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PubMed2000

Vacuolar H(+)-pyrophosphatase.

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria. This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple. Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function. The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+). In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane. The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed.

Amino Acid Sequence↗

PubMed1996

Potassium and voltage dependence of the inorganic pyrophosphatase of intact vacuoles from Chenopodium rubrum.

The activity and the voltage dependence of the inorganic pyrophosphatase (PPase) was measured on intact vacuoles of Chenopodium rubrum cells using the patch-clamp technique. With K+ at the cytoplasmic side a negative current representing the forward mode of the pump was measured after addition of pyrophosphate (PPi). The pump was reversed and a positive current was detected after addition of orthophosphate (Pi) in the presence of K+ at the vacuolar side when a pH gradient across the tonoplast was applied. The PPase operates as a constant current source, because no voltage dependence was observed (-60 to 60 mV). The K+ dependence of the PPi-induced current was investigated by substitution of cytoplasmic K+ by other cations. The selectivity sequence was: K+ > or = Rb+ > NH4+ = Cs+ > Na+ > Li+ = choline+, and was independent of the membrane voltage and pHcyt. With Cs+ or Li+ in the bath and K+ inside the vacuole the PPi-induced current became voltage-dependent, and positive currents were observed even if the pump was geared to operate in the forward mode. We suggest a "tunneling' effect through a channel-like domain in the PPase molecule which, under defined electrochemical gradient conditions and in the presence of PPi, allows K+ ions to cross the energy barrier usually separating the cytoplasmic from the vacuolar face of the pump.

Cations, Monovalent↗

PubMed2002

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa.

Proton-translocating inorganic pyrophosphatases (H(+)-PPase, EC 3.6.1.1) are integral membrane proteins that have been extensively studied in higher plants, the photosynthetic bacterium Rhodospirillum rubrum and, more recently, in some human pathogenic protozoa. By using a PCR-based approach, fragments of genes coding for H(+)-PPases in a number of protists, both free-living and parasites of animals and plants, that belong to diverse taxonomic groups (trypanosomatids, ciliates, apicomplexans, euglenoids, amoeboid mycetozoa, heterokonts) have been isolated. The experimental procedure involved the use of degenerate oligonucleotides designed from protein domains conserved in H(+)-PPases from plants and bacteria. The PCR-amplified DNA fragments exhibited the characteristic genomic structure and codon usage of the corresponding protozoan group. Paralogous genes were found in some species suggesting the occurrence of protein isoforms. These results indicate that H(+)-PPases are more widely distributed among protozoa than previously thought.

Amino Acid Sequence↗

PubMed1992

Computer modeling of two inorganic pyrophosphatases.

The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related. One, PPA1, is a cytoplasmic enzyme. The other, PPA2, is located in the mitochondria and appears to be energy-linked. The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50%. All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2. The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1. Two cysteines in PPA2 and one in the S. pombe enzyme are located at the catalytic cleft. Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme.

Amino Acid Sequence↗

PubMed2000

Upregulation of vacuolar H(+)-translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures.

The influence of phosphate (Pi) deprivation on the vacuolar H(+)-translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed. Pi starvation significantly elevated the ratios of PPi-:ATP-dependent H(+) translocation rate and H(+)-PPiase:H(+)-ATPase hydrolytic activities. These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi-starved cells. Immunoblotting indicated that Pi starvation also induced a two-fold increase in the amount of H(+)-PPiase protein, whereas the amount of H(+)-ATPase remained unchanged. It is proposed that H(+)-PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress.

Acid Phosphatase↗

PubMed1998

Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes.

An open reading frame located in the COTF-TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+-activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.

Amino Acid Sequence↗

PubMed1999

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps.

Vacuolar-type H(+)-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H(+)-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M(r) 64¿ omitted¿000 membrane polypeptide that specifically catalyzes Mg(2+)-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H(+)-translocation is phylogenetically more deeply rooted than previously thought.

Amino Acid Sequence↗

PubMed1999

Interaction of Escherichia coli inorganic pyrophosphatase active sites.

Escherichia coli inorganic pyrophosphatase (PPase) is a hexamer of identical subunits. This work shows that trimeric form of PPase exhibits the interaction of the active sites in catalysis. Some trimer subunits demonstrate high substrate binding affinity typical for hexamer whereas the rest of subunits reveal more than 300-fold substrate affinity decrease. This fact indicates the appearance of negative cooperativity of trimer subunits upon substrate binding. Association of the wild-type (WT) trimer with catalytically inactive, but still substrate binding mutant trimer into hexameric chimera restores the high activity of the first trimer, characteristic of trimer incorporated in the hexamer of WT PPase. Interaction of PPase active sites suggests that there are pathways for information transmission between the active sites, providing the perfect organization and concerted functioning of the hexameric active sites in catalysis.

Binding Sites↗

PubMed1999

Vacuolar H(+)-pyrophosphatase purified from pear fruit.

A vacuolar H(+)-translocating inorganic pyrophosphatase was purified from pear fruit through selective detergent treatments, Superose 6 and Mono Q column chromatography. The specific activity of the purified enzyme was 850 mumol h-1 mg protein-1. The Mr of V-PPase was 66 kDa by SDS-PAGE and the polypeptide cross-reacted with the antiserum against V-PPase of mung bean. The purified V-PPase was stimulated by potassium and inhibited by calcium and N, N'-dicyclohexylcarbodiimide.

Blotting, Western↗

PubMed1976

Distribution of adenosine triphosphatase and thamine pyrophosphatase in the digestive system of Heteropneustes fossils.

Histochemical localization of adenosine triphosphatase and thiamine pyrophosphatase in the digestive system of the teleost fish, Heteropneustes fossilis has been studied. In the stomach, ATPase activity is observed in the mucosa, gastric glands and muscularis. The activity is stronger in the muscularis. Very weak TPPase activity is localized only in the mucosa and gastric glands. In the intestinal mucosa ATPase activity is stronger especially, along the brush border. Mild activity is also found in the connective tissue network and their nuclei, muscularis and serosa. In the posterior portion of the intestine and rectum, the localization pattern is similar to that of intestine but the activity is weaker. TPPase activity in the intestine and rectum is restricted only to the goblet shaped mucus secreting cells. In the liver, strong activity of ATPase and moderate activity of TPPase are found in the cytoplasm as well as the nuclei of the hepatic cells.

Adenosine Triphosphatases↗

PubMed1977

Histochemistry and cytochemistry of thiamine pyrophosphatase (TPP-ase 3.6.1.) in the rat gastric mucosa.

The paper deals with the histochemical and cytochemical distribution of thiamine pyrophosphatase (TPP-ase 3.6.1.) in the normal rat gastric mucosa using the method of NOVIKOFF and GOLDFISCHER (1961). In light microscopy, activity was demonstrated only in the GOLGI apparatus of the foveolar mucous cells; in the other specialized gastric mucosal cells no activity was established. At electron microscopic level, the reaction product was localized in the membranes of the cisternae of the GOLGI apparatus of foveolar mucous cells, undifferentiated neck cells, parietal cells, chief cells and in endocrine cells. The reaction product was not found in the endoplasmic reticulum of any of the above mentioned types of cells with the exception of parietal cells. The deposits of the reaction product in the cytoplasmic membrane, in the capillary endothelium and on the surface of the plasma membrane of foveolar mucous cells represents the sites of activity of alkaline phosphatase. The examination of the TPP-ase in the gastric mucosa inables us to estimate the activity of the GOLGI apparatus of gastric mucosal cells in the norm and in different pathological conditions connected with its changes.

Animals↗

PubMed1977

Cytochemical localization of adenosine triphosphatase and thiamine pyrophosphatase in the synapases of rat's cerebral cortex.

Ultrastructural distribution of adenosine triphosphatase and thiamine pyrophosphatase in synapses of rat's cerebral cortex was studied. Adenosine triphosphatase activity in some synaptic vesicles and mitochondria, on pre- and postsynaptic membranes, as well as in the postsynaptic thickening was established. The reaction specificity was proved by means of some controls: various concentrations of ouabain, NaF, NiCl2, cysteine, substrate free medium and non-specific substrates - cocarboxylase and beta-glycerophosphate. At the thiamine pyrophatase reaction, the enzyme positive product was found on the membrane of some clear synaptic vesicles, on the singl sacs of smooth endoplasmic reticulum in the axon terminal, and bouton cell membrane. Substrate free medium, addition of cystein and substitution of orininal substrate with adenosine triphosphate and beta-glycerophosphate as controls were used. The fine structure localization of both enzymes in synaptic structures suggests their important role in the synaptic function.

Adenosine Triphosphatases↗

PubMed1982

Thiamine pyrophosphatase activity of the prenatal mouse molar. A histochemical investigation.

Thiamine pyrophosphatase (TPPase), an enzyme associated with the Golgi apparatus, has been implicated in the regulation of cellular oxidation as well as of transport across cell membranes. This enzyme has been localized in odontogenic tissues of the postnatal mouse and it was the intent of the present study to localize TPPase during prenatal odontogenesis. Mouse fetuses (CDI, Charles River) 14 through 19 days postconception were decapitated, the heads were frozen and mounted on the chuck of a cryostat. Frontal sections, 14 micrometers thick, were air-dried and incubated for TPPase activity. Subsequent to incubation the activity was visualized by immersion in 1% ammonium sulfide. The degree of enzyme activity varied not only with the chronological age of the fetus but also as a function of the tissue's metabolic state. Regions, such as the dental lamina, evidenced decreased TPPase activity with increasing age, while tissue layers such as the IEE displayed greater enzyme activity with increasing age.

Animals↗

PubMed1984

Ultrastructural localization of thiamine pyrophosphatase activity in the intestinal epithelial cells of adult rats.

Ultrastructural localization of thiamine pyrophosphatase (TPPase) activity was studied in the small and large intestinal epithelial cells of adult rats. In the duodenal and jejunal absorptive cells, TPPase activity was localized on the microvilli, on the lateral cell membrane, and on the lysosome-like bodies. In the ileal epithelial cells, TPPase activity was detected on the lysosome-like bodies, and weak phosphatase activity was observed on the microvilli and on the cisternae of the rough-surfaced endoplasmic reticulum (rER). In the principal cells of the caecum, the ascending colon, and the descending colon, the lysosome-like bodies and the cisternae of the rER showed TPPase activity. Almost all Golgi lamellae, vesicles and GERL elements (NOVIKOFF 1964) in the duodenal absorptive cells were strongly TPPase positive. The 1st to 3rd of the inner Golgi lamellae and the vesicles of the jejunal, ileal, and large intestinal epithelial cells usually showed positive TPPase activity, while the GERL elements were totally negative.

Animals↗

PubMed1985

Demonstration of thiamine pyrophosphatase in human germ cells and Sertoli cells, a histochemical study.

The distribution of thiamine pyrophosphatase (TPPase) was studied in the human testicular biopsy tissue with the help of a modified histochemical gel method. The spermatogonia showed a large round strongly positive reaction zone in the supranuclear region. The pachytene spermatocytes exhibited a large semicircular TPPase reaction zone around the nucleus directing towards the lumen. The early round spermatids were characterized by a small, round, and weak supranuclear reaction zone. The elongated late spermatids were devoid of TPPase activity. The Sertoli cells possessed a "streamer" like formation of strong TPPase activity spreading from basal to apical portion.

Histocytochemistry↗

PubMed1999

Identification of the gene structure and promoter region of H+-translocating inorganic pyrophosphatase in rice (Oryza sativa L. ).

In order to determine the gene structure and promoter region of vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), we isolated the genomic clones using a rice BAC library and probes derived from rice V-PPase cDNA (OVP1). The entire OVP1 gene is approx. 5.4 kb in length, and seven introns interrupt the coding sequence of OVP1. The first intron is extremely large (1869 bp), while the other introns are between 82 and 170 bp. A transcription initiation site, identified by a primer extension analysis, indicated the first exon to be 366 bp. A 1.1 kb fragment containing the 5'-flanking region of the first exon with the GUS reporter gene showed specific promoter activity in rice cells. These data show that the OVP1 gene is composed of eight exons and seven introns, and regulatory elements are present within 1.1 kb upstream from the first exon.

Amino Acid Sequence↗

PubMed1996

The structural basis for pyrophosphatase catalysis.

BACKGROUND: Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable. RESULTS: We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions. CONCLUSIONS: Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

Alkaline Phosphatase↗

PubMed2000

Fluoride effects along the reaction pathway of pyrophosphatase: evidence for a second enzyme.pyrophosphate intermediate.

The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase). Fluoride action on yeast PPase during PP(i) hydrolysis involves rapid and slow phases, the latter being only slowly reversible [Smirnova, I. N., and Baykov, A. A. (1983) Biokhimiya 48, 1643-1653]. A similar behavior is observed during yeast PPase catalyzed PP(i) synthesis. The amount of enzyme.PP(i) complex formed from solution P(i) exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme. The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution. These data show that fluoride binds to an enzyme.PP(i) intermediate during the slow phase and to an enzyme.P(i) intermediate during the rapid phase of the inhibition. In Escherichia coli PPase, the enzyme.PP(i) intermediate binds F(-) rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme.PP(i) intermediate formed during PP(i) hydrolysis binds fluoride much faster (yeast PPase) or tighter (E. coli PPase) than the similar complex existing at equilibrium with P(i). It is concluded that PPase catalysis involves two enzyme.PP(i) intermediates, of which only one (immediately following PP(i) addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme.PP(i) intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.

Catalysis↗