Characterization of a unique class C acid phosphatase from Clostridium perfringens.

Science.gov (United States)

Reilly, Thomas J; Chance, Deborah L; Calcutt, Michael J; Tanner, John J; Felts, Richard L; Waller, Stephen C; Henzl, Michael T; Mawhinney, Thomas P; Ganjam, Irene K; Fales, William H

2009-06-01

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

Science.gov (United States)

Oguro, Ami; Imaoka, Susumu

2012-03-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

Influence of acid phosphatase activity on the saccharification of potato maltodextrins by Aspergillus niger glucoamylase

Energy Technology Data Exchange (ETDEWEB)

Zyla, K. (Akademia Rolnicza, Cracow (Poland). Dept. of Biotechnology)

1990-01-01

A preparation of Aspergillus niger acid phosphatase, which had the temperature optimum 60deg C, pH optimum 1.8-3.0; good stability at pH 4-5, the ability to hydrolyze glucose-6-phosphate at a high rate, and substantial lack of glucogenic activities, was used simultaneously with a glucoamylase in order to learn its influence on the saccharification of potato maltodextrins. The addition of the acid phosphatase activity in amounts that gave the 50 fold increase, as compared to phosphatase activity which naturally occurs in the gluocoamylase (GA) preparation 'AMG-200', was found to influence on the DE level, mainly at the high substrate concentration (40% d.s.) and low glucoamylase dosage (60-100 GAU/kg d.s.). It may also be possible, when using the acid phosphatase addition, to shorten the saccharification time. (orig.).

Phosphotyrosine as a substrate of acid and alkaline phosphatases.

Science.gov (United States)

Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

1985-01-01

A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

Study on alkaline and acid phosphatase activity in acute uranium intoxication

International Nuclear Information System (INIS)

Bokova, N.V.; Pavlova, V.B.; Stancheva, Yu.A.; Khadzhirusev, S.B.; Kiradzhiev, G.D.

1975-01-01

The protective potential of diethyl barbituric acid sodium salt is studied, in comparison with that of acetazolamide, on kidneys under acute uranium intoxication. Experiments involved rats given intraperitoneal injections with uranyl acetate on 12 successive days up to a total dose of 0.5, 2.0 or 7.0 mg/kg. The resulting effects are measured by chemical assays of serum and urine for alkaline and acid phosphatase and histochemical assays for phosphatase activities in kidneys, kinetics being followed over a 30-day period after total dose administration. Protection of kidneys from toxic uranium effects was found to be of about the same degree with sodium diethyl barbiturate as with acetazolamide. (A.B.)

Purification and properties of acid phosphatase from Avena elatior L. seeds

Directory of Open Access Journals (Sweden)

E. Wieczorek

2015-01-01

Full Text Available Acid phosphatase F1 from Avena elatior seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn2+, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

Acid phosphatase and lipid peroxidation in human cataractous lens epithelium

Directory of Open Access Journals (Sweden)

Vasavada Abhay

1993-01-01

Full Text Available The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium. In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium. From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

Science.gov (United States)

Kaur, Parvinder; Satyanarayana, T.

The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

Phosphatase activity of Poa pratensis seeds. III. Effect of fluoride, citrate, urea and other substances on the activity of acid phosphatase Ia2 and Ia3

Directory of Open Access Journals (Sweden)

Irena Lorenc-Kubis

2015-01-01

Full Text Available Effects of fluoride, citrate, urea and other substances on the activity of acid phosphatase a2 and a3 toward p-nitrophenylphosphate and phenylphosphate were investigated. Both enzymes were inhibited by fluoride, p-chloromercuribenzoate and oxalate. Fluoride inhibited acid phosphatase a2 noncorapetitively with p-mitrophenylphosphate, whereas acid phosphatase a3 showed inhibition of mixed type. Hydrolysis of phenylphosphate by both acid phosphatases was activated by citrate. Cytosine and uridine inhibited the activity of phosphatase a2 toward p-nitrophenylphosphate and phenylphosphate, but no effect was observed in case of acid phosphatase a3. After 30 min. incubation with 4 M urea both enzymes lost about 30% of activity.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

Science.gov (United States)

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens▿

Science.gov (United States)

Reilly, Thomas J.; Chance, Deborah L.; Calcutt, Michael J.; Tanner, John J.; Felts, Richard L.; Waller, Stephen C.; Henzl, Michael T.; Mawhinney, Thomas P.; Ganjam, Irene K.; Fales, William H.

2009-01-01

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class. PMID:19363079

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

Science.gov (United States)

Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

1996-06-01

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

International Nuclear Information System (INIS)

Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

1988-01-01

Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis

International Nuclear Information System (INIS)

Singh, Harkewal; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

2011-01-01

Methods for the expression, purification and crystallization of the class C acid phosphatase from M. bovis are reported. This enzyme is atypical in that it is nearly 20 kDa larger than other known class C acid phosphatases. Class C acid phosphatases (CCAPs) are 25–30 kDa bacterial surface proteins that are thought to function as broad-specificity 5′,3′-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers

The tillage effect on the soil acid and alkaline phosphatase activity

Directory of Open Access Journals (Sweden)

Lacramioara Oprica

2011-12-01

Full Text Available Phosphatases (acid and alkaline are important in soils because these extracellular enzymes catalyze the hydrolysis of organic phosphate esters to orthophosphate; thus they form an important link between biologically unavailable and mineral phosphorous. Phosphatase activity is sensitive to environmental perturbations such as organic amendments, tillage, waterlogging, compaction, fertilizer additions and thus it is often used as an environmental indicator of soil quality in riparian ecosystems. The aim of the study was to assess the effect of tillage systems on phosphatases activity in a field experiment carried out in Ezăreni farm. The phosphatase activitiy were determined at two depths (7-10 cm and 15-25cm layers of a chernozem soil submitted to conventional tillage (CT in a fertilised and unfertilised experiment. Monitoring soil alkaline phosphatase activity showed, generally, the same in fertilized soil profiles collected from both depths; the values being extremely close. In unfertilized soils, alkaline phosphatase activity is different only in soils that were exposed to unconventional work using disc harrows and 30cm tillage. Both works type (no tillage and conventional tillage cause an intense alkaline phosphatase activity in 7-10 cm soil profile. Acid phosphatase activity is highly fluctuating in both fertilized as well unfertilized soil, this enzyme being influenced by the performed works.

[Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

Science.gov (United States)

Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

2004-11-01

Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

Effect of vanadium compounds on acid phosphatase activity

OpenAIRE

Vescina, Cecilia M.; Sálice, Viviana C.; Cortizo, Ana María; Etcheverry, Susana B.

1996-01-01

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activi...

Phosphatase activity of Poa pratensis seeds. II. Purification and characterization of acid phosphatase Ia2 and Ia3

Directory of Open Access Journals (Sweden)

I. Lorenc-Kubis

2015-01-01

Full Text Available Two acid phosphatases (Ia2, Ia3 have been isolated from Poa pratensis seeds and partially purified. Both enzymes showed maximal activity at pH 4,9. They exhibited high activity towards p-nitrophenyl phosphate, inorganic pyrophosphate and phenyl phosphate, much less activity towards glucose-6 phosphate, and mononucleotides. Phosphatases a2 and a3 differed in their activity towards ADP. Orthophosphate, fluoride and Zn2+ were effective inhibitors. EDTA, β-mercaptoethanol and Mg2+ activated phophatase a2 but had no effect on phosphatase a3. Zn2+ inhibited the activity of phosphatase a2 noncompetitively, whereas phosphatase a3 showed inhibition of mixed type. Trypsin, chymotrypsin and pronase had no effect on the enzyme activities of both molecular forms.

Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

Science.gov (United States)

Lee, Eunhee; Stafford, Walter F

2015-01-01

Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

Directory of Open Access Journals (Sweden)

Ely Nahas

2015-10-01

Full Text Available ABSTRACTThis work studied the acid phosphatase (APase activity from culture medium (extracellular, eAPase and mycelial extract (intracellular, iAPase ofAspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with Vmax = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH 2PO4 and 5 mM (NH42MoO4; eAPase was also inhibited by 1 mM CoCl2.

Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

Science.gov (United States)

Waymack, P P; Van Etten, R L

1991-08-01

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

Science.gov (United States)

Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

2010-01-01

This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

Acid phosphatase from stored Poa pratensis caryopses and its ability for binding to lectins

Directory of Open Access Journals (Sweden)

Irena Lorenc-Kubis

2014-01-01

Full Text Available The effect of the storage period of Poa pratensis caryopses on acid phosphatase activity and on the ability of this enzyme to interact with lectins has been studied. It has been shown that after ten years of caryopses storage, the activity of acid phosphatase decreased about 50 per cent, while the content of proteins and carbohydrates did not change. The decrease of enzyme activity during the long period of storage was found only in seeds, but not in chaffs. Acid phosphatase was isolated from caryopses stored one, two, three, five and ten years. The enzyme showed the ability to bind to immoblized as well as to free conA during the whole period of storage, hut did not react with Wheat Germen Agglutinin (WGA. The activation of acid phosphatase by binding to conA decreased with the length of storage period.

Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

Energy Technology Data Exchange (ETDEWEB)

Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

1991-09-11

The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

Phosphate activity of Poa pratensis seeds. III. Effect of fluoride, citrate, urea and other substances on the activity of acid phosphatase Ia/sub 2/ and Ia/sub 3/

Energy Technology Data Exchange (ETDEWEB)

Lorenc-Kubis, I.; Morawiecka, B.

1978-01-01

Effects of fluoride, citrate, urea and other substances on the activity of acid phosphatase a/sub 2/ and a/sub 3/ toward p-nitrophenylphosphate and phenylphosphate were investigated. Both enyzmes were inhibited by fluoride, p-chloro-mercuribenzoate and oxalate. Fluoride inhibited acid phosphatase a/sub 2/ non-competitively with p-nitrophenylphosphate, whereas acid phosphatase a/sub 3/ showed mixed type inhibition. Hydrolysis of phenylphosphate by both acid phosphatases was activated by citrate. Cytosine and uridine inhibited the activity of phosphatase a/sub 2/ toward p-nitrophenylphosphate and phenylphosphate, but no effect was observed in case of acid phosphatase a/sub 3/. After 30 min. incubation with 4 M urea both enzymes lost about 30% of their activity. 11 references, 5 figures, 1 table.

Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India

Directory of Open Access Journals (Sweden)

B.C. Behera

2017-06-01

Full Text Available Phosphorus is an essential element for all life forms. Phosphate solubilizing bacteria are capable of converting phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-37, was isolated from mangrove soil of the Mahanadi river delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Serratia sp. The maximum phosphate solubilizing activity of the strain was determined to be 44.84 μg/ml, accompanied by a decrease in pH of the growth medium from 7.0 to 3.15. During phosphate solubilization, various organic acids, such as malic acid (237 mg/l, lactic acid (599.5 mg/l and acetic acid (5.0 mg/l were also detected in the broth culture through HPLC analysis. Acid phosphatase activity was determined by performing p-nitrophenyl phosphate assay (pNPP of the bacterial broth culture. Optimum acid phosphatase activity was observed at 48 h of incubation (76.808 U/ml, temperature of 45 °C (77.87 U/ml, an agitation rate of 100 rpm (80.40 U/ml, pH 5.0 (80.66 U/ml and with glucose as a original carbon source (80.6 U/ml and ammonium sulphate as a original nitrogen source (80.92 U/ml. Characterization of the partially purified acid phosphatase showed maximum activity at pH 5.0 (85.6 U/ml, temperature of 45 °C (97.87 U/ml and substrate concentration of 2.5 mg/ml (92.7 U/ml. Hence the present phosphate solubilizing and acid phosphatase production activity of the bacterium may have probable use for future industrial, agricultural and biotechnological application.

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

Science.gov (United States)

Zylka, Mark J.; Sowa, Nathaniel A.; Taylor-Blake, Bonnie; Twomey, Margaret A.; Herrala, Annakaisa; Voikar, Vootele; Vihko, Pirkko

2008-01-01

SUMMARY Thiamine monophosphatase (TMPase, also known as Fluoride-Resistant Acid Phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of Prostatic Acid Phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent anti-nociceptive, anti-hyperalgesic and anti-allodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5’-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. PMID:18940592

Diagnosis of prostate cancer using a radioimmunoassay for prostatic acid phosphatase in serum

International Nuclear Information System (INIS)

Lea, O.A.; Hoeisaeter, P.Aa.

1981-01-01

The paper describes the development and evaluation of a specific radioimmunoassay for the determination of prostatic acid phosphatase in serum as a useful aid in the detection of prostatic cancer. (Auth.)

Comparative studies on the carbohydrate, protein and acid phosphatase contents in seeds of some rye (Secale cereale varieties

Directory of Open Access Journals (Sweden)

B. Morawiecka

2015-01-01

Full Text Available The contents of sugars. proteins and acid phosphatase extracted with 0.1 M acetate buffer, pH 5.1, from some rye varieties were determined. The total sugar level amounted to 3.25-9.70 g per 100 g of seeds; the estimates for pentoses were 1.7-2.9 g and those for proteins 0.91-1.60 g per 100 g of seeds. Acid phosphatase showed and activity level between 0.18 and 1.26 units/mg protein. After disc electrophoresis proteins were separated into 10 to 11 bands: at pH 9.4 or into 4-7 bands at pH 3.8. Essential variety differences were expressed in protein patterns after electrophoresis at pH 3.8. Acid phosphatase was separated into 5 and 4 activity bands at pH 9.4 and 3.3,. respectively. No variations in zymogram patterns were observed in respect to variety differences or cultivation in various climate and soil conditions.

Effect of vanadium compounds on acid phosphatase activity.

Science.gov (United States)

Vescina, C M; Sálice, V C; Cortizo, A M; Etcheverry, S B

1996-01-01

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.

The effect of potassium iodide on the production of acid phosphatase by Sporothrix schenckii

Directory of Open Access Journals (Sweden)

P. S. Grover

2003-06-01

Full Text Available The present study was undertaken to find out the in vitro effect of potassium iodide (KI on the production of acid phosphatase by fully characterized strain of S.schenckii isolated from a patient of Cutaneous Sporotrichosis. The enzyme acid phosphatase was estimated during the 3 phases of growth of S.schenckii, without and with three concentrations of KI incorporated in the culture medium. In the control and in the test proper, with various concentrations of KI, no adverse effect of KI was observed on the production of acid phosphatase in early and mid log phase of fungal growth. Whereas in the exponential phase in test proper, there was a statistical significant decrease in the enzyme production with 0.8% and 3.2% of KI. The low activity at 0.8% and 3.2% KI indicates that KI has inhibitory effect on the growth of S.schenckii and has led to decrease in the activity of the enzyme. (Med J Indones 2003; 12: 65-8 Keywords: S.schenckii, acid phosphatase, potassium iodide

Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone

Science.gov (United States)

Cotter, David A.; Martel, Anita J.; MacDonald, Paul

1975-01-01

Decryptification of acid phosphatase in Geotrichum sp. arthrospores was accomplished using acetone or dimethyl sulfoxide treatment. Both dimethyl sulfoxide and acetone irreversibly destroyed the integrity of the spore membranes without solubilizing acid phosphatase. PMID:1167386

Acid phosphatase patterns in microfilariae of Onchocerca volvulus s.l. from the Upper Orinoco Basin, Venezuela.

Science.gov (United States)

Yarzàbal, L; Petralanda, I; Arango, M; Lobo, L; Botto, C

1983-06-01

The patterns of acid phosphatase in strains of Onchocerca volvulus s.l. which parasitize an Amerindian population (Yanomami) in Venezuela's Upper Orinoco Basin were examined by using the naphthol AS-TR phosphate method. The study sample consisted of 40 Yanomami inhabiting a savannah area at 950 m above sea level and 21 Yanomami residents of a tropical rainforest area at an altitude of 250 m. Stained intrauterine microfilariae, still within the egg case, exhibited a diffuse distribution of the enzyme in the early stages of embryonic development and a negative reaction at a more developed stage. Four of the five enzyme staining patterns described by Omar (1978) were found in the 3157 microfilariae examined from skin snips. Their distribution was: Type I--17.2%, Type III--0.5%, Type IV--75.6% and Type V--6.6%. No examples of Type II were observed. The results indicate that acid phosphatase patterns of the Upper Orinoco Onchocerca strain most resemble those of strains from Guatemala and Yemen, and are different from the African strains found in Upper Volta and Liberia. The relative frequency of acid phosphatase patterns was modified by cryopreservation of microfilariae.

Identification of genes required for secretion of the Francisella oxidative burst-inhibiting acid phosphatase AcpA

Directory of Open Access Journals (Sweden)

John S Gunn

2016-04-01

Full Text Available Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.

Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay

International Nuclear Information System (INIS)

McCarthy, R.C.; Jakubowski, H.V.; Markowitz, H.

1983-01-01

Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with α-naphthyl phosphate at 30 0 C and para-nitrophenyl phosphate at 37 0 C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 μg of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9-7.7% with serum at 1.3 to 5.6 μg PAP/l. (Auth.)

Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

Science.gov (United States)

Imai, K; Yoshimura, T

1994-08-01

Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

Science.gov (United States)

Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

1993-06-01

The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain.

Directory of Open Access Journals (Sweden)

Marie-Claude Gingras

Full Text Available The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP. To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported.Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status.In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

Purification of acidic phosphatase from mustard seedlings

OpenAIRE

sprotocols

2014-01-01

### INTRODUCTION Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases...

EFEITO DE FATORES AMBIENTAIS DA FOSFATASE ÁCIDA NO FEIJOEIRO EFFECTS OF ENVIRONMENTAL FACTORS ON THE ACTIVITY OF ACID PHOSPHATASE IN COMMON BEAN

Directory of Open Access Journals (Sweden)

José Renato de Freitas

2007-09-01

Full Text Available

Plantas com 15 dias após a germinação foram colhidas em experimentos de campo com a finalidade de conhecer o pH, temperatura e tempo necessários para melhor expressar a atividade da fosfatase ácida em três variedades do feijoeiro (Phaseolus vulgaris L., Carioca, EMP-84 e CNF-l0, na presença e na ausência de fósforo. Os maiores valores de atividade da fosfatase ácida foram observadas quando as plantas foram colocadas em solução em pH 5,5 durante 120 minutos à temperatura de 30°C. A utilização de substâncias tamponantes como PNPP + Triton X-100 expressaram melhor a atividade da fosfatase ácida. As condições de vácuo constituíram um fator positivo para a atividade da fosfatase ácida. As plantas desenvolvidas sob estresse hídrico apresentaram menor atividade da fosfatase ácida. A relação folha-raiz da atividade da fosfatase ácida atingiu 5,72 para a variedade Carioca, 4,91 para a variedade EMP-84 e 4,36 para a variedade CNF-10.

PALAVRAS-CHAVE: pH; temperatura; solução tamponada; tempo de reação; Phaseolus vulgaris.

Plants with 15 days after the germination were picked in field experiments with the purpose of knowing the best pH, temperature and the necessary time to express the activity of the phosphatase acid in three bean varieties (Phaseolus vulgaris L., Carioca, EMP-84 and CNF-10, in the presence and in the phosphorus absence. The largest values of activity of the phosphatase acid were observed when the plants were tested in pH 5.5 solution during 120 minutes at the temperature of 30°C. The use of buffer substances as PNPP + Triton X-100 expressed better the activity of the phosphatase acid. The vacuum condition constituted a positive factor to express the activity of the phosphatase acid. The plants

Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

Directory of Open Access Journals (Sweden)

Norris-Mullins Brianna

2014-01-01

Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

Inhibition of hydrolytic enzymes by gold compounds. I. beta-Glucuronidase and acid phosphatase by sodium tetrachloroaurate (III) and potassium tetrabromoaurate (III).

Science.gov (United States)

Lee, M T; Ahmed, T; Friedman, M E

1989-01-01

Purified bovine liver beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32) and wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) were inhibited with freshly dissolved and 24 h aquated tetrahaloaurate (III) compounds. Rate and equilibrium inhibition constants were measured. From this data two acid phosphatases species were observed. Equilibrium inhibition constants ranged from 1 to 12.5 microM for the various gold compounds toward both enzymes. The first order rate constants ranged between 0.005 and 0.04 min.-1 for most reactions with the exception of the fast reacting acid phosphatase which had values as high as 2.6 and 2.8 min.-1. It is observed that the beta-glucuronidase is rapidly inhibited during the equilibrium phase before the more slower reaction covalent bond formation takes place. The acid phosphatases form the covalent bonds more rapidly, especially the faster reacting species suggesting a unique difference in the active site geometry to that of the more slowly reacting species. The tightly bonded gold (III)-enzyme complex is probably the reason for its toxicity and non-anti-inflammatory use as a drug.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

OpenAIRE

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

The IBO germination quantitative trait locus encodes a phosphatase 2C-related variant with a nonsynonymous amino acid change that interferes with abscisic acid signaling.

Science.gov (United States)

Amiguet-Vercher, Amélia; Santuari, Luca; Gonzalez-Guzman, Miguel; Depuydt, Stephen; Rodriguez, Pedro L; Hardtke, Christian S

2015-02-01

Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0). IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

Science.gov (United States)

Srivastava, Pramod Kumar; Anand, Asha

2015-01-01

Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

Science.gov (United States)

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Evidence That Speciation of Oxovanadium Complexes Does Not Solely Account for Inhibition of Leishmania Acid Phosphatases

Science.gov (United States)

Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

2018-01-01

Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios. PMID:29707535

Evidence that Speciation of Oxovanadium Complexes does not Solely Account for Inhibition of Leishmania Acid Phosphatases

Science.gov (United States)

Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

2018-04-01

Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios.

Effects of sub-lethal dose of gamma-irradiation on levels of acid phosphatase in cerebellum of pigeons

International Nuclear Information System (INIS)

Shah, V.C.; Gadhia, P.K.

1980-01-01

The changes in the activities of acid phosphatase in the sham-irradiated and γ-irradiated cerebellum of pigeons have been studied both biochemically as well as histochemically after 400 rads. The specific activity of acid phosphatase decreased significantly after 48h and 72h of irradiation. The histochemical observations following total body irradiation confirmed the results obtained by quantitative biochemical studies. (author)

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

Science.gov (United States)

Koller, E; Wolfbeis, O S

1984-11-15

A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

Increased amount of phosphorylated proinflammatory osteopontin in rheumatoid arthritis synovia is associated to decreased tartrate-resistant acid phosphatase 5B/5A ratio.

Directory of Open Access Journals (Sweden)

Jani Luukkonen

Full Text Available Osteopontin (OPN is an immunoregulatory protein which production increases in both rheumatoid arthritis (RA and osteoarthritis (OA. Phosphorylated osteopontin (Phospho-OPN is known to increase macrophage and osteoclast activation, this process is controlled by extracellular tartrate-resistant acid phosphatase (TRAcP, also a biomarker for RA. Here, we evaluated the phosphorylation status of OPN in RA and OA synovia, as well as its correlation with TRAcP isoforms.Synovial tissue and fluid were obtained from 24 RA (14 seropositive and 10 seronegative and 24 OA patients. Western blotting was used to analyze the extent of OPN phosphorylation. TRAcP isoforms were measured in synovial fluid using ELISA; immunohistochemistry assessed the distribution of OPN and TRAcP expressing cells in the synovial tissue, especially distinguishing between the TRAcP isoforms.Full-length OPN was more phosphorylated in RA than in OA (p<0.05. The thrombin cleaved C-terminal end of OPN was also more phosphorylated in RA (p<0.05. RA patients had a lower concentration of TRAcP 5B and higher concentration of less active 5A in their synovial fluid compared to OA patients. The TRAcP 5B/5A ratio was decreased in RA and correlated negatively with the amount of phospho-OPN (p<0.05. TRAcP positive cells for both isoforms were found all along the synovial lining; OPN antibody staining was localized in the extracellular matrix.Our data suggests that in RA the synovial fluid contains insufficient amounts of TRAcP 5B which increase levels of the proinflammatory phospho-OPN. This may lead to increased macrophage and osteoclast activation, resulting in the increased local inflammation and bone resorption present in RA joints.

The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

Directory of Open Access Journals (Sweden)

Bergamaschi Antonio

2008-09-01

Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

Cloning and expression of a widely expressed receptor tyrosine phosphatase

DEFF Research Database (Denmark)

Sap, J; D'Eustachio, P; Givol, D

1990-01-01

We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

Activity of acid phosphatase in tissues of dogs exposed to γ-radiation at low dose rates and treated with adenosine triphosphate

International Nuclear Information System (INIS)

Bichejkina, N.I.; Tikhomirova, M.V.; Romantsev, E.F.; Rogozkin, V.D.

1979-01-01

A study was made of the activity of acid phosphatase in the liver, spleen and blood of dogs under various experimental conditions: (a) exposure to γ-rays at low dose rates, (b) preventive and therapeutic application of ATP and (c) administration of ATP to intact animals. It was demonstrated that the activity of acid phosphatase in the liver and spleen was invariable after the first 24 h and decreased after 72 h of observation in each of the experimental variants. Preventive and therapeutic administration of ATP to dogs not substantially influence the activity of acid phosphatase throughout the entire period of observation

Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

Science.gov (United States)

Cao, Heping; Sethumadhavan, Kandan; Grimm, Casey C; Ullah, Abul H J

2014-01-01

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

Science.gov (United States)

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

Science.gov (United States)

Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

2016-01-01

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

Histochemical study on effects of low dose γ-irradiation on acid phosphatase in liver of the pigeons Columbus livia intermedia Strickland

International Nuclear Information System (INIS)

Gadhia, P.K.; Shah, V.C.

1982-01-01

Effects of total body γ-irradiation with sub-lethal dose (400 rads) on acid phosphatase have been studied in the liver of pigeon. The histochemical study showed increased activity of acid phosphatase in liver after 48 hr and 72 hr of irradiation. (author)

Biosynthesis of acid phosphatase of baker's yeast . Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme

NARCIS (Netherlands)

Boer, Pieter; Rijn, Herman J.M. van; Reinking, A.; Steyn-Parvé, Elizabeth P.

1975-01-01

1. 1.|Yest protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M.; Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431–441). The major part

The effects of drought stress on the activity of acid phosphatase and ...

African Journals Online (AJOL)

A model of drought was created on pigweed and the effects of drought stress on the activity of acid phosphatase and its protective enzymes were examined. The pot-cultured pigweeds were divided into 4 groups (ten plants per group) when they reached 6 leaves. (1) In the control group, the culture media contained 70 ...

Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells.

Science.gov (United States)

Lu, Gang; Sun, Haipeng; She, Pengxiang; Youn, Ji-Youn; Warburton, Sarah; Ping, Peipei; Vondriska, Thomas M; Cai, Hua; Lynch, Christopher J; Wang, Yibin

2009-06-01

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

The effect of water and salt stresses on the phosphorus content and acid phosphatase activity in oilseed rape

Directory of Open Access Journals (Sweden)

Stanisław Flasiński

2014-01-01

Full Text Available Oilseed rape plants responded to water and salt stresses (-0.5 MPa, PEG 6000 and NaCI by reduction of the fresh and dry weights of shoots and roots. When PEG was used, the ratio of dry weights of roots:shoots surpassed that of controls. The leaf protein content increased considerably. The phosphorus content decreased only in the roots, most significantly after three days of stress. Immediately after the stresses were induced, an increase in the acid phosphatase (AP activity was noted. Water and salt stresses caused four- and two-fold increases in AP activity in leaves, respectively. Changes in the enzyme activity were negligible in stems and roots. There are nine forms of AP in young leaves of oilseed rape. In the stressed plants, from No. 5 revealed lower activity and forms Nos 8 and 9, higher activities than in the control. The increase in AP activity was directly accompanied by the decrease in the water potential of the tissues. Oilseed rape is considerably less sensitive to salt stress than to water stress, which is manifested as the lower inhibition of plant growth and also by a smaller increase in acid phosphatase activity.

Studies on alkaline and acid phosphatase activity of neutrophil leukicytes, 2

International Nuclear Information System (INIS)

Niki, Yoko

1983-01-01

With a view to analyzing the inhibiting effect of anticancer drugs and irradiation on hematopoiesis in rabbits neutrophil (pseudoeosinophil) counts and the neutrophilic activities of alkaline phosphatase (AP) and acid phosphatase (SP) were serially followed up after drug administration or irradiation. The enzym activity was estimated histochemically, using azo-dye staining. Each rabbit was given cyclophosphamid (CP) (25mg/kg x 10, at intervals of 5 - 7 days ; 50mg/kg x 5, every day; or 100mg/kg x 1, i.m.), Thio-TEPA (4mg/kg x 1, i.m.), Vinblastin (VBT) (1mg/kg x 1, i.v.), 6MP (25mg/kg x 1, p.o.), or Mitomycin C (MMC) (1.5mg/kg x 1, i.v.). The results obtained were as follows : 1) The neutrophil counts became slightly elevated at 24 hrs, reached their nadir at 48 to 72 hrs, and recovered to normal in 5 to 6 days thereafter, except with 6 MP which produced no significant change but for a temporary elevation after dosages. 2) Except in the group administrated 6MP, which caused no significant hematorogical changes, the AP changes were similar in all of the animal groups : after temporary depression, it became elevated for 5 to 6 days, and recovered to normal about 9 days thereafter. 3) SP showed no changes in the 25mg/kg x 10 CP and the 6MP groups, it became elevated in 2 or 3 days after the administration of MMC, VBT, or Thio-TEPA to recover to normal in 5 to 10 days thereafter. 4) 60 Co irradiation (1,000 rad/whole body x 1) led to a temporary ascent in phil count followed by a descent from the 6th day on, and then a slow recovery to normal. AP was elevated from the third to the sixth days, and, after a depression on the tenth day, it returned to normal 24 days after irradiation, while SP showed a continued elevation from the 2nd to the 13th day. (author)

A Novel Phytase with Sequence Similarity to Purple Acid Phosphatases Is Expressed in Cotyledons of Germinating Soybean Seedlings 1

Science.gov (United States)

Hegeman, Carla E.; Grabau, Elizabeth A.

2001-01-01

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases. PMID:11500558

Simplified preparation of a phosphatase inhibitor and further studies of its action.

Science.gov (United States)

Coburn, S P; Schaltenbrand, W E

1978-05-01

1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.

The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

Science.gov (United States)

Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

2015-01-01

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

Alkaline and Acid Phosphatase Activity in Blood Plasma of Chickens Irradiated by Low dose Gamma Radiation

International Nuclear Information System (INIS)

Petar, K.; Marinko, V.; Saveta, M.; Miljenko, S.

2004-01-01

In our previous paper (Kraljevic et, al, 2000; Kraljevic et al 2002) we showed that the growth of the chickens hatched from eggs irradiated with 0.15 Gy gamma-rays before incubation was significantly higher than in controls during the fattening period (1-42 days). The concentration of total protein, glucose and cholesterol in the blood plasma of the same chickens was also significantly changed. In this paper an attempt was made to determine the effect of irradiation of eggs by low dose ionizing radiation before incubation upon activity of alkaline and acid phosphatase in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breeding chickens were irradiated by dose of 0.15 Gy gamma radiation (60 Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups. After hatching, blood samples were taken from the wing vein on days 1, 3, 5, 6, 10, 20, 30 and 42. The activity of both enzymes was determined spectrophotometrically by using Boehring Mannheim GmbH optimized kits. the activity of alkaline phosphatase in blood plasma was decreased on days 42, and the activity of acid phosphatase in the blood plasma of the same chickens was increased on day 42. Obtained results confirm our early obtained results that low dose of gamma radiation has effects upon metabolic processes in the chickens hatched from eggs irradiated before incubation. (Author)

Detection of phosphatase activity in aquatic and terrestrial cyanobacterial strains

Directory of Open Access Journals (Sweden)

Babić Olivera B.

2013-01-01

Full Text Available Cyanobacteria, as highly adaptable microorganisms, are characterized by an ability to survive in different environmental conditions, in which a significant role belongs to their enzymes. Phosphatases are enzymes produced by algae in relatively large quantities in response to a low orthophosphate concentration and their activity is significantly correlated with their primary production. The activity of these enzymes was investigated in 11 cyanobacterial strains in order to determine enzyme synthesis depending on taxonomic and ecological group of cyanobacteria. The study was conducted with 4 terrestrial cyanobacterial strains, which belong to Nostoc and Anabaena genera, and 7 filamentous water cyanobacteria of Nostoc, Oscillatoria, Phormidium and Microcystis genera. The obtained results showed that the activity of acid and alkaline phosphatases strongly depended on cyanobacterial strain and the environment from which the strain originated. Higher activity of alkaline phosphatases, ranging from 3.64 to 85.14 μmolpNP/s/dm3, was recorded in terrestrial strains compared to the studied water strains (1.11-5.96 μmolpNP/s/dm3. The activity of acid phosphatases was higher in most tested water strains (1.67-6.28 μmolpNP/s/dm3 compared to the activity of alkaline phosphatases (1.11-5.96 μmolpNP/s/dm3. Comparing enzyme activity of nitrogen fixing and non-nitrogen fixing cyanobacteria, it was found that most nitrogen fixing strains had a higher activity of alkaline phosphatases. The data obtained in this work indicate that activity of phosphatases is a strain specific property. The results further suggest that synthesis and activity of phosphatases depended on eco-physiological characteristics of the examined cyanobacterial strains. This can be of great importance for the further study of enzymes and mechanisms of their activity as a part of cyanobacterial survival strategy in environments with extreme conditions. [Projekat Ministarstva nauke Republike

Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

International Nuclear Information System (INIS)

Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

1988-01-01

Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area

Phosphatase activity and culture conditions of the yeast Candida mycoderma sp. and analysis of organic phosphorus hydrolysis ability.

Science.gov (United States)

Yan, Mang; Yu, Liufang; Zhang, Liang; Guo, Yuexia; Dai, Kewei; Chen, Yuru

2014-11-01

Orthophosphate is an essential but limiting macronutrient for plant growth. About 67% cropland in China lacks sufficient phosphorus, especially that with red soil. Extensive soil phosphorus reserves exist in the form of organic phosphorus, which is unavailable for root uptake unless hydrolyzed by secretory acid phosphatases. Thus, many microorganisms with the ability to produce phosphatase have been exploited. In this work, the activity of an extracellular acid phosphatase and yeast biomass from Candida mycoderma was measured under different culture conditions, such as pH, temperature, and carbon source. A maximal phosphatase activity of 8.47×10(5)±0.11×10(5)U/g was achieved by C. Mycoderma in 36 hr under the optimal conditions. The extracellular acid phosphatase has high activity over a wide pH tolerance range from 2.5 to 5.0 (optimum pH3.5). The effects of different phosphorus compounds on the acid phosphatase production were also studied. The presence of phytin, lecithin or calcium phosphate reduced the phosphatase activity and biomass yield significantly. In addition, the pH of the culture medium was reduced significantly by lecithin. The efficiency of the strain in releasing orthophosphate from organic phosphorus was studied in red soil (used in planting trees) and rice soil (originating as red soil). The available phosphorus content was increased by 230% after inoculating 20 days in rice soil and decreased by 50% after inoculating 10 days in red soil. This work indicates that the yeast strain C. mycoderma has potential application for enhancing phosphorus utilization in plants that grow in rice soil. Copyright © 2014. Published by Elsevier B.V.

Acid and Alkaline Phosphatase Levels in GCF during Orthodontic Tooth Movement

Science.gov (United States)

Farahani, Mohammad; Safavi, Seyed Mohammadreza; Dianat, Omid; Khoramian Tusi, Somayeh; Younessian, Farnaz

2015-01-01

Statement of the Problem The present constituents of gingival crevicular fluid (GCF) can reflect the changes occurring in underlying tissues. Considering variety of biologic bone markers, alkaline phosphatase and acid phosphatase have been examined as bone turn over markers in orthodontic tooth movement. Purpose The current study designed in a longitudinal pattern to determine the changes of acid and alkaline phosphatase (ACP & ALP) in GCF during orthodontic tooth movement. Materials and Method An upper canines from twelve patients (mean age: 14±2 years) undergoing extraction orthodontic treatment for distal movement served as the test tooth (DC), and its contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in orthodontic appliance without orthodontic force; the AC was free from any orthodontic appliance. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance placement (T0), and 14 (T2) and 28 days (T3) after it and ALP and ACP concentration were determined spectrophotometrically. Results ALP concentration was elevated significantly in DC and CC groups at days 14 and 28 compared with the AC. In DC group, the ALP was significantly greater in mesial sites than distal site, while no significant changes were found between both sites of CC. The peak level of ALP was observed in mesial sites of DC at T2. Regarding ACP, significant elevation of this enzyme was seen in DC group both in mesial and distal sites at T2 and T3. The peak level of this enzyme was seen at T2. Conclusion Monitoring simultaneous changes of ALP and ACP levels in GCF can reflect the tissue responses occur in periodontium during bone formation and bone resorption during orthodontic tooth movement, respectively. PMID:26535403

Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

Science.gov (United States)

Tomaszewski, M; Buchowicz, J

1971-08-01

The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

The manometric determination of thiamine pyrophosphate and the inhibition of the acid yeast phosphatase

NARCIS (Netherlands)

Steyn-Parvé, Elizabeth P.

1962-01-01

Sodium molybdate is a powerful inhibitor of the acid yeast phosphatase in both fresh baker's yeast and dried brewer's yeast, provided that the yeast is suspended in a suitable buffer. It displays no action in citrate or phosphate buffers, but is active in acetate or maleate buffers, both at the

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

Science.gov (United States)

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

Effect of PUFAs from Pteleopsis suberosa stem bark on androgenic enzymes, cellular ATP and prostatic acid phosphatase in mercury chloride – Exposed rat

Directory of Open Access Journals (Sweden)

J.K. Akintunde

2017-09-01

Full Text Available Occupational and environmental exposure to mercury causes varieties of adverse reproductive disorders in mammals. The present study was designed to investigate the unsaturated fatty acids of Pteleopsis suberosa stem bark extract (PTSSBE, evaluate its antioxidant properties and examine its biochemical targets on sub-acute mercury-induced testicular dysfunctions. Rats were divided into five groups of 10 animals each. Group I was given distilled water; group II, III, IV and V was orally administered with mercury at a dose of 3.75 mg/kg body weight. Group III, IV and V were co-treated with PTSSBE of 25, 50 and 100 mg/kg body weight respectively, for 10 days. Rats exposed to mercury significantly decreased the activities of catalase (CAT, lactate dehydrogenase (LDH, and the level of reduced glutathione (GSH, while the formation of malondialdehyde (MDA was increased. There was also a marked significant decrease (p < 0.05 in testicular activities of Δ5-3β-hydroxysteroid dehydrogenase and Δ5 17β-hydroxysteroid dehydrogenase. Moreover, the activities of prostatic acid phosphatase, total acid phosphatase and prostatic alkaline phosphatase, were significantly (p < 0.05 elevated in mercury treated rats. These effects were prevented by co-treatment with PTSSBE in mercury-induced testicular toxicity in rats. Aphrosidiac effects of Pteleopsis suberosa, may find clinical application in reproductive abnormalities. Isolation and translation of individual active ingredient would help to find new drugs to cure and/or prevent male infertility among mercury exposed workers.

Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae.

Directory of Open Access Journals (Sweden)

Kentaro Nishida

Full Text Available ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E and prostatic acid phosphatase (PAP were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience. On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice.

Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

International Nuclear Information System (INIS)

Dowling, J.N.; Saha, A.K.; Glew, R.H.

1987-01-01

Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

Energy Technology Data Exchange (ETDEWEB)

Pradalier, N; Canal, P; Pujol, A; Fregevu, Y [Groupe de Recherches du Centre Claudius-Regaud, Toulouse (France); Soula, G [Faculte des Sciences Pharmaceutiques, Toulouse (France)

1982-01-01

We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma.

An Additional Method for Analyzing the Reversible Inhibition of an ?Enzyme Using Acid Phosphatase as a Model

OpenAIRE

Baumhardt, Jordan M.; Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

2015-01-01

Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent const...

The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold

Directory of Open Access Journals (Sweden)

Svetlana Vladimirovna Antonyuk

2014-03-01

Full Text Available Phosphatases function in the production, transport and recycling of inorganic phosphorus, which is crucial for cellular metabolism and bioenergetics, as well as in bacterial killing, since they are able to generate reactive oxygen species via Fenton chemistry. Diphosphonucleotide phosphatase/phosphodiesterase (PPD1, a glycoprotein plant purple acid phosphatase (PAP from yellow lupin seeds, contains a bimetallic Fe–Mn catalytic site which is most active at acidic pH. Unlike other plant PAPs, PPD1 cleaves the pyrophosphate bond in diphosphonucleotides and the phosphodiester bond in various phosphodiesters. The homohexameric organization of PPD1, as revealed by a 1.65 Å resolution crystal structure and confirmed by solution X-ray scattering, is unique among plant PAPs, for which only homodimers have previously been reported. A phosphate anion is bound in a bidentate fashion at the active site, bridging the Fe and Mn atoms in a binding mode similar to that previously reported for sweet potato PAP, which suggests that common features occur in their catalytic mechanisms. The N-terminal domain of PPD1 has an unexpected and unique fibronectin type III-like fold that is absent in other plant PAPs. Here, the in vitro DNA-cleavage activity of PPD1 is demonstrated and it is proposed that the fibronectin III-like domain, which `overhangs' the active site, is involved in DNA selectivity, binding and activation. The degradation of DNA by PPD1 implies a role for PPD1 in plant growth and repair and in pathogen defence.

Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

Science.gov (United States)

Hurt, Julie K; Coleman, Jennifer L; Fitzpatrick, Brendan J; Taylor-Blake, Bonnie; Bridges, Arlene S; Vihko, Pirkko; Zylka, Mark J

2012-01-01

Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/-) mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

Directory of Open Access Journals (Sweden)

Julie K Hurt

Full Text Available Thiamine (Vitamin B1 is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP can dephosphorylate BT in vitro, in dorsal root ganglia (DRG neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT then decomposes to O-benzoylthiamine (O-BT and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/- mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

Membrane interaction and functional plasticity of inositol polyphosphate 5-phosphatases.

Science.gov (United States)

Braun, Werner; Schein, Catherine H

2014-05-06

In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. Copyright © 2014 Elsevier Ltd. All rights reserved.

Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

International Nuclear Information System (INIS)

Pradalier, N.; Canal, P.; Pujol, A.; Fregevu, Y.; Soula, G.

1982-01-01

We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma [fr

Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

Science.gov (United States)

Majumder, Shyam Prasad; Das, Amal Chandra

2016-04-01

An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

Science.gov (United States)

Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparat...

Radioimmunoassay of human prostate-specific acid phosphatase in the diagnosis and follow-up of therapy of prostatic cancer

International Nuclear Information System (INIS)

Vihko, P.

1981-01-01

The author describes the development of a radioimmunoassay for the determination of serum prostate-specific acid phosphatase and studies its application to the diagnosis and follow-up of therapy of prostatic carcinoma. (Auth./C.F.)

Lowering of phytic acid content by enhancement of phytase and acid phosphatase activities during sunflower germination

Directory of Open Access Journals (Sweden)

Juliana da Silva Agostini

2010-08-01

Full Text Available The objective of this work was to investigate the germination of hybrid sunflowers BRS191 and C11 as a means of lowering phytic acid (PA content by enhancing the activity of endogenous phytase and acid phosphatase. The concentration of PA in hybrid sunflower achenes varied from 2.16 to 2.83g/100g of sample (p O objetivo deste trabalho foi investigar a germinação de girassóis híbridos BRS 191 e C11 com finalidade de reduzir o teor de AF e aumentar as atividades de phytases e fosfatases endógenas. A concentração do AF nos aquênios de girassóis híbridos variou de 2,16 a 2,83 g /100g de amostra (p< 0,005. As atividades de fitases e fosfatases de girassóis BRS191 e C11 foram elevadas no 4º e 5º dia de germinação, respectivamente, com liberação do fósforo necessário para o desenvolvimento da semente. Estes resultados indicam que o AF do girassol hibrido reduz e a atividade de phytase aumenta em períodos distintos da germinação, possibilitando assim a aplicação desta enzima no controle do teor de AF em cereais, melhorando o seu valor nutricional.

Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

Science.gov (United States)

Seymen, Figen; Kim, Youn Jung; Lee, Ye Ji; Kang, Jenny; Kim, Tak-Heun; Choi, Hwajung; Koruyucu, Mine; Kasimoglu, Yelda; Tuna, Elif Bahar; Gencay, Koray; Shin, Teo Jeon; Hyun, Hong-Keun; Kim, Young-Jae; Lee, Sang-Hoon; Lee, Zang Hee; Zhang, Hong; Hu, Jan C-C; Simmer, James P; Cho, Eui-Sic; Kim, Jung-Wook

2016-11-03

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

Science.gov (United States)

Collins, Juliet; Cilibrizzi, Agostino; Fedorova, Marina; Whyte, Gillian; Mak, Lok Hang; Guterman, Inna; Leatherbarrow, Robin; Woscholski, Rudiger; Vilar, Ramon

2016-04-28

Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains.

Science.gov (United States)

Jacewicz, Agata; Shuman, Stewart

2015-08-01

Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3'-OH end and at least one or two ribonucleotides on the 5'-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. Rnh

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

Science.gov (United States)

Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

2015-06-01

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

International Nuclear Information System (INIS)

Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

2011-01-01

Research highlights: → Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. → However, it is not clear exactly how PP5 participates in this process. → Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

Domain-to-domain coupling in voltage-sensing phosphatase.

Science.gov (United States)

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP).

Science.gov (United States)

Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J; Sasaki, Takehiko; Okamura, Yasushi

2012-06-19

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain. Possible dual specificity.

Science.gov (United States)

Frank, D W; Waechter, C J

1998-05-08

Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid

Defects in the acid phosphatase ACPT cause recessive hypoplastic amelogenesis imperfecta.

Science.gov (United States)

Smith, Claire El; Whitehouse, Laura LE; Poulter, James A; Brookes, Steven J; Day, Peter F; Soldani, Francesca; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2017-08-01

We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

Effect of mercuric chloride on cellular morphology and acid phosphatase of tissue culture cells cultivated in suspension

Energy Technology Data Exchange (ETDEWEB)

Li, M F; Traxler, G S

1974-01-01

Cells exposed to HgCl/sub 2/ (0.5 mg/liter) increased dramatically in size and stained poorly with May-Grunwald Giemsa stain and exhibited incompleteness in cell division. When the cell DNA was stained by the Feulgen technique, many multinucleated cells were apparent in the cultures treated with HgCl/sub 2/. Additionally, enlargement and alteration of the nucleoli were evident. Electron-micrographs of the experimental cells revealed that microvilli, ribosomes, mitochondria, and endoplasmic reticula were abundant in the control cells, but in contrast a scarcity of these organelles was observed together with notable cytoplasmic vacuolation in the HgCl/sub 2/-treated cells. In addition the nucleolini of the treated cells were enlarged and had begun to fuse, producing a mulberry appearance. Electronmicroscopic detection of acid phosphatase activity in the cells indicated that the periplasmic enzyme activity was present in control cells, but not in the cells exposed to HgCl/sub 2/. The possible reaction of Hg/sup + +/ with deoxyribonucleic acid and disulfides is discussed with respect to the observed cytopathic effect and impaired enzyme activity. 10 references, 5 figures.

Effect of perfluorooctane sulfonate on the conformation of wheat germ acid phosphatase.

Science.gov (United States)

Xu, Dongmei; Jin, Jianchang; Shen, Tong; Wang, Yanhua

2013-11-01

Fluorescence spectroscopy was used to study the quenching mechanism, the type of force and the binding sites of perfluorooctane sulfonate (PFOS) on wheat germ acid phosphatase (ACPase). The results showed that the quenching effect of PFOS on ACPase was mainly due to a static quenching mechanism that occurred via the formation of hydrogen bonds and van der Waals forces. The results from synchronous fluorescence spectroscopy demonstrated that PFOS interacts with ACPase close to the tryptophan residues. In addition, synchronous fluorescence spectroscopy also showed that PFOS increases the hydrophobicity of the microenvironment of the tyrosine residues, hence decreasing the local polarity.

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings.

Science.gov (United States)

Cheng, H F; Tao, M

1989-10-19

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.

Inhibition of Protein Tyrosine Phosphatase 1B by Aurintricarboxylic Acid and Methylenedisalicylic Acid: Polymer versus Monomer

International Nuclear Information System (INIS)

Shrestha, Suja; Lee, Keun Hyeung; Cho, Hyeong Jin

2004-01-01

In this study, we examined whether the in vitro inhibitory activity of ATA against PTPases resides in the monomer or high molecular weight components. Not to mention commercial ATA, the ATA sample synthesized according to the method previously reported to produce monomer was also found to contain polymeric materials as described below. Therefore, monomeric component of ATA was prepared absolutely free of polymer. Also synthesized in a pure form was methylenedisalicylic acid (MDSA), one of the low molecular weight components formed in the conventional preparation of ATA. Commercial MDSA was also proved to contain polymeric substances. The inhibitory potency of ATA and MDSA synthesized in a polymer-free form was evaluated against human protein tyrosine phosphatase 1B (PTP1B). Commercial ATA, however, contains significant amounts of polymeric materials schematically represented as. In general, ATA is prepared by condensation of salicylic acid with formaldehyde and the branching reaction results in the formation of polymers of molecular weights up to several thousands Dalton

Secretion of acid phosphatase by axenic Entamoeba histolytica NIH-200 and properties of the extracellular enzyme.

Science.gov (United States)

Agrawal, A; Pandey, V C; Kumar, S; Sagar, P

1989-01-01

Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupric chloride, ethylene diamine-tetra acetic acid, ammonium molybdate and cysteine; however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.

Ontogeny and distribution of alkaline and acid phosphatases in the digestive system of California halibut larvae (Paralichthys californicus).

Science.gov (United States)

Zacarias-Soto, Magali; Barón-Sevilla, Benjamín; Lazo, Juan P

2013-10-01

Studies aimed to assess the digestive physiology of marine fish larvae under culture conditions are important to further understand the functional characteristics and digestive capacities of the developing larvae. Most studies to date concentrate on intestinal lumen digestion and little attention to the absorption process. Thus, the objectives of this study were to histochemically detect and quantify some of the enzymes responsible for absorption and intracellular digestion of nutrients in the anterior and posterior intestine of California halibut larvae. Alkaline and acid phosphatases were detected from the first days post-hatch (dph). Alkaline phosphatase maintained a high level of activity during the first 20 dph in both intestinal regions. Thereafter, a clear intestinal regionalization of the activity was observed with the highest levels occurring in the anterior intestine. Acid phosphatase activity gradually increased in both intestinal regions during development, and a regionalization of the activity was not observed until late in development, once the ocular migration began. Highest levels were observed in the anterior intestine at the end of metamorphosis concomitant with the stomach development. The results from this study show some morphological and physiological changes are occurring during larval development and a clear regionalization of the absorption process as the larvae develops. These ontological changes must be considered in the elaboration of diets according to the digestive capacity of the larvae.

Phosphatase activity in Antarctica soil samples as a biosignature of extant life

Science.gov (United States)

Sato, Shuji; Itoh, Yuki; Takano, Yoshinori; Fukui, Manabu; Kaneko, Takeo; Kobayashi, Kensei

Microbial activities have been detected in such extreme terrestrial environments as deep lithosphere, a submarine hydrothermal systems, stratosphere, and Antarctica. Microorganisms have adapted to such harsh environments by evolving their biomolecules. Some of these biomolecules such as enzymes might have different characteristics from those of organisms in ordinary environments. Many biosignatures (or biomarkers) have been proposed to detect microbial activities in such extreme environments. A number of techniques are proposed to evaluate biological activities in extreme environments including cultivation methods, assay of metabolism, and analysis of bioorganic compounds like amino acids and DNA. Enzyme activities are useful signature of extant life in extreme environments. Among many enzymes, phosphatase could be a good indicator of biological activities, since phosphate esters are essential for all the living terrestrial organisms. In addition, alkaline phosphatase is known as a typical zinc-containing metalloenzyme and quite stable in environments. We analyzed phosphatase activities in Antarctica soil samples to see whether they can be used as biosignatures for extant life. In addition, we characterized phosphatases extracted from the Antarctica soil samples, and compared with those obtained from other types of environments. Antarctica surface environments are quite severe environments for life since it is extremely cold and dry and exposed to strong UV and cosmic rays. We tried to evaluate biological activities in Antarctica by measuring phosphatase activities. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) are measured spectrophotometrically after mixing the powdered sample and p-nitrophenyl phosphate solution (pH 6.5 for ACP, pH 8.0 for ALP). ALP was characterized after extraction from soils with

Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

Science.gov (United States)

Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

2011-07-01

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

Diversity and Gene Expression of Phosphatase Genes Provide Insight into Soil Phosphorus Dynamics in a New Zealand Managed Grassland

Science.gov (United States)

Dunfield, K. E.; Gaiero, J. R.; Condron, L.

2017-12-01

Healthy and diverse communities of soil organisms influence key soil ecosystem services such as carbon sequestration, water quality protection, climate regulation and nutrient cycling. Microbially driven mineralization of organic phosphorus is an important contributor to plant available inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs) which act on common forms of organic phosphorus (P). Our current understanding of P turnover in soils has been limited by lack of research tools capable of targeting these genes. Thus, we developed a set of oligonucleotide PCR primers that targeted bacteria with the genetic potential for acid phosphatase production. A long term randomized-block pasture trial was sampled following 22 years of continued aerial biomass removal and retention. Primers were used to target genes encoding alkaline phosphatase (phoD) and the three classes (CAAP, CBAP, CCAP) of non-specific acid phosphatases. PCR amplicons targeting total genes and gene transcripts were sequenced using Illumina MiSeq to understand the diversity of the bacterial phosphatase producing communities. In general, the majority of operational taxonomic units (OTUs) were shared across both treatments and across metagenomes and transcriptomes. However, analysis of DNA OTUs revealed significantly different communities driven by treatment differences (P reduced Olsen P levels (15 vs. 36 mg kg-1 in retained treatment). Acid phosphatase activity was measured in all samples, and found to be highest in the biomass retained treatment (16.8 vs. 11.4 µmol g-1 dry soil h-1), likely elevated due to plant-derived enzymes; however, was still correlated to bacterial gene abundances. Overall, the phosphatase producing microbial communities responded to the effect of consistent P limitation as expected, through alteration in the composition of the community structure and through increased levels of gene expression of the phosphatase genes.

Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

International Nuclear Information System (INIS)

Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

1987-01-01

C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

DEFF Research Database (Denmark)

Jespersen, Thomas; Gavillet, Bruno; van Bemmelen, Miguel X

2006-01-01

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull......-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn...... does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5....

Age-related changes of serum tartrate-resistant acid phosphatase 5b and the relationship with bone mineral density in Chinese women

Institute of Scientific and Technical Information of China (English)

Yue-juan QIN; Zhen-lin ZHANG; Hao ZHANG; Wei-wei HU; Yu-juan LIU; Yun-qiu HU; Miao LI; Jie-mei GU; Jin-wei HE

2008-01-01

Aim: Ostcoclastic activity is mainly assessed by measurement of urinary markers (eg C-terminal cross-linked telopeptides of type I collagen, N-terminal cross-linked telopeptides of type I collagen etc), the levels of which could often be affected by renal clearance. Recently, serum tartrate-resistant acid phosphatase 5b (TRACP5b) has been used as an alternative serum marker to evaluate osteoclastic activity. We investigated the age-related changes of TRACP5b level and its association with bone mineral density (BMD) in Chinese women. Methods: Seven-hundred and twenty-two Chinese mainland women aged 20-79 years were recruited in the study. Serum TRACP5b level was measured using immunoassay to evaluate the state of bone resorption. Bone mineral density (BMD) (g/cm2) at lumbar spine 1-4 and proximal femur were measured by duel-energy X-ray absorptiometry. Results: The serum TRACP5b level reached a bottom value in premenopausal women aged 30-39, gradually increased in women aged 40-49, rapidly rose in women aged 50-59, and culminated with a maximum value in women aged 60-69 before a slow drop in women aged 70-79. The average level of TRACPSb was significantly higher in postmenopausal women [(3.29±1.07) U/L] than in premenopausal women ([1.70±0.59] U/L). The levels of TRACP5b were inversely correlated with BMD at all measured sites (P<0.001). Furthermore, the level of TRACP5b was obviously higher in women with osteoporosis and osteopenia than those with normal bone mass (P<0.001). Conclusion: We have established the reference values of serum TRACPSb in Chinese mainland women, and found that postmenopausal women had higher TRACP5b concentration than younger women. The results showed that serum TRACPSb was a sensitive and useful parameter for the evaluation of age-related changes of bone absorption.

Voltage-sensing phosphatase: its molecular relationship with PTEN.

Science.gov (United States)

Okamura, Yasushi; Dixon, Jack E

2011-02-01

Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

Detection of Ca2+-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

Directory of Open Access Journals (Sweden)

Tigran R Petrosyan

2016-01-01

Full Text Available The study aims to confirm the neuroregenerative effects of bacterial melanin (BM on central nervous system injury using a special staining method based on the detection of Ca2+-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex (group I; n = 12 or unilateral rubrospinal tract transection at the cervical level (C3–4 (group II; n = 12. In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution (BM subgroup and the remaining six rats were intramuscularly injected with saline (saline subgroup. Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca2+-dependent acid phosphatase activity detection in combination with Chilingarian's calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca2+-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

Prognostic role of serum prostatic acid phosphatase for 103Pd-based radiation for prostatic carcinoma

International Nuclear Information System (INIS)

Dattoli, Michael; Wallner, Kent; True, Lawrence; Sorace, Richard; Koval, John; Cash, Jennifer; Acosta, Rudolph; Biswas, Mohendra; Binder, Michael; Sullivan, Brent; Lastarria, Emilio; Kirwan, Novelle; Stein, Douglas

1999-01-01

Purpose: To establish the prognostic role of serum enzymatic prostatic acid phosphatase (PAP) in patients treated with palladium ( 103 Pd) and supplemental external beam irradiation (EBRT) for clinically localized, high-risk prostate carcinoma. Methods and Materials: One hundred twenty-four consecutive patients with Stage T2a-T3 prostatic carcinoma were treated from 1992 through 1995. Each patient had at least one of the following risk factors for extracapsular disease extension: Stage T2b or greater (100 patients), Gleason score 7-10 (40 patients), pretreatment prostate specific antigen (PSA) > 15 ng/ml (32 patients), or elevated serum PAP (25 patients). Patients received 41 Gy conformal EBRT to a limited pelvic field, followed 4 weeks later by a 103 Pd boost (prescription dose 80 Gy). Biochemical failure was defined as a PSA greater than 1 ng/ml (normal < 4 ng/ml). Results: The overall, actuarial freedom from biochemical failure at 4 years after treatment was 79%. In Cox-proportional hazard multivariate analysis, the strongest predictor of failure was elevated pretreatment acid phosphatase (p = 0.02), followed by Gleason score (p = 0.1), and PSA (p = 0.14). Conclusion: PAP was the strongest predictor of long-term biochemical failure. It may be a more accurate indicator of micrometastatic disease than PSA, and as such, we suggest that it be reconsidered for general use in radiation-treated patients

A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions.

Science.gov (United States)

Orlando, Kelly A; Iosue, Christine L; Leone, Sarah G; Davies, Danielle L; Wykoff, Dennis D

2015-10-15

Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2. © 2015 Authors; published by Portland Press Limited.

Localization of acid phosphatase activity in the apoplast of root nodules of pea (Pisum sativum

Directory of Open Access Journals (Sweden)

Marzena Sujkowska

2011-01-01

Full Text Available Changes in the activity of acid phosphatase (AcPase in the apoplast of pea root nodule were investigated. The activity was determined using lead and cerium methods. The results indicated a following sequence of AcPase activity appearance during the development of the infection thread: 1 low AcPase activity appears in the outer part of cells of symbiotic bacteria; 2 bacteria show increased AcPase activity, and the enzyme activity appears in the thread walls; 3 activity exhibits also matrix of the infection thread; 4 bacteria just before their release from the infection threads show high AcPase activity; 5 AcPase activity ceases after bacteria transformation into bacteroids. The increase in bacterial AcPase activity may reflect a higher demand for inorganic phosphorus necessary for propagation of the bacteria within the infection threads and/or involved in bacteria release from the infection threads.

Soil properties influence kinetics of soil acid phosphatase in response to arsenic toxicity.

Science.gov (United States)

Wang, Ziquan; Tan, Xiangping; Lu, Guannan; Liu, Yanju; Naidu, Ravi; He, Wenxiang

2018-01-01

Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As). However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the Michaelis constant (K m ) and maximum reaction velocity (V max ) values of soil ACP ranged from 1.18 to 3.77mM and 0.025-0.133mMh -1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed differently with As contamination. The K m remained unchanged and V max decreased with increase of As concentration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in alkaline soils, the K m increased linearly and V max decreased with increase of As concentration, indicating a mixed inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (K ic ) and noncompetitive inhibition constant (K iu ) varied among soils and ranged from 0.38 to 3.65mM and 0.84-7.43mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a difference of As toxicity to soil ACP. Catalytic efficiency (V max /K m ) of soil ACP was a sensitive kinetic parameter to assess the ecological risks of soil As contamination. Copyright © 2017 Elsevier Inc. All rights reserved.

Presence of multiple acid phosphatases activity in seedlings of cucumber, radish and rocket salad Presença de atividade de múltiplas fosfatases ácidas em plântulas de pepino, rabanete e rúcula

Directory of Open Access Journals (Sweden)

Luciane Almeri Tabaldi

2008-06-01

Full Text Available Acid phosphatases (3.1.3.2 are a group of enzymes widely distributed in nature, which catalyze the hydrolysis of a variety of phosphate esters in the pH range of 4-6. We confirmed the presence of acid phosphatases in seedlings of cucumber (Cucumis sativus, radish (Raphanus sativus and rocket salad (Eruca vesicaria under different assay conditions using a rapid and simple preparation. The results showed that the optimum pH and temperature used for all species were close to 5.5 and 35°C, respectively. The enzyme was inhibited by molybdate, fluoride, azide, levamisole, orthovanadate, Zn2+ and Cu2+. Suramin had no effect on enzyme activity. The acid phosphatase from cucumber, radish and rocket salad hydrolyzed a wide variety of phosphate esters and the highest activity was observed with PPi, ATP and GTP. These results demonstrate that the enzyme investigated in this study is different from well known ester phosphate cleaving plant enzymes (apyrase and inorganic pyrophosphatases and this preparation could be a useful tool to future toxicological studies and to study initially all isoforms of acid phosphatase.As fosfatases ácidas (3.1.3.2 são um grupo de enzimas amplamente distribuídas na natureza, as quais catalisam a hidrólise de uma variedade de ésteres de fosfato com uma variação de pH entre quatro e seis. Foi confirmada a presença de fosfatases ácidas em plântulas de pepino (Cucumis sativus, rabanete (Raphanus sativus e rúcula (Eruca vesicaria sob diferentes condições de ensaio usando uma preparação rápida e simples. Os resultados mostraram que o pH e a temperatura ótimos para todas as espécies foram 5,5 e 35°C, respectivamente. A enzima foi inibida por molibdato, fluoreto, azida, levamisole, ortovanadato, Zn2+ e Cu2+. O inibidor suramim não afetou a atividade enzimática. As fosfatases ácidas de pepino, rabanete e rúcula hidrolisaram uma ampla variedade de ésteres de fosfato e a maior atividade foi observada com PPi, ATP

Role of Ocrl1 and Inpp5E in primary cilia assembly and maintenance: a phosphatidylinositol phosphatase relay system?

Directory of Open Access Journals (Sweden)

Madhivanan K

2016-02-01

Full Text Available Kayalvizhi Madhivanan,* Swetha Ramadesikan,* R Claudio Aguilar Department of Biological Sciences, Purdue University, West Lafayette, IN, USA *These authors contributed equally to this work Abstract: The primary cilium (PC is a plasma membrane-derived structure of great importance for cell and organismal physiology. Indeed, abnormalities in assembly or function of the PC trigger the onset of a group of genetic diseases collectively known as ciliopathies. In recent years, it has become evident that the integrity and function of the PC depends substantially on signaling elements such as phosphoinositides (PI and their regulators. Because phospholipids such as PI(4,5P2 constitute recruitment platforms for cytoskeleton, signaling, and trafficking machinery, control over their levels is critical for PC function. Although information about phosphoinositol phosphate (PIP kinases in the PC is scarce, a growing body of evidence supports a role for PIP phosphatases in cilia assembly/maintenance. Indeed, deficiencies in two 5′ PIP phosphatases, Inpp5E and Ocrl1, are clearly linked to ciliopathies like Joubert/MORM syndromes, or ciliopathy-associated diseases like Lowe syndrome. Here, we review the unique roles of these proteins and their specific site of action for ensuring ciliary integrity. Further, we discuss the possibility that a phosphatase relay system able to pass PI control from a preciliary to an intraciliary compartment is in place to ensure PC integrity/function. Keywords: primary cilia, Ocrl1, Inpp5E, Pip2, Pip3

Disruption of a Guard Cell–Expressed Protein Phosphatase 2A Regulatory Subunit, RCN1, Confers Abscisic Acid Insensitivity in Arabidopsis

Science.gov (United States)

Kwak, June M.; Moon, Ji-Hye; Murata, Yoshiyuki; Kuchitsu, Kazuyuki; Leonhardt, Nathalie; DeLong, Alison; Schroeder, Julian I.

2002-01-01

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell–expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca2+ increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling. PMID:12417706

Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation

Science.gov (United States)

1991-06-26

extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM...diacylglycerol (DAG) 2. TPA 3. Indolactam Figure 6. Chemical structure of: 1. H-7 (A kinase inhibitor) 2. okadaic acid (A phosphatase inhibitor) Figure 7...elevating agents: Forskolin and Cholera toxin Figure 17. Down-regulation of 2-5A-depRNase by Okadaic 77 acid : A phosphatase inhibitor Figure 18

Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes metastasis-related properties via TGFβ2/TβR and CD44 in MDA-MB-231 breast cancer cells.

Science.gov (United States)

Reithmeier, Anja; Panizza, Elena; Krumpel, Michael; Orre, Lukas M; Branca, Rui M M; Lehtiö, Janne; Ek-Rylander, Barbro; Andersson, Göran

2017-09-15

Tartrate-resistant acid phosphatase (TRAP/ACP5), a metalloenzyme that is characteristic for its expression in activated osteoclasts and in macrophages, has recently gained considerable focus as a driver of metastasis and was associated with clinically relevant parameters of cancer progression and cancer aggressiveness. MDA-MB-231 breast cancer cells with different TRAP expression levels (overexpression and knockdown) were generated and characterized for protein expression and activity levels. Functional cell experiments, such as proliferation, migration and invasion assays were performed as well as global phosphoproteomic and proteomic analysis was conducted to connect molecular perturbations to the phenotypic changes. We identified an association between metastasis-related properties of TRAP-overexpressing MDA-MB-231 breast cancer cells and a TRAP-dependent regulation of Transforming growth factor (TGFβ) pathway proteins and Cluster of differentiation 44 (CD44). Overexpression of TRAP increased anchorage-independent and anchorage-dependent cell growth and proliferation, induced a more elongated cellular morphology and promoted cell migration and invasion. Migration was increased in the presence of the extracellular matrix (ECM) proteins osteopontin and fibronectin and the basement membrane proteins collagen IV and laminin I. TRAP-induced properties were reverted upon shRNA-mediated knockdown of TRAP or treatment with the small molecule TRAP inhibitor 5-PNA. Global phosphoproteomics and proteomics analyses identified possible substrates of TRAP phosphatase activity or signaling intermediates and outlined a TRAP-dependent regulation of proteins involved in cell adhesion and ECM organization. Upregulation of TGFβ isoform 2 (TGFβ2), TGFβ receptor type 1 (TβR1) and Mothers against decapentaplegic homolog 2 (SMAD2), as well as increased intracellular phosphorylation of CD44 were identified upon TRAP perturbation. Functional antibody-mediated blocking and chemical

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

Science.gov (United States)

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-07-05

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Science.gov (United States)

Sakata, Souhei; Jinno, Yuka; Kawanabe, Akira; Okamura, Yasushi

2016-01-01

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane. PMID:27330112

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1.

Science.gov (United States)

Suleimanova, Aliya D; Beinhauer, Astrid; Valeeva, Liia R; Chastukhina, Inna B; Balaban, Nelly P; Shakirov, Eugene V; Greiner, Ralf; Sharipova, Margarita R

2015-10-01

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of N-type glycosylation sites and glycan structures of Purple Acid Phosphatase Phytases from Wheat (Triticum aestivum L.)

DEFF Research Database (Denmark)

Dionisio, Giuseppe; Brinch-Pedersen, Henrik; Welinder, Karen Gjesing

2011-01-01

Wheat (Triticum aestivum L.) possesses preformed phytase activity in the grain that is essential to make phosphate available to cell metabolism and in food and feed (Brejnholt S. et al., 2011). Cereals contain the purple acid phosphatase type of phytases, PAPhy (Dionisio G. et al., 2011a). Mature......., Skov L. Brinch-Pedersen H. (2011). The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin. J. Sci. Food Agri. (in press). Dionisio G., Madsen C.K., Holm P.B., Welinder K.G., Jørgensen M., Stoger E., Arcalis E., Brinch-Pedersen H. (2011a......) Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat (Triticum aestivum L.), Barley (Hordeum vulgare L.), Maize (Zea maize L.) and Rice (Oryza sativa L.). Plant Physiol. [in press, Jan 10, Epub ahead of print] Dionisio G., Brinch-Pedersen H., Welinder K.G., Jørgensen M. (2011b...

Vanillic acid derivatives from the green algae Cladophora socialis as potent protein tyrosine phosphatase 1B inhibitors.

Science.gov (United States)

Feng, Yunjiang; Carroll, Anthony R; Addepalli, Rama; Fechner, Gregory A; Avery, Vicky M; Quinn, Ronald J

2007-11-01

A novel vanillic acid derivative (1) and its sulfate adduct (2) were isolated from a green algae, Cladophora socialis. The structures of 1 and 2 were elucidated from NMR and HRESIMS experiments. Both compounds showed potent inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), an enzyme involved in the regulation of insulin cell signaling. Compounds 1 and 2 had IC50 values of 3.7 and 1.7 microM, respectively.

Research on Phosphatases of Belladona Leaves and Their Purification

Directory of Open Access Journals (Sweden)

M. Khorsand

1957-01-01

Full Text Available Through experimentation with several leaves it has been possible for us to point out the existance of two different acid phosphatases. We have studied in more detail the phosphatases of belldon a leaves (Atropa Belladona L. Solanacees. The great part of the phosphatase activity is water extractable. We have compared the activity of the soluble fraction with that not directly extractable by means of water. The insoluble fraction could not be solubilized in a satisfaetC'fY m.anner.The digestion by papaine produced a slight solubilizing effect; on the other hand salt solutions, neutral or alkaline, or water glycerol mixtures had no solubilizing effect on the enzyme, It has been possible to demonstrate the existence of two different phosphatases in the insoluble fraction: the first of the type II,

Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival.

Science.gov (United States)

McRae, Steven; Pagliai, Fernando A; Mohapatra, Nrusingh P; Gener, Alejandro; Mahmou, Asma Sayed Abdelgeliel; Gunn, John S; Lorca, Graciela L; Gonzalez, Claudio F

2010-02-19

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.

Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe.

Directory of Open Access Journals (Sweden)

Na Luo

Full Text Available Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm's canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer's vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

DEFF Research Database (Denmark)

Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

2011-01-01

this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture...... mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked...... enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from...

Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

Directory of Open Access Journals (Sweden)

Pernilla Lång

2008-03-01

Full Text Available Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer.Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity.Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

Chromatographic separation of alkaline phosphatase from dental enamel

DEFF Research Database (Denmark)

Moe, D; Kirkeby, S; Salling, E

1989-01-01

Alkaline phosphatase (AP) was prepared from partly mineralized bovine enamel by extraction in phosphate buffer, centrifugation and various chromatographic techniques. Chromatofocusing showed that the enamel enzyme possessed five isoelectric points at the acid pH level ranging from pH 5.7 to pH 4.......4. Three enzyme peaks were eluted using low pressure chromatography with a Bio-gel column. With a HPLC gel filtration column the separation of the enamel extract resulted in only one peak with AP activity. The fractions of this peak were used to produce an antibody against bovine AP....

Insertion of a specific fungal 3'-phosphoadenosine-5'-phosphatase motif into a plant homologue improves halotolerance and drought tolerance of plants.

Science.gov (United States)

Gašparič, Meti Buh; Lenassi, Metka; Gostinčar, Cene; Rotter, Ana; Plemenitaš, Ana; Gunde-Cimerman, Nina; Gruden, Kristina; Zel, Jana

2013-01-01

Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3'-phosphoadenosine-5'-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

Science.gov (United States)

Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

2010-01-01

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

Science.gov (United States)

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 o...

Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes.

Science.gov (United States)

Bozzo, Gale G; Singh, Vinay K; Plaxton, William C

2004-08-27

Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP). The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa. In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells. The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins.

Reduction of the degradation activity of umami-enhancing purinic ribonucleotide supplement in miso by the targeted suppression of acid phosphatases in the Aspergillus oryzae starter culture.

Science.gov (United States)

Marui, Junichiro; Tada, Sawaki; Fukuoka, Mari; Wagu, Yutaka; Shiraishi, Yohei; Kitamoto, Noriyuki; Sugimoto, Tatsuya; Hattori, Ryota; Suzuki, Satoshi; Kusumoto, Ken-Ichi

2013-09-02

Miso (fermented soybean paste) is a traditional Japanese fermented food, and is now used worldwide. The solid-state culture of filamentous fungus, Aspergillus oryzae, grown on rice is known as rice-koji, and is important as a starter for miso fermentation because of its prominent hydrolytic enzyme activities. Recently, commercial miso products have been supplemented with purinic ribonucleotides, such as inosine monophosphate (IMP) and guanine monophosphate, to enhance the characteristic umami taste of glutamate in miso. Because the purinic ribonucleotides are degraded by enzymes such as acid phosphatases in miso, heat inactivation is required prior to the addition of these flavorings. However, heat treatment is a costly process and reduces the quality of miso. Therefore, an approach to lower acid phosphatase activities in koji culture is necessary. Transcriptional analysis using an A. oryzae KBN8048 rice-koji culture showed that eight of the 13 acid phosphatase (aph) genes were significantly down-regulated by the addition of phosphoric acid in the preparation of the culture in a concentration-dependent manner, while aphC expression was markedly up-regulated under the same conditions. The eight down-regulated genes might be under the control of the functional counterpart of the Saccharomyces cerevisiae transcriptional activator Pho4, which specifically regulates phosphatase genes in response to the ambient phosphate availability. However, the regulatory mechanism of aphC was not clear. The IMP dephosphorylation activities in rice-koji cultures of KBN8048 and the aphC deletion mutant (ΔaphC) were reduced by up to 30% and 70%, respectively, in cultures with phosphoric acid, while protease and amylase activity, which is important for miso fermentation, was minimally affected. The miso products fermented using the rice-koji cultures of KBN8048 and ΔaphC prepared with phosphoric acid had reductions in IMP dephosphorylation activity of 80% and 90%, respectively, without

Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

Science.gov (United States)

Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

2000-01-01

The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase.

Science.gov (United States)

Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M

2015-12-15

Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All

Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

Directory of Open Access Journals (Sweden)

Janetti R Signorelli

Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

Science.gov (United States)

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

International Nuclear Information System (INIS)

Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

1990-01-01

The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

Direct determination of phosphatase activity from physiological substrates in cells.

Directory of Open Access Journals (Sweden)

Zhongyuan Ren

Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene

DEFF Research Database (Denmark)

Madsen, Claus Krogh; Dionisio, Giuseppe; Holme, Inger

2013-01-01

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley...

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: Implications for immunoassays

DEFF Research Database (Denmark)

Jørgensen, Kaj Anker; Karamperis, Nikolaos; Koefoed-Nielsen, Pernille Bundgaard

2006-01-01

by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

DEFF Research Database (Denmark)

Karamperis, N; Koefoed-Nielsen, PB; Brahe, P

2006-01-01

by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

Effect of Exogenous Phytase Addition on Soil Phosphatase Activities: a Fluorescence Spectroscopy Study.

Science.gov (United States)

Yang, Xiao-zhu; Chen, Zhen-hua; Zhang, Yu-lan; Chen, Li-jun

2015-05-01

The utilization of organic phosphorus (P) has directly or indirectly improved after exogenous phytase was added to soil. However, the mechanism by which exogenous phytase affected the soil phosphatases (phosphomonoesterase and phosphodiesterase) activities was not clear. The present work was aimed to study red soil, brown soil and cinnamon soil phosphomonoesterase (acid and alkaline) (AcP and AlP) and phosphodiesterase (PD) activities responding to the addition of exogenous phytase (1 g phytase/50 g air dry soil sample) based on the measurements performed via a fluorescence detection method combined with 96 microplates using a TECAN Infinite 200 Multi-Mode Microplate Reader. The results indicated that the acid phosphomonoesterase activity was significantly enhanced in red soil (p≤0. 01), while it was significantly reduced in cinnamon soil; alkaline phosphomonoesterase activity was significantly enhanced in cinnamon soil (p≤ 0. 01), while it was significantly reduced in red soil; phosphodiesterase activity was increased in three soils but it was significantly increased in brown soil (p≤0. 01) after the addition of exogenous phytase. The activities still remained strong after eight days in different soils, which indicated that exogenous phytase addition could be enhance soil phosphatases activities effectively. This effect was not only related to soil properties, such as pH and phosphorus forms, but might also be related to the excreted enzyme amount of the stimulating microorganism. Using fluorescence spectroscopy to study exogenous phytase addition influence on soil phosphatase activities was the first time at home and abroad. Compared with the conventional spectrophotometric method, the fluorescence microplate method is an accurate, fast and simple to use method to determine the relationships among the soil phosphatases activities.

P depletion and activity of phosphatases in the rhizosphere of mycorrhizal and non-mycorrhizal cucumber (Cucumis Sativus L.)

DEFF Research Database (Denmark)

Joner, E.J.; Magid, J.; Gahoonia, T.S.

1995-01-01

An experiment was set up to test the ability of arbuscular mycorrhizal (AM) roots and hyphae to produce extracellular phosphatases and to study the relationship between phosphatase activity and soil organic P (P-o). Non-mycorrhizal cucumber and cucumber in symbiosis with either of two mycorrhizal...... fungi were grown in a sandy loam-sand mixture in three-compartment pots. Plant roots were separated from two consecutively adjoining compartments, first by a 37 m mesh excluding roots and subsequently by a 0.45 m membrane excluding mycorrhizal hyphae. Soil from the two root-free compartments...... was sectioned in a freezing microtome and analyzed for extracellular acid (pH 5.2) and alkaline (pH 8.5) phosphatase activity as well as depletion of NaHCO-3-extractable inorganic P (P-i) and P-o. Roots and mycorrhizal hyphae depleted the soil of P-i but did not influence the concentration of P-o in spite...

Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

Directory of Open Access Journals (Sweden)

Sosanka Protim SANDILYA

2014-12-01

Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

Growth and extracellular phosphatase activity of arbuscular mycorrhizal hyphae as influenced by soil organic matter

DEFF Research Database (Denmark)

Joner, E.J.; Jakobsen, I.

1995-01-01

Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus...... additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may...

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

Science.gov (United States)

Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

2005-11-01

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

Science.gov (United States)

Magnusson, P; Farley, J R

2002-12-01

High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P acid residues compared with B/I, which mainly explains the apparent differences in molecular weight. Future investigations will focus on the clinical and functional significance of the revealed differences in sialic acid residues.

A novel fluorescence biosensor for sensitivity detection of tyrosinase and acid phosphatase based on nitrogen-doped graphene quantum dots.

Science.gov (United States)

Qu, Zhengyi; Na, Weidan; Liu, Xiaotong; Liu, Hua; Su, Xingguang

2018-01-02

In this paper, we developed a sensitive fluorescence biosensor for tyrosinase (TYR) and acid phosphatase (ACP) activity detection based on nitrogen-doped graphene quantum dots (N-GQDs). Tyrosine could be catalyzed by TYR to generate dopaquinone, which could efficiently quench the fluorescence of N-GQDs, and the degree of fluorescence quenching of N-GQDs was proportional to the concentration of TYR. In the presence of ACP, l-Ascorbic acid-2-phosphate (AAP) was hydrolyzed to generate ascorbic acid (AA), and dopaquinone was reduced to l-dopa, resulting in the fluorescence recovery of the quenched fluorescence by dopaquinone. Thus, a novel fluorescence biosensor for the detection of TYR and ACP activity based on N-GQDs was constructed. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of TYR and ACP in the range of 0.43-3.85 U mL -1 and 0.04-0.7 mU mL -1 with a detection limit of 0.15 U mL -1 and 0.014 mU mL -1 , respectively. The feasibility of the proposed biosensor in real samples assay was also studied and satisfactory results were obtained. Copyright © 2017 Elsevier B.V. All rights reserved.

Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity

Directory of Open Access Journals (Sweden)

Francisco Cen-Pacheco

2018-04-01

Full Text Available The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+-longilene peroxide (1, longilene (2, and (+-prelongilene (3 that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A was evaluated, showing that (+-longilene peroxide (1 inhibited PP2A (IC50 11.3 μM. In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

Study of possible changes brought about by plutonium oxide in the acid phosphatase activity of alveolar macrophages of the rabbit

International Nuclear Information System (INIS)

Rouvroy, Huguette

1970-06-01

This report describes the various techniques used for determining the acid phosphatase activity of alveolar rabbit macrophages after inhalation of radioactive plutonium oxide particles, exposure of the animals, removal and sampling of the alveolar cells, and technical dosage. The results obtained are presented; they do not make it possible, in this particular case, to affirm that an important change in the enzymatic activity studied occurs. (author) [fr

Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

Energy Technology Data Exchange (ETDEWEB)

Kumar M.; Sharma M.K.; Saxena P.S.; Kumar A. [Rajasthan Univ., Jaipur (India)

2003-03-01

The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

International Nuclear Information System (INIS)

Kumar, M.; Sharma, M.K.; Saxena, P.S.; Kumar, A.

2003-01-01

The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

Directory of Open Access Journals (Sweden)

Gilbert Christophe

2008-04-01

Full Text Available Abstract Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP, with an optimal activity in presence of bovine serum albumin (BSA 1% at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.

Behavior of soluble and immobilized acid phosphatase in hydro-organic media.

Science.gov (United States)

Wan, H; Horvath, C

1975-11-20

The hydrolysis of p-nitrophenyl phosphate by wheat germ acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) has been investigated in mixtures of aqueous buffers with acetone, dioxane and acetonitrile. The enzyme was either in free solution or immobilized on a pellicular support which consisted of a porous carbonaceous layer on solid glass beads. The highest enzyme activity was obtained in acetone and acetonitrile mixed with citrate buffer over a wide range of organic solvent concentration. In 50% (v/v) acetone both V and Km of the immobilized enzyme were about half of the values in the neat aqueous buffer, but the Ki for inorganic phosphate was unchanged. In 50% (v/v) mixtures of various solvents and citrate buffers of different pH, the enzymic activity was found to depend on the pH of the aqueous buffer component rather than the pH of the hydro-organic mixture as measured with the glass-calomel electrode. The relatively high rates of p-nitrophenol liberation in the presence of glucose even at high organic solvent concentrations suggest that transphosphorylation is facilitated at low water activity.

Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases.

Science.gov (United States)

Siniossoglou, Symeon

2013-03-01

Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. Copyright © 2012 Elsevier B.V. All rights reserved.

Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize and Rice

DEFF Research Database (Denmark)

Dionisio, Giuseppe; Madsen, Claus Krogh; Holm, Preben Bach

2011-01-01

development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed......Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here......, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic...

Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract

Directory of Open Access Journals (Sweden)

RATKO M. JANKOV

2002-09-01

Full Text Available An acid phosphatase from an extract of mugwort (Artemisia vulgaris pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE (under reducing and non-reducing conditions, respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands, pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16 mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and a-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM, molybdate (Ki = 0.055 mM and pyrophosphate (Ki = 6.7 mM and non-competitively by fluoride (Ki = 9.8 mM. Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

Growth, extracellular alkaline phosphatase activity, and kinetic characteristic responses of the bloom-forming toxic cyanobacterium, Microcystis aeruginosa, to atmospheric particulate matter (PM2.5, PM2.5-10, and PM>10).

Science.gov (United States)

Xu, Ziran; Wang, Shoubing; Wang, Yuanan; Zhang, Jie

2018-03-01

Atmospheric particulate matter (APM), commonly seen and widely excited in environment, appears great enough to influence the biochemical processes in aquatic microorganisms and phytoplankton. Understanding the response of cyanobacteria to various factors is fundamental for eutrophication control. To clarify the response of cyanobacteria to APM, the effects of PM 2.5 , PM 2.5-10 , and PM >10 on Microcystis aeruginosa were researched. Variabilities in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular activity, and kinetic parameters of alkaline phosphatase were evaluated by lab-cultured experiments. Results showed that the PM 2.5 had a slight stimulation impact on the growth and enhanced both of the 48- and 72-h extracellular alkaline phosphatase activity (APA), the affinity of alkaline phosphatase for substrate, and the 72-h maximum enzymatic reaction velocity (V max ). Moreover, the stimulations in extracellular APA and V max enhanced with the increasing exposure concentrations. We also found there were no obvious distinctions on the effects of growth and alkaline phosphatase in M. aeruginosa between PM 2.5-10 and PM >10 exposure groups. Obviously, inhibitory effects on growth existed in 4.0 and 8.0 mg/L PM 2.5-10 and 8.0 mg/L PM >10 at 120 h. Furthermore, PM 2.5-10 and PM >10 exerted inhibitory effects on the extracellular APA during the 72-h exposure. Simultaneously, the V max was notably inhibited and the affinity of alkaline phosphatase for substrate was more inseparable compared with control in PM 2.5-10 and PM >10 treatments. Nevertheless, the inhibitors in extracellular APA and kinetic parameters were unrelated to PM 2.5-10 and PM >10 exposure concentrations. Two-way ANOVA results revealed that there were significant interactions between exposure concentration and diameter of APM on the 120-h cell density, soluble protein content, APA, and 72 h APA of M. aeruginosa. These results in our study would be meaningful to further

The human tartrate-resistant acid phosphatase (TRAP): involvement of the hemin responsive elements (HRE) in transcriptional regulation.

Science.gov (United States)

Fleckenstein, E C; Dirks, W G; Drexler, H G

2000-02-01

The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

Autophagy Signaling in Prostate Cancer: Identification of a Novel Phosphatase

Science.gov (United States)

2013-01-01

chloroquine treatment] and LC3 accumulation used as a measure of autophagic flux (Tanida et al., 2005). We found that both knockdown of PTPs and amino acid...6 Jeffrey P. MacKeigan,3 Kyle A. Furge,2 H. Eric Xu1,7†D ow nloaded The antimalaria drug chloroquine has been used as an anti-inflammatory agent for...verified and characterized for phosphatase activity in vitro (task 2). In addition to inserting these constructs into mammalian expression vectors, used

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Science.gov (United States)

Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

2015-01-01

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

Science.gov (United States)

Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

2014-06-01

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048

NARCIS (Netherlands)

van Dijk, T. H.; van der Sluijs, F. H.; Wiegman, C. H.; Baller, J. F.; Gustafson, L. A.; Burger, H. J.; Herling, A. W.; Kuipers, F.; Meijer, A. J.; Reijngoud, D. J.

2001-01-01

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats - A pharmacological study with the chlorogenic acid derivative S4048

NARCIS (Netherlands)

van Dijk, TH; van der Sluijs, FH; Wiegman, CH; Baller, JFW; Gustafson, LA; Burger, HJ; Herling, AW; Kuipers, F; Meijer, AJ; Reijngoud, DJ

2001-01-01

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in

Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

DEFF Research Database (Denmark)

Jiang, Y P; Wang, H; D'Eustachio, P

1993-01-01

We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, ...

Acid phosphatase activity in the liver of Indian desert gerbil (Meriones hurrianae, Jerdon) exposed to internal β-irradiation

International Nuclear Information System (INIS)

Gupta, N.K.; Kumar, A.; Sharma, S.

1985-01-01

Alterations in acid phosphatase activity in the liver of Indian desert gerbil after administration of 45 Ca are reported. There is an increase in the enzyme activity during early periods after 45 Ca administration. The response of the enzyme to internal irradiation was dose dependent. The enzyme activity remained elevated for a longer period in animals given the higher dose. With starting repair, the activity declined and control values were nearly obtained on the 28 posttreatment day in gerbils having received 37 kBq/g body weight of 45 Ca. Animals with higher doses could not survive till this period

Relation of fatty acid composition in lead-exposed mallards to fat mobilization, lipid peroxidation and alkaline phosphatase activity

Science.gov (United States)

Mateo, R.; Beyer, W.N.; Spann, J.W.; Hoffman, D.J.

2003-01-01

The increase of n-6 polyunsaturated fatty acids (PUFA) in animal tissues has been proposed as a mechanism of lead (Pb) poisoning through lipid peroxidation or altered eicosanoids metabolism. We have studied fatty acid (FA) composition in liver and brain of mallards (Anas platyrhynchos) feeding for 3 weeks on diets containing combinations of low or high levels of vitamin E (20 or 200 UI/kg) and Pb (0 or 2 g/kg). Saturated FA, n-6 PUFA and total concentrations of FA were higher in livers of Pb-exposed mallards, but not in their brains. The percentage of n-6 PUFA in liver and brain was slightly higher in Pb-exposed mallards. The increase of n-6 PUFA in liver was associated with decreased triglycerides and increased cholesterol in plasma, thus could be in part attributed to feed refusal and fat mobilization. The hepatic ratios between adrenic acid (22:4 n-6) and arachidonic acid (20:4 n-6) or between adrenic acid and linoleic acid (18:2 n-6) were higher in Pb exposed birds, supporting the existing hypothesis of increased fatty acid elongation by Pb. Among the possible consequences of increased n-6 PUFA concentration in tissues, we found increased lipid peroxidation in liver without important histopathological changes, and decreased plasma alkaline phosphatase activity that may reflect altered bone metabolism in birds.

Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

Energy Technology Data Exchange (ETDEWEB)

Sakisaka, Yukihiko [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tsuchiya, Masahiro [Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tohoku Fukushi University, Sendai 989-3201 (Japan); Nakamura, Takashi [Department of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Liason Center for Innovative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tamura, Masato [Department of Biochemistry and Molecular Biology, Hokkaido University Graduate School of Dentistry, Sapporo 060-8586 (Japan); Shimauchi, Hidetoshi [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@dent.tohoku.ac.jp [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

2015-08-01

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

International Nuclear Information System (INIS)

Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

2015-01-01

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression

Elevated Serum Level of Human Alkaline Phosphatase in Obesity

International Nuclear Information System (INIS)

Khan, A. R.; Awan, F. R.; Najam, S. S.; Islam, M.; Siddique, T.; Zain, M.

2015-01-01

Objective: To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. Methods: The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass index: normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Results: Of the 197 subjects, 97(49 percent) were obese and 100(51 percent) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Conclusion: Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity. (author)

Increase in tartrate-resistant acid phosphatase of bone at the early stage of ascorbic acid deficiency in the ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rat.

Science.gov (United States)

Goto, A; Tsukamoto, I

2003-08-01

The effect of ascorbic acid deficiency on bone metabolism was evaluated using the ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rat model. Ascorbic acid (Asc)-deficient rats gained body weight in a manner similar to Asc-supplemented rats (control) during 3 weeks, but began to lose weight during the 4th week of Asc deficiency. The tartrate-resistant acid phosphatase (TRAP) activity in serum increased to about 2-fold the control value in the rats fed the Asc-free diet for 2, 3, and 4 weeks (AscD2, AscD3, and AscD4), while a decrease in the alkaline phosphatase (ALP) activity was observed only in AscD4 rats. The serum pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) level significantly increased to 1.3-, 1.4-, and 1.9-fold of that in the controls in AscD2, D3, and D4, respectively. The ALP activity in the distal femur was unchanged in AscD1, D2, and D3, but decreased to 50% of the control level in AscD4 rats. The TRAP activity in the distal femur increased to about 2-fold of that in the controls in the AscD2 and D3 and decreased to the control level in the AscD4 rats. The amount of hydroxyproline in the distal femur significantly decreased to about 80%, 70%, and 60% of the control in AscD2, D3, and D4 rats, respectively. These decreases were associated with a similar reduction in the calcium content of the distal femur. Histochemical analysis of the distal femur showed an increase in TRAP-positive cells in AscD2 and AscD3 rats and a decrease in the trabecular bone in AscD2, D3, and D4 rats. These results suggested that a deficiency of Asc stimulated bone resorption at an early stage, followed by a decrease in bone formation in mature ODS rats which already had a well-developed collagen matrix and fully differentiated osteoblasts.

A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

International Nuclear Information System (INIS)

Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y.

2013-01-01

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer

A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

Directory of Open Access Journals (Sweden)

Z. Wen

2013-08-01

Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Directory of Open Access Journals (Sweden)

Bruno L Bozaquel-Morais

Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

Directory of Open Access Journals (Sweden)

Alessandra Pasquo

Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK

Directory of Open Access Journals (Sweden)

felipe Nael Seixas

2014-02-01

Full Text Available This study evaluated the performance of strips for colorimetric detection of alkaline phosphatase and peroxidase in milk, comparing them with a kit of reagents for alkaline phosphatase and the official methodology for peroxidase. The samples were analyzed at the Laboratory Inspection of Products of Animal Origin, State University of Londrina. For the comparison tests for the detection of alkaline phosphatase four treatments were made by adding different percentages of raw milk (1%, 2%, 5% and 10% in the pasteurized milk, plus two control treatments. Thirty-eight samples triplicate for each treatment were analyzed. To compare the performance of tests for peroxidase 80 pasteurized milk samples were evaluated simultaneously by official methodology and by colorimetric strips. The performance of the alkaline phosphatase were different for the treatments with 1% and 2% of raw milk which had all the strips change color as the reagent kit showed the presence of phosphatase in just 2.63% and 5.26% the cases, respectively for each treatment. The colorimetric strips for alkaline phosphatase are more sensitive for the identification of small quantities compared to the reagent kit. The performance of tests for peroxidase showed no difference. The strips for the detection of peroxidase or alkaline phosphatase were effective and can replace traditional methods.

Genetic variation of an acid phosphatase (Acp-2) in the laboratory rat: possible homology with mouse AP-1 and human ACP2.

Science.gov (United States)

Bender, K; Bissbort, S; Kuhn, A; Nagel, M; Günther, E

1986-02-01

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in the rat (Rattus norvegicus) is described. The locus, designed Acp-2, is not expressed in erythrocytes but is expressed in all other tissues studied. The product of Acp-2 hydrolyzes a wide variety of phosphate monoesters and is inhibited by L(+)-tartaric acid. Inbred rat strains have fixed either allele Acp-2a or allele Acp-2b. Codominant expression is observed in the respective F1 hybrids. Backcross progenies revealed the expected 1:1 segregation ratio. Possible loose linkage was found between the Acp-2 and the Pep-3 gene loci at a recombination frequency of 0.36 +/- 0.06.

Low Soil Phosphorus Availability Increases Acid Phosphatases Activities and Affects P Partitioning in Nodules, Seeds and Rhizosphere of Phaseolus vulgaris

Directory of Open Access Journals (Sweden)

Jean-Jacques Drevon

2012-06-01

Full Text Available The effect of phosphorus (P deficiency on phosphatases activities in N2-fixing legumes has been widely studied in hydroponic culture. However, the response of acid phosphatase (APase and phytase in rhizosphere, nodules and seeds of Phaseolus vulgaris to low soil’s P-availability is not yet fully understood. In this study, six genotypes of N2-fixing P. vulgaris were grown under contrasting soil P-availabilities; i.e., low  (4.3 mg P kg−1 and sufficient (16.7 mg P kg−1 in the Haouz region of Morocco. At flowering and maturity stages, plants were harvested and analyzed for their phosphatases activities, growth and P content. Results show that, low P decreased nodulation, growth, P uptake and N accumulation in all the genotypes, but to a greater extent in the sensitive recombinant inbreed line 147. In addition, while seed P content was slightly reduced under low P soil; a higher P was noticed in the Flamingo and Contender large seeded-beans (6.15 to 7.11 mg g−1. In these latter genotypes, high APase and phytase activities in seeds and nodules were associated with a significant decline in rhizosphere’s available P. APase activity was mainly stimulated in nodules, whereas phytase activity was highly induced in seeds (77%. In conclusion, the variations of APase and phytase activities in nodules and seeds depend on genotype and can greatly influence the internal utilization of P, which might result in low P soil tolerance in N2-fixing legumes.

The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

Science.gov (United States)

Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

2016-03-01

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.

Serum Bone Resorption Markers after Parathyroidectomy for Renal Hyperparathyroidism: Correlation Analyses for the Cross-Linked N-telopeptide of Collagen I and Tartrate-Resistant Acid Phosphatase

Directory of Open Access Journals (Sweden)

Kuo-Chin Hung

2012-01-01

Full Text Available Patients on long-term dialysis may develop secondary hyperparathyroidism (SHPT with increased serum concentrations of bone resorption markers such as the cross-linked N-telopeptide of type I collagen (NTX and type-5b tartrate-resistant acid phosphatase (TRAP. When SHPT proves refractory to treatment, parathyroidectomy (PTX may be needed. Renal patients on maintenance HD who received PTX for refractory SHPT (n=23 or who did not develop refractory SHPT (control subjects; n=25 were followed prospectively for 4 weeks. Serum intact parathyroid hormone (iPTH, NTX, TRAP, and bone alkaline phosphatase (BAP concentrations were measured serially and correlation analyses were performed. iPTH values decreased rapidly and dramatically. BAP values increased progressively with peak increases observed at 2 weeks after surgery. NTX and TRAP values decreased concurrently and progressively through 4 weeks following PTX. A significant correlation between TRAP and NTX values was observed before PTX but not at 4 weeks after PTX. Additionally, the fractional changes in serum TRAP were larger than those in serum NTX at all times examined after PTX. Serum iPTH, TRAP, and NTX values declined rapidly following PTX for SHPT. Serum TRAP values declined to greater degrees than serum NTX values throughout the 4-week period following PTX.

Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

Directory of Open Access Journals (Sweden)

Daniel Bini

Full Text Available ABSTRACT: Arbuscular mycorrhizal fungi (AMF are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.

Ethylene signalling is involved in regulation of phosphate starvation-induced gene expression and production of acid phosphatases and anthocyanin in Arabidopsis

KAUST Repository

Lei, Mingguang

2010-11-30

With the exception of root hair development, the role of the phytohormone ethylene is not clear in other aspects of plant responses to inorganic phosphate (Pi) starvation. The induction of AtPT2 was used as a marker to find novel signalling components involved in plant responses to Pi starvation. Using genetic and chemical approaches, we examined the role of ethylene in the regulation of plant responses to Pi starvation. hps2, an Arabidopsis mutant with enhanced sensitivity to Pi starvation, was identified and found to be a new allele of CTR1 that is a key negative regulator of ethylene responses. 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, increases plant sensitivity to Pi starvation, whereas the ethylene perception inhibitor Ag+ suppresses this response. The Pi starvation-induced gene expression and acid phosphatase activity are also enhanced in the hps2 mutant, but suppressed in the ethylene-insensitive mutant ein2-5. By contrast, we found that ethylene signalling plays a negative role in Pi starvation-induced anthocyanin production. These findings extend the roles of ethylene in the regulation of plant responses to Pi starvation and will help us to gain a better understanding of the molecular mechanism underlying these responses. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

International Nuclear Information System (INIS)

Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

2004-01-01

Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

Directory of Open Access Journals (Sweden)

Consuelo Giménez-Pardo

2004-03-01

Full Text Available Cholinesterase and acid phosphatase (AP, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

Science.gov (United States)

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

Serum alkaline phosphatase screening for vitamin D deficiency states

International Nuclear Information System (INIS)

Shaheen, S.; Barrakzai, Q.

2012-01-01

Objective: To determine whether serum vitamin D levels are correlated with serum levels of alkaline phosphatase or not. Study Design: Cross-sectional, observational study. Place and Duration of Study: Multi-centre study, conducted at Liaquat National Hospital and Medical College, National Medical Centre and Medicare Hospital, Karachi, from January to October 2009. Methodology: Patients attending the Orthopaedic OPDs with complaints of pain in different body regions and serum vitamin D/sub 3/ levels of greater or equal to 30 ng/ml were included in the study. Patients with vitamin D deficiency were further categorized into mild deficiency or insufficiency (vit. D/sub 3/ = 20-29 ng/ml), moderate deficiency (vit. D/sub 3/ = 5 - 19 ng/ml) and severe deficiency forms (vit. D/sub 3/ < 5 ng/ml). Pearson correlation was applied to test the correlation of serum alkaline phosphatase levels with serum vitamin D/sub 3/ levels. P-value < 0.05 was considered to be significant. Results: Out of 110 samples, 26 had mild (23%), 61 had moderate (55%) and 21 had severe (19.1%) vitamin D deficiencies. All of the patients in the three groups had alkaline phosphatase with in normal limits and the total mean value of the enzyme was 135.97 +- 68.14I U/L. The inter group comparison showed highest values of alkaline phosphatase in the moderate vitamin D deficiency group. The correlation coefficient of alkaline phosphatase and serum vitamin D/sub 3/ levels was r =0.05 (p =0.593). Conclusion: Serum vitamin D/sub 3/ levels may not be correlated with increased serum alkaline phosphatase levels. Therefore, alkaline phosphatase may not be used as a screening test to rule out vitamin D deficiency. (author)

Hemagglutinating and acid phosphatase (AcPASE activities in developing seedlings of four species of Cucurbitaceae

Directory of Open Access Journals (Sweden)

Irena Lorenc-Kubis

2014-01-01

Full Text Available The acid phosphatase and hemagglutinating activities of four species of Cucurbitaceae were determined during seeds germination and seedlings development. In all cases traces of enzyme and hemagglutinating activities were found in dry and imbibided seeds. In developing seedlings of Cucumis sativus the activities increased to maximum on the 3rd day while in other species on the 6th day of germination and than fell down. Dot blot and Western blot techniques have shown that in seeds and seedlings of all investigated species present were proteins which cross-reacted with antibodies raised against lectins: CLBa and Con A. It has been shown that proteins from seeds and seedlings of Cucurbita maxima var. bambino, Cucurbita pepo var. giromontia and Cucumis sativus had more pronounced antigenical similarity to lectin CLBa (from Cucurbitaceae than Con A, while proteins from cotyledons of Cucurbita pepo var. patissonina reacted better with antibodies raised against Con A (the lectin from Papilionaceae than with CLBa.

TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

Directory of Open Access Journals (Sweden)

Emmanuelle Dubots

Full Text Available The evolutionarily conserved target of rapamycin complex 1 (TORC1 controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

Application of ascorbic acid 2-phosphate as a new voltammetric substrate for alkaline phosphatase determination in human serum

Directory of Open Access Journals (Sweden)

Wei Sun

2005-12-01

Full Text Available An electrochemical assay of the enzyme alkaline phosphatase (ALP using ascorbic acid 2-phosphate (AAP as a new voltammetric substrate has been described in this paper. In the alkaline buffer solution the ALP enzymatic hydrolysis product of AAP was ascorbic acid (AA, which was an electro-active substance and had a sensitive differential pulse voltammetric (DPV oxidative response on glassy carbon electrode (GCE at +380 mV (versus Ag/AgCl, so the activity of ALP could be monitored voltammetrically of the oxidative peak current of AA. The electrochemical behaviours of AA were carefully studied and the AA standard solution could be measured by DPV method in the linear range from 10.0 to 1000.0 μmol/L with the detection limit of 8.0 μmol/L. The optimal conditions for ALP enzymatic reaction and the voltammetric detection were optimized. Under the optimal conditions the calibration curve for ALP assay exhibited a linear range from 0.4 to 2000.0 U/L with a detection limit of 0.3 U/L. This proposed method was further applied to determine the ALP content in healthy human serum and the results were in good agreement with the traditional p-nitrophenyl phosphate spectrophotometric method. The kinetic constants of enzymatic reaction were also investigated with the apparent kinetic constant Km as 2.77 mmol/L and the maximum velocity Vmax as 0.33 mol/min.

The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress

Directory of Open Access Journals (Sweden)

Shiori Sekine

2016-03-01

Full Text Available Phosphoglycerate mutase family member 5 (PGAM5 is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT, a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21 that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.

Impact of ionic aluminium on extracellular phosphatases in acidified lakes

Czech Academy of Sciences Publication Activity Database

Bittl, T.; Vrba, Jaroslav; Nedoma, Jiří; Kopáček, Jiří

2001-01-01

Roč. 3, č. 9 (2001), s. 578-587 ISSN 1462-2912 R&D Projects: GA ČR GA206/97/0072; GA ČR GA206/00/0063 Keywords : acid phosphatases * pH effect * inhibition Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.276, year: 2001

Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

Directory of Open Access Journals (Sweden)

Kirstin eHobiger

2015-02-01

Full Text Available The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs. Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs. In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs. Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

Simultaneous demonstration of acid phosphatase and glucose-6-phosphate dehydrogenase in mouse hepatocytes. A novel electron-microscopic dual staining enzyme-cytochemistry

Directory of Open Access Journals (Sweden)

S Matsubara

2010-01-01

Full Text Available Acid phosphatase (ACPase and glucose-6-phosphate dehydrogenase (G6PD play important roles in cell biology/disease pathophysiology in various organs including the liver. The purpose of the present report is to introduce a new enzymecytochemical method to simultaneously demonstrate the subcellular localization of ACPase and G6PD within the same hepatocyte in the mouse liver. The ultrastructural localization of ACPase and G6PD were demonstrated, with concomitant use of the cerium method and the copper-ferrocyanide method, respectively. ACPase labelings were localized in the lysosomes, and G6PD labelings were visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of the hepatocyte. This novel double staining procedure may be a useful histochemical tool for the study of liver functions in both physiological and pathological conditions.

Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli

International Nuclear Information System (INIS)

Zheng, Jimin; Lee, Daniel C.; Jia, Zongchao

2009-01-01

Isocitrate dehydrogenase kinase/phosphatase has been crystallized in three different crystal forms. Data were collected from each crystal form for structure determination. The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P4 1 2 1 2, P3 2 21 and P2 1 2 1 2 1 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively

Phosphoprotein phosphatase of bovine spleen cell nuclei: physicochemical properties

International Nuclear Information System (INIS)

Rezyapkin, V.I.; Leonova, L.E.; Komkova, A.I.

1986-01-01

The physicochemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were studied. The enzyme possesses broad substrate specificity and catalyzes the dephosphorylation of phosphocasein, ATP, ADP, and p-nitrophenyl phosphate (pNPP). K/sub m/ for ATP, ADP, and pNPP are equal to 0.44, 0.43, and 1.25 mM, respectively. M/sub r/ of the enzyme, according to the data of gel filtraction of Sephadex G-75 and electrophoresis in polyacrylamide gel of various concentrations is ∼ 33,000. In electrophoresis in the presence of SDS, two protein bands with M/sub r/ 12,000 and 18,000 are detected. In the enzyme molecule, acid amino acid residues predominate; two free SH groups and two disulfide bridges are detected. Phosphoprotein phosphatase is a glycoprotein, containing ∼ 22% carbonhydrates. The protein possesses a supplementary absorption maximum at 560 nm. Ammonium molybdate is a competitive inhibitor with K/sub i/ 0.37 μM, while sodium fluoride is a noncompetitive inhibitor with K/sub i/ 1.3 mM. Incubation in the presence of 2 mM phenylmethylsulfonyl fluoride for 25 h leads to a loss of ∼ 46% of the enzymatic activity. Ammonium molybdate, sodium fluoride, and PMSF are reversible inhibitors. Modifications of the SH groups, NH 2 groups, and histidine leads to a decrease in the enzymatic activity. Incubation of phosphoprotein phosphatase with [γ- 32 P]ATP leads to the incorporation of 0.33 mole 33 P per mole of the enzyme. The mechanism of hydrolysis of the phosphodiester bond, catalyzed by the enzyme, is discussed

Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

Science.gov (United States)

Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-12-26

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

A generally applicable sequential alkaline phosphatase immunohistochemical double staining

NARCIS (Netherlands)

van der Loos, Chris M.; Teeling, Peter

2008-01-01

A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

OpenAIRE

Hoopman, Todd C.; Wang, Wei; Brautigam, Chad A.; Sedillo, Jennifer L.; Reilly, Thomas J.; Hansen, Eric J.

2007-01-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation dom...

Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

Directory of Open Access Journals (Sweden)

Guillaume Manat

Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

Energy Technology Data Exchange (ETDEWEB)

Martinez, Robert J.; Beazley, Melanie J.; Wilson, Jarad J.; Taillefert, Martial; Sobecky, Patricia A.

2005-04-05

The overall goal of this project is to examine the role of nonspecific phosphohydrolases present in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, we hypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbial release and/or accumulation of PO{sub 4}{sup 3-}. During this phase of the project we have been conducting assays to determine the effects of pH, inorganic anions and organic ligands on U(VI) mineral formation and precipitation when FRC bacterial isolates were grown in simulated groundwater medium. The molecular characterization of FRC isolates has also been undertaken during this phase of the project. Analysis of a subset of gram-positive FRC isolates cultured from FRC soils (Areas 1, 2 and 3) and background sediments have indicated a higher percentage of isolates exhibiting phosphatase phenotypes (i.e., in particular those surmised to be PO{sub 4}{sup 3-}-irrepressible) relative to isolates from the reference site. A high percentage of strains that exhibited such putatively PO{sub 4}{sup 3-}-irrepressible phosphatase phenotypes were also resistant to the heavy metals lead and cadmium. Previous work on FRC strains, including Arthrobacter, Bacillus and Rahnella spp., has demonstrated differences in tolerance to U(VI) toxicity (200 {micro}M) in the absence of organophosphate substrates. For example, Arthrobacter spp. exhibited the greatest tolerance to U(VI) while the Rahnella spp. have been shown to facilitate the precipitation of U(VI) from solution and the Bacillus spp. demonstrate the greatest sensitivity to acidic conditions and high concentrations of U(VI). PCR-based detection of FRC strains are being conducted to determine if non-specific acid phosphatases of the known molecular classes [i.e., classes A, B and C] are present in these FRC isolates. Additionally, these

Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells.

Science.gov (United States)

Botos, Erzsébet; Turi, Agnes; Müllner, Nándor; Kovalszky, Ilona; Tátrai, Péter; Kiss, Anna L

2007-01-01

The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

Probing protein phosphatase substrate binding

DEFF Research Database (Denmark)

Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

2012-01-01

Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

Directory of Open Access Journals (Sweden)

Bennett Hayley J

2010-08-01

Full Text Available Abstract Background Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. Results We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. Conclusion This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases

A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis.

Science.gov (United States)

Beresford, Nicola J; Saville, Charis; Bennett, Hayley J; Roberts, Ian S; Tabernero, Lydia

2010-08-02

Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides

Phosphatases in Cancer : Shifting the balance

NARCIS (Netherlands)

E. Hoekstra (Elmer)

2015-01-01

markdownabstractAbstract The role of phosphatases in cancer is an ignored research field, mostly based on the dogma that phosphatases function as tumor suppressor genes. However, in our opinion dephosphorylation events by phosphatases can also enhance signaling in cancer. The current research

Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

Science.gov (United States)

Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

2018-04-24

The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

International Nuclear Information System (INIS)

Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

2012-01-01

Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Liu, Xin, E-mail: fangfei6073@126.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn [Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150040 (China); Cai, Hua, E-mail: small-big@sohu.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Ji, Wei, E-mail: iwei_j@hotmail.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Luo, Xiao, E-mail: luoxiao2010@yahoo.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Li, Jing, E-mail: lijing@neau.edu.cn [Plant Secondary Metabolism Laboratory, Northeast Agricultural University, Harbin 150030 (China); Bai, Xi, E-mail: baixi@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China)

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver.

Science.gov (United States)

Kubicz, A; Szalewicz, A; Chrambach, A

1991-01-01

1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

Science.gov (United States)

Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

1999-01-01

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

Loss of SYNJ1 dual phosphatase activity leads to early onset refractory seizures and progressive neurological decline

DEFF Research Database (Denmark)

Hardies, Katia; Cai, Yiying; Jardel, Claude

2016-01-01

SYNJ1 encodes a polyphosphoinositide phosphatase, synaptojanin 1, which contains two consecutive phosphatase domains and plays a prominent role in synaptic vesicle dynamics. Autosomal recessive inherited variants in SYNJ1 have previously been associated with two different neurological diseases...... with intractable epilepsy and tau pathology. We performed whole exome or genome sequencing in three independent sib pairs with early onset refractory seizures and progressive neurological decline, and identified novel segregating recessive SYNJ1 defects. A homozygous missense variant resulting in an amino acid...

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

Directory of Open Access Journals (Sweden)

Heidi O Nousiainen

Full Text Available Prostatic acid phosphatase (PAP, the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG, but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP in the brain by utilizing mice deficient in TMPAP (PAP-/- mice. Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

Science.gov (United States)

Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

2008-06-27

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

International Nuclear Information System (INIS)

Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Sakaguchi, Nobuo

2006-01-01

BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM lo IgD hi B-cells but not in AICD susceptible immature IgM hi IgD lo B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23 hi B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack of G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells

The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

Science.gov (United States)

Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

2012-11-01

Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid, drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate accumulation.

Science.gov (United States)

Ma, Lichao; Wang, Yanrong; Liu, Wenxian; Liu, Zhipeng

2014-11-01

GDP-mannose 3', 5'-epimerase (GME) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (ascorbic acid) biosynthetic pathway in higher plants. In this study, a novel cDNA fragment (MsGME) encoding a GME protein was isolated and characterised from alfalfa (Medicago sativa). An expression analysis confirmed that MsGME expression was induced by salinity, PEG and acidity stresses. MsGME overexpression in Arabidopsis enhanced tolerance of the transgenic plants to salt, drought and acid. Real-time PCR analysis revealed that the transcript levels of GDP-D-mannose pyrophosphorylase (GMP), L-galactose-phosphate 1-P phosphatase (GP) and GDP-L-galactose phosphorylase (GGP) were increased in transgenic Arabidopsis (T3 generation). Moreover, the ascorbate content was increased in transgenic Arabidopsis. Our results suggest that MsGME can effectively enhance tolerance of transgenic Arabidopsis to acid, drought and salt by increasing ascorbate accumulation.

Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells.

Directory of Open Access Journals (Sweden)

Soung-Hoon Lee

Full Text Available Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA, a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo.Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP. VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX, a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice.Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.

Structural comparisons of two allelic variants of human placental alkaline phosphatase.

Science.gov (United States)

Millán, J L; Stigbrand, T; Jörnvall, H

1985-01-01

A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

DEFF Research Database (Denmark)

Daum, G; Regenass, S; Sap, J

1994-01-01

Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

Science.gov (United States)

Rajadurai, Charles V; Havrylov, Serhiy; Coelho, Paula P; Ratcliffe, Colin D H; Zaoui, Kossay; Huang, Bruce H; Monast, Anie; Chughtai, Naila; Sangwan, Veena; Gertler, Frank B; Siegel, Peter M; Park, Morag

2016-09-12

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease. © 2016 Rajadurai et al.

Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

Directory of Open Access Journals (Sweden)

Julieta B Carrillo

Full Text Available Ostreococcus tauri, the smallest free-living (non-symbiotic eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP, homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.

A vaccine strategy with multiple prostatic acid phosphatase-fused cytokines for prostate cancer treatment.

Science.gov (United States)

Fujio, Kei; Watanabe, Masami; Ueki, Hideo; Li, Shun-Ai; Kinoshita, Rie; Ochiai, Kazuhiko; Futami, Junichiro; Watanabe, Toyohiko; Nasu, Yasutomo; Kumon, Hiromi

2015-04-01

Immunotherapy is one of the attractive treatment strategies for advanced prostate cancer. The US Food and Drug Administration (FDA) previously approved the therapeutic vaccine, sipuleucel-T, which is composed of autologous antigen-presenting cells cultured with a fusion protein [prostatic acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GMCSF)]. Although sipuleucel-T has been shown to prolong the median survival of patients for 4.1 months, more robust therapeutic effects may be expected by modifying the vaccination protocol. In the present study, we aimed to develop and validate a novel vaccination strategy using multiple PAP-fused cytokines for prostate cancer treatment. Using a super gene expression (SGE) system that we previously established to amplify the production of a recombinant protein, significant amounts of PAP-fused cytokines [human GMCSF, interleukin-2 (IL2), IL4, IL7 and mouse GMCSF and IL4] were obtained. We examined the activity of the fusion proteins in vitro to validate their cytokine functions. A significant upregulation of dendritic cell differentiation from monocytes was achieved by PAP-GMCSF when used with the other PAP-fused cytokines. The PAP-fused human IL2 significantly increased the proliferation of lymphocytes, as determined by flow cytometry. We also investigated the in vivo therapeutic effects of multiple PAP-fused cytokines in a mouse prostate cancer model bearing prostate-specific antigen (PSA)- and PAP-expressing tumors. The simultaneous intraperitoneal administration of PAP-GMCSF, -IL2, -IL4 and -IL7 significantly prevented tumor induction and inhibited the tumor growth in the PAP-expressing tumors, yet not in the PSA-expressing tumors. The in vivo therapeutic effects with the multiple PAP-fused cytokines were superior to the effects of PAP-GMCSF alone. We thus demonstrated the advantages of the combined use of multiple PAP-fused cytokines including PAP-GMCSF, and propose a promising prostatic

The protein histidine phosphatase LHPP is a tumour suppressor.

Science.gov (United States)

Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

2018-03-29

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

Posttranslational heterogeneity of bone alkaline phosphatase in metabolic bone disease.

Science.gov (United States)

Langlois, M R; Delanghe, J R; Kaufman, J M; De Buyzere, M L; Van Hoecke, M J; Leroux-Roels, G G

1994-09-01

Bone alkaline phosphatase is a marker of osteoblast activity. In order to study the posttranscriptional modification (glycosylation) of bone alkaline phosphatase in bone disease, we investigated the relationship between mass and catalytic activity of bone alkaline phosphatase in patients with osteoporosis and hyperthyroidism. Serum bone alkaline phosphatase activity was measured after lectin precipitation using the Iso-ALP test kit. Mass concentration of bone alkaline phosphatase was determined with an immunoradiometric assay (Tandem-R Ostase). In general, serum bone alkaline phosphatase mass and activity concentration correlated well. The activity : mass ratio of bone alkaline phosphatase was low in hyperthyroidism. Activation energy of the reaction catalysed by bone alkaline phosphatase was high in osteoporosis and in hyperthyroidism. Experiments with neuraminidase digestion further demonstrated that the thermodynamic heterogeneity of bone alkaline phosphatase can be explained by a different glycosylation of the enzyme.

Genetic evaluations of Chinese patients with odontohypophosphatasia resulting from heterozygosity for mutations in the tissue-non-specific alkaline phosphatase gene.

Science.gov (United States)

Wan, Jia; Zhang, Li; Liu, Tang; Wang, Yewei

2017-08-01

Hypophosphatasia is a rare heritable metabolic disorder characterized by defective bone and tooth mineralization accompanied by a deficiency of tissue-non-specific (liver/bone/kidney) isoenzyme of alkaline phosphatase activity, caused by a number of loss-of-function mutations in the alkaline phosphatase liver type gene. We seek to explore the clinical manifestations and identify the mutations associated with the disease in a Chinese odonto- hypophosphatasia family. The proband and his younger brother affected with premature loss of primary teeth at their 2-year-old. They have mild abnormal serum alkaline phosphatase and 25-hydroxy vitamin D values, but the serum alkaline phosphatase activity of their father, mother and grandmother, who showed no clinical symptoms of hypophosphatasia, was exhibited significant decreased. In addition to premature loss of primary teeth, the proband and his younger brother showed low bone mineral density, X-rays showed that they had slight metaphyseal osteoporosis changes, but no additional skeletal abnormalities. Deoxyribonucleic acid sequencing and analysis revealed a single nucleotide polymorphism c.787T>C (p.Y263H) in exon 7 and/or a novel mutation c.-92C>T located at 5'UTR were found in the affected individuals. We examined all individuals of an odonto- hypophosphatasia family by clinical and radiographic examinations as well as laboratory assays. Furthermore, all 12 exons and the exon-intron boundaries of the alkaline phosphatase liver type gene were amplified and directly sequenced for further analysis and screened for mutations. Our present findings suggest the single nucleotide polymorphism c.787T>C and c.-92C>T should be responsible for the odonto- hypophosphatasia disorders in this family.

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

Science.gov (United States)

Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

2016-09-01

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS+ for Diabetic and Its Complication

Directory of Open Access Journals (Sweden)

Seung Hwan Hwang

2016-09-01

Full Text Available Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β, advanced glycation end products (AGEs, and ABTS+ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM, PTP1β (1.88 μM, AGEs (82.79 μM, and ABTS+ (6.03 μM. This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively. In addition, 3,5-di-O-CQA (88.14 μM and protocatechuic acid (32.93 μM had a considerable inhibitory effect against α-glucosidase and ABTS+. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.

[Effect of elevated atmospheric CO2 on soil urease and phosphatase activities].

Science.gov (United States)

Chen, Lijun; Wu, Zhijie; Huang, Guohong; Zhou, Likai

2002-10-01

The response of soil urease and phosphatase activities at different rice growth stages to free air CO2 enrichment (FACE) was studied. The results showed that comparing with the ambient atmospheric CO2 concentration (370 mumol.mol-1), FACE (570 mumol.mol-1) significantly increased the urease activity of 0-5 cm soil layer at the vigorous growth stage of rice, whole that of 5-10 cm layer had no significant change during the whole growing season. Phosphatase activity of 0-5 cm and 5-10 cm soil layers significantly increased, and the peak increment was at the vigorous growth stage of rice.

A novel 35 kDa frog liver acid metallophosphatase.

Science.gov (United States)

Szalewicz, A; Radomska, B; Strzelczyk, B; Kubicz, A

1999-04-12

The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases.

An Experimental Insight into Extracellular Phosphatases – Differential Induction of Cell-Specific Activity in Green Algae Cultured under Various Phosphorus Conditions

Directory of Open Access Journals (Sweden)

Jaroslav Vrba

2018-02-01

Full Text Available Extracellular phosphatase activity (PA has been used as an overall indicator of P depletion in lake phytoplankton. However, detailed insights into the mechanisms of PA regulation are still limited, especially in the case of acid phosphatases. The novel substrate ELF97 phosphate allows for tagging PA on single cells in an epifluorescence microscope. This fluorescence-labeled enzyme activity (FLEA assay enables for autecological studies in natural phytoplankton and algal cultures. We combined the FLEA assay with image analysis to measure cell-specific acid PA in two closely related species of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta isolated from two acidic lakes with distinct P availability. The strains were cultured in a mineral medium supplied with organic (beta-glycerol phosphate or inorganic (orthophosphate P at three concentrations. Both strains responded to experimental conditions in a similar way, suggesting that acid extracellular phosphatases were regulated irrespectively of the origin and history of the strains. We found an increase in cell-specific PA at low P concentration and the cultures grown with organic P produced significantly higher (ca. 10-fold PA than those cultured with the same concentrations of inorganic P. The cell-specific PA measured in the cultures grown with the lowest organic P concentration roughly corresponded to those of the original Coccomyxa population from an acidic lake with impaired P availability. The ability of Coccomyxa strains to produce extracellular phosphatases, together with tolerance for both low pH and metals can be one of the factors enabling the dominance of the genus in extreme conditions of acidic lakes. The analysis of frequency distribution of the single-cell PA documented that simple visual counting of ‘active’ (labeled and ‘non-active’ (non-labeled cells can lead to biased conclusions regarding algal P status because the actual PA of the ‘active’ cells can vary from

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

Science.gov (United States)

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

Phosphatase activity in sandy soil influenced by mycorrhizal and non-mycorrhizal cover crops

Directory of Open Access Journals (Sweden)

Alceu Kunze

2011-06-01

Full Text Available Cover crops may difffer in the way they affect rhizosphere microbiota nutrient dynamics. The purpose of this study was to evaluate the effect of mycorrhizal and non-mycorrhizal cover crops on soil phosphatase activity and its persistence in subsequent crops. A three-year experiment was carried out with a Typic Quartzipsamment. Treatments were winter species, either mycorrhizal black oat (Avena strigosa Schreb or the non-mycorrhizal species oilseed radish (Raphanus sativus L. var. oleiferus Metzg and corn spurry (Spergula arvensis L.. The control treatment consisted of resident vegetation (fallow in the winter season. In the summer, a mixture of pearl millet (Pennisetum americanum L. with sunnhemp (Crotalaria juncea L. or with soybean (Glycine max L. was sown in all plots. Soil cores (0-10 cm and root samples were collected in six growing seasons (winter and summer of each year. Microbial biomass P was determined by the fumigation-extraction method and phosphatase activity using p-nitrophenyl-phosphate as enzyme substrate. During the flowering stage of the winter cover crops, acid phosphatase activity was 30-35 % higher in soils with the non-mycorrhizal species oilseed radish, than in the control plots, regardless of the amount of P immobilized in microbial biomass. The values of enzyme activity were intermediate in the plots with corn spurry and black oat. Alkaline phosphatase activity was 10-fold lower and less sensitive to the treatments, despite the significant relationship between the two phosphatase activities. The effect of plant species on the soil enzyme profile continued in the subsequent periods, during the growth of mycorrhizal summer crops, after completion of the life cycle of the cover crops.

Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS⁺ for Diabetic and Its Complication.

Science.gov (United States)

Hwang, Seung Hwan; Wang, Zhiqiang; Yoon, Ha Na; Lim, Soon Sung

2016-09-16

Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA) derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β), advanced glycation end products (AGEs), and ABTS⁺ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM), PTP1β (1.88 μM), AGEs (82.79 μM), and ABTS⁺ (6.03 μM). This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively). In addition, 3,5-di-O-CQA (88.14 μM) and protocatechuic acid (32.93 μM) had a considerable inhibitory effect against α-glucosidase and ABTS⁺. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.

Identification of human pulmonary alkaline phosphatase isoenzymes.

Science.gov (United States)

Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

1997-04-01

An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli

DEFF Research Database (Denmark)

Brøndsted, Lone; Atlung, Tove

1996-01-01

The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene has been investigated during different environmental conditions using single copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase...... of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high expression of the operon was obtained in glucose medium supplemented with formate, where E.coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented...... in this paper indicate a clear difference in the regulation of the cyx-appA operon compared to the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function at even more oxygen limiting conditions than cytochrome d oxidase. The expression of the app...

Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

Science.gov (United States)

Sarma, Bani Kanta

2013-09-01

The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

Science.gov (United States)

Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

2014-02-14

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

Synthesis of diadenosine 5', 5'''-P1, P4-tetraphosphate (AP4A) in sea urchin embryos

International Nuclear Information System (INIS)

Morioka, Mizue; Shimada, Hiraku

1984-01-01

Sea urchin embryos were labeled with ( 3 H)adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5', 5'''-P 1 , P 4 -tetraphosphate (AP 4 A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP 4 A fraction. The present result suggests that AP 4 A is actively synthesized in the sea urchin embryos. (author)

5′-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion

Science.gov (United States)

Zaoui, Kossay; Huang, Bruce H.; Sangwan, Veena; Gertler, Frank B.

2016-01-01

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5′-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2–Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease. PMID:27597754

Transmembrane prostatic acid phosphatase (TMPAP interacts with snapin and deficient mice develop prostate adenocarcinoma.

Directory of Open Access Journals (Sweden)

Ileana B Quintero

Full Text Available The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP, a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP and a transmembrane type-I (TMPAP. The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/- with C57BL/6J background. The PAP(-/- mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

Science.gov (United States)

Quintero, Ileana B.; Herrala, Annakaisa M.; Araujo, César L.; Pulkka, Anitta E.; Hautaniemi, Sampsa; Ovaska, Kristian; Pryazhnikov, Evgeny; Kulesskiy, Evgeny; Ruuth, Maija K.; Soini, Ylermi; Sormunen, Raija T.; Khirug, Leonard; Vihko, Pirkko T.

2013-01-01

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma. PMID:24039861

The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

DEFF Research Database (Denmark)

Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

2016-01-01

Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN...

Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

Directory of Open Access Journals (Sweden)

Delyan P Ivanov

Full Text Available Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

OpenAIRE

Zhang, Ju-Yuan; Zou, Jie; Bao, Qiyu; Chen, Wen-Li; Wang, Li; Yang, Huanming; Zhang, Cheng-Cai

2006-01-01

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to th...

Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

Science.gov (United States)

Chang, Heng-Kwei

2015-01-01

Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329

Science.gov (United States)

Sánchez-Martín, Pablo; Raththagala, Madushi; Bridges, Travis M.; Husodo, Satrio; Gentry, Matthew S.; Sanz, Pascual; Romá-Mateo, Carlos

2013-01-01

Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization. PMID:23922729

Purification and characterization of three lowmolecular- weight acid ...

African Journals Online (AJOL)

SDS-PAGE of the purified acid phosphatase PI resolved two closely protein bands that migrated to approximately 14 and 12 kDa. ... The three enzymes had a similar optima pH (5.0) and temperature (55°C), and appeared to be stable in the presence of non-ionic detergents such as Triton X-100, Nonidet P 40 as well as Na+ ...

5,5-Dihydroxybarbituric acid 1,4-dioxane hemisolvate

Directory of Open Access Journals (Sweden)

Thomas Gelbrich

2010-05-01

Full Text Available The asymmetric unit of the title compound,, C4H4N2O5·0.5C4H8O2, contains one molecule of 5,5-dihydroxybarbituric acid with a nearly planar barbiturate ring and half a molecule of 1,4-dioxane. The geometry of the centrosymmetric dioxane molecule is close to an ideal chair conformation. The crystal structure exhibits a complex three-dimensional hydrogen-bonded network. Barbiturate molecules are connected to one another via N—H...O=C, O—H...O=C and N—H...O(hydroxy interactions, while the barbituric acid molecule is linked to dioxane by an O—H...O contact.

An Additional Method for Analyzing the Reversible Inhibition of an 
Enzyme Using Acid Phosphatase as a Model.

Science.gov (United States)

Baumhardt, Jordan M; Dorsey, Benjamin M; McLauchlan, Craig C; Jones, Marjorie A

2015-08-01

Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: K max , K min , and K inflect . K max and K min represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. K inflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

Science.gov (United States)

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

Science.gov (United States)

Singh, Neetu; Heneberg, Petr; Rathaur, Sushma

2014-10-01

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 μM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 μM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.

Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

International Nuclear Information System (INIS)

Hill, Jennifer J.; Callaghan, Deborah A.; Ding Wen; Kelly, John F.; Chakravarthy, Balu R.

2006-01-01

Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in Brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3β, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is First report of a phosphorylation site on complexin 2

The activity state of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Science.gov (United States)

Wagenmakers, A J; Schepens, J T; Veldhuizen, J A; Veerkamp, J H

1984-05-15

An assay is described to define the proportion of the branched-chain 2-oxo acid dehydrogenase complex that is present in the active state in rat tissues. Activities are measured in homogenates in two ways: actual activities, present in tissues, by blocking both the kinase and phosphatase of the enzyme complex during homogenization, preincubation, and incubation with 1-14C-labelled branched-chain 2-oxo acid, and total activities by blocking only the kinase during the 5 min preincubation (necessary for activation). The kinase is blocked by 5 mM-ADP and absence of Mg2+ and the phosphatase by the simultaneous presence of 50 mM-NaF. About 6% of the enzyme is active in skeletal muscle of fed rats, 7% in heart, 20% in diaphragm, 47% in kidney, 60% in brain and 98% in liver. An entirely different assay, which measures activities in crude tissue extracts before and after treatment with a broad-specificity protein phosphatase, gave similar results for heart, liver and kidney. Advantages of our assay with homogenates are the presence of intact mitochondria, the simplicity, the short duration and the high sensitivity. The actual activities measured indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and kidney and is limited in skeletal muscle in the fed state.

Increased liver alkaline phosphatase and aminotransferase ...

African Journals Online (AJOL)

The effect of daily, oral administration of ethanolic extract of Khaya senegalensis stem bark (2mg/kg body weight) for 18days on the alkaline phosphatase, aspartate and alanine aminotransferase activities of rat liver and serum were investigated. Compared with the control, the activities of liver alkaline phosphatase (ALP), ...

Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

Directory of Open Access Journals (Sweden)

Fang Li

2006-03-01

Full Text Available Abstract Background Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in

Modelando a coordenação de fosfato com o sítio ativo das fosfatases ácidas púrpuras Modeling the coordination of phosphate to the active site of purple acid phosphatases

OpenAIRE

Marcos Aires de Brito; Ademir Neves; Luiz R. Zilli

1997-01-01

We present a new binuclear complex, Fe2III(BBPMP)(OH)(O2 P(OPh)2) ClO4.CH3OH, 3, where BBPMP is the anion of 2,6-bis(2-hydroxybenzyl)(2-pyridylmethyl) aminomethyl-4-methylphenol, as a suitable model for the chromophoric site of purple acid phosphatases coordinated to phosphate. The complex was obtained by the reaction of complex 2, Fe2III(BBPMP)(O2P(OPh) 2)2 ClO4.H2O, in CH3CN with one equivalent of triethylamine. Based on the chromophoric properties of the model complex, lmax = 560 nm/ e = 4...

Improvement of Student Understanding of How Kinetic Data Facilitates the Determination of Amino Acid Catalytic Function through an Alkaline Phosphatase Structure/Mechanism Bioinformatics Exercise

Science.gov (United States)

Grunwald, Sandra K.; Krueger, Katherine J.

2008-01-01

Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was…

Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism

Energy Technology Data Exchange (ETDEWEB)

Erickson, Anna I.; Sarsam, Reta D.; Fisher, Andrew J., E-mail: ajfisher@ucdavis.edu

2014-05-10

The cysQ gene from Mycobacterium tuberculosis was cloned and the expressed protein, a 3′-phosphoadenosine-5′’-phosphatase, was purified and crystallized. X-ray diffraction data were collected to 1.7 Å resolution.

Electro-chemical coupling in the voltage-dependent phosphatase Ci-VSP

Science.gov (United States)

Kohout, Susy C.; Bell, Sarah C.; Liu, Lijun; Xu, Qiang; Minor, Daniel L.; Isacoff, Ehud Y.

2010-01-01

In the voltage sensing phosphatase, Ci-VSP, a voltage sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we find that the inter-domain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays show that the linker is also needed for activity in the isolated PD. We identify a late step of VSD motion in the full-length protein that depends on the linker. Strikingly, this VSD motion is found to require PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme. PMID:20364128

Persistently increased intestinal fraction of alkaline phosphatase

DEFF Research Database (Denmark)

Nathan, E; Baatrup, G; Berg, H

1984-01-01

Persistent elevation of the intestinal fraction of the alkaline phosphatase (API) as an isolated finding has to our knowledge not been reported previously. It was found in a boy followed during a period of 5.5 years. The only symptom was transient periodic fatigue observed at home, but not apparent...... during hospitalization. His blood type was O, RH+, Le (a-, b+) and he was a secretor of H-substance, which may be associated with rising API activity after fat-loading. In this case API was unchanged after fat-loading. Neither intestinal nor liver diseases were found, and no other cause for the elevated...

Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil

Directory of Open Access Journals (Sweden)

Ying Nie

2016-01-01

Full Text Available The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling.

 Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis.

Science.gov (United States)

Hilscher, Moira; Enders, Felicity B; Carey, Elizabeth J; Lindor, Keith D; Tabibian, James H

2016-01-01

 Introduction. Recent studies suggest that serum alkaline phosphatase may represent a prognostic biomarker in patients with primary sclerosing cholangitis. However, this association remains poorly understood. Therefore, the aim of this study was to investigate the prognostic significance and clinical correlates of alkaline phosphatase normalization in primary sclerosing cholangitis. This was a retrospective cohort study of patients with a new diagnosis of primary sclerosing cholangitis made at an academic medical center. The primary endpoint was time to hepatobiliaryneoplasia, liver transplantation, or liver-related death. Secondary endpoints included occurrence of and time to alkaline phosphatase normalization. Patients who did and did not achieve normalization were compared with respect to clinical characteristics and endpoint-free survival, and the association between normalization and the primary endpoint was assessed with univariate and multivariate Cox proportional-hazards analyses. Eighty six patients were included in the study, with a total of 755 patient-years of follow-up. Thirty-eight patients (44%) experienced alkaline phosphatase normalization within 12 months of diagnosis. Alkaline phosphatase normalization was associated with longer primary endpoint-free survival (p = 0.0032) and decreased risk of requiring liver transplantation (p = 0.033). Persistent normalization was associated with even fewer adverse endpoints as well as longer survival. In multivariate analyses, alkaline phosphatase normalization (adjusted hazard ratio 0.21, p = 0.012) and baseline bilirubin (adjusted hazard ratio 4.87, p = 0.029) were the only significant predictors of primary endpoint-free survival. Alkaline phosphatase normalization, particularly if persistent, represents a robust biomarker of improved long-term survival and decreased risk of requiring liver transplantation in patients with primary sclerosing cholangitis.

Screening a library of household substances for inhibitors of phosphatases: An introduction to high-throughput screening.

Science.gov (United States)

Taylor, Ann T S

2005-01-01

Library screening methods are commonly used in industry and research. This article describes an experiment that screens a library of household substances for properties that would make a good "drug," including enzyme inhibition, neutral pH, and nondenaturing to proteins, using wheat germ acid phosphatase as the target protein. An adaptation of the experiment appropriate for lower level biochemistry or outreach is also described. This work was supported by Wabash College through the Haines Fund for the Study of Biochemistry and the National Science Foundation through Grant DUE 0126242. Copyright © 2005 International Union of Biochemistry and Molecular Biology, Inc.

A new highly selective metabotropic excitatory amino acid agonist: 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid

DEFF Research Database (Denmark)

Bräuner-Osborne, Hans; Sløk, F A; Skjaerbaek, N

1996-01-01

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5......-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N......-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was...

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions.

Science.gov (United States)

Tan, Hao; Wu, Xiang; Xie, Liyuan; Huang, Zhongqian; Peng, Weihong; Gan, Bingcheng

2016-01-01

Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in Escherichia coli and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 μmol/min/mg at the physiological temperature of 37°C and 3,989 μmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feeding. © 2016 S. Karger AG, Basel.

Further characterization of serum alkaline phosphatase from male and female beagle dogs.

Science.gov (United States)

Amacher, D E; Higgins, C V; Schomaker, S J; Clay, R J

1989-01-01

Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

Science.gov (United States)

Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

1999-01-01

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains

Directory of Open Access Journals (Sweden)

Huxley-Jones Julie

2007-11-01

Full Text Available Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

Science.gov (United States)

Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

1996-09-01

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Pharmacology of (S)-homoquisqualic acid and (S)-2-amino-5-phosphonopentanoic acid [(S)-AP5] at cloned metabotropic glutamate receptors

DEFF Research Database (Denmark)

Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

1998-01-01

1 In this study we have determined the pharmacological profile of (S)-quisqualic acid, (S)-2-amino-4-phosphonobutyric acid ((S)-AP4) and their higher homologues (S)-homoquisqualic acid, (S)-2-amino-5-phosphonopentanoic acid ((S)-AP5), respectively, and (R)-AP5 at subtypes of metabotropic (S)-glutamic...... demonstrate that incorporation of an additional carbon atom into the backbone of (S)-glutamic acid and its analogues, to give the corresponding homologues, and replacement of the terminal carboxyl groups by isosteric acidic groups have profound effects on the pharmacological profiles at mGlu receptor subtypes...... acid (mGlu) receptors expressed in Chinese hamster ovary cells. 2 (S)-Quisqualic acid was a potent mGlu1/mGlu5 agonist (EC50 values of 1.1 microM and 0.055 microM, respectively) showing no activity at mGlu2 and weak agonism at mGlu4 (EC50 approximately 1000 microM). 3 (S)-Homoquisqualic acid displayed...

Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

Science.gov (United States)

Tang, Wei; Lin, Jinxing; Newton, Ronald J

2007-05-01

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

Directory of Open Access Journals (Sweden)

Charlene Desbonnet

2016-04-01

Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

Defining Starch Binding by Glucan Phosphatases

DEFF Research Database (Denmark)

Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

2015-01-01

Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch...... is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

Phosphoinositide 5-phosphatase Fig 4p is required for both acute rise and subsequent fall in stress-induced phosphatidylinositol 3,5-bisphosphate levels.

Science.gov (United States)

Duex, Jason E; Nau, Johnathan J; Kauffman, Emily J; Weisman, Lois S

2006-04-01

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P(2)) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P(2), vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P(2) vacuole volume is restored. We show that Fig 4p, consistent with its proposed role as a PI3,5P(2) 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P(2). Surprisingly, we find that Fig 4p is also required for the hyperosmotic shock-induced increase in PI3,5P(2) levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P(2) and further suggest that Fig 4p is important in regulating both the acute rise and subsequent fall in PI3,5P(2) levels.

Synthesis and phosphatase activity of a Cobalt(II) phenanthroline ...

Indian Academy of Sciences (India)

MAMONI GARAI

2017-09-19

Sep 19, 2017 ... Synthesis and phosphatase activity of a Cobalt(II) phenanthroline complex. MAMONI GARAIa ... tion, cobalt complexes have gained importance because of their application as ... 2.3 Physical measurements. Infrared spectrum ...

A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary Cholangitis

NARCIS (Netherlands)

Nevens, Frederik; Andreone, Pietro; Mazzella, Giuseppe; Strasser, Simone I.; Bowlus, Christopher; Invernizzi, Pietro; Drenth, Joost P. H.; Pockros, Paul J.; Regula, Jaroslaw; Beuers, Ulrich; Trauner, Michael; Jones, David E.; Floreani, Annarosa; Hohenester, Simon; Luketic, Velimir; Shiffman, Mitchell; van Erpecum, Karel J.; Vargas, Victor; Vincent, Catherine; Hirschfield, Gideon M.; Shah, Hemant; Hansen, Bettina; Lindor, Keith D.; Marschall, Hanns-Ulrich; Kowdley, Kris V.; Hooshmand-Rad, Roya; Marmon, Tonya; Sheeron, Shawn; Pencek, Richard; MacConell, Leigh; Pruzanski, Mark; Shapiro, David; Angus, Peter; Roberts, Stuart; Vogel, Wolfgang; Graziadei, Ivo; de Lédinghen, Victor; Berg, Thomas; Gotthardt, Daniel; Hartmann, Heinz; Kremer, Andreas E.; Lammert, Frank; Manns, Michael P.; Rust, Christian; Schramm, Christoph; Trautwein, Christian; Zeuzem, Stefan; Carbone, Marco; van Nieuwkerk, Carin C. M. J.; Celinski, Krzysztof; Gonciarz, Maciej; Hartleb, Marek; Milkiewicz, Piotr; Parés, Albert; Bramley, Peter; Thorburn, Douglas; Mookerjee, Rajeshwar P.; Burroughs, Andrew; Chapman, Roger; Dillon, John F.; Greer, John A.; Tripathi, Dhiraj; McCune, Anne; Ryder, Stephen; Bacon, Bruce R.; Naik, Jahnavi; Wang, Lan Sun; Bodenheimer, Henry C.; Bowlus, Christopher L.; Chalasani, Naga; Forman, Lisa M.; Gordon, Stuart C.; Luketic, Velimir A.; Mayo, Marlyn; Muir, Andrew J.; Reddy, K. Gautham; Talwalker, Jayant T.; Vierling, John M.

2016-01-01

BACKGROUND Primary biliary cholangitis ( formerly called primary biliary cirrhosis) can progress to cirrhosis and death despite ursodiol therapy. Alkaline phosphatase and bilirubin levels correlate with the risk of liver transplantation or death. Obeticholic acid, a farnesoid X receptor agonist, has

Amino acid limitation induces down-regulation of WNT5a at transcriptional level

International Nuclear Information System (INIS)

Wang Zuguang; Chen Hong

2009-01-01

An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear β-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

Science.gov (United States)

Graser, Stephanie; Mentrup, Birgit; Schneider, Doris; Klein-Hitpass, Ludger; Jakob, Franz; Hofmann, Christine

2015-10-01

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies. Copyright © 2015 Elsevier Inc. All rights reserved.

NEW METABOLITES OF THE DRUG 5-AMINOSALICYLIC ACID .2. N-FORMYL-5-AMINOSALICYLIC ACID

DEFF Research Database (Denmark)

Tjornelund, J.; Hansen, S. H.; Cornett, Claus

1991-01-01

1. A new metabolite of the drug 5-aminosalicylic acid (5-ASA) has been found in urine from pigs and in plasma of humans. The metabolite has been isolated from pig urine using an XAD-2 column and purified using preparative h.p.l.c. 2. The metabolite has been identified as N-formyl-5-ASA (5-formami...

[Phenolic acid derivatives from Bauhinia glauca subsp. pernervosa].

Science.gov (United States)

Zhao, Qiao-Li; Wu, Zeng-Bao; Zheng, Zhi-Hui; Lu, Xin-Hua; Liang, Hong; Cheng, Wei; Zhang, Qing-Ying; Zhao, Yu-Ying

2011-08-01

To study the chemical constituents of Bauhinia glauca subsp. pernervosa, eleven phenolic acids were isolated from a 95% ethanol extract by using a combination of various chromatographic techniques including column chromatography over silica gel, ODS, MCI, Sephadex LH-20, and semi-preparative HPLC. By spectroscopic techniques including 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS, these compounds were identified as isopropyl O-beta-(6'-O-galloyl)-glucopyranoside (1), ethyl O-beta-(6'-O-galloyl)-glucopyranoside (2), 3, 4, 5-trimethoxyphenyl-(6'-O-galloyl)-O-beta-D-glucopyranoside (3), 3, 4, 5-trimethoxyphenyl-beta-D-glucopyranoside (4), gallic acid (5), methyl gallate (6), ethyl gallate (7), protocatechuic acid (8), 3, 5-dimethoxy-4-hydroxybenzoic acid (9), erigeside C (10) and glucosyringic acid (11). Among them, compound 1 is a new polyhydroxyl compound; compounds 2, 10, and 11 were isolated from the genus Bauhinia for the first time, and the other compounds were isolated from the plant for the first time. Compounds 6 and 8 showed significant protein tyrosine phosphatase1B (PTP1B) inhibitory activity in vitro with the IC50 values of 72.3 and 54.1 micromol x L(-1), respectively.

INFLUENCE OF LIMING AND WASTE ORGANIC MATERIALS ON THE ACTIVITY OF PHOSPHATASE IN SOIL CONTAMINATED WITH NICKEL

Directory of Open Access Journals (Sweden)

Beata Kuziemska

2014-10-01

Full Text Available A study was carried out on soil following a two-year pot experiment that was conducted in 2009–2010, in three repetitions in Siedlce. The experiment included the following factors: 1 – amount of Ni in soil (0, 75, 150 and 225 mg·kg-1 soil by applying an aqueous NiSO4·7H2O solution; 2 – liming (0 and Ca according to 1 Hh as CaCO3; 3 – organic waste products (rye straw at a dose of 4 t·ha-1 and brown coal at a dose of 40 t·ha-1. In each experimental year, orchard grass was the test plant and four swaths were harvested. The activities of acidic and alkaline phosphatase, pH and the content of carbon in organic compounds were determined in the soil samples collected after each grass swath and in each experimental year. It was found that Ni at 75 mg·kg-1 soil activated the enzymes under study, whereas higher doses caused their statistically-confirmed inactivation. The lowest activity of the investigated enzymes was detected in soil supplemented with 225 Ni·kg-1 soil. Liming caused an increase in the activity of alkaline phosphatase and a reduction in the activity of acidic phosphatase. Straw and brown coal induced a substantial increase in the activity of both enzymes in the tested soil samples. Both liming and straw and carbon eliminated the negative effect of higher nickel doses on the activity of the enzymes under study.

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

Science.gov (United States)

Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L; Thiel, Gerhard; Moroni, Anna

2013-03-01

The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

Association of canalicular membrane enzymes with bile acid micelles and lipid aggregates in human and rat bile.

Science.gov (United States)

Accatino, L; Pizarro, M; Solís, N; Koenig, C S

1995-01-18

This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the celular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.

Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

International Nuclear Information System (INIS)

Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

2012-01-01

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

Energy Technology Data Exchange (ETDEWEB)

Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

2014-10-02

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

Overexpression of a Protein Phosphatase 2C from Beech Seeds in Arabidopsis Shows Phenotypes Related to Abscisic Acid Responses and Gibberellin Biosynthesis1

Science.gov (United States)

Reyes, David; Rodríguez, Dolores; González-García, Mary Paz; Lorenzo, Oscar; Nicolás, Gregorio; García-Martínez, José Luis; Nicolás, Carlos

2006-01-01

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis. PMID:16815952

Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

Directory of Open Access Journals (Sweden)

Kayla A Boortz

Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

Protein kinase and phosphatase activities of thylakoid membranes

International Nuclear Information System (INIS)

Michel, H.; Shaw, E.K.; Bennett, J.

1987-01-01

Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

PrognosticValue of PINP,BoneAlkaline Phosphatase, CTX-I, andYKL-40 in Patients With Metastatic Prostate Carcinoma

DEFF Research Database (Denmark)

Brasso, Klaus; Christensen, Ib Jarle; Johansen, Julia S

2006-01-01

Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma. Prostate. 2006 Apr 1;66(5):503-13. PMID: 16372331 [PubMed - indexed for MEDLINE]......Prognostic value of PINP, bone alkaline phosphatase, CTX-I, and YKL-40 in patients with metastatic prostate carcinoma. Prostate. 2006 Apr 1;66(5):503-13. PMID: 16372331 [PubMed - indexed for MEDLINE]...

Cdc14 phosphatase

DEFF Research Database (Denmark)

Machín, Félix; Quevedo Rodriguez, Oliver; Ramos-Pérez, Cristina

2016-01-01

and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only...

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

Science.gov (United States)

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Inactivation of peroxidase, pectinesterase and alkaline phosphatase in polymers as a model for irradiation of dried foodstuffs

NARCIS (Netherlands)

Roozen, J.P.

1972-01-01

This thesis consists of a summary only of 6 refereed scientific papers published in Lebensmittel -Wissenschaft und -Technologie 3, 37-40 (1970); 4, 24-26, 93-96, 158-162, 196-200 (1971); 5, 128-131 (1972).

Aqueous solutions containing an enzyme (peroxidase, alkaline phosphatase,

The impact of phosphatases on proliferative and survival signaling in cancer.

Science.gov (United States)

Narla, Goutham; Sangodkar, Jaya; Ryder, Christopher B

2018-05-03

The dynamic and stringent coordination of kinase and phosphatase activity controls a myriad of physiologic processes. Aberrations that disrupt the balance of this interplay represent the basis of numerous diseases. For a variety of reasons, early work in this area portrayed kinases as the dominant actors in these signaling events with phosphatases playing a secondary role. In oncology, these efforts led to breakthroughs that have dramatically altered the course of certain diseases and directed vast resources toward the development of additional kinase-targeted therapies. Yet, more recent scientific efforts have demonstrated a prominent and sometimes driving role for phosphatases across numerous malignancies. This maturation of the phosphatase field has brought with it the promise of further therapeutic advances in the field of oncology. In this review, we discuss the role of phosphatases in the regulation of cellular proliferation and survival signaling using the examples of the MAPK and PI3K/AKT pathways, c-Myc and the apoptosis machinery. Emphasis is placed on instances where these signaling networks are perturbed by dysregulation of specific phosphatases to favor growth and persistence of human cancer.

The oxygen isotope composition of phosphate released from phytic acid by the activity of wheat and Aspergillus niger phytase

Science.gov (United States)

von Sperber, C.; Tamburini, F.; Brunner, B.; Bernasconi, S. M.; Frossard, E.

2015-07-01

Phosphorus (P) is an essential nutrient for living organisms. Under P-limiting conditions plants and microorganisms can exude extracellular phosphatases that release inorganic phosphate (Pi) from organic phosphorus compounds (Porg). Phytic acid (myo-inositol hexakisphosphate, IP6) is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields available Pi and less phosphorylated inositol derivates as products. The hydrolysis of organic P compounds by phosphatases leaves an isotopic imprint on the oxygen isotope composition (δ18O) of released Pi, which might be used to trace P in the environment. This study aims at determining the effect of phytase on the oxygen isotope composition of released Pi. For this purpose, enzymatic assays with histidine acid phytases from wheat and Aspergillus niger were prepared using IP6, adenosine 5'-monophosphate (AMP) and glycerophosphate (GPO4) as substrates. For a comparison to the δ18O of Pi released by other extracellular enzymes, enzymatic assays with acid phosphatases from potato and wheat germ with IP6 as a substrate were prepared. During the hydrolysis of IP6 by phytase, four of the six Pi were released, and one oxygen atom from water was incorporated into each Pi. This incorporation of oxygen from water into Pi was subject to an apparent inverse isotopic fractionation (ϵ ~ 6 to 10 ‰), which was similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ~ 7 ‰), where less than three Pi were released. The incorporation of oxygen from water into Pi during the hydrolysis of AMP and GPO4 by phytase yielded a normal isotopic fractionation (ϵ ~ -12 ‰), similar to values reported for acid phosphatases from potato and wheat germ. We attribute this similarity in ϵ to the same amino acid sequence motif (RHGXRXP) at the active site of these enzymes, which leads to similar reaction mechanisms. We suggest that the striking

A randomized trial of obeticholic acid monotherapy in patients with primary biliary cholangitis.

Science.gov (United States)

Kowdley, Kris V; Luketic, Velimir; Chapman, Roger; Hirschfield, Gideon M; Poupon, Raoul; Schramm, Christoph; Vincent, Catherine; Rust, Christian; Parés, Albert; Mason, Andrew; Marschall, Hanns-Ulrich; Shapiro, David; Adorini, Luciano; Sciacca, Cathi; Beecher-Jones, Tessa; Böhm, Olaf; Pencek, Richard; Jones, David

2018-05-01

Obeticholic acid (OCA), a potent farnesoid X receptor agonist, was studied as monotherapy in an international, randomized, double-blind, placebo-controlled phase 2 study in patients with primary biliary cholangitis who were then followed for up to 6 years. The goals of the study were to assess the benefit of OCA in the absence of ursodeoxycholic acid, which is relevant for patients who are intolerant of ursodeoxycholic acid and at higher risk of disease progression. Patients were randomized and dosed with placebo (n = 23), OCA 10 mg (n = 20), or OCA 50 mg (n = 16) given as monotherapy once daily for 3 months (1 randomized patient withdrew prior to dosing). The primary endpoint was the percent change in alkaline phosphatase from baseline to the end of the double-blind phase of the study. Secondary and exploratory endpoints included change from baseline to month 3/early termination in markers of cholestasis, hepatocellular injury, and farnesoid X receptor activation. Efficacy and safety continue to be monitored through an ongoing 6-year open-label extension (N = 28). Alkaline phosphatase was reduced in both OCA groups (median% [Q1, Q3], OCA 10 mg -53.9% [-62.5, -29.3], OCA 50 mg -37.2% [-54.8, -24.6]) compared to placebo (-0.8% [-6.4, 8.7]; P OCA improved many secondary and exploratory endpoints (including γ-glutamyl transpeptidase, alanine aminotransferase, conjugated bilirubin, and immunoglobulin M). Pruritus was the most common adverse event; 15% (OCA 10 mg) and 38% (OCA 50 mg) discontinued due to pruritus. OCA monotherapy significantly improved alkaline phosphatase and other biochemical markers predictive of improved long-term clinical outcomes. Pruritus increased dose-dependently with OCA treatment. Biochemical improvements were observed through 6 years of open-label extension treatment. (Hepatology 2018;67:1890-1902). © 2017 The Authors. Hepatology published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.

Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.

Science.gov (United States)

Villalba-Galea, Carlos A; Miceli, Francesco; Taglialatela, Maurizio; Bezanilla, Francisco

2009-07-01

The Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

An evolutionarily conserved phosphatidate phosphatase maintains lipid droplet number and endoplasmic reticulum morphology but not nuclear morphology

Directory of Open Access Journals (Sweden)

Anoop Narayana Pillai

2017-11-01

Full Text Available Phosphatidic acid phosphatases are involved in the biosynthesis of phospholipids and triacylglycerol, and also act as transcriptional regulators. Studies to ascertain their role in lipid metabolism and membrane biogenesis are restricted to Opisthokonta and Archaeplastida. Here, we report the role of phosphatidate phosphatase (PAH in Tetrahymena thermophila, belonging to the Alveolata clade. We identified two PAH homologs in Tetrahymena, TtPAH1 and TtPAH2. Loss of function of TtPAH1 results in reduced lipid droplet number and an increase in endoplasmic reticulum (ER content. It also results in more ER sheet structure as compared to wild-type Tetrahymena. Surprisingly, we did not observe a visible defect in the nuclear morphology of the ΔTtpah1 mutant. TtPAH1 rescued all known defects in the yeast pah1Δ strain and is conserved functionally between Tetrahymena and yeast. The homologous gene derived from Trypanosoma also rescued the defects of the yeast pah1Δ strain. Our results indicate that PAH, previously known to be conserved among Opisthokonts, is also present in a set of distant lineages. Thus, a phosphatase cascade is evolutionarily conserved and is functionally interchangeable across eukaryotic lineages.

Curcumin Inhibits NTHi-Induced MUC5AC Mucin Overproduction in Otitis Media via Upregulation of MAPK Phosphatase MKP-1

Directory of Open Access Journals (Sweden)

Anuhya Sharma Konduru

2017-01-01

Full Text Available Otitis media (OM, characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

Synthesis and evaluation of mutual azo prodrug of 5-aminosalicylic acid linked to 2-phenylbenzoxazole-2-yl-5-acetic acid in ulcerative colitis

Directory of Open Access Journals (Sweden)

Jilani JA

2013-07-01

Full Text Available Jamal A Jilani,1 Maha Shomaf,2 Karem H Alzoubi3 1Department of Medicinal Chemistry and Pharmacognosy, Jordan University of Science and Technology, Irbid, Jordan; 2Department of Pathology, Jordan University, Amman, Jordan; 3Department of Clinical Pharmacy, Jordan University of Science and Technology, Irbid, Jordan Abstract: In this study, the syntheses of 4-aminophenylbenzoxazol-2-yl-5-acetic acid, (an analogue of a known nonsteroidal anti-inflammatory drug [NSAID] and 5-[4-(benzoxazol-2-yl-5-acetic acidphenylazo]-2-hydroxybenzoic acid (a novel mutual azo prodrug of 5-aminosalicylic acid [5-ASA] are reported. The structures of the synthesized compounds were confirmed using infrared (IR, hydrogen-1 nuclear magnetic resonance (1H NMR, and mass spectrometry (MS spectroscopy. Incubation of the azo compound with rat cecal contents demonstrated the susceptibility of the prepared azo prodrug to bacterial azoreductase enzyme. The azo compound and the 4-aminophenylbenzoxazol-2-yl-5-acetic acid were evaluated for inflammatory bowel diseases, in trinitrobenzenesulfonic acid (TNB-induced colitis in rats. The synthesized diazo compound and the 4-aminophenylbenzoxazol-2-yl-5-acetic acid were found to be as effective as 5-aminosalicylic acid for ulcerative colitis. The results of this work suggest that the 4-aminophenylbenzoxazol-2-yl-5-acetic acid may represent a new lead for treatment of ulcerative colitis. Keywords: benzoxazole acetic acid, azo prodrug, colon drug delivery

YbiV from E. coli K12 is a HAD phosphatase

Energy Technology Data Exchange (ETDEWEB)

Roberts, Anne; Lee, Seok-Yong; McCullagh, Emma; Silversmith, Ruth E.; Wemmer, David E.

2004-03-16

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

International Nuclear Information System (INIS)

Hristov, K.; Mitev, V.; Knox, K.

2006-01-01

Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

International Nuclear Information System (INIS)

Volakakis, Nikolaos; Joodmardi, Eliza; Perlmann, Thomas

2009-01-01

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

Science.gov (United States)

Schröter, W; Neuvians, M

1970-12-01

Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

Biochemical effects of ethylene diamine tetra-acetic acid (EDTA) on ...

African Journals Online (AJOL)

mamus

2016-04-13

Apr 13, 2016 ... fresh weight, level of lipid per oxidation, alkaline phosphatase, acid phosphatase, super oxide dismutase ... Its effects on plants are manifested by inhibition of the normal uptake and utilization of mineral nutrients (Liu et al., 2003). ... reactive oxygen species (ROS) indirectly by production of disturbances in ...

Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

Science.gov (United States)

Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

2013-01-01

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

Directory of Open Access Journals (Sweden)

Andaleeb Sajid

2011-03-01

Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

Regulation of Akt/Protein Kinase B Signaling by a Novel Protein Phosphatase in Breast Cancer Cells

National Research Council Canada - National Science Library

Brognard, John; Newton, Alexandra

2008-01-01

...: cell proliferation, growth, and apoptosis. Finally, since this phosphatase resides in a location of frequent loss of heterozygosity in breast cancer, we sought to determine if this phosphatase played a role in breast tumorigenesis...

Biotransformation of 5-hydroxy-methylfurfural into 2,5-furan-dicarboxylic acid by bacterial isolate using thermal acid algal hydrolysate.

Science.gov (United States)

Yang, Chu-Fang; Huang, Ci-Ruei

2016-08-01

Thermal acid hydrolysis is often used to deal with lignocellulosic biomasses, but 5-hydroxy-methylfurfural (5-HMF) formed during hydrolysis deeply influences downstream fermentation. 2,5-Furan-dicarboxylic acid (FDCA), which is in the list of future important biomass platform molecules can be obtained using 5-HMF biotransformation. Based on the connection between 5-HMF removal in acid hydrolysate and FDCA production, the optimum thermal acid hydrolysis condition for macroalgae Chaetomorpha linum was established. Potential microbes capable of transforming 5-HMF into FDCA were isolated and characterized under various parameters and inoculated into algal hydrolysate to perform 5-HMF biotransformation. The optimum hydrolysis condition was to apply 0.5M HCl to treat 3% algal biomass under 121°C for 15min. Isolated Burkholderia cepacia H-2 could transform 2000mg/L 5-HMF at the initial pH of 7 at 28°C and 1276mg/L FDCA was received. Strain B. cepacia H-2 was suitable for treating the algal hydrolysate without dilution, receiving 989.5mg/L FDCA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Osteocalcin and bone-specific alkaline phosphatase in Sickle cell ...

African Journals Online (AJOL)

specific alkaline phosphatase (b-AP) total protein levels were evaluated as indicators of bone turnover in twenty patients with sickle cell haemoglobinopathies and in twenty normal healthy individuals. The serum bonespecific alkaline phosphatase ...

Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA.

Science.gov (United States)

Brelle, Solène; Baronian, Grégory; Huc-Brandt, Sylvaine; Zaki, Laila Gannoun; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

2016-01-15

Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public-health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

Energy Technology Data Exchange (ETDEWEB)

Volakakis, Nikolaos; Joodmardi, Eliza [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); The Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm (Sweden)

2009-12-25

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

Comparative acute nephrotoxicity of salicylic acid, 2,3-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid in young and middle aged Fischer 344 rats.

Science.gov (United States)

McMahon, T F; Stefanski, S A; Wilson, R E; Blair, P C; Clark, A M; Birnbaum, L S

1991-03-11

Experimental evidence suggests that the oxidative metabolites 2,3- and 2,5-dihydroxybenzoic acid (DIOH) may be responsible for the nephrotoxicity of salicylic acid (SAL). In the present study, enzymuria in conjunction with glucose (GLU) and protein (PRO) excretion were used as endpoints to compare the relative nephrotoxicity of SAL with 2,3- and 2,5-DIOH. In addition, the effect of age on enzymuria and GLU and PRO excretion following treatment with SAL or 2,3- and 2,5-DIOH was investigated because the elderly are at greater risk for SAL-induced nephrotoxicity. Three and 12-month male Fischer 344 rats were administered either no treatment, vehicle, SAL, 2,3-DIOH, or 2,5-DIOH at 500 mg/kg p.o. in 5 ml/kg corn oil/DMSO (5:1). Effects of these treatments on functional integrity of renal tissue was assessed from 0--72 h after dosing by measurement of urinary creatinine, GLU, and PRO, as well as excretion of proximal and distal tubular renal enzymes. Enzymes measured as indicators of proximal tubular damage were N-acetyl-beta-glucosaminidase (NAG), gamma glutamyltransferase (GGT), alanine aminotransferase (ALT), and alkaline phosphatase (AP), while urinary lactate dehydrogenase (LD) and aspartate aminotransferase (AST) were measured as indicators of distal tubular damage. In comparison to 3-month vehicle-treated rats, 2,3- and 2,5-DIOH caused a significant increase between 0-8 h in excretion of urinary GLU and activities of AST, NAG, and LD, with peak effects occurring between 4-8 h. Toxic effects of either metabolite were not evident beyond 24 h, and toxicity of 2,5-DIOH was significantly greater in comparison to 2,3-DIOH. SAL treatment resulted in similar effects on enzymuria as well as GLU and PRO excretion, but peak effects did not occur until 16-24 h, and often persisted until 72 h after dosing. Maximal enzymuria in response to SAL treatment was significantly greater in 12- vs. 3-month rats for AST, NAG, and LD. In response to 2,3-DIOH treatment, the maximal

Alkaline phosphatase as a screening test for osteomalacia.

Science.gov (United States)

Chinoy, Muhammad Amin; Javed, Muhammad Imran; Khan, Alamzeb; Sadruddin, Nooruddin

2011-01-01

Vitamin D deficiency remains common in children and adults in Pakistan despite adequate sunlight exposure. Diagnosis in adults is usually delayed and is made following pathological fractures that result in significant morbidity. The objective of this study was to see whether Serum Alkaline Phosphatase levels could be used as a screening test for osteomalacia. The Study was conducted at Fatima Hospital, Baqai Medical University, Gadap, Karachi, between July 2002 and June 2005. Serum calcium levels are commonly used to screen patients suspected of osteomalacia, and raised serum alkaline phosphatase (SALP) is considered a diagnostic finding. We used SALP to screen patients who presented with back or non-specific aches and pain of more than six months duration. Three hundred thirty-four (334) patients were screened of which 116 (35%) had raised SALP. Osteomalacia was diagnosed in 92 (79.3%) of these 116 either by plain radiographs, bone biopsy or isotope bone scan. Fifty-four (53.4%) of the 101 cases had a normal level of serum calcium. Osteomalacia is likely to be missed if only serum calcium is used to screen patients. Serum Alkaline Phosphate should be used as the preferred method for screening these patients.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

Science.gov (United States)

Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

1997-04-15

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine

Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

Science.gov (United States)

Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

2013-02-08

The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter, leading to FASN-Shp2-COP1 complex formation and FASN degradation mediated by ubiquitination pathway. By fasting p38 is activated and stimulates FASN protein degradation in mice. Consistently, the FASN protein levels are dramatically elevated in mouse liver and pancreas in which Shp2/Ptpn11 is selectively deleted. Thus, this study identifies a new activity for Shp2 in lipid metabolism.

Root-exuded acid phosphatase and 32Pi-uptake kinetics of wheat, rye and triticale under phosphorus starvation

International Nuclear Information System (INIS)

Pandey, Renu

2006-01-01

A nutrient culture experiment was conducted with cereal species viz., wheat (Triticum aestivum L.) cv. PBW-343), rye (Secale cereale L cv. R-308) and triticale (Triticale octoploide L. cv. DT-46), a hybrid of wheat and rye, to examine the genetic variation in root-exuded acid phosphatase (ACPase) activity and kinetics of 32 Pi-uptake under P deficient condition. The ACPase activity was assayed in the extract (intra-) and on surface (extra-cellular) or root, using p-nitrophenyl phosphate as substrate. Significantly higher ACPase activity was observed in wheat followed by rye and triticale both on the root surface and in root extract. In general, root surface ACPase activity was 2.2-fold higher than that in root extract. A strong correlation (r 2 = 0.87**) between extra and intra-cellular ACPase activity was observed. In terms of kinetic parameters, it was observed that 32 Pi uptake and I max values were significantly higher in rye while C min and K m were lowest compared to wheat and triticale. The dry weights of shoot, root and total plant were significantly higher in rye compared to wheat and triticale. Rye also had higher amount of total plant P content The superiority of rye over wheat and triticale in P uptake was observed mainly due to efficient Pi-uptake system, which needs further studies to ascertain the enhancement of Pi-induced high-affinity P transporter in these cereals. (author)

Osteomalacia with low alkaline phosphatase: a not so rare condition with important consequences.

Science.gov (United States)

Belkhouribchia, Jamal; Bravenboer, Bert; Meuwissen, Marije; Velkeniers, Brigitte

2016-01-28

Hypophosphatasia is a genetic disorder, characterised by a dysfunctional tissue-non-specific isoenzyme of alkaline phosphatase that impacts bone metabolism and predisposes to osteomalacia or rickets. The clinical presentation is very diverse, depending on the age of onset and the severity of the disease. Several forms of hypophosphatasia are recognised. We present a case of a 50-year-old woman with low impact fractures and loss of teeth at a young age. She also had a low alkaline phosphatase and was diagnosed with adult hypophosphatasia. Although the severe forms of hypophosphatasia are rather rare, the adult form is thought to occur quite frequently. As this condition is not well known by healthcare professionals, the time to diagnosis and initiation of adequate treatment is often postponed. When encountering a patient with low alkaline phosphatase, low bone density or a history of bone fractures, the possibility of hypophosphatasia should be considered. 2016 BMJ Publishing Group Ltd.

Dynamic Changes in Yeast Phosphatase Families Allow for Specialization in Phosphate and Thiamine Starvation.

Science.gov (United States)

Nahas, John V; Iosue, Christine L; Shaik, Noor F; Selhorst, Kathleen; He, Bin Z; Wykoff, Dennis D

2018-05-10

Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of Saccharomycetaceae yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In Candida glabrata , loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, CgPMU3 , as a thiamine phosphatase. We further determine that the 81% identical paralog CgPMU2 does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources. Copyright © 2018, G3: Genes, Genomes, Genetics.

Serum creatinine and alkaline phosphatase levels are associated with severe chronic periodontitis.

Science.gov (United States)

Caúla, A L; Lira-Junior, R; Tinoco, E M B; Fischer, R G

2015-12-01

Periodontitis may alter systemic homeostasis and influence creatinine and alkaline phosphatase levels. Therefore, the aim of this study was to evaluate the relationship between severe chronic periodontitis and serum creatinine and alkaline phosphatase levels. One hundred patients were evaluated, 66 with severe chronic periodontitis (test group) and 34 periodontally healthy controls (control group). Medical, demographic and periodontal parameters were registered. Blood sample was collected after an overnight fast and serum creatinine and alkaline phosphatase levels were determined. There were significant differences between test and control groups in ethnicity, gender and educational level (p creatinine level (p creatinine and alkaline phosphatase levels. Severe chronic periodontitis was associated to lower creatinine and higher alkaline phosphatase levels. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Hydrogen Bonded Structures of Two 5-Bromobarbituric Acids and Analysis of Unequal C5–X and C5–X′ Bond Lengths (X = X′ = F, Cl, Br or Me in 5,5-Disubstituted Barbituric Acids

Directory of Open Access Journals (Sweden)

Thomas Gelbrich

2016-04-01

Full Text Available The crystal structure of the methanol hemisolvate of 5,5-dibromobarbituric acid (1MH displays an H-bonded layer structure which is based on N–H∙∙∙O=C, N–H∙∙∙O(MeOH and (MeOHO–H∙∙∙O interactions. The barbiturate molecules form an H-bonded substructure which has the fes topology. 5,5′-Methanediylbis(5-bromobarbituric acid 2, obtained from a solution of 5,5-dibromobarbituric acid in nitromethane, displays a N–H···O=C bonded framework of the sxd type. The conformation of the pyridmidine ring and the lengths of the ring substituent bonds C5–X and C5–X′ in crystal forms of 5,5-dibromobarbituric acid and three closely related analogues (X = X′ = Br, Cl, F, Me have been investigated. In each case, a conformation close to a C5-endo envelope is correlated with a significant lengthening of the axial C5–X′ in comparison to the equatorial C5–X bond. Isolated molecule geometry optimizations at different levels of theory confirm that the C5-endo envelope is the global conformational energy minimum of 5,5-dihalogenbarbituric acids. The relative lengthening of the axial bond is therefore interpreted as an inherent feature of the preferred envelope conformation of the pyrimidine ring, which minimizes repulsive interactions between the axial substituent and pyrimidine ring atoms.

5'Azido-N-1-napthylphthalamic acid, a photolabile analog of N-1-naphthylphthalamic acid

International Nuclear Information System (INIS)

Voet, J.G.; Howley, K.S.; Shumsky, J.S.

1987-01-01

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [ 3 H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K/sub 0.5/ = 2.8 x 10 -7 molar. The K/sub 0.5/ for NPA under these conditions is 2 x 10 -8 molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [ 3 H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [ 3 H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment

Effect of Soil Amendments on Microbial Resilience Capacity of Acid Soil Under Copper Stress.

Science.gov (United States)

Mounissamy, Vassanda Coumar; Kundu, Samaresh; Selladurai, Rajendiran; Saha, Jayanta Kumar; Biswas, Ashish Kumar; Adhikari, Tapan; Patra, Ashok Kumar

2017-11-01

An incubation study was undertaken to study microbial resilience capacity of acid soil amended with farmyard manure (FYM), charcoal and lime under copper (Cu) perturbation. Copper stress significantly reduced enzymatic activities and microbial biomass carbon (MBC) in soil. Percent reduction in microbial activity of soil due to Cu stress was 74.7% in dehydrogenase activity, 59.9% in MBC, 48.2% in alkaline phosphatase activity and 15.1% in acid phosphatase activity. Soil treated with FYM + charcoal showed highest resistance index for enzymatic activities and MBC. Similarly, the highest resilience index for acid phosphatase activity was observed in soil amended with FYM (0.40), whereas FYM + charcoal-treated soil showed the highest resilience indices for alkaline, dehydrogenase activity and MBC: 0.50, 0.22 and 0.25, respectively. This investigation showed that FYM and charcoal application, either alone or in combination, proved to be better than lime with respect to microbial functional resistance and resilience of acid soil under Cu perturbation.

Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects.

Science.gov (United States)

Somoza, R; Beutler, E

1983-10-01

Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.

Protein tyrosine phosphatases: regulatory mechanisms.

NARCIS (Netherlands)

den Hertog, J.; Ostman, A.; Bohmer, F.D.

2008-01-01

Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections.

Science.gov (United States)

Veh, R W