial amino acid sequence analyses. All the catalytically important residues were conserved. A unique 17-base-pair sequence motif was found to be repeated four times in frame in the gene, encoding a cluster of acidic amino acids essential for the function. Although the codon usage of the gene was quite different from that of Escherichia coli, the gene was effectively expressed in E. coli. Coexpression of tRNA(Arg), cognate for the rare codon AGA in E. coli, however, further improved the production of the enzyme, which occupied more than 85% of the soluble proteins obtained after removal of heat denatured E. coli proteins.

Amino Acid Sequence↗

PubMed1983

A new sensitive, thiol tolerant method for the determination of inorganic pyrophosphatase.

In this work a new sensitive, thiol tolerant method for the determination of inorganic pyrophosphatase (EC 3.6.1.1) was developed. It is based on the formation of phosphomolybdate-triethylamine complex, which in acid-acetone mixture forms a white, homogeneous precipitate, the turbidity of which can accurately measured spectrophotometrically at 355 nm. This method can be easily applied to all the Pi-producing enzymes whose substrates are stable enough in our acidic experimental conditions.

Drug Stability↗

PubMed1999

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions.

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.

Aspartic Acid↗

PubMed2000

Engineering a new magnesium binding site in the subunit contact region of Escherichia coli inorganic pyrophosphatase.

Three Gln-80 residues belonging to different subunits of homohexameric Escherichia coli pyrophosphatase are separated by only one water molecule to which they are hydrogen bonded. Substitution of Glu for Gln-80 stabilizes quaternary structure of the enzyme but has only a small effect on enzyme activity. The substitution stimulates Mg2+ binding and changes the appearance of the Mg2+ concentration dependence of the rate constant for the trimer --> hexamer transition. These data suggest that a new Mg2+ binding site is formed in the intersubunit contact region as a result of the substitution. Three-dimensional modeling of the mutated protein showed that a chelate complex might form involving two of the three Glu-80 residues.

Amino Acid Substitution↗

PubMed1992

[Functionally important tyrosine residues in Saccharomyces cerevisiae pyrophosphatase. I. Chemical modification and localization in the primary structure].

Inorganic pyrophosphatase (PPase) of S. cerevisiae is effectively inactivated by 7-chloro-4-nitrobenzofuran; the CaPP1 substrate analog has a protective effect. The modified enzyme separated from low molecular weight contaminants has an adsorption maximum at 345 nm. Preliminary modification of PPase SH-groups does not influence the enzyme binding to the inhibitor. The PPase activity is reconstituted by beta-mercapto-ethanol; hence, the inhibiting effect of the reagent is due to modification of tyrosine residues. A single reagent-containing peptide was isolated by specific adsorption from the tryptic hydrolysate of modified PPase. Within the primary structure of PPase, this peptide occupies positions 82-111 and contains two tyrosine residues. Hydrolysis of the isolated peptide by chymotrypsin and determination of the structure of fragments obtained by mass spectrometry and automated sequencing revealed that inactivation of PPase is due to selective modification of Tyr89.

Amino Acid Sequence↗

PubMed1992

[A functionally important Tyr-89 residue in Saccharomyces cerevisiae pyrophosphatase. II. A possible role in the mechanism of enzyme action].

Earlier it has been demonstrated that inactivation of inorganic pyrophosphatase (PPase) of S. cerevisiae by 7-chloro-4-nitronbenzofurasane is due to modification of Tyr89. The effect of pH and active center ligands on this reaction has been studied. It was found that pK for Tyr89 does not exceed 8.5; the phosphate-metal complex binding to the high affinity center protects Tyr89 from inactivation. Activating ions (Mg2+ and Zn2+) do not influence the inactivation, whereas the PPase inhibitor, Ca2+, enhances this process after saturation of the high affinity binding site. Saturation of two binding sites with Ca2+ has a protective effect on the enzyme. An increase in the rate of Tyr89 binding to the inhibitor in the presence of low concentrations of Ca2+ is due to the decrease of Tyr89 pK. The data obtained suggest that Tyr89 is located near the high affinity binding site for phosphate. The high reactivity of Tyr89 and its tight binding in the active center point to the presence of a hydrogen bondage with the substrate and suggest a role of a proton donor whose acceptor is the product of the enzymatic reaction, i.e., phosphate.

Benzofurans↗

PubMed1992

Reconstitution of transport function of vacuolar H(+)-translocating inorganic pyrophosphatase.

A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.

Biological Transport↗

PubMed1977

Subcellular distribution of thiamine-pyrophosphokinase and thiamine-pyrophosphatase activities in rat isolated enterocytes.

Starting from mucosal cells, isolated from rat small intestine, subcellular fractionation was carried out. Four fractions were prepared and characterized by marker enzymes and electron microscopic examination: nuclei plus microvilli, mitochondria, microsomes and supernatant. Thiamine-pyrophosphokinase activity was localized mainly in the supernatant fraction, with minor amount in mitchondria and microsomes. On the contrary thiamine-pyrophosphatase activity was present only in the particulate fractions (especially in microsomes), being completely absent from the supernatant.

Animals↗

PubMed1988

A lead conversion method for staining thiamine pyrophosphatase and other phosphatases in rat brain after thin layer polyacrylamide gel isoelectric focusing.

Rat brain thiamine pyrophosphatase (TPPase) was separated by thin layer polyacrylamide gel isoelectric focusing (IF) and stained with a modification of the lead conversion method of Allen and Hyncik (1963). 10 bands with TPPase activity were observed in the pH range 4.6-7.1. The overall IF pattern of TPPase was similar to that of uridine diphosphatase and inosine diphosphatase but was clearly different from that of adenosine diphosphatase, p-nitrophenyl phosphatase, alpha-naphthyl phosphatase and thiamine monophosphatase. A semiquantitative assessment of TPPase isoenzymes has been performed using laser densitometry.

Animals↗

PubMed1987

[Ultrastructural localization of thiamine pyrophosphatase activity in the epitheliocytes of duodenal biopsies in peptic ulcer].

Using electron microscopic studies the location of thiamine pyrophosphatase (TPPase) activity in the epithelial cells of duodenal mucosa was investigated in 39 patients with ulcer exacerbation and during the disease remission. The biopsy material was studied before and after the administration of physiological and therapeutic doses of thiamine. TPPase activity was detected on the membranes of Golgi complex, lateral cell membrane and microvilli. The possibility of intra- and extracellular localization of TPPase activity and the activity of various enzymes depending on the mechanism of thiamine absorption across the epithelial barrier is discussed.

Adult↗

PubMed1987

[Functionally important lysine residues in inorganic pyrophosphatase from E. coli. II. Isolation and characteristics of modified tryptic peptide and analysis of the functional role of lysine residues controlling enzyme activity].

Inactivation of inorganic pyrophosphatase from E. coli by pyridoxal-5'-phosphate is due to the modification of a lysine residue located in the tryptic peptide with the Asp-Leu-Pro-Glu N-terminal sequence. In course of the enzymatic process this lysine-residue appears to be in the protonated state and either operators as the proton donor for the product of the enzymatic reaction or is involved in stabilization of the transition state.

Amino Acid Sequence↗