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Sample records for acid phosphatase activity

  1. Acid phosphatase purified from Mycoplasma fermentans has protein tyrosine phosphatase-like activity.

    Shibata, K; Noda, M.; Sawa, Y; Watanabe, T.

    1994-01-01

    Acid phosphatase purified from Mycoplasma fermentans dephosphorylated phosphotyrosine-containing lysozyme and Raytide, a peptide substrate for protein tyrosine phosphatases. The optimum pH for Raytide was about 5.5. Raytide phosphatase activity was inhibited by potassium fluoride, sodium molybdate, and sodium orthovanadate and was found to exist in some mycoplasmas.

  2. Phosphatase activity of Poa pratensis seeds. I. Preliminary studies on acid phosphatase II

    I. Lorenc-Kubis

    2015-05-01

    Full Text Available Acid phosphatase (EC 3.1.3.2 was extracted with 0.1 M sodium acetate buffer pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction Il-b (acid phosphatase II. The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward β-naphtyl phosphate and phenyl phosphate, very low activity towards β-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca2+ and fluoride, but activated by Mg2+. EDTA had no influence on the activity of the enzyme.

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

    Juliana da Silva Agostini; Rosicler Balduíno Nogueira; Elza Iouko Ida

    2010-01-01

    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 < 0.05). The phytase and acid phosphatase activities of sunflowers BRS191 and C11 were the highest on the 4th and 5th days of germination, respectively, with the release of the phosphor...

  4. Prostatic acid phosphatase is the main acid phosphatase with 5'-ectonucleotidase activity in the male mouse saliva and regulates salivation.

    Araujo, César L; Quintero, Ileana B; Kipar, Anja; Herrala, Annakaisa M; Pulkka, Anitta E; Saarinen, Lilli; Hautaniemi, Sampsa; Vihko, Pirkko

    2014-06-01

    We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts with snapin (a SNARE-associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity and elicits antinociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP(-/-) and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry, and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and that it is expressed only in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in male PAP(-/-) mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMGs. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP(-/-) mice, SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice, PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds. PMID:24717577

  5. Effects of precipitation on soil acid phosphatase activity in three successional forests in southern China

    W. Huang; Liu, J; Zhou, G.; Zhang, D; Deng, Q

    2011-01-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of organic P mineralization potential in soils. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment with precipitation t...

  6. Effects of multivalent cations on cell wall-associated acid phosphatase activity

    Tu, S.I.; Brouillette, J.N.; Nagahashi, G.; Kumosinski, T.F.

    1988-09-01

    Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu/sup 2 +/, Mg/sup 2 +/, Za/sup 2 +/, and Mn/sup 2 +/; unaffected by Ba/sup 2 +/, Cd/sup 2 +/, and Pb/sup 2 +/; and inhibited by Al/sup 3 +/. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg/sup 2 +/. On the other hand, in the case of corn root cells walls (CCW), only inhibition of the bound acid phosphatase by Al/sup 3 +/ and Hg/sup 2 +/ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg/sup 2 +/. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca/sup 2 +/ significantly reduced the effects of Hg/sup 2 +/ or Al/sup 3 +/, but not Mg/sup 2 +/, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg/sup 2 +/ or Al/sup 3 +/ which caused a Ca/sup 2 +/-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.

  7. Effects of precipitation on soil acid phosphatase activity in three successional forests in southern China

    W. Huang

    2011-07-01

    Full Text Available Phosphorus (P is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of organic P mineralization potential in soils. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment with precipitation treatments (no precipitation, natural precipitation and doubled precipitation in three successional forests in southern China was carried out. The three forests include Masson pine forest (MPF, coniferous and broad-leaved mixed forest (MF and monsoon evergreen broad-leaved forest (MEBF. Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, soil acid phosphatase activity was depressed by no precipitation treatment in the three forests. However, doubled precipitation treatment exerted a significantly negative effect on it only in MEBF. These results indicate that the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. A decrease in organic P turnover would occur in the three forests if there was a drought in a whole year in the future. More rainfall in the wet season would also be adverse to organic P turnover in MEBF due to its high soil moisture.

  8. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  9. Nitrate reductase and acid phosphatase activities as affected by inorganic phosphate in corn roots

    Marie Kummerova; Józef Buczek

    2014-01-01

    The deficieny of inorganic phosphate in nutrient solution reduces by about 50 per cent NO3- absorption in corn seedlings, it decreases both in vitro and in vivo nitrate reductase (NR) activity, as well the potential and actual NR level and has a very weak effect on NR induction. Acid phosphatases activities increase in corn roots when the plants are grown in nutrient solution without phosphorus. We suggest that inorganic phosphate is required mainly for maintenance of NR activity rather, than...

  10. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

    Smyth, Susan S; Sciorra, Vicki A; Sigal, Yury J; Pamuklar, Zehra; Wang, Zuncai; Xu, Yong; Prestwich, Glenn D; Morris, Andrew J

    2003-10-31

    Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production. PMID:12909631

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

    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.

  12. Study on alkaline and acid phosphatase activity in acute uranium intoxication

    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.)

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

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

    2004-07-01

    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)

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

    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)

  15. Effect of gamma-irradiation on nucleic acids, proteins, respiration and phosphatase activity of carrot callus cultures

    Callus tissue cultures were subjected to 60Co qamma irradiation at 0.5 Krad and analysed for nucleic acids, proteins, respiration rate and phosphatase activity on 0, 10, 20 and 30 days. The RNA contents and respiratory rates were enhanced as a result of irradiation. The RNA contents were reduced than their non-irradiated counterparts. The acid phosphatase activity was enhanced immediately after irradiation, declined on 10th and 20th day and more thereafter. (author)

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

    Marzena Sujkowska

    2011-04-01

    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.

  17. Effecf of pH and some cations on activity of acid phosphatase secreted from Ustilago sp. isolated from acid sulphate soil

    Chairatana Nilnond

    2007-03-01

    Full Text Available Acid phosphatase secreted from Ustilago sp. is able to hydrolyze organic phosphorus. These soil yeast microorganisms were isolated from rice roots grown in acid sulphate soil that generally contains highamount of aluminum (Al, iron (Fe and manganese (Mn ions. Therefore, the objectives of this study were to examine the effect of pH and some cations on acid phosphatase activity. Two isolates of Ustilago sp., AR101and AR102, were cultured in 100 mL of modified Pikovskaya's broth containing Na-phytate, pH 4, and acid phosphatase activity was determined at pH 2.0-7.0. Effect of Al, Fe, and Mn, including calcium (Ca ions,on growth of AR101 and AR102, secreted acid phosphatase activity, and the ability of acid phosphatase on the phosphorus release from Na-phytate by Ustilago sp. were investigated. It was found that the optimum pH for acid phosphatase activity was 3.5-4.5. The activity of acid phosphatase secreted from AR101 (3,690nmol min-1 mL-1 was remarkably higher than that from AR102 (956 nmol min-1 mL-1. Aluminum, iron, manganese and calcium ions in the medium did not affect the growth of either isolate. The activity of secretedacid phosphatase of AR101 was inhibited by Al and Ca ion, and synthesis of acid phosphatase of Ustilago sp. AR102 was possibly stimulated by Fe ion. Both AR101 and AR102 solubilized Na-phytate, resulting in therelease of P. However, some amount of released P was then precipitated with Al and Fe ions as the highly insoluble Fe- or Al- phosphate.

  18. Effect of copper on acid phosphatase activity in yeast Yarrowia lipolytica

    Ito, Hiroyasu; Inouhe, Masahiro; Tohoyama, Hiroshi; Joho, Masanori [Ehime Univ., Matsuyama (Japan). Dept. of Biology

    2007-01-15

    Acid phosphatase (APase) activity of the yeast Yarrowia lipolytica increased with increasing Cu{sup 2+} concentrations in the medium. Furthermore, the enzyme in soluble form was stimulated in vitro by Cu{sup 2+}, Co{sup 2+}, Ni{sup 2+}, Mn{sup 2+} and Mg{sup 2+} and inhibited by Ag{sup +} and Cd{sup 2+}. The most effective ion was Cu{sup 2+}, especially for the enzyme from cultures in medium containing Cu{sup 2+}, whereas APase activity in wall-bound fragments was only slightly activated by Cu{sup 2+}. The content of cellular phosphate involving polyphosphate was decreased by adding Cu{sup 2+}, regardless of whether or not the medium was rich in inorganic phosphate. Overproduction of the enzyme stimulated by Cu{sup 2+} might depend on derepression of the gene encoding the APase isozyme. (orig.)

  19. Origin and production of phosphatases in the acid Lake Gardsjoen

    Olsson, H.

    1983-01-01

    The activity of acid phosphatases was followed for one year in Lake Gardsjoen as well as in the inlet and the outlet of the lake. A budget of the phosphatases was calculated, including an estimation of the production of phosphatases. The phosphatase activity was also measured in two basins upstream of L. Gardsjoen: the north basin and the south basin of L. Stora Haestevatten. The acid phosphatase activity was very high compared with reported alkaline phosphatase activities in other lakes. About 95% of the phosphatases in L. Gardsjoen was produced in the lake, and the production was highest in early summer. Small Chrysophyceae (< 10 ..mu..m) probably produced the majority of the acid phosphatases in the investigated lakes, and accordingly could be favoured in environments with low phosphorus supply due to their ability to produce large amounts of phosphatases. 10 references, 8 figures, 2 tables.

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

    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) 60Co 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)

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

    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)

  2. Potentiometric assay for acid and alkaline phosphatase

    Simple potentiometric kinetic assay for evaluation of acid and alkaline phosphatase activity has been developed. Enzymatically catalyzed hydrolysis of monofluorophosphate, the simplest inorganic compound containing P-F bond, has been investigated as the basis of the assays. Fluoride ions formed in the course of the hydrolysis of this specific substrate have been detected using conventional fluoride ion-selective electrode based on membrane made of lanthanum fluoride. The key analytical parameters necessary for sensitive and selective detection of both enzymes have been assessed. Maximal sensitivity of the assays was observed at monofluorophosphate concentration near 10-3 M. Maximal sensitivity of acid phosphatase assay was found at pH 6.0, but pH of 4.8 is recommended to eliminate effects from alkaline phosphatase. Optimal pH for alkaline phosphatase assay is 9.0. The utility of the developed substrate-sensor system for determination of acid and alkaline phosphatase activity in human serum has been demonstrated

  3. The Effects of Two Species of Daphne, Betulin and Betulinic Acid on Alkaline Phosphatase Activity in Two Human Cancer Cell lines, K562 and MCF-7

    E Panahi Kokhdan

    2014-02-01

    Full Text Available Abstract Background & aim: Changes of alkaline phosphatase activity is one of the symptoms of many diseases. The aim of this study was to evaluate the effect of two types of Daphne, Betulin and Betulinic acid, on alkaline phosphatase activity in K562 and MCF-7 cell lines, respectively. Methods: In this study, 106 cancer cell lines of K562 and MCF-7 were cultured in presence of 5% carbon dioxide at 37 ° C. at doses near the IC50. The viability of cells, inside and outside alkaline phosphatase activity and the amount of total protein in each treatment were studied. The collected data was analyzed with a multivariate analysis of variance (Nested Design and Dunnett test. Results: The intracellular alkaline phosphatase activity of the cells showed different behavior compared to the extracellular alkaline phosphatase activity (p< 0.01. The highest increase of alkaline phosphatase activity in two cell lines (K562 and MCF-7 were 339% and 236% which was related to the treatment by macronata daphne. Conclusion: Unexpected increase in intracellular alkaline phosphatase activity in D. mucronata, D. oleides, Betulin, and Betulinic acid treatment may be due to changes in the composition of plasma membrane component and an increase the non-connected membrane of the protein which is due to the creation of more active proteins. Keywords: Daphne mucronata, Daphne oleoides, Alkaline Phosphatase, Betulinic Acid, Betulin

  4. Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua)

    Asgeirsson, Bjarni; Nielsen, Berit Noesgaard; Højrup, Peter

    2003-01-01

    Atlantic cod is a marine fish that lives at low temperatures of 0-10 degrees C and contains a cold-adapted alkaline phosphatase (AP). Preparations of AP from either the lower part of the intestines or the pyloric caeca area were subjected to proteolytic digestion, mass spectrometry and amino acid...... sequencing by Edman degradation. The primary structure exhibits greatest similarity to human tissue non-specific AP (80%), and approximately 30% similarity to AP from Escherichia coli. The key residues required for catalysis are conserved in the cod AP, except for the third metal binding site, where cod AP...

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

    Soung-Hoon Lee

    Full Text Available BACKGROUND: 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. METHODOLOGY/ PRINCIPAL FINDINGS: 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. CONCLUSIONS/ SIGNIFICANCE: 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.

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

    Stanisław Flasiński

    2014-02-01

    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.

  7. Phosphate status and acid phosphatase activity in soil and ectomycorrhizas in two mature stands of scots pine (Pinus sylvestris L.) exposed to different levels of anthropogenic pollution

    Barbara Kieliszewska-Rokicka

    2014-01-01

    The relations between anthropogenic environmental pollution and the level of inorganic phosphorus in soil, enzyme activities of extracellular soil acid phosphatase and the surface acid phosphatase of excised ectomycorrhizas of Scots pine (Pinus sylvestris L.) were studied. Soil and root samples were taken from two Scots pine stands in central Poland: a polluted site exposed to long-term pollution from a steelworks and the city of Warsaw and a reference plot (control) free from direct impact o...

  8. Castor oil increases intestinal formation of platelet-activating factor and acid phosphatase release in the rat.

    Pinto, A; Calignano, A; Mascolo, N; Autore, G; Capasso, F

    1989-01-01

    1. When castor oil was administered by gavage to rats, the duodenum and jejunum but not ileum and colon produced large amounts (5-6 fold greater than control) of platelet activating factor (Paf). 2. Intraluminal release of acid phosphatase (AP) was also markedly increased (5-6 fold greater than control) in the duodenum and jejunum of castor oil-treated rats and there was a correlation between the elevated release of AP and intestinal hyperaemia. 3. These findings support a role for Paf as a m...

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

    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.

  10. Changes of the Biomass and Acid Phosphatase Activity in Maize (Zea mays L.) Lines Under Low-P Stress

    YAO Qilun

    2008-01-01

    A pot culture trial was conducted to investigate the changes of the biomass and acid phosphatase (APase) activity in 10 maize lines under low-P stress. P-deficiency significantly decreased the biomass, but induced the significant enhancement of the APase activity. Since P-deficiency had smaller effects on the low-P tolerant maize lines compared with P-sensitive lines, it was demonstrated that differences of tolerance to P-deficiency existed among 10 different maize lines. In addition, the relative biomass and APase activity changed during the vegetative stage of development, and there existed a significant correlation between the biomass and APase activity under low-P stress. These results suggest that the biomass and APase activity can be regarded as indicative traits of maize lines for tolerance to low-P stress at seedling stage.

  11. Primary structure of rat secretory acid phosphatase and comparison to other acid phosphatases.

    Roiko, K; Jänne, O A; Vihko, P

    1990-05-14

    Overlapping cDNA clones encoding rat prostatic acid phosphatase (rPAP) were isolated by using two human prostatic acid phosphatase (hPAP)-encoding cDNAs to screen rat prostatic cDNA libraries. The isolated cDNAs encompassed a total of 1626 nucleotides (nt), of which 1143 nt corresponded to the protein coding sequence encoding a mature polypeptide of 350 amino acids (aa) and a 31-aa long signal peptide-like sequence. The deduced Mr of the mature rPAP was 40,599. RNA blot analysis indicated the presence of three mRNA species (4.9, 2.3 and 1.5 kb in size) in the rat prostate. The deduced aa sequences of rPAP and hPAP show 75% identity, whereas the similarity between rPAP and human lysosomal acid phosphatase (hLAP) is only 45%. Furthermore, the sequence similarity between rPAP and rat lysosomal acid phosphatase (rLAP) is 46% at the aa level. Similar to hPAP, but unlike hLAP and rLAP, the rPAP sequence lacks a membrane-anchoring domain indicating the secretory character of this phosphatase. All six cysteines present in the overlapping areas of the mature rPAP, hPAP, rLAP and hLAP proteins are positionally conserved, suggesting that these residues are important for the tertiary structure of acid phosphatases (APs). The previously reported active site residues, two arginines and one histidine, are also conserved in these APs. PMID:2373368

  12. Radiation-induced alterations in splenic acid phosphatase of pigeons

    The effect of total body ν-irradiation with sub-lethal dose (400 rad) on acid phosphatase has been studied in spleen of pigeons. The specific activity of acid phosphatase increased significantly 48 hr and 72 hr after irradiation. This increase was accompanied by a substantial reduction in per cent 'bound' activity. The histochemical observation after irradiation confirmed the result obtained by quantitative biochemical study. This increase in acid phosphatase activity may be attributed to an increased permeability of lysosomal membrane caused by damaged lymphocytes (lymphocytolysis) after ν-irradiation. (author)

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

    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 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 three 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 increased triglycerides and 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.

  14. Evaluating the levels of salivary alkaline and acid phosphatase activities as biochemical markers for periodontal disease: A case series

    Sarita Dabra

    2012-01-01

    Full Text Available Background: The purpose of this study was to determine the salivary levels of alkaline phosphatase (ALP and acid phosphatase (ACP activities in patients with periodontal disease and to evaluate the use of these enzymes as biochemical markers for periodontal tissue damage. Materials and Methods: In this prospective analytical study, we examined the activities of salivary ALP and ACP in patients with periodontal disease, before and after periodontal treatment. The experimental groups consisted of 20 gingivitis patients and 20 periodontitis patients and the control group had healthy subjects (20 samples. The stimulated saliva of the patient was collected in a sterile test tube and analyzed using Hitachi′s Diagnostic Automatic Analyser. Periodontal disease was determined based on clinical parameters such as gingival index, probing depth and clinical attachment loss. Patients with periodontal disease were under conventional periodontal treatment. The statistical analysis applied was Student′s t-test. Probabilities less than 0.05 (P < 0.05 were considered significant. Results: The obtained results showed statistically significant increased activities of ALP and ACP in saliva from patients with periodontal disease in relation to control group. A significant reduction in the enzyme levels was seen after conventional periodontal therapy. Conclusions: Based on these results, salivary ALP and ACP can be considered to be the biomarkers for evaluating periodontal tissue damage.

  15. Effect of x-irradiation on the acid and alkaline phosphatase activities in the south Indian scorpion Heterometrus fulvipes (C.L. Koch)

    Variations in the activity levels of acid phosphatase and alkaline phosphatase in the tissues of scorpion Heterometrus fulvipes after whole-body irradiation with X-rays are studied. The animals were exposed to sublethal dose (1/3 of LD50) of radiation for different periods of time ranging from 1/2h to 4h. Tissue homogenates were also irradiated and studied. The activity of phosphatases was found to decrease with increase in exposure time both at in vivo and in vitro, the per cent decrease being higher at in vitro. The activity levels of phosphatases in different tissues were in the order of hepato pancreas > heart pedipalpal > muscle > nervous tissue. (M.G.B.)

  16. Amylase and acid phosphatase activity in potato tubers treated with gibberellic acid and stored at 2°C and 8°C

    M. Bielińska-Czarnecka; K. Białek

    2015-01-01

    Amylase activity was higher in tubers stored at 2°C and mare marked in the soaked ones (both in water and in GA3). In the late and difficult-sprouting cv. Uran, sokaing resulted in increased amylolytic activity also at 8°C stored tubers. On the contrary, the acid phosphatase activity was a little higher at 8°C than at 2°C stored tubers. At the former temperature two peaks of activity were marked:, in November–December and February–March.

  17. Phosphate status and acid phosphatase activity in soil and ectomycorrhizas in two mature stands of scots pine (Pinus sylvestris L. exposed to different levels of anthropogenic pollution

    Barbara Kieliszewska-Rokicka

    2014-02-01

    Full Text Available The relations between anthropogenic environmental pollution and the level of inorganic phosphorus in soil, enzyme activities of extracellular soil acid phosphatase and the surface acid phosphatase of excised ectomycorrhizas of Scots pine (Pinus sylvestris L. were studied. Soil and root samples were taken from two Scots pine stands in central Poland: a polluted site exposed to long-term pollution from a steelworks and the city of Warsaw and a reference plot (control free from direct impact of pollution. The polluted site was characterised by high concentration of trace elements (Cd, Pb, Cu, Zn, Mn, Cr and low level of inorganic phosphate in soil. This site had significantly lower enzyme activities of soil acid phosphatase (0.54 µmoles p-nitrophenol released g-1 dry weight h-1 and surface acid phosphatase of pine ectomycorrhizas (3.37 µmoles p-nitrophenol released g-1 fresh weight h-1 than the control site (1.36 µmoles p-nitrophenol released g-1 dry weight h-1 and 12.46 µmoles p-nitrophenol released g-1 fresh weight h-1, respectively. The levels of phosphate, carbon and nitrogen in pine fine roots were also analysed. Low concentrations of P04-P and high N: P ratio in pine fine roots from polluted site were found. The results suggest that soil pollutants may have a negative effect on the extracellular acid phosphatase of soil and Scots pine ectomycorrhizas and on the phosphorus status in fine roots of the plant.

  18. Effect of noise exposure (85 dB ) on testicular adrenocortical steroidogenic key enzymes, acid and alkaline phosphatase activities of sex organs in mature albino rats

    2000-01-01

    Changes in the activities of △5-3β-hydroysteroid dehydrogenase (HSD) in testis and adrenal gland, 17β-hydroxysteroid dehydrogenase in testis, acid and alkaline phosphatase in testis, prostate and seminal vesicle were observed in noise exposed mature rats at the intensity of 85 dB for 8 h/day for 45 days. The results indicated that noise exposed group showed a significant diminution in the activities of androgenic key enzymes △5-3β and 17β-HSD, acid phosphatase in testis, prostate and seminal vesicle. There was a significant elevation in the activities of adrenal △5-3β-HSD, alkaline phosphatase in testis and other accessory sex organ in noise exposed group. Gonadosomatic, prostatosomatic and seminal vesiculo-somatic indexes were decreased significantly in noise exposed group. Therefore, it is evident that noise exposure at 85dB exerts a deleterious effect on testicular and adrenocortical activities.

  19. Revisiting histidine-dependent acid phosphatases: a distinct group of tyrosine phosphatases

    Veeramani, Suresh; Lee, Ming-Shyue; Lin, Ming-Fong

    2009-01-01

    Although classical protein tyrosine phosphatase (PTP) superfamily members are cysteine-dependent, emerging evidence shows that many acid phosphatases (AcPs) function as histidine-dependent PTPs in vivo. These AcPs dephosphorylate phospho-tyrosine substrates intracellularly and could have roles in development and disease. In contrast to cysteine-dependent PTPs, they utilize histidine, rather than cysteine, for substrate dephosphorylation. Structural analyses reveal that active site histidine, ...

  20. Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake

    Nedoma, Jiří; Vrba, Jaroslav

    2006-01-01

    Roč. 8, č. 7 (2006), s. 1271-1279. ISSN 1462-2912 R&D Projects: GA AV ČR(CZ) IAA6017202 Institutional research plan: CEZ:AV0Z60170517 Keywords : alkaline phosphatase * bacterial morphorypes * acid ified lake Subject RIV: CE - Biochemistry Impact factor: 4.630, year: 2006

  1. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium

    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.

  2. Catalytic DNA with phosphatase activity

    Chandrasekar, Jagadeeswaran; Silverman, Scott K.

    2013-01-01

    Catalytic DNA sequences (deoxyribozymes, DNA enzymes, or DNAzymes) have been identified by in vitro selection for various catalytic activities. Expanding the limits of DNA catalysis is an important fundamental objective and may facilitate practical utility of catalysts that can be obtained from entirely unbiased (random) sequence populations. In this study, we show that DNA can catalyze Zn2+-dependent phosphomonoester hydrolysis of tyrosine and serine side chains (i.e., exhibit phosphatase ac...

  3. Phosphorus compounds, proteins, nuclease and acid phosphatase activities in isolated spinach chloroplasts

    E. Mikulska

    2015-05-01

    Full Text Available This paper deals with attempts to elaborate a simple method of spinach chloroplast isolation ensuring a high proportion of intact chloroplasts. We obtained 3 preparations of isolated chloroplasts. Several preliminary analyses of the obtained chloroplast fraction were also performed. Phosphorus compounds, total protein and the enzyme activities of RNase, DNase and GPase were determined. We found: 0,36-0,59% of RNA, 0,19-0,24% of DNA, 2,1-2,9% of phospholipids and 26-28% of protein. RNase activity was very high.

  4. Association of erythrocyte acid phosphatase phenotypes with myopia

    Himabindu P

    2005-01-01

    Full Text Available Acid phosphatase is a polymorphic nonspecific orthophosphate monoesterase which catalyses the cleaving of phosphoric acid and subsequent breakdown of several monophosphoric esters under acidic pH conditions. Acid phosphatase has a physiologic function as a flavin mononucleotide phosphatase (FMN and regulates the intracellular concentrations of flavin coenzymes that are electron carriers in the oxidative phosphorylation pathway. Myopia or nearsightedness is caused by both environmental and genetic factors. Myopic eyes when subjected to excessive oxidative stress results in retinal detachments .In the present study there is a significant elevation of AA phenotype in myopes when compared to controls. The AA phenotype is more susceptible to oxidative stress and its lower enzyme activity is known to be associated with increased intrauterine growth that further results in increased axial length in progressive myopia. The AA phenotype also confers risk for myopia development in males, early age group and cases with parental consanguinity.

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

    Tigran R Petrosyan; Anna S Ter-Markosyan; Anna S Hovsepyan

    2016-01-01

    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 ifbers and dilated the capillaries in the brain and spinal cord. These results sug-gest 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 regenera-tion-promoting effects of BM on the injured central nervous system.

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

    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.

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

    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

  8. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    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…

  9. [Phosphatase activity in Amoeba proteus at pH 9.0].

    Sopina, V A

    2007-01-01

    In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1). PMID:17933343

  10. Direct determination of phosphatase activity from physiological substrates in cells.

    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.

  11. Mammalian-like Purple Acid Phosphatases in Plants

    2006-01-01

    @@ Introduction Purple acid phosphatases (PAPs) comprise of a family of binuclear metal-containing hydrolases, some members of which have been isolated and characterized from animal, plant and fungal sources[1]. PAPs not only catalyze the hydrolyses of a wide range of phosphate esters and anhydrides under acidic reaction conditions,but also catalyze the generation of hydroxyl radicals in a Fenton-like reaction, by virtue of the presence of a redox-active binuclear metal center.

  12. Detection of phosphatase activity in aquatic and terrestrial cyanobacterial strains

    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

  13. Effect of phenylmercuric acetate injections on phosphatase activity in chickens resistant and susceptible to Leukosis

    Miller, V.L.; Bearse, G.E.; Csonka, E.

    1972-01-01

    The weighted means of liver and kidney alkaline phosphatase activity was greater in three strains of chickens classified as susceptible to limphoid leukosis than in five strains classified as resistant. On the same basis, four strains classified as susceptible to Marek's disease had more liver alkaline phosphatase activity than four strains classified as resistant. The weighted means of liver and kidney acid phosphatase activity were not different among the same strains of chickens classified similarly. Kidney alkaline phosphatase activity was the most generally inhibited by phenylmercuric acetate injections, followed by liver acid and alkaline phosphatase. Kidney acid phosphatase activity was enhanced by phenylmercuric acetate injections in three strains of chickens classified as resistant to both lymphoid leukosis and Marek's disease. Liver acid phosphatase activity was depressed in three strains classed as resistant to lymphoid leukosis.

  14. Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

    Lång, Pernilla; van Harmelen, Vanessa; Rydén, Mikael; Kaaman, Maria; Parini, Paolo; Carneheim, Claes; Cassady, A. Ian; Hume, David A.; Andersson, Göran; Arner, Peter

    2008-01-01

    Background 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. Principal Findings Using mice over expressing TRAP...

  15. Tartrate resistant acid phosphatase in the immune and nervous system : Distribution and pathophysiological implications

    Lång, Pernilla

    2007-01-01

    Tartrate resistant acid phosphatase (TRAP) belongs to the family of purple acid phosphatases (PAP). It is a glycoprotein synthesized as a monomer with low enzyme activity containing a redox active diiron centre in the active site. Post-translational proteolytic processing of this monomer into a dimeric protein increases the enzyme activity. Traditionally, TRAP has been used as a marker for bone resorbing cells but the biological function of TRAP is still not fully elucidated...

  16. Testicular acid phosphatase induces odontoblast differentiation and mineralization.

    Choi, Hwajung; Kim, Tak-Heun; Yun, Chi-Young; Kim, Jung-Wook; Cho, Eui-Sic

    2016-04-01

    Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration. PMID:26547858

  17. CONTROL OF ALKALINE PHOSPHATASE ACTIVITY IN C3H10T1/2 CELLS: ROLE OF RETINOIC ACID AND CELL DENSITY

    The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. ecause retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have sugge...

  18. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz; E. Wieczorek; B. Morawiecka

    2015-01-01

    Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.). The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance ...

  19. Assessing the Biological Activity of the Glucan Phosphatase Laforin.

    Romá-Mateo, Carlos; Raththagala, Madushi; Gentry, Mathew S; Sanz, Pascual

    2016-01-01

    Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity. PMID:27514803

  20. The spatial distribution of acid phosphatase activity in ectomycorrhizal tissues depends on soil fertility and morphotype, and relates to host plant phosphorus uptake.

    Alvarez, Maricel; Huygens, Dries; Díaz, Leila Milena; Villanueva, Claudia Añazco; Heyser, Wolfgang; Boeckx, Pascal

    2012-01-01

    Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACP(myc)), the mantle (ACP(mantle)) and the Hartig net region (ACP(Hartig)) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzyme-labelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACP(myc), ACP(mantle) and ACP(Hartig), while soil fertility had a significant effect on ACP(myc) and ACP(Hartig). The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACP(Hartig) and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus. PMID:21902696

  1. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  2. Effect of colchicine on the Golgi apparatus and on GERL of rat jejunal absorptive cells. Ultrastructural localization of thiamine pyrophosphatase and acid phosphatase activity.

    Pavelka, M; Ellinger, A

    1981-04-01

    Ultrastructural localization of thiamine pyrophosphatase (TTP) and acid phosphatase (AcPase) activity was performed on jejunal absorptive cells of rats pretreated with the antimicrotubular agent colchicine and of control animals. Demonstration of TPP activity showed that most of the dislocated Golgi stacks after colchicine application lacked positively staining cisternae of the mature side. This cytochemical finding is in agreement with the morphologically demonstrable changes of the Golgi stacks resulting in a loss of polarity and give evidence for a colchicine-induced deficiency of the Golgi apparatus. The cytochemical localization of AcPase activity showed deposits of reaction product over lysosomes and GERL and demonstrated a dislocation of GERL occurring concomitantly with the changes of the Golgi apparatus. The antimicrotubular effect of colchicine is well documented; thus the morphological and cytochemical changes of the Golgi apparatus and of GERL might be due to a disturbed microtubular function after application of this agent suggesting an influence of microtubules in the maintenance of the integrity of these organelles. This hypothesis includes the possibility of an involvement of microtubules in formation and differentiation of Golgi stacks and GERL as well as a kind of "skeletal"function being responsible for their characteristic structure and fashion. PMID:6113143

  3. Plasma acid and alkaline phosphatase in patients with breast cancer.

    Nguyen, M; Bonneterre, J; Hecquet, B; Desoize, B; Demaille, A

    1991-01-01

    Acid and alkaline phosphatase were determined in 107 breast cancer patients to study their potential value in case of bone metastases. The patients were divided into 4 groups: A, patients without metastases (n = 34); B, metastatic patients without bone lesions (n = 37); C, patients with metastases in and outside of bones (n = 24), D, patients with bone-only metastases (n = 12). Tartrate resistant acid phosphatase (TR-ACP), and bone alkaline phosphatase (bone-ALP) were significantly higher in patients with metastases than in patients without. However, no difference in TR-ACP was observed between subgroups of metastatic patients. PMID:2064338

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

    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)

  5. Activation of Calf Intestinal Alkaline Phosphatase by Trifluoroethanol

    曹志方; 徐真; 朴龙斗; 周海梦

    2001-01-01

    Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (0 - 60%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α-helix content. We conclude that the activation of ClAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of ClAP induced by TFE results in the enhancement of ClAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of ClAP can be attained with various secondary structures.

  6. Alkaline phosphatase for immunocytochemical labelling: problems with endogenous enzyme activity.

    Bulman, A. S.; Heyderman, E

    1981-01-01

    Alkaline phosphatase may be used as a label for immunocytochemistry and can be demonstrated in tissue sections using the single step naphthol phosphate method. Endogenous enzyme activity may not be destroyed by fixation in formalin, formol alcohol, Carnoy's or Baker's solutions and should be inhibited before results are assessed. Either Bouin's solution or periodic acid followed by potassium borohydride are satisfactory inhibitor and do not adversely affect immunocytochemical results.

  7. Methods to distinguish various types of protein phosphatase activity

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on 32P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of 32P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble [32P]phosphate

  8. Porównanie aktywności peroksydazy, katalazy i fosfatazy kwaśnej w liściach pora w okresie intensywnego wzrostu i pod koniec wegetacji roślin w rożnych warunkach uprawy [Dynamics of the changes of peroxidase, catalase, and acid phosphatase activities in leeks under the influence of nitrogen fertilization and irrigation

    E. Gurgul; E. Kołota; D. Ściążko

    2015-01-01

    Results obtained in a field experiment showed a high influence of irrigation and nitrogen fertilization on the activity of peroxidase, while acid phosphatase activity showed only small differences. The peroxidase activity depended to a large degree on the leeks growth stage. Maximum peroxidase, catalase and - in some cases – acid phosphatase activity were found at nitrogen doses higher than optimal for the plant growth and yield.

  9. Porównanie aktywności peroksydazy, katalazy i fosfatazy kwaśnej w liściach pora w okresie intensywnego wzrostu i pod koniec wegetacji roślin w rożnych warunkach uprawy [Dynamics of the changes of peroxidase, catalase, and acid phosphatase activities in leeks under the influence of nitrogen fertilization and irrigation

    E. Gurgul

    2015-06-01

    Full Text Available Results obtained in a field experiment showed a high influence of irrigation and nitrogen fertilization on the activity of peroxidase, while acid phosphatase activity showed only small differences. The peroxidase activity depended to a large degree on the leeks growth stage. Maximum peroxidase, catalase and - in some cases – acid phosphatase activity were found at nitrogen doses higher than optimal for the plant growth and yield.

  10. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases

    The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading ac...

  11. Wpływ nawożenia mineralnego i nawadniania na aktyumość peroksydazy, katalazy i fosfatazy kwaśnej w dwóch fazach wzrostu kapusty i porów [The influence of mineral fertilization and irrigation on the activity of peroxidase, catalase and acid phosphatase of cabbage and leek in two stages of growth

    E. Gurgul; E. Kołota

    2015-01-01

    During the growth of plant, the very distinct increase of enzymatic activity of peroxidase and catalase was observed, but in case of acid phosphatase in smaller degree. An irrigation caused the decreasing of activity of all tested enzymes in both stages of cabbage growth. However, in case of leeks leaves sprinkling irrigation stimulated activity of catalase and acid phosphatase in both stages and peroxidase in the second stage of growth. The effectiveness of the mineral nutritive was differen...

  12. Prostatic acid phosphatase, purification and iodination using Iodogen

    Prostatic acid phosphatase was purified from prostatic adenomas. The procedure involved chromatography on Concanavalin A-Sepharose, DEAE-cellulose, Bio-Gel P-150 and L-tartrate-Sepharose. The purified phosphatase hydrolyzed p-nitrophenyl phosphate at a rate of 270 μmol.mg-1.min-1 (250C) and showed homogeneity upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The final prostatic acid phosphatase preparation was pure and the antisera were monospecific as judged by the highly-sensitive technique of crossed immunoelectrophoresis. Of the procedures evaluated for the radioiodination of the purified enzyme with iodine 125, oxidation with Iodogen was found to give the best radioiodinated product, to be used in radioimmunoassay. (Auth.)

  13. Lipophosphoglycan and secreted acid phosphatase of Leishmania tropica share species-specific epitopes.

    Jaffe, C L; Perez, L; Schnur, L F

    1990-06-01

    Several species-specific monoclonal antibodies (T11, T13-T15) which only react with Leishmania tropica, recognize phosphorlated carbohydrate epitopes on lipophosphoglycan and the structurally related molecule, phosphoglycan, which is shed by promastigotes into spent culture medium. During immunoaffinity isolation of [32P]orthophosphate-labeled phosphoglycan on monoclonal antibody T15 conjugated to Sepharose 4B, a high-Mr component (approx. 200,000) was co-purified. The latter material is metabolically labeled with [35S]methionine and [3H]glucosamine. This glycoprotein was separated from phosphoglycan by chromatography on lentil lectin resin. The glycoprotein exhibited a L-tatrate-sensitive acid phosphatase activity, typical of secreted acid phosphatase (EC 3.1.3.2) from Leishmania. Monospecific antibodies to Leishmania donovani-secreted acid phosphatase selectively precipitated the L. tropica enzyme from immunoaffinity purified mixtures of the two antigens, and monoclonal antibodies to lipophosphoglycan precipitate the pure enzyme. Species-specific monoclonal antibodies to L. major lipophosphoglycan also recognized both L. tropica antigens. Treatment of the acid phosphatase with periodate or phosphodiesterase I abolished binding by the monoclonal antibodies to the pure enzyme. These results demonstrate that the two major secreted glycoconjugates of Leishmania tropica, the lipophosphoglycan and the acid phosphatase, share species-specific phosphorylated carbohydrate epitope(s). PMID:1697935

  14. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

    Romero-Montoya, Lydia; Martínez-Rodríguez, Hugo; Pérez, Miguel Antonio; Argüello-García, Raúl

    2011-03-20

    In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all

  15. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose.

    Hibbs, John B; Vavrin, Zdenek; Cox, James E

    2016-08-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces. PMID:26895212

  16. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a λgt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed

  17. The development of determining human prostatic acid phosphatase by radioimmunoassay

    We purified human prostatic acid phosphatase (hPAP) from prostatic tissues by affinity chromatography, DEAE cellulose and gel filtration and also examined physicochemical properties of highly purified PAP. We developed a double-antibody radioimmunoassay for hPAP in serum, with use of antiserum raised in rabbit against highly purified PAP. The antiserum did not cross react with acid phosphatase from platelets and red blood cells. Experimental detail are outlined to assess the reproducibility and reliability of the method under various conditions. The upper limit of the serum PAP levels in the present assay was set at 3.0 ng/ml by 162 determinations of samples. The serum PAP levels of 2 untreated patients with prostatic carcinoma were higher than 3.0 ng/ml and 39 patients with benign prostatic hyperplasia were an average value of 1.9 ng/ml. (author)

  18. Phosphorus resorption by young beech trees and soil phosphatase activity as dependent on phosphorus availability.

    Hofmann, Kerstin; Heuck, Christine; Spohn, Marie

    2016-06-01

    Motivated by decreasing foliar phosphorus (P) concentrations in Fagus sylvatica L. forests, we studied P recycling depending on P fertilization in mesocosms with juvenile trees and soils of two contrasting F. sylvatica L. forests in a greenhouse. We hypothesized that forests with low soil P availability are better adapted to recycle P than forests with high soil P availability. The P resorption efficiency from senesced leaves was significantly higher at the P-poor site (70 %) than at the P-rich site (48 %). P fertilization decreased the resorption efficiency significantly at the P-poor site to 41 %, while it had no effect at the P-rich site. Both acid and alkaline phosphatase activity were higher in the rhizosphere of the P-poor than of the P-rich site by 53 and 27 %, respectively, while the activities did not differ in the bulk soil. Fertilization decreased acid phosphatase activity significantly at the P-poor site in the rhizosphere, but had no effect on the alkaline, i.e., microbial, phosphatase activity at any site. Acid phosphatase activity in the P-poor soil was highest in the rhizosphere, while in the P-rich soil, it was highest in the bulk soil. We conclude that F. sylvatica resorbed P more efficiently from senescent leaves at low soil P availability than at high P availability and that acid phosphatase activity in the rhizosphere but not in the bulk soil was increased at low P availability. Moreover, we conclude that in the P-rich soil, microbial phosphatases contributed more strongly to total phosphatase activity than plant phosphatases. PMID:26875186

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

    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

  20. 基于不同方法测定土壤酸性磷酸酶活性的比较%Comparison of soil acid phosphatase activity determined by different methods

    李莹飞; 耿玉清; 周红娟; 杨英

    2016-01-01

    Method, edited by Songyin Guan, for measurement method of soil acid phosphatase activity based on phenyl phosphate disodium salt substrate. In contrast, researchers outside China mainly cite the book entitled Methods of Soil Enzymology, edited by Dick, that was based on disodium p-Nitrophenyl phosphate tetrahydrate (PNPP) substrate. However, non-conspicuous coloration has existed for the measurement of products based on phenyl phosphate disodium salt substrate. Furthermore, it has been difficult for researchers to select an optimal method for determining acid phosphatase activity since these methods use different substrates. To determine the optimal method for measuring soil acid phosphatase activity, three different methods were used to measure the acid phosphatase activity of 10 soil samples of acid, neutral and alkaline soils, respectively. The three selected methods were 1) based on phenyl phosphate disodium salt substrate and colored using pH 5.0 acetate buffer (DPP 1);2) based on phenyl phosphate disodium salt substrate and colored using pH 9.4 borate buffer (DPP 2) during chromogenic process;or 3) the PNPP method. Furthermore, the study analyzed the effects of different pH buffers and phenol concentrations on product absorbance. The results showed that chromogenic reaction of phenol with 2,6-dibromchinone-chlorimide was colorless within pH≤6 buffer solution with phenyl phosphate disodium salt as the substrate. In contrast, the above chromogenic reaction was observed under alkaline buffer (pH≥ 8) in all the samples. And there were significant differences in the Pearson correlation coefficient (R2) between phenol concentration and product absorbance at 0.01 level. Therefore, pH was a significant factor in determining the coloration between phenol and 2,6-dibromchinone-chlorimide. Furthermore, when acid phosphatase activity was determined using the PNPP method, the coefficient of variation of acid phosphatase activities in the 10 soil samples increased by 70

  1. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    The overall objective of this project is to examine the activity 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 PO43- as a means to detoxify radionuclides and heavy metals. An experimental approach was designed to determine the extent of phosphatase activity in bacteria previously isolated from contaminated subsurface soils collected at the ERSP Field Research Center (FRC) in Oak Ridge, TN. Screening of 135 metal resistant isolates for phosphatase activity indicated the majority (75 of 135) exhibited a phosphatase-positive phenotype. During this phase of the project, a PCR based approach has also been designed to assay FRC isolates for the presence of one or more classes of the characterized non-specific acid phophastase (NSAP) genes likely to be involved in promoting U(VI) precipitation. Testing of a subset of Pb resistant (Pbr) Arthrobacter, Bacillus and Rahnella strains indicated 4 of the 9 Pbr isolates exhibited phosphatase phenotypes suggestive of the ability to bioprecipitate U(VI). Two FRC strains, a Rahnella sp. strain Y9602 and a Bacillus sp. strain Y9-2, were further characterized. The Rahnella sp. exhibited enhanced phosphatase activity relative to the Bacillus sp. Whole-cell enzyme assays identified a pH optimum of 5.5, and inorganic phosphate accumulated in pH 5.5 synthetic groundwater (designed to mimic FRC conditions) incubations of both strains in the presence of a model organophosphorus substrate provided as the sole C and P source. Kinetic experiments showed that these two organisms can grow in the presence of 200 (micro)M dissolved uranium and that Rahnella is much more efficient in precipitating U(VI) than Bacillus sp. The

  2. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    Robert J. Martinez; Melanie J. Beazley; Samuel M. Webb; Martial Taillefert (co-PI); and Patricia A. Sobecky

    2007-04-19

    The overall objective of this project is to examine the activity 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 PO4 3- as a means to detoxify radionuclides and heavy metals. An experimental approach was designed to determine the extent of phosphatase activity in bacteria previously isolated from contaminated subsurface soils collected at the ERSP Field Research Center (FRC) in Oak Ridge, TN. Screening of 135 metal resistant isolates for phosphatase activity indicated the majority (75 of 135) exhibited a phosphatase-positive phenotype. During this phase of the project, a PCR based approach has also been designed to assay FRC isolates for the presence of one or more classes of the characterized non-specific acid phophastase (NSAP) genes likely to be involved in promoting U(VI) precipitation. Testing of a subset of Pb resistant (Pbr) Arthrobacter, Bacillus and Rahnella strains indicated 4 of the 9 Pbr isolates exhibited phosphatase phenotypes suggestive of the ability to bioprecipitate U(VI). Two FRC strains, a Rahnella sp. strain Y9602 and a Bacillus sp. strain Y9-2, were further characterized. The Rahnella sp. exhibited enhanced phosphatase activity relative to the Bacillus sp. Whole-cell enzyme assays identified a pH optimum of 5.5, and inorganic phosphate accumulated in pH 5.5 synthetic groundwater (designed to mimic FRC conditions) incubations of both strains in the presence of a model organophosphorus substrate provided as the sole C and P source. Kinetic experiments showed that these two organisms can grow in the presence of 200 μM dissolved uranium and that Rahnella is much more efficient in precipitating U(VI) than Bacillus sp. The

  3. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura

    Edwin, E.; P Vasantha-Srinivasan; A Thanigaivel; A Ponsankar; S Selin-Rani; K. Kalaivani; WB Hunter; Duraipandiyan, V; NA AlDhabi

    2016-01-01

    Andrographolide was isolated from ethanol extraction of Andrographis paniculata by column chromatography. Evaluation of larvicidal efficacy, enzymatic changes and cytotoxic activities against Spodoptera litura were conducted across a range of concentrations. The compound showed significant larvicidal activity between 5 - 25 ppm, post ingestion. Morphological deformities observed in larval-pupal stages. Enzymatic profiles were altered by reduction in acid phosphatase, ACP activity ...

  4. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz

    2015-05-01

    Full Text Available Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.. The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance of these bands. RNase activity was detected in 4 main zones, in some of them additional subbands were visible. Differences between the three examined varieties were reflected in the occurence of the particular activity zones or their subbands.

  5. High acid phosphatase level in the gingival tissues of periodontitis subjects

    Pushparani, D. S.

    2015-01-01

    Aim: Periodontitis is one of the major problems slowly progressing and could affect 70% of the global population. The prevalence of periodontitis differs from mild to moderate forms of race and geographic region. The aim of this study is to determine the acid phosphatase (ACP) activity in the gingival tissues of periodontitis subjects. In this study, the activity of ACP in the gingival tissue of subjects with periodontitis was examined. Materials and Methods: A total of 30 subjects were selec...

  6. Direct Electrochemistry of Porcine Purple Acid Phosphatase (Uteroferrin)

    Bernhardt, Paul V; Schenk, Gerhard; Wilson, Gregory J.

    2004-01-01

    Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIII-FeII f FeIII-FeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Ufo) have been determined. The effect of pH on the redox potent...

  7. Acid phosphatase localization in neurons of Bulla gouldiana (Gastropoda: Opisthobranchia.

    Robles, L J; Fisher, S K

    1975-01-01

    The organization of the ganglia and the ultrastructure of the neurons of Bulla gouldiana are similar to those described for other molluscs. Acid phosphatase positive reactions were found in the large pigmented granules, small dense bodies, multivesicular bodies, and Golgi lamellae and associated vesicles. The small dense bodies and multivesicular bodies may be stages in the formation of the larger pigmented granules which are interpreted as lysosomes. Comparison is made between the pigmented granules in Bulla and the lipofuscin bodies of vertebrate neurons. The possible involvement of these pigmented granules in the hyperpolarization of Bulla and Aplysia neurons to light is discussed. PMID:1122539

  8. Study on prostatic acid phosphatase (PAP) immunoradiometric assay kit

    This coat-antibody-count PAP IRMA is a solid-phase immunoradiometric assay based on two strains of monoclonal antibodies, designed for the quantitative measurement of prostatic acid phosphatase (PAP) in serum. The minimal detectable concentration is 0.1 μg/L. The intra and inter coefficients of variation are 8.8%-9.6% and 7.7%-12.3%, respectively. The recovery is 96.3%-105.0% and the range of detection is 2.5-200.0 μg/L

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

    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.

  10. Wpływ nawożenia mineralnego i nawadniania na aktyumość peroksydazy, katalazy i fosfatazy kwaśnej w dwóch fazach wzrostu kapusty i porów [The influence of mineral fertilization and irrigation on the activity of peroxidase, catalase and acid phosphatase of cabbage and leek in two stages of growth

    E. Gurgul

    2015-06-01

    Full Text Available During the growth of plant, the very distinct increase of enzymatic activity of peroxidase and catalase was observed, but in case of acid phosphatase in smaller degree. An irrigation caused the decreasing of activity of all tested enzymes in both stages of cabbage growth. However, in case of leeks leaves sprinkling irrigation stimulated activity of catalase and acid phosphatase in both stages and peroxidase in the second stage of growth. The effectiveness of the mineral nutritive was differentiated, and often correlated with a level of soil moisture, kind of plant and its stage of growth.

  11. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor

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

    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)

  13. Cervical acid phosphatase detection: A guide to abnormal cells in cytology smear screening for cervical cancer

    Deb Prabal; Iyer Venkateswaran; Bhatla Neerja; Markovic O; Verma Kusum

    2008-01-01

    Background: Cervical acid phosphatase-Papanicolaou (CAP-PAP) test has recently been described for detection of acid phosphatase enzyme in abnormal squamous cells, and has been proposed as a biomarker-based technology for the screening of cervical cancer. Materials and Methods: Eighty-one consecutive cervical smears were subjected to routine Papanicolaou (Pap) staining as well as CAP-PAP, which combined cytochemical staining for acid phosphatase with modified Pap stain. Statistical evaluation ...

  14. High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase.

    Peters, C; Geier, C; Pohlmann, R; Waheed, A; von Figura, K; Roiko, K; Virkkunen, P; Henttu, P; Vihko, P

    1989-02-01

    Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes. PMID:2706086

  15. Evaluation of the Staphylococcus aureus Class C Nonspecific Acid Phosphatase (SapS) as a Reporter for Gene Expression and Protein Secretion in Gram-Negative and Gram-Positive Bacteria▿

    Du Plessis, Erika; Theron, Jacques; Berger, Eldie; Louw, Maureen

    2007-01-01

    A phosphatase secreted by Staphylococcus aureus strain 154 has previously been characterized and classified as a new member of the bacterial class C family of nonspecific acid phosphatases. As the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. The S. aureus acid phosphatase (sapS) gene has been cloned and expressed f...

  16. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods. PMID:15797237

  17. Comparison of alkaline phosphatase activity of MC3T3-E1 cells cultured on different Ti surfaces: modified sandblasted with large grit and acid-etched (MSLA), laser-treated, and laser and acid-treated Ti surfaces

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

  18. Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

    Solberg, Lene B.; Brorson, Sverre-Henning; Stordalen, Gunhild A.; Bækkevold, Espen S; Andersson, Göran; Reinholt, Finn P.

    2014-01-01

    Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were...

  19. The activity of some phosphatases in tissues of adult Hymenolepis nana Siebold (Csetoda).

    Humiczewska, M

    1989-01-01

    Histochemical methods were used to study the localization and activity of acid and alkaline phosphatases, ATP-ase, 5-nucleotidase, and glucose-6-phosphatase in tissues of the mature form of Hymenolepis nana. Considerable differences in activity and localization of particular enzymes were observed in the organs of the parasite. The results obtained permit the statement that the integument is the most active enzymatically; in connection with the literature data, this gives grounds for the thesis that the integument of the cestodes functions as an absorbent-digestive organ. PMID:2558920

  20. 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

    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.......ArcA and AppY activated transcription of the cyx-appA operon during entry into stationary phase and under anaerobic growth conditions. The expression of the cyx-appA operon was affected by the anaerobic energy metabolism.The presence of the electron acceptors nitrate and fumarate repressed the expression...... 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...

  1. Acid phosphatase complex from the freshwater snail Viviparus viviparus L. under standard conditions and intoxication by cadmium ions.

    Tsvetkov, I L; Popov, A P; Konichev, A S

    2003-12-01

    Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water. PMID:14756629

  2. 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

    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

  3. Phosphoglycosylation of a secreted acid phosphatase from Leishmania donovani.

    Lippert, D N; Dwyer, D W; Li, F; Olafson, R W

    1999-06-01

    The secreted acid phosphatase (SAcP) of L.donovani is a heterogeneous glycoprotein that displays a wide array of N- and O-linked glycosylations. The O-linked sugars are of particular interest due to their similarity to the phosphoglycan structures of the major lipophosphoglycan surface antigen and released phosphoglycan (Turco et al., 1987; Greis et al., 1992). This study describes a structural analysis of the SAcP O-linked glycosylations using mass spectroscopy, amino acid sequencing, and enzymatic carbohydrate sequencing. Analysis of glycan chain lengths and peptide glycosylation site distribution was performed, revealing that the average O-linked structure was approximately 32 repeat units in length. Amino acid sequence analysis of glycosylated peptides showed that phosphoglycosylations did not occur randomly but were localized to specific serine residues within an array of degenerate serine/threonine-rich repeat sequences localized in the C-terminus. No evidence was obtained for modification of threonine residues. The observed pattern suggested that a consensus sequence may exist for localization of phosphoglycan structures. PMID:10336996

  4. Novel 2,7-Substituted (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acids: Peroxisome Proliferator-Activated Receptor γ Partial Agonists with Protein-Tyrosine Phosphatase 1B Inhibition.

    Otake, Kazuya; Azukizawa, Satoru; Takeda, Shigemitsu; Fukui, Masaki; Kawahara, Arisa; Kitao, Tatsuya; Shirahase, Hiroaki

    2015-01-01

    A novel series of 2,7-substituted 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-(2-Furylacryloyl)-7-[2-(2-methylindane-2-yl)-5-methyloxazol-4-yl]methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid tert-butylamine salt (13jE) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ)-selective agonist (EC50=85 nM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC50=1.0 µM). Compound 13jE partially activated PPARγ, but not PPARα or PPARδ, and antagonized farglitazar, a full PPARγ agonist. Cmax after the oral administration of 13jE at 10 mg/kg was 28.6 µg/mL (53 µM) in male Sprague-Dawley (SD) rats. Repeated administration of 13jE and rosiglitazone for 14 d at 10 mg/kg/d decreased plasma glucose and triglyceride levels significantly in male KK-A(y) mice. Rosiglitazone, but not 13jE, significantly increased the plasma volume and liver weight. In conclusion, 13jE showed stronger hypoglycemic and hypolipidemic effects and weaker hemodilution and hepatotoxic effects than rosiglitazone, suggesting that its safer efficacy may be due to its partial PPARγ agonism and PTP-1B inhibition. PMID:26633022

  5. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

    Harris, Thurl E; Huffman, Todd A; Chi, An; Shabanowitz, Jeffrey; Hunt, Donald F; Kumar, Anil; Lawrence, John C

    2007-01-01

    Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity. PMID:17105729

  6. Ecto-phosphatase activity on the external surface of Rhodnius prolixus salivary glands: modulation by carbohydrates and Trypanosoma rangeli.

    Gomes, Suzete A O; Fonseca de Souza, André L; Kiffer-Moreira, Tina; Dick, Claudia F; dos Santos, André L A; Meyer-Fernandes, José R

    2008-05-01

    The salivary glands of insect's vectors are target organs to study the vectors-pathogens interactions. Rhodnius prolixus an important vector of Trypanosoma cruzi can also transmit Trypanosoma rangeli by bite. In the present study we have investigated ecto-phosphatase activity on the surface of R. prolixus salivary glands. Ecto-phosphatases are able to hydrolyze phosphorylated substrates in the extracellular medium. We characterized these ecto-enzyme activities on the salivary glands external surface and employed it to investigate R. prolixus-T. rangeli interaction. Salivary glands present a low level of hydrolytic activity (4.30+/-0.35 nmol p-nitrophenol (p-NP)xh(-1)xgland pair(-1)). The salivary glands ecto-phosphatase activity was not affected by pH variation; and it was insensitive to alkaline inhibitor levamisole and inhibited approximately 50% by inorganic phosphate (Pi). MgCl2, CaCl2 and SrCl2 enhanced significantly the ecto-phosphatase activity detected on the surface of salivary glands. The ecto-phosphatase from salivary glands surface efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate release when phospho-tyrosine is used as a substrate. This ecto-phosphatase activity was inhibited by carbohydrates as d-galactose and d-mannose. Living short epimastigotes of T. rangeli inhibited salivary glands ecto-phosphatase activity at 75%, while boiled parasites did not. Living long epimastigote forms induced a lower, but significant inhibitory effect on the salivary glands phosphatase activity. Interestingly, boiled long epimastigote forms did not loose the ability to modulate salivary glands phosphatase activity. Taken together, these data suggest a possible role for ecto-phosphatase on the R. prolixus salivary glands-T. rangeli interaction. PMID:18407240

  7. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the nonspecificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination

  8. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  9. Diagnostic value of prostatic acid phosphatase as determined by radioimmunoassay

    Serum concentrations of prostatic acid phosphatase (PAP) were determined with 4 different radioimmunoassays and with the standard enzymatic method (p-nitrophenylphosphate) in 35 patients with prostatic carcinoma. Staging of localized tumors was based on histopathological evaluation after radial prostatectomy and pelvic lymphnode dissection (pTsub(1-3), pN0). In tumor lesions Tsub(1-2) N0 M0 elevated PAP-serum concentrations were found by RIA-determination in only one patient. Increased PAP serum levels were observed in 43-78% of carcinomas stage T3 N0 M0 and in 54-83% in stage Tsub(2-4) Nsub(x) M1 tumors, depending on the test kit used for the PAP determination. Concentrations for PAP obtained with the 4 different RIA-kits used, varied significantly and thus are not comparable. No false positive results were observed in sera of 9 patients with benign prostatic hyperplasia. Elevated PAP serum levels were found in a significantly higher frequency when determined by radioimmunoassay than by the enzymatic method. The results clearly indicate, that PAP is of no value for early recognition of carcinoma of the prostate even when measured by radioimmunoassay. However, the RIA-method seems to be of clinical importance in estimating the course of advanced local and metastasizing carcinoma of the prostate. (orig.)

  10. Radioimmunoassay for human prostatic acid phosphatase: Pt.4

    After PAP RIA has been established, serum prostatic acid phosphatase concentration was measured in 40 healthy males, 20 healthy females, 57 patients with benign prostatic hyperplasia, 20 patients with prostate cancers at various stages, 11 patients with cancers after prostatectomy or orchiectomy, and 36 patients with cancers other than prostate cancer. An upper cutoff value was calculated from the x + 2S of healthy males, which yielded a value of 2.2 μg/L of serum. More male patients with cancers other than prostate cancer had serum PAP values of less than 2.2 g/L. 91% (52/57) of BPH patients had normal value, 9% (5/57) exhibited elevated serum PAP levels. If cutoff the limit of x + 2S, the calculated value from 57 BPH patients was used, i.e. 3.0 μg/L, as hormal limit, the false positives presented by BPH were almost eliminated. 95% (1/20) patients with prostate cancers demonstrated an evidently elevated PAP, only one of those patients had a normal PAP value. After prostatectomy of prostate cancer, PAP declined to normal range or near upper cutoff value. The values obtained by this PAP assay were able to distinguish patients with prostate cancer from those with benign prostatic hyperplasia, and the course of disease could be monitored by the assay. Intraindividual sequential studies of PAP could be used to evaluate therapeutic response and prognosis

  11. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-01

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au+ complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe3+ with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe3+, and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs.A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by

  12. Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida

    Singh, Harkewal; Malinski, Thomas J.; Reilly, Thomas J.; Henzl, Michael T.; Tanner, John J.

    2011-01-01

    Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85 Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the pr...

  13. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes

    Solberg, L. B.; Stang, E.; Brorson, S.-H.; Andersson, G; Reinholt, F.P.

    2014-01-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in o...

  14. Partial Purification and Properties of an Acid Phosphatase from Pearl Oyster Pinctada Fucata

    柴云峰; 谢莉萍; 张荣庆

    2003-01-01

    Acid phosphatases (ACPs) are marker enzymes for the detection of lysosomes in cell fractions.However, ACPs in sea creatures are less studied than those on land.An acid phosphatase was partially purified from pearl oyster Pinctada fucata by chromatography on Sephadex G-150 and Con A-Sepharose 4B.The specific activity was 1719 U*mg-1 and with optimum pH (5.0) and temperature (60℃).The enzyme was strongly inhibited competitively by product analog WO3-4 and MoO3-4, but less inhibited by product analog AsO3-4.The enzyme could also be strongly inhibited by heavy metal ions, such as Ag+ and Cu2+, but was not affected by Pb2+.High concentrations of ethanol (64%) and NaF (10-3 mol·L-1) could inhibit the enzyme while low concentration of NaF (<10-4 mol·L-1) could slightly activate the enzyme.Other haloids (Cl-, Br-, I-) and EDTA did not have any effect on this enzyme, while tartrate and some chemical modification reagents (bromoacetic acid, formaldehyde and dithiothreitol) could inhibit the enzyme.It is concluded that the properties of the enzyme are different from many fresh water mollusks.

  15. Use of acid phosphatase as biomarker during the castor bean seeds germination (ricinus communis

    Carmen Ferreira Veríssima

    2008-12-01

    Full Text Available One of the main oil crop of prominent social and economic importance is to mamoneira (Ricinus communis L.; with countless application in the industry and agricultural. Broadly it distributed in Brazil; his cultivation can be an alternative of sustainability in the Brazilian northeast. It know the physiological and biochemical mechanisms of the germination they are important for the best utilization of the plant. The objective of this work was use acid phosphatase as biomarker during the germination. In the rough extract occurred the dosage of the activity for pNPP; Tyr-Pi and PPi; determination of protein and inorganic phosphatse. The peak of activity for pNPP was in the seventh day; for PPi and Tyr-Pi in the ninth and for PEP in the fifth. The concentration of protein increased according to the days of germination; with peak of activity in the eighth day; being coincidental with the peaks of the activities for the substrates. The content of inorganic phosphate diminished with the time of germination and after the third day occurred a fall accentuated of its concentration. We concluded that acid phosphatase is important for the germination of the seeds and his paper is related with the mobilization of inorganic phosphate; the main nutrients for the development.

  16. Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

    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.

  17. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis. PMID:25201349

  18. Sensitive and selective determining ascorbic acid and activity of alkaline phosphatase based on electrochemiluminescence of dual-stabilizers-capped CdSe quantum dots in carbon nanotube-nafion composite.

    Ma, Xiaolong; Zhang, Xin; Guo, Xinli; Kang, Qi; Shen, Dazhong; Zou, Guizheng

    2016-07-01

    Sensitive and selective determining bio-related molecule and enzyme play an important role in designing novel procedure for biological sensing and clinical diagnosis. Herein, we found that dual-stabilizers-capped CdSe quantum dots (QDs) in composite film of multi-walled carbon nanotubes (CNTs) and Nafion, displaying eye-visible monochromatic electrochemiluminescence (ECL) with fwhm of 37nm, which offers promising ECL signal for detecting ascorbic acid (AA) as well as the activity of alkaline phosphatase (ALP) in biological samples. It was also shown that the dual-stabilizers-capped CdSe QDs can preserve their highly passivated surface states with prolonged lifetime of excited states in Nafion mixtures, and facilitate electron-transfer ability of Nafion film along with CNTs. Compared with the QDs/GCE, the ECL intensity is enhanced 1.8 times and triggering potential shifted to lower energy by 0.12V on the CdSe-CNTs-Nafion/GCE. The ECL quenching degree increases with increasing concentration of AA in the range of 0.01-30nM with a limit of detection (LOD) of 5pM. The activity of ALP was determined indirectly according to the concentration of AA, generated in the hydrolysis reaction of l-ascorbic acid 2-phosphate sesquimagnesium (AA-P) in the presence of ALP as a catalyst, with an LOD of 1μU/L. The proposed strategy is favorable for developing simple ECL sensor or device with high sensitivity, spectral resolution and less electrochemical interference. PMID:27154663

  19. Prostatic Acid Phosphatase Is Expressed in Peptidergic and Nonpeptidergic Nociceptive Neurons of Mice and Rats

    Taylor-Blake, Bonnie; Zylka, Mark J.

    2010-01-01

    Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-...

  20. Purification and characterization of 29 kda acid phosphatase from germinating melon seeds

    Not much progress on the purification and characterization of low molecular weight acid phosphatases from plants has been made as yet. In the current study a low molecular weight acid phosphatase from seedling of melon was purified about 114-fold with specific activity of 45 U/ mg of protein and a recovery of 3 %. The enzyme was found to be homogeneous and showed a single band corresponding to 29 kDa on SDS-polyacrylamide gel electrophoresis. The Km for p-nitrophenyl phosphate was found to be 0.175 mM and Vmax was 42 micro mol of substrate hydrolyzed /min/mg of protein at pH 5.5 and at 37 degree C. The enzyme showed its optimum activity at pH 5.0 and 50 degree C. The enzyme was thermostable and it retained 70 % activity for 45 min at 60 degree C. The pH stability was 4.8-6.0. Phosphate, vanadate, molybdate and fluoride acted as strong inhibitors. Metal ions such as Zn /sup +2/, Cu /sup +2/, Ag /sup +2/ and Hg /sup +2/ deactivated the enzyme while other divalent ions such as Ca /sup +2/ and Mg /sup +2/ had no effect. (author)

  1. The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

    Papadaki, Amalia; Politou, Anastasia S; Smirlis, Despina; Kotini, Maria P; Kourou, Konstadina; Papamarcaki, Thomais; Boleti, Haralabia

    2015-05-01

    Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence. PMID:25695743

  2. Activation of SPS from darkened spinach leaves by an endogenous protein phosphatase

    Sucrose-phosphate synthase from darkened spinach leaves has a low activation state but can undergo a time-dependent activation in desalted leaf extracts that is inhibited by Pi, molybdate, okadaic acid and vanadate, but stimulated by fluoride. SPS labeled in vivo with [32P]Pi in excised leaves in the dark loses incorporated 32P with time when extracts are incubated at 25 degree C. This loss is largely prevented by vanadate, suggesting that an endogenous protein phosphatase can use SPS as substrate. Changes in phosphorylation state are closely paralleled by changes in SPS activation state. The spontaneous activation achieved in the extracts can be reversed by addition of 2 mM MgATP. Feeding okadaic acid to darkened leaves prevents light activation of SPS suggesting that the endogenous protein phosphatase is similar to the type-1 enzyme of animal tissues. Overall, the results are consistent with the notion that light activation of SPS involves dephosphorylation of inhibitory phosphorylation site(s). Regulation of the protein phosphatase by Pi may be of physiological significance

  3. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura

    E Edwin

    2016-05-01

    Full Text Available Andrographolide was isolated from ethanol extraction of Andrographis paniculata by column chromatography. Evaluation of larvicidal efficacy, enzymatic changes and cytotoxic activities against Spodoptera litura were conducted across a range of concentrations. The compound showed significant larvicidal activity between 5 - 25 ppm, post ingestion. Morphological deformities observed in larval-pupal stages. Enzymatic profiles were altered by reduction in acid phosphatase, ACP activity by 69.18 %, alkaline phosphatase, ALP activity 75.3 % and 74.9 % reduction in ATPase. Binding affinity to midgut epithelium cells suggests disintegration of cellular organelles observed was directly associated with ingestion of the compound. The results suggest that andrographolide has potential for development as a significant inhibitor of development against the pest Spodoptera litura.

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

    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.

  5. Metavanadate at the active site of the phosphatase VHZ.

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  6. ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY

    KOSINIAK-KAMYSZ K.

    2007-01-01

    Full Text Available The investigation was performed to evaluate the dog semen freezability and itsquality after thawing allowing its use for artificial insemination (AI. On the basis ofsperm motility, concentration and alkaline phosphatase (AP activity in semenplasma it was possible to establish that AP activity corresponds with the basic factorof semen examination. Significant statistical differences occurred between thequality of ejaculates which were qualified or disqualified to deep freezing and AI.These results show that AP activity in raw dog semen plasma can be used as amarker for the dog semen qualification for deep freezing and AI with 95%probability of the prognosis of the results.

  7. PROTEN TYROSINE PHOSPHATASE ACTIVITY IN RAT ASCITES HEPATOMA CELLS

    M.Saadat

    1998-10-01

    Full Text Available Protein tyrosine phosphatases (PTPases regulate tyrosine phosphorylation of target proteins involved in several aspects of cellular functions. Enzyme activities of the PTPases in cytosolic and particulate fractions of rat ascites hepatoma cell lines were determined and compared with those of normal rat liver. Our present data revealed that although there was no neoplatic-specific alteration of the PTPase activity in examined hepatomas, the activity in particulate fractions of island type of hepatomas was remarkably decreased compared with either rat liver or free type hepatomas.

  8. ALKALINE PHOSPHATASE ACTIVITY AS A MARKER OF DOG SEMEN FREEZABILITY

    K. KOSINIAK-KAMYSZ

    2013-12-01

    Full Text Available The investigation was performed to evaluate the dog semen freezability and itsquality after thawing allowing its use for artificial insemination (AI. On the basis ofsperm motility, concentration and alkaline phosphatase (AP activity in semenplasma it was possible to establish that AP activity corresponds with the basic factorof semen examination. Significant statistical differences occurred between thequality of ejaculates which were qualified or disqualified to deep freezing and AI.These results show that AP activity in raw dog semen plasma can be used as amarker for the dog semen qualification for deep freezing and AI with 95%probability of the prognosis of the results.

  9. Overexpression of OsPAP10a, A Root-Associated Acid Phosphatase, Increased Extracellular Organic Phosphorus Utilization in Rice

    Jingluan Tian; Chuang Wang; Qian Zhang; Xiaowei He; James Whelan; Huixia Shou

    2012-01-01

    Phosphorus (P) deficiency is a major limitation for plant growth and development.Among the wide set of responses to cope with low soil P,plants increase their level of intracellular and secreted acid phosphatases (APases),which helps to catalyze inorganic phosphate (Pi) hydrolysis from organophosphates.In this study we characterized the rice (Oryza sativa) purple acid phosphatase 10a (OsPAP10a).OsPAP10a belongs to group la of purple acid phosphatases (PAPs),and clusters with the principal secreted PAPs in a variety of plant species including Arabidopsis.The transcript abundance of OsPAP10a is specifically induced by Pi deficiency and is controlled by OsPHR2,the central transcription factor controlling Pi homeostasis.In gel activity assays of root and shoot protein extracts,it was revealed that OsPAP10a is a major acid phosphatase isoform induced by Pi starvation.Constitutive overexpression of OsPAP10a results in a significant increase of phosphatase activity in both shoot and root protein extracts.In vivo root 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) assays and activity measurements on external media showed that OsPAP10a is a root-associated APase.Furthermore,overexpression of OsPAP10a significantly improved ATP hydrolysis and utilization compared with wild type plants.These results indicate that OsPAP10a can potentially be used for crop breeding to improve the efficiency of P use.

  10. Binuclear Metal Centers in Plant Purple Acid Phosphatases: Fe-Mn in Sweet Potato and Fe-Zn in Soybean

    Schenk, Gerhard; Ge, Yubin; Carrington, Lyle E; Wynne, Ceridwen J.; Searle, Iain R.; Carroll, Bernard J; Hamilton, Susan E.; de Jersey, John

    1999-01-01

    Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII–ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the pu...

  11. Inhibition of acid, alkaline, and tyrosine (PTP1B) phosphatases by novel vanadium complexes.

    McLauchlan, Craig C; Hooker, Jaqueline D; Jones, Marjorie A; Dymon, Zaneta; Backhus, Emily A; Greiner, Bradley A; Dorner, Nicole A; Youkhana, Mary A; Manus, Lisa M

    2010-03-01

    In the course of our investigations of vanadium-containing complexes for use as insulin-enhancing agents, we have generated a series of novel vanadium coordination complexes with bidentate ligands. Specifically we have focused on two ligands: anthranilate (anc(-)), a natural metabolite of tryptophan, and imidizole-4-carboxylate (imc(-)), meant to mimic naturally occurring N-donor ligands. For each ligand, we have generated a series of complexes containing the V(III), V(IV), and V(V) oxidation states. Each complex was investigated using phosphatase inhibition studies of three different phosphatases (acid, alkaline, and tyrosine (PTP1B) phosphatase) as prima facia evidence for potential use as an insulin-enhancing agent. Using p-nitrophenyl phosphate as an artificial phosphatase substrate, the levels of inhibition were determined by measuring the absorbance of the product at 405nm using UV/vis spectroscopy. Under our experimental conditions, for instance, V(imc)(3) appears to be as potent an inhibitor of alkaline phosphatase as sodium orthovanadate when comparing the K(cat)/K(m) term. VO(anc)(2) is as potent an inhibitor of acid phosphatase and tyrosine phosphatase as the Na(3)VO(4). Thus, use of these complexes can increase our mechanistic understanding of the effects of vanadium in vivo. PMID:20071031

  12. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    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 PO43-. 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 PO43--irrepressible) relative to isolates from the reference site. A high percentage of strains that exhibited such putatively PO43--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 amplified phosphatases are being

  13. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    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

  14. An Approach to More Accurate Model Systems for Purple Acid Phosphatases (PAPs).

    Bernhardt, Paul V; Bosch, Simone; Comba, Peter; Gahan, Lawrence R; Hanson, Graeme R; Mereacre, Valeriu; Noble, Christopher J; Powell, Annie K; Schenk, Gerhard; Wadepohl, Hubert

    2015-08-01

    The active site of mammalian purple acid phosphatases (PAPs) have a dinuclear iron site in two accessible oxidation states (Fe(III)2 and Fe(III)Fe(II)), and the heterovalent is the active form, involved in the regulation of phosphate and phosphorylated metabolite levels in a wide range of organisms. Therefore, two sites with different coordination geometries to stabilize the heterovalent active form and, in addition, with hydrogen bond donors to enable the fixation of the substrate and release of the product, are believed to be required for catalytically competent model systems. Two ligands and their dinuclear iron complexes have been studied in detail. The solid-state structures and properties, studied by X-ray crystallography, magnetism, and Mössbauer spectroscopy, and the solution structural and electronic properties, investigated by mass spectrometry, electronic, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and Mössbauer spectroscopies and electrochemistry, are discussed in detail in order to understand the structures and relative stabilities in solution. In particular, with one of the ligands, a heterovalent Fe(III)Fe(II) species has been produced by chemical oxidation of the Fe(II)2 precursor. The phosphatase reactivities of the complexes, in particular, also of the heterovalent complex, are reported. These studies include pH-dependent as well as substrate concentration dependent studies, leading to pH profiles, catalytic efficiencies and turnover numbers, and indicate that the heterovalent diiron complex discussed here is an accurate PAP model system. PMID:26196255

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

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

    1995-01-01

    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 of......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...... increased phosphatase activity in soil influenced by roots. Phosphatase activity at both pH values was highest in soil influenced by uncolonized roots, but this was attributed to higher root length densities as compared to mycorrhizal roots. Mycorrhizal hyphae showed no influence on soil phosphatase...

  16. Members of a unique histidine acid phosphatase family are conserved amongst a group of primitive eukaryotic human pathogens.

    Shakarian, Alison M; Joshi, Manju B; Yamage, Mat; Ellis, Stephanie L; Debrabant, Alain; Dwyer, Dennis M

    2003-03-01

    Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism. In this report, to further examine this hypothesis, we assessed whether members of the L. donovani histidine-acid phosphatase enzyme family were conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Such phylogenetic conservation would clearly indicate an evolutionary selection for this family of enzymes and strongly suggest and support an important functional role for acid phosphatases to the survival of these parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that homologs of both the Ld SAcPs and Ld MAcP were present in each of the visceral and cutaneous Leishmania species examined (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of enzyme assays showed that all of these organisms expressed both tartrate-sensitive and tartrate-resistant acid phosphatase activities. In addition, homologs of both the Ld SAcPs and Ld MAcP genes and their corresponding enzyme activities were also identified in two Crithidia species (C. fasciculata and C. luciliae) and in Leptomonas seymouri. In contrast, Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens had only very-low levels of such enzyme activities. Cumulatively, results of this study showed that homologs of the Ld SAcPs and Ld MAcP are conserved amongst all pathogenic Leishmania sps. suggesting

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

    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.

  18. Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes.

    Bates, P A; Hermes, I; Dwyer, D M

    1990-03-01

    Monensin, an inhibitor of Golgi function, was used to investigate the role of this cell compartment in the glycosylation of Leishmania donovani promastigote secretory acid phosphatase (EC 3.1.3.2). Monensin-treated cells demonstrated morphological changes in the Golgi complex and secreted enzyme with an altered electrophoretic mobility: two discrete bands of approximately 95 and 110 kDa were found, as compared to the heterodisperse nature of the enzyme from untreated controls. Chemical deglycosylation by mild acid hydrolysis resulted in a similar effect on the electrophoretic mobility of purified extracellular enzyme. Acid phosphatase was also treated with N-glycosidase F (EC 3.5.1.52) to remove N-linked oligosaccharides. The altered lectin-binding properties of the enzyme after these two treatments demonstrated that an unusual type of galactose-containing acid-labile carbohydrate was present in secretory acid phosphatase in addition to the N-linked oligosaccharides. Further, experiments with 32P-labelled enzyme indicated that phosphodiester bonds were the structural component responsible for the sensitivity of this carbohydrate to mild acid hydrolysis. Cumulatively, these results demonstrated that a novel form of Golgi-mediated posttranslational modification had occurred to the secretory acid phosphatase presumably by the addition of an acid-labile phosphoglycan. PMID:2320058

  19. Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.

    Hsing, W; Silhavy, T J

    1997-01-01

    EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the ph...

  20. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P41212, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P41212, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  1. Effects of synthetic detergents on in vivo activity of tissue phosphatases and succinic dehydrogenase from Mystus vittatus

    Mohan, D.; Verma, S.R.

    1981-05-01

    African catfish (Mystus vittatus) were exposed to three sub-lethal concentrations of Swascofix E45 (13.8, 9.2 and 4.6 mg/l) and Swascol 3L (69.3, 46.2 and 23.1 mg/l) for 15 and 30 days, and their effects on alkaline and acid phosphatase, and succinic dehydrogenase in liver, kidney and intestine were measured. The enzymes were found to be inhibited in all the tissues. Maximum inhibition (38.44%) was observed in liver alkaline phosphatase activity after 30 days with the highest concentration of Swascofix E45 and the lowest inhibition (0.118%) was found in kidney acid phosphatase activity with the lowest concentration of Swascol 3L after 15 days. Insignificant enzyme stimulation in some cases was also observed.

  2. Strigolactone regulates anthocyanin accumulation, acid phosphatases production and plant growth under low phosphate condition in Arabidopsis.

    Shinsaku Ito

    Full Text Available Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.

  3. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  4. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2005-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P41212, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2.

  5. Activity of Follicular Fluid Phosphatases and Their Correlation with Levels of Serum Esteroidal Hormones and Gonadotropins

    Sh Byranvand

    2006-10-01

    Full Text Available Introduction: The aim of this study was to evaluate the correlation between serum levels of ovarian and gonadotropin hormones, age and number of follicles with follicular alkaline and acid phosphatase activity in infertile women under controlled ovarian hyperstimulation. Methods: After collection of follicular fluid and calculation of the number of follicles, the specific activity of alkaline (ALP and acid phosphatase (ACP was determined according to the total protein in 19 women at the time of puncture. Also at that time, the levels of progesterone, estradiol, and follicle stimulating hormone (FSH and leuteinizing hormone (LH of their sera were measured. The correlation of follicular ALP and ACP with each serum hormone levels, women age and number of follicles was calculated using non-parametric analysis. Results: The ALP has a correlation with progesterone (P=0.01 levels but doesn’t have any correlation with the other factors. However, the ACP activity has a correlation not only with follicular number but also with estradiol and progesterone levels (P=0.05. Conclusion: Thus ACP activity is more affected by ovarian hormone than ALP and it can affect the ovarian microenvironment and oocyte development.

  6. The genomic complement of purple acid phosphatase phytases in the Triticeae

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

    2011-01-01

    , PAPhy_b promoters contain elements typical of gibberellic acid induced germination related hydrolases. PAPhy_a promoters in contrast possess elements known from storage protein promoters. **Dionisio G, Madsen CK, Holm PB, Welinder KG, Jørgensen M, Stoger E, Arcalis E, Brinch-Pedersen H. 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. 2011a, Jan 10.[Epub ahead of print]...

  7. Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase.

    Vihko, P; Virkkunen, P; Henttu, P; Roiko, K; Solin, T; Huhtala, M L

    1988-08-29

    lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases. PMID:2842184

  8. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    Sobecky, Patricia A. [Univ. of Alabama, Tuscaloosa, AL (United States)

    2015-04-06

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  9. Sensitive optical detection of alkaline phosphatase activity with quantum dots

    Ren, Xiangling [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China); The State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096 (China); Chen, Zhenzhen; Chen, Xiaoying; Liu, Jing [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China); Tang, Fangqiong, E-mail: tangfq@mail.ipc.ac.cn [Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No. 29, Zhongguancun East Road, Haidian District, Beijing 100190 (China)

    2014-01-15

    A simple method has been developed to detect the activity of alkaline phosphatase (ALP) by the changing of fluorescence intensities of the quantum dots (QDs). In this system, the fluorescence intensities of the QDs were quenched by p-nitrophenol (pNP) which was produced in the process of ALP catalytic reaction. A series of linear calibration curves of the activity of ALP were obtained in different pH buffer solutions. The wide linear range was 3–1000 U L{sup −1} and the detection limit was 3 U L{sup −1} (S/N=3). Furthermore, the experimental conditions of biosensor were optimized, and anti-interference ability was presented. The activity of ALP was also detected in serum and the recovery of ALP in serum samples was more than 95%. The excellent performance of this biosensor indicates that it can be used in practice detection of ALP. -- Highlights: • A sensitive ALP biosensor is constructed based on QDs without complex processes. • The analysis processing is very convenient, simple and rapid. • The detection mechanism of the ALP biosensor is studied by XPS. • The paper proposes a feasible approach for some substrates or enzymes detecting.

  10. Sensitive optical detection of alkaline phosphatase activity with quantum dots

    A simple method has been developed to detect the activity of alkaline phosphatase (ALP) by the changing of fluorescence intensities of the quantum dots (QDs). In this system, the fluorescence intensities of the QDs were quenched by p-nitrophenol (pNP) which was produced in the process of ALP catalytic reaction. A series of linear calibration curves of the activity of ALP were obtained in different pH buffer solutions. The wide linear range was 3–1000 U L−1 and the detection limit was 3 U L−1 (S/N=3). Furthermore, the experimental conditions of biosensor were optimized, and anti-interference ability was presented. The activity of ALP was also detected in serum and the recovery of ALP in serum samples was more than 95%. The excellent performance of this biosensor indicates that it can be used in practice detection of ALP. -- Highlights: • A sensitive ALP biosensor is constructed based on QDs without complex processes. • The analysis processing is very convenient, simple and rapid. • The detection mechanism of the ALP biosensor is studied by XPS. • The paper proposes a feasible approach for some substrates or enzymes detecting

  11. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  12. Alteração da atividade enzimática em organismos aquáticos por poluentes de origem agrícola: uma abordagem geral e sobre a suscetibilidade da fosfatase ácida Alteration of enzymatic activity in aquatic organisms by agricultural pollutants: a general approach and the susceptibility of the acid phosphatase

    Claudio Martín Jonsson

    2010-01-01

    Full Text Available The input of agrochemicals in the aquatic compartment can results in biochemical injuries for living organisms. In this context, the knowledge of alterations of enzymatic activities due the presence of agriculture pollutants contributes for the elucidation of the mechanisms of toxicity, implementation of economic methods for monitoring purposes and establishment of maximum allowed concentrations. In the present work, the above considerations are discussed, and data concerning changes in enzymatic function by pesticides and fertilizer contaminants are reviewed. Also, we focused on the acid phosphatase due its susceptibility to several pollutants and diversity in cellular functions.

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

    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......) 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...

  14. Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

    Płachno, B J; Adamec, L; Lichtscheidl, I K; Peroutka, M; Adlassnig, W; Vrba, J

    2006-11-01

    A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients. PMID:16865659

  15. How Should an Increase in Alkaline Phosphatase Activity Be Interpreted?

    Low-level laser therapy, commonly known as LLLT, is the application of low power, monochromatic, and coherent light to injuries and lesions to stimulate healing and give pain relief. There are conflicting reports in the literature regarding the role of ALP. Objective: this study aimed to compare the cellular responses of wounded human skin fibroblasts exposed to doses of 0.5 J/cm2, 2.5 J/cm2, 5 J/cm2, or 16 J/cm2 using LLLT with a Helium-Neon laser (632.8 nm, 18.8 mW power output, 2.07 mW/cm2 power density, and 3.4 cm diameter spot size or area 9.1?cm2) to elucidate the role of alkaline phosphatase (ALP) in cell proliferation. Methods: cellular responses to laser irradiation were evaluated using ALP enzyme activity, LDH membrane integrity, neutral red for cell proliferation, optical density at 540?nm, and basic fibroblast growth factor (bFGF) expression. Results: results suggest that an increase in ALP is negatively correlated with cell growth depending on the concentration of growth factors in the medium. Results also indicate that an increase in ALP may be related to cellular damage. Conclusion: since the exact role of ALP is unknown, the ALP enzyme activity assay should be considered in conjunction with other cell proliferation assays such as neutral red, optical density, or more specifically bFGF expression.

  16. Optimal level of Purple Acid Phosphatase5 is required for maintaining complete resistance to Pseudomonas syringae

    Sridhar eRavichandran

    2015-08-01

    Full Text Available Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host’s innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5 lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000. Here, we report that an optimal level of PAP5 is crucial for mounting complete basal resistance. Overexpression of PAP5 impaired ICS1, PR1 expression and salicylic acid (SA accumulation similar to pap5 knockout mutant plants. Moreover, plant overexpressing PAP5 was impaired in H2O2 accumulation in response to Pst DC3000. PAP5 is localized in to peroxisomes, a known site of generation of reactive oxygen species for activation of defense responses. Taken together, our results demonstrate that optimal levels of PAP5 is required for mounting resistance against Pst DC3000 as both knockout and overexpression of PAP5 lead to compromised basal resistance.

  17. Tunable phosphatase-sensitive stable prodrugs of 5-aminolevulinic acid for tumor fluorescence photodetection.

    Babič, Andrej; Herceg, Viktorija; Ateb, Imène; Allémann, Eric; Lange, Norbert

    2016-08-10

    5-Aminolevulinic acid (5-ALA) has been at the forefront of small molecule based fluorescence-guided tumor resection and photodynamic therapy. 5-ALA and two of its esters received marketing authorization but suffer from several major limitations, namely low stability and poor pharmacokinetic profile. Here, we present a new class of 5-ALA derivatives aiming at the stabilization of 5-ALA by incorporating a phosphatase sensitive group, with or without self-cleavable linker. Compared to 5-ALA hexyl ester (5-ALA-Hex), these compounds display an excellent stability under acidic, basic and physiological conditions. The activation and conversion into the 5-ALA is controlled and can be structure-tailored. The prodrugs display reduced acute toxicity compared to 5-ALA-Hex with superior dose response profiles of protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro. Clinically relevant fluorescence kinetics in vivo shown in U87MG glioblastoma spheroid tumor model in chick embryos provide a solid basis for their further development and translation to clinical fluorescence guided tumor resection and photodynamic therapy. PMID:27235981

  18. Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan. Partial characterization of private and public epitopes.

    Ilg, T; Harbecke, D; Wiese, M; Overath, P

    1993-10-15

    -6[Glc beta 1,3]Gal beta 1,4Man alpha 1 repeats while six mAbs react with the unmodified repeats. Two antibodies specific for Leishmania major recognize Gal beta 1,3-substituted repeats unique for lipophosphoglycan from this species. Analysis by immunoblotting indicates that the high-molecular-mass proteo-phosphoglycan of L. mexicana secreted acid phosphatase carries epitopes for all anti-lipophosphoglycan mAbs suggesting the presence of capped phosphosaccharide repeats while the enzymically active glycoprotein subunit is modified by caps but probably not by repeats. In the case of Leishmania donovani secreted acid phosphatase, the enzymically active polypeptide may be directly modified by repeats. The mAbs are used to characterize changes in lipophosphoglycan structure, which occur in culture during the transition of promastigotes from the logarithmic to the stationary growth phase.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7693464

  19. Alkaline Phosphatase Activity in San Francisco and Monterey Bays

    Nicholson, D. P.

    2002-12-01

    Phosphorus (P) is an essential nutrient utilized by all living organisms, and has been recognized as a limiting nutrient in some oceanic systems (Cotner et al., 1997; Karl et al., 1995; Michaels et al., 1996; Wu et al., 2000). However, relatively little is known about the extent of P limitation in natural environments, how P limitation varies spatially and temporally, and what determines how and when P becomes limiting (Benitez-Nelson, 2000). A more direct estimate of the degree of P limitation in a variety of oceanic systems is needed to better understand P cycling and dynamics within the ocean and how these have and will change in response to global climate and environmental perturbation. Accordingly, the objective this study is to assess the P-status of marine planktonic communities in Monterey and San Francisco Bays using the activity of alkaline phosphatase in the water column. Alkaline phosphatase (AP) is the most widely used enzyme that marine organisms use to hydrolize organic P compounds to biologically available orthophosphate. Accordingly it is expected that in areas where P is a limiting nutrient organisms will produce and release more AP to seawater so they can utilize the dissolved and particulate organic P compounds. Indeed it has been suggested that the AP activity is a reliable indicator of P-availability to planktonic communities (Ammerman and Azam, 1985; Cotner and Wetzel, 1991; Hong et al., 1998). High enzyme activities indicate low dissolved inorganic phosphate (DIP) availability while low levels suggest that DIP supply satisfies the community P-demand. This study examines AP activity in San Francisco and Monterey Bays over a 12 month period, from November, 2001 through November, 2002 using two enzyme assays. The study encompasses data from a three-station transect in Monterey Bay, at depths ranging from 0-60 meters. The stations range from coastal waters to open ocean depths of several thousand meters. In San Francisco Bay, surface water from

  20. Purification of a tartrate-resistant acid phosphatase (TrACP) from bovine cortical bone matrix

    It has been previously demonstrated that a partially purified bovine skeletal TrACP showed protein phosphatase (P'ase) activity that was specific for phosphotyrosyl (Ptyr) proteins. They have now purified TrACP activity from bovine cortical bone matrix to apparent homogeneity. The purification procedures included CM-Sepharose ion-exchange, cellulose phosphate affinity, sephacryl S-300 gel filtration and phenyl sepharose affinity chromatographies. Overall yield was > 25% and purification was approximately 2000-fold with a specific activity of 8.15 umol pNPP hydrolyzed/min/mg protein at 370C. The purified enzyme was judged to be homogeneous based on: (i) appearance as a single protein band on SDS-PAGE (silver staining technique) and (ii) distribution analysis of radioiodinated purified TrACP after SDS-PAGE revealing one band of radioactivity at the same positions as the TrACP protein band. M.W. of TrACP was 34,600 as assessed by gel filtration and 32,500 by SDS-PAGE, suggesting that bovine skeletal TrACP exists as active monomer. Analysis of the purified TrACP by isoelectric focusing showed at least 9 bands of enzyme activities with pIs between 4 and 5, indicating micro-heterogenecity. Substrate specificity analyses revealed that the purified TrACP also hydrolyzed nucleotide tri- and di-phosphates, but not monophosphates or other low M.W. phosphoryl esters, and was also capable of hydrolyzing phosphotyrosine (Tyr(P)) and Ptyr proteins with little activity toward other phosphoamino acids or phosphoseryl proteins. Optimal pH was 5.5 for TrACP activity, 6.0 for Tyr(P) P'ase activity and 7.0 for Ptyr protein P'ase activity. Results of these studies represent the first purification of a skeletal TrACP to apparent homogeneity

  1. Changes in Phosphorus Fractions, pH, and phosphatase Activity in rhizosphere of Two Rice Genotypes

    LI Yong-Fu; LUO An-Cheng; WEI Xing-Hua; YAO Xu-Guo

    2008-01-01

    A rhizobox experiment with two phosphorus (P) treatments, zero-P (0 mg P kg-1) and plus-P (100 mg P kg-1) as Ca(H2PO4)2·H2O, was conducted to study the chemical and biochemical properties in the rhizosphere of two rice genotypes (cv. Zhongbu 51 and Pembe) different in P uptake ability and their relationship with the depletion of soil P fractions. Plant P uptake, pH, phosphatase activity, and soil P fractions in the rhizcephere were measured. Both total dry weight and total P uptake of Pembe were significantly (P < 0.05) higher than those of Zhongbu 51 in the zero-P and plns-P treatments. Significant depletions of resin-Pi, NaHCO3-Pi, NaHCO3-Po, and NaOH-Pi, where Pi stands for inorganic P and Po for organic P, were observed in the rhizosphere of both Zhongbu 51 and Pembe under both P treatments. Pembe showed a greater ability than Zhongbu 51 in depleting resin-Pi, NaHCO2-Pi, NaHCO3-Po, NaOH-Pi, and NaOH-Po in the rhizosphere. HCl-Pi and residual-P were not depleted in the rhizosphere of both genotypes, regardless of P treatments despite significant acidification in the rhizosphere of Pembe under zero-P treatment. Higher acid phosphatase (AcPME) activity and alkaline phosphatase (A1PME) activity were observed in the rhizosphere of both Zhongbu 51 and Pembe compared to the corresponding controls without plant. AcPME activity was negatively (P < 0.01) correlated to NaHCO3-Po concentration in the rhizosphere of both Zhongbu 51 and Pembe, suggesting that AcPME was associated with the mineralization of soil organic P.

  2. Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface

    Taillefert, Martial [Georgia Tech Research Corporation, Atlanta, GA (United States)

    2015-04-01

    This project investigated the geochemical and microbial processes associated with the biomineralization of radionuclides in subsurface soils. During this study, it was determined that microbial communities from the Oak Ridge Field Research subsurface are able to express phosphatase activities that hydrolyze exogenous organophosphate compounds and result in the non-reductive bioimmobilization of U(VI) phosphate minerals in both aerobic and anaerobic conditions. The changes of the microbial community structure associated with the biomineralization of U(VI) was determined to identify the main organisms involved in the biomineralization process, and the complete genome of two isolates was sequenced. In addition, it was determined that both phytate, the main source of natural organophosphate compounds in natural environments, and polyphosphate accumulated in cells could also be hydrolyzed by native microbial population to liberate enough orthophosphate and precipitate uranium phosphate minerals. Finally, the minerals produced during this process are stable in low pH conditions or environments where the production of dissolved inorganic carbon is moderate. These findings suggest that the biomineralization of U(VI) phosphate minerals is an attractive bioremediation strategy to uranium bioreduction in low pH uranium-contaminated environments. These efforts support the goals of the SBR long-term performance measure by providing key information on "biological processes influencing the form and mobility of DOE contaminants in the subsurface".

  3. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using [32P]phosphorylase α as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at ∼5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase

  4. Changes phosphorus associated to phosphatase activity because of application of carbon, nitrogen and manure

    Paredes, Cecilia; Gianfreda, Liliana; Mora, María de la Luz

    2015-04-01

    The Chilean Andisols are of great importance in the economy of southern Chile supporting the bulk of agricultural production. The major characteristics of Chilean volcanic soils are the high adsorption capacity of P with a concomitant low P availability to plants. Studies preliminary using dairy cattle dung suggest that we can improve P availability using organic P sources within the soil because of microorganism. Phosphorous solubilization by microorganisms is a complex phenomenon, which depends on many factors such as nutritional, physiological and growth condition of the culture. The principal mechanism for mineral phosphate solubilization is the production of organic acids where the enzyme phosphatases play a major role in the mineralization of organic phosphorous in soil. The objective of this study was to evaluate changes in soil phosphorus fractions due to application the cattle dung, glucose, nitrogen (N) and phosphorus (P). In this experiment we incubated soil samples with 300 g of cattle dung, 30 mg kg-1 of N and P and 1000 mg glucose kg-1. The soil samples were moistened to field capacity and incubated in plastic bags to room temperature by different time. The changes in P forms in soil were monitored through the Hedley fractionation procedure and phosphatase activity. Our preliminary results indicated that the application of cattle dung, glucose nitrogen and phosphorus, caused the increased phosphatase activity until to 7 days and then apparently return to normal values. Interestingly, we observed a rise in the inorganic P fraction extracted by NaHCO3 in the same period. In summary, the increase biological activity by carbon and nitrogen increase P availability. Acknowledgements: The authors thank Fondecyt 1141247 Project.

  5. Comparative theoretical studies of the phosphomonoester hydrolysis mechanism by purple acid phosphatases.

    Retegan, M; Milet, A; Jamet, H

    2010-07-01

    We present here the first ONIOM (our own n-layered integrated molecular orbital + molecular mechanics method) studies of a purple acid phosphatase enzyme. Our study focused on the structures of the red kidney bean PAP (kbPAP) complexed with phosphate and with phenyl phosphate and on the mechanism of the phenyl phosphate hydrolysis by the enzyme. Density functional theory (DFT) calculations were also performed using models of different sizes for comparison purpose. Results show that the inclusion of three histidine residues, His202, His295, and His296, with their protein surrounding, is crucial to properly describe the coordination of the substrates. They induce a conformation with the substrate closer to the nucleophilic mu-hydroxyde bridge. In the mechanistic study, a transition state is stabilized by a strong hydrogen bond between His202 and the leaving group of the substrate. Consequently, a smaller value for the activation energy barrier is obtained from DFT calculations including this histidine to the same calculations without this histidine. Using the ONIOM method, this activation energy barrier is even more reduced. So the mechanism, which considers the hydroxo group bridging the two metal ions as nucleophile, becomes really convincing, contrary to the results obtained with a small model at the DFT level. PMID:20550096

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

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

    2011-01-01

    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......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....... Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only...

  7. Optimal level of purple acid phosphatase5 is required for maintaining complete resistance to Pseudomonas syringae

    Ravichandran, Sridhar; Stone, Sophia L.; Benkel, Bernhard; Zhang, Junzeng; Berrue, Fabrice; Prithiviraj, Balakrishnan

    2015-01-01

    Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host's innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5) lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Here, we report that an optimal level of PAP5 is crucial for mounting complete basal re...

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

    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

  9. Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml-1], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first...

  10. Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

    Qin, S.P.; C. S. Hu; Oenema, O.

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio = 1/8 (w/v) and power density = 15 watt ml(-1)], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first p...

  11. Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir

    Tseng, Y.

    2011-12-01

    This research was conducted by downscaling study to understand phosphorus (P)-deficient status of different plankton and the role of alkaline phosphatase activity (APA) in subtropical Feitsui Reservoir. Results from field survey showed that bulk APA (1.6~95.2 nM h-1) was widely observed in the epilimnion (0~20 m) with an apparent seasonal variations, suggesting that plankton in the system were subjected to P-deficient seasonally. Mixed layer depth (an index of phosphate availability) is the major factor influencing the variation of bulk APA and specific APA (124~1,253 nmol mg C-1 h-1), based on multiple linear regression analysis. Size-fractionated APA assays showed that picoplankton (size 0.2~3 um) contributed most of the bulk APA in the system. In addition, single-cell APA detected by enzyme-labeled fluorescence (ELF) assay indicated that heterotrophic bacteria are the major contributors of APA. Thus, we can infer that bacteria play an important role in accelerating P-cycle within P-deficient systems. Light/nutrient manipulation bioassays showed that bacterial growth was directly controlled by phosphate, while picocyanobacterial growth is controlled by light and can out-compete bacteria under P-limited condition with the aid of light. Further analysis revealed that the strength of summer typhoon is a factor responsible for the inter-annual variability of bulk and specific APA. APA study demonstrated the episodic events (e.g. strong typhoon and extreme precipitation) had significant influence on APA variability in sub-tropical to tropical aquatic ecosystems. Hence, the results herein will allow future studies on monitoring typhoon disturbance (intensity and frequency) as well as the APA of plankton during summer-to-autumn in subtropical systems.

  12. Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus

    Jørgensen, Kaj Anker; Koefoed-Nielsen, P.B.; Karamperis, N.

    2003-01-01

    The mode of immunosuppressive action of tacrolimus (FK506) and cyclosporin A has been elucidated. Both drugs bind to proteins in the cytoplasm to form complexes, which in turn inhibit the phosphatase activity of calcineurin, an important limiting step in the activation of T cells. The association...

  13. Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

    Katarzyna Bednarska; Magdalena Klink; Zofia Sulowska

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104–106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using...

  14. Chemical properties population of nitrites oxidizers, urease and phosphatase activities in sewage sludge-amended soils

    Bonmati Pont, Manuel; Pujolà Cunill, Montserrat; Saña Vilaseca, Josep; Soliva Torrentó, Montserrat; Felipó, Ma. Teresa (María Teresa); Garau, M; B. Ceccanti; P. Nannipieri

    1985-01-01

    The aim of this work has been to determine the effect of sterilized and non-sterilized, aerobically or anaerobically digested sewage sludges on urease and phosphatase aetivities, on populations of nitrite oxidizers and on some chemical properties in laboratory conditions and for long incubation periods. Both urease an phosphatase activities were affected when anaerobic sludges were added to the soil. The inhibitory effects on both enzyme activities attributed to the presence of...

  15. Alkaline phosphatase activity in plasma and liver of rats submitted to chronic exposure to fluoride

    Mileni da Silva Fernandes

    2011-12-01

    Full Text Available The aim of this study was to compare the effect of fluoride (F on alkaline phosphatase activity in the liver and plasma of the rats. Four groups of male Wistar rats (n=6, which received drinking water containing 5, 15 or 50 ppm F or deionized water (control throughout the experiment were included in the study. The animals were euthanized and had their tissues and blood plasma collected for the analysis of fluoride and alkaline phosphatase. There was an increase in F concentration in most tissues in the animals treated with higher F concentrations, except for the heart. The alkaline phosphatase assay showed an increase in the activity in the liver and blood plasma of the animals treated with fluoride concentrations of 15 and 50 ppm (p<0.05. This study suggested that F at a concentration of 50 ppm in drinking water promotes increased the activity of alkaline phosphatase in the liver and blood plasma.

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

    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.

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

    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.

  18. Single and Combined Effects of As (III) and Acetochlor on Phosphatase Activity in Soil

    ZHANG Yun; ZHANG Feng; ZHANG Guan-cai; GUAN Lian-zhu

    2013-01-01

    The actions and interactions of acetochlor and As on the soil phosphatase activity were investigated after 1, 3, 6, 10, 15, 30 and 60 d of exposure under control conditions. The soils were exposed to various concentrations of acetochlor and As individually and simultaneously. The results showed that acetochlor, As only, and combined pollution all clearly inhibited soil phosphatase activity. The maximum inhibition ratios of soil phosphatase activity by acetochlor, As only and combined pollution were 36.44, 74.12 and 61.29%, respectively. Two kinetic models,ν=c/(1+bi) (model 1) andν=c(1+ai)/(l+bi) (model 2), were used to describe the relationship between the concentrations of As and acetochlor and the activity of soil phosphatase. The semi-effect dose (ED50) values induced by As and acetochlor stress based on the inhibition of soil phosphatase were 18.1 and 33.11 mg kg-1, respectively, according to calculation by model 1. The interactive effect of acetochlor with As on soil phosphatase primarily consisted of significant antagonism effects at the higher concentrations tested. The step regression results show that the toxicity order was As (III)>acetochlor>As (III)×acetochlor throughout the incubation period.

  19. Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils

    Supernatants prepared from disrupted Coxiella burnetii posses acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 grad C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 grad C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-liked glycan residues since its treatment with N-glycosidase F decreased its Mr to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phospho-compounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP >5'-AMP. The ACP also dephosphorylated 32P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C.burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells. (author)

  20. The maize (Zea mays ssp. mays var. B73 genome encodes 33 members of the purple acid phosphatase gene family

    Eliécer eGonzález Muñoz

    2015-05-01

    Full Text Available Purple acid phosphatases (PAPs play an important role in plant phosphorus nutrition, both by liberating phosphorus from organic sources in the soil and by modulating distribution within the plant throughout growth and development. Furthermore, members of the PAP protein family have been implicated in a broader role in plant mineral homeostasis, stress responses and development. We have identified 33 candidate PAP encoding gene models in the maize (Zea mays ssp. mays var. B73 reference genome. The maize Pap family includes a clear single-copy ortholog of the Arabidopsis gene AtPAP26, shown previously to encode both major intracellular and secreted acid phosphatase activities. Certain groups of PAPs present in Arabidopsis, however, are absent in maize, while the maize family contains a number of expansions, including a distinct radiation not present in Arabidopsis. Analysis of RNA-sequencing based transcriptome data revealed accumulation of maize Pap transcripts in multiple plant tissues at multiple stages of development, and increased accumulation of specific transcripts under low phosphorus availability. These data suggest the maize PAP family as a whole to have broad significance throughout the plant life cycle, while highlighting potential functional specialization of individual family members.

  1. Generic phosphatase activity detection using zinc mediated aggregation modulation of polypeptide-modified gold nanoparticles

    Selegård, Robert; Enander, Karin; Aili, Daniel

    2014-11-01

    A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme

  2. Tyrosine Phosphatase TpbA of Pseudomonas aeruginosa Controls Extracellular DNA via Cyclic Diguanylic Acid Concentrations

    Ueda, Akihiro; Wood, Thomas K.

    2010-01-01

    Inactivating the tyrosine phosphatase TpbA of Pseudomonas aeruginosa PA14 induces biofilm formation by 150-fold via increased production of the second messenger cyclic diguanylic acid (c-di-GMP). Here, we show the tpbA mutation reduces extracellular DNA (eDNA) and that increased expression of tpbA increases eDNA; hence, eDNA is inversely proportional to c-di-GMP concentrations. Mutations in diguanylate cyclases PA0169, PA4959, and PA5487 and phosphodiesterase PA4781 corroborate this trend. Th...

  3. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446.

    Masuda, Kouhei; Shima, Hiroshi; Katagiri, Chiaki; Kikuchi, Kunimi

    2003-08-22

    We previously showed that MKP-7 suppresses MAPK activation in COS-7 cells in the order of selectivity, JNK > p38 > ERK, but interacts with ERK as well as JNK and p38. In this study we found that, when expressed in COS-7 cells with HA-ERK2, the mobility of FLAG-MKP-7 was decreased on SDS-PAGE gels depending on several stimuli, including phorbol 12-myristate 13-acetate, fetal bovine serum, epidermal growth factor, H2O2, and ionomycin. By using U0126, a MEK inhibitor, and introducing several point mutations, we demonstrated that this upward mobility shift is because of phosphorylation and identified Ser-446 of MKP-7 as the phosphorylation site targeted by ERK activation. To determine how MKP-7 interacts with MAPKs, we identified three domains in MKP-7 required for interaction with MAPKs, namely, putative MAP kinase docking domains (D-domain) I and II and a long COOH-terminal stretch unique to MKP-7. The D-domain I is required for interaction with ERK and p38, whereas the D-domain II is required for interaction with JNK and p38, which is likely to be important for MKP-7 to suppress JNK and p38 activations. The COOH-terminal stretch of MKP-7 was shown to determine JNK preference for MKP-7 by masking MKP-7 activity toward p38 and is a domain bound by ERK. These data strongly suggested that Ser-446 of MKP-7 is phosphorylated by ERK. PMID:12794087

  4. Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

    Wang, Huanchen; Nair, Vasudha S; Holland, Ashley A; Capolicchio, Samanta; Jessen, Henning J; Johnson, Michael K; Shears, Stephen B

    2015-10-27

    Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity. PMID:26422458

  5. Association of acid phosphatase locus 1*C allele with the risk of cardiovascular events in rheumatoid arthritis patients

    Teruel, María; Martín, J. E.; González-Juanatey, C.; López-Mejias, Raquel; Miranda-Filloy, J. A.; Blanco, Ricardo; Balsa, A.; Pascual-Salcedo, Dora; Rodríguez-Rodríguez, Luis; Fernández-Gutiérrez, B.; Ortiz, A M; González-Alvaro, I.; Gómez-Vaquero, C.; Bottini, N.; Llorca, Javier

    2011-01-01

    Abstract Introduction Acid phosphatase locus 1 (ACP1) encodes a low molecular weight phosphotyrosine phosphatase implicated in a number of different biological functions in the cell. The aim of this study was to determine the contribution of ACP1 polymorphisms to susceptibility to rheumatoid arthritis (RA), as well as the potential contribution of these polymorphisms to the increased risk of cardiovascular disease (CV) observed in RA patients. Methods A set of 1,603 Spanish RA patients and 1,...

  6. 钾水平对巴西橡胶树幼苗磷组分和酸性磷酸酶活性的影响%Effects of Different Potassium Levels on Components of Phosphorus and Activities of the Acid Phosphatase in H.brasiliensis Seedlings

    吴敏; 何鹏; 韦家少; 吴炳孙

    2011-01-01

    [Objective] The aim was to study the effects of different potassium levels on components of phosphorus and activities of acid phosphatase in different organs of H. Brasiliensis seedling. [ Method] Seedlings derived from seeds of rubber trees of Clone RRIM600 ( H. Brasiliensis ) were grown under hydroponic culture at different levels of potassium (K2O concentration was 0, 1, 10, 50, 250 mg/L) to determine their components of phosphorus and the activities of the acid phosphatase. [ Result] The results indicated that the contents of the soluble phosphoruses and the ratios were decreased under the deficiency of the potassium nutrients, but increased the contents and the ratios of the insoluble phosphorus. The results of significance test of difference indicated that the contents of the soluble phosphorus which were the highest in roots, secondly in skins of the stem, thirdly in leaves were significantly different in different organs, but the contents of the insoluble phosphorus in the roots were significantly greater than the contents in the leaves and the skins of the stem, moreover the contents of the insoluble phosphorus were not significant different between the roots and the skins of the stem. The activities of the acid phosphatase were highly significantly different among the different organs, but oppositely in same organs under the different levels of potassium. [ Conclusion] The study reveals the components characters of phosphorus and activities change law of the acid phosphatase in different organs of H. Brasiliensis seedling under different potassium levels, can lay theoretic basis for reasonable application of potassium and phosphorus during the growth periods of H. Brasiliensis.%[目的]研究不同钾水平对巴西橡胶树(Hevea brasiliensis)幼苗各器官磷组分和酸性磷酸酶活性的影响.[方法]采用水培试验.试验材料为巴西橡胶树RRIM600种子实生幼苗,K2O浓度分别为0、1、10、50、250 mg/L.[结果]缺钾降低了橡

  7. PTEN Protein Phosphatase Activity Correlates with Control of Gene Expression and Invasion, a Tumor-Suppressing Phenotype, But Not with AKT Activity

    Tibarewal, P; Zilidis, G; Spinelli, L.; et al.

    2012-01-01

    The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has a well-characterized lipid phosphatase activity and a poorly characterized protein phosphatase activity. We show that both activities are required for PTEN to inhibit cellular invasion and to mediate most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at threonine 366, and mutation of this site makes lipid phosphatase activity sufficient for PTEN to inhibit invasion. We p...

  8. Protein kinase and phosphatase activities of thylakoid membranes

    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 Mg2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg2+ 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

  9. Application of intracellular alkaline phosphatase activity measurement in detection of neutrophil adherence in vitro.

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (10(4)-10(6)). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

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

    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

  11. Cloning and Characterization of a Novel Purple Acid Phosphatase Gene (MtPAP1) from Medicago truncatula Barrel Medic

    2006-01-01

    A novel purple acid phosphatase gene (MtPAP1) was isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp in length with an open reading frame (ORF) of 1 398 bp capable of encoding an N-terminal signal peptide of 23 amino acids. The transcripts of MtPAP1 were mainly detected in leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced in leaves and increased in roots, with the strongest hybridization signal detected in roots. A chimeric gene construct fusing MtPAP1 and GFPwas made in which the fusion was driven by the CaMV35S promoter. Transgenlc Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal microscopic analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptide at the N-terminal. The coding region of MtPAP1 without signal peptide was inserted into the prokaryotic expression vector pET-30a (+) and overexpressed in Escherlchia coll BL21(DE3). The acid phosphatase (APase) proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An enzyme activity assay demonstrated that the APase activity in the transformed bacteria was 3.16-fold higher than that of control. The results imply that MtPAP1 functions to improve phosphorus acquisition in plants under conditions of phosphorus (P) stress.

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

    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.

  13. Benomyl inhibits phosphorus transport but not fungal alkaline phosphatase activity in a Glomus–cucumber symbiosis

    Larsen, John; Thingstrup, Ida; Jakobsen, Iver;

    1996-01-01

    Short-term effects of benomyl on the arbuscular mycorrhizal fungus Glomus caledonium (Nicol. & Gerd.) Trappe and Gerdeman associated with Cucumis sativus L. were studied by measuring effects on fungal P transport and on fungal alkaline phosphatase activity. Mycorrhizal plants were grown in three...... when benomyl was applied to the HC at 10 µg g-1 soil, whereas the uptake of 32P from RHC I roots + hyphae) was reduced only at the highest dose of application to the RHC (100 µ g g-1 soil). In contrast to the marked reduction of benomyl on fungal P transport, the activity of fungal alkaline phosphatase...

  14. Phosphatase activity in the rhizosphere and root of mycorrhizal teak seedlings with three levels of NPK fertilization

    CORRYANTI; J SOEDARSONO; B RADJAGUKGUK; S M WIDYASTUTI

    2007-01-01

    To examine the phosphatase alkaline activity of VA mycorrhizal fungi in the rizhosphere and in root, teak seedlings inoculated spores of VA mycorrhizal fungi were grown in sterilized soils. Teak seedlings were fertilized with NPK fertilizer consisting three levels, i.e. 0; 0.0625; 0.125 g per seedling. Phosphatase alkaline in rizhosphere was measured in terms of pNP on soil dry weight basis, meanwhile alkaline phosphatase activity in roots were quantified in using method developed by Tissera...

  15. Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp.

    Allan, Victoria J M; Callow, Maureen E; Macaskie, Lynne E; Paterson-Beedle, Marion

    2002-01-01

    A phosphatase-overproducing Citrobacter sp. (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen. Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation. However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition. Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface. PMID:11782520

  16. Adsorption of Acid Phosphatase on Minerals and Soil Colloids in Presence of Citrate and Phosphate

    2002-01-01

    The aim of this work was to study the influence of phosphate and citrate, which are common inorganic andorganic anions in soils, on the adsorption of acid phosphatase by kaolin, goethite and the colloids separatedfrom yellow-brown soil (YBS) and latosol (LS) in central-south China. The YBS colloid has the major claymineral composition of 1.4 nm mineral, illite and kaolinite while the LS colloid mainly contains kaolinite andoxides. The adsorption isotherm of acid phosphatase on the examined soil colloids and minerals fitted tothe Langmuir model. The amount of enzyme adsorbed in the absence of ligands was in the order of YBScolloid >LS colloid>kaolin≈goethite. In the presence of phosphate or citrate, the amounts of the enzymeadsorbed followed the sequence YBS colloid>kaolin>LS colloid>goethite. The presence of ligands alsodecreased the binding energy between the enzyme and soil colloids or minerals. With the increase of ligandconcentration from 10 mmol L-1 to 400 m mol L-1, different behaviors for the adsorption of enzyme werefound in the colloid and mineral systems studied. A sharp decrease in enzyme adsorption was observed ongoethite while gradual decreases of enzyme adsorption were recorded in the two soil colloid systems. However,no any decrease was found for the amount of enzyme adsorbed on kaolin at higher ligand concentrations.When phosphate or citrate was introduced to the system before the addition of enzyme, the ligands usuallyenhanced the adsorption of enzyme. The results obtained in this study suggested the important role ofkaolinite mineral in the adsorption of enzyme molecules in acidic soils in the presence of various ligands.

  17. Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38

    Lasa, Marina; Abraham, Sonya M.; Boucheron, Christine; Saklatvala, Jeremy; Clark, Andrew R.

    2002-01-01

    The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibiti...

  18. Control of Sty1 MAPK activity through stabilisation of the Pyp2 MAPK phosphatase.

    Kowalczyk, Katarzyna M; Hartmuth, Sonya; Perera, David; Stansfield, Peter; Petersen, Janni

    2013-08-01

    In all eukaryotes tight control of mitogen-activated protein kinase (MAPK) activity plays an important role in modulating intracellular signalling in response to changing environments. The fission yeast MAPK Sty1 (also known as Spc1 or Phh1) is highly activated in response to a variety of external stresses. To avoid segregation of damaged organelles or chromosomes, strong Sty1 activation transiently blocks mitosis and cell division until such stresses have been dealt with. MAPK phosphatases dephosphorylate Sty1 to reduce kinase activity. Therefore, tight control of MAPK phosphatases is central for stress adaptation and for cell division to resume. In contrast to Pyp1, the fission yeast Pyp2 MAPK phosphatase is under environmental control. Pyp2 has a unique sequence (the linker region) between the catalytic domain and the N-terminal MAPK-binding site. Here we show that the Pyp2 linker region is a destabilisation domain. Furthermore, the linker region is highly phosphorylated to increase Pyp2 protein stability and this phosphorylation is Sty1 dependent. Our data suggests that Sty1 activation promotes Pyp2 phosphorylation to increase the stability of the phosphatase. This MAPK-dependent Pyp2 stabilisation allows cells to attenuate MAPK signalling and resume cell division, once stresses have been dealt with. PMID:23690545

  19. Optimization of the chloramine T method for radioiodination of prostate gland by acid phosphatase

    Optimal conditions for preparation of [125I]-labelled human prostatic acid phosphatase (PAP) by chloramin T method were developed which make it possible to increase the radiochemical yield and immunoreactivity of labelled PAP. The influence of buffer system, pH of reaction mixture and molarity of borate buffer was investigated. Iodination in 0.2 M borate buffer, pH 8.0 make it possible to prepare [125I]-PAP with 90-98 immunoreactivity. Under storage of labelled PAP during 2 month immunoreactivity is decreased only by 10%. On the base of prepared 125I]-PAP high-sensitive competitive radioimmunoassay of PAP in human serum have developed

  20. Radioimmunological and enzymatic assay for prostatic acid phosphatase (PAP) in prostatic cancer

    Serum prostatic acid phosphatase (PAP) level was determined by radioimmunoassay (RIA) in 272 patients. For comparative purposes PAP was also measured by an enzymatic assay. In prostate adenocarcinoma 55% of the values were elevated. In early stages (A and B) 17% of patients were found to be positive; at later stages (C and D) the percentage increased to 78%. The enzymatic method yielded 46% positive values in these patients: 17% in the former group (A+B), and 64% in the latter one (C+D). False positive values were observed in 10% of the patients with prostate adenoma, and 22% of the patients with prostatis. The data confirm the low sensitivity of RIA (and enzymatic assay) for detecting early intracapsular disease. RIA determination of PAP is a good diagnostic tool for advanced cancer of the prostate. (author)

  1. Phytase, Phosphatase Activity and P-Nutrition of Soybean as Influenced by Inoculation of Bacillus

    A. Ramesh; Sushil K. Sharma; Joshi, O. P.; Khan, I. R.

    2011-01-01

    The efficiency of different Bacillus isolates on rhizosphere soil enzyme activities and P-nutrition of soybean was carried out under microcosm conditions. Significant increase in enzyme activities viz., fluorescein diacetate activity, phosphatase and phytase activity and consequent effects on P-nutrition were observed with the inoculation of Bacillus isolates over uninoculated control. Among the isolates, BD-3-1B, KHBD-6, BDKH-3, Bacillus amyloliquefacians, and Bacillus cereus were found to b...

  2. Analysis of the contribution of acid phosphatase to P efficiency in Brassica napus under low phosphorus conditions

    2010-01-01

    To understand whether genotypic variation in acid phosphatase (APase) activity in rapeseed (Brassica napus L.) induced by phosphorus (P) deficiency has impact on P efficiency,soil APase activity in the rhizosphere for rapeseed P-efficient genotype 102 and P-inefficient genotype 105 was measured against organic and inorganic P sources in the pot experiment,and the activities of root-secreted APase and leaf intracellular APase were investigated in different P-starvation periods in the nutrient solution.Higher activity of root-secreted APase in B.napus was induced under low P conditions.However,P nutrition and P uptake efficiency of the plants supplied with organic P were not directly related to the activity of root-secreted APase due to several confounding factors affecting APase availability.The higher activity of leaf APase improved P remobilization in plants and played important roles in enhancing P use efficiency,shown by the significant correlation between leaf APase activity and P use efficiency in a rapeseed recombinant inbred population of 135 lines.

  3. A study of acid phosphatase locus 1 in women with high fat content and normal body mass index.

    De Lorenzo, Antonino; Di Renzo, Laura; Puja, Alberto; Saccucci, Patrizia; Gloria-Bottini, Fulvia; Bottini, Egidio

    2009-03-01

    De Lorenzo and coworkers have recently described a class of women with normal body mass index (BMI) and high fat content (normal weight obese syndrome [NWO]). This observation prompted us to study the possible role of acid phosphatase locus 1 (ACP(1)) in the differentiation of this special class of obese subjects. Acid phosphatase locus 1 is a polymorphic gene associated with severe obesity and with total cholesterol and triglycerides levels. The enzyme is composed by 2 isoforms--F and S--that have different biochemical properties and probably different functions. The sample study was composed of 130 white women from the population of Rome. Total fat mass and percentage of fat mass were measured by dual-energy x-ray absorptiometry. Thirty-six women had a BMI less than 25 and percentage of fat mass greater than 30 (high fat, normal BMI [HFHB]), and 94 women showed a BMI greater than 25 and a percentage of fat mass greater than 30 (high fat, high BMI [HFHB]). In the whole sample, the proportion of low-activity ACP(1) genotypes (*A/*A and *B/*A) was higher than in controls. However, whereas HFNB showed a very high frequency of ACP(1) *A/*A genotype, high-fat, high-BMI women showed an increase of *B/*A genotype. These 2 genotypes differ in the concentration of F isoform and the F/S ratio, which are lower in ACP(1)*A/*A genotype than in ACP(1)*B/*A genotype. The genetic differentiation of the class of women with normal BMI and high fat content from the class showing a concordant level of the 2 parameters supports the hypothesis that HFNB class represents a special cluster of obese subjects not revealed by BMI evaluation. Because ACP(1) is present in adipocytes, the present observation suggests that F isoform may have a specific role in the regulation of quantity of adipose tissue. PMID:19217450

  4. Alkaline phosphatase activity and some minerals concentration in canine blood plasma with radiation combined injuries

    Alkaline phosphatase activity, Ca, P and Mg concentration were investigated in the blood plasma of dogs with a bone fracture as well as in those ones with a bone fracture combined with irradiation (2Gy). Obtained results show that none of the investigated parameters were significantly changed during the experiment. (author) 6 refs.; 1 tab

  5. Phosphatase activities in soil after repeated untreated and alum-treated poultry litter applications

    Repeated additions of untreated and aluminum sulfate (alum)-treated poultry litter to soil affect ecology and consequent nutrient dynamics. The objective of this study was to determine how repeated annual poultry litter additions affected phosphatase activities in concert with changes in soil phosph...

  6. Serum proteins, trace metals and phosphatases in psoriasis

    Bhatnagar M

    1994-01-01

    Full Text Available Serum proteins, zinc, copper, acid phosphatase (AcPase and alkaline phosphatase (AlPase were studied in both active and remission phases of psoriasis. Data were compared with healthy controls, ?1, ? and ? globulins were high in active phase while ?1 and ? globulins were at par in remission phase. Serum copper was low but zinc and alkaline phosphatase were significantly high in both active and remission phases of the disease. Acid phosphatase level was at par in all the experimental groups. Study suggests a positive correlation of globulin, zinc and Alpase in active and remission phase of psoriasis.

  7. Fósforo da biomassa microbiana e atividade de fosfatases ácidas durante a diminuição do fósforo disponível no solo Soil microbial biomass phosphorus and activity of acid phosphatases during decline of soil available phosphorus

    Luciano Colpo Gatiboni

    2008-08-01

    Full Text Available O objetivo deste trabalho foi avaliar o conteúdo de fósforo armazenado na biomassa microbiana e a atividade de fosfatases ácidas, durante a diminuição dos teores de fósforo disponível no solo, causado por cultivos sucessivos com plantas. Foram utilizadas amostras de Latossolo Vermelho distroférrico típico, com adição prévia de fosfatos solúveis (0, 180, 360, 540 e 720 kg ha-1 de P2O5, aplicados em seis anos consecutivos. Efetuaram-se 15 cultivos sucessivos com diferentes plantas, em casa de vegetação, sem a reposição do fósforo absorvido pelas plantas. Após cada três cultivos sucessivos, foram determinados: o teor de fósforo disponível por resina trocadora de ânions, o fósforo microbiano e a atividade de fosfatases ácidas. Com a diminuição da disponibilidade de fósforo do solo, a quantidade de fósforo armazenada na biomassa microbiana do solo diminuiu, e a atividade de fosfatases ácidas aumentou. Em solos com baixo teor de fósforo e de resíduos de plantas, o P microbiano tem pouca importância para a nutrição das plantas.The objective of this work was to evaluate the content of phosphorus stored in the soil microbial biomass and the activity of acid fosfatases, during the decline of soil available phosphorus, caused by successive crops in pot experiment. Samples of Oxisol were utilized with previous addition of soluble phosphates (0, 180, 360, 540, and 720 kg ha-1 of P2O5, applied in six consecutive years. The soil samples were submitted to 15 successive crops in greenhouse, without replacement of absorbed phosphorus by plants. After each three successive crops, soil was sampled, and the following variables were determined: the available phosphorus by anion exchange resin, phosphorus stored in the soil microbial biomass and the activity of acid phosphatases. As a consequence of the reduction of the soil available phosphorus, the amount of microbial phosphorus decreased, and the activity of phosphatases increased

  8. Phosphatase activity in the rhizosphere and root of mycorrhizal teak seedlings with three levels of NPK fertilization

    CORRYANTI

    2007-07-01

    Full Text Available To examine the phosphatase alkaline activity of VA mycorrhizal fungi in the rizhosphere and in root, teak seedlings inoculated spores of VA mycorrhizal fungi were grown in sterilized soils. Teak seedlings were fertilized with NPK fertilizer consisting three levels, i.e. 0; 0.0625; 0.125 g per seedling. Phosphatase alkaline in rizhosphere was measured in terms of pNP on soil dry weight basis, meanwhile alkaline phosphatase activity in roots were quantified in using method developed by Tisserant. The results showed that alkaline phosphatase activity increased on inoculated seedlings compare to with uninoculated. NPK fertilization of 0.0625 g per seedling and inoculation on teak seedlings showed alkaline phosphatase activity in range 90-201 EU, and in roots indicated in range 14-72%. Gigaspora sp inoculation on teak seedlings was showing the highest of alkaline phosphatase activity. Increasing phosphatase alkaline activity relevant to hyphae growth, and increasing of root infection decreased alkaline phosphatase activity. Arbuscular mycorrhizal inoculation increased seedling dry weight.

  9. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. PMID:27043172

  10. Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

    De Leon, Gabriel; Sherry, Tara C; Krucher, Nancy A

    2008-06-01

    There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. PMID:18360108

  11. 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

    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

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

    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 32Pi-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 (r2 = 0.87**) between extra and intra-cellular ACPase activity was observed. In terms of kinetic parameters, it was observed that 32Pi uptake and Imax values were significantly higher in rye while Cmin and Km 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)

  13. Assessment and kinetics of soil phosphatase in Brazilian Savanna systems.

    Ferreira, Adão S; Espíndola, Suéllen P; Campos, Maria Rita C

    2016-05-31

    The activity and kinetics of soil phosphatases are important indicators to evaluate soil quality in specific sites such as the Cerrado (Brazilian Savanna). This study aimed to determine the activity and kinetic parameters of soil phosphatase in Cerrado systems. Soil phosphatase activity was assessed in samples of native Cerrado (NC), no-tillage (NT), conventional tillage (CT) and pasture with Brachiaria brizantha (PBb) and evaluated with acetate buffer (AB), tris-HCl buffer (TB), modified universal buffer (MUB) and low MUB. The Michaelis-Menten equation and Eadie-Hofstee model were applied to obtain the kinetic parameters of soil phosphatase using different concentrations of p-nitrophenol phosphate (p-NPP). MUB showed the lowest soil phosphatase activity in all soils whereas AB in NC and NT presented the highest. Low MUB decreased interferences in the assessment of soil phosphatase activity when compared to MUB, suggesting that organic acids interfere on the soil phosphatase activity. In NC and NT, soil phosphatase activity performed with TB was similar to AB and low MUB. Km values from the Michaels-Menten equation were higher in NC than in NT, which indicate a lower affinity of phosphatase activity for the substrate in NC. Vmax values were also higher in NC than in NT. The Eadie-Hofstee model suggests that NC had more phosphatase isoforms than NT. The study showed that buffer type is of fundamental importance when assessing soil phosphatase activity in Cerrado soils. PMID:27254453

  14. Comparison of Activities of Amylase, Esterase, Acid Phosphatase in Ginseng Radix et Rhizoma from Different Origins and Different Growth Periods%不同产地、不同年限人参中淀粉酶、酯酶、酸性磷酸酯酶的活力比较

    邢楠楠; 赵雨; 刘宏; 张惠; 李红艳

    2011-01-01

    目的 对不同产地、不同生长年限人参进行淀粉酶(AMY)、酯酶(Est)和酸性磷酸酯酶(ACP)活力比较.方法 用中性缓冲溶液提取总蛋白,应用3,5-二硝基水杨酸比色法测定AMY活力;乙酸-α-萘酯比色法测定Est活力;对硝基酚比色法测定ACP活力.结果 不同产地人参AMY、Est、ACP活力均存在很大差异.取不同产地、相同生长年限酶活力平均值进行不同年限的酶活力比较,可知4年与5年生人参3种水解酶活力均无明显差异.结论 AMY、Est、ACP活力可以作为评价人参质量的内在指标之一.%OBJECTIVE To compare activities of Ginseng Radix et Rhizoma amylase(AMY), esterase (Est) and acid phosphatase (ACP) from different origins and different growth periods. METHODS Total protein was extracted using a neutral buffer solution. Determination of AMY activity applied 3,5-dinitrosalicylic acid colorimetry. Determination of Est activity was using acetic acid-α-naphthyl ester colorimetry. Determination of ACP activity was using p-nitrophenol colorimetry. RESULTS There was a significant difference in activities of AMY, Est and ACP in Ginseng Radix et Rhizoma from different origins. Take the average value for enzyme activity in Ginseng Radix et Rhizoma of different origin and the same growth period, purpose was to compare the enzyme activity at different ages. We can know that activities of three kinds of enzyme hydrolysis were not significantly different in Ginseng Radix et Rhizoma from 4 years old and 5 years old. CONCLUSION Activities of AMY, Est,ACP can be used as one of the intrinsic indicators to assess the quality of Ginseng Radix et Rhizoma.

  15. Dual 4- and 5-phosphatase activities regulate SopB-dependent phosphoinositide dynamics to promote bacterial entry.

    Piscatelli, Heather L; Li, Menghan; Zhou, Daoguo

    2016-05-01

    Salmonella are able to invade non-phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4-phosphatase and 5-phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4-phosphatase activity of SopB is required for PtdIns-3-phosphate (PtdIns(3)P) accumulation and SopB-mediated invasion. We show here that both the 4-phosphatase as well as the 5-phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5-phosphatase activity of SopB is likely responsible for generating PtdIns-3,4-bisphosphate (PtdIns(3,4)P2 ) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4-phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P2 into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities led to SNX9-mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4- and 5-phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry. PMID:26537021

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

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

    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...

  17. Surface alkaline phosphatase activities of macroalgae on coral reefs of the central Great Barrier Reef, Australia

    Schaffelke, B.

    2001-05-01

    Inshore reefs of the Great Barrier Reef (GBR) are subject to episodic nutrient supply, mainly by flood events, whereas midshelf reefs have a more consistent low nutrient availability. Alkaline phosphatase activity (APA) enables macroalgae to increase their phosphorus (P) supply by using organic P. APA was high (~4.0 to 15.5 µmol PO4 3- g DW-1 h-1) in species colonising predominantly inshore reefs and low (human activity, which currently is a global problem.

  18. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Anoushka Davé; Carol Cooley; Mansi Garg; Alessandro Bianchi

    2014-01-01

    Summary The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early re...

  19. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

  20. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    Becq, F; Jensen, T J; Chang, X B; Savoia, A.; Rommens, J M; Tsui, L C; Buchwald, M; Riordan, J R; Hanrahan, J W

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epi...

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

    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 (S...

  2. Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

    Wiese, M; Ilg, T; Lottspeich, F; Overath, P

    1995-03-15

    The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, lmsap1 and lmsap2, were cloned and sequenced. lmsap1 predicts a protein with features characteristic of acid phosphatases and a remarkable serine- and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsap1 encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase. PMID:7720697

  3. Association of Protein Phosphatase 1γ1 with Spinophilin Suppresses Phosphatase Activity in a Parkinson Disease Model*

    Brown, Abigail M.; Baucum, Anthony J.; Bass, Martha A.; Roger J Colbran

    2008-01-01

    Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr286 phosphorylation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks...

  4. The content of macro- and microelements and the phosphatase activity of soils under a varied plant cultivation technology

    Bartkowiak, A.; Lemanowicz, J.; Kobierski, M.

    2015-12-01

    The paper presents the results of the analyses of selected physicochemical properties and the activity of alkaline and acid phosphatase in the soils which differed in terms of plant cultivation technology. Profile sI represented arable land in the crop rotation with cereals dominating (medium intensive technology), without irrigation, while profile sII—represented arable land with vegetable crops cultivation (intensive technology), intensively fertilized and irrigated. The content of available phosphorus in the two soil profiles investigated ranged from 6.6 to 69.1 mg/kg. The highest contents of phosphorus available to plants were reported in the plough horizon of both soils, while the abundance of potassium and magnesium was highest in the illuvial horizon of both soils. The soil profiles investigated showed a significant variation in terms of the cultivation technologies applied. The contents of plant-available Cu and Zn in soil were low and they resulted in the inhibition of neither alkaline nor acid phosphatase. The intensive vegetable crops cultivation technology decreased the content of organic matter and increased the content of the nutrients in soil. Using the Ward method, it was found that relatively similar physicochemical and chemical properties were reported for the genetic horizons of both soil profiles, especially Ap horizon of the soil representing arable land with intensive cultivation of vegetable crops.

  5. CD45 Phosphatase Inhibits STAT3 Transcription Factor Activity in Myeloid Cells and Promotes Tumor-Associated Macrophage Differentiation.

    Kumar, Vinit; Cheng, Pingyan; Condamine, Thomas; Mony, Sridevi; Languino, Lucia R; McCaffrey, Judith C; Hockstein, Neil; Guarino, Michael; Masters, Gregory; Penman, Emily; Denstman, Fred; Xu, Xiaowei; Altieri, Dario C; Du, Hong; Yan, Cong; Gabrilovich, Dmitry I

    2016-02-16

    Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy. PMID:26885857

  6. Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity.

    Hernández-Sotomayor, S M; Arteaga, C L; Soler, C. (Carlos); Carpenter, G

    1993-01-01

    This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphory...

  7. The Effect of Titanium Dioxide Nanoparticles on Salivary Alkaline Phosphatase Activity

    Eaman A. S. AL-Rubaee; Suha T. Abd; Nadia M. Kadim

    2015-01-01

    The structural and optical properties of the titanium dioxide nanoparticles [TiO2 NPs] have been investigated using [UV-Vis] spectrophotometer and SEM. The produced nanoparticles show small and about sharp round peaks around 220 nm. The produced nanoparticles have a spherical shape with an average particle size ˂50 nm. The effect of titanium dioxide NPs was studied on the activity of Alkaline Phosphatase [ALP] in the saliva of 25 patients with gingivitis in comparison to 20 healthy subjects w...

  8. Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

    Schmidt, A.; Rutledge, S J; Endo, N; Opas, E E; Tanaka, H; Wesolowski, G.; Leu, C T; Huang, Z; Ramachandaran, C; Rodan, S B; Rodan, G A

    1996-01-01

    Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot ...

  9. Regulation of glucose homeostasis by small RNA mediated activation of sugar phosphatase mRNA

    Papenfort, Kai; Sun, Yan; Miyakoshi, Masatoshi; Vanderpool, Carin K.; Vogel, Jörg

    2013-01-01

    Glucose homeostasis is strictly controlled in all domains of life. Bacteria that are unable to balance intracellular sugar levels and deal with potentially toxic phosphosugars cease growth and risk being outcompeted. Here, we identify the conserved haloacid dehalogenase (HAD)-like enzyme YigL as the previously hypothesized phosphatase for detoxification of phosphosugars, and reveal that its synthesis is activated by an Hfq dependent small RNA in Salmonella typhimurium. We show that the glucos...

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

    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.

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

    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.

  12. Fundamental studies of three radioimmunoassay kits measuring prostatic acid phosphatase (PAP) concentrations

    Measurement of prostatic acid phosphatase (PAP) concentration is useful for the diagnosis and treatment of prostatic cancer. Fundamental studies of measurement of PAP concentration by radioimmunoassay were performed and values determined by three commercially available kits were compared, those are, PAP EIKEN RIA Kit (E kit), RIA Quant P.A.P. (M kit) and GammaDab PAP RIA Kit (C kit). Upper limits of normal PAP concentration were 3 ng/ml by E and M kits and 2 ng/ml by C kits, respectively. The reproducibility and the recovery studies of three kits were satisfactory. However, dilution curve of some patients was not strait. PAP concentration of 27 patients measured by three kits were correlated well to each other, but the discrepancy of values was noticed in high PAP concentrations. PAP concentration measured by RIA is more reliable than that by enzyme immunoassay. When keeping serum samples, the effect of time, temperature and freeze and thawing on PAP values was obvious. It is recommended that serum is separated at 40C as soon as possible after collecting blood and kept frozen until use. (author)

  13. 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

    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 pl

  14. Phosphatase activity and organic phosphorus turnover on a high Arctic glacier

    M. Stibal

    2009-05-01

    Full Text Available Arctic glacier surfaces harbour abundant microbial communities consisting mainly of heterotrophic and photoautotrophic bacteria. The microbes must cope with low concentrations of nutrients and with the fact that both the dissolved and debris-bound nutrient pools are dominated by organic phases. Here we provide evidence that phosphorus (P is deficient in the supraglacial environment on a Svalbard glacier, we quantify the enzymatic activity of phosphatases in the system and we estimate the contribution of the microbes to the cycling of the dominant organic P in the supraglacial environment. Incubation of cryoconite debris revealed significant phosphatase activity in the samples (19–67 nmol MUP g−1 h−1. It was inhibited by inorganic P during incubations and had its optimum at around 30°C. The phosphatase activity measured at near-in situ temperature and substrate concentration suggests that the available dissolved organic P can be turned over by microbes within ~3–11 h on the glacier surface. By contrast, the amount of potentially bioavailable debris-bound organic P is sufficient for a whole ablation season. However, it is apparent that some of this potentially bioavailable debris-bound P is not accessible to the microbes.

  15. The effects of Zinc supplementation on serum zinc, alkaline phosphatase activity and fracture healing of bones

    Objective was to determine the effect of zinc supplementation on callus information, serum zinc and alkaline phosphatase activity in humans. This randomized, double-blind, placebo controlled clinical trial was conducted on 60 patients with traumatic bone fracture referred to Shohada Hospital of Tabriz, Iran from August to December 2007. Subjects were randomly divided into 2 groups: cases (n=30), receiving one capsule of zinc sulfate consists of 50 mg zinc each day and the controls (n=30), receiving placebo for 60 days. Individual and clinical information was determined by a questionnaire: nutritional intake by 3 days food records at the beginning and the end of trial. Serum zinc and alkaline phosphatase was measured by atomic absorption spectroscopy and by enzymatic method. Callus information during fracture healing was evaluated by radiography of the bone. There was no significant difference in physical activity, gender, age, type of fractures and nutrient intake, between the 2 groups. The administration of zinc caused a significant elevation of serum zinc and alkaline phosphatase activity. Assessment of bone x-rays showed a significant progress in callus formation in cases compared to the controls. This study shows that zinc supplementation can stimulate fracture healing, however, it needs further study. (author)

  16. The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis

    Johnson, Kim L.; Ramm, Sascha; Kappel, Christian; Ward, Sally; Leyser, Ottoline; Sakamoto, Tomoaki; Kurata, Tetsuya; Bevan, Michael W.; Lenhard, Michael

    2015-01-01

    Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that c...

  17. Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

    DONG Hong-bo; LI Guo-dong; WANG Shao-feng; FU Xue-qi; ZHAO Zhi-zhuang Joe

    2011-01-01

    Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

  18. Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

    Swingle, M. R.; Honkanen, R.; Ciszak, E.

    2004-01-01

    Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

  19. Tartrate resistant acid phosphatase 5a : a potential regulator of adipocyte cell number and differentiation in white adipose tissue

    Patlaka, Christina

    2015-01-01

    Tartrate- resistant acid phosphatase (TRAP) exists in two isoforms, TRAP 5a which is monomeric and TRAP 5b which is a dimer generated by proteolytic cleavage of TRAP 5a, that exhibit different functions and localizations. TRAP 5a is expressed by adipose tissue macrophages and secreted into the extracellular environment and has been shown to lead to hyperplastic insulin- sensitive obesity when over-expressed in mice. In bone, TRAP is suggested to interact with the heparan sulfat...

  20. Biomineralization of uranium by PhoY phosphatase activity aids cell survival in Caulobacter crescentus.

    Yung, Mimi C; Jiao, Yongqin

    2014-08-01

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria. PMID:24878600

  1. Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

    Yung, M C [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jiao, Y [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-07-22

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.

  2. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  3. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

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

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  4. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  5. Effect of phosphoric fertilizer and starter rates of nitrogen fertilizers on the phosphatase activity in the rhizosphere soil and nonlignified soybean roots under drought conditions

    Emnova, E. E.; Daraban, O. V.; Bizgan, I. V.; Toma, S. I.

    2014-02-01

    In a small-plot field experiment, two soybean ( Glycine max L.) cultivars were grown on a calcareous chernozem under the drought conditions of 2012 with the preplanting application of simple superphosphate (Ps) at 60 kg/ha, urea (Nu) at 10 and 20 kg/ha, and ammonium nitrate (Nan) at 20 kg/ha. The phosphatase activity was measured in the rhizosphere soil (0- to 20-cm layer) and the fine nonlignified roots of soybean plants at the blossoming and pod-formation stages (the soil water content was 19 and 33% of the total water capacity, respectively). The maximum content of available phosphorus in the rhizosphere of both soybean cultivars (4.3-4.8 mg/100 g dry soil) was found at the simultaneous application of Ps and Nu20. Higher activities of the predominant phosphatases (alkaline phosphatase in the rhizosphere and acid phosphatase in the roots) were observed in the root-inhabited zone of the soil under the Indra cultivar compared to the Aura cultivar, which correlated with the lower content of available phosphorus in the rhizosphere soil (especially at the simultaneous application of Ps and Nu20) and the higher productivity of this cultivar in this treatment.

  6. Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase.

    Du, Juan; Say, Rafael F; Lü, Wei; Fuchs, Georg; Einsle, Oliver

    2011-10-27

    Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3 Å. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality. PMID:21983965

  7. Mitogen-activated Protein Kinase Phosphatase (Mkp)-1 Protects Mice against Acetaminophen-induced Hepatic Injury

    Wancket, Lyn M.; Meng, Xiaomei; Rogers, Lynette K.; Liu, Yusen

    2012-01-01

    c-Jun N-terminal kinase (JNK) activation promotes hepatocyte death during acetaminophen overdose, a common cause of drug-induced liver failure. While mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1 is a critical negative regulator of JNK MAPK, little is known about the role of Mkp-1 during hepatotoxicity. In this study, we evaluated the role of Mkp-1 during acute acetaminophen toxicity. Mkp-1+/+ and Mkp-1−/− mice were dosed ip with vehicle or acetaminophen at 300 mg/kg (for mechan...

  8. Effect of Combined Heavy Metal Pollution on Nitrogen Mineralization Potential,Urease and Phosphatase Activities in a Typic Udic Ferrisol

    ZHENGCHUNRONG; TUCONG; 等

    1999-01-01

    Individual and combined effects of Cu,Pb,Zn and Cd on N mineralization,urease and phosphatase were examined in a Typic Udic Ferrisol in laboratory by employing and uniform design and a single factor design,Soil pollution caused by heavy metals inhibited N mineralization (N0 value)and urease and phosphatase activities.The combined pollution of metals alleviated their toxicity to N mineralization to some extent whereas aggravated the toxicity to urease and phosphatase.Phosphorous application could mitigat the toxic effect of heavy metals on phosphatase activities,while alleviating effect of N application on the toxicity of heavy metals to urease was inconsistent.However,the mitigating effect of the fertilizers was limited in heavily polluted soils.

  9. Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

    Palma, E.; Ragozzino, D. A.; Di Angelantonio, S.; Spinelli, G.; Trettel, F.; Martinez-Torres, A.; Torchia, G.; Arcella, A.; Di Gennaro, G.; Quarato, P. P.; Esposito, V.; Cantore, G.; Miledi, R.; Eusebi, F.

    2004-01-01

    The properties of γ-aminobutyric acid (GABA) type A receptors (GABAA receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GABAA receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat α1β2γ2-GABA receptors exhibited a much less pronounced GABA-current run-down. Moreover, the GABAA receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GABAA receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GABAA-receptor β1, β2, β3, and γ2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GABAA receptors. Blockage of phosphatases stabilizes the TLE GABAA receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. PMID:15218107

  10. Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

    Adams, S P; Laws, G M; Selden, J R; Nichols, W W

    1994-05-15

    Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling. PMID:8059938

  11. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.;

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

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

    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...... precipitation leached through the soil, or indoor at constant moisture) with or without 9% (w/w) chopped wheat straw plus mineral N. Then the soils were partially sterilized and placed in two-compartment pots where mycorrhizal or non-mycorrhizal cucumber plants were grown in one root compartment (RC), and soils...

  13. Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

    Yung, Mimi C.; Jiao, Yongqin

    2014-01-01

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the ...

  14. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  15. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  16. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Anoushka Davé

    2014-04-01

    Full Text Available The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

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

    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.

  18. Identification of Genes Required for Secretion of the Francisella Oxidative Burst-Inhibiting Acid Phosphatase AcpA.

    Hoang, Ky Van; Chen, Carolyn G; Koopman, Jacob; Moshiri, Jasmine; Adcox, Haley E; Gunn, John S

    2016-01-01

    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. PMID:27199935

  19. Acid phosphatase-1 1, a molecular marker tightly linked to root-knot nematode resistance in tomato.

    Aarts, J.M.M.J.G.

    1993-01-01

    Root knot nematode resistance in tomato is a genetic trait which is determined by a single dominant gene ( Mi ) on chromosome 6 of tomato. Information about the mRNA or protein product is completely lacking, which precludes the cloning of Mi by conventional strategies based on gene expression. However, an acid phosphatase-1 allozyme marker ( Aps-1 1 ) is known, which shows tight genetic linkage to the root knot nematode resistance trait. With a view to isolating Mi nucleotide sequences by a p...

  20. Ionizing Groups at the Active Site of the Alkaline Phosphatase from Ostrea cucullate

    CHEN Qiao; WANG Qin; LIAO Jinhua; CHEN Qingxi

    2006-01-01

    The ionizing groups at the active site of alkaline phosphatase (ALP, EC 3.1.3.1) from Ostrea cucullate were studied using kinetic methods. The ionization constant, pKe, of the ionizing groups at the active site of the enzyme was found to be 10.11 at 37.0℃ and the organic solvent, dioxin, had no effect on the pKe. The standard dissociation enthalpy (△Ho) was determined to be 10.57 kcal/mol (1 cal=4.18 J). The results show that the dissociation group of the enzyme active center is the з-NH3+ of lysine. Chemical modification of the enzyme by acetic anhydride and succinic anhydride demonstrates that the amino group is one of the enzyme's functional groups.

  1. Alkaline phosphatase activity and the phosphorus mineralization rate of Lake Taihu

    2006-01-01

    The phosphorus fractions, the alkaline phosphatase activity (APA) and other water chemical parameters were concomitantly monitored from April 2003 to October 2004 in different ecotype sites of Lake Taihu. During the stages of algae growth, the phosphorus fractions and their relationships with APA in different ecotype sites were discussed and the phosphorus mineralization rate was calculated. In the water of Lake Taihu, most of the phosphorus (70.2%) could be attributed to the suspended particulate phosphorus, while the dissolved reactive phosphorus (DRP) seems to contribute less than 7%. About 58% of the total phosphorus, however, can be hydrolyzed as inorganic phosphate to compensate for phosphorus deficiency of algae and bacteria growth. During the different algae growth stages, the APA and its Kinetic parameters were varied significantly between different ecotype sites of Lake Taihu. This trend is also visible by comparing the phosphorus mineralization rate,and the most rapidly phosphorus turnover time is only several minutes. The fast recycle of phosphorus can, to some extent, be explained that the phosphorus source of algal blooms. The phytoplankton seems to compensate for phosphorus deficiency by using the alkaline phosphatase to hydrolyze phosphomonoesters.

  2. Procyanidins Negatively Affect the Activity of the Phosphatases of Regenerating Liver.

    Sven Stadlbauer

    Full Text Available Natural polyphenols like oligomeric catechins (procyanidins derived from green tea and herbal medicines are interesting compounds for pharmaceutical research due to their ability to protect against carcinogenesis in animal models. It is nevertheless still unclear how intracellular pathways are modulated by polyphenols. Monomeric polyphenols were shown to affect the activity of some protein phosphatases (PPs. The three phosphatases of regenerating liver (PRLs are close relatives and promising therapeutic targets in cancer. In the present study we show that several procyanidins inhibit the activity of all three members of the PRL family in the low micromolar range, whereas monomeric epicatechins show weak inhibitory activity. Increasing the number of catechin units in procyanidins to more than three does not further enhance the potency. Remarkably, the tested procyanidins showed selectivity in vitro when compared to other PPs, and over 10-fold selectivity toward PRL-1 over PRL-2 and PRL-3. As PRL overexpression induces cell migration compared to control cells, the effect of procyanidins on this phenotype was studied. Treatment with procyanidin C2 led to a decrease in cell migration of PRL-1- and PRL-3-overexpressing cells, suggesting the compound-dependent inhibition of PRL-promoted cell migration. Treatment with procyanidin B3 led to selective suppression of PRL-1 overexpressing cells, thereby corroborating the selectivity toward PRL-1- over PRL-3 in vitro. Together, our results show that procyanidins negatively affect PRL activity, suggesting that PRLs could be targets in the polypharmacology of natural polyphenols. Furthermore, they are interesting candidates for the development of PRL-1 inhibitors due to their low cellular toxicity and the selectivity within the PRL family.

  3. Increased tartrate-resistant acid phosphatase (TRAP) expression in malignant breast, ovarian and melanoma tissue: an investigational study

    Tartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies. TRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells. A marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines. Overexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies

  4. Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

    Ryzinska-Paier, G.; Sommer, R.; Haider, J.M.; Knetsch, S.; Frick, C.; Kirschner, A.K.T.; Farnleitner, A.H.

    2011-01-01

    Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method. PMID:21872622

  5. Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol

    Phosphotyrosyl-protein phosphatase activity of human erythrocyte cytosol can be resolved into two fractions by DEAE-cellulose chromatography followed by P-cellulose chromatography. Both 32P-Tyr-phosphatases are able to dephosphorylate 32P-Tyr of poly (Glu-Tyr) 4:1 but no angiotensin II and synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Gly, previously phosphorylated on tyrosine residues by rat spleen tyrosine-protein kinase. Both 32P-Tyr-phosphatase activities distinctly differ from either 32P-Ser-casein phosphatase activity or acid and alkaline p-nitrophenylphosphatase activities with regard to catalytic and physico-chemical properties such as substrate specificity, chromatographic behavior, response to various effectors

  6. Soil acid phosphomonoesterase activity and phosphorus forms in ancient and post-agricultural black alder [Alnus glutinosa (L.) Gaertn.] woodlands

    Anna Orczewska; Anna Piotrowska; Joanna Lemanowicz

    2012-01-01

    Black alder, an N-fixing tree is considered to accelerate the availability of phosphorus in soils due to the increased production of phosphatase enzymes, which are responsible for the P release from the litter. Acid phosphatase activity plays a pivotal role in organic P mineralization in forest soils and in making P available to plants. In order to check whether Alnus glutinosa stimulates acid phosphomonoesterase (PHACID) activity, we compared enzyme activities, total P concentration (PTOT), ...

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

    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 (70%

  8. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32P-ACC phosphorylated by the casein kinases was identified

  9. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis

    Vishakha Grover

    2016-01-01

    Full Text Available Context: Current clinical periodontal diagnostic techniques emphasize the assessment of clinical and radiographic signs of periodontal diseases which can provide a measure of history of disease. Hence, new methodologies for early identification and determination of periodontal disease activity need to be explored which will eventually result in expedited treatment. Aim: To evaluate the correlation of alkaline phosphatase (ALP activity in gingival crevicular fluid (GCF to clinical parameters of periodontal inflammation in smokers with chronic periodontitis. Materials and Methods: Study population included 15 smoker male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present. Following parameters were evaluated at baseline, 1 month and 3 months after scaling and root planing: plaque index, bleeding index, probing pocket depth (PD, relative attachment level (RAL, and GCF ALP activity. Statistical Analysis Used: Independent variables for measurements over time were analyzed by using Wilcoxon signed rank test. Results: A statistically significant reduction in all the clinical parameters and GCF ALP activity was observed from baseline to 1 month and 3 months. A correlation was observed between change in GCF ALP activity and PD reduction as well as gain in RAL at 3 months. Conclusion: The present study emphasizes that total ALP activity could be used as a marker for periodontal disease activity in smokers. Estimation of changes in the levels of this enzyme has a potential to aid in the detection of progression of periodontal disease and monitoring the response to periodontal therapy.

  10. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis

    Grover, Vishakha; Malhotra, Ranjan; Kapoor, Anoop; Bither, Rupika; Sachdeva, Sonia

    2016-01-01

    Context: Current clinical periodontal diagnostic techniques emphasize the assessment of clinical and radiographic signs of periodontal diseases which can provide a measure of history of disease. Hence, new methodologies for early identification and determination of periodontal disease activity need to be explored which will eventually result in expedited treatment. Aim: To evaluate the correlation of alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) to clinical parameters of periodontal inflammation in smokers with chronic periodontitis. Materials and Methods: Study population included 15 smoker male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present. Following parameters were evaluated at baseline, 1 month and 3 months after scaling and root planing: plaque index, bleeding index, probing pocket depth (PD), relative attachment level (RAL), and GCF ALP activity. Statistical Analysis Used: Independent variables for measurements over time were analyzed by using Wilcoxon signed rank test. Results: A statistically significant reduction in all the clinical parameters and GCF ALP activity was observed from baseline to 1 month and 3 months. A correlation was observed between change in GCF ALP activity and PD reduction as well as gain in RAL at 3 months. Conclusion: The present study emphasizes that total ALP activity could be used as a marker for periodontal disease activity in smokers. Estimation of changes in the levels of this enzyme has a potential to aid in the detection of progression of periodontal disease and monitoring the response to periodontal therapy. PMID:27563197

  11. Redox Modulation of PTEN Phosphatase Activity by Hydrogen Peroxide and Bisperoxidovanadium Complexes.

    Lee, Chang-Uk; Hahne, Gernot; Hanske, Jonas; Bange, Tanja; Bier, David; Rademacher, Christoph; Hennig, Sven; Grossmann, Tom N

    2015-11-01

    PTEN is a dual-specificity protein tyrosine phosphatase. As one of the central tumor suppressors, a thorough regulation of its activity is essential for proper cellular homeostasis. The precise implications of PTEN inhibition by reactive oxygen species (e.g. H2 O2 ) and the subsequent structural consequences remain elusive. To study the effects of PTEN inhibition, bisperoxidovanadium (bpV) complexes serve as important tools with the potential for the treatment of nerve injury or cardiac ischemia. However, their mode of action is unknown, hampering further optimization and preventing therapeutic applications. Based on protein crystallography, mass spectrometry, and NMR spectroscopy, we elucidate the molecular basis of PTEN inhibition by H2O2 and bpV complexes. We show that both molecules inhibit PTEN via oxidative mechanisms resulting in the formation of the same intramolecular disulfide, therefore enabling the reactivation of PTEN under reductive conditions. PMID:26418532

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

    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...... differing in organic matter were placed in six parallel hyphal compartments (HC) separated from the RC with a 37 mu m mesh. In the first experiment, using Glomus caledonium, hyphal length densities were measured in the HC after 31 days. Added straw increased hyphal length densities by 34 and 62% for soil...... kept outdoors and indoors, respectively. In the second experiment, using G. invermaium and only soil kept outdoors, three treatments were included: soil with no added straw with or without a new addition of 0.5% (w/w) of ground clover leaves, and soil with 9% straw plus mineral N. After 41 days hyphal...

  13. Phosphatases in plants.

    Schweighofer, Alois; Meskiene, Irute

    2015-01-01

    Reversible protein phosphorylation is an essential posttranslational modification mechanism executed by opposing actions of protein phosphatases and protein kinases. About 1,000 predicted kinases in Arabidopsis thaliana kinome predominate the number of protein phosphatases, of which there are only ~150 members in Arabidopsis. Protein phosphatases were often referred to as "housekeeping" enzymes, which act to keep eukaryotic systems in balance by counteracting the activity of protein kinases. However, recent investigations reveal the crucial and specific regulatory functions of phosphatases in cell signaling. Phosphatases operate in a coordinated manner with the protein kinases, to execute their important function in determining the cellular response to a physiological stimulus. Closer examination has established high specificity of phosphatases in substrate recognition and important roles in plant signaling pathways, such as pathogen defense and stress regulation, light and hormonal signaling, cell cycle and differentiation, metabolism, and plant growth. In this minireview we provide a compact overview about Arabidopsis protein phosphatase families, as well as members of phosphoglucan and lipid phosphatases, and highlight the recent discoveries in phosphatase research. PMID:25930691

  14. The extended human PTPome: a growing tyrosine phosphatase family.

    Alonso, Andrés; Pulido, Rafael

    2016-04-01

    Tyr phosphatases are, by definition, enzymes that dephosphorylate phospho-Tyr (pTyr) from proteins. This activity is found in several structurally diverse protein families, including the protein Tyr phosphatase (PTP), arsenate reductase, rhodanese, haloacid dehalogenase (HAD) and His phosphatase (HP) families. Most of these families include members with substrate specificity for non-pTyr substrates, such as phospho-Ser/phospho-Thr, phosphoinositides, phosphorylated carbohydrates, mRNAs, or inorganic moieties. A Cys is essential for catalysis in PTPs, rhodanese and arsenate reductase enzymes, whereas this work is performed by an Asp in HAD phosphatases and by a His in HPs, via a catalytic mechanism shared by all of the different families. The category that contains most Tyr phosphatases is the PTP family, which, although it received its name from this activity, includes Ser, Thr, inositide, carbohydrate and RNA phosphatases, as well as some inactive pseudophosphatase proteins. Here, we propose an extended collection of human Tyr phosphatases, which we call the extended human PTPome. The addition of new members (SACs, paladin, INPP4s, TMEM55s, SSU72, and acid phosphatases) to the currently categorized PTP group of enzymes means that the extended human PTPome contains up to 125 proteins, of which ~ 40 are selective for pTyr. We set criteria to ascribe proteins to the extended PTPome, and summarize the more important features of the new PTPome members in the context of their phosphatase activity and their relationship with human disease. PMID:26573778

  15. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine label from the purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase

  16. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  17. Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    Della Monica, Rosa; Visconti, Roberta; Cervone, Nando; Serpico, Angela Flavia; Grieco, Domenico

    2015-01-01

    During cell division, progression through mitosis is driven by a protein phosphorylation wave. This wave namely depends on an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1 while activities of major protein phosphatases, like PP1 and PP2A, appear directly or indirectly repressed by Cdk1. However, how Cdk1 inactivation is coordinated with reactivation of major phosphatases at mitosis exit still lacks substantial knowledge. We show here that activation of PP2A-B55, a major mitosis exit phosphatase, required the phosphatase Fcp1 downstream Cdk1 inactivation in human cells. During mitosis exit, Fcp1 bound Greatwall (Gwl), a Cdk1-stimulated kinase that phosphorylates Ensa/ARPP19 and converts these proteins into potent PP2A-B55 inhibitors during mitosis onset, and dephosphorylated it at Cdk1 phosphorylation sites. Fcp1-catalyzed dephosphorylation drastically reduced Gwl kinase activity towards Ensa/ARPP19 promoting PP2A-B55 activation. Thus, Fcp1 coordinates Cdk1 and Gwl inactivation to derepress PP2A-B55, generating a dephosphorylation switch that drives mitosis progression. DOI: http://dx.doi.org/10.7554/eLife.10399.001 PMID:26653855

  18. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    Larissa Balabanova; Vasily Golotin; Svetlana Kovalchuk; Alexander Bulgakov; Galina Likhatskaya; Oksana Son; Valery Rasskazov

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa...

  19. Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition

    Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 μg/cm2 DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that is independent of the organic content of these particles. These data show that DEP-induced EGFR phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity

  20. Effects of overlying water aeration on phosphorus fractions and alkaline phosphatase activity in surface sediment

    Jianjun Chen; Shaoyong Lu; Yikun Zhao; Wei Wang; Minsheng Huang

    2011-01-01

    Microbial activity may influence phosphorus (P) deposit and release at the water sediment interface.The properties of DO (dissolved oxygen), pH, P fractions (TP, Ca-P, Fe-P, OP, IP), and APA (alkaline phosphatase activity) at the water sediment interface were measured to investigate microbial activity variations in surface sediment under conditions of two-month intermittent aeration in overlying water.Results showed that DO and TP of overlying water increased rapidly in the first week and then decreased gradually after 15 day of intermittent aeration.Microorganism metabolism in surface sediment increased pH and decreased DO and TP in the overlying water.After two-month intermittent aeration, APA and OP from surface sediment (0-2 crm) were both significantly higher than those from bottom sediment (6-8 cm) (p < 0.05), and surface sediment Fe-P was transferred to OP during the course of microorganism reproduction on the surface sediment.These results suggest that microbial activity and microorganism biomass from the surface sediment were higher than those from bottom sediment afar two-month intermittent aeration in the overlying water.

  1. The effect of 1,25-dihydroxycholecalciferol on the multiple forms of alkaline phosphatase and the sialic acid incorporation into microsomes of chick duodenum

    Polyacrylamide disc gel electrophoresis of n-butanol solubilized alkaline phosphatase from chick duodenum revealed that the change of alkaline phosphatase induced by 1,25-(OH)2D3 involved the transformation of desialoenzyme to sialoenzyme. The initial stimulation by 1,25-(OH)2D3 of the incorporation of sialic acid into duodenal microsomes corresponded with the initial increase in calcium absorption. After this initial stimulation, there was a rapid decline in sialic acid incorporation into microsomes decreasing below control levels at 24 hr. Calcium concentration in the microsomes followed a pattern similar to the incorporation of sialic acid into microsomes. The depressed sialic acid incorporation was reversed by the addition of calcium in vitro. These results suggest that the initial action of 1,25-(OH)2D3 is to change the membrane permeability to calcium and to change the subcellular distribution of calcium in the small intestine. The accumulated calcium in the microsomes then stimulates the sialic acid incorporation into desialoenzyme. This results in the changes of isozyme pattern of alkaline phosphatase, viz, the transformation of desialoenzyme to sialoenzyme. The transformed alkaline phosphatase might be one of the factors involved more directly in the regulation of calcium transport in intestine. (auth.)

  2. The wheat MAP kinase phosphatase 1 alleviates salt stress and increases antioxidant activities in Arabidopsis.

    Zaidi, Ikram; Ebel, Chantal; Belgaroui, Nibras; Ghorbel, Mouna; Amara, Imène; Hanin, Moez

    2016-04-01

    Mitogen-activated protein kinase phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, development and stress responses. We have previously shown that the heterologous expression of a durum wheat MKP, TMKP1, results in increased tolerance to salt stress in yeast but its particular contribution in salt stress tolerance in plants was not investigated. Here, TMKP1 was overexpressed in Arabidopsis thaliana and physiological changes were assessed in transgenic plants exposed to stress conditions. Under salt stress and especially LiCl, the TMKP1 overexpressors displayed higher germination rates in comparison to wild type plants. The enhancement of salt stress tolerance was accompanied by increased antioxidant enzyme activities, namely superoxide dismutase, catalase and peroxydases. Such increases in antioxidant activities were concomitant with lower malondialdehyde, superoxide anion O2(-) and hydrogen peroxide levels in the TMKP1 transgenic seedlings. Moreover, we provide evidence that, in contrast to the Arabidopsis ortholog AtMKP1, TMKP1 acts as a positive regulator of salt stress tolerance via its ectopic expression in the Arabidopsis mkp1 mutant. PMID:26927025

  3. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    ). Our group previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in S. cerevisiae. Here we show the conservation of this pathway in mammalian cells. In response to DNA damage, ATM (ataxia telangiectasia mutated) phosphorylates the Chk2 tumor suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by auto-phosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and de-phosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. The recombinant Chk2 protein is fully phosphorylated and activated, due to the high protein concentrations obtained during production. In vitro, Wip 1 de-phosphorylates the phospho-T68 of Chk2, but does not reduce Chk2 kinase activity on its usual GST-CDC25C substrate. These observations suggest that Wip1 phosphatase controls Chk2 activation rather than its enzymatic activity that relies on phosphorylations in the T-loop. The physiological consequences of Wip1 overexpression were tested in human adenocarcinoma cells: the HCT15 cell line. The specificities of this cell line are (i ) the absence of functional p53 proteins, leading to a G2 delay in response to a genotoxic stress, and (ii) the absence of functional Chk2 proteins, because of one CHK2 allele being unexpressed and because the second allele codes for a mutated protein that is unstable and inactive. The HCT15 cell line was complemented by a functional form of HA-Chk2 and the selected clone expresses the protein to a level similar to that observed in other cell lines. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip 1 modest overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to DNA

  4. Insulin resistance is associated with reduced fasting and insulin-stimulated glycogen synthase phosphatase activity in human skeletal muscle.

    Kida, Y; Esposito-Del Puente, A; Bogardus, C; Mott, D M

    1990-01-01

    Insulin-stimulated glycogen synthase activity in human skeletal muscle correlates with insulin-mediated glucose disposal rate (M) and is reduced in insulin-resistant subjects. We have previously reported reduced insulin-stimulated glycogen synthase activity associated with reduced fasting glycogen synthase phosphatase activity in skeletal muscle of insulin-resistant Pima Indians. In this study we investigated the time course for insulin stimulation of glycogen synthase and synthase phosphatas...

  5. Increased tartrate-resistant acid phosphatase (TRAP expression in malignant breast, ovarian and melanoma tissue: an investigational study

    Eck M

    2006-07-01

    Full Text Available Abstract Background Tartrate-resistant acid phosphatase (TRAP is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies. Methods TRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells. Results A marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines. Conclusion Overexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies.

  6. Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes.

    Ma, Xue-Feng; Wright, Elane; Ge, Yaxin; Bell, Jeremey; Xi, Yajun; Bouton, Joseph H; Wang, Zeng-Yu

    2009-04-01

    Phosphate is one of the least available macronutrients restricting crop production in many ecosystems. A phytase gene (MtPHY1) and a purple acid phosphatase gene (MtPAP1), both isolated from the model legume Medicago truncatula, were introduced into white clover (Trifolium repens L.) by Agrobacterium-mediated transformation. The transgenes were driven by the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Transcripts were detected in roots of the transgenic plants. Phytase or acid phosphatase (APase) activities in root apoplasts of the transgenic plants were increased up to three-fold compared to the wild type control. After the plants were grown 80 days in sand pots supplied with organic phosphorus (Po) as the sole P source, dry weights of shoot tissues of the best performing transgenic plants almost doubled that of the control and were comparable to the counterparts supplied with inorganic phosphorus (Pi). Relative biomass production of the transgenics under Po treatment was over 90% and 80% of that from the Pi treatment when the plants were grown in hydroponics (40 days) and sand pots (80 days), respectively. In contrast, biomass of the wild type controls under Po treatment was only about 50% of the Pi treatment in either hydroponic cultures or sand pots. In addition, shoot P concentrations of the transgenic plants were significantly increased compared to the control. Transgenic plants accumulated much higher amounts of total P (up to 2.6-fold after 80 days of growth) than the control in Po supplied sand pots. The results showed that transgenic expression of MtPHY1 or MtPAP1 in white clover plants increased their abilities of utilizing organic phosphorus in response to P deficiency. PMID:26493137

  7. How nitrogen and sulphur addition, and a single drought event affect root phosphatase activity in Phalaris arundinacea.

    Robroek, Bjorn J M; Adema, Erwin B; Venterink, Harry Olde; Leonardson, Lars; Wassen, Martin J

    2009-03-15

    Conservation and restoration of fens and fen meadows often aim to reduce soil nutrients, mainly nitrogen (N) and phosphorus (P). The biogeochemistry of P has received much attention as P-enrichment is expected to negatively impact on species diversity in wetlands. It is known that N, sulphur (S) and hydrological conditions affect the biogeochemistry of P, yet their interactive effects on P-dynamics are largely unknown. Additionally, in Europe, climate change has been predicted to lead to increases in summer drought. We performed a greenhouse experiment to elucidate the interactive effects of N, S and a single drought event on the P-availability for Phalaris arundinacea. Additionally, the response of plant phosphatase activity to these factors was measured over the two year experimental period. In contrast to results from earlier experiments, our treatments hardly affected soil P-availability. This may be explained by the higher pH in our soils, hampering the formation of Fe-P or Fe-Al complexes. Addition of S, however, decreased the plants N:P ratio, indicating an effect of S on the N:P stoichiometry and an effect on the plant's P-demand. Phosphatase activity increased significantly after addition of S, but was not affected by the addition of N or a single drought event. Root phosphatase activity was also positively related to plant tissue N and P concentrations, plant N and P uptake, and plant aboveground biomass, suggesting that the phosphatase enzyme influences P-biogeochemistry. Our results demonstrated that it is difficult to predict the effects of wetland restoration, since the involved mechanisms are not fully understood. Short-term and long-term effects on root phosphatase activity may differ considerably. Additionally, the addition of S can lead to unexpected effects on the biogeochemistry of P. Our results showed that natural resource managers should be careful when restoring degraded fens or preventing desiccation of fen ecosystems. PMID:19101022

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

    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.

  9. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  10. Phosphatase Activities of Endolithic Communities in Rocks of the Antarctic Dry Valleys.

    Banerjee; Whitton; Wynn-Williams

    2000-01-01

    Phosphorus is scarce in Beacon Sandstone of the McMurdo Dry Valleys, Antarctica, and any input from precipitation is minimal. In endolithic microbial communities recycling of P by the action of phosphatases may therefore be important. The phosphatase activities of three different types of endolithic communities in the McMurdo Dry Valley, Antarctica, were studied in the laboratory. The dominant phototrophs were Chroococcidiopsis, mixed Gloeocapsa and Trebouxia, and Trebouxia. Bacteria were also visually conspicuous in the latter two communities, and the Trebouxia in both cases formed a lichenized association with fungal hyphae. In each case marked phosphomonoesterase (PMEase) activity was found in assays with 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate as substrate, and phosphodiesterase activity with bis-p-nitrophenyl phosphate as substrate. The pH optimum of PMEase (assayed at 0.5 pH intervals) of the Chroococcidiopsis, Gloeocapsa-Trebouxia, and Trebouxia communities was 9.5, 5.5, and 8.0, respectively. These values are similar for aqueous extracts of the respective rocks (pH 9.2, 6.2, 7.5). All three communities showed significantly higher PMEase activity at 5 degrees than 1 degrees C, and the first two also showed much higher activity at 5 degrees than 10 degrees C. All three communities also showed slightly lower activity in the light (7 µmol photon m(-2) s(-1)) than the dark; this was found with all substrates and substrate concentrations. Prior exposure of a moistened sample to light for 2 h led to a reduction in activity even when the subsequent assay was done in the dark. The rate of PMEase activity (using 100 µM MUP) in the Gloeocapsa-Trebouxia and Trebouxia communities was approximately linear with time up to 24 h, whereas the Chroococcidiopsis community showed a marked decrease after 6 h. At least part of this was due to retention of the 4-methylumbelliferone (MU) hydrolysis product. In spite of the assays being conducted on a whole

  11. In vivo detection of prostatic carcinoma with antibodies against prostatic acid phosphatase

    Serum prostatic acid phosphates (PAP) immunoassay is used to evaluate patients with prostatic carcinoma; however, as with other tumor markers, the enzyme levels do not necessarily reflect the presence or extent of tumor. The authors investigated the use of radiolabeled PAP antibodies for the in vivo detection of prostatic carcinoma by external scintillation imaging. Nine patients with prostatic carcinoma were entered into the study. Each received from 2.0 to 2.5 mCi of I-131 labeled antibody to PAP, administered i.v. The immunogen (PAP) was purified from normal human seminal fluid. Antiserum was prepared in rabbits by injecting the purified PAP. The antibodies were labeled with I-131 by chloramine-T method (10 to 20 Ci/g of IgG). Total body images were obtained at 24 and 48 hrs following administration of the labeled antibody. Nontarget I-131 activity was diminished by computer processing. Tumor sites detected by I-131 antibodies were correlated with other diagnostic procedures. In 7 of 9 patients primary and metastatic sites of cancer were detected by antibody imaging, however, no bone lesions were detected (6 cases). In 3 patients with concomitant pulmonary tumors, one was identified as of prostate origin. The serum PAP was normal in 4 patients; however, the primary tumor was identified in 3 of these. These findings suggest that the localization of prostatic carcinoma by means of in-vivo imaging of labeled antibodies to PAP is feasible and offers diagnostic opportunities based upon the functional characteristics

  12. Molecular Differences between a Mutase and a Phosphatase: Investigations of the Activation Step in Bacillus cereus Phosphopentomutase

    Iverson, T.M.; Panosian, Timothy D.; Birmingham, William R.; Nannemann, David P.; Bachmann, Brian O. (Vanderbilt)

    2012-05-09

    Prokaryotic phosphopentomutases (PPMs) are di-Mn{sup 2+} enzymes that catalyze the interconversion of {alpha}-D-ribose 5-phosphate and {alpha}-D-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPM{sup T85E} phosphomimetic variant and the neutral corollary PPM{sup T85Q} determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator {alpha}-D-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to {alpha}-D-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM{sup D156A} variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.

  13. Activity and isoenzyme spectrum of alkaline phosphatase in liver and blood plasma of gamma-irradiated hen and chick embryos

    Twelve day and 20 day-old embryos and one-day old chicks were gamma-irradiated with a single dose of 100 rad. On the 1st, 2nd and 72nd hours after treatment the alkaline phosphatase (AP) activity and isozyme spectrum in liver homogenates and blood plasma were determined. AP in the liver was demonstrated histochemically as well. The results show that the initial damage of the liver parenchyma by irradiation treatment is characterized by a slight decrease of liver AP activity and a marked increase in total plasma enzyme activity. The isozyme spectrum changes (increase of AP1 in the early period and increase of AP1 and AP5 in the later period) show that the initial liver parenchymal damage is followed by bile duct damage as well. Comparison of the results of biochemical and histochemical studies indicate the presence of direct correlation between serum and liver alkaline phosphatase activities. (A.B.)

  14. The small chemical enzyme inhibitor 5-phenylnicotinic acid/CD13 inhibits cell migration and invasion of tartrate-resistant acid phosphatase/ACP5-overexpressing MDA-MB-231 breast cancer cells.

    Krumpel, Michael; Reithmeier, Anja; Senge, Teresa; Baeumler, Toni Andreas; Frank, Martin; Nyholm, Per-Georg; Ek-Rylander, Barbro; Andersson, Göran

    2015-11-15

    Tartrate-resistant acid phosphatase (TRAP/ACP5/uteroferrin/purple acid phosphatase/PP5) has received considerable attention as a newly discovered proinvasion metastasis driver associated with different malignancies. This renders TRAP an interesting target for novel anti-cancer therapy approaches. TRAP exists as two isoforms, 5a and 5b, where the 5a isoform represents an enzymatically less active monomeric precursor to the more enzymatically active 5b isoform generated by proteolytic excision of a repressive loop domain. Recently, three novel lead compounds were identified by fragment-based screening and demonstrated to be efficient TRAP enzyme inhibitors in vitro. We conclude that one of the three compounds i.e. 5-phenylnicotinic acid (CD13) was efficient as a TRAP inhibitor with Kic values in the low micromolar range towards the TRAP 5b isoform, but was not able to inhibit the TRAP 5a isoform. Structure-based docking revealed similar interactions of CD13 with the active site in both TRAP isoforms. In stably TRAP-overexpressing MDA-MB-231 breast cancer cells, CD13 inhibited intracellular TRAP activity and showed no cytotoxicity at 200 µM. Furthermore, CD13 selectively blocked the TRAP 5b isoform compared to the TRAP 5a in cultured cells, indicating the usefulness of CD13 for assessing the different biological functions of the two TRAP isoforms 5a and 5b in cell systems. Moreover, inhibition of cell migration and invasion of stably TRAP-overexpressing MDA-MB-231 by CD13 was observed. These data establish a proof of principle that a small chemical inhibitor of the TRAP enzyme can block TRAP-dependent functions in cancer cells. PMID:26428664

  15. Alkaline phosphatase activity at the southwest coast of India: A comparison of locations differently affected by upwelling..

    Mamatha, S.S.; Malik, A.; Varik, S.; Parvathi, V.; Jineesh, V.K.; Gauns, M.; LokaBharathi, P.A.

    thrice with Milli-Q water and completely dried before use. Bottles were also rinsed with sample before collection. Samples for DO were collected in 125 mL stoppered glass bottles avoiding air bubbles and were immediately fixed with Winkler’s reagents..., including nucleotides, proteins and alkaloids. Phosphatase activity degrades not only particulate P containing matter but also high molecular dissolved organic matter. This enzyme activity has been suggested as one of the possible mechanisms in the marine...

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

    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 initia...

  17. Fosfatasa ácida en Oxisoles bajo cultivo de tabaco Acid phosphatase in Oxisols under tobacco cropping

    Toledo Marcela

    2010-07-01

    Full Text Available En suelos ácidos de trópicos y subtrópicos, caracterizados por una baja disponibilidad de P para las plantas, el papel de las fosfatasas ácidas en la mineralización del P orgánico es fundamental, constituyendo una variable promisoria para estimar la calidad del suelo. El objetivo del trabajo fue evaluar la actividad de la fosfatasa ácida en Oxisoles bajo uso tabacalero, como indicador sensible de calidad. En la provincia de Misiones ubicada al nordeste de la República Argentina, se estableció un ensayo sobre Eutrudoxes Ródicos, familia arcillosa fina, hipertérmica, aplicándose un diseño con cuatro bloques completos aleatorizados. Se establecieron 2 tratamientos: selva subtropical (Sv y uso tabacalero (Ta. Se tomaron muestras compuestas a 3 profundidades: 0-10; 10-20; 20-30 cm. Se determinaron las siguientes variables: actividad de la fosfatasa ácida (APA, pH, contenido de arcilla, carbono orgánico edáfico (CO, nitrógeno total (N, fósforo asimilable (P, materia orgánica particulada (MOP, y respiración del suelo (RES. En los casos estudiados, la APA fue mayor en los primeros diez centímetros de suelo, y fue disminuyendo con el aumento de la profundidad del perfil, en estrecha relación con los contenidos orgánicos del suelo. El 70% de la variabilidad de la APA se explicó por el nitrógeno total, íntimamente relacionado con la materia orgánica del suelo (pSoil biological parameters are of great value as sensitive indicators of transformations occurring under different uses and management practices (Mijangos et al., 2006. The aim of this study was to evaluate the activity of the acid phosphatase enzyme in Oxisols under tobacco cropping. The experimental design was in randomized complete blocks, with two treatments: subtropical rainforest (Sv and tobacco cropping (Ta (Nicotiana tabacum L.. Soil samples were taken from 0-10, 10 -20 and 20 -30 cm-deep layers. The variables measured were: APA, pH, clay content, total nitrogen (N

  18. Interactive effects of temperature, ultraviolet radiation and food quality on zooplankton alkaline phosphatase activity.

    Wolinski, Laura; Modenutti, Beatriz; Souza, Maria Sol; Balseiro, Esteban

    2016-06-01

    Ultraviolet Radiation (UVR) is a stressor for aquatic organisms affecting enzyme activities in planktonic populations because of the increase in reactive oxygen species. In addition, UVR exposure combined with other environmental factors (i.e. temperature and food quality) could have even higher detrimental effects. In this work, we aimed to determine the effect of UVR on somatic Alkaline Phosphatase Activity (APA) and Glutathione S-Transferase (GST) activity on the cladoceran Daphnia commutata under two different temperatures (10 °C and 20 °C) and under three food qualities (carbon:phosphorus ratios: 1150, 850 and 550). APA is a biomarker that is considered as a P deficiency indicator in zooplankton. Since recovery from UVR damage under dark conditions is an ATP depending reaction we also measured APA during recovery phases. We carried out a laboratory experiment combining different temperatures and food qualities with exposition to UVR followed by luminic and dark phases for recovery. In addition, we exposed organisms to H2O2, to establish if the response on APA to UVR was a consequence of the reactive oxygen species produced these short wavelengths. Our results showed that somatic APA was negatively affected by UVR exposure and this effect was enhanced under high temperature and low food quality. Consistently, GST activity was higher when exposed to UVR under both temperatures. The H2O2 experiments showed the same trend as UVR exposure, indicating that APA is affected mainly by oxidative stress than by direct effect of UVR on the enzyme. Finally, APA was affected in the dark phase of recovery confirming the P demands. These results enlighten the importance of food quality in the interacting effect of UVR and temperature, showing that C:P food ratio could determine the success or failure of zooplanktonic populations in a context of global change. PMID:26895537

  19. Alkaline Phosphatase Activity : an overlooked player on the phosphate behavior in macrotidal estuaries

    Delmas, Daniel; Labry, Claire; Youenou, Agnes; Quere, Julien; Auguet, Jean Christophe; Montanie, Helene

    2014-05-01

    The non-conservative behavior of phosphate within the estuarine salinity gradient is essentially assigned to physico-chemical processes, such as desorption at low salinity and to benthic exchanges. Microbial phosphatase activity (APA), generally related to phosphate deficiency, is seldom studied in phosphate rich estuarine waters. In order to address the impact of microbial activity (bacterial abundance, production BSP, APA) on phosphate behavior, we studied these activities on a seasonal basis within the salinity gradient of two macrotidal estuaries presenting different levels of suspended solids. Whatever the season the Charente estuary is characterized by high levels of Suspended Particulate Matter (SPM > 1g.L-1), particularly in the Maximum Turbidity Zone (MTZ) located at the 5-10 psu. In this area characterized by high BSP and APA there is a significant increase of PO4 levels especially during summer. In the Aulne estuary the particle load is significantly lower (1/10) but high BSP and APA are equally recorded. In the highly turbid waters of the Charente estuary, active phytoplankton is virtually absent as pheopigments constitute up to 80% of the total pigments, particularly in the MTZ, therefore APA may essentially have a bacterial origin. In the Aulne estuary attached bacteria are dominant, both in numbers and production, and their distribution along the haline gradient perfectly follows those of APA and phosphate levels. These observations, associated with the very close relationships observed between APA, SPM and BSP, suggest that APA derive mainly from bacterial (attached) origin and operate at the expense of particulate phosphorus and hence contribute to PO4 regeneration, especially in spring and summer. Finally, as APA increased as PO4, whereas the reverse is observed in both fresh and marine waters, an original scheme for APA regulation, related to the large dominance of attached bacteria can be described for the estuarine waters.

  20. Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease.

    Liu, Xue-Guo; Xing, Xiao-Jing; Li, Bo; Guo, Yong-Ming; Zhang, Ye-Zhen; Yang, Yan; Zhang, Lian-Feng

    2016-07-15

    A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3'-end (F-DNA) and its complementary sequence modified with a 5'-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5'-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field. PMID:27015149

  1. The influence of ionizing radiation at the alkaline phosphatase activity and calcium 45 transport under hypothyroid conditions

    The experiments with 2-month-old hypothyroid male rats (mercasolil 10 mg/kg per os during 21 days) have shown that total gamma-irradiation ( 0,5 Gy) caused phased changes of alkaline phosphatase - its reduction in the first dates of the investigation (3, 10 and 30 days), normal activity in a 90 days and repeated drop of the activity at the end of the experiment (180 days). In a 3 and 10 days after irradiation it was revealed the diminish of the calcium-accumulated ability of the duodenum mucous membrane in the hypothyroid rats. So it was concluded that combined action of radiation and mercasolil leaded to a decrease in calcium 45 transport in the duodenum and reduction in the activity of thermo labile isoenzyme of alkaline phosphatase in blood serum

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

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

    2011-01-01

    Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this......-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 the...... brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft...

  3. [Influences of uncommon isoenzymes on determination of alkaline phosphatase activity by dry-chemistry analyzers].

    Tozawa, T; Hashimoto, M

    2001-04-01

    Dry-chemistry(DC) analysis may be influenced by some matrix effects for measuring uncommon isoenzyme forms. Placental and intestinal alkaline phosphatase(AP) are overestimated by the VITROS DC, compared with results obtained with the wet-chemistry(WC) method of Bretaudiere, et al. using 2-amino-2-methyl-1-propanol (AMP) buffer, however, no such discrepancy between AP results in any DC method and that with a routine WC method recommended by Japanese Society of Clinical Chemistry in that 2-ethylaminoethanol(EAE) buffer is used, has been demonstrated. The type of buffer used affects differently the rates of AP isoenzymes activities. We therefore examined whether the presence of uncommon AP isoenzyme forms in serum caused aberrant DC results for AP in comparison with a routine WC method using EAE buffer. Here, serum samples with only liver AP and bone AP(n : 32); high-molecular-mass AP(n : 11); placental AP(n : 12); intestinal AP(n : 13) and immunoglobulin (Ig) bound AP(n : 12) were analyzed for total AP activity on three different DC analyzers: VITROS 700XR, FUJIDRYCHEM 5000, SPOTCHEM 4410 and a WC analyzer: HITACHI 7350. Values obtained in all of the DCs for sera containing only liver/bone AP agreed with those with the WC method. For sera containing placental AP, the VITROS values were higher than those with the WC method, while the FUJIDRYCHEM values and the SPOTCHEM values were lower. The VITROS values and the FUJIDRYCHEM values for sera containing intestinal AP were lower than those with the WC method, while the SPOTCHEM values were higher. All of the DCs did not affect high-molecular-mass AP and Ig bound liver/bone AP types of macro AP, but underestimated Ig bound intestinal type. Ig bound intestinal AP may be sieved by DC multilayer elements. PMID:11391954

  4. Alkaline phosphatase activity related to phosphorus stress of microphytoplankton in different trophic conditions

    Ivančić, Ingrid; Pfannkuchen, Martin; Godrijan, Jelena; Djakovac, Tamara; Marić Pfannkuchen, Daniela; Korlević, Marino; Gašparović, Blaženka; Najdek, Mirjana

    2016-08-01

    The northern Adriatic (NA) is a favorable basin for studying the adaptive strategies of plankton to a variety of conditions along the steep gradients of environmental parameters over the year. Earlier studies identified phosphorus (P)-limitation as one of the key stresses within the NA that shape the biological response in terms of biodiversity and metabolic adjustments. A wide range of reports supports the notion that P-limitation is a globally important phenomenon in aquatic ecosystems. In this study P stress of marine microphytoplankton was determined at species level along a trophic gradient in the NA. In P-limitation all species with considerable contributions to the diatom community expressed alkaline phosphatase activity (APA), compared to only a few marginal dinoflagellate species. Nevertheless, APA expressing species did not always dominate the phytoplankton community, suggesting that APA is also an important strategy for species to survive and maintain active metabolism outside of their mass abundances. A symbiotic relationship could be supposed for diatoms that did not express APA themselves and probably benefited from APA expressed by attached bacteria. APA was not expressed by any microphytoplankton species during the autumn when P was not limiting, while most of the species did express APA during the P-limitation. This suggests that APA expression is regulated by orthophosphate availability. The methods employed in this study allowed the microscopic detection of APA for each microphytoplankton cell with simultaneous morphologic/taxonomic analysis. This approach uncovered a set of strategies to compete in P-limited conditions within the marine microphytoplankton community. This study confirms the role of P-limitation as a shaping factor in marine ecosystems.

  5. Enzyme Activities in Perfluorooctanoic Acid (PFOA)-Polluted Soils

    ZHANG Wei; LIN Kuang-Fei; YANG Sha-Sha; ZHANG Meng

    2013-01-01

    Perfluorooctanoic acid (PFOA) is a popular additive of the chemical industry; its effect on activities of important soil enzymes is not well understood.A laboratory incubation experiment was carried out to analyze the PFOA-induced changes in soil urease,catalase,and phosphatase activities.During the entire incubation period,the activities of the three soil enzymes generally declined with increasing PFOA concentration,following certain dose-response relationships.The values of EC10,the contaminant concentration at which the biological activity is inhibited by 10%,of PFOA for the soil enzyme activity calculated from the modeling equation of the respective dose-response curve suggested a sensitivity order of phosphatase > catalase > urease.The effect of PFOA on soil enzyme activities provided a basic understanding of the eco-toxicological effect of PFOA in the environment.Results of this study supported using soil phosphatase as a convenient biomarker for ecological risk assessment of PFOA-polluted soils.

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

    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.

  7. A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

    Balabanova, Larissa; Golotin, Vasily; Kovalchuk, Svetlana; Bulgakov, Alexander; Likhatskaya, Galina; Son, Oksana; Rasskazov, Valery

    2014-01-01

    A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens. PMID:25397876

  8. A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

    Larissa Balabanova

    Full Text Available A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian's lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

  9. Alkaline phosphatase activity in the subtropical ocean: insights from nutrient, dust and trace metal addition experiments

    Claire eMahaffey

    2014-12-01

    Full Text Available Phosphorus is an essential nutrient for all life on earth. In the ocean, the most bioavailable form of phosphorus is inorganic phosphate, but in the extensive subtropical gyres, phosphate concentrations can be chronically low and limit primary productivity and nitrogen fixation. In these regions, organisms produce hydrolytic enzymes, such as alkaline phosphatase (AP, that enable them to utilize the more replete dissolved organic phosphorus (DOP pool to meet their cellular phosphorus demands. In this study, we synthesized data from 14 published studies and present our own findings from two research cruises (D326 and D361 in the eastern subtropical Atlantic to explore the relationship between AP activity (APA and nutrients, Saharan dust and trace metals. We found that below a threshold phosphate concentration of ~ 30 nM, APA increased with an inverse hyperbolic relationship with phosphate concentration. Meanwhile, DOP concentrations decreased with enhanced APA, indicating utilization of the DOP pool. We found APA rates were significantly higher in the subtropical Atlantic compared to the subtropical Pacific Ocean, even over the same low phosphate concentration range (0 to 50 nM. While the phosphate concentration may have a first order control on the APA rates, we speculate that other factors influence this basin scale contrast. Using bioassay experiments, we show that the addition of Saharan dust and zinc significantly increased the rate of APA. To our knowledge, our results are the first direct field-based evidence that APA is limited by zinc in the subtropical ocean. Further work is required to explore the relationship between trace metals such as iron and zinc, which are co-factors of phosphohydrolytic enzymes, specifically PhoX and PhoA, respectively, and APA in the ocean.

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

    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.

  11. Molecular Characterization and Functional Analysis of a New Acid Phosphatase Gene (Ha-acp1) from Heterodera avenae

    LIU Yan-ke; HUANG Wen-kun; LONG Hai-bo; PENG Huan; HE Wen-ting; PENG De-liang

    2014-01-01

    For sedentary endo-parasitic nematodes, parasitism genes encoding secretory protein expressed in the subventral glands cells always play an important role during the early parasitic process. A new acid phosphatase gene (Ha-acp1) expressed in the subventral glands of the cereal cyst nematode (Heterodera avenae) was cloned and the characteristics of the gene were analyzed. Results showed that the gene had a putative signal peptide for secretion and in situ hybridization showed that the transcripts of Ha-acp1 accumulated speciifcally in the subventral gland cells of H. avenae. Southern blot analysis suggested that Ha-acp1 belonged to a multigene family. RT-PCR analysis indicated that this transcription was strong at the pre-parasitic juveniles. Knocking down Ha-acp1 using RNA interference technology could reduce nematode infectivity by 50%, and suppress the development of cyst. Results indicated that Ha-acp1 could play an important role in destroying the defense system of host plants.

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

    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

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

    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y. [Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing (China)

    2013-08-10

    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.

  14. Experimental studies on bone with focus on tartrate-resistant acid phosphatase and bone remodeling

    2014-01-01

    List of papers. Papers I and III are removed from the thesis due to publisher restrictions. Paper I : Melhus G, Solberg LB, Dimmen S, Madsen JE, Nordsletten L, Reinholt FP. Experimental osteoporosis induced by ovariectomy and vitamin D deficient diet does not markedly affect fracture healing in rats Acta Orthop 2007 Jun;78(3):393-403 doi:10.1080/17453670710013988 Paper II: Solberg LB, Brorson SH, Stordalen GA, Bækkevold E, Andersson G, Reinholt FP. Increased tartrate-resistant acid ph...

  15. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton. Cytometry Part A, 85A: 841–853

    Diaz-de-Quijano, D.; Palacios, P.; Horňák, Karel; Felip, M.

    85A, č. 10 (2014), s. 841-853. ISSN 1552-4922 Institutional support: RVO:60077344 Keywords : 3D fluorescence microscopy * deconvolution * ELF phosphate * phosphatase activity * phytoplankton Subject RIV: EE - Microbiology, Virology Impact factor: 2.928, year: 2014

  16. DevS/DosS sensor is bifunctional and its phosphatase activity precludes aerobic DevR/DosR regulon expression in Mycobacterium tuberculosis.

    Kaur, Kohinoor; Kumari, Priyanka; Sharma, Saurabh; Sehgal, Snigdha; Tyagi, Jaya Sivaswami

    2016-08-01

    Two-component systems, comprising histidine kinases and response regulators, empower bacteria to sense and adapt to diverse environmental stresses. Some histidine kinases are bifunctional; their phosphorylation (kinase) and dephosphorylation (phosphatase) activities toward their cognate response regulators permit the rapid reversal of genetic responses to an environmental stimulus. DevR-DevS/DosR-DosS is one of the best-characterized two-component systems of Mycobacterium tuberculosis. The kinase function of DevS is activated by gaseous stress signals, including hypoxia, resulting in the induction of ~ 48-genes DevR dormancy regulon. Regulon expression is tightly controlled and lack of expression in aerobic Mtb cultures is ascribed to the absence of phosphorylated DevR. Here we show that DevS is a bifunctional sensor and possesses a robust phosphatase activity toward DevR. We used site-specific mutagenesis to generate substitutions in conserved residues in the dimerization and histidine phosphotransfer domain of DevS and determined their role in kinase/phosphatase functions. In vitro and in vivo experiments, including a novel in vivo phosphatase assay, collectively establish that these conserved residues are critical for regulating kinase/phosphatase functions. Our findings establish DevS phosphatase function as an effective control mechanism to block aerobic expression of the DevR dormancy regulon. Asp-396 is essential for both kinase and phosphatase functions, whereas Gln-400 is critical for phosphatase function. The positive and negative functions perform opposing roles in DevS: the kinase function triggers regulon induction under hypoxia, whereas its phosphatase function prevents expression under aerobic conditions. A finely tuned balance in these opposing activities calibrates the dormancy regulon response output. PMID:27327040

  17. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs; Nouveaux procedes pour mesurer les activites synthase et phosphatase de la bisphosphoglycerate mutase. Interet pour le developpement de drogues therapeutiques

    Ravel, P.; Garel, M.C. [Hopital Henri-Mondor, 94 - Creteil (France); Toullec, D. [Laboratoire Glaxo Wellcome, 91- Les Ulis (France)

    1997-12-31

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  18. The informative value of the radioimmunological determination of acid prostate phosphatase for the diagnostic of prostate carcinoma: A comparison of various reagent kits

    Patients of the university urological clinic in Freiburg gave, in all, 89 serum samples for the determination of acid prostate phosphatase using four different methods (3 radioimmunological methods and 1 enzymatic procedure) in order to compare the informative ability of these procedures in the diagnostic of prostate carcinoma. The results of the radioimmunological determinations agreed mostly with one another. They showed a higher sensitivity than the enzymatic method, but a lower specificity. The rate of false positive as well as false negative results is with both procedures too large, so that a reliable early diagnosis is not possible. Not until after metastasis of the carcinoma do all four methods give clearly pathological values. The acid prostate phosphatase determination is therefore suited for progress and therapy control. The values sink with successful therapy, rise again with recitivism. (TRV)

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

    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.

  20. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

    Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

    2004-01-01

    Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  1. Phosphate solubilizing bacteria and alkaline phosphatase activity in coastal waters off Trivandrum

    Mamatha, S.S.; Gobika, A.; Janani, P.

    of Oceanography, Dona Paula, Goa. * The paper was presented in the MoES-sponsored National Seminar on Coastal and Island Ecosystems: Conservation and Management at National Institute of Oceanography (NIO), Goa, February 16-17, 2012. Jour. Coast. Env., Vol. 3, No... of heterotrophic and phosphate solubilizing bacteria from Chennai. Southeast coast of India. Indian Journal of Marine Science. 31. 69-72. Siuda, W. 1984. Phosphatases and their role in organic phosphorus transformation in natural waters : A review. Polish...

  2. Osteoblast response (initial adhesion and alkaline phosphatase activity following exposure to a barrier membrane/enamel matrix derivative combination

    Thangakumaran S

    2009-01-01

    Full Text Available Background and Objective: The enamel matrix derivative (EMD has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP activity of an osteoblast cell line (SaOS-2 when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior. Materials and Methods: 5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1 a non cross-linked porcine Type I and III collagen membrane (BG, 2 a weakly cross-linked Type I collagen membrane (HG, 3 a glutaraldehyde cross-linked bovine Type I collagen (BM, and 4 a resorbable polymer membrane (CP. Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 µg/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated. Results: The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows: EMD alone (75.59±2

  3. Phosphatase activities in rice-planting meadow brown soil and their responses to fertilization%草甸棕壤水稻田磷酸酶活性及对施肥措施的响应

    沈菊培; 陈振华; 陈利军

    2005-01-01

    This study is aimed to investigate the activities of phosphomonoesterase (acid-, neutral-, and alkaline-), phosphodiesterase and phosphotriesterase in a rice-planting meadow brown soil at the lower reach of Liao River, and their responses to different fertilization treatments. The results showed that there was no significant difference in soil total P and organic P contents among all treatments, but soil available P content was significantly higher in treatment OM than in other treatments. Soil acid-and neutral phosphomonoesterase had a higher activity than alkaline phosphomonoesterase and phosphodiesterase, while phosphotriesterase had the lowest activity. No significant difference was found in phosphatase activities between different fertilization treatments. Soil acid phosphomonoesterase activity had a significant correlation with soil total P and available P contents, while soil phosphodiesterase activity significantly correlated with soil organic P content.

  4. Modulators of intestinal alkaline phosphatase.

    Bobkova, Ekaterina V; Kiffer-Moreira, Tina; Sergienko, Eduard A

    2013-01-01

    Small molecule modulators of phosphatases can lead to clinically useful drugs and serve as invaluable tools to study functional roles of various phosphatases in vivo. Here, we describe lead discovery strategies for identification of inhibitors and activators of intestinal alkaline phosphatases. To identify isozyme-selective inhibitors and activators of the human and mouse intestinal alkaline phosphatases, ultrahigh throughput chemiluminescent assays, utilizing CDP-Star as a substrate, were developed for murine intestinal alkaline phosphatase (mIAP), human intestinal alkaline phosphatase (hIAP), human placental alkaline phosphatase (PLAP), and human tissue-nonspecific alkaline phosphatase (TNAP) isozymes. Using these 1,536-well assays, concurrent HTS screens of the MLSMR library of 323,000 compounds were conducted for human and mouse IAP isozymes monitoring both inhibition and activation. This parallel screening approach led to identification of a novel inhibitory scaffold selective for murine intestinal alkaline phosphatase. SAR efforts based on parallel testing of analogs against different AP isozymes generated a potent inhibitor of the murine IAP with IC50 of 540 nM, at least 65-fold selectivity against human TNAP, and >185 selectivity against human PLAP. PMID:23860652

  5. Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: Identification of a myotubularin-related protein lacking catalytic activity

    Nandurkar, H. H.; Caldwell, K K; Whisstock, J C; Layton, M. J.; Gaudet, E. A.; Norris, F. A.; Majerus, P W; Mitchell, C. A.

    2001-01-01

    The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP ...

  6. Alkaline Phosphatase in Stem Cells

    Kateřina Štefková

    2015-01-01

    Full Text Available Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

  7. Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa.

    Bucci, Diego; Giaretta, Elisa; Spinaci, Marcella; Rizzato, Giovanni; Isani, Gloria; Mislei, Beatrice; Mari, Gaetano; Tamanini, Carlo; Galeati, Giovanna

    2016-01-15

    Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm-oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen-thawed samples compared with freshly ejaculated ones (n = 21), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen-thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing. PMID:26433714

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

    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.

  9. Direct and indirect effects of ammonia, ammonium and nitrate on phosphatase activity and carbon fluxes from decomposing litter in peatland

    Here we investigate the response of soils and litter to 5 years of experimental additions of ammonium (NH4), nitrate (NO3), and ammonia (NH3) to an ombrotrophic peatland. We test the importance of direct (via soil) and indirect (via litter) effects on phosphatase activity and efflux of CO2. We also determined how species representing different functional types responded to the nitrogen treatments. Our results demonstrate that additions of NO3, NH4 and NH3 all stimulated phosphatase activity but the effects were dependent on species of litter and mechanism (direct or indirect). Deposition of NH3 had no effect on efflux of CO2 from Calluna vulgaris litter, despite it showing signs of stress in the field, whereas both NO3 and NH4 reduced CO2 fluxes. Our results show that the collective impacts on peatlands of the three principal forms of nitrogen in atmospheric deposition are a result of differential effects and mechanisms on individual components. - We found that nitrogen deposition affects microbial activity associated with litter through both indirect and direct mechanisms, but these effects were dependent on the chemical form of inorganic nitrogen compounds.

  10. Inhibition of osteoblast activity by zoledronic acid

    Fernanda Gonçalves Basso

    2013-10-01

    Full Text Available INTRODUCTION: Patients treated with nitrogen-containing bisphosphonates, such as zoledronic acid (ZA, have frequently shown oral bone exposure areas, termed osteonecrosis. In addition, these patients may also present low repair and regeneration potential, mainly after tooth extractions. These side-effects caused by bisphosphonates may be due to their inhibitory effects on oral mucosa and local bone cells. OBJECTIVE: To evaluate the effects of ZA on the mineralization capacity of cultured osteoblasts. MATERIALS AND METHODS: Human immortalized osteoblasts (SaOs-2 were grown in plain culture medium (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum [FBS] in wells of 24-well plates. After 48-hour incubation, the plain DMEM was replaced by a solution with ZA at 5 µM which was maintained in contact with cells for seven, 14 or 21 days. After these periods, cells were evaluated regarding alkaline phosphatase (ALP activity and mineral nodule formation (alizarin red. Data were statistically analyzed by Mann-Whitney test, at 5% of significance level. RESULTS: ZA caused significant reduction on ALP activity and mineral nodules formation by cultured osteoblasts in all evaluated periods (p < 0.05. CONCLUSION: These data indicate that ZA causes inhibition on the osteogenic phenotype of cultured human osteoblasts, which, in turn, may reduce bone repair in patients subjected to ZA therapy.

  11. Variations of alkaline phosphatase activity and P fractions in sediments of a shallow Chinese eutrophic lake (Lake Taihu)

    The distribution of alkaline phosphatase activity (APA) and P fractions in sediment cores and the relationship between them were studied in a shallow Chinese freshwater lake (Lake Taihu). Sediment cores were collected from four sites, characterized by different degrees of eutrophication in June 2004. Sediment P was fractionated into Fe/Al-P, Ca-P, organic P (OP), inorganic P (IP) and total P (TP). The former two species made the largest contribution to the sediment P pool. Results show that trophic status and hydrological conditions have great impact on the APA of the sediments. The order of the APA in sediments was conjectured to be: macrophyte dominated lake > transitional lake > algal dominated lake. APA profiles follow a similar downcore decreasing trend. There was a positive relationship between the APA and the TP, IP. The multiple linear regression equation of the APA and P fractions is: APA = -97 + 0.768TP - 0.985Fe/Al-P. - Characteristics of the alkaline phosphatase activity and P fractions in sediments of different trophic status lake were studied in Lake Taihu

  12. 18O isotope effect in 13C nuclear magnetic resonance spectroscopy. Part 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

    The 18O isotope-induced shifts in 13C and 31P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the 18O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, [α-13C,ester-18O]benzyl phosphate and [ester-18O]benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 750C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 2H]Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables

  13. Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

    Zhu Li

    2002-01-01

    Full Text Available Protein-tyrosine phosphatases (PTPases have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible fraction of the endogenous PTPase pool.

  14. Effect of Phosphatases Activity in the Hepatopancreas and Muscle of the Fresh Water Female Field Crab, Spiralothelphusa hydrodroma (Herbst Treated with Cypermethrin

    R. S. Sreenivasan

    2011-04-01

    Full Text Available The fresh water field crab, Spiralothelphusa hydrodroma is an important human food source in parts of South India and the crab is constantly exposed to pesticides, which are used extensively to control agricultural pests. Evaluation of the toxic effect of cypermethrin on the experimental crab for the LC₅₀ value was carried out. Effect of cypermethrin on the biochemical changes in the hepatopancreas and muscle was observed. Quantitative study of biochemical changes of acid phosphatase and alkaline phosphatase were undertaken.

  15. PHARMACOLOGICAL ACTIVITIES OF PROTOCATECHUIC ACID.

    Khan, Abida Kalsoom; Rashid, Rehana; Fatima, Nighat; Mahmood, Sadaf; Mir, Sadullah; Khan, Sara; Jabeen, Nyla; Murtaza, Ghulam

    2015-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a simple phenolic acid. It is found in a large variety of edible plants and possesses various pharmacological activities. This article aims to review the modern trends in phytochemical isolation and extraction of PCA from plants and other natural resources. Moreover, this article also encompasses pharmacological and biological activities of PCA. It is well known to have anti-inflammatory, antioxidant, anti-hyperglycemia, antibacterial, anticancer, anti-ageing, anti-athro- genic, anti-tumoral, anti-asthma, antiulcer, antispasmodic and neurological properties. PMID:26647619

  16. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

    Lee Jee

    2012-02-01

    Full Text Available Abstract Background The peroxisome proliferator-activated receptor (PPAR-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA, is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. Methods To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1 were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. Results We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK phosphorylation and activator protein 1 (AP1 activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. Conclusion ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism

  17. Comparison of the expression, activity, and fecal concentration of intestinal alkaline phosphatase between healthy dogs and dogs with chronic enteropathy.

    Ide, Kaori; Kato, Kazuki; Sawa, Yuki; Hayashi, Akiko; Takizawa, Rei; Nishifuji, Koji

    2016-07-01

    OBJECTIVE To compare expression, activity, and fecal concentration of intestinal alkaline phosphatase (IAP) between healthy dogs and dogs with chronic enteropathy (CE). ANIMALS 9 healthy university-owned Beagles and 109 healthy client-owned dogs (controls) and 28 dogs with CE (cases). PROCEDURES Cases were defined as dogs with persistent (> 3 weeks) gastrointestinal signs that failed to respond to antimicrobials and anti-inflammatory doses of prednisolone or dietary trials, did not have mechanical gastrointestinal abnormalities as determined by abdominal radiography and ultrasonography, and had a diagnosis of lymphoplasmacytic enteritis or eosinophilic gastroenteritis on histologic examination of biopsy specimens. Duodenal and colonic mucosa biopsy specimens were obtained from the 9 university-owned Beagles and all cases for histologic examination and determination of IAP expression (by real-time quantitative PCR assay) and activity (by enzyme histochemical analysis). Fecal samples were obtained from all dogs for determination of fecal IAP concentration by a quantitative enzyme reaction assay. RESULTS For dogs evaluated, IAP expression and activity were localized at the luminal side of epithelial cells in the mucosa and intestinal crypts, although both were greater in the duodenum than in the colon. Active IAP was detected in the feces of all dogs. Intestinal alkaline phosphatase expression and activity were lower for cases than for controls, and fecal IAP concentration for dogs with moderate and severe CE was lower than that for dogs with mild CE. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CE had impaired IAP expression and activity. Additional research is necessary to elucidate the role of IAP in the pathogenesis of CE. PMID:27347825

  18. Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate

    HeLa S3 cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-[35S]methionine into enzyme protein and active-site labeling. The 64.5K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5K to 64.5K monomer was accelerated, and the presence of the 61.5 precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA is untreated HeLa S3 cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. The data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S3 cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product

  19. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  20. A fluorometric assay for alkaline phosphatase activity based on β-cyclodextrin-modified carbon quantum dots through host-guest recognition.

    Tang, Cong; Qian, Zhaosheng; Huang, Yuanyuan; Xu, Jiamin; Ao, Hang; Zhao, Meizhi; Zhou, Jin; Chen, Jianrong; Feng, Hui

    2016-09-15

    A convenient, reliable and highly sensitive assay for alkaline phosphatase (ALP) activity in the real-time manner is developed based on β-cyclodextrin-modified carbon quantum dots (β-CD-CQDs) nanoprobe through specific host-guest recognition. Carbon quantum dots were first functionalized with 3-aminophenyl boronic acid to produce boronic acid-functionalized CQDs, and then further modified with hydropropyl β-cyclodextrins (β-CD) through B-O bonds to form β-CD-CQDs nanoprobe. p-Nitrophenol phosphate disodium salt is used as the substrate of ALP, and can hydrolyze to p-nitrophenol under the catalysis of ALP. The resulting p-nitrophenol can enter the cavity of β-CD moiety in the nanoprobe due to their specific host-guest recognition, where photoinduced electron transfer process between p-nitrophenol and CQDs takes place to efficiently quench the fluorescence of the probe. The correlation between quenched fluorescence and ALP level can be used to establish quantitative evaluation of ALP activity in a broad range from 3.4 to 100.0U/L with the detection limit of 0.9U/L. This assay shows a high sensitivity to ALP even in the presence of a very high concentration of glucose. This study demonstrates a good electron donor/acceptor pair, which can be used to design general detection strategy through PET process, and also broadens the application of host-guest recognition for enzymes detection in clinical practice. PMID:27132001

  1. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    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.

  2. pH dependence of the acetylcholinesterase-catalyzed oxygen exchange between acetate and water/purification and characterization of the low-molecular-weight acid phosphatase from bovine liver: Clarification of the roles of cysteine and histidine at the active site

    Acetylcholinesterase (AChE) catalyzes ester hydrolysis through nucleophilic attack of an active-site serine on the acyl carbon of the substrate, acetylcholine, leading to the formation of an acylenzyme intermediate, with hydrolysis resulting in the production of choline and acetic acid. The first half of the reverse reaction-AChE-catalyzed attack on acetate-was studied over a wide range in pH. Homogeneous enzyme was obtained after chromatography using the synthetic affinity resin 9-(5-carboxypentylamino)acridine. The pH-dependence of AChE-catalyzed acetylthiocholine hydrolysis and its inhibition by acetate implicated one, or possibly two, active site residues in deacylation with pK/sub a/ values of 6.7 and 5.0. The pH-dependence of the enzyme-catalyzed exchange of oxygen between sodium [1-13C, 18O2] acetate and water suggested acetic acid as the preferred substrate for exchange, while rate data supported the role of an induced-fit rate-limiting step involving a virtual transition state in catalysis

  3. Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

    Olsen, Line; Bressendorff, Simon; Troelsen, Jesper T;

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the...... of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear...... extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4...

  4. INHIBITION OF PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden particulate matter inhibits protein tyrosine phosphatase activity in HAEC and leads to Src-dependent activation of EGFR sign...

  5. Activity of alkaline phosphatase and its fractions in blood serum of rats with experimental hyperthyroidism, kept in a radionuclide contaminated area

    In studies on male rats, with experimentally induced thyrotoxicosis a modifying effect of the stay in radioactively contaminated zone on the total alkaline phosphatase activity was shown. The data indicate disturbances in calcium-phosphoric metabolism, resulting in pronounced deviations from the normal range in the activity of thermolabile (bone) fraction (authors)

  6. Is the release of acid phosphatases by ectomycorrhizal fungi a matter of environmental conditions or species in situ?

    Plassard, Claude; Ali, Muhhammad A.; Duchemin, Myriam; Legname-Vonarx, Elvira; Louche, Julien; Cloutier-Hurteau, Benoît

    2011-01-01

    Ectomycorrhizal (ECM) fungi are able to release significant amounts of phosphatases (Pases) in their environment. These enzymes, by releasing the phosphate group of organic P, may play an important role in the recycling of P in forest soil. However, whether this enzyme release depends on the fungal diversity and / or the nutrient availability is not known. We addressed this question in the maritime pine (Pinus pinaster) forest, the first planted area in France on sandy podzol very poor in ...

  7. Integrin α1β1 Promotes Caveolin-1 Dephosphorylation by Activating T Cell Protein-tyrosine Phosphatase*

    Borza, Corina M.; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R.; Zent, Roy; Pozzi, Ambra

    2010-01-01

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  8. Integrin {alpha}1{beta}1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase.

    Borza, Corina M; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R; Zent, Roy; Pozzi, Ambra

    2010-12-17

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  9. Alkaline phosphatase activity at the southwest coast of India: A comparison of locations differently affected by upwelling

    Mamatha, S. S.; Malik, Ashish; Varik, Sandesh; Parvathi, V.; Jineesh, V. K.; Gauns, Mangesh U.; LokaBharathi, P. A.

    2015-01-01

    The realization of the potential importance of phosphorus (P) as a limiting nutrient in marine ecosystem is increasing globally. Hence, the contribution of biotic variables in mobilizing this nutrient would be relevant especially in productive coastal waters. As alkaline phosphatase activity (APA) indicates the status of P for primary production in aquatic environments, we asked the following question: is the level of APA indicative of P sufficiency or deficiency in coastal waters, especially, where upwelling is a regular phenomenon? Therefore, we have examined the total APA, chlorophyll a along with phosphatase producing bacteria (PPB) and related environmental parameters from nearshore to offshore in coastal waters off Trivandrum and Kochi regions differently affected by upwelling during the onset of monsoon. Off Trivandrum, APA in the offshore waters of 5-m layer at 2.23 μM P h- 1 was > 4 times higher than nearshore. Thus, low APA could be indicative of P sufficiency in coastal waters and higher activity suggestive of deficiency in offshore waters off Trivandrum. In contrast, there was less difference in APA between near and offshore surface waters off Kochi. Our results show that the regions differently affected by upwelling respond differently according to ambient P concentration, distance from shore or depth of water. These observations could apparently be applicable to other coastal systems as well, where gradients in upwelling and phosphate runoff have been noticed. Further studies on other transects would throw more light on the extent and direction of the relationship between APA and ambient P concentration. Such studies would help in understanding the level of control of this nutrient on the productivity of coastal waters.

  10. Effects of nitrogen fertilization on soil nutrient concentration and phosphatase activity and forage nutrient uptake from a grazed pasture system.

    Dillard, Sandra Leanne; Wood, Charles Wesley; Wood, Brenda Hall; Feng, Yucheng; Owsley, Walter Frank; Muntifering, Russell Brian

    2015-05-01

    Over a 3-year period, the effect of differing N-application regimes on soil extractable-P concentration, soil phosphatase activity, and forage P uptake in a P-enriched grazed-pasture system was investigated. In the fall of each year, six 0.28-ha plots were overseeded with triticale ( × Triticosecale rimpaui Wittm.) and crimson clover (Trifolium incarnatum) into a tall fescue (Lolium arundinacea)/bermudagrass (Cynodon dactylon) sod and assigned to 1 of 3 N-fertilizer treatments (n = 2): 100% of N recommendation in a split application (100N), 50% in a single application (50N), and 0% of N recommendation (0N) for triticale. Cattle commenced grazing the following spring and grazed until May. In the summer, plots were overseeded with cowpea (Vigna unguiculata), fertilized at the same rates by reference to N recommendations for bermudagrass, and grazed by cattle until September. There were no effects of N fertilization on soil phosphatase activity, electrical conductivity, or concentrations of water-soluble P. Concentrations of extractable P decreased in plots receiving 50N, but increasing N fertilization to 100N resulted in no further reduction in extractable P. Forage biomass, foliar P concentrations, and forage P mass were not affected by N fertilization rates at the plant-community level, but responses were observed within individual forage species. Results are interpreted to mean that N fertilization at 50% of the agronomic recommendation for the grass component can increase forage P mass of specific forages and decrease soil extractable P, thus providing opportunity for decreasing P losses from grazed pasture. PMID:25728918

  11. Nutrient addition modifies phosphatase activities along an altitudinal gradient in a tropical montane forest in Southern Ecuador

    Karla eDietrich

    2016-02-01

    Full Text Available Atmospheric nutrient deposition and climate change are expected to endanger the diversity of tropical forest ecosystems. Nitrogen (N deposition might influence nutrient fluxes beyond the N cycle by a concomitant increased demand for other nutritional elements such as phosphorus (P. Organisms might respond to the increased P demand by enhanced activity of enzymes involved in releasing inorganic P from organic matter (OM. Our aims were to assess the effect of i climate shifts (approximated by an altitudinal gradient, and ii nutrient addition (N, P, N+P on phosphatase activity (PA in organic layer and mineral soil of a tropical montane rainforest in Southern Ecuador. A nutrient manipulation experiment (NUMEX was set up along an altitudinal gradient (1000, 2000, and 3000 m a.s.l.. We determined PA and inorganic and total P concentrations. PA at 1000 m was significantly lower (mean ± standard error: 48 ± 20 µmol p-NP g-1 dm h-1 as compared to 2000 m and 3000 m (119 ± 11 and 137 ± 19, respectively. One explanation might be that very rapid decomposition of OM at 1000 m results in very thin organic layers reducing the stabilization of enzymes and thus, resulting in leaching loss of enzymes under the humid tropical climate. We found no effect of N addition on PA neither in the organic layer nor in mineral soil, probably because of the low nutrient addition rates that showed ambiguous results so far on productivity measures as a proxy for P demand. In the organic layers of P and N+P treatments, we found decreased PA and increased concentrations of inorganic P. This indicates that the surplus of inorganic P reduced the biosynthesis of phosphatase enzymes. PA in megadiverse montane rainforests is likely to be unaffected by increased atmospheric N deposition but reduced upon atmospheric P deposition.

  12. A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2 beta)

    Drake, P.G.; Peters, Günther H.j.; Andersen, H.S.;

    2003-01-01

    Islet-cell antigen 512 (IA-2) and phogrin (IA-2) are atypical members of he receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent studies...... indicate that IA-2 may be involved in granule trafficking and exocytosis. To further 9 understand their function, we have embarked upon developing low-molecular-mass inhibitors of IA-2 and IA-2. Previously, we have shown that a general PTP inhibitor, 2-(oxalylamino)benzoic acid (OBA), can be developed into...

  13. Alterations in Co2 fixation enzymes, Phosphatase Activity and Endogenous Phytohormones in P-deficient Callus induced from phloem of carrot (Daccus Carota) Roots

    A carrot callus liquid medium culture experiment was conducted to investigate the effects of P-deficiency on cellular responses separate from the whole plant response. Carrot (Daccus carota L.) callus was induced from the secondary phloem of the tap root. When explants were supplied with one-tenth the amount of Pi supplied to control explants (40 ppm), the concentration of P in callus was reduced by about 68% in a period of three weeks. This reduction in callus P was correlated with 48% reduction in callus fresh and dry weights. This effect was mediated through a reduction in cell number/callus by 48%. Meanwhile, the cell number/mg f.wt. of callus tissue was not affected in P-deficient treatment comparing to P-sufficient one, which might refer to a direct role of P-deficiency on the reduction of cell division. Although total N and soluble protein concentrations were not affected in P-deficient callus, chlorophyll concentration was reduced. In addition higher activity of acid phosphatase was obtained in P-deficient tissue reaching about 41% over its activity in P-sufficient callus which in turn could increase recycling process of P to spare available P for the newly formed cells. This was supported by the higher value of P utilization efficiency (d.wt. produced per unit P taken up) obtained from P-deficient callus

  14. Facile and Sensitive Fluorescence Sensing of Alkaline Phosphatase Activity with Photoluminescent Carbon Dots Based on Inner Filter Effect.

    Li, Guoliang; Fu, Huili; Chen, Xuejie; Gong, Peiwei; Chen, Guang; Xia, Lian; Wang, Hua; You, Jinmao; Wu, Yongning

    2016-03-01

    A simple and sensitive fluorescent assay for detecting alkaline phosphatase (ALP) based on the inner filter effect (IFE) has been proven, which is conceptually different from the previously reported ALP fluorescent assays. In this sensing platform, N-doped carbon dots (CDs) with a high quantum yield of 49% were prepared by one-pot synthesis and were directly used as a fluorophore in IFE. p-Nitrophenylphosphate (PNPP) was employed to act as an ALP substrate, and its enzyme catalytic product (p-nitrophenol (PNP)) was capable of functioning as a powerful absorber in IFE to influence the excitation of fluorophore (CDs). When in the presence of ALP, PNPP was transformed into PNP and induced the absorption band transition from 310 to 405 nm, which resulted in the complementary overlap between the absorption of PNP and the excitation of CDs. Because of the competitive absorption, the excitation of CDs was significantly weakened, resulting in the quenching of CDs. The present IFE-based sensing strategy showed a good linear relationship from 0.01 to 25 U/L (R(2) = 0.996) and provided an exciting detection limit of 0.001 U/L (signal-to-noise ratio of 3). The proposed sensing approach was successfully applied to ALP sensing in serum samples, ALP inhibitor investigation and phosphatase cell imaging. The presented IFE-based CDs fluorescence sensing strategy gives new insight on the development of the facile and sensitive optical probe for enzyme activity assay because the surface modification or the linking between the receptor and the fluorophore is no longer required. PMID:26820049

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

    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.

  16. Studies on the Consequences of the Administration of Aqueous Extracts of Azadiracta Indica and Morinda Lucida on the Heamatological Parameter and the Activities of Phosphatases in Rats Cellular Tissues

    Ejembi Daniel

    2012-08-01

    Full Text Available Studies on the effects of the combination of aqueous extracts of Azadiracta indica and Morinda lucida on packed cell volume(PCV and the activities of phosphatases of rat cellular tissues was investigated. Twenty eight (28 albino rats of six month old of an average weight 182.4g were grouped in to four groups; A, B, C and D. All the rats in the four groups were fed with fowl formulation feeds obtained from poultry store in VOM Jos, Plateau State, Nigeria and water was provided ad libitum for 18 days. Rats in group A, B, C were administered 0.5ml/kg body wt each of A.indica extract, only M.lucida extract, and combination of A.indica and M.lucida extracts orally respectively for 18 days. Group D contained seven rats designated “control”. One of the rats from the control was sacrificed at day one, its cellular tissues (liver, kidney, small intestine, and large intestine were collected and analyzed for PCV, Alkaline Phosphatase (ALP and Acid Phosphatase (ACP activities. One of the rats from each of the groups A, B, C and D were sacrificed at every three days interval and these tissues were also collected and analyzed for PCV, ALP, and ACP activities. The result showed a significant decrease in the activities of these enzymes as compared to the control values. The trends of the activities of these enzymes were similar following repeated administration of the extracts, and thereby caused alterations in the activities of both enzymes. This trend when subjected to student t-test analysis, showed significant difference at P>0.05.

  17. Monitoring the activity and inhibition of alkaline phosphatase via quenching and restoration of the fluorescence of carbon dots

    We report that the fluorescence of carbon dots (C-dots) in water is quenched by the addition of Cu2+ ions, and that the subsequent addition of pyrophosphate (PPi) restores fluorescence. This is likely to be due to the coordination of Cu2+ by PPi. This effect forms the basis for a method to determine the activity and inhibition of the enzyme alkaline phosphatase (ALP). If ALP is added to a system composed of C-dots, Cu2+ and PPi, fluorescence will decrease over time because ALP catalyzes the hydrolysis of PPi to form orthophosphate (Pi). This results in a release of the quencher Cu2+. The decrease in fluorescence is related to the activity of ALP. The method is simple and displays good sensitivity (with a limit of detection of 1 units per L) and selectivity. The method was successfully applied to the determination of ALP in serum samples. We also have studied the inhibitory effect of Pi on the activity of ALP. We presume that this method holds a large potential in terms of diagnosis of ALP-related diseases, to evaluate the function of ALP in biological systems and in screening for potential inhibitors of ALP. (author)

  18. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  19. Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

    Knodler, Leigh A

    2009-11-01

    The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

  20. Assessment of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase activities in cow's milk as an indicator of subclinical mastitis.

    Babaei, H; Mansouri-Najand, L; Molaei, M M; Kheradmand, A; Sharifan, M

    2007-05-01

    This study examined the activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in the milk of lactating Holstein cows in association with subclinical mastitis (SCM). A total of 94 milk samples were collected from 58 lactating dairy cows representing stages of lactation from the second to the tenth week after calving. Those which were classified as positive by California mastitis test (CMT) were deemed to have subclinical mastitis. All the milk samples were skimmed by centrifugation at 10 000g at 0 degrees C and were used for enzyme activities estimations. The mean activities of LDH and ALP were higher in the milk from udders with SCM than in the milk from healthy udders (p CMT results and LDH and ALP values were seen at thresholds of > 180 IU/L and > 40 IU/L respectively (kappa values 0.65 and 0.79, respectively). However, the sensitivity of the tests for identifying SCM at these thresholds was higher for ALP (96.4%) than for LDH (68.5%). In this study, LDH and ALP tests were standardized for cow's milk and results showed that only the ALP test was reliable in the early diagnosis of subclinical mastitis. PMID:17268916

  1. The mitogen-activated protein kinase (MAPK) cascade controls phosphatase and tensin homolog (PTEN) expression through multiple mechanisms.

    Ciuffreda, Ludovica; Di Sanza, Cristina; Cesta Incani, Ursula; Eramo, Adriana; Desideri, Marianna; Biagioni, Francesca; Passeri, Daniela; Falcone, Italia; Sette, Giovanni; Bergamo, Paola; Anichini, Andrea; Sabapathy, Kanaga; McCubrey, James A; Ricciardi, Maria Rosaria; Tafuri, Agostino; Blandino, Giovanni; Orlandi, Augusto; De Maria, Ruggero; Cognetti, Francesco; Del Bufalo, Donatella; Milella, Michele

    2012-06-01

    The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent "escape" mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. PMID:22215152

  2. Leishmanial phosphatase hydrolyzes phosphoproteins and inositol phosphates

    An extensively purified preparation of the predominant, tartrate-resistant acid phosphatase (ACP) from the external surface of Leishmania donovani promastigotes form catalyzes the dephosphorylation of several phosphoproteins; these include: pyruvate kinase, phosphorylase kinase and histones. However, the protein phosphatase activity of ACP is very low compared with that of other protein phosphates known to be involved in regulating various metabolic pathways. 32P-labelled inositoltriphosphate (IP3), a well-established second messenger derived from phosphatidylinositol-4,5-diphosphate (PIP2), was a substrate for the leishmanial acid phosphatase; incubation of the IP3 preparation with 13.2 milliunits (1 unit equals 1 μmol 4-methylumbelliferyl phosphate (MUP) cleaved per min at pH 5.5) of ACP at pH 5.5 for 4 hr resulted in hydrolysis of 75% of the radiolabelled substrate resulting in a mixture of inositoldiphosphate and inositolmonophosphate. In addition PIP2 was hydrolyzed rapidly by ACP at pH 5.5 (V/sub max/, 71 units/mg protein; k/sub m/, 4.16 μM). In contrast, to MUP which is hydrolzyed most rapidly at pH 5.5, PIP2 hydrolysis was optimal at pH 6.8. These observations raise the possibility that ACP could play a role in the host-phagocyte interaction by degrading the precursor of the second messenger, PIP2 or the second messenger itself, IP3

  3. Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua.

    Alvarez, Maricel; Godoy, Roberto; Heyser, Wolfgang; Härtel, Steffen

    2004-01-01

    We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. SBP activity was detected at different pH values (3-7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). In the cases of D. antartica, A. boletinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per μm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the discussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi. PMID:21148871

  4. Adiponectin haploinsufficiency promotes mammary tumor development in MMTV-PyVT mice by modulation of phosphatase and tensin homolog activities.

    Janice B B Lam

    Full Text Available BACKGROUND: Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, MMTV-polyomavirus middle T antigen (MMTV-PyVT transgenic mice with reduced adiponectin expressions were established and the stromal effects of adiponectin haploinsufficiency on mammary tumor development evaluated. In mice from both FVB/N and C57BL/6J backgrounds, insufficient adiponectin production promoted mammary tumor onset and development. A distinctive basal-like subtype of tumors, with a more aggressive phenotype, was derived from adiponectin haplodeficient MMTV-PyVT mice. Comparing with those from control MMTV-PyVT mice, the isolated mammary tumor cells showed enhanced tumor progression in re-implanted nude mice, accelerated proliferation in primary cultures, and hyperactivated phosphatidylinositol-3-kinase (PI3K/Akt/beta-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN activities. Further analysis revealed that PTEN was inactivated by a redox-regulated mechanism. Increased association of PTEN-thioredoxin complexes was detected in tumors derived from mice with reduced adiponectin levels. The activities of thioredoxin (Trx1 and thioredoxin reductase (TrxR1 were significantly elevated, whereas treatment with either curcumin, an irreversible inhibitor of TrxR1, or adiponectin largely attenuated their activities and resulted in the re-activation of PTEN in these tumor cells. Moreover, adiponectin could inhibit TrxR1 promoter-mediated transcription and restore the mRNA expressions of TrxR1. CONCLUSION: Adiponectin haploinsufficiency facilitated mammary tumorigenesis by down-regulation of PTEN activity and activation of PI3K

  5. Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis

    Ulm, Roman; Revenkova, Ekaterina; Di Sansebastiano, Gian-Pietro; Bechtold, Nicole; Paszkowski, Jerzy

    2001-01-01

    Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein k...

  6. Uranium Biomineralization As a Result of Bacterial Phosphatase Activity: Insights From Bacterial Isolates From a Contaminated Subsurface

    Uranium contamination is an environmental concern at the Department of Energy's Field Research Center in Oak Ridge, Tennessee. In this study, we investigated whether phosphate biomineralization, or the aerobic precipitation of U(VI)-phosphate phases facilitated by the enzymatic activities of microorganisms, offers an alternative to the more extensively studied anaerobic U(VI) bioreduction. Three heterotrophic bacteria isolated from FRC soils were studied for their ability to grow and liberate phosphate in the presence of U(VI) and an organophosphate between pH 4.5 and 7.0. The objectives were to determine whether the strains hydrolyzed sufficient phosphate to precipitate uranium, to determine whether low pH might have an effect on U(VI) precipitation, and to identify the uranium solid phase formed during biomineralization. Two bacterial strains hydrolyzed sufficient organophosphate to precipitate 73-95% total uranium after 120 h of incubation in simulated groundwater. The highest rates of uranium precipitation and phosphatase activity were observed between pH 5.0 and 7.0. EXAFS spectra identified the uranyl phosphate precipitate as an autunite/meta-autunite group mineral. The results of this study indicate that aerobic heterotrophic bacteria within a uranium-contaminated environment that can hydrolyze organophosphate, especially in low pH conditions, may play an important role in the bioremediation of uranium

  7. Protein phosphatase 2A in stretch-induced endothelial cell proliferation

    Murata, K.; Mills, I.; Sumpio, B. E.

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  8. Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

    Valsala Haridas

    Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

  9. Adiponectin Increases Skeletal Muscle Mitochondrial Biogenesis by Suppressing Mitogen-Activated Protein Kinase Phosphatase-1

    Qiao, Liping; Kinney, Brice; Yoo, Hyung sun; Lee, Bonggi; Schaack, Jerome; Shao, Jianhua

    2012-01-01

    Adiponectin enhances mitochondrial biogenesis and oxidative metabolism in skeletal muscle. This study aimed to investigate the underlying mechanisms through which adiponectin induces mitochondrial biogenesis in skeletal muscle. Mitochondrial contents, expression, and activation status of p38 mitogen-activated protein kinase (MAPK) and PPARγ coactivator 1α (PGC-1α) were compared between skeletal muscle samples from adiponectin gene knockout, adiponectin-reconstituted, and control mice. Adenovi...

  10. Alkaline phosphatase and transaminase activity in rat liver and blood serum at delayed periods following external γ-irradiation combined with internal exposure to plutonium 239

    A study was made of activity of alkaline phosphatase and alanine- and aspartate aminotransferase in rat liver and blood serum at remote times after external γ-irradiation combined with internal exposure to 239Pu nitrate delivered in two chronically effective doses. The radionuclide was shown to be mainly responsible for the changes observed in activity of the enzymes under study. The degree to which the changes were manifest depended upon dose of plutonium administered

  11. Novel Mechanism for Suppression of Hyperpolarization-activated Cyclic Nucleotide-gated Pacemaker Channels by Receptor-like Tyrosine Phosphatase-α*

    Huang, Jianying; Huang, Aijie; Zhang, Qi; Lin, Yen-Chang; Yu, Han-Gang

    2008-01-01

    We have previously reported an important role of increased tyrosine phosphorylation activity by Src in the modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we provide evidence showing a novel mechanism of decreased tyrosine phosphorylation on HCN channel properties. We found that the receptor-like protein-tyrosine phosphatase-α (RPTPα) significantly inhibited or eliminated HCN2 channel expression in HEK293 cells. Biochemical eviden...

  12. Radioprotective activity of folic acid

    The radioprotective activity of folic acid has been studied using rat liver mitochondria membrane, protein and superoxide dismutase (SOD) as well as pBR 322 plasmid DNA as the model in vitro systems. The vitamin could effectively prevent the γ-ray induced lipid peroxidation as assessed by measuring thiobarbituric acid reactive substrates and protein carbonyl formation effectively. It also could also prevent radiation-induced damage of mitochondrial SOD and restore its level to normalcy Likewise; it prevented radiation-induced DNA strand breaks in a concentration dependent manner. The radioprotective activity could be attributed to its ability to scavenge the hydroxyl and superoxide radicals wherein its pseudo-phenolic moiety and C-9 methylene group play the key role. Radioprotective activity of a polysaccharide preparation from the Indian medicinal plant, Tinospora cordifolia Miers has been established using Saccharomyces cerevisiae X2180 strain as the in vivo test model. The entire activity could be attributed to the radical scavenging capacity of the preparation, as it did not enhance the expression of the protective enzymes, catalase and superoxide dismutase in the yeast cells. (author)

  13. Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

    Dhaese, Ingeborg; Lefebvre, Romain A

    2009-03-15

    The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP. PMID:19374871

  14. Seasonal dynamics, alkaline phosphatase activity and phosphate uptake of adnate and loosely attached epiphytes in an oligotrophic lake

    Burkholder, J.M.

    1986-01-01

    This research characterizes the seasonal dynamics of algal epiphytes throughout an annual cycle and examines potential major sources of algal phosphorus. In a phosphorus-limited hardware lake, epiphytic microflora consisted almost entirely of diatoms and blue-green algae, and community structure fluctuated temporally and spatially (with age of macrophyte tissue). Epiphytic ATP was greatest during initial colonization of new macrophyte ramets, and was not correlated with algal biomass except on oldest leaf tissue. Epiphytic chlorophyll was determined primarily by blue-green algal biomass, except on oldest leaf tissue and during colder periods. Epiphytic alkaline phosphatase activity on new and aging macrophyte leaves was significantly less than that on artificial plants, indicating that algae on natural plants were less phosphorus-limited and had obtained a portion of their phosphorus from the macrophytes. Collaborative research quantified the importance of macrophytes as a phosphorus source for algal epiphytes. The author examined the importance of position in determining nutrient availability within the epiphyte matrix. Following short-term assays, scanning electron microscopy (SEM)- and LM-autoradiography were used to directly compare uptake of /sup 33/P-phosphate from the water by intact adnate vs. loosely attached algae, respectively. Loosely attached algae took up significantly more phosphate from the water than did underlying adnate cells, after long-term community acclimation to either phosphorus-poor or phosphorus-enriched conditions.

  15. Distinct expression of alkaline phosphatase activity in epilimnetic bacteria: Implication for persistent DOC consumption in a P-limited reservoir

    Tseng, Y.; Kao, S.; Shiah, F.

    2013-12-01

    In a P-deficient system, P availability usually controls the microbial activity and thus the ecosystem function. Thingstad et al. (1997) first addressed a 'Malfunctioning Microbial-loop' theory, which stated that low bacterial production (BP) caused by insufficient nutrient supply would result in DOC accumulation in an oligotrophic ecosystem. In this study we re-examined the theory by conducting seasonal patterns and correlations among soluble reactive phosphate (SRP) and DOC, microbial abundances (picocyanobacteria, bacteria, and heterotrophic nanoflagellate; HNF) and activities (primary production, bacterial production, and alkaline phosphatase activity; APA) coupled with enzyme-labeled fluorescence (ELF) assays on bacterioplankton in a subtropical reservoir sharing the common features, nitrate-replete and P-deficient, with most natural freshwater system during Oct 2007-Oct 2008. Persistently high APA was recorded during most of time, implying that the system was P-deficient. Size fractionated APA and ELF assay revealed that bacteria were the major APA contributor. However, significantly low epilimnion DOC was recorded during the stratified summer season accompanying with high BP and APA as well as high PP, implying that heterotrophic bacteria can well sustain in P-deficient system by utilizing DOP to rapidly lower down DOC under relatively high PP. Such findings oppose the 'Malfunctioning Microbial-loop' theory. On the other hand, strong epilimnetic DOC accumulation occurred in Oct 2007 under low light and low PP condition accompanying with high abundance of HNF, implying that HNF grazing may contribute to a certain degree of DOC accumulation. Correlation matrix supported our suggestions. This study testified the DOC dynamics in P-deficient ecosystem are tightly coupled with the source (PP and grazing) and sink (BP). We also suggested that in SRP-limited freshwater systems bacteria are capable of breaking down autochthonous DOC to reduce the chance of DOC

  16. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

    2016-01-19

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  17. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

    2016-01-01

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  18. Amino Acid Decarboxylase Activity of Some Lactic Acid Bacteria

    Pelin ERTÜRKMEN; Turhan, İlkay; Öner, Zübeyde

    2015-01-01

    Microorganisms which have decarboxylase activity can form biogenic amine by enzymatic decarboxylation of amino acids in foods. Histamine poisoning results from consumption of foods typically certain types of fish and cheeses that contain unusually high levels of histamine. Therefore, decarboxylase activity is an important problem at the selection of lactic acid bacteria as a starter culture in fermented products. In this study, decarboxylase activities of 161 lactic acid bacteria (LAB) strain...

  19. Alkaline phosphatase activity in blood serum of dogs exposed to a mixture of external γ-radiation and internal α-radiation

    Alkaline phosphatase activity in dog blood serum was studied for two years following separate and combined exposure to gamma radiation (6.45 to 51.6 mc/kg) and inhaled submicron 239Pu oxide containing 25% 241Am in chronically effective amounts (approx. 7-10 kBq/kg). Alkaline phosphatase activity was of an ondulatory nature and the significance of changes depended on the kind and the level of radiation as well as the time lapsed from the start of the expose. With the combined exposure to gamma and alpha radiation in the doses used no enhancement of the effec twaas noted as compared with the action of each factor applied separately

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

    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

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

    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

  2. Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis)

    Guha, Anirban; Gera, Sandeep; Sharma, Anshu

    2012-01-01

    Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis ...

  3. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

    Seo, Hyun-Ju; Cho, Young-Eun; Kim, Taewan; Shin, Hong-In; Kwun, In-Sook

    2010-01-01

    Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentra...

  4. Evaluation of alkaline phosphatase activity and availability of various P fractions for bean (Phaseolus vulgaris in some calcareous soils amended with municipal sewage sludge

    T. Raeisi

    2014-07-01

    Full Text Available To evaluate the relationship of various P fractions and alkaline phosphatase activity with bean indices growing in 10 calcareous soils, amended with municipal sewage sludge from Chaharmahal-Va-Bakhtiari province, a greenhouse research was carried out. Soil samples were incubated for one month with sludge at a rate equivalent to 1% (w/w. Then, the P fractions, including P adsorbed by Fe and Al oxides (]NaOH+CB]-P, occluded P (CBD-P and P absorbed by Ca (HCl-P, were determined by Olsen and Summers' sequential fractionation procedure. Furthermore, total P, organic P and residual P were determined. Also, alkaline phosphatase activity was measured. A pot experiment in a completely randomized design with three replications in the ten soils was done to evaluate the bean plant indices. The results showed that the amount of P fractions decreased in the following order: HCl-P>residual-P>]NaOH+CB]-P > OP>CBD-P. The results also indicated that alkaline phosphatase activity was significantly correlated with CBD-P fraction, organic P and total P. In addition, significant correlations were found between ([NaOH+CB]-P and HCl-P and plant shoots. In general, the results of this research showed that P fractionation method appears to be a powerful tool to identify the P status and availability in the soils amended with sewage sludge.

  5. O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

    Ilg, T; Overath, P; Ferguson, M A; Rutherford, T; Campbell, D G; McConville, M J

    1994-09-30

    The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of

  6. An evaluation on the activity level of Aspartate aminotransferase and Alkaline phosphatase nzymes in peri-implant sulcus fluid

    Paknegad M. Assistant Professor

    2003-07-01

    Full Text Available Statement of Problem: The correlation between the activity of aspartate aminotransferase (AST and alkaline phosphatase (ALP enzymes in gingival sulcular fluid (GCF with inflammation and periodontal attachment loss has been proved, however there are not adequate studies about dental implants. Purpose: The aim of present study was to investigate the presence and activity level of AST & ALP and their correlation with pocket depth (PD and bleeding of peri-implant slcular fluid (PISF, and to evaluate the possibility of using these assessments as a diagnostic index in oral implantology. Material and Methods: In this study, 41 implants as test group and 41 contralateral teeth as control group, in 21 patients were evaluated. At first visit, the general information about implants and the values of pocket probing depth (PPD, modified sulcus bleeding index (mSBl and modified plaque index (mPI were recorded. At the second visit, samples of GCF/PISF were collected. AST & ALP activity was determined spectrophotometrically and data were analyzed by "t", "Mann-Whitney" tests and Pearson Spearman correlation coefficient."nResults: The results showed that there was a significant difference in the activity of AST between two study groups (P<0.0001. The average activity of ALP in test group was more than control group but the difference was not significant. After elimination of the confounding variables, the average AST in test group was 54.6 (S£=2.3 and in control groups was 44.8 (SE=2.3 (P=0.004. The average ALP in test group (SE=2.2 and in control (SE=2.2 were 36.6 and 35.4, respectively. Values of AST and ALP were positively correlated with other clinical parameters such as PD and mSBI which was significant in test group."nConclusion: The present study suggests that PISF analysis could be considered as a proper diagnostic strategy in the evaluation of dental implant success.

  7. ALP (Alkaline Phosphatase) Test

    ... Also known as: ALK PHOS; Alkp Formal name: Alkaline Phosphatase Related tests: AST ; ALT ; GGT ; Bilirubin ; Liver Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP All content on ...

  8. Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

    Ndong Christian

    2012-04-01

    Full Text Available Abstract Background Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide. Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. Conclusions Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

  9. Protein tyrosine phosphatase 1B inhibitory activity of Indonesian herbal medicines and constituents of Cinnamomum burmannii and Zingiber aromaticum.

    Saifudin, Azis; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2013-04-01

    We screened water and methanol extracts of 28 Indonesian medicinal plants for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Nine water extracts, i.e., Alstonia scholaris leaf, Blumea balsamifera, Cinnamomum burmannii, Cymbopogon nardus, Melaleuca leucadendra, Phyllanthus niruri, Piper nigrum, Syzygium aromaticum, and Sy. polyanthum, exhibited ≥70 % inhibition at 25 μg/mL, whereas 11 methanol extracts, i.e., Als. scholaris, Andrographis paniculata, B. balsamifera, Ci. burmannii, Curcuma heyneana, Glycyrrhiza glabra, M. leucadendra, Punica granatum, Rheum palmatum, Sy. polyanthum, and Z. aromaticum, exhibited ≥70 % inhibition at 25 μg/mL. Water extracts of B. balsamifera (IC50, 2.26 μg/mL) and M. leucadendra (IC50, 2.05 μg/mL), and methanol extracts of Ci. burmannii (IC50, 2.47 μg/mL), Pu. granatum (IC50, 2.40 μg/mL), and Sy. polyanthum (IC50, 1.03 μg/mL) exhibited strong inhibitory activity, which was comparable with that of the positive control, RK-682 (IC50, 2.05 μg/mL). The PTP1B inhibitory activity of the constituents of Ci. burmannii and Z. aromaticum was then evaluated. 5'-Hydroxy-5-hydroxymethyl-4″,5″-methylenedioxy-1,2,3,4-dibenzo-1,3,5-cycloheptatriene (2; IC50, 29.7 μM) and trans-cinnamaldehyde (5; IC50, 57.6 μM) were the active constituents of Ci. burmannii, while humulatrien-5-ol-8-one (21; IC50, 27.7 μM), kaempferol-3,4'-di-O-methyl ether (32; IC50, 17.5 μM), and (S)-6-gingerol (33; IC50, 28.1 μM) were those of Z. aromaticum. These results suggest that these medicinal plants may contribute to the treatment and/or prevention of type II diabetes and/or obesity through PTP1B inhibition. PMID:22645080

  10. Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells

    Movahedi Najafabadi, Bent-al-hoda; Abnosi, Mohammad Hussein

    2016-01-01

    Objective Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). Materials and Methods In this experimental study, BMSCs were extracted and expanded to the 3rdpassage, then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 µg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. Results Although 6 µg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. Conclusion Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture. PMID:27054120

  11. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    Einstein, R.; Gabel, C.A. (Columbia Univ. College of Physicians and Surgeons, NY (USA))

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  12. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated

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

    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…

  14. Expression and Characterization of Recombinant Thermostable Alkaline Phosphatase from a Novel Thermophilic Bacterium Thermus thermophilus XM

    Jianbo LI; Limei XU; Feng YANG

    2007-01-01

    A gene (tap) encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co2+, Fe2+, Mg2+, or Mn2+ but was strongly inhibited by 2.0 mM Fe2+. Under optimal conditions, the Michaelis constant (Km) for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.

  15. Acid Rain: Activities for Science Teachers.

    Johnson, Eric; And Others

    1983-01-01

    Seven complete secondary/college level acid rain activities are provided. Activities include overview; background information and societal implications; major concepts; student objectives; vocabulary/material lists; procedures; instructional strategies; and questions/discussion and extension suggestions. Activities consider effects of acid rain on…

  16. Biological activities of substituted trichostatic acid derivatives

    Cédric Charrier; Joëlle Roche; Jean-Pierre Gesson; Philippe Bertrand

    2009-07-01

    New substituted trichostatic acid derivatives have been synthesized and evaluated for their biological activities towards the H661 non-small lung cancer cell line. These syntheses were achieved by alkylation of propiophenones to introduce the side chain with a terminal precursor of hydroxamic acid and aminobenzamide derivatives. The first fluorinated derivatives of trichostatic acid are described, such as 6-fluoro trichostatin A, with antiproliferative activities in the micromolar range and with histone deacetylase inhibitory activity.

  17. ANTIMICROBIAL ACTIVITY OF LACTIC ACID BACTERIAL ISOLATES

    Utkarsha S. Shivsharan

    2013-08-01

    Full Text Available Micro-organisms have tendency to produce antimicrobial substances which show biological activity against other kind of micro-organisms. This phenomenon of bacterial antagonism is observed in lactic acid bacteria with competitive advantages. The lactic acid bacteria are commonly present in many fermented products, fruits and milk products. The variety of antimicrobial substances produced by lactic acid bacteria showing good inhibition capacity include production of lactic acid, acetic acid, hydrogen peroxide, carbon dioxide, diacetyl and bacteriocin. Bacteriocins produced by lactic acid bacteria are the subject of intense research because of their antimicrobial activity against food born bacteria such as Listeria monocytogenes, staphylococcus aureus, Bacillus cereus, Clostridium botulinum and several others .Bacteriocins may be bacteriostatic or bactericidal with narrow or broad range of activity. The main of the study was to study the antimicrobial activity of such lactic acid bacterial isolates.

  18. Comparison of the effects of eldecalcitol with either raloxifene or bisphosphonate on serum tartrate resistant acid phosphatase-5b, a bone resorption marker, in postmenopausal osteoporosis

    Takada, Junichi; Ikeda, Satoshi; Kusanagi, Tetsuya; Mizuno, Satoshi; Wada, Hiroshi; Iba, Kousuke; Yoshizaki, Takashi; Yamashita, Toshihiko

    2016-01-01

    Summary Objective This study analyzes whether concomitant raloxifene (RLX) or bisphosphonates (BP) plus eldecalcitol (ELD) has excessive suppressive effects on a bone resorption marker during the first 6 months of treatment in postmenopausal women in real-world setting. Methods 285 postmenopausal osteoporotic patients who had been treated with RLX or BP plus ELD were evaluated the bone resorption marker, serum tartrate resistant acid phosphatase-5b (TRACP-5b), during the first 6 months of treatment. Results In drug-naïve group (not received osteoporosis medications before the administration, n=70), the concomitant RLX or BP with ELD significantly decreased levels of TRACP-5b without severe suppression. In vitamin D switch group [RLX or BP plus alfacalcidol (ALF) and then switched to RLX or BP plus ELD, n=215], the replacing ALF with ELD further and significantly decreased TRACP-5b and tertile analyses based on baseline values were significantly decreased far more in the highest, compared with the lowest tertile in the ELD+RLX and ELD+BP groups. Conclusion ELD combined with RLX or BP administered for 6 months to postmenopausal women with osteoporosis who were drug-naïve or who had switched medications significantly reduced and maintained TRACP-5b values within the reference range. PMID:27252739

  19. Estudio de la fosfatasa ácida y alcalina en suelos de la Región Pampeana Norte del área sojera argentina Study of acid and alkaline phosphatase in soils of the Pampean North Region from argentine soybean area

    Leticia Andrea Fernández

    2008-07-01

    ón de ambos métodos, es posible estudiar la fosfatasa ácida y alcalina de un suelo y obtener información sobre el potencial del mismo para movilizar Po.Transformation of organic phosphorus (Po into soluble inorganic phosphorus (Pi is called mineralization and is carried out by phosphatase enzymes. The present research focuses on the study of the phosphatase activity of five soils from the soybean area of the Northern Pampean region, by evaluating the phosphatase activity in soil samples and the number of bacteria and fungi with that activity. Soil samples were collected and the total number and phosphatase activity of cultivated heterotrophic aerobic bacteria (CHAB and cultivated fungi (CF was assessed. No significant differences were observed in the numbers of CHAB and CH between the studied soils. The number of bacteria with acid phosphatase activity was 6.85 10(5 CFU g-1 soil, while alkaline activity was 5.80 10(5 CFU g-1 soil. In contrast, the number of fungi with acid phosphatase activity was 1.78 10³ CFU g-1 soil and with alkaline activity was 1.77 10³ CFU g-1 soil. No significant differences were observed in the number of bacteria and fungi with both enzymes. However, acid activity was higher than alkaline activity in soil samples. Alkaline phosphatase activity ranged from 5.72 to 15.5 mg p- nitrofenol kg-1 soil h-1 while acid activity varied from 27.4 to 10(5 mg p-nitrofenol kg-1 soil h-1. There were significant differences in phosphatase activity between the soybean soils. Our results show that the mineralization activities of Po sources are in agreement with other cultivated soils. On the other hand, the number of bacteria and fungi complements the information on soil phosphatase activity. Clearly, both methods allow the study of alkaline and acid phosphatase activity in soil and give information about the soil potential to mobilize Po.

  20. Biochemical effect of a histidine phosphatase acid (phytase) of Aspergillus japonicus var. Saito on performance and bony characteristics of broiler.

    Maller, Alexandre; de Quadros, Thays Cristina Oliveira; Junqueira, Otto M; Graña, Alfredo Lora; de Lima Montaldi, Ana Paula; Alarcon, Ricardo Fernandes; Jorge, João Atílio; de Lourdes T M Polizeli, Maria

    2016-01-01

    Phytases are enzymes that hydrolyze the ester linkage of phytic acid, releasing inositol and inorganic phosphate. The phytic acid (phytate) is a major form of phosphorus in plant foods. Knowing that diet for animal of production has the cereal base (corn and soybean), primarily, broilers need for an alternative to use of the phosphate present in these ingredients, since it does not naturally produce the enzyme phytase, which makes it available. The aims of this work was studding the safe supplementation of Aspergillus japonicus var. Saito crude phytase in feeding broilers and check the biochemical effect on performance and bones of these animals. The enzymatic extract did not have aflatoxins B1, B2, G2 and G1 and zearalenone and ochratoxin, and low concentrations of this extract did not have cytotoxic effects on cells derived from lung tissue. The in vivo experiments showed that the phytase supplied the available phosphate reduction in the broiler feed formulation, with a live weight, weight gain, feed intake, feed conversion, viability, productive efficiency index and carcass yield similar to the control test. Furthermore, the phytase supplementation favored the formation of bone structure and performance of the broilers. The results show the high biotechnological potential of A. japonicus phytase on broiler food supplementation to reduce phosphorus addition in the food formulation. So, this enzyme could be used as a commercial alternative to animal diet supplementation. PMID:27625972

  1. Theophylline Represses IL-8 Secretion from Airway Smooth Muscle Cells Independently of Phosphodiesterase Inhibition. Novel Role as a Protein Phosphatase 2A Activator.

    Patel, Brijeshkumar S; Rahman, Md Mostafizur; Rumzhum, Nowshin N; Oliver, Brian G; Verrills, Nicole M; Ammit, Alaina J

    2016-06-01

    Theophylline is an old drug experiencing a renaissance owing to its beneficial antiinflammatory effects in chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Multiple modes of antiinflammatory action have been reported, including inhibition of the enzymes that degrade cAMP-phosphodiesterase (PDE). Using primary cultures of airway smooth muscle (ASM) cells, we recently revealed that PDE4 inhibitors can potentiate the antiinflammatory action of β2-agonists by augmenting cAMP-dependent expression of the phosphatase that deactivates mitogen-activated protein kinase (MAPK)-MAPK phosphatase (MKP)-1. Therefore, the aim of this study was to address whether theophylline repressed cytokine production in a similar, PDE-dependent, MKP-1-mediated manner. Notably, theophylline did not potentiate cAMP release from ASM cells treated with the long-acting β2-agonist formoterol. Moreover, theophylline (0.1-10 μM) did not increase formoterol-induced MKP-1 messenger RNA expression nor protein up-regulation, consistent with the lack of cAMP generation. However, theophylline (at 10 μM) was antiinflammatory and repressed secretion of the neutrophil chemoattractant cytokine IL-8, which is produced in response to TNF-α. Because theophylline's effects were independent of PDE4 inhibition or antiinflammatory MKP-1, we then wished to elucidate the novel mechanisms responsible. We investigated the impact of theophylline on protein phosphatase (PP) 2A, a master controller of multiple inflammatory signaling pathways, and show that theophylline increases TNF-α-induced PP2A activity in ASM cells. Confirmatory results were obtained in A549 lung epithelial cells. PP2A activators have beneficial effects in ex vivo and in vivo models of respiratory disease. Thus, our study is the first to link theophylline with PP2A activation as a novel mechanism to control respiratory inflammation. PMID:26574643

  2. Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model

    Karamperis, N; Koefoed-Nielsen, P B; Marcussen, N;

    2008-01-01

    it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various...... as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection....... postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity...

  3. Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF.

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K

    2016-01-01

    Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events. PMID:27078017

  4. Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

    Zhang Xue

    2006-07-01

    Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

  5. Sorafenib Inhibits Signal Transducer and Activator of Transcription-3 Signaling in Cholangiocarcinoma Cells by Activating the Phosphatase Shatterproof 2

    Blechacz, Boris R. A.; Smoot, Rory L.; Bronk, Steven F; Werneburg, Nathan W.; Sirica, Alphonse E.; Gores, Gregory J.

    2009-01-01

    The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is one of the key signaling cascades in cholangiocarcinoma (CCA) cells, mediating their resistance to apoptosis. Our aim was to ascertain if sorafenib, a multikinase inhibitor, may also inhibit JAK/STAT signaling and, therefore, be efficacious for CCA. Sorafenib treatment of three human CCA cell lines resulted in Tyr705 phospho-STAT3 dephosphorylation. Similar results were obtained with the Raf-kinase inhibit...

  6. A Chronoamperometric Screen Printed Carbon Biosensor Based on Alkaline Phosphatase Inhibition for W(VI Determination in Water, Using 2-Phospho-l-Ascorbic Acid Trisodium Salt as a Substrate

    Ana Lorena Alvarado-Gámez

    2015-01-01

    Full Text Available This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3, a repeatability of 9.4% (n = 3 and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition.

  7. Lipoxygenase inhibitory activity of anacardic acids.

    Ha, Tae Joung; Kubo, Isao

    2005-06-01

    6[8'(Z)-pentadecenyl]salicylic acid, otherwise known as anacardic acid (C15:1), inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) with an IC50 of 6.8 microM. The inhibition of the enzyme by anacardic acid (C15:1) is a slow and reversible reaction without residual activity. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:1) is a competitive inhibitor and the inhibition constant, KI, was obtained as 2.8 microM. Although anacardic acid (C15:1) inhibited the linoleic acid peroxidation without being oxidized, 6[8'(Z),11'(Z)-pentadecadienyl]salicylic acid, otherwise known as anacardic acid (C15:2), was dioxygenated at low concentrations as a substrate. In addition, anacardic acid (C15:2) was also found to exhibit time-dependent inhibition of lipoxygenase-1. The alk(en)yl side chain of anacardic acids is essential to elicit the inhibitory activity. However, the hydrophobic interaction alone is not enough because cardanol (C15:1), which possesses the same side chain as anacardic acid (C15:1), acted neither as a substrate nor as an inhibitor. PMID:15913294

  8. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  9. Using "On/Off" (19)F NMR/Magnetic Resonance Imaging Signals to Sense Tyrosine Kinase/Phosphatase Activity in Vitro and in Cell Lysates.

    Zheng, Zhen; Sun, Hongbin; Hu, Chen; Li, Gongyu; Liu, Xiaomei; Chen, Peiyao; Cui, Yusi; Liu, Jing; Wang, Junfeng; Liang, Gaolin

    2016-03-15

    Tyrosine kinase and phosphatase are two important, antagonistic enzymes in organisms. Development of noninvasive approach for sensing their activity with high spatial and temporal resolution remains challenging. Herein, we rationally designed a hydrogelator Nap-Phe-Phe(CF3)-Glu-Tyr-Ile-OH (1a) whose supramolecular hydrogel (i.e., Gel 1a) can be subjected to tyrosine kinase-directed disassembly, and its phosphate precursor Nap-Phe-Phe(CF3)-Glu-Tyr(H2PO3)-Ile-OH (1b), which can be subjected to alkaline phosphatase (ALP)-instructed self-assembly to form supramolecular hydrogel Gel 1b, respectively. Mechanic properties and internal fibrous networks of the hydrogels were characterized with rheology and cryo transmission electron microscopy (cryo-TEM). Disassembly/self-assembly of their corresponding supramolecular hydrogels conferring respective "On/Off" (19)F NMR/MRI signals were employed to sense the activity of these two important enzymes in vitro and in cell lysates for the first time. We anticipate that our new (19)F NMR/magnetic resonance imaging (MRI) method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance. PMID:26901415

  10. VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

    James D Swarbrick

    Full Text Available VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+ and Zn(2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

  11. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes.

    Ahmad, Jawid Nazir; Cerny, Ondrej; Linhartova, Irena; Masin, Jiri; Osicka, Radim; Sebo, Peter

    2016-03-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host. PMID:26334669

  12. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone

  13. Rapid kinetics of liver microsomal glucose-6-phosphatase. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity

    Rapid kinetics of both glucose-6-P uptake and hydrolysis in fasted rat liver microsomes were investigated with a recently developed fast-sampling, rapid-filtration apparatus. Experiments were confronted with both the substrate transport and conformational models currently proposed for the glucose-6-phosphatase system. Accumulation in microsomes of 14C products from [U-14C]glucose-6-P followed biexponential kinetics. From the inside to outside product concentrations, it could be inferred that mostly glucose should accumulate inside the vesicles. While biexponential kinetics are compatible with the mathematical predictions of a simplified substrate transport model, the latter fails in explaining the burst in total glucose production over a similar time scale to that used for the uptake measurements. Since the initial rate of the burst phase in untreated microsomes exactly matched the steady-state rate of glucose production in detergent-treated vesicles, it can be definitely concluded that the substrate transport model does not describe adequately our results. While the conformational model accounts for both the burst of glucose production and the kinetics of glucose accumulation into the vesicles, it cannot explain the burst in 32Pi production from [32P]glucose-6-P measured under the same conditions. Since the amplitude of the observed bursts is not compatible with a presteady state in enzyme activity, we propose that a hysteretic transition best explains our results in both untreated and permeabilized microsomes, thus providing a new rationale to understand the molecular mechanism of the glucose-6-phosphatase system

  14. Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde: Involvement of Lysine and Histidine Residues at the Active Site

    CHEN Hongtao; XIE Liping; YU Zhenyan; ZHANG Rongqing

    2005-01-01

    Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde (OPA). The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 (mmol/L)-1·min-1 at pH 7.5 and 25°C. A Tsou's plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.

  15. EFFECTS OF GLYPHOSATE AMMONIUM SALT ON THE BIOAVAILABLE PHOSPHORUS CONTENT AND THE ACTIVITY OF SELECTED PHOSPHATASES IN LOAMY SAND

    Maciej Płatkowski

    2015-07-01

    Full Text Available The aim of this study was to determine the effects of glyphosatę ammonium salt on the activity of some enzymes involved in the metabolism of phosphorus in the soil: acid phosphomonoeaterase (EC 3.1.3.2, alkaline phosphomonoeaterase (EC 3.1.3.1, phosphotrieaterase (EC 3.1.5.1, inorganic pyrophosphatase (EC 3.1.6.1, and a phosphorus content in a form available to plants. The experiment was carried out on loamy sand samples with organic carbon content 8.7 g kg-1. Into soil samples the aqueous solutions of Avans Premium 360 SC (360 g glyphosate ammonium salt in 1 dm3 were added. The amount of introduced glyphosate ammonium salt was 0 (control, 1, 10, 50 and 100 mg·kg-1, on days 0 (1 hour after glyphosate application, 7, 14, 28 and 56 measured parameters were determined spectrophotometrically. The obtained results showed that the application of glyphosate ammonium salt resulted in changes of available phosphorus content and the activity of enzymes involved in the metabolism of this element in loamy sand. The effects glyphosate ammonium salt dosage and effect of day of experiment were ambiguous. Among the determined parameters the most sensitive to the presence of the glyphosate ammonium was alkaline phosphomonoesterase.

  16. Effects of sediment and turbulence on alkaline phosphatase activity and photosynthetic activity of phytoplankton in the shallow hyper-eutrophic Lake Taihu, China.

    Ding, Yanqing; Qin, Boqiang; Xu, Hai; Wang, Xiaodong

    2016-08-01

    Sediments play important roles, as nutrient reservoir, especially in shallow lake ecosystem. The water column of large shallow lakes is often stable but also disturbed by turbulence causing resuspension of sediments. While considerable research has been carried out to investigate the influence of sediment resuspension on nutrient release, fewer studies have been done to understand the contribution of alkaline phosphatase activity (APA) in water as a response to the two conditions (turbulence and stability). Also, effects of the two lake conditions on photosynthetic efficiency of phytoplankton are still poorly understood. This study will evaluate the effect of these two conditions on photosynthetic efficiency and APA. Sediments used in the indoor experiments were collected from Zhushan Bay in Lake Taihu. Turbulence was generated by rotors to simulate the strong wind-induced disturbance in Lake Taihu. Results of the experiments showed that TN and TP in the stable and episodically turbulent conditions were not significantly different, with TN ranging from 1.34 to 1.90 mg/L and TP from 0.08 to 0.18 mg/L. Whereas, the soluble reactive phosphorus in the episodically turbulent condition was significantly higher than in the stable condition. Episodic turbulence could enhance P cycling by resuspending sediment-associated P, which alleviated algal P limitation. In stable conditions, P deficiency induced the production of high APA, which enhanced the availability of P. Although episodic turbulence could also cause increased algal biomass, photosynthetic efficiency of the algae was also affected not only by the nutrients but also by many other factors, especially light availability. Our results suggest that episodic turbulence is an important driver of biogeochemical cycling in large shallow hypertrophic lake ecosystem. PMID:27151245

  17. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    Xi'en Chen

    2013-12-01

    Full Text Available Protein phosphatase 5 (PP5 is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5 was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5 was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

  18. Capric Acid Inhibits NO Production and STAT3 Activation during LPS-Induced Osteoclastogenesis

    Park, Eun-Jung; Kim, Sun A.; Choi, Yong-Min; Kwon, Hyuk-Kwon; Shim, Wooyoung; Lee, Gwang; Choi, Sangdun

    2011-01-01

    Capric acid is a second medium-chain fatty acid, and recent studies have shown that fatty acids are associated with bone density and reduce bone turnover. In this study, we investigated the effects of capric acid on lipopolysaccharide (LPS)-induced osteoclastogenesis in RAW264.7 cells. After treatment with capric acid (1 mM), the number of tartrate resistant acid phosphatase (TRAP)-positive cells decreased significantly. Capric acid reduced LPS-induced TRAP expression, an osteoclast different...

  19. Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

    CHAO Chen; HUANG Gan; LI Xia; YANG Lin; LIN Jian; JIN Ping; LUO Shuo-ming

    2013-01-01

    Background Glutamic acid decarboxylase antibody (GADA) and protein tyrosine phosphatase antibody (IA-2A) are two major autoantibodies,which exert important roles in the process of type 1 diabetes mellitus (T1D).Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.Methods Two hundreds and forty-seven Chinese newly diagnosed acute-onset T1D patients were consecutively recruited.GADA and IA-2A were detected at the time of diagnosis,one year later,3-5 years later after diagnosis during the follow-up; all the clinical data were recorded and analyzed as well.Results During the course of acute-onset T1D,the majority of patients remained stable for GADA or IA-2A,however,a few patients changed from positivity to negativity and fewer patients converted from negativity to positivity.The prevalence of GADA was 56.3% at diagnosis,decreasing to 50.5% one year later,and 43.3% 3-5 years later while the corresponding prevalence of IA-2A were 32.8%,31.0% and 23.3%,respectively.The median GADA titers were 0.0825 at diagnosis,declining to 0.0585 one year later and 0.0383 3-5 years later (P <0.001),while the corresponding median titers were 0.0016,0.0010,0.0014 for IA-2A,respectively.Fasting C-peptide (FCP) and postprandial C-peptide 2 hours (PCP2h)levels of GADA or IA-2A negativity persistence patients were higher than those of positivity persistence and negativity conversion patients (P <0.05) which indicated GADA or IA-2A negativity persistence T1D patients had a less loss of β cell function.Conclusion Our data suggest that repeated detection of GADA and IA-2A are necessary for differential diagnosis of autoimmune diabetes and the indirect prediction of the β cell function in Chinese patients.

  20. Searching for the role of protein phosphatases in eukaryotic microorganisms

    da-Silva A.M.

    1999-01-01

    Full Text Available Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively. Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

  1. Effect of three insecticides on soil phosphatase activity%3种杀虫剂对土壤磷酸酶活性的影响

    郭明; 张小萍; 曹玉

    2001-01-01

    Inhibiting effect of three pesticides (furadan,aldicarb,bifenthrin) on soil phosphatase activity was studied.The different dose,incubating time,phenyl phosphate dinatrium dose levels and adding buffer solution with different PН were experimented and analyzed.The results showed that all the three treatme nts can inhibit the activity of soil phosphatase,and the activity of soil ph osphatase was significantly affected by the doses of pesticides and phenyl phosphate dinatrium (R>0.990 0).It also implied that there was a possibility that agricu ltural chemicals would affect the agrological environment.%研究了呋喃丹、铁灭克、天王星3种农药对土壤磷酸酶活性的抑制作用。结果表明,3种农药在不同农药用量、培养时间、磷酸苯二钠用量及外加不同pН值缓冲溶液时,对磷酸酶活性均有不同程度的抑制作用。同时,在该条件下进行回归分析建模时发现不同农药用量、磷酸苯二钠用量对磷酸酶活性影响的相关系数均大于\\{0.990 0\\}。证明了在本地区特有的农业环境条件下,这3种农药可能会影响和破坏农业生态环境。

  2. Variation of Alkaline Phosphatase Activity in Sediments of Shrimp Culture Ponds and Its Relationship with the Contents of C, N and P

    SU Yuepeng; MA Shen; DONG Shuanglin

    2005-01-01

    Nine enclosures (5 m × 5 m) were built in a Fenneropenaeus chinensis culture pond of Rushan Gulf in April, 2001.The probiotics and BIO ENERGIZER solution were applied for disparate treatments. Variations of alkaline phosphatase activity (APA) and its relationship with the contents of C, N and P in sediments were studied. Results show that APA of sediments increases from 3.096 nmol g-1min-1 to 5.407 nmol g-1nin-1 in culture period; the bacteria biomass is not the only factor to determine APA; the contents of total P and total organic carbon have a significant positive correlation with APA, while that of total nitrogen has a negative correlation. In addition, the contents of inorganic P and organic P are not regular with APA. By comparison, TOC shows a more significant coherence with APA, meaning that organic pollution in sediments affects APA remarkably.

  3. Concentrations of testosterone, luteal hormone and prolactin in the serum as well as comparisons of sensitivity between radioimmunoassays and enzyme assays for the detection of acid prostate phosphatase in the presence of carcinomas of the prostate

    The relationship between carcinomas of the prostate and the plasma levels of testosterone, luteal hormone and prolactin as well as the possible influence of these neoplasms on the testosterone binding capacity and free testosterone index are investigated for various tumour stages and degrees of histological differentiation, in connection with several forms of local therapy as well as a variety of contrasexual methods. The sensitivity of enzyme assays and radioimmunoassays for the detection of acid prostate phosphatase is evaluated within the framework of this study. (MBL)

  4. A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

    Mukhopadhyay, Archana; Kennelly, Peter J.

    2011-01-01

    The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylate...

  5. Effects of tumour mass and circulating antigen on the biodistribution of 111In-labelled F(ab')2 fragments of human prostatic acid phosphatase monoclonal antibody in nude mice bearing PC-82 human prostatic tumor xenografts

    We have evaluated the effects of tumour mass and circulating antigen (prostatic acid phosphatase, PAP) on the biodistribution and the incorporation of 111In-labelled F(ab')2 monoclonal antibody (MoAb) fragments directed against human PAP into human prostatic tumours (PC-82; 0.1-8.9 g) growing in nude mice. The radioactivities in the blood, liver, spleen, kidney and tumour were compared at 1, 3, 4 and 6 days after the intravenous administration of the antibody fragments. There was a significant correlation between the tumour size and the serum PAP concentration in the model employed. Even tissue of a small tumour (111In-labelled F(ab')2 fragments. This relationship had levelled off by 72 h and most likely reflected a better vascularisation of the smaller tumours. Our results show that the increase in tumour size and in the concentration of circulating antigen in the blood led to decreased tumour-to-blood ratios, since there was a tendency for higher blood activities in mice with larger tumours and higher serum PAP concentrations. There was no correlation between tumour size and label uptake by the liver during the follow-up over 144 h, although serum PAP concentrations ranged from 3.1 μg/l to 352 μg/l. On the other hand, when compared with our previous data obtained with non-tumour-bearing mice, there was a significant increase in the uptake by the liver and spleen. These results indicate that even a small concentration of circulating antigen was able to trigger an abnormal change in the biodistribution of MoAbs. (orig.)

  6. PTP-PEST phosphatase variations in human cancer.

    Streit, Sylvia; Ruhe, Jens Ernst; Knyazev, Pjotr; Knyazeva, Tatjana; Iacobelli, Stefano; Peter, Stephan; Hoefler, Heinz; Ullrich, Axel

    2006-10-01

    Signal transduction via tyrosine phosphorylation, normally fine-tuned by the concerted action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key mechanism in tumorigenesis. PTP-PEST, a ubiquitously expressed cytoplasmic tyrosine phosphatase, is thought to play an important role in cell adhesion and motility, and may be involved in metastasis. A search for sequence variations within the gene PTPN12 (alias PTP-PEST) was performed in breast cancer cell lines, leading to the identification of three amino acid substitutions at positions 322, 573, and 709. These alterations were also found in squamous cell carcinoma cell lines and could be verified in primary human breast and kidney tumor samples. Analysis of peripheral blood samples confirmed the germline origin of these alterations. Furthermore, functional characterization of the Ile322 and Ala573 PTP-PEST mutants revealed an enhancement of in vitro phosphatase activity, whereas the Lys709 variant showed reduced catalytic activity. These data demonstrate the existence of PTP-PEST variants that might be meaningful for human cancer and underscore the need for further characterizing PTP-PEST and its signaling pathways in context of this disease. PMID:16965954

  7. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  8. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  9. Alkaline phosphatase activity of marine bacteria studied with ELF 97 substrate: success and limits in the P-limited Mediterranean Sea

    Van Wambeke, F.; Nedoma, Jiří; Duhamel, S.; Lebaron, P.

    2008-01-01

    Roč. 52, č. 3 (2008), s. 245-251. ISSN 0948-3055 Grant ostatní: MŠMT(CZ) PAI Barrande 2005-06-009-01 Institutional research plan: CEZ:AV0Z60170517 Keywords : marine bacteria * alkaline phosphatase * ELF97 phosphatase substrate Subject RIV: DA - Hydrology ; Limnology Impact factor: 2.190, year: 2008

  10. [Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

    Sibirnyi, A A; Shavlovskii, G M

    1978-01-01

    The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed. PMID:208203

  11. S. cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided and Gln3 Phosphorylation Levels Become Nitrogen Source-Responsive In a sit4 Deleted Strain

    Tate, Jennifer J.; Feller, André; Dubois, Evelyne; Cooper, Terrance G.

    2006-01-01

    Tor1,2 control of type-2A-related phosphatase activities in S. cerevisiae has been reported to be responsible for the regulation of Gln3 phosphorylation and intracellular localization in response to the nature of the nitrogen source available. According to the model, excess nitrogen stimulates Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then complexes with and inactivates Sit4 phosphatase, thereby preventing it from dephosphorylating Gln3. Phosphorylated Gln3 complexes with Ure2 and is ...

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

    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

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

    C. v. Sperber

    2015-03-01

    Full Text Available 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 (IP6 is an important form of Porg in many soils. The enzymatic hydrolysis of IP6 by phytase yields plant available inorganic phosphate (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 substrate were prepared. During the hydrolysis of IP6 by phytase, four Pi are released, and one oxygen atom from water is incorporated into each Pi. This incorporation of oxygen from water into Pi is subject to an apparent inverse isotopic fractionation (ϵ ∼ 6 to 10‰, which is similar to that imparted by acid phosphatase from potato during the hydrolysis of IP6 (ϵ ∼ 7‰ where less than three Pi are 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‰, again 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 substrate

  14. Chromatographic separation of alkaline phosphatase from dental enamel

    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....

  15. Comparative genetic analysis of Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

    Liangsheng Wang; Shan Lu; Ye Zhang; Zheng Li; Xiaoqiu Du; Dong Liu

    2014-01-01

    Induction and secretion of acid phosphatases (APases) is thought to be an adaptive mechanism that helps plants survive and grow under phosphate (Pi) deprivation. In Arabidopsis, there are 29 purple acid phosphatase (AtPAP) genes. To systematical y investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for al 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAP10 is mainly a secreted APase. On Pi-deficient (P-) medium or P-medium supplemented with the organophosphates ADP and fructose-6-phosphate (Fru-6-P), growth of atpap10 was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type (WT). Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P-or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essential y the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.

  16. Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+

    Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 μM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure

  17. A new monoterpene acid from Marrubium vulgare with potential antihepatotoxic activity.

    Ahmed, Bahar; Masoodi, Mubashir H; Siddique, Anwarul H; Khan, Shamshir

    2010-11-01

    The whole plant of Marrubium vulgare L. afforded a new terpenoid, characterised as p-menthane-5,6-dihydroxy-3-carboxylic acid (1), which has been designated as marrubic acid. Its structure has been elucidated on the basis of spectral and chemical analyses. The compound (1) also exhibited a significant antihepatotoxic activity by reducing the elevated levels of serum enzymes such as serum glutamate oxaloacetate transaminase (SGOT) by 40.16%, serum glutamate pyruvate oxaloacetate transaminase (SGPT) by 35.06%, and alkaline phosphatase (ALP) by 30.51%. On the other hand, total protein (TP) levels were increased by 34.07%, as compared to the standard drug silymarin, which decreased SGOT by 53.04%, SGPT by 55.96%, ALP by 35.87% and increased TP levels by 59.59%. These biochemical observations were also supplemented by histopathological examinations of liver sections. PMID:20628963

  18. Serum total and bone alkaline phosphatase and tartrate-resistant acid phosphatase activities for the assessment of bone fracture healing in dogs

    C. Sousa

    2011-08-01

    Full Text Available O objetivo deste trabalho foi estudar o padrão de variação da atividade sérica da fosfatase alcalina total (tALP, da isoenzima óssea da fosfatase alcalina (BALP e da fosfatase ácida resistente ao tartarato (TRAP, assim como a variação da concentração dos minerais séricos durante o processo de cicatrização de fraturas ósseas no cão. A variação sérica destes marcadores do metabolismo ósseo foi avaliada em nove cães com fraturas diafisárias fechadas de ossos longos, submetidas a tratamento cirúrgico para osteosíntese. Durante o período pós-operatório, sete animais evoluíram no sentido de uma normal união óssea, sendo que dois deles desenvolveram um processo de não união óssea. Foram observados, relativamente à BALP, valores de actividade sérica mais elevados e com diferença estatística (P<0,05 no grupo de animais que evoluiu no sentido de uma normal união óssea, comparativamente ao grupo de animais que evoluiu no sentido do processo de não união. No grupo de animais que evoluiu para a completa união óssea foram, adicionalmente, observados valores diminuidos (P<0,05 da atividade sérica da TRAP, até ao dia 60 do período pós-operatório seguido de uma elevação estatisticamente significativa após este período. Em conclusão, os biomarcadores do metabolismo ósseo poderão vir a constituir um método auxiliar de diagnóstico na monitorização do processo de cicatrização de fracturas ósseas, possibilitando, a detecção precoce de complicações pós-operatórias.

  19. On the anticonvulsant activity of kaurenic acid.

    Daló, Nelson L; Sosa-Sequera, Miriam C; Usubillaga, Alfredo

    2007-09-01

    Kaurenic acid [(-)-kaur-16-en-19-oic acid] is a diterpene isolated from the aerial parts of Espeletia semiglobulata, one of 85 species of Espeletiinae found in Venezuela. Its anticonvulsive activity was studied using two different models of experimental seizures: spinal seizures induced by sudden cooling (SSSC) in amphibians and seizures induced by pentylenetetrazol (PTZ) in mice. In SSSC, kaurenic acid (KA) inhibited the tonic hind-limb extension with an ED50 of 2.5 mg/kg. It was 4-fold more potent than known anticonvulsant drugs such as carbamazepine and phenytoin and 100-fold more potent than valproic acid. However, KA as well as valproic acid were ineffective against the clonic phase of SSSC. In the PTZ-induced seizures, KA at doses of 0.625 and 1.25 mg/kg increased the latency of seizure onset and protected against generalized clonic-tonic seizures by 45% and 65%, respectively. The sedative effects of KA had an ED50 of 8.5 mg/kg in mice and 75 mg/kg in amphibians. This work provides experimental evidence supporting the potential value of kaurenic acid as an anticonvulsive drug. PMID:17853794

  20. Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

    Moutahir-Belqasmi, F; Balmain, N; Lieberrher, M; Borzeix, S; Berland, S; Barthelemy, M; Peduzzi, J; Milet, C; Lopez, E

    2001-01-01

    The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (pproduction in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration. PMID:15348370

  1. Glucose-6-phosphatase deficiency

    Labrune Philippe

    2011-05-01

    Full Text Available Abstract Glucose-6-phosphatase deficiency (G6P deficiency, or glycogen storage disease type I (GSDI, is a group of inherited metabolic diseases, including types Ia and Ib, characterized by poor tolerance to fasting, growth retardation and hepatomegaly resulting from accumulation of glycogen and fat in the liver. Prevalence is unknown and annual incidence is around 1/100,000 births. GSDIa is the more frequent type, representing about 80% of GSDI patients. The disease commonly manifests, between the ages of 3 to 4 months by symptoms of hypoglycemia (tremors, seizures, cyanosis, apnea. Patients have poor tolerance to fasting, marked hepatomegaly, growth retardation (small stature and delayed puberty, generally improved by an appropriate diet, osteopenia and sometimes osteoporosis, full-cheeked round face, enlarged kydneys and platelet dysfunctions leading to frequent epistaxis. In addition, in GSDIb, neutropenia and neutrophil dysfunction are responsible for tendency towards infections, relapsing aphtous gingivostomatitis, and inflammatory bowel disease. Late complications are hepatic (adenomas with rare but possible transformation into hepatocarcinoma and renal (glomerular hyperfiltration leading to proteinuria and sometimes to renal insufficiency. GSDI is caused by a dysfunction in the G6P system, a key step in the regulation of glycemia. The deficit concerns the catalytic subunit G6P-alpha (type Ia which is restricted to expression in the liver, kidney and intestine, or the ubiquitously expressed G6P transporter (type Ib. Mutations in the genes G6PC (17q21 and SLC37A4 (11q23 respectively cause GSDIa and Ib. Many mutations have been identified in both genes,. Transmission is autosomal recessive. Diagnosis is based on clinical presentation, on abnormal basal values and absence of hyperglycemic response to glucagon. It can be confirmed by demonstrating a deficient activity of a G6P system component in a liver biopsy. To date, the diagnosis is most

  2. Biodistribution in normal mice of an 111In-labelled prostatic acid phosphatase-specific antibody and its F(ab')2 fragments derivatized site-specifically or via bicyclic diethylenetriaminepentaacetic acid anhydride

    We examined the optimization of derivatization of monoclonal antibodies and their fragments intended for use as radiopharmaceutical in radioimaging and/or radioimmunotherapy of prostatic cancer. Two different principles were used to conjugate (DTPA) to a monoclonal antibody (Mab, subclass IgG1) raised against human prostatic acid phosphatase (PAP). In addition, the F(ab')2 fragments of this Mab were also derivatized. The biodistribution of the 111In-labelled derivatives was investigated in normal mice. All the derivatives of IgG1 demonstrated a slower blood clearance than the corresponding derivatives of the F(ab')2 fragments. This property was particularly pronounced in the site-specifically conjugated derivatives of IgG1. All the derivatives studied accumulated in the liver, kidney, and spleen. The CA-DTPA derivatives of F(ab')2 fragments showed the highest kidney-to-blood ratios of radioactivity. The derivatives of IgG1 showed a higher percentage of the injected dose in liver and spleen tissues than the derivatives of the F(ab')2 fragments. The F(ab')2 fragments studied also gave rise to site-specific derivatives, which demonstrated that carbohydrates were also present in this part of the molecule. They behaved similarly to the CA-DTPA F(ab')2 derivative in other respects, but the kidney accumulation was lower at 72 and 120 h. The F(ab')2 fragments studied would be better suited for radioimaging than the derivatives of the IgG1 studied. In contrast, the derivatives of IgG1, especially the p-NH2-Bz-DTPA conjugate, might be more suitable candidates for the development of therapeutic agents. (orig.)

  3. Interleukin 2 induces a transient downregulation of protein phosphatase 1 and 2A activity in human T cells

    Brockdorff, J; Nielsen, M; Dobson, P; Geisler, C; Röpke, C; Svejgaard, A; Odum, N

    Stimulation of human CD4+ T cell lines with interleukin 2 (IL-2) induces tyrosine, serine and threonine phosphorylation of a series of proteins involved in the IL-2 receptor (IL-2R) signaling pathway. Here, we examined whether IL-2 induces changes in the activity of protein serine/threonine phosp......Stimulation of human CD4+ T cell lines with interleukin 2 (IL-2) induces tyrosine, serine and threonine phosphorylation of a series of proteins involved in the IL-2 receptor (IL-2R) signaling pathway. Here, we examined whether IL-2 induces changes in the activity of protein serine...... activity (p < 0.0005, n = 17) and a seven percent decrease in PP1 activity (p < 0.00005, n = 17). Cytokine-induced downregulation of PP2A activity reaches a maximum 60 min after IL-2R ligation, and returns to baseline levels within two hours. Downregulation of PPI activity reaches a maximum after 30 min...

  4. Leucine aminopeptidase, beta-glucosidase and alkaline phosphatase activity rates and their significance in nutrient cycles in some coastal Mediterranean sites.

    Caruso, Gabriella

    2010-01-01

    In aquatic microbial ecology, knowledge of the processes involved in the turnover of organic matter is of utmost importance to understand ecosystem functioning. Microorganisms are major players in the cycling of nutrients (nitrogen, phosphorus) and carbon, thanks to their enzymatic activities (leucine aminopeptidase, LAP, alkaline phosphatase, AP, and beta-glucosidase, beta-GLU) on organic polymers (proteins, organic phosphates and polysaccharides, respectively). Estimates of the decomposition rates of organic polymers are performed using fluorogenic compounds, whose hydrolysis rate allow us to obtain information on the "potential" metabolic activity of the prokaryotic community. This paper refers the enzyme patterns measured during recent oceanographic cruises performed in some coastal Mediterranean sites, not yet fully investigated in terms of microbial biogeochemical processes. Mean enzyme activity rates ranged from 5.24 to 5558.1 nM/h, from 12.68 to 244.73 nM/h and from 0.006 to 9.51 nM/h for LAP, AP and beta-GLU, respectively. The highest LAP and AP activity rates were measured in the Gulf of Milazzo (Tyrrhenian Sea) and in the Straits of Messina, in association with the lowest bacterioplankton abundance; in contrast, the lowest ones were found in the northern Adriatic Sea. beta-GLU was more active in the Straits of Messina. Activity rates were analysed in relation to the main environmental variables. Along the northern Adriatic coastal side affected by the Po river, significant inverse relationships linked LAP and AP with salinity, pointing out that fluvial inputs provided organic substrates for microbial metabolism. Both in the Gulf of Manfredonia and in the Straits of Messina, LAP and AP levels were inversely related with the concentration of nitrate and inorganic phosphorus, respectively. In the Gulf of Milazzo, high cell-specific AP measured in spite of phosphorus availability suggested the role of this enzyme not only in phosphorus, but also in carbon

  5. Leucine Aminopeptidase, β-Glucosidase and Alkaline Phosphatase Activity Rates and Their Significance in Nutrient Cycles in Some Coastal Mediterranean Sites

    Gabriella Caruso

    2010-03-01

    Full Text Available In aquatic microbial ecology, knowledge of the processes involved in the turnover of organic matter is of utmost importance to understand ecosystem functioning. Microorganisms are major players in the cycling of nutrients (nitrogen, phosphorus and carbon, thanks to their enzymatic activities (leucine aminopeptidase, LAP, alkaline phosphatase, AP, and β-glucosidase, β-GLU on organic polymers (proteins, organic phosphates and polysaccharides, respectively. Estimates of the decomposition rates of organic polymers are performed using fluorogenic compounds, whose hydrolysis rate allow us to obtain information on the “potential” metabolic activity of the prokaryotic community. This paper refers the enzyme patterns measured during recent oceanographic cruises performed in some coastal Mediterranean sites, not yet fully investigated in terms of microbial biogeochemical processes. Mean enzyme activity rates ranged from 5.24 to 5558.1 nM/h, from 12.68 to 244.73 nM/h and from 0.006 to 9.51 nM/h for LAP, AP and β-GLU, respectively. The highest LAP and AP activity rates were measured in the Gulf of Milazzo (Tyrrhenian Sea and in the Straits of Messina, in association with the lowest bacterioplankton abundance; in contrast, the lowest ones were found in the northern Adriatic Sea. β-GLU was more active in the Straits of Messina. Activity rates were analysed in relation to the main environmental variables. Along the northern Adriatic coastal side affected by the Po river, significant inverse relationships linked LAP and AP with salinity, pointing out that fluvial inputs provided organic substrates for microbial metabolism. Both in the Gulf of Manfredonia and in the Straits of Messina, LAP and AP levels were inversely related with the concentration of nitrate and inorganic phosphorus, respectively. In the Gulf of Milazzo, high cell-specific AP measured in spite of phosphorus availability suggested the role of this enzyme not only in phosphorus, but also

  6. Cdc14 phosphatase

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

    2016-01-01

    Cycling events in nature start and end to restart again and again. In the cell cycle, whose purpose is to become two where there was only one, cyclin-dependent kinases (CDKs) are the beginning and, therefore, phosphatases must play a role in the ending. Since CDKs are drivers of the cell cycle...

  7. The antioxidant effects of vitamin C on liver enzymes: aspartate aminotransferase, alanine aminotranferease, alkaline phosphatase and gamma-glutamyltransferase activities in rats under Paraquat insult

    Benjamin Nnamdi Okolonkwo

    2013-06-01

    Full Text Available Paraquat (PQ is a bipyridylium herbicide; applied around trees in orchards and between crop rows to control broad-leaved and grassy weeds. Its oxidation results in the formation of superoxides which causes damage to cellular components. In this study, we determined the antioxidant effect vitamin C has on the liver enzymes [aspartate aminotransferase (SGOT, alanine aminotranferease (SGPT, alkaline phosphatase (ALP, and gamma-glutamyltransferase (GGT] of rats under this toxic insult. Male rats in groups (A, B, C and D were intraperitoneally injected with different sublethal increasing doses (0, 0.02, 0.04 and 0.06 g/kg body weigh of PQ respectively on monthly basis. Subsequently, the subgroups (A2, B2, C2 and D2 were given orally, 200 mg/L vitamin C, while the subgroups A1, B1, C1, and D1, received only water. Four animals per subgroup were decapitated on monthly basis and blood samples taken for enzyme assay. The parameters studied were - SGOT, SGPT, ALP and GGT - liver enzymes. The dose and time dependent PQ toxicity effect resulted in highly elevated Liver enzymes activities. The subgroups on vitamin C had significantly lower enzyme activities when compared to the same subgroups on only PQ insult. But the values were high when compared to the control subgroups (A1 and A2. These results were indication that vitamin C when given at moderate doses and maintained for a longer period could be a life saving adjunct to toxic insult.

  8. Copper(II) complexes with cyanoguanidine and o-phenanthroline: Theoretical studies, in vitro antimicrobial activity and alkaline phosphatase inhibitory effect

    Martínez Medina, Juan J.; Islas, María S.; López Tévez, Libertad L.; Ferrer, Evelina G.; Okulik, Nora B.; Williams, Patricia A. M.

    2014-01-01

    Calculations based on density functional methods are carried out for two Cu(II) complexes with cyanoguanidine (cnge) and o-phenanthroline (o-phen): [Cu(o-phen)2(cnge)](NO3)2ṡ2H2O (1) and [Cu(o-phen)(cnge)(H2O)(NO3)2] (2). The calculated geometrical parameters are in agreement with the experimental values. The results of Atoms in Molecules (AIM) topological analysis of the electron density indicate that the Cu-N(phen) bonds in complex (1) have lower electron density, suggesting that those bonds are stronger in complex (2). Moreover, the ionic character of the Cu-N bond in the complex (1) is slightly stronger than that in the complex (2) and this situation would explain the fact that only complex (2) was stable in water solution. For this reason, the antimicrobial and enzymatic assays were performed using complex (2). It is well known that the increased use of antibiotics has resulted in the development of resistant bacterial and fungal strains. In this context, the study of novel antimicrobial agents has an enormous importance and metal complexes represent an interesting alternative for the treatment of infectious diseases. The aim of this work is to prove the modification of some biological properties like antimicrobial activity or alkaline phosphatase inhibitory activity upon copper complexation.

  9. Lithocholic acid and derivatives: Antibacterial activity.

    do Nascimento, Patrícia G G; Lemos, Telma L G; Almeida, Macia C S; de Souza, Juliana M O; Bizerra, Ayla M C; Santiago, Gilvandete M P; da Costa, José G M; Coutinho, Henrique D M

    2015-12-01

    In order to develop bioactive lithocholic acid derivatives, we prepared fifteen semi-synthetic compounds through modification at C-3 and/or C-24. The reactions showed yields ranging from 37% to 100%. The structures of all compounds obtained were identified on the basis of their spectral data (IR, MS, 1D- and 2D-NMR). The activity of lithocholic acid and derivatives was evaluated against the growth of Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa. The derivative 3α-formyloxy-5β-cholan-24-oic acid (LA-06) showed the best activity, with MIC values of 0.0790 mM against E. coli (Ec 27) and B. cereus in both cases, and 0.0395 mM against S. aureus (ATCC 12692). Lithocholic acid and the derivatives with MIC⩽1.2 mM were evaluated on the susceptibility of some bacterial pathogens to the aminoglycoside antibiotics neomycin, amikacin and gentamicin was evaluated. There are no previously reported studies about these compounds as modifiers of the action of antibiotics or any other drugs. PMID:26216208

  10. Modulation of antioxidant and phosphatase enzymes by beta-carotene against gamma radiation induced testicular disorders in albino rats

    Beta-carotene is a group of plant compounds called carotenoids. It is a precursor for vitamin A and an important antioxidant. This study aimed to evaluate the radioprotective efficacy of β-carotene against gamma radiation induced disorders in the testis of male albino rats, it included 4 groups: control group, treated group; animals of this group received a daily oral dose of β-carotene (30 mg/kg body wt) for 1 week, irradiated group; animals of this group were subjected to whole body gamma irradiation at a dose level of 6 Gy, and treated-irradiated group; animals received a daily oral dose of β-carotene (30 mg/ kg body wt) for 1 week before exposure to whole body gamma irradiation at a dose level of 6 Gy. 6 animals of each group were autopsied at 1, 3 and 5 days after β-carotene treatment and/ or irradiation. All animals were subjected to the following investigations: acid phosphatase, alkaline phosphatase, glutathione and glutathione peroxidase in testis homogenate. In irradiated animals there was a highly significant decrease in testis alkaline phosphatase, glutathione and glutathione peroxidase activity. On the other hand, significant increase in acid phosphatase activity was observed. Treatment with β-carotene before irradiation causes significant increase in alkaline phosphatase, glutathione and glutathione peroxidase activity and significant decrease in acid phosphatase activity compared to the irradiated group. The results of the present study indicated that β-carotene ameliorated oxidative stress and the loss of cellular antioxidants and suggest that β-carotene may reduce the radiation damage in testis of male albino rats

  11. Caffeic Acid Inhibits NFkappaB Activation of Osteoclastogenesis Signaling Pathway

    Ferry Sandra

    2011-12-01

    Full Text Available BACKGROUND: Caffeic acid (3,4-dihydroxycinnamic acids is involved in various green plants. Based on our previous report, a major component of sweet potato extracts, possibly caffeic acid, was shown as a promising inhibitor of osteoclastogenesis. However, the effect of caffeic acid in inhibiting osteoclastogenesis needs to be confirmed. The underlying mechanism needs to be disclosed as well. METHODS: Caffeic acid in various concentrations was added to in vitro osteoclastogenesis of receptor activator nuclear factor kB ligand (RANKL-tumor necrosis factor alpha (TNF-α-macrophage colony stimulating factor (M-CSF-induced bone marrow-derived monocyte/macrophage precursor cells (BMMs and RANKL-TNF-α-induced RAW264 cells D-Clone (RAW-D cells. Tartrate resistant acid phosphatase (TRAP staining was performed and TRAP-positive polynucleated cells (PNCs were counted. For apoptosis analysis, caffeic acid-treated BMMs, RAW-D cells and osteoclast-like PNCs were subjected to Sub-G1 Apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays. To measure NFkB activity, RAW-D cells were transfected with pNFkB-TA-Luc and subjected to Dual Luciferase Reporter Assay System. RESULTS: Caffeic acid inhibited osteoclastogenesis of RANKL-TNF-α-M-CSF-induced BMMs as well as RANKL-TNF-α-induced RAW-D cells in a dose dependent manner. Caffeic acid did not induce apoptosis in BMMs, RAW-D cells and osteoclast-like PNCs. RANKL-TNF-α-induced NFkB activity in RAW-D was diminished by caffeic acid in a dose dependent manner. Significant NFkB activity inhibtion was observed starting from 1µg/mL caffeic acid. CONCLUSIONS: Caffeic acid could be a potent osteoclastogenesis inhibitor through inhibition of NFkB activity. Our present study should be further followed up to disclose caffeic acid's possible overlying signaling pathways in inhibiting osteoclastogenesis. KEYWORDS: caffeic acid, osteoclastogenesis, NFkB, RANKL, TNF-α.

  12. Synthesis and anticonvulsant activity of novel bicyclic acidic amino acids

    Conti, Paola; De Amici, Marco; Joppolo Di Ventimiglia, Samuele;

    2003-01-01

    Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them...

  13. Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores

    Aguilar, Jorge L.; Kulkarni, Ritwij; Randis, Tara M.; Soman, Sandeep; Kikuchi, Alexander; Yin, Yuxin; Ratner, Adam J.

    2009-01-01

    Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This acti...

  14. The effect of phosphours and water deficit on phosphatase activity and proline accumulation in seedling cotyledons and roots of oilseed rape as compared to that of excised cotyledons and roots

    Stanisław Flasiński; Janina Rogozińska; Lucyna Drozdowska

    2014-01-01

    Oilseed rape seedlings and excised cotyledons and roots were exposed to phosphorus and osmotic stress (-1 MPa: NaCl or PEG). The stress factors limited the growth of the seedlings and inhibited the growth of the excised roots and cotyledons. The phosphorus content in the cotyledons and roots depended on its level in the media and on the stress factors used. Phosphorus deficiency differentiated total phosphatase activity in seedling cotyledons and increased the activity in the excised cotyledo...

  15. Ginkgetin Blocks Constitutive STAT3 Activation and Induces Apoptosis through Induction of SHP-1 and PTEN Tyrosine Phosphatases.

    Baek, Seung Ho; Lee, Jae Hwi; Ko, Jeong-Hyeon; Lee, Hanwool; Nam, Dongwoo; Lee, Seok Geun; Yang, Woong Mo; Um, Jae-Young; Lee, Junhee; Kim, Sung-Hoon; Shim, Bum Sang; Ahn, Kwang Seok

    2016-04-01

    Ginkgetin, a biflavone from Ginkgo biloba leaves, is known to exhibit antiinflammatory, antifungal, neuroprotective, and antitumor activities, but its precise mechanism of action has not been fully elucidated. Because the aberrant activation of STAT3 has been linked with regulation of inflammation, proliferation, invasion, and metastasis of tumors, we hypothesized that ginkgetin modulates the activation of STAT3 in tumor cells. We found that ginkgetin clearly suppressed constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK1 and c-Src kinases and nuclear translocation of STAT3 on both A549 and FaDu cells. Treatment with sodium pervanadate reversed the ginkgetin-induced down-modulation of STAT3, thereby indicating a critical role for a PTP. We also found that ginkgetin strongly induced the expression of the SHP-1 and PTEN proteins and its mRNAs. Further, deletion of SHP-1 and PTEN genes by siRNA suppressed the induction of SHP-1 and PTEN, and reversed the inhibition of STAT3 activation. Ginkgetin induced apoptosis as characterized by an increased accumulation of cells in subG1 phase, positive Annexin V binding, loss of mitochondrial membrane potential, down-regulation of STAT3-regulated gene products, and cleavage of PARP. Overall, ginkgetin abrogates STAT3 signaling pathway through induction of SHP-1 and PTEN proteins, thus attenuating STAT3 phosphorylation and tumorigenesis. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27059688

  16. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of...... tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of...

  17. Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase.

    Mustelin, T; Coggeshall, K M; Altman, A

    1989-01-01

    T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endo...

  18. Effects of Lanthanum on Hydrolytic Enzyme Activities in Red Soil

    褚海燕; 朱建国; 谢祖彬; 李振高; 曹志洪; 曾青; 林先贵

    2002-01-01

    The effects of La on some hydrolytic enzyme activities in red soil were studied in incubation and pot culture experiments. In the incubation experiment, La slightly stimulates the activities of urease and acidic phosphatase in soil and strongly stimulates sucrase activity in soil. In the pot culture experiment, La stimulates the activities of urease, acidic phosphatase and sucrase to different degrees. The stimulative effects of rare earth elements (REE) on hydrolytic enzyme activities in soil may result in increasing yield of crops.

  19. New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

    Yamagishi, J; Furutani, Y; Inoue, S.; Ohue, T; Nakamura, S; Shimizu, M

    1981-01-01

    In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were ...

  20. Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

    Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported. The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found

  1. Extracellular phosphatase activity of freshwater phytoplankton exposed to different .i.in situ./i. phosphorus concentrations

    Štrojsová, Alena; Vrba, Jaroslav; Nedoma, Jiří; Šimek, Karel

    2005-01-01

    Roč. 56, č. 4 (2005), s. 417-424. ISSN 1323-1650. [Symposium for European Freshwater Sciences /4./. Krakow, 22.08.2005-26.08.2005] R&D Projects: GA ČR(CZ) GA206/02/0003; GA AV ČR(CZ) IAA6017202 Grant ostatní: FRVŠ(CZ) G4 1841 Institutional research plan: CEZ:AV0Z60170517 Keywords : ELF97 phosphate * image cytometry * species-specific activity Subject RIV: EH - Ecology, Behaviour Impact factor: 1.478, year: 2005

  2. Yam (Dioscorea batatas) Root and Bark Extracts Stimulate Osteoblast Mineralization by Increasing Ca and P Accumulation and Alkaline Phosphatase Activity

    Kim, Suji; Shin, Mee-Young; Son, Kun-Ho; Sohn, Ho-Yong; Lim, Jae-Hwan; Lee, Jong-Hwa; Kwun, In-Sook

    2014-01-01

    Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within 0~10 μg/mL during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineral...

  3. Gamma-ray irradiation induce suppression of TNF-α production via up-regulation of maitogen-activated protein kinase phosphatase-1

    Complete text of publication follows. Ionizing irradiation induces DNA damage and activates a lot of signalling pathways, such as ATM and p53, due to repair the DNA damage. On the other hand, irradiation also induces activation of extracellular signal regulated protein kinase (ERK1/2) through trans-activation of EGF receptor. However, EGF-receptor-independent signalling pathways induced by irradiation are unclear. Here, we studied gamma-ray irradiation-induced signaling pathways focusing mitogen-activated kinase (MAPK), such as ERK1/2 and p38 MAPK in human keratinocyte HaCat cells, which express EGF receptor, and mouse macrophage RAW264.7 cells, which express EGF receptor at low level. These cells were irradiated by gamma-ray (0.05-2.5 Gy) from 137Cs source (0.96 Gy/min), and phosphorylated MAPKs were detected by immune blotting. Gamma-ray irradiation (0.1- 2.5Gy) induced phosphorylation of ERK1/2 in HaCat cells. However, dephosphorylation of p38 MAPK was occurred 15 min after the irradiation, indicating activation of MAPK phosphatase (MKP). On the other hand, dephosphorylation of not only p38 MAPK but also ERK1/2 were induced 15 min after irradiation (0.5 Gy) in RAW264.7 cells. At the same time point, expression of MKP-1, which dephosphorylates ERK1/2 and p38 MAPK, was significantly increased. Up-regulation of MKP-1 and dephosphorylation of p38 MAPK were also observed in irradiated mouse peritoneal macrophage. Because phosphorylation of p38 MAPK mediates pro-inflammatory cytokines, such as TNF-α , we examined the change in production of TNF-α after irradiation. Production of TNF-α was suppressed in 0.5 Gy irradiated RAW264.7 cells. In conclusion, our results suggest that gamma-ray irradiation induces up-regulation of MKP-1, leading to dephosphorylation of p38 MAPK and suppression of TNF-α production in RAW264.7cells, though ERK1/2 is activated through activation of EGF receptor in HaCat cells.

  4. Protein kinase C (PKC) phosphorylates human platelet inositol trisphosphate 5/sup +/-/-phosphomonoesterase (IP3 5'-p'tase) increasing phosphatase activity

    Phosphoinositide breakdown in response to thrombin stimulation of human platelets generates messenger molecules that activate PKC (diglyceride) and mobilize Ca++ (inositol tris-phosphates). The water soluble products of phospholipase C-mediated metabolism of phosphatidylinositol 4,5-diphosphate are inositol 1,4,5 P3 (IP3) and inositol 1:2-cyclic 4,5 P3 (cIP3). A specific phosphatase, IP3 5'-p'tase, cleaves the 5 phosphate from IP3 or cIP3 to form IP2 or cIP2 and P/sub i/, none of which mobilizes Ca++. Thus, the IP3 5'-p'tase may regulate cellular responses to IP3 or cIP3. The authors find that IP3 5'-p'tase isolated from human platelets is phosphorylated by rat brain PKC, resulting in a 4-fold increase in IP3 5'-p'tase activity. The authors phosphorylated IP3 5'-p'tase using γ 32P-ATP and found that the labeled enzyme comigrated on SDS-PAGE with the previously described 40K protein phosphorylated in response to thrombin stimulation of platelets. The similarity of the PKC-phosphorylated IP3 5'-p'tase observed in vitro and the thrombin-stimulated phosphorylated 40K protein known to be phosphorylated by PKC in vivo, suggests that these proteins may be the same. These results suggest that platelet Ca++ mobilization maybe regulated by PKC phosphorylation of the IP3 5'-p'tase and can explain the observation that phorbol ester treatment of intact human platelets results in decreased production of IP3 and decreased Ca++ mobilization upon subsequent thrombin addition

  5. Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.

    Romá-Mateo, Carlos; Sacristán-Reviriego, Almudena; Beresford, Nicola J; Caparrós-Martín, José Antonio; Culiáñez-Macià, Francisco A; Martín, Humberto; Molina, María; Tabernero, Lydia; Pulido, Rafael

    2011-04-01

    Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including

  6. Protein tyrosine phosphatase 1B (PTP1B) inhibitors from Morinda citrifolia (Noni) and their insulin mimetic activity.

    Nguyen, Phi-Hung; Yang, Jun-Li; Uddin, Mohammad N; Park, So-Lim; Lim, Seong-Il; Jung, Da-Woon; Williams, Darren R; Oh, Won-Keun

    2013-11-22

    As part of our ongoing search for new antidiabetic agents from medicinal plants, we found that a methanol extract of Morinda citrifolia showed potential stimulatory effects on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3-8, 11-13, and 15). The absolute configurations of compounds 9, 10, and 14 were determined by ECD spectra analysis. Compounds 3, 6, 7, and 15 showed inhibitory effects on PTP1B enzyme with IC50 values of 21.86 ± 0.48, 15.01 ± 0.20, 16.82 ± 0.42, and 4.12 ± 0.09 μM, respectively. Furthermore, compounds 3, 6, 7, and 15 showed strong stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. This study indicated the potential of compounds 3, 6, 7, and 15 as lead molecules for antidiabetic agents. PMID:24224843

  7. FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Aps-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively

    Zhong, X.B.; Bodeau, J.; Fransz, P.F.; Williamson, V.M.; Kammen, van A.; Jong, de J.H.; Zabel, P.

    1999-01-01

    The root-knot nematode resistance gene Mi-1 in tomato has long been thought to be located in the pericentromeric heterochromatin region of the long arm of chromosome 6 because of its very tight genetic linkage (approx. 1 cM) to the markers Aps-1 (Acid phosphatase 1) and yv (yellow virescent). Using

  8. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    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.

  9. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    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.

  10. Long-acting β2-agonists increase fluticasone propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1 in airway smooth muscle cells.

    Melanie Manetsch

    Full Text Available Mitogen-activated protein kinase phosphatase 1 (MKP-1 represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol, alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone in primary human airway smooth muscle (ASM cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6 and interleukin 8 (IL-8 secretion. As IL-6 expression (like MKP-1 is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα. Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.

  11. Sodium selenate retards epileptogenesis in acquired epilepsy models reversing changes in protein phosphatase 2A and hyperphosphorylated tau.

    Liu, Shi-Jie; Zheng, Ping; Wright, David K; Dezsi, Gabi; Braine, Emma; Nguyen, Thanh; Corcoran, Niall M; Johnston, Leigh A; Hovens, Christopher M; Mayo, Jamie N; Hudson, Matthew; Shultz, Sandy R; Jones, Nigel C; O'Brien, Terence J

    2016-07-01

    There are no treatments in clinical practice known to mitigate the neurobiological processes that convert a healthy brain into an epileptic one, a phenomenon known as epileptogenesis. Downregulation of protein phosphatase 2A, a protein that causes the hyperphosphorylation of tau, is implicated in neurodegenerative diseases commonly associated with epilepsy, such as Alzheimer's disease and traumatic brain injury. Here we used the protein phosphatase 2A activator sodium selenate to investigate the role of protein phosphatase 2A in three different rat models of epileptogenesis: amygdala kindling, post-kainic acid status epilepticus, and post-traumatic epilepsy. Protein phosphatase 2A activity was decreased, and tau phosphorylation increased, in epileptogenic brain regions in all three models. Continuous sodium selenate treatment mitigated epileptogenesis and prevented the biochemical abnormalities, effects which persisted after drug withdrawal. Our studies indicate that limbic epileptogenesis is associated with downregulation of protein phosphatase 2A and the hyperphosphorylation of tau, and that targeting this mechanism with sodium selenate is a potential anti-epileptogenic therapy. PMID:27289302

  12. Structural mechanisms of plant glucan phosphatases in starch metabolism.

    Meekins, David A; Vander Kooi, Craig W; Gentry, Matthew S

    2016-07-01

    Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode two glucan phosphatases, called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases, and outlines how they are uniquely adapted to perform their cellular functions. We outline the physical mechanisms used by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan-binding platform comprising its dual-specificity phosphatase domain and carbohydrate-binding module, while LSF2 utilizes surface binding sites. SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic dual-specificity phosphatase domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2, and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism and protein-glucan interaction, thereby providing a framework for their application in both agricultural and industrial settings. PMID:26934589

  13. Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode*

    Ishida, Hiroaki; Rainaldi, Mario; Vogel, Hans J.

    2009-01-01

    The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca2+-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca2+-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of b...

  14. Studies on the acid activation of Brazilian smectitic clays

    Francisco R. Valenzuela Díaz; Pérsio de Souza Santos

    2001-01-01

    Fuller's earth and acid activated smectitic clays are largely used as bleaching earth for the industrial processing of vegetable, animal and mineral oils and waxes. The paper comments about the nomenclature used for these materials, the nature of the acid activation of smectitic clays (bentonites), activation laboratory procedures and presents a review of the acid activation of bentonites from 20 deposits from several regions of Brazil. The activated clays were tested and show good decolorizi...

  15. Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

    Mao, Liang; Tang, Dong; Feng, Haiwei; Gao, Yang; Zhou, Pei; Xu, Lurong; Wang, Lumei

    2015-12-01

    Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil. PMID:26286803

  16. The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site

    Ettrich, Rüdiger

    2004-01-01

    Roč. 271, č. 19 (2004), s. 3923-3939. ISSN 0014-2956 Institutional research plan: CEZ:AV0Z5011922; MSM113100003 Keywords : phosphatase Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.260, year: 2004

  17. Detection of extracellular phosphatase activity at the single-cell level by Enzyme-Labeled Fluorescence and flow cytometry: The importance of time kinetics in ELFA labeling

    Duhamel, S.; Gregori, G.; Van Wambeke, F.; Nedoma, Jiří

    75A, č. 2 (2009), s. 163-168. ISSN 1552-4922 Grant ostatní: MŠMT(CZ) PAI Barrande 2005-06-009-01 Institutional research plan: CEZ:AV0Z60170517 Keywords : alkaline phosphatase * ELF phosphate * heterotrophic bacteria Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.032, year: 2009

  18. Mineral nutrient uptake from prey and glandular phosphatase activity as dual test of carnivory in semidesert plants with glandular leaves suspected of carnivory

    Plachno, B.J.; Adamec, Lubomír; Huet, H.

    2009-01-01

    Roč. 104, č. 4 (2009), s. 649-654. ISSN 0305-7364 Institutional research plan: CEZ:AV0Z60050516 Keywords : mineral nutrient uptake * phosphatases * glandular leaves Subject RIV: EF - Botanics Impact factor: 3.501, year: 2009

  19. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    Göttlicher, M; Widmark, E; Q. Li; Gustafsson, J.A.

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring acti...

  20. Effect of organic/inorganic compounds on the enzymes in soil under acid rain stress

    LIU Guang-shen; XU Dong-mei; WANG Li-ming; LI Ke-bin; LIU Wei-ping

    2004-01-01

    The main effects of pollutions including acid rain, Cu2+, atrazine and their combined products on theactivities of urease, invertin, acid phosphatase and catalase were studied by means of orthogonal test. The resultsshowed that H + and Cu2+ had significant influence on the activities of four enzymes and the ability of their inhibitingfollowed the order: H+ > Cu2+ . Al3+ and atrazine only had litter effects on the activity of urease and phosphatase,respectively. Furthermore, interaction analysis revealed that Cu2+ -H+ affected on the activity of acid phosphatasesignificantly and antagonism on invertin and urease, Cu2+ -atrazine only exhibited the synergism on the activity ofacid phosphatase. But atrazine-H+ had non-interaction within the investigated concentration range. Among fourenzymes, acid phosphatase was the most sensitive one to the contaminations.

  1. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  2. CAMBIOS EN LAS PROPIEDADES QUÍMICAS Y EN LA ACTIVIDAD DE LAS FOSFATASAS EN SUELOS CULTIVADOS CON MAÍZ DULCE (ZEA MAYS L. FERTILIZADOS CON VINAZA CHANGES IN CHEMICAL PROPERTIES AND PHOSPHATASES ACTIVITY IN SOILS UNDER SWEET CORN (ZEA MAYS L. CULTIVATION FERTILIZED WITH VINASSE

    Magda Narváez Castillo

    2010-12-01

    Full Text Available Se evalúo el efecto de la aplicación de vinaza en las propiedades químicas y la actividad de las fosfatasas ácidas y alcalinas de dos suelos del Valle del Cauca, Colombia (Typic Argiudoll y Fluventic Haplustoll en condiciones controladas. Se establecieron seis tratamientos y cinco repeticiones distribuidos en un diseño completamente al azar, cuatro de ellos buscaban suplir los requerimientos de K+ del cultivo maíz dulce de la siguiente forma: T1, 100% del K+ con vinazas, T2 100% de K+ con KCl, T3 50% de K+ con Vinaza y 50% con KCl, y T4 75% del K+ con vinaza y 25% con KCl, T5 fue el testigo absoluto sin planta y T6 el testigo más planta. Las evaluaciones se realizaron en un periodo de 76 días y las muestras se tomaron entre 0-5 cm de profundidad. El pH, K+, B y relación Ca + Mg/K disminuyeron, muy significativamente (PIt was evaluated the effect of vinasse application on chemical properties and activity of acid and alkaline phosphatases of two soils of Valle del Cauca Colombia (Typic Argiudoll and Fluventic Haplustoll under controlled conditions. Six treatments and five replicates arranged in a completely randomized design were stablished; four of them, sought meet the requirements of K+ crop sweet corn as follows: T1, 100% of K+ with vinasse, T2 100% K+ with KCl T3 50% of K+ with vinasse and 50% KCl, and T4 75% of K+ with vinasse and 25% with KCl, T5 was the absolute control without plant and T6 the witness more plant. The evaluations were conducted over a period of 76 days and samples were taken from 0 - 5 cm deep. The pH, K+, B and Ca + Mg / K decreased significantly (P<0.01, while the contents of K+ and B were increased in both soils The P, Cu, Na and Fe increased differently in each soil. Alkaline phosphatase was significantly higher in the Fluventic Haplustoll and maximum activity occurred at 10 days after sowing (DAS; however, no significant differences between soils with reference to acid phosphatase and increased activity

  3. Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase.

    Uchiya, K I; Tohsuji, M; Nikai, T.; Sugihara, H.; Sasakawa, C

    1996-01-01

    A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus....

  4. Acaricidal activity of usnic acid and sodium usnic acid against Psoroptes cuniculi in vitro.

    Shang, Xiaofei; Miao, Xiaolou; Lv, Huiping; Wang, Dongsheng; Zhang, Jiqin; He, Hua; Yang, Zhiqiang; Pan, Hu

    2014-06-01

    Usnic acid, a major active compound in lichens, was first isolated in 1884. Since then, usnic acid and its sodium salt (sodium usnic acid) have been used in medicine, perfumery, cosmetics, and other industries due to its extensive biological activities. However, its acaricidal activity has not been studied. In this paper, we investigated the acaricidal activity of usnic acid and sodium usnic acid against Psoroptes cuniculi in vitro. After evaluating the acaricidal activity and toxicity of usnic acid and sodium usnic acid in vitro, the results showed that at doses of 250, 125, and 62.5 mg/ml, usnic acid and sodium usnic acid can kill mites with 91.67, 85.00, and 55.00% and 100, 100, and 60.00% mortality after treatment 24 h. The LT50 values were 4.208, 8.249, and 16.950 h and 3.712, 7.339, and 15.773 h for usnic acid and sodium usnic acid, respectively. Sodium usnic acid has a higher acaricidal activity than usnic acid, which may be related to the difference in their structures. PMID:24770718

  5. Purification and properties of alkaline phosphatase of silkworm Bombyx mori

    TANG Yunming; CEN Liang; CHU Bo; LI Changchun; XU Min; LUO Ying; LU Cheng

    2006-01-01

    Alkaline phosphatase(AKP),from the succus entericus of silkworm,was purified using 10%-50% ammonium sulfate fractions,ion exchange chromatography Of DEAE-Sepharose,and size exclusion chromatography of Sephacryl S-200.The purification fold was 464 times and specified activity was 3936 U/mg.Optimum pH value of the phosphatase was 10.5,and was stable between pH 7.5 and 11.The optimum temperature of the phosphatase was 40℃ and it was unstable over 50℃.Km value of the phosphatase was 1.25 mmol/L.In a given condition,the phosphatase was selectively modified by PCMB,NBS,PMSE TNBS,SUAN,DTT,BrAc,and IAc,the results indicate that PMSF,SUA,BrAc,IAc,and TNBS could Obviously inhibit the activity of the phosphatase,and the degree of inhibition depended on the concentration of these reagents.There was little effect on the activity of phosphatase after treatment by PMSF,DTT,and NBT.We primarily conclude that mercapto and imidazole are essential for AKP from silkworm.Also,Lys residue and disulfide bands are necessary to protect the catalysis of the AKP.

  6. Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

    Burtis, C A; Seibert, L E; Baird, M A; Sampson, E J

    1977-09-01

    The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed. PMID:19164

  7. Protein phosphatase 2A associates with Rb2/p130 and mediates retinoic acid-induced growth suppression of ovarian carcinoma cells

    Vuocolo, Scott; Purev, Enkhtsetseg; Zhang, Dongmei;

    2003-01-01

    Levels of Rb2/p130 protein are increased 5-10-fold following all-trans-retinoic acid (ATRA) treatment of the retinoid-sensitive ovarian adenocarcinoma cell line CAOV3, but not the retinoid-resistant adenocarcinoma cell line SKOV3. We found that this increase in Rb2/p130 protein levels in ATRA...

  8. Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

    V. Bertone

    2011-02-01

    Full Text Available Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT. Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS. However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20 that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1, frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa, using as controls lean Zucker (fa/+ and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but

  9. Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonas aeruginosa

    Koveal, Dorothy; Clarkson, Michael W.; Wood, Thomas K.; Page, Rebecca; Peti, Wolfgang

    2013-01-01

    TpbA is a periplasmic dual specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium, Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA ad...

  10. The antioxidant effects of vitamin C on liver enzymes: aspartate aminotransferase, alanine aminotranferease, alkaline phosphatase and gamma-glutamyltransferase activities in rats under Paraquat insult

    Benjamin Nnamdi Okolonkwo; Edna Ogechi Nwachuku

    2013-01-01

    Paraquat (PQ) is a bipyridylium herbicide; applied around trees in orchards and between crop rows to control broad-leaved and grassy weeds. Its oxidation results in the formation of superoxides which causes damage to cellular components. In this study, we determined the antioxidant effect vitamin C has on the liver enzymes [aspartate aminotransferase (SGOT), alanine aminotranferease (SGPT), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT)] of rats under this toxic insult. Male ...

  11. Isolation and characterization of a neutral phosphatase from wheat seedlings

    A neutral phosphatase was purified to homogeneity from wheat seedlings. The enzyme was a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 26 A, and sedimentation coefficient of 3.2 S. That the enzyme was a glycoprotein was surmised from its chromatographic property on Concanavalin A-Sepharose column. The phosphatase activity was assayed using either fructose-2,6-bisphosphate or p-nitrophenyl phosphate as substrate. The phosphatase activity was not affected by high concentrations of chelating agents and did not require the addition of Mg+2 or Ca+2 for its activity. Molybdate, orthovanadate, Zn+2, and Hg+2 were all potent inhibitors of the phosphatase activity. The inhibition by Hg+2 was reversed by dithiothreitol. The enzyme activity was stimulated by Mn+2 about 2-fold. On the other hand, 3-phosphoglycerate, fructose-6-P and Pi as well as polyamines inhibited the enzyme activity. The ability of the neutral phosphatase to dephosphorylate protein phosphotyrosine was also investigated. The phosphotyrosyl-substrates, such as [32P] phosphotyrosyl-poly(Glu, Tyr)n, -alkylated bovine serum albumin, -angiotensin-1, and -band 3 of erythrocytes, were all substrates of the phosphatase. On the other hand, the enzyme had no activity toward protein phosphoserine and protein phosphothreonine

  12. Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

    Jones, N L; Jerome, W. G.; Lewis, J. C.

    1991-01-01

    Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermedia...

  13. Sulfuric acid leaching of mechanically activated manganese carbonate ore

    Kenan Yıldız

    2010-01-01

    Acidic leaching of mechanically activated manganese ore from Denizli Tavas was investigated. The ore was activated mechanically in a planetary mill and the amorphisation in manganese structure was analyzed with X-ray diffraction. The parameters in acidic leaching of the ore were milling time, acid concentration and time. All experiments were performed at 25°C with solid to liquid ratio: 1/10. The activation procedure led to amorphization and structural disordering in manganese ore and accele...

  14. Sulfuric acid leaching of mechanically activated manganese carbonate ore

    Yıldız, Kenan

    2000-01-01

    Acidic leaching of mechanically activated manganese ore from Denizli – Tavas was investigated. The ore was activated mechanically in a planetary mill and the amorphisation in manganese structure was analyzed with X-ray diffraction. The parameters in acidic leaching of the ore were milling time, acid concentration and time. All experiments were performed at 25°C with solid to liquid ratio: 1/10. The activation procedure led to amorphization and structural disordering in manganese ore and acce...

  15. The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

    MacConnell, W P; Kaplan, N O

    1982-05-25

    The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes. PMID:6121796

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

    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.

  17. Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2.

    Sun, Liyun; Gu, Shaohua; Sun, Yaqiong; Zheng, Dan; Wu, Qihan; Li, Xin; Dai, Jianfeng; Dai, Jianliang; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2005-04-01

    This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56-1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney. PMID:16010976

  18. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  19. Radioimmunodetection of lymph node invasion in prostatic cancer. The use of iodine-123 (123I)-labeled monoclonal anti-prostatic acid phosphatase (PAP) 227 A F(ab')2 antibody fragments in vivo

    The therapeutic indications in prostatic cancer depend on the regional and distant extension of the cancer and are difficult to assess before lymphadenectomy. Radioimmunodetection of lymph node involvement with monoclonal anti-prostatic acid phosphatase (PAP) antibodies can be proposed as a noninvasive alternative to lymphadenectomy. Fifteen patients with various stages of histologically proven prostatic cancer were examined by immunolymphoscintigraphy (ILS) before treatment to detect lymph node metastases. These patients had Stage A (n = 7), Stage B (n = 3), Stage C (n = 2), and Stage D (n = 3) tumors. They received between 100 and 400 micrograms of monoclonal antibody 227 A in the form of F(ab')2 fragments labeled with iodine-123 (123I). The antibody was injected directly into the periprostatic area. ILS images were obtained after 1, 3, 6, and 24 hours. Three days later, each patient underwent a lymphadenectomy for histologic examination. The results of the histologic examination and ILS were compared. In ten patients, the examination did not show any images capable of being interpreted as lymphadenopathy and histologic examination confirmed the integrity of the nodes examined. In five cases, scintigraphy suggested the presence of lymph node invasion by prostatic cancer and this was confirmed by histologic examination in three of the five cases. Overall, in terms of lymphadenopathy, this examination had a sensitivity of 100% and a specificity of 83%. Therefore, ILS appears to be capable of detecting lymph node metastases in prostatic cancer

  20. Activation of p38 mitogen-activated protein kinase contribute to BMP4-induced alkaline phosphatase expression in MC3T3-E1 preosteoblast

    YUAN Ye; Wu Zhi-jun; YAO Hui-yu; YU Xiao-dan; GUO Zi-kuan; CHEN Xiao-san; TANG Pei-xian; MAO Ning

    2006-01-01

    @@ Bone morphogenetic proteins (BMPs) induce ectopic bone formation and promote osteoblast differentiation.1 It has been documented that Smad transcriptional factors function as primary mediators of BMPs activity. Receptor-regulated Smad (Smad1, 5, 8) could be phosphorylated by activated BMPR-I and form complex with Smad4. The Smad complex translocates to the nucleus and regulate target gene transcription.2