SEARCH · PubMed Health

Results for “Pyrophosphatases”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources

PubMed1995

[Synthesis of pyrophosphate by rat liver cytoplasmic and mitochondrial pyrophosphatases].

The kinetics of pyrophosphate synthesis catalyzed by rat liver cytosolic and mitochondrial pyrophosphatase in the presence of Mg2+ as cofactor has been studied. According to the kinetic model, this reaction proceeds through two parallel pathways: pathway I utilizing two magnesium phosphate molecules and pathway II utilizing both magnesium phosphate and free phosphate. Pyrophosphate formation is greatly facilitated in the active sites of the enzymes ([E.PPi]/[E.2Pi] = 0.11-0.24). Both enzymes are characterized by high efficiency in pyrophosphate synthesis in solution: the catalytic constants for the cytosolic and mitochondrial pyrophosphatases are 14 and 9.3 s-1, respectively (9 and 16% relative to those for PPi hydrolysis). The results obtained demonstrate that enzyme-catalyzed synthesis of pyrophosphate can proceed at a high rate in the absence of any external energy input, such as that provided by the proton-motive force in membrane systems.

Animals↗

PubMed1976

Cr(III) complexes as active site probes of yeast inorganic pyrophosphatase.

The inert coordination complex of Cr(III) with PP1 (CrPP1) is not a substrate for yeast inorganic pyrophosphatase and does not inhibit the Mg2+-dependent activity of this enzyme. These results, when compared with the effects of CrPP1 on other enzymes and with the effects of other divalent metal ions on this enzyme, suggest that yeast inorganic pyrophosphatase binds substraather than the phosphate groups. Mg2+ partially protects against a slow inactivation of the enzyme by Cr(H2O)6(3)+; this exchange inert ion may provide a means of labeling the metal ion binding site of this enzyme.

Binding Sites↗

PubMedDate not supplied

[An ultramicro method for determining pyrophosphatase using malachite green B].

A modified sensitive micromethod is described for estimation of pyrophosphatase activity both in homogenate and suspensions of nuclear and mitochondrial fractions of rat liver tissue using a kit of reagents containing malachite green B. The analysis may be carried out in the preparation containing only 0.2 mg of biological material; the procedure is reproducible, rapid and allows to the requisite amount of reagents to be decreased. The activity of pyrophosphatase in homogenate, nuclear and mitochondrial fractions of rat liver tissue was higher at acid pH values as compared with that in the neutral medium; the highest enzymatic activity was detected in the nuclear fraction.

Animals↗

PubMed1995

Induction of geranyl pyrophosphate pyrophosphatase activity by cholesterol-suppressive isoprenoids.

Diets supplemented (1 mmol/kg) with thymol, carvacrol, and beta-ionone significantly decreased the serum cholesterol levels of cockerels. These mevalonate-derived end products of plant secondary metabolism (isoprenoids) had no impact on two cytosolic prenyl alcohol (and ethanol) dehydrogenase activities; each treatment increased microsomal geranyl pyrophosphate pyrophosphatase activity by greater than twofold. The structural diversity of the isoprenoids which suppress cholesterol synthesis may be reconciled by their ability to increase pyrophosphatase activity, thus leading to the production of the endogenous, post-transcriptional regulator of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Animals↗

PubMed1970

Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes.

Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.

Animals↗

PubMed1979

Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence.

Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species-specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence.

Journal Article↗

PubMed1977

Characterization of the m7G(5')pppN-pyrophosphatase activity from HeLa cells.

The m7(G(5')pppN-pyrophosphatase activity previously detected in HeLa cells has been further characterized. Results from DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one enzyme activity in HeLa cell extracts which was capable of selectively hydrolyzing m7G(5')pppN to yield m7pG + ppN (where N = 2'-O-methylated or unmethylated ribonucleosides or oligonucleotides of up to 8 to 10 nucleosides in length). The majority (approximately 95%) of this activity was found in the cytoplasmic extract but appeared not to be associated with the lysosomal fraction. m7G(5')pppG was hydrolyzed by the partially purified enzyme in the absence of divalent cations at a pH optimum of 7.5 and a temperature optimum of 45 degrees, with a Michaelis constant (Km) of 1.7 micronM. Sedimentation analysis and gel filtration showed the molecular weight of the enzyme as approximately 81,000. Inhibition studies testing the effect of a number of prospective substrates on the rate of m7G(5')pppG hydrolysis have confirmed the importance of the methyl moiety at the N7 position of guanosine for enzyme-substrate interaction. Furthermore, the trimethylated guanosine-containing 5'-terminal structure derived from U-2 RNA was found not to serve as substrate, and 7-methylinosine, unlike 7-methylguanosine, was not an effective inhibitor of m7G(5')pppG hydrolysis. Thus, the 2-amino group of the 7-methylguanosine portion of m7G(5')pppN is also important for substrate interaction with this specific pyrophosphatase.

Cytoplasm↗

PubMed1993

Pyrophosphatase-based enzyme-linked immunosorbent assay of total IgE in serum.

Human IgE was purified to near homogeneity by a two-step procedure consisting of immunoaffinity chromatography and high-performance liquid chromatography. Mouse hybridoma cell lines secreting antibodies against IgE were generated. Monoclonal and polyclonal antibodies were used to develop a "sandwich"-type ELISA for determining total IgE in human serum. Inorganic pyrophosphatase (EC 3.6.1.1), an enzyme having a high turnover number, was used as the label. The mean analytical recovery of our ELISA was 95.2% and the results showed good linear correlation with an established RIA of IgE. We found pyrophosphatase to be a good alternative label for use in immunoassays.

Animals↗

PubMed1963

Localization of thiamine pyrophosphatase activity in the golgi apparatus of a mollusc, Helix aspersa.

Spermatids of the snail Helix aspersa were studied after fixation in buffered osmium tetroxide and after applying Novikoff and Goldfischer's method (15) for demonstrating thiamine pyrophosphatase (TPPase) activity both with the light and the electron microscope. The appearance of cells in the light microscope after localizing the enzyme is very similar to the appearance after the application of classical Golgi techniques. The electron microscope shows the "dictyosomes" to consist of non-granular membranes, vesicles, and vacuoles typical of the ultrastructure of the Golgi apparatus. Sites of TPPase activity are localized by deposits of lead phosphate, and are found between the membranes of the Golgi apparatus, in the small vesicles, in multivesicular bodies often found associated with it, but not within the large Golgi vacuoles. Heavy deposits are found on the caudal part of the nuclear envelope, but not in the acrosomal granule. It is suggested that TPPase may act as an intermediary in acrosome formation by the Golgi apparatus or "acroblast" of this cell. The finding of diphosphatase activity in the Golgi apparatus of an invertebrate is suggested as additional evidence for the existence of a homology between the Golgi apparatus of all animal cells.

Animals↗

PubMed1983

Histochemical studies on the distribution of thiamine pyrophosphatase and enzymes related to carbohydrate metabolism in the intercalated neurons of the rat supraoptic nucleus.

Histochemical studies have been conducted by applying hexokinase (HK), aldolase (AD), glyceraldehyde-3-phosphate dehydrogenase (G3), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PD), and thiamine pyrophosphatase (TPPase) methods, as well as Nissl staining and Gomori's chrome-alum-hematoxylin-phloxine (CHP) methods to intercalated neurons of the supraoptic nucleus (SO) on Wistar strain rats. Intercalated neurons reacted weakly to the AD, G3, G6PD, and SDH tests, indicating that they belong to the category of ordinary neurons with low carbohydrate metabolism. Many fibrous astrocytes showing strong HK reactions surround neurosecretory neurons. However, they do not surround intercalated neurons with mild HK activity. These results indicate that the latter receive a poor supply of energy from glucose in the circulating blood in contrast to the former. Intercalated neurons are very rich in Nissl substance but lack CHP-positive material. They may have a high potential for synthesizing protein. The principal morphological features of the TPPase-positive Golgi material are peculiar and heterogeneous shape and poor development. These findings together with mild G6PD activity suggest that intercalated neurons are very likely to have poor synthesizing activity.

Animals↗

PubMed1981

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and cytidine monophosphatase (CMPase) in the Golgi apparatus of early spermatids of the rat.

At the early steps 3-7 of spermiogenesis the hemispherical Golgi apparatus elaborates and is closely associated to the acrosomic system which grows at the surface of the spermatid's nucleus. It shows two distinct zones, a cortex made up of flattened saccules and related membranous tubules, and a medulla containing various types of vesicular profiles. The various components of the cortex of the Golgi apparatus were tested for their reactivity to three phosphatases. Nicotinamide adenine dinucleotide phosphatase activity (NADPase, Smith, 1980) was observed in the middle two to six saccules in the stack with a midsaccule being more reactive than the saccules above and below. A weak and spotty reaction was also noted in the remaining saccules on the trans-face of the stack and in the thick elements making up the GERL on the trans aspect of the stacks of saccules. Thiamine pyrophosphatase activity (TPPase, Novikoff and Goldfisher, 1961) was found in one or two saccules on the trans-face of the stacks but was absent from the other Golgi components. Cytidine monophosphatase activity (CMPase, Novikoff, 1967) was observed in the GERL, in vesicles of the medulla and in the developing acrosomic system. In the intersaccular regions of the cortex the branching membranous tubules showed the same reactivity for the phosphatases to that of the saccules to which they are connected. ER cisternae associated with the Golgi apparatus, anastomotic membranous tubules seen in the peripheral Golgi region, small vesicles, as well as the first saccule on the cis-face of the stacks were all negative for the three enzymes studied. These data indicated that in the cortex of the Golgi apparatus there were several distinct compartments that could be distinguished on the basis of structural and cytochemical features.

Animals↗

PubMed1995

H+/PPi stoichiometry of membrane-bound pyrophosphatase of Rhodospirillum rubrum.

Two kinetic methods have been used to measure the H+/PPi stoichiometry in the chromatophores of the photosynthetic bacteria Rhodospirillum rubrum. In the first method, the fluorescent probe acridine orange was employed to infer the proton pump activity at the steady state of the delta pH generation. At this point the translocation of protons by the H(+)-PPiase in one direction is balanced exactly by the leak of protons in the opposite direction. Pyrophosphatase activity was then quickly stopped by adding EDTA, producing a relaxation of delta pH. From the initial rate of this relaxation and the rate of PPi hydrolysis measured under the same condition, the H+/PPi stoichiometry was obtained. In the second method, a mathematical model was used to describe the time course of delta pH formation. In the two methods an apparent H+/PPi stoichiometry of nearly 2 was obtained. The H+/ATP stoichiometry was determined also as an internal control, giving a value of nearly 3.6, which is in agreement with the value in different F-type H(+)-ATPases.

Adenosine Triphosphate↗

PubMed1997

Purification and characterization of thylakoid membrane-bound inorganic pyrophosphatase from Spinacia oleracia L.

An inorganic pyrophosphatase (PPase) was purified from thylakoid membrane of spinach leaves to electrophoretic purity by methods including detergent solubilization, ammonium sulfate fractionation, and successive chromatographic techniques. Current protocol yielded about 10% recovery of total activity with a 30-fold purification. The specific activity of the purified enzyme was approximately 400 micromol PPi consumed/mg protein x h. This enzyme is a monomer with a molecular mass of 55 kDa. Several properties, including subunit composition, substrate specificity, ion requirements, inhibitor sensitivities, and amino acid composition, have been studied. Mg2+ is an essential cofactor for the thylakoid PPase. The preferred substrate for the hydrolytic reaction of PPase appears to be dimagnesium pyrophosphate. K+ could not stimulate the enzymatic activity of thylakoid PPase, while F- was a potent inhibitor. Group-specific modification of the thylakoid PPase demonstrates possible involvement of carboxylate residues in the enzymatic activity. Furthermore, antibodies raised against thylakoid PPase in a rabbit could inactivate the PPi hydrolysis of thylakoid and the purified enzyme, but not that of vacuolar H+-PPase, indicating both PPi hydrolases are structurally distinct.

Amino Acids↗

PubMed1997

Purification and characterization of hepatic inorganic pyrophosphatase hydrolyzing imidodiphosphate.

A 56-kDa inorganic pyrophosphatase consisting of 33-kDa subunits was purified from bovine liver almost to homogeneity. This hydrolase required divalent cations such as MgCl2, CoCl2, and MnCl2 to hydrolyze PPi and was insensitive to 2 mM sodium fluoride. The purified hydrolase released 2.1 mumol Pi from PPi per minute per milligram of protein in the presence of 1 mM MgCl2, and the Km for PPi was 0.14 mM. It also hydrolyzed imidodiphosphate to yield Pi and ammonia even in the absence of a divalent cation. The purified hydrolase liberated 2.2 mumol Pi from imidodiphosphate per minute per milligram of protein and the Km for imidodiphosphate was 0.12 mM. Addition of 80 microM MgCl2, CoCl2, or MnCl2 to the reaction mixture increased the hydrolysis rate of imidodiphosphate by 1.5-, 2.0-, and 3.4-fold, respectively. In the absence of divalent cations, the enzymatic hydrolysis of imidodiphosphate was inhibited competitively by PPi (Ki = 0.13 mM). Moreover, one-half of the maximal hydrolysis of imidodiphosphate was inhibited by 10 microM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid or 45 microM p-chloromercuriphenyl sulfonate. When the hydrolase was treated with heat or SDS, two activities capable of hydrolyzing PPi and imidodiphosphate gave similar inactivation profiles, indicating that one hydrolase participated in the hydrolysis of both substrates.

Animals↗

PubMed1994

Inorganic pyrophosphatase-based detection systems. II. Detection and quantification of cell lysis and cell-lysing activity.

A novel technique, useful for detection of cell lysis and cell-lysing activity, has been developed. The method can be used for detection of cell lysis, both induced and natural, in all types of cells. The technique can be used for detection and quantification of all types of cell-lysing activities, e.g., cell wall hydrolases, toxicants, phospholipases, and antibiotics. The method relies on the detection, by a very sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA; P. Nyrén and A. Lundin (1985) Anal. Biochem. 151, 504-509) of an enzyme, inorganic pyrophosphatase, which is constitutively expressed in all cells. The fraction of lysed cells in a sample can be assessed by determining the activity in the absence and in the presence of total lysing activity. The technique was used for determination of the effect of storage conditions on the intactness of two different cells, Micrococcus luteticus and Saccharomyces cerevisiae. In a model system, the approach was also used for detection of cell-lysing activity. The activity of a cell wall hydrolase (lysozyme) and a surfactant (Triton X-100) was quantified. M. luteticus cells were incubated with a specific buffer containing the lysing activity and inorganic pyrophosphate. The amount of unhydrolyzed PPi was determined by the ELIDA. The amount of PPi hydrolyzed was proportional to the amount of lysozyme present. The sensitivity of the assay was dependent on several factors, such as amount of cells used, incubation time, and incubation temperature. Lysozyme at concentrations below 5 ng/ml (< 50 pg) could be detected. The possibility of using the approach for detection of other types of cell-lysing activities is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriolysis↗

PubMed2002

Bacillus subtilis inorganic pyrophosphatase: the C-terminal signature sequence is essential for enzyme activity and conformational integrity.

Bacillus subtilis inorganic pyrophosphatase is the first member of a newly identified Family II of PPases. To examine the role of a signature sequence found near the C-terminus, two truncated variants and a series of site-specific mutants were produced. A truncation of 17 residues (17AATR) but also single alanine substitutions, R295A and K296A, produced inactive enzyme. Removal of 5 nonconserved terminal residues (5AATR) markedly affected enzyme stability. Replacing S294 with A, T, C, or V decreased activity, the latter two mutations showing the greatest effect. Substitutions V299I and V300I had no or minor effects, whereas V300W and V299G/V300W significantly reduced activity. The sizes of truncated proteins and the full-length PPase were indistinguishable by gel-filtration. We conclude that the C-terminus has no role in multimerization, while both its conserved and nonconserved regions are essential for full enzyme activity. The signature sequence is required for both the conformation and composition of the active site.

Bacillus subtilis↗

PubMed1998

The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications.

We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 A and R-factors of 19.5% and 18.3%, respectively. In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile. The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site. These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the "two-metal ion" mechanism of polymerases.

Amino Acid Sequence↗

PubMed2000

High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example.

This paper describes the development of a new, malachite green based, enzymatic assay for the identification of specific inhibitors of inorganic pyrophosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug discovery. The human iPPase was used as counterscreen. The coding regions of both enzymes were amplified, cloned into a vector providing a His-tag at the C-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. Since the complete human sequence had not been published previously, the human iPPase was cloned on the basis of expressed sequence tag data. The human sequence was confirmed and showed about 55% amino acid identity with the yeast enzyme and 95% identity with an already published bovine enzyme. Both recombinant iPPases were characterized with regard to their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spectrophotometrically. The assay was adapted to a microtiter plate format. Thus, it is possible to screen a large compound pool for iPPase inhibitors in a short period of time.

Amino Acid Sequence↗