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PubMed1981

Galactosyltransferase activities in rat intestinal mucosa. Inhibition of nucleotide pyrophosphatase.

Nucleotide pyrophosphatase, a Zn2+-dependent enzyme catalyzing the hydrolysis of sugar nucleotides, generally interferes with glycosyltransferase assays. Certain tissues such as intestinal mucosa are particularly rich sources of nucleotide pyrophosphatase activity. The inhibition of nucleotide pyrophosphatase without affecting glycosyltransferase activities by the removal of Zn2+ allows for the assay of galactosyltransferases in rat intestinal particulate fractions. Inactivation of nucleotide pyrophosphatase was achieved by preincubation of particulate enzyme preparations in the presence of EDTA. Addition of Mn2+, required for the galactosyltransferase assays, did not reactivate the nucleotide pyrophosphatase. The presence of 0.05% (w/v) soybean trypsin inhibitor was required during the preparation of intestinal mucosa homogenates and enzyme assays to protect the UDP-galactose: N-acetylglucosaminyl galactosyltransferase from inactivation.

Animals↗

PubMed1990

Phosphorylation of rat liver inorganic pyrophosphatase by ATP in the absence and in the presence of protein kinase.

Cytoplasmic inorganic pyrophosphatase of rat liver can be phosphorylated by cAMP-dependent protein kinase on a serine residue with a concomitant increase in enzymic activity. Phosphorylation is also observed in the absence of protein kinase, but in this case much higher concentrations of ATP are required and the stability characteristics of the phosphoenzyme resemble those of an acyl phosphate. Kinase-free phosphorylation of the animal inorganic pyrophosphatase, unlike that of microbial pyrophosphatases, does not activate the enzyme. Pyrophosphatase may thus provide a new example of an enzyme whose evolution involves convergence of regulatory phosphorylation mechanisms.

Adenosine Triphosphate↗

PubMed1985

Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase.

In this work the two interconvertible forms of inorganic pyrophosphatase (EC 3.6.1.1) of Streptococcus faecalis were shown to differ in kinetics. The highly active form of the enzyme was more sensitive to the changes in the Mg2+ concentration, and thus also more sensitive to the inhibition caused by ATP, which competes with PPi for the chelation of Mg2+ ions. We have previously described a kinetic model for the less-active form of S. faecalis inorganic pyrophosphatase [Lahti & Jokinen (1985) Biochemistry 24, 3526-3530]. The kinetic model of the highly active enzyme form is proposed to be a modification of the model of the less-active form in which enzyme activation by free Mg2+ is necessary for the reaction to occur. In this model the enzyme exists in two states, referred to as R- and T-states. In the absence of ligands the enzyme is in the T-state. R-state, i.e. the catalytically active state, exists only in the presence of free Mg2+. Mg1PPi2- is the primary substrate, and free pyrophosphate is a weak inhibitor that cannot serve as a substrate for the highly active form of S. faecalis inorganic pyrophosphatase. This model closely resembles that previously presented for yeast inorganic pyrophosphatase.

Adenosine Triphosphate↗

PubMed1988

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase.

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.

Acid Anhydride Hydrolases↗

PubMed1998

Inorganic pyrophosphatase is a component of the Drosophila nucleosome remodeling factor complex.

The Drosophila nucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.

Amino Acid Sequence↗

PubMed1975

Histochemical localization of adenosine triphosphatase and thiamine pyrophosphatase in the digestive system of a teleost fish, Ophiocephalus punctatus.

The localization of ATPase and thiamine pyrophosphatase in the digestive system of Ophiocephalus punctatus has been studied. In stomach ATPase is found in the free border of the mucosa, gastric glands, submucosal connective tissue nuclei and muscularis. Thiamine pyrophosphatase is localized only in the mucosa and gastric gland cells. In the intestine, pyloric caeca and rectum, ATPase is distributed along the brush border of the columnar epithelial cells, their nuclei and cytoplasm. Mild activity is also found in the nuclei of submucosa and muscularis. The activity is stronger in the intestine than in the other portions. Thiamine pyrophosphatase activity in these portions is restircted only to the goblet cells. In the liver ATPase activity is associated both with the cytoplasm and nucleus of the hepatic cells. Thiamine pyrophosphatase activity is maximum in the centro-lobular portion.

Adenosine Triphosphatases↗

PubMed1992

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning.

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.

Base Sequence↗

PubMed1992

[Functionally important residues of glutamic acid in E. coli pyrophosphatase. I. Chemical modification and localization in the primary structure].

Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor. The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation. A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity. This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues. A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8. This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure. It is concluded that inactivation of E. coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.

Amino Acid Sequence↗

PubMedDate not supplied

[Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates].

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.

Animals↗

PubMed1983

[Subcellular and submitochondrial localization of inorganic pyrophosphatase in the mammalian heart and liver].

The rat heart and liver homogenates contain 3 forms of inorganic pyrophosphatase designated as I, II and III in accordance with a decrease of their electrophoretic mobility. Separation of the homogenates by stepped density gradient differential centrifugation demonstrated that forms I and II are localized in the hyaloplasm, while form III --in the mitochondria. Liver mitochondria contain in addition some amount of pyrophosphatase II. The hyaloplasm and mitochondria of bovine heart contain 2 enzyme forms, whose mobilities are identical to those of forms I and II. Upon destruction of rat mitochondria by detergent (digitonin, Lubrol WX) treatment, sonication or osmotic shock followed by a freezing-thawing precedure pyrophosphatase II remains bound to the inner membrane fragments. The experimental data provide direct evidence for the existence of a specific membrane pyrophosphatase in the mitochondria.

Animals↗

PubMed1994

Modulation of ecto-nucleoside triphosphate pyrophosphatase activity of human osteoblast-like bone cells by 1 alpha,25-dihydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, parathyroid hormone, and dexamethasone.

Extracellular inorganic pyrophosphate (PPi) is involved in the regulation of mineralization, and there is evidence that the cell surface enzyme, NTP pyrophosphatase, is a major source of this metabolite in bone. Osteotrophic agents that influence bone turnover may exert their effects, in part, by modulating the activity of ecto-NTP pyrophosphatase in bone cells. We investigated the effect of 1, 25(OH)2 D3, 24, 25(OH)2D3, dexamethasone, and parathyroid hormone (PTH) on the activity of this enzyme in cultured human trabecular bone-derived osteoblast-like cells. 1,25(OH)2D3 at 10(-11)-10(-9) M induced a dose- and time-dependent increase in activity (at 96 h; maximum 10(-9) M, p < 0.001), whereas higher concentrations (10(-8) and 10(-7) M) had no effect. In contrast, 24,25(OH)2D3 was effective only at 10(-8) and 10(-6) M (at 96 h; p < 0.01). Dexamethasone (10(-9)-10(-7) M) caused a dose-dependent decrease in ecto-NTP pyrophosphatase activity (10(-7) M, p < 0.001); concentrations higher than 10(-7) M did not evoke greater inhibition. This effect became apparent by 48 h and was significantly enhanced after 72 h. The response to dexamethasone was attenuated by cycloheximide, indicating a requirement for de novo protein synthesis. Interestingly, the stimulatory effect of 10(-9) M 1,25(OH)2D3 on ecto-NTP pyrophosphatase activity was significantly enhanced in the presence of dexamethasone (10(-9)-10(-7) M). Human PTH(1-34) and bovine PTH(1-34) in the range 10(-10)-10(-7) M had no effect on enzyme activity over a 72 h period.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitriol↗

PubMed2001

Urea increases tolerance of yeast inorganic pyrophosphatase activity to ethanol: the other side of urea interaction with proteins.

Ethanol is the major product of yeast sugar fermentation and yet, at certain concentrations, it is very toxic to yeast cells. The major targets for ethanol's toxicity are the plasma membrane and the cytosolic enzymes: ethanol alters membrane organization and permeability and inactivates and unfolds globular cytosolic enzymes. The effects of ethanol on the plasma membrane are attenuated by the presence of trehalose, a disaccharide of glucose that is accumulated simultaneously with urea. The data presented in this paper show that trehalose is not effective at protecting yeast cytosolic inorganic pyrophosphatase against the inactivation of its catalytic activity promoted by alcohols. In contrast, 1 M trehalose increased the toxicity of alcohols against pyrophosphatase by at least 34%. On the other hand, 1.5 M urea attenuated the inactivation of pyrophosphatase promoted by alcohols by approximately 50%. Here we propose that, in the presence of alcohols, urea functions as a molecular filter, enriching the vicinity of the protein with water and excluding alcohol molecules. Conversely, trehalose tends to increase the interaction of alcohols with protein molecules, by withdrawing water, leading to a stronger inactivation promoted for a given concentration of alcohol in the bulk solution on pyrophosphatase activity.

1-Propanol↗

PubMed1986

Localization of nucleotide pyrophosphatase in the rat kidney.

Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freeze-dried sections. Nucleotide pyrophosphatase activity, expressed in mumol X min-1 X mg protein-1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichment of the activity during the purification of brush border vesicles was measured. A ten-fold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.

Animals↗

PubMed1984

A pyrophosphatase which degrades NAD+ is located on the external surface of cultured fibroblasts: evidence that NAD+ is not extruded during treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

The Harvey sarcoma virus-transformed derivative of normal rat kidney cells contains a very large membrane-associated NAD+ pyrophosphatase activity. Exogenous NAD+ was metabolized into NMN, demonstrating that the enzyme was located, at least in part, on the external surface of the cell. This external location of the pyrophosphatase was used to evaluate possible extrusion of NAD+ during treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); i.e., extruded NAD+ would be degraded to NMN. NAD+ levels quickly decreased following MNNG addition. The nicotinamide moiety of degraded NAD+ was accounted for as nicotinamide in the culture medium, and no NMN was detectable. Thus, NAD+ was not extruded during MNNG treatment. Additional studies were done to determine which enzyme system was responsible for the rapid fall in NAD+ in these cells. MNNG treatment increased (ADP-rib)n synthetase activity; moreover, this increase in activity was sufficient to account for the loss in intracellular NAD+. Also, the decrease in NAD+ during treatment with MNNG was prevented by 3-aminobenzamide, an agent which inhibited (ADP-rib)n synthetase but not NAD+ glycohydrolase or pyrophosphatase. MNNG had no effect on NAD+ glycohydrolase or pyrophosphatase activities. The results are consistent with the proposal that NAD+ is not extruded but rather is rapidly metabolized by the nuclear (ADP-rib)n synthetase during MNNG treatment.

Animals↗

PubMed1989

Kinetic models for the action of cytosolic and mitochondrial inorganic pyrophosphatases of rat liver.

Initial rates of PPi hydrolysis by cytosolic and mitochondrial inorganic pyrophosphatases of rat liver have been measured in the presence of 0.2-100 microM MgPPi and 0.01-50 mM Mg2+ at pH 7.2 to 9.3. The apparently simplest model consistent with the data for both enzymes implies that they bind substrate, in the form of MgPPi, and three Mg2+ ions, of which two are absolutely required for activity. The third metal ion facilitates substrate binding but decreases maximal velocity for the cytosolic enzyme, while substrate binding is only modulated for the mitochondrial enzyme. The model is also applicable to bovine heart mitochondrial pyrophosphatases. The active form of the substrate for the cytosolic pyrophosphatase is MgP2O7(-2); the catalytic and metal-binding steps require a protonated group with pKa = 9.2 and an unprotonated group with pKa = 8.8, respectively. The results indicate that the mitochondrial pyrophosphatase is more sensitive to variations of Mg2+ concentration in rat liver cells than is the cytosolic one.

Animals↗

PubMed1992

A two-step mechanism of fluoride inhibition of rat liver inorganic pyrophosphatase.

Product formation curves for inorganic pyrophosphatase-catalyzed hydrolysis of pyrophosphate in the presence of fluoride were analyzed in order to get insight into the mechanism of its inhibitory action on this enzyme. The enzymatic reaction was monitored with a phosphate analyzer operating on the time scale of seconds. Inhibition patterns were virtually identical for cytosolic and mitochondrial pyrophosphatases. The effect of fluoride was biphasic: it caused a rapid (t 1/2 less than 1 s) decrease in the initial velocity of the reaction followed by slow (t 1/2 greater than or equal to 4 s) inactivation of the enzyme during catalysis. The slow phase resulted in trapping intact substrate at the active site, and the resulting complex could be isolated by gel filtration. Pyrophosphatase remained active when incubated with fluoride in the absence of pyrophosphate or in the presence of its bisphosphonate analogs, which are bound to but not hydrolyzed by this enzyme. These features of the inhibition are consistent with the mechanism in which rapid binding of the inhibitor to the enzyme.substrate complex is followed by its slow isomerization. Kinetic parameters obtained in this work indicate that appreciable inactivation of pyrophosphatase can occur at fluoride concentrations found in human plasma. This effect may therefore be one of the major factors contributing to fluoride toxicity.

Animals↗

PubMed1977

Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule.

A presumed pyrophosphoryl-enzyme intermediate of the reaction catalyzed by bakers' yeast inorganic pyrophosphatase pyrophosphate phosphohydrolase, EC 3.6.1.1) has been isolated using fluoride-mediated inactivation of the enzyme during catalysis. The analysis of the F--inactivated pyrophosphatase revealed the presence of one molecule of PPi and one atom of fluoride per active site. The incubation of the inactivated enzyme at 25 degrees C and pH 7.2 resulted in gradual recovery of catalytic activity and concomitant removal of PPi by a first-order reaction with tau1/2 of 1 h. The digestion of the F--treated pyrophosphatase with pepsin yielded phosphorous-containing peptides, which were reduced with NaBH4 and gave homoserine and homoserine lactone after acid hydrolysis. This suggests that the PPi residue is linked to the protein through a bond of an acyl phosphate type involving the beta-COOH function of aspartic acid. Together with the results of the kinetic studies of fluoride inhibition of pyrophosphatase reported in accompanying papers, these findings strongly indicate that the enzyme-substrate compound stabilized by fluoride is a transient of the catalytic reaction.

Binding Sites↗

PubMed1977

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.

Carbodiimides↗