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Sample records for acid phosphatase gene

  1. The discovery of the fat-regulating phosphatidic acid phosphatase gene

    PubMed Central

    CARMAN, George M.

    2011-01-01

    Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid (substrate) and diacylglycerol (product), which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol. Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, secretion, and endocytosis. While this important enzyme has been known for several decades, its gene was only identified recently from yeast. This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans. PMID:21785579

  2. The discovery of the fat-regulating phosphatidic acid phosphatase gene.

    PubMed

    Carman, George M

    2011-05-01

    Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid (substrate) and diacylglycerol (product), which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol. Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, secretion, and endocytosis. While this important enzyme has been known for several decades, its gene was only identified recently from yeast. This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.

  3. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes.

    PubMed

    Thaller, M C; Lombardi, G; Berlutti, F; Schippa, S; Rossolini, G M

    1995-01-01

    The gene encoding a minor phosphate-irrepressible acid phosphatase (named NapA) of Morganella morganii was cloned and sequenced, and its product characterized. NapA is a secreted acid phosphatase composed of four 27 kDa polypeptide subunits. The enzyme is active on several organic phosphate monoesters but not on diesters, and is also endowed with transphosphorylating activity from organic phosphoric acid esters to nucleosides and other compounds with free hydroxyl groups. Its activity is inhibited by EDTA, inorganic phosphate, nucleosides and Ca2+, but not by fluoride or tartrate, and is enhanced by Mg2+, Co2+ and Zn2+. At the sequence level, the NapA enzyme did not show similarities to any other sequenced bacterial phosphatases. However, a search for homologous genes in sequence databases allowed identification of two open reading frames located within sequenced regions of the Escherichia coli and Proteus mirabilis genomes respectively, encoding proteins of unknown function which are highly homologous to the Morganella enzyme. Moreover, the properties of the NapA enzyme are very similar to those reported for the periplasmic nonspecific acid phosphatase II of Salmonella typhimurium (for which no sequence data are available). These data point to the existence of a new family of bacterial acid phosphatases, which we propose designating class B bacterial acid phosphatases.

  4. Structure of Acid phosphatases.

    PubMed

    Araujo, César L; Vihko, Pirkko T

    2013-01-01

    Acid phosphatases are enzymes that have been studied extensively due to the fact that their dysregulation is associated with pathophysiological conditions. This characteristic has been exploited for the development of diagnostic and therapeutic methods. As an example, prostatic acid phosphatase was the first marker for metastatic prostate cancer diagnosis and the dysregulation of tartrate resistant acid phosphatase is associated with abnormal bone resorption linked to osteoporosis. The pioneering crystallization studies on prostatic acid phosphatase and mammalian tartrate-resistant acid phosphatase conformed significant milestones towards the elucidation of the mechanisms followed by these enzymes (Schneider et al., EMBO J 12:2609-2615, 1993). Acid phosphatases are also found in nonmammalian species such as bacteria, fungi, parasites, and plants, and most of them share structural similarities with mammalian acid phosphatase enzymes. Acid phosphatase (EC 3.1.3.2) enzymes catalyze the hydrolysis of phosphate monoesters following the general equation. Phosphate monoester + H2O -->/<-- alcohol + phosphate. The general classification "acid phosphatase" relies only on the optimum acidic pH for the enzymatic activity in assay conditions using non-physiological substrates. These enzymes accept a wide range of substrates in vitro, ranging from small organic molecules to phosphoproteins, constituting a heterogeneous group of enzymes from the structural point of view. These structural differences account for the divergence in cofactor dependences and behavior against substrates, inhibitors, and activators. In this group only the tartrate-resistant acid phosphatase is a metallo-enzyme whereas the other members do not require metal-ion binding for their catalytic activity. In addition, tartrate-resistant acid phosphatase and erythrocytic acid phosphatase are not inhibited by L-(+)-tartrate ion while the prostatic acid phosphatase is tartrate-sensitive. This is an important

  5. Position effect variegation of an acid phosphatase gene in Drosophila melanogaster.

    PubMed

    Frisardi, M C; MacIntyre, R J

    1984-01-01

    X-ray mutagenesis has produced a series of deficiencies in a duplication of part of the third chromosome containing the acid phosphatase gene (Acph-1) in Drosophila melanogaster. In one of these deficiencies, Acph-1 is shown to be undergoing position effect variegation. Naturally occurring electrophoretic variants of the enzyme were used to visualize and determine quantitatively the extent of variegation of the allele which is cis to the heterochromatic breakpoint. Alteration of genotypic background and temperature provided further evidence for position effect. Rocket immunoelectrophoresis was used to correlate the levels of acid phosphatase activity and protein in flies containing the deficiency. A novel result indicates that the variegation is not the consequence of an averaging of active and inactive cells, but rather due to a quantitative alteration of gene activity within at least some individual cells.

  6. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-09

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  7. Uteroferrin and intracellular tartrate-resistant acid phosphatases are the products of the same gene.

    PubMed

    Ling, P; Roberts, R M

    1993-04-05

    Uteroferrin (Uf) is a purple acid phosphatase with a bi-iron center. It is the major secretory product of the porcine uterus under the influence of progesterone and supplies iron to the developing fetuses during pregnancy. Tartrate-resistant acid phosphatases (TRAP) are clearly similar to Uf in many of their properties but are generally located intracellularly in lysosomes. To determine whether Uf and intracellular TRAP are distinct gene products, cDNA for the TRAP from pig spleen were compared with Uf cDNA. Although no full-length cDNA for the former were isolated, a TRAP cDNA of 1.1 kilobases was identical in nucleotide sequence to a Uf cDNA (1.42 kilobases) in the region of overlap, which included the entire 3'-end of the transcript and most of the open reading frame. TRAP purified from porcine spleen also had an NH2-terminal amino acid sequence that corresponded to that of Uf purified from uterine secretions and was also similar in sequence to intracellular TRAP isolated from tissues of other species, including ones from human osteoclastomas and spleen. Finally, Southern hybridization analysis with two probes specific for exons 1 and 2 of the Uf gene strongly suggested the presence of only a single gene for acid phosphatases of this class in the pig. A similar analysis performed on human DNA with an exon-specific probe for human TRAP was also consistent with a single gene. It is concluded that the difference in trafficking between a secreted TRAP, such as Uf, and TRAP located in lysosomes is not the result of distinctive primary sequence of the polypeptides and that the variability within species ascribed to such enzymes is most likely the result of minor posttranslational changes.

  8. The Saccharomyces cerevisiae PHM8 gene encodes a soluble magnesium-dependent lysophosphatidic acid phosphatase.

    PubMed

    Reddy, Venky Sreedhar; Singh, Arjun Kumar; Rajasekharan, Ram

    2008-04-04

    Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.

  9. Cloning and characterization of drought-stimulated phosphatidic acid phosphatase genes from Vigna unguiculata.

    PubMed

    França, Marcel Giovanni Costa; Matos, Ana Rita; D'arcy-Lameta, Agnès; Passaquet, Chantal; Lichtlé, Christiane; Zuily-Fodil, Yasmine; Pham-Thi, Anh Thu

    2008-12-01

    Under environmental stresses, several lipolytic enzymes are known to be activated and to contribute to membrane lipid turnover and generation of second messengers. In animal cells, phosphatidic acid phosphatase (PAP, EC 3.1.3.4), which dephosphorylates phosphatidic acid generating diacylglycerol, is long known as an enzyme involved in lipid synthesis and cell signalling. However, knowledge on PAP in plants remains very limited. The aim of this work was to isolate and characterize PAP genes in the tropical legume Vigna unguiculata (cowpea), and to study their expression under different stress conditions. Two cDNAs designated as VuPAPalpha and VuPAPbeta were cloned from the leaves of cowpea. Both proteins share sequence homology to animal type 2 PAP, namely, the six transmembrane regions and the consensus sequences corresponding to the catalytic domain of the phosphatase family, like the recently described Arabidopsis LPP (Lipid Phosphate Phosphatase) proteins. The recombinant protein VuPAPalpha expressed in Escherichia coli cells was able to convert phosphatidic acid into diacylglycerol. Unlike VuPAPbeta, VuPAPalpha has an N-terminal transit peptide and was addressed to chloroplast in vitro. Both genes are expressed in several cowpea organs and their transcripts accumulate in leaves in response to water deficit, including progressive dehydration of whole plants and rapid desiccation of detached leaves. No changes in expression of both genes were observed after wounding or by treatment with jasmonic acid. Furthermore, the in silico analysis of VuPAPalpha promoter allowed the identification of several putative drought-related regulatory elements. The possible physiological role of the two cloned PAPs is discussed.

  10. Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii.

    PubMed

    Wang, Q H; Jiao, Y; Du, X D; Zhao, X X; Huang, R L; Deng, Y W; Yan, F

    2015-01-26

    Purple acid phosphatases (PAPs), also known as type 5 acid phosphatases, are widely present in animals, plants, and fungi. In mammal, PAP was reported to participate in immune defense and bone resorption. In this study, the characteristics and potential functions of a PAP gene from pearl oyster Pinctada martensii (pm-PAP) were examined. The Pm-PAP cDNA was found to be 2777 base pairs, containing a 1581-base pair open reading fragment encoding for 526 amino acids with an estimated molecular mass of 60.1 kDa and theoretical isoelectric point of 5.82. One signal peptide and five conserved motifs [GDXX/GDXXY/GNH(D/E)/XXXH/(A/G)HXH] were present in the entire sequence. Tissue expression profile analysis showed that pm-PAP mRNA was constitutively expressed in all tissues studied with abundant mRNA found in mollusk defense system, including hepatopancreas, gill, and hemocytes. After lipopolysaccharide stimulation, the expression of pm-PAP mRNA in hemocytes was dramatically upregulated at 2 h and achieved the highest level at 36 h. Additionally, pm-PAP mRNA expression was significantly increased and achieved the highest level at 2 days after the surgical implantation during pearl production. These results suggest that pm-PAP is a constitutive and inducible protein that may be involved in the immune defense of pearl oyster.

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

    PubMed Central

    Li, Chengchen; Gui, Shunhua; Yang, Tao; Walk, Thomas; Wang, Xiurong; Liao, Hong

    2012-01-01

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. Methods BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. Key Results In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. Conclusions Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions. PMID:21948626

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

    PubMed

    Li, Chengchen; Gui, Shunhua; Yang, Tao; Walk, Thomas; Wang, Xiurong; Liao, Hong

    2012-01-01

    Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions.

  13. Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells.

    PubMed

    Kaida, Rumi; Sage-Ono, Kimiyo; Kamada, Hiroshi; Okuyama, Hidetoshi; Syono, Kunihiko; Kaneko, Takako S

    2003-01-27

    Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.

  14. Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter.

    PubMed

    Reddy, S V; Hundley, J E; Windle, J J; Alcantara, O; Linn, R; Leach, R J; Boldt, D H; Roodman, G D

    1995-04-01

    Tartrate-resistant acid phosphatase (TRAP) is an iron-binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5' and 3' deletions of a 1.8 kb fragment containing the 5'-flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P1) was located within the region from -881 bp to -463 bp relative to the ATG, while the downstream promoter (P2) was located between -363 bp to -1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P2 promoter at -1846 bp to -1240 bp and a putative enhancer region at -962 bp to -881 bp relative to the ATG were identified. PCR analysis of promoter-specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin-bound iron increased P1 promoter activity 2.5-fold and hemin decreased P1 promoter activity, but neither had any effect on P2 activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.

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

    USDA-ARS?s Scientific Manuscript database

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

  16. Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron.

    PubMed

    Alcantara, O; Reddy, S V; Roodman, G D; Boldt, D H

    1994-03-01

    Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881

  17. Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron.

    PubMed Central

    Alcantara, O; Reddy, S V; Roodman, G D; Boldt, D H

    1994-01-01

    Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881

  18. Soybean root nodule acid phosphatase.

    PubMed Central

    Penheiter, A R; Duff, S M; Sarath, G

    1997-01-01

    Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism. PMID:9193092

  19. Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

    PubMed

    Bostian, K A; Lemire, J M; Halvorson, H O

    1983-05-01

    We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

  20. Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

    PubMed Central

    Bostian, K A; Lemire, J M; Halvorson, H O

    1983-01-01

    We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed. Images PMID:6346058

  1. Acid phosphatase/phosphotransferases from enteric bacteria.

    PubMed

    Mihara, Y; Utagawa, T; Yamada, H; Asano, Y

    2001-01-01

    We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.

  2. The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

    PubMed

    San Miguel, S M; Goseki-Sone, M; Sugiyama, E; Watanabe, H; Yanagishita, M; Ishikawa, I

    1998-10-01

    Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

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

    PubMed Central

    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

  4. The Acid Phosphatase-Encoding Gene GmACP1 Contributes to Soybean Tolerance to Low-Phosphorus Stress

    PubMed Central

    Hao, Derong; Wang, Hui; Kan, Guizhen; Jin, Hangxia; Yu, Deyue

    2014-01-01

    Phosphorus (P) is essential for all living cells and organisms, and low-P stress is a major factor constraining plant growth and yield worldwide. In plants, P efficiency is a complex quantitative trait involving multiple genes, and the mechanisms underlying P efficiency are largely unknown. Combining linkage analysis, genome-wide and candidate-gene association analyses, and plant transformation, we identified a soybean gene related to P efficiency, determined its favorable haplotypes and developed valuable functional markers. First, six major genomic regions associated with P efficiency were detected by performing genome-wide associations (GWAs) in various environments. A highly significant region located on chromosome 8, qPE8, was identified by both GWAs and linkage mapping and explained 41% of the phenotypic variation. Then, a regional mapping study was performed with 40 surrounding markers in 192 diverse soybean accessions. A strongly associated haplotype (P = 10−7) consisting of the markers Sat_233 and BARC-039899-07603 was identified, and qPE8 was located in a region of approximately 250 kb, which contained a candidate gene GmACP1 that encoded an acid phosphatase. GmACP1 overexpression in soybean hairy roots increased P efficiency by 11–20% relative to the control. A candidate-gene association analysis indicated that six natural GmACP1 polymorphisms explained 33% of the phenotypic variation. The favorable alleles and haplotypes of GmACP1 associated with increased transcript expression correlated with higher enzyme activity. The discovery of the optimal haplotype of GmACP1 will now enable the accurate selection of soybeans with higher P efficiencies and improve our understanding of the molecular mechanisms underlying P efficiency in plants. PMID:24391523

  5. Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana.

    PubMed

    Reddy, Venky Sreedhar; Rao, D K Venkata; Rajasekharan, Ram

    2010-04-01

    Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.

  6. Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

    PubMed

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

    2008-02-01

    Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

  7. Biology of tartrate-resistant acid phosphatase.

    PubMed

    Lamp, E C; Drexler, H G

    2000-11-01

    Tartrate-resistant acid phosphatase (TRAP) is a member of the ubiquitously expressed enzyme family of the acid phosphatases. Nearly 30 years ago, TRAP became known to hematologists as cytochemical marker enzyme of hairy cell leukemia. Physiologically, TRAP is primarily a cytochemical marker of macrophages, osteoclasts and dendritic cells. TRAP is localized intracellularly in the lysosomal compartment. Recent data suggest also secretion of TRAP by some cell types, in particular by osteoclasts. Human, mouse and rat TRAP are biochemically well characterized. While the complete genomic sequence of TRAP has been elucidated, only limited information on the genetic details of the gene and its regulation is available. It appears that the intracellular iron content is involved in the regulation of the enzyme. The physiological substrates for this enzyme have not been identified yet and consequently the functional role of TRAP remains completely unknown, though some hypotheses have been forwarded, e.g. involvement in bone resorption and iron homeostasis (transport, metabolism). Taken together, research on the biology of TRAP has been intensive and has led to considerable progress on a number of fronts, including the cloning of the gene. Further studies are, however, still required to determine the role of TRAP in vivo.

  8. GmPAP4, a novel purple acid phosphatase gene isolated from soybean (Glycine max), enhanced extracellular phytate utilization in Arabidopsis thaliana.

    PubMed

    Kong, Youbin; Li, Xihuan; Ma, Jun; Li, Wenlong; Yan, Guijun; Zhang, Caiying

    2014-04-01

    GmPAP4 , a novel plant PAP gene in soybean, has phytase activity. Over-expressing GmPAP4 can enhance Arabidopsis growth when phytate is the sole P source in culture. Phosphorus (P) is an important macronutrient for plant growth and development. However, most of the total P in soils is fixed into organic phosphate (Po). Purple acid phosphatase (PAP) can hydrolyze Po in the soil to liberate inorganic phosphate and enhance plant P utilization. We isolated a novel PAP gene, GmPAP4, from soybean (Glycine max). It had an open reading frame of 1,329 bp, encoding 442 amino acid residues. Sequence alignment and phylogenetics analysis indicated that GmPAP4 was similar to other plant PAPs with large molecular masses. Quantitative real-time PCR analysis showed that the induced expression of GmPAP4 was greater in P-efficient genotype Zhonghuang15 (ZH15) than in P-inefficient genotype Niumaohuang (NMH) during the periods of flowering (28-35 days post phytate stress; DPP) and pod formation (49-63 DPP). Moreover, peak expression, at 63 DPP, was about 3-fold higher in 'ZH15' than in 'NMH'. Sub-cellular localization showed that GmPAP4 might be on plasma membrane or in cytoplasm. Over-expressing GmPAP4 in Arabidopsis resulted in significant rises in P acquisition and utilization compared with the wild-type (WT). Under phytate condition, transgenic Arabidopsis plants showed increases of approximately 132.7 % in dry weight and 162.6 % in shoot P content compared with the WT. Furthermore, when phytate was added as the sole P source in cultures, the activity of acid phosphatase was significantly higher in transgenic plants. Therefore, GmPAP4 is a novel PAP gene that functions in plant's utilization of organic phosphate especially under phytate condition.

  9. Expression patterns of purple acid phosphatase genes in Arabidopsis organs and functional analysis of AtPAP23 predominantly transcribed in flower.

    PubMed

    Zhu, Huifen; Qian, Weiqiang; Lu, Xuzhong; Li, Dongping; Liu, Xin; Liu, Kunfan; Wang, Daowen

    2005-11-01

    Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

  10. A STRESS-RESPONSIVE NAC1-Regulated Protein Phosphatase Gene Rice Protein Phosphatase18 Modulates Drought and Oxidative Stress Tolerance through Abscisic Acid-Independent Reactive Oxygen Species Scavenging in Rice1[W][OPEN

    PubMed Central

    You, Jun; Zong, Wei; Hu, Honghong; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2014-01-01

    Plants respond to abiotic stresses through a complexity of signaling pathways, and the dephosphorylation mediated by protein phosphatase (PP) is an important event in this process. We identified a rice (Oryza sativa) PP2C gene, OsPP18, as a STRESS-RESPONSIVE NAC1 (SNAC1)-regulated downstream gene. The ospp18 mutant was more sensitive than wild-type plants to drought stress at both the seedling and panicle development stages. Rice plants with OsPP18 suppressed through artificial microRNA were also hypersensitive to drought stress. Microarray analysis of the mutant revealed that genes encoding reactive oxygen species (ROS) scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant exhibited reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses. Overexpression of OsPP18 in rice led to enhanced osmotic and oxidative stress tolerance. The expression of OsPP18 was induced by drought stress but not induced by abscisic acid (ABA). Although OsPP18 is a typical PP2C with enzymatic activity, it did not interact with SNF1-RELATED PROTEIN KINASE2 protein kinases, which function in ABA signaling. Meanwhile, the expression of ABA-responsive genes was not affected in the ospp18 mutant, and the ABA sensitivities of the ospp18 mutant and OsPP18-overexpressing plants were also not altered. Together, these findings suggest that OsPP18 is a unique PP2C gene that is regulated by SNAC1 and confers drought and oxidative stress tolerance by regulating ROS homeostasis through ABA-independent pathways. PMID:25318938

  11. Characterization of Arabidopsis Acid Phosphatase Promoter and Regulation of Acid Phosphatase Expression

    PubMed Central

    Haran, Shoshan; Logendra, Sithes; Seskar, Mirjana; Bratanova, Margarita; Raskin, Ilya

    2000-01-01

    The expression and secretion of acid phosphatase (APase) was investigated in Indian mustard (Brassica juncea L. Czern.) plants using sensitive in vitro and activity gel assays. Phosphorus (P) starvation induced two APases in Indian mustard roots, only one of which was secreted. Northern-blot analysis indicated transcriptional regulation of APase expression. Polymerase chain reaction and Southern-blot analyses revealed two APase homologs in Indian mustard, whereas in Arabidopsis, only one APase homolog was detected. The Arabidopsis APase promoter region was cloned and fused to the β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS expression was first evident in leaves of the P-starved Arabidopsis plants. In P-starved roots, the expression of GUS initiated in lateral root meristems followed by generalized expression throughout the root. GUS expression diminished with the addition of P to the medium. Expression of GFP in P-starved roots also initiated in the lateral root meristems and the recombinant GFP with the APase signal peptide was secreted by the roots into the medium. The APase promoter was specifically activated by low P levels. The removal of other essential elements or the addition of salicylic or jasmonic acids, known inducers of gene expression, did not activate the APase promoter. This novel APase promoter may be used as a plant-inducible gene expression system for the production of recombinant proteins and as a tool to study P metabolism in plants. PMID:11027712

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

    PubMed

    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.

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

    PubMed Central

    Brinch-Pedersen, Henrik

    2013-01-01

    The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a. PMID:23918958

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

    PubMed

    Madsen, Claus Krogh; Dionisio, Giuseppe; Holme, Inger Bæksted; Holm, Preben Bach; Brinch-Pedersen, Henrik

    2013-08-01

    The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a.

  15. Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

    PubMed Central

    Shekels, L. L.; Smith, A. J.; Van Etten, R. L.; Bernlohr, D. A.

    1992-01-01

    We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins. PMID:1304913

  16. Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.

    PubMed

    Tanaka, Masayuki; Kishi, Yasuhiro; Takanezawa, Yasukazu; Kakehi, Yoshiyuki; Aoki, Junken; Arai, Hiroyuki

    2004-07-30

    Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.

  17. Cloning and characterization of phosphorus starvation inducible Brassica napus PURPLE ACID PHOSPHATASE 12 gene family, and imprinting of a recently evolved MITE-minisatellite twin structure.

    PubMed

    Lu, Kun; Chai, You-Rong; Zhang, Kai; Wang, Rui; Chen, Li; Lei, Bo; Lu, Jun; Xu, Xin-Fu; Li, Jia-Na

    2008-10-01

    Purple acid phosphatase (PAP) is important for phosphorus assimilation and in planta redistribution. In this study, seven Brassica napus PAP12 (BnPAP12) genes orthologous to Arabidopsis thaliana PAP12 (AtPAP12) are isolated and characterized. NCBI BLASTs, multi-alignments, conserved domain prediction, and featured motif/residue characterization indicate that all BnPAP12 members encode dimeric high molecular weight plant PAPs. BnPAP12-1, BnPAP12-2, BnPAP12-3 and BnPAP12-7 (Group I) have six introns and encode 469-aa polypeptides structurally comparable to AtPAP12. BnPAP12-4 and BnPAP12-6 (Group II) have seven introns and encode 526-aa PAP12s. Encoding a 475-aa polypeptide, BnPAP12-5 (Group III) is evolved from a chimera of 5' part of Group I and 3' part of Group II. Sequence characterization and Southern detection suggest that there are about five BnPAP12 alleles. Homoeologous non-allelic fragment exchanges exist among BnPAP12 genes. BnPAP12-4 and BnPAP12-6 are imprinted with a Tourist-like miniature inverted-repeat transposable element (MITE) which is tightly associated with a novel minisatellite composed of four 36-bp tandem repeats. Existing solely in B. rapa/oleracea lineage, this recently evolved MITE-minisatellite twin structure does not impair transcription and coding capacity of the imprinted genes, and could be used to identify close relatives of B. rapa/oleracea lineage within Brassica. It is also useful for studying MITE activities especially possible involvement in minisatellite formation and gene structure evolution. BnPAP12-6 is silent in transcription. All other BnPAP12 genes basically imitate AtPAP12 in tissue specificity and Pi-starvation induced expression pattern, but divergence and complementation are distinct among them. Alternative polyadenylation and intron retention also exist in BnPAP12 mRNAs.

  18. Penicillin inhibitors of purple acid phosphatase.

    PubMed

    Faridoon; Hussein, Waleed M; Ul Islam, Nazar; Guddat, Luke W; Schenk, Gerhard; McGeary, Ross P

    2012-04-01

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  19. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  20. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  1. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    PubMed

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  2. Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

    PubMed

    Cottrill, Michael A; Golovan, Serguei P; Phillips, John P; Forsberg, Cecil W

    2002-09-01

    When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.

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

    PubMed

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

    2015-06-01

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

  4. The Glycosylphosphatidylinositol-Anchored Phosphatase from Spirodela oligorrhiza Is a Purple Acid Phosphatase1

    PubMed Central

    Nakazato, Hiroshi; Okamoto, Takashi; Nishikoori, Miwa; Washio, Kenji; Morita, Naoki; Haraguchi, Kensaku; Thompson, Guy A.; Okuyama, Hidetoshi

    1998-01-01

    We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP. PMID:9808746

  5. The cytochemistry of tartrate-resistant acid phosphatase. Technical considerations.

    PubMed

    Janckila, A J; Li, C Y; Lam, K W; Yam, L T

    1978-07-01

    Cytochemical demonstration of tartrate-resistant acid phosphatase activity is essential for the diagnosis of leukemic reticuloendotheliosis. In order to perform this test correctly and to interpret the results propertly, it is necessary to understand the technical details of the cytochemical methods thoroughly. The method using naphthol--ASBI phosphoric acid--fast garnet GBC is recommended for this purpose, and factors crucial to the cytochemical study, such as fixation, substrate, coupler, pH and temperature of incubation buffer, counterstains, and mounting media are examined and discussed. Conventional methods for acid phosphatase in the presence and absence of L(+) tartaric acid are also critically examined. The naphthol--ASBI phosphoric acid--fast garnet GBC method is sensitive, technically simple and easily reproducible. Its reaction product is highly chromogenic and is most suitable for cytochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase activity in cytologic preparations. The naphthol--ASBI phosphoric acid--pararosaniline method is highly specific and is best for histochemical demonstration of acid phosphatase and tartrate-resistant acid phosphatase in tissue sections.

  6. Emerging Roles of Human Prostatic Acid Phosphatase

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2013-01-01

    Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed. PMID:24009853

  7. Inhibition of purple acid phosphatase with alpha-alkoxynaphthylmethylphosphonic acids.

    PubMed

    McGeary, Ross P; Vella, Peter; Mak, Jeffrey Y W; Guddat, Luke W; Schenk, Gerhard

    2009-01-01

    Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The K(i) values of these compounds are as low as 4 microM, the lowest reported to date for a PAP inhibitor.

  8. The acid phosphatases of Thermoascus crustaceus, a thermophilic fungus.

    PubMed

    Arnold, W N; Garrison, R G; Mann, L C; Wallace, D P

    1988-01-01

    Thermoascus crustaceus, a filamentous, thermophilic ascomycete with pathogenic potential was cultured on Sabouraud's liquid medium at temperatures from 27 to 47 degrees C for periods up to 7 days. Growth rate and yield were optimal at 37 degrees C. Morphological changes were confined to the cell walls, the thickness being greatest at 47 degrees C, which were also more resistant to mechanical disruption. Significant amounts of acid phosphatase (EC 3.1.3.2) activity occurred in the spent media of all cultures but were greatest at 37 degrees C. The proportions of acid phosphatase activity which were operationally defined as soluble or bound were also documented; the optimum pH for acid phosphatase activity in all fractions was 5.0. Extracts were subjected to polyacrylamide gel electrophoresis under non-denaturing conditions and the gels were stained for acid phosphatase activity. This revealed four electrophoretically distinct acid phosphatases which had different susceptibilities to inhibition by fluoride, phosphate, or tartrate. Effects of growth temperature, or phosphate supplement in the culture medium, on the acid phosphatase isoenzyme pattern were judged to be minor. Cytochemistry at the electron microscope level indicated acid phosphatase activity on the surface, in the periplasmic space, and in the cytoplasm, but no trends with regard to growth conditions. A substantial temperature range can be tolerated by this species but it is concluded that neither the general shape of the cells nor the acid phosphatase isoenzyme pattern changes substantially; this contrasts with previously documented differences for this class of enzyme in dimorphic Sporotrix schenckii.

  9. Synthesis of repressible acid phosphatase in Saccharomyces cerevisiae under conditions of enzyme instability.

    PubMed Central

    Bostian, K A; Lemire, J M; Halvorson, H O

    1982-01-01

    The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme. Images PMID:7050664

  10. OBSERVATIONS ON THE ACID PHOSPHATASES OF EUGLENA GRACILIS

    PubMed Central

    Blum, Jacob J.

    1965-01-01

    When a bleached strain of Euglena is maintained in a medium containing very low con centrations of phosphate, the acid phosphatase activity increases. The increase in acid phosphatase activity is prevented by Actinomycin D and by p-fluorophenylalanine (PFA), indicating that the increased activity is due to de novo synthesis of acid phosphatase. When phosphate is replenished, the acid phosphatase activity decreases to the level characteristic of uninduced cells before there is any appreciable cell division. When cell division resumes in the presence of PFA, the level of acid phosphatase activity remains approximately constant. This indicates that there are two different phosphatases: a constitutive enzyme, whose synthesis is insensitive to the presence of PFA, and an induced enzyme, whose synthesis is sensitive to PFA. These enzymes are not equally sensitive to changes in pH and in fluoride concentration, thus permitting them to be assayed individually in whole toluene-treated cells. Induced cells also acquire the ability to remove phosphate from the medium very rapidly. PMID:14326108

  11. Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

    PubMed Central

    Hadler, Kieran S; Huber, Thomas; Cassady, A Ian; Weber, Jane; Robinson, Jodie; Burrows, Allan; Kelly, Gregory; Guddat, Luke W; Hume, David A; Schenk, Gerhard; Flanagan, Jack U

    2008-01-01

    Background Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. Findings A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. Conlusion The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism. PMID:18771593

  12. Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

    PubMed

    Hadler, Kieran S; Huber, Thomas; Cassady, A Ian; Weber, Jane; Robinson, Jodie; Burrows, Allan; Kelly, Gregory; Guddat, Luke W; Hume, David A; Schenk, Gerhard; Flanagan, Jack U

    2008-09-04

    Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

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

    PubMed

    Vasavada, A R; Thampi, P; Yadav, S; Rawal, U M

    1993-12-01

    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.

  14. Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

    PubMed

    Weinberg, R A; Zusman, D R

    1990-05-01

    One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and

  15. Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase.

    PubMed

    Ek-Rylander, B; Barkhem, T; Ljusberg, J; Ohman, L; Andersson, K K; Andersson, G

    1997-01-15

    The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat

  16. Acid phosphatase activities during the germination of Glycine max seeds.

    PubMed

    dos Prazeres, Janaina Nicanuzia; Ferreira, Carmen Veríssima; Aoyama, Hiroshi

    2004-01-01

    In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.

  17. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  18. Differential expression of three purple acid phosphatases from potato.

    PubMed

    Zimmermann, P; Regierer, B; Kossmann, J; Frossard, E; Amrhein, N; Bucher, M

    2004-09-01

    Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Désirée) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration.

  19. Studies on the catalytic mechanism of pig purple acid phosphatase.

    PubMed

    Wynne, C J; Hamilton, S E; Dionysius, D A; Beck, J L; de Jersey, J

    1995-05-10

    Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.

  20. New form of acid phosphatase during lysosome biogenesis.

    PubMed Central

    Rao, G R; Aithal, H N; Toback, F G; Getz, G S

    1981-01-01

    Lysosome formation was induced in cells of the renal medulla by feeding rats on a K+-deficient diet. The role of the endoplasmic reticulum in the production of acid phosphatase, a typical lysosomal enzyme, was examined. Lysosomal and microsomal fractions were prepared for study by differential centrifugation of homogenates of renal papilla and inner stripe of red medulla. Acid phosphatase activity in the microsomal fraction was distinguished from the activity in the lysosomal fraction in normal tissue by differences in pH optima, tartrate inhibition, distribution of multiple forms after polyacrylamide-gel electrophoresis and detergent-sensitivity. During progressive K+ depletion, acid phosphatase activity in both microsomal and lysosomal fractions of the tissue increased 3-fold. In the lysosomes, K+ depletion was associated with the appearance of a new band of acid phosphatase. The neuraminidase-sensitivity of this band on polyacrylamide-gel electrophoresis indicated that the enzyme protein had been modified by the addition of sialic acid residues. K+ depletion also altered the lysosomal enzyme so that thiol compounds were able to stimulate its activity. Images Fig. 4. PMID:7326004

  1. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    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…

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

    NASA Astrophysics Data System (ADS)

    Kaur, Parvinder; Satyanarayana, T.

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

  3. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    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…

  4. Cloning of the canine glucose-6-phosphatase gene

    SciTech Connect

    Kishnani, P.; Bao, Y.; Brix, A.E.

    1994-09-01

    Two Maltese puppies with massive hepatomegaly and failure to thrive were found to have a markedly reduced Glucose-6-phosphatase (G-6-Pase) activity in the liver and kidney. Deficiency of G-6-Pase activity causes type 1a glycogen storage disease in humans. To further study the mutation responsible for the disease in dog, we cloned G-6-Pase canine cDNA from normal mixed breed dog liver RNA using reverse transcriptase and PCR amplification using primers derived from the published murine G-6-Pase gene sequence. Sequencing revealed an open reading frame of 1071 nucleotides that encodes a predicted 357 amino acid polypeptide in the canine G-6-Pase gene, same as mouse and human. We found more than 90% sequence homology between dog and human G-6-Pase sequence. Hydropathy analysis of the deduced canine G-6-Pase polypeptide shows six transmembrane-spanning segments similar to those seen in human and mouse. Endoplasmic reticulum (ER) localization is similarly predicted by the presence of the ER protein retention signal KK positioned 3 and 4 amino acids from the carboxy terminal. Potential asparagine-linked glycosylation sites are identified at positions 96, 203, and 276. Northern blot analysis revealed increased G-6-Pase mRNA in the deficient dog liver compared to control. This could possibly reflect upregulation of transcription due to the persistent hypoglycemic state. Further studies are directed at the identification of the mutation involved in this deficient dog strain. Characterization of the G-6-Pase gene and protein in the deficient dog model can pave the way for new understanding in the pathophysiology of this disease and for the trials of novel therapeutic approaches including gene therapy.

  5. Phosphotyrosine as a substrate of acid and alkaline phosphatases.

    PubMed

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

    1985-01-01

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

  6. Effect of vanadium compounds on acid phosphatase activity.

    PubMed

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

    1996-01-01

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

  7. Purple acid phosphatase in the walls of tobacco cells.

    PubMed

    Kaida, Rumi; Hayashi, Takahisa; Kaneko, Takako S

    2008-10-01

    Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220kDa homotetramer composed of 60kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k(cat)/K(m)) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120kDa dimer in the cytoplasm and as a 220kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.

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

    PubMed Central

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

    1975-01-01

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

  9. Identification of rice purple acid phosphatases related to phosphate starvation signalling.

    PubMed

    Zhang, Q; Wang, C; Tian, J; Li, K; Shou, H

    2011-01-01

    Purple acid phosphatases (PAPs) are a family of metallo-phosphoesterases involved in a variety of physiological functions, especially phosphate deficiency adaptations in plants. We identified 26 putative PAP genes by a genome-wide analysis of rice (Oryza sativa), 24 of which have isolated EST sequences in the dbEST database. Amino acid sequence analysis revealed that 25 of these genes possess sets of metal-ligating residues typical of known PAPs. Phylogenetic analysis classified the 26 rice and 29 Arabidopsis PAPs into three main groups and seven subgroups. We detected transcripts of 21 PAP genes in roots or leaves of rice seedlings. The expression levels of ten PAP genes were up-regulated by both phosphate deprivation and over-expression of the transcription factor OsPHR2. These PAP genes all contained one or two OsPHR2 binding elements in their promoter regions, implying that they are directly regulated by OsPHR2. Both acid phosphatase (AP) and surface secretory acid phosphatase (SAP) activity assays showed that the up-regulation of PAPs by Pi starvation, OsPHR2 over-expression, PHO2 knockout or OsSPX1 RNA interference led to an increase in AP and SAP activity in rice roots. This study reveals the potential for developing technologies for crop improvement in phosphorus use efficiency. © 2010 German Botanical Society and The Royal Botanical Society of the Netherlands.

  10. The five glucose-6-phosphatase paralogous genes are differentially regulated by insulin alone or combined with high level of amino acids and/or glucose in trout hepatocytes.

    PubMed

    Lucie, Marandel; Weiwei, Dai; Stéphane, Panserat; Sandrine, Skiba-Cassy

    2016-04-01

    A recent analysis of the newly sequenced rainbow trout (Oncorhynchus mykiss) genome suggested that duplicated gluconeogenic g6pc paralogues, fixed in this genome after the salmonid-specific 4th whole genome duplication, may have a role in the setting up of the glucose-intolerant phenotype in this carnivorous species. This should be due to the sub- or neo-functionalization of their regulation. In the present short communication we thus addressed the question of the regulation of these genes by insulin, hormone involved in the glucose homeostasis, and its interaction with glucose and amino acids in vitro. The stimulation of trout hepatocytes with insulin revealed an atypical up-regulation of g6pcb2 ohnologues and confirmed the sub- or neo-functionalization of the five g6pc genes at least at the regulatory level. Intriguingly, when hepatocytes were cultured with high levels of glucose and/or AAs in presence of insulin, most of the g6pc paralogues were up-regulated. It strongly suggested a cross-talk between insulin and nutrients for the regulation of these genes. Moreover these results strengthened the idea that g6pc duplicated genes may significantly contribute to the setting up of the glucose-intolerant phenotype in trout via their atypical regulation by insulin alone or in interaction with nutrients. These findings open new perspectives to better understand in vivo glucose-intolerant phenotype in trout fed a high carbohydrate diet.

  11. Arabidopsis Vegetative Storage Protein Is an Anti-Insect Acid Phosphatase

    PubMed Central

    Liu, Yilin; Ahn, Ji-Eun; Datta, Sumana; Salzman, Ron A.; Moon, Jaewoong; Huyghues-Despointes, Beatrice; Pittendrigh, Barry; Murdock, Larry L.; Koiwa, Hisashi; Zhu-Salzman, Keyan

    2005-01-01

    Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the protein's secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity. PMID:16258019

  12. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

    PubMed Central

    Sneddon, A A; Cohen, P T; Stark, M J

    1990-01-01

    Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell. Images Fig. 5. PMID:2176150

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

    SciTech Connect

    Chou, J.Y.; Takahashi, S.

    1987-06-16

    HeLa S/sub 3/ 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-(/sup 35/S)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 S/sub 3/ 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 S/sub 3/ cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product.

  14. Purification and Characterization of Acid Phosphatase V from Aspergillus nidulans

    PubMed Central

    Harsanyi, Zsolt; Dorn, Gordon L.

    1972-01-01

    Acid phosphatase V of Aspergillus nidulans was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzyme demonstrated a charge microheterogeneity on starch and acrylamide gel electrophoresis, but proved to be homogeneous on ultracentrifugation and gel filtration. Phosphatase V was found to be a classic acid orthophosphoric monoester phosphohydrolase, and it cleaved p-nitrophenylphosphate, glucose-6-phosphate, and uridine-5′-monophosphate at maximal rates. It was inhibited by fluoride, borate, and molybdate ions, and demonstrated end-product inhibition by inorganic phosphate. Metallic ions or cofactors were not required for activity. The molecular weight was estimated to be 100,000, the S20,w was calculated to be 4.1, and the pH optimum was found to be 6.1. Images PMID:4552990

  15. Cytochemical localization of acid phosphatase in Leishmania mexicana amazonensis.

    PubMed

    Pimenta, P F; De Souza, W

    1986-01-01

    Acid phosphatase was cytochemically detected at the ultrastructural level in infective and non-infective promastigotes and in amastigotes of the parasitic protozoan Leishmania mexicana amazonensis. Cerium chloride was used as the capture agent of the phosphate liberated during the hydrolysis of the substrate (Na-beta-glycerophosphate). Reaction product, indicative of enzyme activity, was seen in the outer face of the plasma membrane of many, but not all, infective and noninfective promastigote forms. No reaction product was seen in the plasma membrane of amastigote forms. Reaction product was seen in the endoplasmic reticulum, in the Golgi complex, in vesicles located close to the flagellar pocket and in cytoplasmic structures which may represent lysosomes. No reaction product was seen when the substrate was omitted from or sodium fluoride was added to the incubation medium. The possible role played by the acid phosphatase present in the plasma membrane of Leishmania parasites is discussed.

  16. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    SciTech Connect

    Ou, Zhonghui; Felts, Richard L.; Reilly, Thomas J.; Nix, Jay C.; Tanner, John J.

    2006-05-01

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P4{sub 2}2{sub 1}2, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

  17. Acid Phosphatase Development during Ripening of Avocado 1

    PubMed Central

    Sacher, Joseph A.

    1975-01-01

    The activity and subcellular distribution of acid phosphatase were assayed during ethylene-induced ripening of whole fruit or thick slices of avocado (Persea americana Mill. var. Fuerte and Hass). The activity increased up to 30-fold during ripening in both the supernatant fraction and the Triton X-100 extract of the precipitate of a 30,000g centrifugation of tissue homogenates from whole fruit or slices ripening in moist air. Enzyme activity in the residual precipitate after Triton extraction remained constant. The development of acid phosphatase in thick slices ripened in moist air was similar to that in intact fruit, except that enzyme development and ripening were accelerated about 24 hours in the slices. The increase in enzyme activity that occurs in slices ripening in moist air was inhibited when tissue sections were infiltrated with solutions, by aspiration for 2 minutes or by soaking for 2 hours, anytime 22 hours or more after addition of ethylene. This inhibition was independent of the presence or absence of cycloheximide or sucrose (0.3-0.5m). However, the large decline in enzyme activity in the presence of cycloheximide, as compared with the controls, indicated that synthesis of acid phosphatase was occurring at all stages of ripening. PMID:16659087

  18. Isolation of Lysophosphatidic Acid Phosphatase from Developing Peanut Cotyledons1

    PubMed Central

    Shekar, Sunil; Tumaney, Ajay W.; Rao, T.J.V. Sreenivasa; Rajasekharan, Ram

    2002-01-01

    The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [3H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min−1 mg−1. The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 ± 1.5 kD. The Km values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 μm, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants. PMID:11891254

  19. Calcitonin releases acid phosphatase from rat ventral prostate explants.

    PubMed

    Latif, A; Nakhla, A M

    1994-01-01

    Inclusion of salmon calcitonin in the culture medium of rat ventral prostate explants diminished l-tartarate-sensitive acid phosphatase activity in the tissues with a concomitant increment of the enzyme activity in the medium. The effect of the hormone was dose-dependent for a dose range of 10(-12)-10(-6) M. Acid phosphatase activity in prostate explants decreased from 38.6 +/- 3.5 to 20.5 +/- 2.8, whereas it increased from 0.60 +/- 0.15 to 2.80 +/- 0.40 nmol p-nitrophenol liberated/mg protein/30 min in the culture medium. Tissues exposed to 10(-6) M salmon calcitonin had higher acetylcholinesterase activity (8.8 +/- 0.7) than non-exposed ones (6.2 +/- 0.5 mumol substrate hydrolyzed/g tissue/min). These results suggest that locally produced calcitonin causes a release for prostatic acid phosphatase from prostate tissues possibly through its interaction with the cholinergic system.

  20. LEPS2, a Phosphorus Starvation-Induced Novel Acid Phosphatase from Tomato1

    PubMed Central

    Baldwin, James C.; Karthikeyan, Athikkattuvalasu S.; Raghothama, Kashchandra G.

    2001-01-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase. PMID:11161030

  1. Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

    NASA Astrophysics Data System (ADS)

    Wang, Yiyan; Sun, Hushan; Wang, Yanjie; Yan, Dongchun; Wang, Lei

    2015-03-01

    In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

  2. Acid phosphatase role in chickpea/maize intercropping.

    PubMed

    Li, S M; Li, L; Zhang, F S; Tang, C

    2004-08-01

    Organic P comprises 30-80 % of the total P in most agricultural soils. It has been proven that chickpea facilitates P uptake from an organic P source by intercropped wheat. In this study, acid phosphatase excreted from chickpea roots is quantified and the contribution of acid phosphatase to the facilitation of P uptake by intercropped maize receiving phytate is examined. For the first experiment using hydroponics, maize (Zea mays 'Zhongdan No. 2') and chickpea (Cicer arietinum 'Sona') were grown in either the same or separate containers, and P was supplied as phytate, KH2PO4 at 0.25 mmol P L(-1), or not at all. The second experiment involved soil culture with three types of root separation between the two species: (1) plastic sheet, (2) nylon mesh, and (3) no barrier. Maize plants were grown in one compartment and chickpea in the other. Phosphorus was supplied as phytate, Ca(H2PO4)2 at 50 mg P kg(-1), or no P added. In the hydroponics study, the total P uptake by intercropped maize supplied with phytate was 2.1-fold greater than when it was grown as a monoculture. In the soil experiment, when supplied with phytate, total P uptake by maize with mesh barrier and without root barrier was 2.2 and 1.5 times, respectively, as much as that with solid barrier. In both experiments, roots of both maize and chickpea supplied with phytate and no P secreted more acid phosphatase than those with KH2PO4 or Ca(H2PO4)2. However, average acid phosphatase activity of chickpea roots supplied with phytate was 2-3-fold as much as maize. Soil acid phosphatase activity in the rhizosphere of chickpea was also significantly higher than maize regardless of P sources. Chickpea can mobilize organic P in both hydroponic and soil cultures, leading to an interspecific facilitation in utilization of organic P in maize/chickpea intercropping.

  3. High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase.

    PubMed

    Wysocki, Paweł; Płucienniczak, Grazyna; Strzezek, Jerzy

    2009-01-01

    Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.

  4. phoD Alkaline Phosphatase Gene Diversity in Soil

    PubMed Central

    Kertesz, Michael A.; Bünemann, Else K.

    2015-01-01

    Phosphatase enzymes are responsible for much of the recycling of organic phosphorus in soils. The PhoD alkaline phosphatase takes part in this process by hydrolyzing a range of organic phosphoesters. We analyzed the taxonomic and environmental distribution of phoD genes using whole-genome and metagenome databases. phoD alkaline phosphatase was found to be spread across 20 bacterial phyla and was ubiquitous in the environment, with the greatest abundance in soil. To study the great diversity of phoD, we developed a new set of primers which targets phoD genes in soil. The primer set was validated by 454 sequencing of six soils collected from two continents with different climates and soil properties and was compared to previously published primers. Up to 685 different phoD operational taxonomic units were found in each soil, which was 7 times higher than with previously published primers. The new primers amplified sequences belonging to 13 phyla, including 71 families. The most prevalent phoD genes identified in these soils were affiliated with the orders Actinomycetales (13 to 35%), Bacillales (1 to 29%), Gloeobacterales (1 to 18%), Rhizobiales (18 to 27%), and Pseudomonadales (0 to 22%). The primers also amplified phoD genes from additional orders, including Burkholderiales, Caulobacterales, Deinococcales, Planctomycetales, and Xanthomonadales, which represented the major differences in phoD composition between samples, highlighting the singularity of each community. Additionally, the phoD bacterial community structure was strongly related to soil pH, which varied between 4.2 and 6.8. These primers reveal the diversity of phoD in soil and represent a valuable tool for the study of phoD alkaline phosphatase in environmental samples. PMID:26253682

  5. In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

    PubMed

    Krebs, H O; Hoffschulte, H K; Müller, M

    1989-05-01

    We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

  6. The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase.

    PubMed

    Reilly, Thomas J; Calcutt, Michael J

    2004-01-01

    The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.

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

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    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.

  8. The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity.

    PubMed

    Dillon, D A; Wu, W I; Riedel, B; Wissing, J B; Dowhan, W; Carman, G M

    1996-11-29

    We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity. DGPP phosphatase activity was absent in pgpB mutant cells and was expressed at high levels in cells carrying the wild-type pgpB gene on a runaway replication plasmid. The pgpB mutant has been primarily characterized by a defect in phosphatidate (PA) phosphatase activity and also exhibits defects in lyso-PA phosphatase and phosphatidylglycerophosphate phosphatase activities. The defective PA phosphatase in the pgpB mutant was shown to be a Mg2+-independent PA phosphatase activity of the DGPP phosphatase enzyme. We characterized DGPP phosphatase activity in membranes from cells overproducing the pgpB gene product. DGPP phosphatase catalyzed the dephosphorylation of the beta phosphate of DGPP to form PA followed by the dephosphorylation of PA to form diacylglycerol. The specificity constant (Vmax/Km) for DGPP was 9.3-fold greater than that for PA. The pH optimum for the DGPP phosphatase reaction was 6. 5. Activity was independent of a divalent cation requirement, was potently inhibited by Mn2+ ions, and was insensitive to inhibition by N-ethylmaleimide. Pure DGPP phosphatase from Saccharomyces cerevisiae was shown to be similar to the E. coli DGPP phosphatase in its ability to utilize lyso-PA and phosphatidylglycerophosphate as substrates in vitro.

  9. Direct electrochemistry of porcine purple acid phosphatase (uteroferrin).

    PubMed

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

    2004-08-17

    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 --> FeII-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 (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.

  10. Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

    PubMed

    Fujimoto, S; Murakami, K; Hosoda, T; Yamamoto, Y; Watanabe, K; Morinaka, Y; Ohara, A

    1992-05-01

    Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.

  11. Molecular cloning and characterization of acid phosphatase in venom of the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

    PubMed

    Zhu, Jia-ying; Ye, Gong-yin; Hu, Cui

    2008-06-15

    The present study describes cDNA cloning, sequencing, enzyme activity determination and localization, and mRNA expression of acid phosphatase in the venom apparatus of an endoparasitoid, Pteromalus puparum. This is the first report of cloning a venom acid phosphatase gene which has been described in parasitoid wasps. The cDNA consisted of 1378bp with 1215bp open reading frame and encoded a sequence of 405 amino acids. A 23 residues N-terminal signal peptide was followed by a short 15 residues (25-39) histidine acid phosphatases phosphohistidine signature and a long 302 residues (24-325) acid phosphatase family domain. The deduced amino acid sequence shared 32-88% identity to its counterparts from other insects. Enzyme activity was measured by using p-nitrophenyl phosphate (p-NPP) as substrate, and a high level of acid phosphatase activity in venom was detected. Optimal pH and temperature for this enzyme activity was 4.8 and 45 degrees C, respectively. Ultracytochemical analyses further revealed that strong enzyme activity was located in the nuclei and secretory vesicles of the venom gland secretory cells. Expression of the acid phosphatase gene was observed to be regulated at different developmental stages by RT-PCR analysis as it expressed immediately with low abundance after adult emergence, then increased to the high level at 2-4 days, followed by a drop to the low abundance after 4 days. Compared to the mRNA expression, a time-course-related enzyme activity in an individual venom apparatus was also found.

  12. Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli

    PubMed Central

    2014-01-01

    Background The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. Results Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 μM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. Conclusion The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops. PMID:24713147

  13. Stimulation of phosphatidylglycerolphosphate phosphatase activity by unsaturated fatty acids in rat heart.

    PubMed

    Cao, S G; Hatch, G M

    1994-07-01

    Phosphatidylglycerolphosphate (PGP) synthase and PGP phosphatase catalyze the sequential synthesis of phosphatidylglycerol from cytidine-5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG) and glycerol-3-phosphate. PGP synthase and PGP phosphatase activities were characterized in rat heart mitochondrial fractions, and the effect of fatty acids on the activity of these enzymes was determined. PGP synthase was observed to be a heat labile enzyme that exhibited apparent Km values for CDP-PG and glycerol-3-phosphate of 46 and 20 microM, respectively. The addition of exogenous oleic acid to the assay mixture did not affect PGP synthase activity. PGP phosphatase was observed to be a heat labile enzyme, and addition of oleic acid to the assay mixture caused a concentration-dependent stimulation of PGP phosphatase activity. Maximum stimulation (1.9-fold) of enzyme activity was observed in the presence of 0.5 mM oleic acid, but the stimulation was slightly attenuated by the presence of albumin in the assay. The presence of oleic acid in the assay mixture caused the inactivation of PGP phosphatase activity to be retarded at 55 degrees C. Stimulation of PGP phosphatase activity was also observed with arachidonic acid, whereas taurocholic, stearic and palmitic acids did not significantly affect PGP phosphatase activity. The activity of mitochondrial phosphatidic acid phosphohydrolase was not affected by inclusion of oleic acid in the incubation mixture. We postulate that unsaturated fatty acids stimulate PGP phosphatase activity in rat heart.

  14. A chemical relaxation study of human prostatic acid phosphatase.

    PubMed

    Shear, D B; Kustin, K

    1968-01-01

    Chemical relaxation methods and a dilution technique were applied to the study of the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase. Although the reaction mechanism was not elucidated, rate constants and equilibrium constants were obtained for the reaction of enzyme and p-nitrophenol to form a complex. A slow, 2-sec relaxation effect which showed no concentration dependence was observed in various reaction mixtures, including some lacking the substrate and products of the hydrolytic reaction. The conclusion drawn is that there are two forms of the prostatic enzyme, which are normally in equilibrium with each other, but which undergo a relatively slow interconversion when this equilibrium is perturbed. A preliminary calculation indicates that these forms are present in the equilibrium ratio of 2:1.

  15. Structure of Thermotoga maritima Stationary Phase Survival Protein SurE: A Novel Acid Phosphatase

    PubMed Central

    Zhang, R.-G.; Skarina, T.; Katz, J.E.; Beasley, S.; Khachatryan, A.; Vyas, S.; Arrowsmith, C.H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.

    2009-01-01

    Summary Background The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase σ subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results The structure of SurE from Thermotoga maritima was determined at 2.0 Å. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5–6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and inter-subunit salt bridges were identified that may explain the SurE thermostability. Conclusions The structure of SurE provided information about the protein’s fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis. PMID:11709173

  16. Structure of thermotoga maritima stationary phase survival protein SurE : a novel acid phosphatase.

    SciTech Connect

    Zhang, R.-G; Skarina, T.; Katz, J. E.; Khachatryan, A; Vyas, S.; Arrowsmith, C. H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Biosciences Division; Univ. of Toronto; Univ. of California; Clinical Genomics Centre /Proteomics, Univ. Health Network

    2001-11-01

    Background: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase {sigma} subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results: The structure of SurE from Thermotoga maritima was determined at 2.0 Angstroms. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. Conclusions: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

  17. Bovine brain low Mr acid phosphatase: purification and properties.

    PubMed

    Saeed, A; Tremori, E; Manao, G; Camici, G; Cappugi, G; Ramponi, G

    1990-01-01

    Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.

  18. Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase.

    PubMed

    Flanagan, J U; Cassady, A I; Schenk, G; Guddat, L W; Hume, D A

    2006-08-01

    Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of approximately 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the approximately 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans.

  19. "Prostatic acid phosphatase?" A comparison of acid phosphatase activities in epithelial cells, granulocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium.

    PubMed Central

    Helms, S. R.; Brattain, M. G.; Pretlow, T. G.; Kreisberg, J. I.

    1977-01-01

    Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells. PMID:560800

  20. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  1. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    PubMed

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

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

    PubMed Central

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

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

  3. Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.

    PubMed

    Zhang, Z Y; Van Etten, R L

    1990-10-01

    A low-molecular-weight acid phosphatase that is representative of a group recently shown to be phosphotyrosyl protein phosphatases was purified to homogeneity from bovine heart. The enzyme was a monomer with a molecular mass of 18 kDa and had an isoelectric point of 7.0. The absorption coefficient, E1% 1cm was 9.65 at 280 nm. The enzyme had pH optima of 5.3 and 6.0 with the substrates p-nitrophenyl phosphate and tyrosine phosphate, respectively. When measured at pH 5 and 37 degrees C, the enzyme had specific activities of 114 and 86 mumol min-1 mg-1 for p-nitrophenyl phosphate and tyrosine O-phosphate, respectively, while the Km values were 0.38 and 14 mM. The enzyme was highly specific for aryl monophosphate esters and showed little or no activity toward aliphatic phosphate esters, with the remarkable exception of flavin mononucleotide (FMN) and certain of its structural analogs. As shown by 31P NMR data, the activity toward FMN was due to the hydrolysis of one of the eight components present in the (commercial) sample. Both molybdate and vanadate were potent inhibitors, with inhibition constants of 37 and 29 microM, respectively; tartrate and fluoride had little effect on enzymatic activity. A two-stage reversible denaturation of the enzyme by guanidine HCl was observed with midpoints of 0.25 and 1.75 M, respectively. The amino acid composition was homologous to the low-molecular-weight acid phosphatases from other tissue. The enzyme showed immunological cross-reactivity against low-molecular-weight human liver acid phosphatase. There were 7 or 8 accessible cysteines on the monomeric protein and at least one was essential for enzyme activity. The enzyme also had phosphotransferase activity, for example transferring phosphate from p-nitrophenyl phosphate to a wide variety of alcohol acceptors.

  4. Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.

    PubMed

    Fagerlund, Katja M; Ylipahkala, Hannele; Tiitinen, Sari L; Janckila, Anthony J; Hamilton, Susan; Mäentausta, Olli; Väänänen, H Kalervo; Halleen, Jussi M

    2006-05-15

    Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.

  5. Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity

    PubMed Central

    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, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. Conclusion Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity. PMID:18320034

  6. Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis.

    PubMed

    Naseri, Joseph Itor; Truong, Ngoc Thanh; Hörentrup, Jens; Kuballa, Petric; Vogel, Andreas; Rompel, Annette; Spener, Friedrich; Krebs, Bernt

    2004-12-01

    Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.

  7. The thermal stability of a castor bean seed acid phosphatase.

    PubMed

    Granjeiro, Paulo Afonso; Cavagis, Alexandre Donizeti Martins; de Campos Leite, Luciana; Ferreira, Carmen Veríssima; Granjeiro, José Mauro; Aoyama, Hiroshi

    2004-11-01

    The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.

  8. Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato.

    PubMed

    Kusudo, Tatsuya; Sakaki, Toshiyuki; Inouye, Kuniyo

    2003-07-01

    Purple acid phosphatase (PAP) was purified from sweet potato dry powder, which is used as a food additive. Spectrometric and enzymatic analyses, and analysis of the amino-terminal sequence indicated that the purified purple acid phosphatase was PAP1. High activity in neutral and acidic conditions, broad substrate specificity, and good thermal stability of PAP1 suggest the possibility of practical applications of PAP1.

  9. Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors.

    PubMed

    Fuchs, Stefan; Tischer, Stefanie V; Wunschel, Christian; Christmann, Alexander; Grill, Erwin

    2014-04-15

    The plant hormone abscisic acid (ABA) acts both as a developmental signal and as an integrator of environmental cues such as drought and cold. ABA perception recruits an ABA-binding regulatory component [regulatory component of ABA receptor (RCAR)/PYR1/PYL] and an associated protein phosphatase 2C (PP2C). Phytohormone binding inactivates the phosphatase activity of the coreceptor, permitting phosphorelay of the ABA signal via downstream protein kinases. RCARs and PP2C coreceptors are represented by small protein families comprising 14 and 9 members in Arabidopsis, respectively. The specificity of the RCAR-PP2C interaction and the constraints contributing to specific combinations are poorly understood. In this contribution, we analyzed RCAR7/PYL13, which is characterized by three variant amino acid residues in the conserved ABA-binding pocket. RCAR7 regulated the phosphatase activity of the PP2Cs ABI1, ABI2, and PP2CA in vitro at nanomolar ABA levels; however, it was unable to regulate the structurally related hypersensitive to ABA 1 (HAB1). Site-directed mutagenesis of HAB1 established ABA-dependent regulation by RCAR7. Conversion of the noncanonical amino acid residues of RCAR7 into the consensus ABA-binding pocket did not perceptibly change receptor function. Ectopic expression of RCAR7 in Arabidopsis resulted in ABA hypersensitivity affecting gene regulation, seed germination, and stomatal closure. The RCAR7 loss-of-function mutant revealed no changes in ABA responses, similar to the RCAR9 knockout line, whereas the combined deficiency of RCAR7 and RCAR9 resulted in ABA-insensitive seed germination. The study shows a role of RCAR7 in early plant development, proves its ABA receptor function, and identifies structural constraints of RCAR7-PP2C interaction.

  10. Purification and properties of an acid phosphatase from Entamoeba histolytica HM-1:IMSS.

    PubMed

    Aguirre-García, M M; Cerbón, J; Talamás-Rohana, P

    2000-04-24

    Entamoeba histolytica contains and secretes acid phosphatase, which has been proposed as a virulence factor in some pathogenic microorganisms. In this work, we purified and characterised a membrane-bound acid phosphatase (MAP) from E. histolytica HM-1:IMSS and studied the effect of different chemical compounds on the secreted acid phosphatase and MAP activities. MAP purification was accomplished by detergent solubilisation, and affinity and ion exchange chromatographies. The enzyme showed a pI of 5.5-6.2, an optimum pH of 5.5, and a Km value of 1.14 mM with p-nitrophenyl phosphate.

  11. Identification, cloning, and expression of Pseudomonas aeruginosa phosphorylcholine phosphatase gene.

    PubMed

    Massimelli, María J; Beassoni, Paola R; Forrellad, Marina A; Barra, José L; Garrido, Mónica N; Domenech, Carlos E; Lisa, Angela T

    2005-05-01

    Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

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

    NASA Astrophysics Data System (ADS)

    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

  13. Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

    PubMed

    Ahlemeyer, B; Selke, D; Schaper, C; Klumpp, S; Krieglstein, J

    2001-10-26

    The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

  14. Acid and alkaline phosphatase localization in the digestive tract mucosa of the Hemisorubim platyrhynchos.

    PubMed

    Faccioli, Claudemir Kuhn; Chedid, Renata Alari; Mori, Ricardo Hideo; Amaral, Antônio Carlos do; Franceschini-Vicentini, Irene Bastos; Vicentini, Carlos Alberto

    2016-09-01

    This cytochemical study investigated the acid and alkaline phosphatase of the digestive tract of Hemisorubim platyrhynchos. Acid phosphatase was detected in the lining epithelium throughout the digestive tract, whereas alkaline phosphatase was only observed in the intestine. In the esophagus, an acid phosphatase reaction occurred in the apical cytoplasm of the epithelial cells and was related to epithelial protection and freeing of superficial cells for sloughing. Similar results were also observed in epithelial cells of gastric epithelium. In the gastric glands, acid phosphatase occurred in lysosomes of the oxynticopeptic cells acting in the macromolecule degradation for use as an energy source, whereas in the vesiculotubular system, its presence could be related to secretion processes. Furthermore, acid phosphatase in the intestine occurred in microvilli and lysosomes of the enterocytes and was correlated to absorption and intracellular digestion. However, no difference was reported among the regions of the intestine. However, alkaline phosphatase reaction revealed a large number of reaction dots in the anterior intestine, with the number decreasing toward the posterior intestine. This enzyme has been related to several functions, highlighting its role in the nutrient absorption primarily in the anterior intestine but also being essential in pH regulation because this is a carnivorous species with many gastric glands with secretions that could damage the intestine.

  15. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    PubMed

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  16. Regulation and expression of type V (tartrate-resistant) acid phosphatase in human mononuclear phagocytes.

    PubMed

    Bevilacqua, M A; Lord, D K; Cross, N C; Whitaker, K B; Moss, D W; Cox, T M

    1991-02-01

    Human type V (tartrate-resistant) acid phosphatase belongs to a unique group of iron-binding proteins that includes uteroferrin and other purple phosphatases. The enzyme is normally restricted to osteoclasts and certain phagocytic cells but its rôle is unknown. We show that phosphatase mRNA is abundant in cells of monohistiocytic phenotype and that enzyme expression in cultured human monocyte-derived macrophages is depressed by gamma-interferon and bacterial lipopolysaccharide, agents that promote functional differentiation in these cells. In contrast, phorbol ester, which stimulates intracellular calcium-mediated events, greatly enhances type V phosphatase expression and mRNA abundance. Lymphokine and phorbol ester-modulated expression of type V acid phosphatase expression thus represents a model system for investigating proliferative responses that are specific to cells of the mononuclear macrophage system.

  17. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase

    DOE PAGES

    Story, Sandra; Brigmon, Robin L.

    2016-12-19

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soilmore » resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5 mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. Finally, these results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.« less

  18. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

    PubMed

    Story, Sandra; Brigmon, Robin L

    2017-03-01

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications. Copyright © 2016. Published by Elsevier Inc.

  19. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase

    SciTech Connect

    Story, Sandra; Brigmon, Robin L.

    2016-12-19

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5 mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. Finally, these results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications.

  20. Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A

    SciTech Connect

    Van Hoof, C.; Cayla, X.; Merlevede, W.; Goris, J.

    1995-07-20

    The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5{prime} flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-{kappa}B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5{prime} of a luciferase reporter gene revealed that the 5{prime} flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis. 28 refs., 8 figs., 1 tab.

  1. A critical evaluation of a specific radioimmunoassay for prostatic acid phosphatase

    SciTech Connect

    Goldenberg, S.L.; Silver, H.K.; Sullivan, L.D.; Morse, M.J.; Archibald, E.L.

    1982-11-01

    A radioimmunoassay (RIA) method for acid phosphatase detection was compared to a standard enzyme assay using sera from 210 normal volunteers and 285 patients with prostatic disease. Statistical and clinical comparisons were made between defined subgroups. All 55 normal females had RIA detectable serum acid phosphatase, implying that this assay cannot be entirely specific for enzyme of prostatic origin. Urinary catheterization did not affect acid phosphatase levels. In all stages of carcinoma there were more acid phosphatase elevations by the RIA method than enzyme method, but neither assay could differentiate intercapsular cancer from benign prostatic hyperplasia. A small number of patients with biopsy proven negative nodules had marginally elevated values, suggesting an obligation for closer follow-up. The RIA method may be superior for monitoring patients with more advanced malignancy. Additional practical advantages of the RIA include relative simplicity and elimination of the special serum handling required for the enzyme assay.

  2. A purple acid phosphatase plays a role in nodule formation and nitrogen fixation in Astragalus sinicus.

    PubMed

    Wang, Jianyun; Si, Zaiyong; Li, Fang; Xiong, Xiaobo; Lei, Lei; Xie, Fuli; Chen, Dasong; Li, Yixing; Li, Youguo

    2015-08-01

    The AsPPD1 gene from Astragalus sinicus encodes a purple acid phosphatase. To address the functions of AsPPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, subcellular localization, and phenotypes associated with its over-expression and RNA interference (RNAi) were investigated. The expression of AsPPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of AsPPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant AsPPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Subcellular localization showed that AsPPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of AsPPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that AsPPD1 overexpression affects the ADP levels in the infected roots and nodules, AsPPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of AsPPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that AsPPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.

  3. Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    PubMed Central

    Lee, Jihye; Kim, Jinhee; Son, Kidong; d’Alexandry d’Orengiani, Anne-Laure Pham Humg; Min, Ji-Young

    2017-01-01

    Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus multi-cycle growth. ACP2 knockdown also decreased replication of seasonal influenza A and B viruses and avian IAVs of the H7 subtype. Interestingly, ACP2 depletion had no effect on the replication of Ebola or hepatitis C virus. Because ACP2 is known to be a lysosomal acid phosphatase, we assessed the role of ACP2 in influenza virus entry. While neither binding of the viral particle to the cell surface nor endosomal acidification was affected in ACP2-depleted cells, fusion of the endosomal and viral membranes was impaired. As a result, downstream steps in viral entry were blocked, including nucleocapsid uncoating and nuclear import of viral ribonucleoproteins. Our results established ACP2 as a necessary host factor for regulating the fusion step of influenza virus entry. PMID:28272419

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

    SciTech Connect

    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.

  5. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

    PubMed

    Yang, T T; Sinai, P; Kain, S R

    1996-10-01

    We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

  6. Potential role for purple acid phosphatase in the dephosphorylation of wall proteins in tobacco cells.

    PubMed

    Kaida, Rumi; Serada, Satoshi; Norioka, Naoko; Norioka, Shigemi; Neumetzler, Lutz; Pauly, Markus; Sampedro, Javier; Zarra, Ignacio; Hayashi, Takahisa; Kaneko, Takako S

    2010-06-01

    It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.

  7. Improvement of an acid phosphatase/DHAP-dependent aldolase cascade reaction by using directed evolution.

    PubMed

    van Herk, Teunie; Hartog, Aloysius F; Babich, Lara; Schoemaker, Hans E; Wever, Ron

    2009-09-04

    To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

  8. Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase

    SciTech Connect

    Zhao, Zhaoyang; Lee, Cheng-Chi; Monckton, D.G.

    1996-07-01

    Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNA encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3{prime}-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11. 31 refs., 6 figs., 1 tab.

  9. Theoretical studies on the mechanism of activation of phosphoprotein phosphatases and purple acid phosphatases suggest an evolutionary strategy to survive in acidic environments.

    PubMed

    Zhang, Hao; Ma, Yingying; Yu, Jian-Guo

    2013-12-01

    Dephosphorylation reactions of phosphoprotein phosphatases (PPPs) share a common catalytic cycle. In one stage of the cycle, the active site is regenerated through formation of a new nucleophilic μ-hydroxy moiety and reprotonation of the proton donor, His125 (numbered according to the protein phosphatase 1 sequence). To date the exact details of the mechanism of this step remain uncertain. On the basis of recurring observations in several crystal structures, we propose an activation mechanism in which dephosphorylation of PPPs proceeds mainly through proton transfer from the water molecule that bridges the metal ions to His125, which is mediated by another water molecule. Our calculations using hybrid density functional theory and B3LYP functionals support this activation mechanism. We also propose that Asp95 facilitates proton transfer by eliminating the energy barrier and the backbone carbonyl oxygen atom of His248 acts mainly to orient and stabilize the μ-hydroxo (or water molecule) through hydrogen bonding. Furthermore, on the basis of the structural similarities of the active sites of purple acid phosphatases (PAPs) and PPPs, we speculate that PAPs are activated by a dual proton transfer mediated by one water molecule. Our calculations support this hypothesis and indicate that the active site of PAPs can still be active in an acidic environment (in agreement with the acid phosphatase activity of PAPs). Therefore, the variant of the activation mechanism from PPPs to PAPs implies an evolutionary adaptation to acidic environments.

  10. Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product.

    PubMed Central

    Icho, T

    1988-01-01

    The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization. Images PMID:2846511

  11. The pH-induced glycosylation of secreted phosphatases is mediated in Aspergillus nidulans by the regulatory gene pacC-dependent pathway.

    PubMed

    Nozawa, S R; Ferreira-Nozawa, M S; Martinez-Rossi, N M; Rossi, A

    2003-08-01

    In this communication, we show that the pacC(c)14 mutation drastically reduced the mannose and N-acetylglycosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans when grown at 22 degrees C, pH 5.0, compared to a control strain. The staining after PAGE was not observed for the pacA-encoded acid phosphatase, while the palD-encoded Pi-repressible alkaline phosphatase had an altered electrophoretic mobility. In addition, the secreted acid phosphatase also had a reduced number of isoforms visualized by staining after IEF and glycosylation had a protective effect against its heat inactivation. We also show that a full-length version of gene pacC-1 cloned from Neurospora crassa complemented the pacC(c)14 mutation of A. nidulans, including the remediation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which indicates that glycosylation of secreted phosphatases is mediated in A. nidulans by the conserved PacC pathway that governs pH-responsive gene expression.

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

    PubMed

    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.

  13. Determination of acid phosphatase in biological fluids using a new substrate, 2,6-dichloro-4-nitrophenyl phosphate.

    PubMed

    Teshima, S; Hayashi, Y; Ando, M

    1987-09-30

    A new substrate, 2,6-dichloro-4-nitrophenyl phosphate (DCNP-P), is used for the determination of acid phosphatase (EC 3.1.3.2) in serum and urine. It was hydrolyzed by acid phosphatase to 2,6-dichloro-4-nitrophenol (DCNP) and phosphoric acid. At a pH of 4.5-6.0, the absorption of DCNP liberated by acid phosphatase was much higher than that of p-nitrophenol, which is commonly used as an aglycone in the acid phosphatase assay. By using DCNP-P as a substrate for acid phosphatase activity, determinations can be made without the colour reaction which requires the addition of an alkaline solution, and can be determined by the rate assay that does not require measurement of sample blanks in serum or urine. This method using DCNP-P is highly sensitive and is the most suitable for the rate assay of acid phosphatase activity in biological fluids.

  14. Eliminating the purple acid phosphatase AtPAP26 in Arabidopsis thaliana delays leaf senescence and impairs phosphorus remobilization.

    PubMed

    Robinson, Whitney D; Carson, Ira; Ying, Sheng; Ellis, Kaya; Plaxton, William C

    2012-12-01

    Limitation of crop productivity by suboptimal phosphorus (P) nutrition is a widespread concern. Enhanced crop P-use efficiency could be achieved by improving P remobilization from senescing leaves to developing tissues and seeds. Transcriptomic studies indicate that hundreds of Arabidopsis thaliana genes are up-regulated during leaf senescence, including that encoding the purple acid phosphatase (PAP) AtPAP26 (At5g34850). In this study, biochemical and functional genomic tools were integrated to test the hypothesis that AtPAP26 participates in P remobilization during leaf senescence. An eightfold increase in acid phosphatase activity of senescing leaves was correlated with the accumulation of AtPAP26 transcripts and immunoreactive AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a > 90% decrease in acid phosphatase activity, markedly impaired P remobilization efficiency and delayed senescence. This was paralleled by reduced seed total P concentrations and germination rates. These results demonstrate that AtPAP26 loss of function causes dramatic effects that cannot be compensated for by any other PAP isozyme, even though Arabidopsis contains 29 different PAP genes. Our current and earlier studies establish that AtPAP26 not only helps to scavenge P from organic P sources when Arabidopsis is cultivated in inorganic orthophosphate (Pi)-deficient soils, but also has an important P remobilization function during leaf senescence. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  15. Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg(2+)-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

  16. Suppression of DS1 Phosphatidic Acid Phosphatase Confirms Resistance to Ralstonia solanacearum in Nicotiana benthamiana

    PubMed Central

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg2+-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum. PMID:24073238

  17. Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization.

    PubMed

    Srivastava, Pramod Kumar; Anand, Asha

    2014-03-01

    Acid phosphatase isolated from Vigna aconitifolia seeds was immobilized onto glutaraldehyde activated chitosan beads by crosslinking method. Chitosan beads activated with 2% of glutaraldehyde have demonstrated maximum immobilization yield (∼ 83%). The immobilized enzyme showed optimum activity at pH 7.0, while soluble form was maximally active in acidic range (pH 5.0). With respect to free form, immobilized acid phosphatase showed better activity in alkaline range. On the other side, immobilization does not affect the optimum temperature range i.e., both, soluble and immobilized acid phosphatase exhibited maximum activity at 60 °C. The Km and Vmax values for the immobilized enzyme were calculated to be 0.37 mM and 13.5 U/mg. The immobilization on chitosan beads enhanced the shelf life of acid phosphatase. The immobilized enzyme retained its more than 50% hydrolytic activity for approximately two months. The immobilized acid phosphatase was reusable for more than 40 cycles of reaction.

  18. Electrophoretic separation of alkaline and acid phosphatase isoenzymes from the pulp of monkey teeth.

    PubMed

    Franzén, A; Hasselgren, G

    1978-01-01

    Monkey pulps were homogenized in a Triton tris solution. After three centrifugation steps (800, 20000, and 105000 g) the supernatant was applied on acryl amide columns at pH 7.5 in a tris-diethyl barbituric acid buffer. Electrophoresis was performed at a constant current of 2.5 mA per gel column at 18--20 degrees C. Incubations for alkaline phosphatase (E.C. 3.1.3.1) were carried out at pH 8.3 using naphthol-AS-MX-phosphate as substrate and Fast Red Violet LB salt as coupler. Incubations for acid phosphatase (E.C. 3.1.3.2) were undertaken at pH 5.0 using alpha-naphtyl phosphate as substrate and hexazotized pararosanilin as coupling agent. After the incubations for alkaline phosphatase as well as acid phosphatase two bands showing enzyme activity were demonstrated. By means of treatment with heat (56 degrees C) prior to incubation or addition of vanadate or pyrophosphate to the incubation medium it was shown that the main part of the fast moving alkaline phosphatase band was sensitive to these procedures. The alkaline phosphatase of the slow moving band appeared to be resistant to heat or the addition of inhibitors.

  19. Purification of the Major Soybean Leaf Acid Phosphatase That Is Increased by Seed-Pod Removal.

    PubMed Central

    Staswick, P. E.; Papa, C.; Huang, J. F.; Rhee, Y.

    1994-01-01

    Fruit removal for 5 weeks after flowering increased acid phosphatase activity 10-fold in soybean (Glycine max L. Merr. Var Hobbit) leaves compared with normal seed-pod-bearing plants. The major acid phosphatase activity in leaves was purified over 2700-fold, yielding a single polypeptide of 51 kD with a specific activity of 1353 units/mg protein using p-nitrophenylphosphate as the substrate. Isoelectric focusing demonstrated that the purified protein co-migrated with a majority of the activity that increased in leaves following seed-pod removal. Immunoblot analysis demonstrated that at least part of the increased activity was due to an increased abundance of the phosphatase protein. In situ enzyme activity staining localized most of the total phosphatase activity to vascular tissues, the leaf paraveinal mesophyll cell layer, and the lower epidermis. This distribution and the response to seed-pod removal paralleled previous results for soybean vegetative storage protein (VSP) [alpha] and [beta]. However, in a native polyacrylamide gel the VSP detected by immunological staining of electrophoretically transferred protein did not migrate with the majority of the phosphatase activity. Fractionation of crude leaf protein on concanavalin A-Sepharose yielded a fraction containing 97% of the total VSP but only 0.1% of the total acid phosphatase activity. PMID:12232060

  20. Phosphatidate Phosphatase Activity Plays Key Role in Protection against Fatty Acid-induced Toxicity in Yeast*

    PubMed Central

    Fakas, Stylianos; Qiu, Yixuan; Dixon, Joseph L.; Han, Gil-Soo; Ruggles, Kelly V.; Garbarino, Jeanne; Sturley, Stephen L.; Carman, George M.

    2011-01-01

    The PAH1-encoded phosphatidate (PA) phosphatase in Saccharomyces cerevisiae is a pivotal enzyme that produces diacylglycerol for the synthesis of triacylglycerol (TAG) and simultaneously controls the level of PA used for phospholipid synthesis. Quantitative lipid analysis showed that the pah1Δ mutation caused a reduction in TAG mass and an elevation in the mass of phospholipids and free fatty acids, changes that were more pronounced in the stationary phase. The levels of unsaturated fatty acids in the pah1Δ mutant were unaltered, although the ratio of palmitoleic acid to oleic acid was increased with a similar change in the fatty acid composition of phospholipids. The pah1Δ mutant exhibited classic hallmarks of apoptosis in stationary phase and a marked reduction in the quantity of cytoplasmic lipid droplets. Cells lacking PA phosphatase were sensitive to exogenous fatty acids in the order of toxicity palmitoleic acid > oleic acid > palmitic acid. In contrast, the growth of wild type cells was not inhibited by fatty acid supplementation. In addition, wild type cells supplemented with palmitoleic acid exhibited an induction in PA phosphatase activity and an increase in TAG synthesis. Deletion of the DGK1-encoded diacylglycerol kinase, which counteracts PA phosphatase in controlling PA content, suppressed the defect in lipid droplet formation in the pah1Δ mutant. However, the sensitivity of the pah1Δ mutant to palmitoleic acid was not rescued by the dgk1Δ mutation. Overall, these findings indicate a key role of PA phosphatase in TAG synthesis for protection against fatty acid-induced toxicity. PMID:21708942

  1. Prostatic acid phosphatase assay with self-indicating substrate 2,6-dichloro-4-acetylphenyl phosphate.

    PubMed

    Osawa, S; Iida, S; Yonemitsu, H; Kuroiwa, K; Katayama, K; Nagasawa, T

    1995-02-01

    We characterized six self-indicating substrates, synthesized as the derivative compounds of acetylphenyl phosphate, for serum prostatic acid phosphatase (PAP) activity. One of the substrates, 2,6-dichloro-4-acetylphenyl phosphate (DCAPP), is superior to others in terms of stability, affinity, and low Km for PAP. The hydrolyzed product, 2,6-dichloro-4-acetylphenol (DCAP), has a maximum absorption at 334.2 nm, a pKa of 4.15, and a molar absorptivity at 340 nm of 21,490 L.mol-1.cm-1 in citrate-HCl buffer, pH 5.4. PAP activity was assessed by subtracting tartaric acid-inhibited acid phosphatase activity from total acid phosphatase activity. Our assay system involving DCAPP is a unique kinetic method that shows good reproducibility, wide analytical dynamic range, and high specificity for PAP. Moreover, it is easily adaptable to automated analyzers because the product, DCAP, can be monitored at 340 nm.

  2. Partial purification and kinetic characterization of acid phosphatase from garlic seedling.

    PubMed

    Yenigün, Begüm; Güvenilir, Yüksel

    2003-01-01

    The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40-80% saturation with (NH4)2SOm4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10- 15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4 degrees C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50 degrees C. The enzyme was stable at pH 4.0-10.0 and 40-60 degrees C

  3. Purple acid phosphatase-like sequences in prokaryotic genomes and the characterization of an atypical purple alkaline phosphatase from Burkholderia cenocepacia J2315.

    PubMed

    Yeung, Sin-Lui; Cheng, Chiwai; Lui, Thomas K O; Tsang, Jimmy S H; Chan, Wing-Tat; Lim, Boon L

    2009-07-01

    Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity.

  4. Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases.

    PubMed

    Valizadeh, Mohsen; Schenk, Gerhard; Nash, Kevin; Oddie, Geoff W; Guddat, Luke W; Hume, David A; de Jersey, John; Burke, Terrence R; Hamilton, Susan

    2004-04-15

    Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.

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

    PubMed

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

    2004-11-01

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

  6. The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

    PubMed Central

    Funk, C R; Zimniak, L; Dowhan, W

    1992-01-01

    To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified. Images PMID:1309518

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    SciTech Connect

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

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, 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 P4{sub 1}2{sub 1}2, 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.

  9. Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation.

    PubMed

    Schmid, Annette C; Wise, Helen M; Mitchell, Christina A; Nussbaum, Robert; Woscholski, Rüdiger

    2004-10-08

    The catalytic properties of the type II phosphoinositide 5-phosphatases of Lowe's oculocerebrorenal syndrome, INPP5B, Synaptojanin1, Synaptojanin2 and SKIP were analysed with respect to their substrate specificity and enzymological properties. Our data reveal that all phosphatases have unique substrate specificities as judged by their corresponding KM and VMax values. They also possessed an exclusive sensitivity towards fatty acid composition, head group phosphorylation and micellar presentation. Thus, the biological function of these enzymes will not just be determined by their corresponding regulatory domains, but will be distinctly influenced by their catalytic properties as well. This suggests that the phosphatase domains fulfil a unique catalytic function that cannot be fully compensated by other phosphatases.

  10. Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2.

    PubMed

    Khare, Sharad; Holgren, Cory; Samarel, Allen M

    2006-12-01

    Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.

  11. Molecular genetic responses to lysergic acid diethylamide include transcriptional activation of MAP kinase phosphatase-1, C/EBP-beta and ILAD-1, a novel gene with homology to arrestins.

    PubMed

    Nichols, Charles D; Sanders-Bush, Elaine

    2004-08-01

    We recently demonstrated that the potent hallucinogenic drug lysergic acid diethylamide (LSD) dynamically influences the expression of a small collection of genes within the mammalian prefrontal cortex. Towards generating a greater understanding of the molecular genetic effects of hallucinogens and how they may relate to alterations in behavior, we have identified and characterized expression patterns of a new collection of three genes increased in expression by acute LSD administration. These genes were identified through additional screens of Affymetrix DNA microarrays and examined in experiments to assess dose-response, time course and the receptor mediating the expression changes. The first induced gene, C/EBP-beta, is a transcription factor. The second gene, MKP-1, suggests that LSD activates the MAP (mitogen activated protein) kinase pathway. The third gene, ILAD-1, demonstrates sequence similarity to the arrestins. The increase in expression of each gene was partially mediated through LSD interactions at 5-HT2A (serotonin) receptors. There is evidence of alternative splicing at the ILAD-1 locus. Furthermore, data suggests that various splice isoforms of ILAD-1 respond differently at the transcriptional level to LSD. The genes thus far found to be responsive to LSD are beginning to give a more complete picture of the complex intracellular events initiated by hallucinogens.

  12. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

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

    PubMed

    Srivastava, Pramod Kumar; Anand, Asha

    2015-01-01

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

  14. Study of Acid Phosphatase in Solubilization of Inorganic Phosphates by Piriformospora indica.

    PubMed

    Seshagiri, Swetha; Tallapragada, Padmavathi

    2017-01-02

    Phosphorus is an essential plant macronutrient present in the soil. Only a small portion of phosphorus in soil is taken up by plants and the rest of it becomes unavailable to plants as it is immobilized. Phosphate solubilizing microorganisms play a vital role in converting the insoluble form of phosphates to the soluble form. The present paper reports the solubilization of tricalcium phosphate, rock phosphate, single super phosphate, zinc phosphate and aluminum phosphate by Piriformospora indica with the production of organic acids as well as acid phosphatase. The amount of phosphate released (4.73 mg ml(-1)) and titratable acidity (0.12%) was found to be the highest in the case of single super phosphate as compared to other phosphate sources. High performance liquid chromatography (HPLC) revealed the presence of oxalic acid, lactic acid, citric acid and succinic acid in the media. Highest phosphatase activity was observed with the cell membrane extract of the organism in the presence of zinc phosphate.

  15. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

    PubMed

    Hegeman, C E; Grabau, E A

    2001-08-01

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

  16. The polymorphism of protein phosphatase Z1 gene in Candida albicans.

    PubMed

    Kovács, László; Farkas, Ilona; Majoros, László; Miskei, Márton; Pócsi, István; Dombrádi, Viktor

    2010-12-01

    The gene of protein phosphatase Z1 (CaPPZ1) that codes a fungus specific regulatory enzyme was investigated in Candida albicans. After cloning and sequencing CaPPZ1 we revealed the heterozygous nature of the ATCC 10231 reference strain, and identified two new alleles termed CaPPZ1-2 and CaPPZ1-3. The genetic polymorphism in CaPPZ1 was extended by finding a fourth allele CaPPZ1-4 in a clinical isolate. Single nucleotide replacements and short in/del mutations were identified in the gene, some of which resulted in amino acid changes in the protein. The analysis of the hypervariable 3'-noncoding gene region in 27 DNA sequences obtained from reference strains and clinical samples confirmed the presence of four distinct DNA sequence-groups that correspond to the four main alleles of CaPPZ1. In addition to the allelic combinations, we detected individual mutations elevating genetic variability of the opportunistic pathogen. We utilized the hypervariable gene region for genotyping C. albicans in clinical isolates by sequencing the cloned amplified region, by direct sequencing of the PCR products, or by RFLP analysis. The comparison of the genotypes of the strains originating from different body parts of the same patient proved to be useful in delineating the origin of the infection.

  17. Comparison of tartrate resistant acid phosphatase in a giant cell bone tumor and a spleen infiltrated with hairy cells.

    PubMed

    Lam, K W; Townsend, D; Garza, A; Li, C Y; Yam, L T

    1990-08-01

    Acid phosphatase (E.C.3.1.3.2) in a giant cell bone tumor and a spleen infiltrated with hairy cells was extracted by citrate buffer and then by 0.3 mol/L NaCl. The cationic acid phosphatase in the crude extract was isolated by CM-cellulose chromatography, and further separated by high pressure liquid chromatography. The majority of the tartrate resistant acid phosphatase in the hairy cell spleen was unabsorbed on CM-cellulose and was insensitive to iron. A much larger portion of the acid phosphatase in the bone tumor, than in the spleen, was cationic and was eluted from the column by 0.8 mol/L NaCl. The cationic acid phosphatase was further separated into consecutive peaks of acid phosphatases with different sensitivity to iron. A major portion of acid phosphatase in the giant cell bone tumor was enhanced by iron, while the amounts of iron-enhanced and iron-insensitive acid phosphatase were about the same in the spleen. The differences of the phosphatases in these two types of pathologic specimens indicate the occurrence of two types of enzymes with different biological significance.

  18. [Effect of acetic acid on adsorption of acid phosphatase by some soil colloids and clay minerals].

    PubMed

    Zhao, Zhenhua; Huang, Qiaoyun; Jiang, Xin; Yu, Guifen; Wang, Fang; Li, Xueyuan

    2004-03-01

    This paper studied the effect of acetic acid with different concentrations and pH values on the adsorption of acid phosphatase by some soil colloids and clay minerals (SCCM). The results showed that the pH values for the maximum adsorption of the enzyme were between the IEP of the enzyme and the PZC of SCCM. In the acetic acid systems, the amount of the enzyme adsorbed by SCCM was in the order of goethite > yellow brown soil > latosol > kaolinite > delta-MnO2. A remarkable influence of acetic acid concentration on the adsorption amount and the binding energy of the enzyme was observed. With the increase of the concentration from 0 to 200 mmol.L-1 in the system, acetic acid exhibited an enhanced effect, followed by an inhibition action on the adsorption of the enzyme on SCCM. The changes of the binding energy (K value) for the enzyme on SCCM were on the contrary to those of the maximum adsorption. The possible mechanisms for the influence of acetic acid on the adsorption of enzyme by SCCM were also discussed.

  19. A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases.

    PubMed

    Calderone, Vito; Forleo, Costantino; Benvenuti, Manuela; Thaller, Maria Cristina; Rossolini, Gian Maria; Mangani, Stefano

    2006-01-27

    The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases.

  20. Cloning, sequencing and characterization of a novel phosphatase gene, phoI, from soil bacterium Enterobacter sp. 4.

    PubMed

    Kang, Seung Ha; Cho, Kwang Keun; Bok, Jin Duck; Kim, Sung Chan; Cho, Jaie Soon; Lee, Peter Chang-Whan; Kang, Sang Kee; Lee, Hong Gu; Woo, Jung Hee; Lee, Hyun Jeong; Lee, Sang Cheol; Choi, Yun Jaie

    2006-04-01

    A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269-amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40 degrees C, respectively. Its specific activity on rho-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40 degrees C. Enzyme activity was inhibited by Al3+, EDTA, and DTT, but fivefold activated by Cu2+ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects.

  1. Genetic instability of constitutive acid phosphatase in shochu and sake yeast.

    PubMed

    Takashita, Hideharu; Kajiwara, Yasuhiro; Shimoda, Masahiko; Matsuoka, Masayoshi; Ogawa, Takahira; Ono, Kazuhisa

    2013-07-01

    Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

  2. Acid- and alkaline phosphatase in amniotic fluid in normal and complicated pregnancy.

    PubMed

    Beckman, G; Beckman, L; Löfstrand, T

    1978-01-01

    171 samples of amniotic fluid were obtained by abdominal amniocentesis from 67 women with complicated pregnancies (isoimmunization, diabetes mellitus or toxaemia). The levels of heat-labile alkaline phosphatase (HLAP), heat-stable alkaline phosphatase (HSAP) and acid phosphatase (AcP) were determined and compared to the enzyme levels in 179 samples from women with normal pregnancies of corresponding gestational ages. HLAP showed two "peaks" of activity, one in the 5th-22nd week and the other at term. HSAP and AcP showed increased activity at term. HSAP was decreased (p less than 0.01) in isoimmunization between the 36th and 40th week. 11 cases of toxaemia with placental insufficiency showed no differences in the levels of HLAP and HSAP compared with normal pregnancy. AcP showed no differences between normal and complicated pregnancy. Samples contaminated by blood showed no significant increase in the acid- and alkaline phosphatase levels. Samples contaminated by meconium showed a complex pattern. Some samples had normal enzyme levels, some had high levels of HLAP only and some had high levels of HSAP and AcP. The origin of the enzymes is not known with certainty. HSAP in amniotic fluid is most likely not of placental but intestinal origin. Determinations of acid- and alkaline phosphatase in amniotic fluid seem to be of little values in the clinical management of complicated pregnancy.

  3. Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei: apparent lack of effect of androgens.

    PubMed

    Wilson, M J; Ahmed, K; Fischbach, T J

    1978-08-03

    A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium flouride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h post-orchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.

  4. Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis.

    PubMed

    Vandesteene, Lies; López-Galvis, Lorena; Vanneste, Kevin; Feil, Regina; Maere, Steven; Lammens, Willem; Rolland, Filip; Lunn, John E; Avonce, Nelson; Beeckman, Tom; Van Dijck, Patrick

    2012-10-01

    Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

  5. In vitro effect of agriculture pollutants and their joint action on Pseudokirchneriella subcapitata acid phosphatase.

    PubMed

    Jonsson, Claudio Martín; Aoyama, Hiroshi

    2007-10-01

    Acid phosphatase plays important roles in algae metabolism such as availability and recycling of inorganic phosphate, autophagic digestive processes and fertilization. Chemicals released into the environment from agriculture activities may impair algae phosphatase activity. The aim of this work was to evaluate the in vitro effect of twenty-four organic compounds and six metals used as pesticides, or present as contaminants in sewage sludge, on the acid phosphatase activity extracted from Pseudokirchneriella subcapitata. Results demonstrated that only the linear surfactant alkyl benzenesulphonate (LAS) and the heavy metals Hg(2+), Al(3+) and Cu(2+) markedly altered (50%) the enzyme activity. Join action inhibition studies indicated that Hg(2+) was more potent inhibitor than Al(3+) or LAS, and that the Hg(2+)+Al(3+) and Hg(2+)+LAS mixtures have, respectively, additive and slight antagonism effects. Copper, which demonstrated an activator effect when preincubated with the enzyme, behaved as a slight antagonist for the inhibitor effect of Hg(2+).

  6. Tartrate-resistant, purple acid phosphatase in Gaucher cells of the spleen. Immuno- and cytochemical analysis.

    PubMed

    Schindelmeiser, J; Radzun, H J; Münstermann, D

    1991-03-01

    Bioptic material from the spleen of a three-year-old boy with a type 1 Gaucher disease was studied by immuno- and cytochemical methods with special regard to the macrophage-derived Gaucher cells. These cells were positive with PAS and Prussian blue staining, and were immuno-positive with the monoclonal 25 F9 antibody, specific to mature, non-inflammatory macrophages. Large Gaucher cells and their postulated small precursor cells revealed strong tartrate-resistant acid phosphatase (TRAP) and unspecific carboxylate esterase activities. Using a polyclonal antibody to bovine spleen purple phosphatase, a lysosomal TRAP from splenic macrophages, the TRAP of the Gaucher cells could be identified to belong to this group of iron-containing, purple acid phosphatases immunocytochemically. The origin of splenic Gaucher cells from blood monocytes and their further development are discussed.

  7. Phosphatidic acid phosphatase and diacylglycerol acyltransferase: potential targets for metabolic engineering of microorganism oil.

    PubMed

    Jin, Hong-Hao; Jiang, Jian-Guo

    2015-04-01

    Oleaginous microorganism is becoming one of the most promising oil feedstocks for biodiesel production due to its great advantages in triglyceride (TAG) accumulation. Previous studies have shown that de novo TAG biosynthesis can be divided into two parts: the fatty acid biosynthesis pathway (the upstream part which generates acyl-CoAs) and the glycerol-3-phosphate acylation pathway (the downstream part in which three acyl groups are sequentially added onto a glycerol backbone). This review mainly focuses on two enzymes in the G3P pathway, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). The former catalyzes a dephosphorylation reaction, and the latter catalyzes a subsequent acylation reaction. Genes, functional motifs, transmembrane domains, action mechanism, and new studies of the two enzymes are discussed in detail. Furthermore, this review also covers diacylglycerol kinase, an enzyme that catalyzes the reverse reaction of diacylglycerol formation. In addition, PAP and DGAT are the conjunction points of the G3P pathway, the Kennedy pathway, and the CDP-diacylglycerol pathway (CDP-DAG pathway), and the mutual transformation between TAGs and phospholipids is discussed as well. Given that both the Kennedy and CDP-diacylglycerol pathways are in metabolic interlock (MI) with the G3P pathway, it is suggested that, via metabolic engineering, TAG accumulation can be improved by the two pathways based on the pivotal function of PAP and DGAT.

  8. [Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase].

    PubMed

    Kasho, V N; Baĭkov, A A; Avaeva, S M

    1982-08-01

    The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

  9. Expression pattern and subcellular localization of Arabidopsis purple acid phosphatase AtPAP9.

    PubMed

    Zamani, Katayoun; Lohrasebi, Tahmineh; Sabet, Mohammad S; Malboobi, Mohammad A; Mousavi, Amir

    2014-01-01

    Purple acid phosphatase (PAP; EC 3.1.3.2) enzymes are metallophosphoesterases that hydrolysis phosphate ester bonds in a wide range of substrates. Twenty-nine PAP-encoding loci have been identified in the Arabidopsis genome, many of which have multiple transcript variants expressed in response to diverse environmental conditions. Having analyzed T-DNA insertion mutants, we have provided strong pieces of evidence that AtPAP9 locus encodes at least two types of transcripts, designated as AtPAP9-1 and AtPAP9-2. These transcript variants expressed distinctly during the course of growth in medium containing sufficient phosphate or none. Further histochemical analysis by the use of AtPAP9-1 promoter fused to β-glucuronidase reporter gene indicated the expression of this gene is regulated in a tissue-specific manner. AtPAP9-1 was highly expressed in stipule and vascular tissue, particularly in response to fungal infection. Subcellular localization of AtPAP9-1:green fluorescent fusion protein showed that it must be involved in plasma membrane and cell wall adhesion. Copyright © 2014. Published by Elsevier B.V.

  10. An Arabidopsis Inositol 5-Phosphatase Gain-of-Function Alters Abscisic Acid Signaling1

    PubMed Central

    Burnette, Ryan N.; Gunesekera, Bhadra M.; Gillaspy, Glenda E.

    2003-01-01

    Signals can be perceived and amplified at the cell membrane by receptors coupled to the production of a variety of second messengers, including inositol 1,4,5-trisphosphate (IP3). We previously have identified 15 putative inositol 5-phosphatases (5PTases) from Arabidopsis and shown that At5PTase1 can hydrolyze IP3. To determine whether At5PTase1 can terminate IP3-mediated signaling, we analyzed transgenic plants ectopically expressing At5PTase1. Stomata from leaves of At5PTase1 transgenic plants were abscisic acid (ABA) and light insensitive, and ABA induction of genes was delayed. Quantification of IP3 in plants exposed to ABA indicated that ABA induced two IP3 increases in wild-type plants. Both of these IP3 increases were reduced in At5PTase1 transgenic plants, indicating that IP3 may be necessary for stomatal closure and temporal control of ABA-induced gene expression. To determine if ABA could induce expression of At5PTase1, we examined RNA and protein levels of At5PTase1 in wild-type plants exposed to ABA. Our results indicate that At5PTase1 is up-regulated in response to ABA. This is consistent with At5PTase1 acting as a signal terminator of ABA signaling. PMID:12805629

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

    EPA Science Inventory

    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 ...

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

    EPA Science Inventory

    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 ...

  13. Nature of mononuclear cells positive for acid phosphatase activity in bone marrow of patients with renal osteodystrophy.

    PubMed Central

    Kaye, M; Henderson, J

    1988-01-01

    Fifty two of 63 patients with renal osteodystrophy had one or more mononuclear cells positive for acid phosphatase in the marrow. These cells are also tartrate resistant and non-specific esterase negative, and are believed to be precursors to osteoclasts and other acid phosphatase positive cells resorbing bone on the trabecular surface. Images Figure PMID:3360955

  14. Prostate cancer derived prostatic acid phosphatase promotes an osteoblastic response in the bone microenvironment

    PubMed Central

    Larson, Sandy R.; Chin, Jessica; Zhang, Xiaotun; Brown, Lisha G.; Coleman, Ilsa M.; Lakely, Bryce; Tenniswood, Martin; Corey, Eva; Nelson, Peter S.; Vessella, Robert L.

    2014-01-01

    Approximately 90 % of patients who die of prostate cancer (PCa) have bone metastases, often promoting osteoblastic lesions. We observed that 88 % of castration-resistant PCa (CRPC) bone metastases express prostatic acid phosphatase (PAP), a soluble secreted protein expressed by prostate epithelial cells in predominately osteoblastic (n = 18) or osteolytic (n = 15) lesions. Additionally, conditioned media (CM) of an osteoblastic PCa xenograft LuCaP 23.1 contained significant levels of PAP and promoted mineralization in mouse and human calvaria-derived cells (MC3T3-E1 and HCO). To demonstrate that PAP promotes mineralization, we stimulated MC3T3-E1 cells with PAP and observed increased mineralization, which could be blocked with the specific PAP inhibitor, phosphonic acid. Furthermore, the mineralization promoted by LuCaP 23.1 CM was also blocked by phosphonic acid, suggesting PAP is responsible for the mineralization promoting activity of LuCaP 23.1. In addition, gene expression arrays comparing osteoblastic to osteolytic CRPC (n = 14) identified betacellulin (BTC) as a gene upregulated during the osteoblastic response in osteoblasts during new bone formation. Moreover, BTC levels were increased in bone marrow stromal cells in response to LuCaP 23.1 CM in vitro. Because new bone formation does occur in osteoblastic and can occur in osteolytic CRPC bone metastases, we confirmed by immunohistochemistry (n = 36) that BTC was highly expressed in osteoblasts involved in new bone formation occurring in both osteoblastic and osteolytic sites. These studies suggest a role for PAP in promoting the osteoblastic reaction in CRPC bone metastases and identify BTC as a novel downstream protein expressed in osteoblasts during new bone formation. PMID:24242705

  15. Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice.

    PubMed

    Dionisio, Giuseppe; Madsen, Claus K; Holm, Preben B; Welinder, Karen G; Jørgensen, Malene; Stoger, Eva; Arcalis, Elsa; Brinch-Pedersen, Henrik

    2011-07-01

    Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic 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 the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.

  16. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  17. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  18. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  19. A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center.

    PubMed

    Schenk, G; Boutchard, C L; Carrington, L E; Noble, C J; Moubaraki, B; Murray, K S; de Jersey, J; Hanson, G R; Hamilton, S

    2001-06-01

    A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.

  20. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  1. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  2. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  3. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  4. Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

    PubMed

    Shao, T C; Kong, A Y; Cunningham, G R

    1987-01-01

    Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

  5. Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

    Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

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

    PubMed

    Tomaszewski, M; Buchowicz, J

    1971-08-01

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

  7. Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

    PubMed Central

    Sarthy, A; Michaelis, S; Beckwith, J

    1981-01-01

    Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported. PMID:6450745

  8. Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle.

    PubMed

    Schenk, Gerhard; Elliott, Tristan W; Leung, Eleanor; Carrington, Lyle E; Mitić, Natasa; Gahan, Lawrence R; Guddat, Luke W

    2008-01-31

    Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood. Red kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 A) and fluoride (2.2 A) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic mu-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this mu-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity. In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

  9. Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

    PubMed

    Andriotis, Vasilios M E; Ross, James D

    2004-06-01

    The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5 +/- 3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/ Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride. Copyright 2004 Springer-Verlag

  10. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

  11. Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities.

    PubMed

    Zhang, Yi-Nan; Zhang, Wei; Hong, Di; Shi, Lei; Shen, Qiang; Li, Jing-Ya; Li, Jia; Hu, Li-Hong

    2008-09-15

    Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K(i) of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.

  12. Arabidopsis purple acid phosphatase 10 is a component of plant adaptive mechanism to phosphate limitation.

    PubMed

    Wang, Liangsheng; Liu, Dong

    2012-03-01

    When grown with inadequate quantities of inorganic phosphate (Pi), plants synthesize and secret acid phosphatases into the rhizosphere. These secreted acid phosphatases are thought to release the Pi group from organophosphates present in the surrounding environment and to thereby increase Pi availability to plants. So far, however, the genetic evidence to support this hypothesis is still lacking. Previously, we showed that overexpression of Arabidopsis purple acid phosphatase 10 (AtPAP10) improved the growth of plants on Pi-deficient medium (P⁻ medium) supplemented with the organophosphate compound ADP; in contrast, the growth of atpap10 mutant lines was reduced on the same medium. In the current research, we determined the growth performance of these lines on P⁻ medium supplemented with four other organophosphates. The results showed that AtPAP10 could utilize rhizosphere organophosphates other than ADP for plant growth but with different utilization efficiencies. This work provides further genetic evidence that AtPAP10 phosphatase is a component of plant adaptive mechanism to Pi limitation.

  13. Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme.

    PubMed

    Malveaux, F J; Clemente, C L

    1969-03-01

    Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000.

  14. Staphylococcal Acid Phosphatase: Extensive Purification and Characterization of the Loosely Bound Enzyme1

    PubMed Central

    Malveaux, F. J.; Clemente, C. L. San

    1969-01-01

    Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000. Images PMID:5776528

  15. Activation of beta-glucan synthases by wall-bound purple acid phosphatase in tobacco cells.

    PubMed

    Kaida, Rumi; Satoh, Yumi; Bulone, Vincent; Yamada, Yohko; Kaku, Tomomi; Hayashi, Takahisa; Kaneko, Takako S

    2009-08-01

    Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.

  16. Okadaic acid-induced inhibition of B-50 dephosphorylation by presynaptic membrane-associated protein phosphatases.

    PubMed

    Han, Y F; Dokas, L A

    1991-10-01

    The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.

  17. Customized Array Comparative Genomic Hybridization Analysis of 25 Phosphatase-encoding Genes in Colorectal Cancer Tissues

    PubMed Central

    LACZMANSKA, IZABELA; SKIBA, PAWEL; KARPINSKI, PAWEL; BEBENEK, MAREK; M. SASIADEK, MARIA

    2016-01-01

    Background/Aim: Molecular mechanisms of alterations in protein tyrosine phosphatases (PTPs) genes in cancer have been previously described and include chromosomal aberrations, gene mutations, and epigenetic silencing. However, little is known about small intragenic gains and losses that may lead to either changes in expression or enzyme activity and even loss of protein function. Materials and Methods: The aim of this study was to investigate 25 phosphatase genes using customized array comparative genomic hybridization in 16 sporadic colorectal cancer tissues. Results: The analysis revealed two unique small alterations: of 2 kb in PTPN14 intron 1 and of 1 kb in PTPRJ intron 1. We also found gains and losses of whole PTPs gene sequences covered by large chromosome aberrations. Conclusion: In our preliminary studies using high-resolution custom microarray we confirmed that PTPs are frequently subjected to whole-gene rearrangements in colorectal cancer, and we revealed that non-polymorphic intragenic changes are rare. PMID:28031238

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

    PubMed

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

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

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

    PubMed Central

    Lee, Eunhee; Stafford, III, Walter F.

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724–837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991–1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998–1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction. PMID:26445108

  20. A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

    PubMed Central

    Feng, Jie; Yuan, Fenghua; Gao, Yin; Liang, Chenggang; Xu, Jin; Zhang, Changling; He, Liyuan

    2003-01-01

    The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants. PMID:12927022

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

    PubMed Central

    Ueda, Akihiro; Wood, Thomas K.

    2010-01-01

    SUMMARY 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. The tpbA mutation also decreases cell lysis while overexpression of tpbA increases cell lysis. To further link c-di-GMP concentrations and eDNA, the gene encoding phosphodiesterase PA2133 was overexpressed which increased eDNA and decreased biofilm formation by decreasing c-di-GMP. Furthermore, the effect of the tpbB mutation along with the tpbA mutation was examined because loss of TpbB restored the phenotypes controlled by enhanced c-di-GMP in the tpbA mutant. The tpbA tpbB double mutations restored eDNA to that of the PA14 wild-type level. These findings suggest that c-di-GMP, rather than TpbA, controls eDNA. Hence, TpbA acts as a positive regulator of eDNA and cell lysis by reducing c-di-GMP concentrations. PMID:21552365

  2. Bioactive enzyme-metal composites: the entrapment of acid phosphatase within gold and silver.

    PubMed

    Ben-Knaz, Racheli; Avnir, David

    2009-03-01

    This paper is concerned with the entrapment of an enzyme within an aggregated metallic matrix and the development of a bioactive enzyme-metal composite. Whereas the use of organic polymers and metal oxides for the preparation of enzymatically active materials is well developed, the third principle enzyme-material combination, namely protein-metal bulk, has not yet been reported. A new methodology for the entrapment of organic molecules and polymers within metals has been employed for the preparation of bioactive acid phosphatase@gold and acid phosphatase@silver, according to which room temperature reduction of the metal cation is carried out in the presence of the enzyme to be entrapped. Protectability of the entrapped enzyme against harsh conditions is shown: the acidic enzyme is kept alive under basic conditions.

  3. An acid phosphatase locus expressed in mouse kidney (Apk) and its genetic location on chromosome 10.

    PubMed

    Womack, J E; Auerbach, S B

    1978-04-01

    A genetic locus controlling the electrophoretic mobility of an acid phosphatase in mouse kidney is described. This locus, called acid phosphatase-kidney (Apk), is not expressed in erythrocytes, liver, spleen, heart, lung, brain, skeletal muscle, stomach, or testes. The product of Apk hydrolyzes the substrate naphthol AS-MX phosphoric acid but is not active on alpha-naphthylphosphate or 4-methylumbelliferylphosphate. It is not inactivated by 50 C for 1 hr, nor is its electrophoretic mobility altered by incubation with neuraminidase. The locus is invariant among 31 inbred strains (Apka), with a variant allele (Apkm) observed only in Mus musculus molossinus. Codominant expression was observed in F1 hybrids of M. m. molossinus and inbred strains. Apk was mapped on Chr 10, near the neurological mutant waltzer (v).

  4. Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

    PubMed

    Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

    2017-01-10

    Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.

  5. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1.

    PubMed

    Maynes, J T; Bateman, K S; Cherney, M M; Das, A K; Luu, H A; Holmes, C F; James, M N

    2001-11-23

    Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.

  6. Rosmarinic acid determination using biomimetic sensor based on purple acid phosphatase mimetic.

    PubMed

    Santhiago, Murilo; Peralta, Rosely A; Neves, Ademir; Micke, Gustavo Amadeu; Vieira, Iolanda Cruz

    2008-04-14

    A new heterodinuclear Fe(III)Zn(II) complex which mimics the active site of the hydrolytic enzyme red kidney bean purple acid phosphatase was synthesized and characterized by IR, CHN and X-ray crystallographic analyses. This complex, [Fe(III)Zn(II)(mu-OH)bpbpmp-CH(3)](ClO(4))(2), containing the ligand (H(2)bpbpmp-CH(3) = {2-[bis(2-pyridylmethyl)aminomethyl]-6-[(2-hydroxy-5-methylbenzyl) (2-pyridyl-methyl) aminomethyl]-4-methyl-phenol}) was employed in the construction of a biomimetic sensor and used in the determination of rosmarinic acid in plant extract samples. The response parameters and optimization of the biomimetic sensor design were evaluated. The best performance of this sensor was obtained for 75:15:10% (w/w/w) of the graphite powder:nujol:Fe(III)Zn(II) complex, 0.1 mol L(-1) phosphate buffer solution (pH 7.5), 1.19x10(-4) mol L(-1) hydrogen peroxide with frequency, pulse amplitude, and scan increment at 30 Hz, 100 mV, and 0.6 mV, respectively. The rosmarinic acid concentration was linear in the range of 2.98x10(-5) to 3.83x10(-4) mol L(-1) (r=0.9991) with a detection limit of 2.30x10(-6) mol L(-1). This biomimetic sensor demonstrated long-term stability (300 days; 900 determinations) and reproducibility, with a relative standard deviation of 12.0%. The recovery study of rosmarinic acid in plant extract samples gave values from 90.3 to 98.3% and the concentrations determined showed agreement when compared with those obtained using capillary electrophoresis at the 95% confidence level.

  7. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  8. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

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

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  9. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

    PubMed

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A

    1981-10-25

    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

  10. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

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

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

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

    PubMed Central

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A. Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J. H.

    2015-01-01

    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. PMID:26042133

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

    PubMed

    González-Muñoz, Eliécer; Avendaño-Vázquez, Aida-Odette; Montes, Ricardo A Chávez; de Folter, Stefan; Andrés-Hernández, Liliana; Abreu-Goodger, Cei; Sawers, Ruairidh J H

    2015-01-01

    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.

  13. Induction of a germination specific, low molecular weight, acid phosphatase isozyme with specific phosphotyrosine phosphatase activity in lentil (Lens esculenta) seeds.

    PubMed

    Bose, S K; Taneja, V

    1998-09-29

    A germination specific isozyme of acid phosphatase (EC 3.1.3.2) hydrolysing O-phospho-L-Tyrosine, pH optima 5.5 is induced in lentil seeds. When seeds at 0 h, 24 h and 36 h of germination are electrophorezed, native PAGE on specific enzyme staining shows several constitutive isozymes of acid phosphatases. At 48 h, an isozyme is induced which gradually decreases and then disappears at 108 h of germination. The short lived, induced isozyme is present in the embryo and seed-coat but not in the plumule and the radical. Induction of this isozyme is inhibited by cycloheximide and actinomycin-D and increased by plant growth regulators such as heteroauxin and gibbrellic acid treatment during germination. The induced isozyme is a single 30 kD polypeptide, with subunit molecular mass of 25 kD, shows activity for O-phospho-L-Tyrosine. It is strongly inhibited by vanadate (microM), molybdate, tungustate as also by iodoacetate, p-chloromercuribenzoate and diethylpyrocarbonate. This study shows for the first time that the germination induced low molecular weight Acid phosphatase is a Tyrosine phosphatase super family class IV enzyme, having a role in cellular differentiation and development during seed germination. Copyright 1998 Academic Press.

  14. Purification, primary structure, and properties of Euphorbia characias latex purple acid phosphatase.

    PubMed

    Pintus, F; Spano, D; Corongiu, S; Floris, G; Medda, R

    2011-06-01

    A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92-99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn2+, which are typical acid phosphatase inhibitors.

  15. Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6.

    PubMed

    Li, Jun; Dong, Yu; Lü, Xingru; Wang, Lu; Peng, Wei; Zhang, Xuejun C; Rao, Zihe

    2013-07-01

    Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

  16. Effect of microbial inoculation during vermicomposting of different organic substrates on microbial status and quantification and documentation of acid phosphatase.

    PubMed

    Pramanik, P; Ghosh, G K; Banik, P

    2009-02-01

    In this experiment, three microbial strains were inoculated in two different organic wastes to study their effect on the humic acids content, acid phosphatase activity and microbial properties of the final stabilized products. Pyrophosphate extract of vermicomposts were analyzed through polyacrylamide gel electrophoresis to study the nature of a isozymes in different treatments. Results suggested that vermicomposting increased humic acids content and acid phosphatase activity in organic substrates and microbial inoculation further enhanced the rate of humification and enzyme activity. Although humic acids content in different microorganism-inoculated vermicomposts were statistically at par, acid phosphatase activity in these treatments was significantly (P<0.05) different. Results revealed that microbial respiration was increased due to vermicomposting, but a reduction in microbial biomass was recorded after stabilization of organic wastes. Although vermicomposting increased the value of microbial quotient (qCO(2)), microbial inoculation did not show any significant effect on qCO(2). The zymogram revealed that two isozymes of acid phosphatase (group II and group III) were present in all vermicompost samples and higher acid phosphatase activity in fungi-inoculated vermicomposts might be due to the presence of an additional isozyme (group I) of acid phosphatase.

  17. Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase.

    PubMed

    Zamudio, M; González, A; Medina, J A

    2001-03-01

    Microbial phytases suitable for food fermentations could be obtained from lactic acid bacteria isolated from natural vegetable fermentations. Phytase activity was evaluated for six lactic acid bacteria cultures. Although the highest activity was found for Lactobacillus plantarum, the phytase activity was very low. Further characterization of the enzyme with phytate-degrading activity showed a molecular weight of 52 kDa and an optimum activity at pH 5.5 and 65 degrees C. Enzyme activity was due to a non-specific acid phosphatase which had a higher hydrolysis rate with monophosphorylated compounds such as acetyl phosphate that could explain the low phytase activity.

  18. An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate.

    PubMed

    Zhang, Wenyan; Gruszewski, Hope A; Chevone, Boris I; Nessler, Craig L

    2008-02-01

    Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.

  19. PP2C gamma: a human protein phosphatase with a unique acidic domain.

    PubMed

    Travis, S M; Welsh, M J

    1997-08-04

    We have cloned a novel cDNA from human skeletal muscle which encodes a protein phosphatase with a unique acidic domain. It is 34% identical to mammalian PP2C alpha and PP2C beta and we call it PP2C gamma. It more closely resembles PP2Cs from Paramecium tetraurelia and Schizosaccharomyces pombe than mammalian PP2Cs. Northern blot analysis shows that PP2C gamma is widely expressed, and is most abundant in testis, skeletal muscle, and heart. Like known PP2Cs, recombinant PP2C gamma requires Mg2+ or Mn2+ for activity. Unlike any other known phosphatase, PP2C gamma has a highly acidic domain: 75% of the 54 residues are glutamate or aspartate.

  20. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    PubMed

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  1. Acid phosphatase positional correlations in solid surface fungal cultivation: a fractal interpretation of biochemical differentiation.

    PubMed

    Jones, C L; Lonergan, G T; Mainwaring, D E

    1995-03-28

    Colour image analysis was used to measure the positional correlation between acid phosphatase intracellular concentration and hyphal cellular differentiation which leads to concentric circular zonal activity patterns. Acid phosphatase is strongly implicated in the biochemical control of hyphal branching, and exo-enzyme secretion, such as laccase in fungi, occurs predominately via the hyphal tips. Different concentrations of an organic dye were used to effect substrate induction of the enzyme response, which was shown to be statistically correlated according to a fractal power law (H approximately 0.39). A self-organized critical state for the molecular responsivity of dissipative enzyme expression is hypothesized as an efficient mechanism for hyphal adaptation, also accounting for the underlying biophysics of the observed pattern formations.

  2. Evolution of acid phosphatase-1 in the genus Drosophilia. Immunological studies.

    PubMed

    MacIntyre, R J; Dean, M R; Batt, G

    1978-12-29

    The enzyme acid phosphatase-1 was partially purified from 10 Drosophila species. Four antisera were produced and the ten enzymes were reacted against each serum. The method used to quantitate the reactions involved the electrophoretic separation of antigen-antibody complexes from uncomplexed enzyme, followed by densitometry of the free enzyme. Immunological distances were used to obtain correlation coefficients for all pairwise combinations of the 10 species. From these correlation coefficients, a dendrogram was constructed which is very similar to one diagramming the presumed phylogenetic relationships of the ten species. In addition, the data indicate acid phosphatase-1 has evolved at different rates in different lineages within the genus. A preliminary estimate of the unit evolutionary period for this enzyme is 3.25 million years. The method of determining immunological distances which was used in this study is compared to the method of microcomplement fixation in the Discussion.

  3. In vivo and in vitro analysis of lysosomes and acid phosphatase activity in human chagasic placentas.

    PubMed

    Fretes, R E; De Fabro, S P

    1995-12-01

    A structural, cytochemical, stereological, and biochemical study of lysosomes and a lysosome marker, the enzyme acid phosphatase, was performed, both in placentas at term from chagasic pregnant women without fetal infection and in normal placentas at term cocultured in vitro with Trypanosoma cruzi. It was found that in placentas from chagasic women lysosomes were normally distributed in the trophoblast. Stereological analysis showed that lysosomes and cytochemical acid phosphatase (AcP) activity were increased in the trophoblast of chagasic placentas. AcP activity increased in subcellular fractions of the isolated trophoblast from chagasic placentas, and the lysosomal fraction of those placentas exhibited the highest value of enzymatic activity in comparison to controls (P < 0.05). No differences in AcP activity were observed between homogenates of normal placentas cocultured with T. cruzi and controls. These data suggest that the placental lysosome population might be involved in the process of placental infection by T. cruzi.

  4. The different iron binding sites of bovine spleen purple acid phosphatase

    NASA Astrophysics Data System (ADS)

    Cichutek, Klaus; Witzel, Herbert; Parak, Fritz

    1988-02-01

    The purple acid phosphatase contains two inequivalent irons which can be removed subsequently. The Mössbauer spectrum of the purple inactive enzyme (oxidized) indicates two high spin ferric irons with antiparallel coupling giving zero effective spin. The active pink enzyme (partly reduced) is obtained by a one electron transfer to the iron which is less stable bound in the protein. The Mössbauer spectra indicate a high spin Fe(2+) antiparallel spin coupled to high spin Fe(3+).

  5. Identification of Aph1, a Phosphate-Regulated, Secreted, and Vacuolar Acid Phosphatase in Cryptococcus neoformans

    PubMed Central

    Lev, Sophie; Crossett, Ben; Cha, So Young; Desmarini, Desmarini; Li, Cecilia; Chayakulkeeree, Methee; Wilson, Christabel F.; Williamson, P. R.; Sorrell, Tania C.

    2014-01-01

    ABSTRACT Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment. PMID:25227465

  6. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme.

    PubMed

    Conklin, Patricia L; Gatzek, Stephan; Wheeler, Glen L; Dowdle, John; Raymond, Marjorie J; Rolinski, Susanne; Isupov, Mikhail; Littlechild, Jennifer A; Smirnoff, Nicholas

    2006-06-09

    In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.

  7. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus

    PubMed Central

    Winkelströter, Lizziane K.; Dolan, Stephen K.; Fernanda dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W.; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H.

    2015-01-01

    Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen. PMID:25943523

  8. Activity of phosphatase-sensitive 5-aminolevulinic acid prodrugs in cancer cell lines.

    PubMed

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

    2017-06-01

    5-aminolevulinc acid (5-ALA)-based photodynamic therapy (PDT) and photodiagnosis (PD) present many advantages over treatments with conventional photosensitizers (PS). It offers great tumor specificity, reduced photosensitivity reactions caused by PS accumulation in non-targeted tissues and also inherent PS metabolism into endogenous non-fluorescent heme. However, chemical instability, low bioavailability and poor pharmacokinetic profile limit systemic efficacy of 5-ALA. Here, we present a comprehensive in vitro evaluation of novel phosphatase-sensitive prodrugs of 5-ALA. These prodrugs are designed to be activated by ubiquitously expressed phosphatases with much improved chemical stability and reduced acute toxicity profile. PpIX kinetic measurements and flow cytometry show accumulation of PpIX upon incubation with phosphatase-sensitive prodrugs in PC3 human prostate cell cancer, MCF7 breast adenocarcinoma, U87MG glioblastoma, T24 bladder cancer and A549 lung carcinoma cells. They revealed a different fluorescence kinetics and dose-response curves for the different types of 5-ALA prodrugs. These experiments have allowed us to identify the most promising cancer cell types for phospho- 5-ALA prodrugs. Confocal fluorescence microscopy provided further evidence of fluorescent protoporphyrin IX accumulation and sub-cellular localisation. These findings, together with the low toxicity profile of phosphatase-sensitive prodrugs of 5-ALA and good response to PDT provide solid basis for future translational development in PC3, MCF7 and U87MG cancer types. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Arsanilic acid modified superparamagnetic iron oxide nanoparticles for Purification of alkaline phosphatase from hen's egg yolk.

    PubMed

    Farzi-Khajeh, Hamed; Safa, Kazem D; Dastmalchi, Siavoush

    2017-09-01

    Recent studies of magnetic carrier technology have focused on its applications in separation and purification technologies, due to easy separation of the target from the reaction medium by applying an external magnetic field. In the present study, Fe3O4 superparamagnetic nanoparticles were prepared to utilize a chemical co-precipitation method, then the surfaces of the nanoparticles were modified with arsanilic acid derivatives which were used as the specific nanocarriers for the affinity purification of alkaline phosphatase from the hen's egg yolk. The six different types of magnetic nanocarriers with varied lengths of the linkers were obtained. All samples were characterized step by step and validated using FTIR, SEM, EDX, VSM and XRD analysis methods As the results were shown, the use of inflexible tags with long linkers on the surface of the nanocarrier could lead to better results for separation of alkaline phosphatase from the hen's egg yolk with 76.2% recovery and 1361.7-fold purification. The molecular weight of the purified alkaline phosphatase was estimated to be 68kDa by SDS-PAGE. The results of this study showed that the novel magnetic nanocarriers were capable of purifying alkaline phosphatase in a practically time and cost effective way. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    1993-06-01

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

  11. Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase

    SciTech Connect

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

    2006-07-01

    Crystallization of a surface-localized acid phosphatase from Bacillus anthracis is reported. Flash annealing increased the high-resolution limit of usable data from 1.8 to 1.6 Å. Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 Å. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6° and a high-resolution limit of 1.8 Å. After flash-annealing, the apparent mosaicity decreased to 0.9° and the high-resolution limit of usable data increased to 1.6 Å. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

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

    PubMed

    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.

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

    PubMed

    Imai, K; Yoshimura, T

    1994-08-01

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

  14. Effect of copper on the activation of the acid phosphatase from the green algae Pseudokirchneriella subcapitata.

    PubMed

    Jonsson, Claudio Martín; Aoyama, Hiroshi

    2010-02-01

    The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of organic phosphate, autophagic digestive process, recycling cellular materials and zygote formation during reproduction. This work describes an in vitro activation effect of copper on the acid phosphatase of the green algae Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) under preincubation condition. Apparent Michaelis constant values of 1.21 and 0.37 mM, and activation energy values of 26.8 and 13.6 kJ mol(-1) were determined in the absence and in the presence of 0.2 mM Cu(2+), respectively. The dissociation constant value for Cu(2+) binding to the enzyme was determined to be 22.04 microM. The decrease of the apparent Michaelis constant (Km) and activation energy values in the presence of Cu(2+) correlates well with its activating effect on the acid phosphatase activity. This propriety could be used as a sensitive bioindicator for copper in environmental samples.

  15. Separation of tissue and serum acid phosphatase isoenzymes by ion-exchange column chromatography.

    PubMed

    Mercer, D W

    1977-01-01

    I describe a simple, rapid ion-exchange column-chromatographic technique for separating the acid phosphatase (EC 3.1.3.2) isoenzymes in human serum and tissue. Extracts of platelets, spleen, liver, erythrocytes, and prostate were used to determine optimum conditions for separating these isoenzymes. Samples layered on mini-colunms of DEAE-Sephadex A-50 were eluted stepwise with sodium chloride (100, 200, and 300 mmol/liter, buffered with tris (hydroxymethyl)aminomethane). Activity in column effluents was measured with p-nitrophenol phosphate as substrate, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Comparision of activity patterns so derived for various tissues revealed prostatic tissue to be a rich source of acid phosphatase isoenzyme 2 activity. Evaluation of sera from six patients with prostatic cancer revealed isoenzyme patterns with prominent amount of isoenzyme 2 (3.8 to 27.6 U/liter). sera from 10 healthy laboratory technicians contained isoenzyme 2 in the range of 0.3-0.5 U/liter. Samples from two patients with abnormally high activity owing to nonprostatic conditions (Gaucher's disease and carcinoma of lung) exhibited less than 2 U of isoenzyme 2 per liter and acid phosphatase isoenzymes 3-5 that were 50- to 100-fold the normal range. Quantification of isoenzyme 2 by DEAE-Sephadex column chromatography as described appears to provide a more sensitive and specific approach to diagnosis of prostatic cancer.

  16. Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone.

    PubMed

    Ek-Rylander, B; Bill, P; Norgård, M; Nilsson, S; Andersson, G

    1991-12-25

    Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N-glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites.

  17. Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

    PubMed

    Feder, Daniel; Hussein, Waleed M; Clayton, Daniel J; Kan, Meng-Wei; Schenk, Gerhard; McGeary, Ross P; Guddat, Luke W

    2012-11-01

    Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 μm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads. © 2012 John Wiley & Sons A/S.

  18. Assessment of bioavailable organic phosphorus in tropical forest soils by organic acid extraction and phosphatase hydrolysis.

    PubMed

    Darch, Tegan; Blackwell, Martin S A; Chadwick, David; Haygarth, Philip M; Hawkins, Jane M B; Turner, Benjamin L

    2016-12-15

    Soil organic phosphorus contributes to the nutrition of tropical trees, but is not accounted for in standard soil phosphorus tests. Plants and microbes can release organic anions to solubilize organic phosphorus from soil surfaces, and synthesize phosphatases to release inorganic phosphate from the solubilized compounds. We developed a procedure to estimate bioavailable organic phosphorus in tropical forest soils by simulating the secretion processes of organic acids and phosphatases. Five lowland tropical forest soils with contrasting properties (pH 4.4-6.1, total P 86-429 mg P kg(- 1)) were extracted with 2 mM citric acid (i.e., 10 μmol g(- 1), approximating rhizosphere concentrations) adjusted to soil pH in a 4:1 solution to soil ratio for 1 h. Three phosphatase enzymes were then added to the soil extract to determine the forms of hydrolysable organic phosphorus. Total phosphorus extracted by the procedure ranged between 3.22 and 8.06 mg P kg(- 1) (mean 5.55 ± 0.42 mg P kg(- 1)), of which on average three quarters was unreactive phosphorus (i.e., organic phosphorus plus inorganic polyphosphate). Of the enzyme-hydrolysable unreactive phosphorus, 28% was simple phosphomonoesters hydrolyzed by phosphomonoesterase from bovine intestinal mucosa, a further 18% was phosphodiesters hydrolyzed by a combination of nuclease from Penicillium citrinum and phosphomonoesterase, and the remaining 51% was hydrolyzed by a broad-spectrum phytase from wheat. We conclude that soil organic phosphorus can be solubilized and hydrolyzed by a combination of organic acids and phosphatase enzymes in lowland tropical forest soils, indicating that this pathway could make a significant contribution to biological phosphorus acquisition in tropical forests. Furthermore, we have developed a method that can be used to assess the bioavailability of this soil organic phosphorus.

  19. Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor.

    PubMed

    Comba, Santiago; Menendez-Bravo, Simón; Arabolaza, Ana; Gramajo, Hugo

    2013-01-29

    Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these

  20. AN ANALYSIS OF CELLULAR AND SUBCELLULAR SYSTEMS WHICH TRANSFORM THE SPECIES CHARACTER OF ACID PHOSPHATASE IN ACETABULARIA

    PubMed Central

    Keck, Konrad; Choules, Elizabeth A.

    1963-01-01

    Several species-specific molecular forms of acid phosphatase are known to exist in the unicellular green alga Acetabularia. In graft combinations between cells of Acetabularia mediterranea (med) and Acicularia Schenckii (acic) the expression of the med phosphatase is dominant over acic phosphatase. There is good evidence that in such grafts the preexisting acic phosphatase is converted on the molecular level via an intermediate form to the med phosphatase. This conversion can be initiated by the transplantation of a med cell nucleus to an anucleate acic cell, but will also take place in grafts between anucleate med and anucleate acic cells, indicating that the direct participation of a cell nucleus is not required. An incomplete conversion of acic phosphatase, which terminates at the intermediate stage, is induced in acic cells by injection of a concentrated homogenate of med cytoplasm. A similar partial conversion occurs in vitro in a mixture of homogenates from med and acic cells. Subcellular particles, such as chloroplasts or mitochondria, can be removed from the homogenates by centrifugation without impairing the reactions leading to the intermediate phosphatase type. Experimental evidence suggests that the transformation of phosphatase types is enzymatically catalyzed and may involve the conjugation of small molecules with the phosphatase protein. It was shown, however, that sialic acid is not involved, since the incubation of med or acic homogenates with neuraminidase did not modify the electrophoretic mobility of either enzyme type. Another type of phosphatase, which occurs in Acetabularia erenulata (cren) and can be distinguished electrophoretically from the aforementioned types, is not subject to interaction. In various mono- and multi-nucleate graft combinations between cren cells on one hand, and med or acic cells on the other hand, the cren phosphatase is synthesized independently of the enzyme of the graft partner. PMID:14079501

  1. Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease Type 1a

    SciTech Connect

    Lei, K.J.; Shelly, L.L.; Pan, C.J.; Sidbury, J.B.; Chou, J.Y. )

    1993-10-22

    Glycogen storage disease (GSD) type 1a is caused by the deficiency of d-glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase are reported. Several mutations in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish the molecular basis of this disease and open the way for future gene therapy.

  2. Crystal structure and tartrate inhibition of Legionella pneumophila histidine acid phosphatase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Reilly, Thomas J; Tanner, John J

    2015-11-01

    Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.

  3. Structural and Functional Characterization of the 2H-Phosphatase Domain of Sts-2 Reveals an Acid-Dependent Phosphatase Activity†

    PubMed Central

    Chen, Yunting; Jakoncic, Jean; Carpino, Nick; Nassar, Nicolas

    2010-01-01

    The suppressors of T cell receptor (TCR) signaling 1 and 2 (Sts-1 and -2, respectively) are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including TCR and the epidermal growth factor receptor (EGFR). Sts-1 was recently shown to be a new type of protein tyrosine phosphatase (PTP), with the phosphatase activity located within its C-terminal phosphoglycerate mutase (PGM) homology domain and key for the regulation of TCR signaling in T cells. The activity of the related Sts-2 enzyme is significantly less than that of Sts-1. Here we investigate the phosphatase activity of the PGM domain of Sts-2, Sts-2PGM. The crystal structure of Sts-2PGM is remarkably similar to Sts-1PGM, including conservation of all catalytic residues. Insight into mechanistic details is provided by the structures of the apo, tungstate-bound, and phosphate-bound enzyme. The active site shows stringent specificity, with the kcat optimum at pH 5.0 suggesting that Sts-2 might function as an acid-dependent phosphatase. Mutation of active site residues Gln372, Ala446, Glu481, Ser552, and Ser582 to their equivalents in Sts-1 increases the phosphatase activity of Sts-2PGM toward model substrates. Overall, our data demonstrate that Sts-2PGM adopts the conformation of an active phosphatase whose activity is fundamentally different from that of Sts-1 despite the strong structural homology. They also demonstrate that nonconserved active site residues are responsible for the difference in activity between the two isoforms. These differences reflect possible distinct physiological substrates. PMID:19196006

  4. Structural and functional characterization of the 2H-phosphatase domain of Sts-2 reveals an acid-dependent phosphatase activity.

    PubMed

    Chen, Yunting; Jakoncic, Jean; Carpino, Nick; Nassar, Nicolas

    2009-03-03

    The suppressors of T cell receptor (TCR) signaling 1 and 2 (Sts-1 and -2, respectively) are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including TCR and the epidermal growth factor receptor (EGFR). Sts-1 was recently shown to be a new type of protein tyrosine phosphatase (PTP), with the phosphatase activity located within its C-terminal phosphoglycerate mutase (PGM) homology domain and key for the regulation of TCR signaling in T cells. The activity of the related Sts-2 enzyme is significantly less than that of Sts-1. Here we investigate the phosphatase activity of the PGM domain of Sts-2, Sts-2(PGM). The crystal structure of Sts-2(PGM) is remarkably similar to Sts-1(PGM), including conservation of all catalytic residues. Insight into mechanistic details is provided by the structures of the apo, tungstate-bound, and phosphate-bound enzyme. The active site shows stringent specificity, with the k(cat) optimum at pH 5.0 suggesting that Sts-2 might function as an acid-dependent phosphatase. Mutation of active site residues Gln372, Ala446, Glu481, Ser552, and Ser582 to their equivalents in Sts-1 increases the phosphatase activity of Sts-2(PGM) toward model substrates. Overall, our data demonstrate that Sts-2(PGM) adopts the conformation of an active phosphatase whose activity is fundamentally different from that of Sts-1 despite the strong structural homology. They also demonstrate that nonconserved active site residues are responsible for the difference in activity between the two isoforms. These differences reflect possible distinct physiological substrates.

  5. Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

    PubMed

    Doronin, K K; Zakharchuk, A N; Grinenko, N F; Yurov, G K; Krougliak, V A; Naroditsky, B S

    1993-04-30

    A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.

  6. OsPAP26 Encodes a Major Purple Acid Phosphatase and Regulates Phosphate Remobilization in Rice.

    PubMed

    Gao, Wenwen; Lu, Linghong; Qiu, Wenmin; Wang, Chuang; Shou, Huixia

    2017-05-01

    During phosphate (Pi) starvation or leaf senescence, the accumulation of intracellular and extracellular purple acid phosphatases (PAPs) increases in plants in order to scavenge organic phosphorus (P). In this study, we demonstrated that a PAP-encoding gene in rice, OsPAP26, is constitutively expressed in all tissues. While the abundance of OsPAP26 transcript is not affected by Pi supply, it is up-regulated during leaf senescence. Furthermore, Pi deprivation and leaf senescence greatly increased the abundance of OsPAP26 protein. Overexpression or RNA interference (RNAi) of OsPAP26 in transgenic rice significantly increased or reduced APase activities, respectively, in leaves, roots and growth medium. Compared with wild-type (WT) plants, Pi concentrations of OsPAP26-overexpressing plants increased in the non-senescing leaves and decreased in the senescing leaves. The increased remobilization of Pi from the senescing leaves to non-senescing leaves in the OsPAP26-overexpressing plants resulted in better growth performance when plants were grown in Pi-depleted condition. In contrast, OsPAP26-RNAi plants retained more Pi in the senescing leaves, and were more sensitive to Pi starvation stress. OsPAP26 was found to localize to the apoplast of rice cells. Western blot analysis of protein extracts from callus growth medium confirmed that OsPAP26 is a secreted PAP. OsPAP26-overexpressing plants were capable of converting more ATP into inorganic Pi in the growth medium, which further supported the potential role of OsPAP26 in utilizing organic P in the rhizosphere. In summary, we concluded that OsPAP26 performs dual functions in plants: Pi remobilization from senescing to non-senescing leaves; and organic P utilization. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

    PubMed

    Henry, Y; Steer, M W

    1985-04-01

    The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

  8. New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity.

    PubMed

    Halbhuber, K J; Zimmermann, N; Linss, W

    1988-01-01

    New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.

  9. Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

    PubMed Central

    Hayman, A R; Warburton, M J; Pringle, J A; Coles, B; Chambers, T J

    1989-01-01

    Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources. Images Fig. 1. Fig. 2. Fig. 4. PMID:2775236

  10. Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

    PubMed

    Hayman, A R; Warburton, M J; Pringle, J A; Coles, B; Chambers, T J

    1989-07-15

    Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.

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

    PubMed

    Waymack, P P; Van Etten, R L

    1991-08-01

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

  12. Molecular characterization of a tomato purple acid phosphatase during seed germination and seedling growth under phosphate stress.

    PubMed

    Suen, Pui Kit; Zhang, Siyi; Sun, Samuel Sai-Ming

    2015-06-01

    SlPAP1 is a phosphate starvation responsive purple acid phosphatase during tomato seed germination. Future research on its family members in tomato might improve the phosphate stress tolerance. Phosphate deficiency is a major constraint upon crop growth and yield. In response to phosphate deficiency, plants secrete acid phosphatases (APases) to scavenge organic phosphate from soil. In this study, we investigated the impact of Pi starvation on germination and seedling growth of tomato, and we then cloned and characterized a phosphate starvation responsive purple APase (SlPAP1) that expressed during tomato seedling growth. Our results showed that phosphate deficiency reduced germination and growth rates of tomato, and also increased intracellular and secretory APase activity in a concentration-dependent manner. An in-gel activity assay found that two APases of 50 and 75 kDa were secreted during conditions of phosphate deficiency. SlPAP1 is a single copy gene belonging to a small gene family. It was expressed as a cDNA of approximately 1.5 kbp encoding a secreted glycoprotein of 470 amino acids. Northern blot analysis showed that SlPAP1 was specifically expressed in root tissue during phosphate deficiency. SlPAP1 had high sequence identity (56-89%) with other plant PAPs and contained highly conserved metal-binding residues. SlPAP1 is the first PAP to be cloned and characterized from tomato. This study provides useful information for future research on PAP family members in tomato, leading to better understanding of phosphate deficiency in this important crop plant.

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

    PubMed

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

    2015-10-15

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

  14. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  15. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  16. Molecular characterization of OsPAP2: transgenic expression of a purple acid phosphatase up-regulated in phosphate-deprived rice suspension cells.

    PubMed

    Hur, Yeon Jae; Jin, Byung Rae; Nam, Jaesung; Chung, Young Soo; Lee, Jai Heon; Choi, Hong Kyu; Yun, Dae Jin; Yi, Gihwan; Kim, Yong Hwan; Kim, Doh Hoon

    2010-01-01

    A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60-63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P( i )). The induced expression of OsPAP2 is a specific response to P( i ) starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P( i )-supply, and transcripts of OsPAP2 were abundant in P( i )-deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P( i )-deficient conditions.

  17. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  18. Investigation of acid phosphatase variants for the synthesis of phosphate monoesters.

    PubMed

    Tasnádi, Gábor; Zechner, Michaela; Hall, Mélanie; Baldenius, Kai; Ditrich, Klaus; Faber, Kurt

    2017-10-01

    The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Is Protein Phosphatase Inhibition Responsible for the Toxic Effects of Okadaic Acid in Animals?

    PubMed Central

    Munday, Rex

    2013-01-01

    Okadaic acid (OA) and its derivatives, which are produced by dinoflagellates of the genera Prorocentrum and Dinophysis, are responsible for diarrhetic shellfish poisoning in humans. In laboratory animals, these toxins cause epithelial damage and fluid accumulation in the gastrointestinal tract, and at high doses, they cause death. These substances have also been shown to be tumour promoters, and when injected into the brains of rodents, OA induces neuronal damage reminiscent of that seen in Alzheimer’s disease. OA and certain of its derivatives are potent inhibitors of protein phosphatases, which play many roles in cellular metabolism. In 1990, it was suggested that inhibition of these enzymes was responsible for the diarrhetic effect of these toxins. It is now repeatedly stated in the literature that protein phosphatase inhibition is not only responsible for the intestinal effects of OA and derivatives, but also for their acute toxic effects, their tumour promoting activity and their neuronal toxicity. In the present review, the evidence for the involvement of protein phosphatase inhibition in the induction of the toxic effects of OA and its derivatives is examined, with the conclusion that the mechanism of toxicity of these substances requires re-evaluation. PMID:23381142

  20. Prostatic Acid Phosphatase Is Required for the Antinociceptive Effects of Thiamine and Benfotiamine

    PubMed Central

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

    2012-01-01

    Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)–a phosphorylated derivative of thiamine–have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine–a compound that is not phosphorylated–were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap−/− mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP. PMID:23119057

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

    PubMed

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

    2012-01-01

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

  2. Trichostrongylus vitrinus (Nematoda: Strongylida): molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene.

    PubMed

    Hu, Min; Abs EL-Osta, Youssef G; Campbell, Bronwyn E; Boag, Peter R; Nisbet, Alasdair J; Beveridge, Ian; Gasser, Robin B

    2007-09-01

    A full-length cDNA (Tv-stp-1) encoding a serine/threonine protein phosphatase (Tv-STP-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 951 nucleotides encoded a predicted protein of 316 amino acids (aa), containing the characteristic motif [LIVMN]-[KR]-G-N-H-E. Comparison with other sequences in non-redundant databases showed that Tv-STP-1 had significant identities/similarities to those from a range of metazoans and protists. Sequence similarity was most pronounced in the central region of the protein, in which the catalytic activity is inferred to be modulated by eight conserved residues (Asp 61, His 63, Asp 92, Asp 95, Asn 121, His 171, His 246 and Tyr 270), known to coordinate the binding of two metal ions (Mn2+ and Fe2+) in various organisms. Phylogenetic analyses of selected amino acid sequence data using the neighbor-joining and maximum parsimony methods revealed Tv-STP-1 to be most closely related to the glc seven-like phosphatases inferred for genes from the free-living nematode Caenorhabditis elegans and the parasitic nematode Oesophagostomum dentatum (order Strongylida). Comparison of the genomic organization of the full-length Tv-stp-1 gene with related molecules from other nematodes revealed substantial variation in the lengths and numbers of the exons and introns. The entire genes Tv-stp-1 (5041-5362 bp; 10 exons and 9 introns) and Od-mpp-1 (10,271 bp; 8 exons and 9 introns) from the parasitic nematodes T. vitrinus and O. dentatum were considerably longer than the C. elegans genes (1222-1603 bp; 3-7 exons and 2-6 introns). Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-stp-1 was transcribed in adult males of T. vitrinus, but not in the adult female or in any larval stages of this species. In spite of considerable variation at the genomic level, the findings of the present

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

    PubMed

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

    2000-02-01

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

  4. Continuous and sensitive acid phosphatase assay based on a conjugated polyelectrolyte.

    PubMed

    Xie, Yonghua; Tan, Ying; Liu, Renxuan; Zhao, Rui; Tan, Chunyan; Jiang, Yuyang

    2012-08-01

    We report a novel continuous and sensitive fluorescence turn-on assay for ACPs, which consists of a cationic conjugated polyelectrolyte (PPE4+) and a commonly used phosphatase substrate p-nitrophenyl phosphate (pNPP). The kinetics of the ACP catalyzed hydrolysis of the substrate pNPP was monitored by the fluorescence change of PPE4+ and corresponding kinetic parameters were derived to be consistent with the literature reports. The applications of PPE4+/pNPP-based ACP assay in high-throughput screening of ACP inhibitors and detection of prostatic acid phosphotase (PAP) in vitro were demonstrated.

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

    PubMed

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

    2013-11-01

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

  6. [Effect of dental alloys on salivary alkaline and acid phosphatase, alpha amylase K+, Na+, and Cl-].

    PubMed

    Todorov, I; Saprjanova, M

    1977-04-01

    Comparative studied were performed in healthy subjects without metals in their oral cavities and in individuals having different metal alloys (gold, steel, amalgam) in their mouths and presenting with various complaints such as xerostomia, burning mucosa, etc. It was found that the contents of alkaline and acid phosphatases, alpha-amylase, K+, Na+ and Cl- in saliva increased significantly with the increase in total corrosion potential when non-precious metal alloys, especially different types of alloys, were present. Parallel to this, the frequency and the intensity of the complaints increased.

  7. Hybrid-DFT study on electronic structures of the active site of sweet potato purple acid phosphatase: the origin of stronger antiferromagnetic couplings than other purple acid phosphatases.

    PubMed

    Koizumi, Kenichi; Yamaguchi, Kizashi; Nakamura, Haruki; Takano, Yu

    2009-04-30

    The electronic structure and magnetic interactions of the active site of sweet potato purple acid phosphatase (PAP) were investigated by using UHF, pure DFT (UBLYP), and hybrid DFT methods (UB3LYP and UB2LYP). PAP catalyzes the hydrolysis of a phosphate ester under acidic conditions and contains a binuclear metal center. Sweet potato PAP provides stronger antiferromagnetic coupling than other PAPs. UB3LYP showed reasonably good agreement with the experimental magnetic coupling, indicating that this stronger antiferromagnetic coupling is caused by a mu-oxo bridge in the Fe(III)-Mn(II) binuclear metal center, which is the origin of the asymmetric spin delocalization. The type of bridging ligand is essential for the reaction mechanism, because the bridging ligand is suggested to function as a nucleophile in the reaction. Analyses of the natural orbital and spin density distributions implied the asymmetric spin delocalization on the bridging oxygen. The mechanism and the pathway of the antiferromagnetic coupling between Fe(III) and Mn(II) were discussed, using chemical indices introduced with the occupation numbers of singly occupied natural orbitals.

  8. Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean.

    PubMed

    Vogel, Andreas; Börchers, Torsten; Marcus, Katrin; Meyer, Helmut E; Krebs, Bernt; Spener, Friedrich

    2002-05-15

    Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents. (c) 2002 Elsevier Science (USA).

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

    PubMed

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

    2007-01-05

    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.

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

    PubMed

    Van Etten, R L; Waymack, P P

    1991-08-01

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

  11. Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.

    PubMed

    Cashikar, A G; Rao, N M

    1996-03-01

    Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.

  12. Design and synthesis of an Fmoc-SPPS-compatible amino acid building block mimicking the transition state of phosphohistidine phosphatase.

    PubMed

    Eerland, Martijn F; Hedberg, Christian

    2012-02-17

    The synthesis of a sulfonamide-based transition-state (TS) analogue of enzymatic phosphohistidine dephosphorylation as an amino acid building block is presented, together with the proof-of-concept of its incorporation into peptides. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The peptides are designed as inhibitors of phosphohistidine phosphatase and as a pull-down probe for identification of phosphohistidine phosphatases, respectively.

  13. Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase.

    PubMed

    Lung, Shiu-Cheung; Leung, Andy; Kuang, Rainbow; Wang, Yu; Leung, Priscilla; Lim, Boon-Leong

    2008-01-01

    Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.

  14. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures.

    PubMed

    Klabunde, T; Sträter, N; Fröhlich, R; Witzel, H; Krebs, B

    1996-06-21

    Purple acid phosphatase is a widely distributed non-specific phosphomonoesterase. X-ray structures of the dimeric 111-kDa Fe(III)-Zn(II) kidney bean purple acid phosphatase (kbPAP) complexed with phosphate, the product of the reaction, and with tungstate, a strong inhibitor of the phosphatase activity, were determined at 2.7 and 3.0 angstroms resolution, respectively. Furthermore the resolution of the unligated enzyme, recently solved at 2.9 angstroms could be extended to 2.65 angstroms with completely new data. The binding of both oxoanions is not accompanied by larger conformational changes in the enzyme structure. Small movements with a maximal coordinate shift of 1 angstroms are only observed for the active site residues His295 and His296. In the inhibitor complex as well as in the product complex, the oxoanion binds in a bidentate bridging mode to the two metal ions, replacing two of the presumed solvent ligands present in the unligated enzyme form. As also proposed for the unligated structure a bridging hydroxide ion completes the coordination spheres of both metal ions to octahedral arrangements. All three structures reported herein support a mechanism of phosphate ester hydrolysis involving interaction of the substrate with Zn(II) followed by a nucleophilic attack on the phosphorus by an Fe(III)-coordinated hydroxide ion. The negative charge evolving at the pentacoordinated transition state is probably stabilized by interactions with the divalent zinc and the imidazole groups of His202, His295, and His296, the latter protonating the leaving alcohol group.

  15. Inhibition kinetics of acid and alkaline phosphatases by atrazine and methomyl pesticides.

    PubMed

    El-Aswad, Ahmed F; Badawy, Mohamed E I

    2015-01-01

    The main objective of this work was to investigate the kinetic characteristics of acid and alkaline phosphatases isolated from different sources and to study the effects of the herbicide atrazine and insecticide methomyl on the activity and kinetic properties of the enzymes. Acid phosphatase (ACP) was isolated from the tomato plant (Solanum lycopersicum L. var. lycopersicum); alkaline phosphatase (ALP) was isolated from two sources, including mature earthworms (Aporrectodea caliginosa) and larvae of the Egyptian cotton leafworm (Spodoptera littoralis). The specific activities of the enzymes were 33.31, 5.56 and 0.72 mmol substrate hydrolyzed per minute per milligram protein for plant ACP, earthworms ALP and cotton leafworm ALP, respectively. The inhibition kinetics indicated that atrazine and methomyl caused competitive-non-competitive inhibition of the enzymes. The relationships between estimates of K(m) and V(max) calculated from the Michaelis-Menten equation have been explored. The extent of the inhibition was different, as estimated by the values of the inhibition constant Ki that were found to be 3.34 × 10(-3), 1.12 × 10(-2) and 1.07 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively, with methomyl. In the case of atrazine, K(i) were found to be 8.99 × 10(-3), 3.55 × 10(-2) and 1.36 × 10(-2) mM for plant ACP, earthworms ALP and cotton leafworm ALP, respectively.

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

    PubMed

    Koller, E; Wolfbeis, O S

    1984-11-15

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

  17. Red cell acid phosphatase types and GC polymorphisms in Mérida, Oaxaca, León, and Saltillo, Mexico.

    PubMed

    Lisker, R; Ramírez, E; Peñaloza, R; Salamanca, F

    1994-12-01

    Red cell acid phosphatase types and GC polymorphisms were studied in Mérida and Oaxaca, Mexico. GC polymorphisms were also investigated in León and Saltillo. The ACP*A, ACP*B, and ACP*C gene frequencies were 0.215, 0.770, and 0.015 respectively, in Mérida and 0.205, 0.788, and 0.002, respectively, in Oaxaca. In Oaxaca the ACP*R gene had a frequency of 0.005. The results are similar to other Mestizo groups studied in Mexico; it is concluded that the ACP*C and ACP*R genes were introduced by admixture of native Amerindians with whites and blacks, respectively. The GC*1S, GC*1F, and GC*2 gene frequencies were 0.489, 0.289 and 0.222, respectively, in León; 0.500, 0.272, and 0.228, respectively, in Mérida; 0.454, 0.337, and 0.209, respectively, in Oaxaca; and 0.505, 0.356, and 0.139, respectively, in Saltillo. These results are similar to what has been obtained in other Mestizo populations and Indian groups in Mexico, probably because the main ethnic component in both is Amerindian.

  18. Inhibition by dithionite and reactivation by iron of the tartrate-resistant acid phosphatase in bone of osteopetrotic (ia) rats.

    PubMed

    Hammarström, L E; Anderson, T R; Marks, S C; Toverud, S U

    1983-10-01

    The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.

  19. Effect of different zinc levels on activity of superoxide dismutases & acid phosphatases and organic acid exudation on wheat genotypes.

    PubMed

    Bharti, Kiran; Pandey, Neha; Shankhdhar, Deepti; Srivastava, P C; Shankhdhar, S C

    2014-01-01

    A field study was carried out to investigate the effect of three Zn levels 0, 20 kg ZnSO4 ha(-1) and 20 kg ZnSO4 ha(-1)+ foliar spray of 0.5 % ZnSO4 on superoxide dismutase activity, acid phosphatase activity and grain yield and a pot experiment to study the effect of zinc deficient and sufficient conditions on organic acid exudation. Increasing Zn levels was established as beneficial in improving the enzyme activities of genotypes. Combined foliar and soil application of Zn proved to be superior of all the treatments. Zinc application resulted in a maximum increment limit of 96.8 % in superoxide dismutase activity, 75.76 % in acid phosphatase activity, and a decrement limit of 88.57 % in oxalic acid exudation irrespective of stages and year of study. The increased enzyme activities had a positive impact on grain yield. As an average of all genotypes an improvement of 19.88 % in 2009 and 21.29 % in 2010 due to soil application while of 16.45 % in 2009 and 13.01 % in 2010 due to combined application was calculated for grain yield. There existed a variation among genotypes in showing responses towards zinc application and the genotypes UP 2584 and PBW 550 were found to be more responsive.

  20. N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase.

    PubMed

    Wang, Yunling; Norgård, Maria; Andersson, Göran

    2005-03-01

    Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.

  1. An 18 kDa acid phosphatase from chicken heart possesses phosphotransferase activity.

    PubMed

    Naz, Rubina; Saeed, Asma; Saeed, Ahmad

    2006-02-01

    A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K (cat)/K (m), 4.3 x 10(4), found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.

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

    PubMed

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

    2016-01-01

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

  3. The Aspergillus nidulans pyrG89 mutation alters glycosylation of secreted acid phosphatase.

    PubMed

    Justino, A; Nozawa, S R; Maccheroni, W; May, G S; Martinez-Rossi, N M; Rossi, A

    2001-03-01

    The glycosylation level of the pacA-encoded acid phosphatase secreted by Aspergillus nidulans was reduced in strains pabaA1 pyroA4and pabaA1 pyroA4 pyrG89, compared to strains carrying these mutations singly. The molecular mass of the enzyme secreted by the triple mutant grown at pH 5.0 was 105 and 45 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. In contrast, the pabaA1 strain secreted acid phosphatases of 119 and 62 kDa. The enzyme also had an altered electrophoretic mobility and glycosylation had a protective effect against its heat inactivation. Thus, this combination of mutants alters glycosylation of the enzyme, leading to changes in their structural properties. In spite of this, no deviation was observed in the apparent optimum pH and Michaelis kinetics for enzymatic hydrolysis of p-nitrophenyl phosphate or alpha-naphthyl phosphate.

  4. Identification of para-Substituted Benzoic Acid Derivatives as Potent Inhibitors of the Protein Phosphatase Slingshot.

    PubMed

    Li, Kang-shuai; Xiao, Peng; Zhang, Dao-lai; Hou, Xu-Ben; Ge, Lin; Yang, Du-xiao; Liu, Hong-da; He, Dong-fang; Chen, Xu; Han, Ke-rui; Song, Xiao-yuan; Yu, Xiao; Fang, Hao; Sun, Jin-peng

    2015-12-01

    Slingshot proteins form a small group of dual-specific phosphatases that modulate cytoskeleton dynamics through dephosphorylation of cofilin and Lim kinases (LIMK). Small chemical compounds with Slingshot-inhibiting activities have therapeutic potential against cancers or infectious diseases. However, only a few Slingshot inhibitors have been investigated and reported, and their cellular activities have not been examined. In this study, we identified two rhodanine-scaffold-based para-substituted benzoic acid derivatives as competitive Slingshot inhibitors. The top compound, (Z)-4-((4-((4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid (D3) had an inhibition constant (Ki) of around 4 μm and displayed selectivity over a panel of other phosphatases. Moreover, compound D3 inhibited cell migration and cofilin dephosphorylation after nerve growth factor (NGF) or angiotensin II stimulation. Therefore, our newly identified Slingshot inhibitors provide a starting point for developing Slingshot-targeted therapies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments. PMID:26938873

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

    PubMed Central

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

    2009-01-01

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

  7. Soybean seed acid phosphatases: unusual optimum temperature and thermal stability studies.

    PubMed

    Ferreira, C V; Granjeiro, J M; Taga, E M; Aoyama, H

    1998-01-14

    In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A, and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80 degrees C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60 degrees C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60 degrees C; the isoforms AP3A and AP3B retained 30% of activity at 70 degrees C after 60 min and all the isoforms were inactivated at 80 degrees C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70 degrees C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.

  8. The glucose-6-phosphatase catalytic subunit gene promoter contains both positive and negative glucocorticoid response elements.

    PubMed

    Vander Kooi, Beth T; Onuma, Hiroshi; Oeser, James K; Svitek, Christina A; Allen, Shelley R; Vander Kooi, Craig W; Chazin, Walter J; O'Brien, Richard M

    2005-12-01

    Glucose-6-phosphatase catalyzes the final step in the gluconeogenic and glycogenolytic pathways. Glucocorticoids stimulate glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription and studies performed in H4IIE hepatoma cells demonstrate the presence of a glucocorticoid response unit (GRU) in the proximal G6Pase promoter. In vitro deoxyribonuclease I footprinting analyses show that the glucocorticoid receptor binds to three glucocorticoid response elements (GREs) in the -231 to -129 promoter region and transfection results indicate all three contribute to glucocorticoid induction of G6Pase gene transcription. Furthermore, binding sites for hepatocyte nuclear factor-1 and -4, CRE binding factors, and FKHR (FOXO1a) are required for the full glucocorticoid response. Chromatin immunoprecipitation assays show that dexamethasone treatment stimulates glucocorticoid receptor and FKHR binding to the endogenous G6Pase promoter. Surprisingly, although glucocorticoids stimulate G6Pase gene transcription, deoxyribonuclease I footprinting and transfection analyses demonstrate the presence of a negative GRE and an associated negative accessory factor element in the -271 to -225 promoter region, which inhibit the glucocorticoid response. This appears to be the first report of a promoter that contains both positive and negative GREs, which function within the same cellular environment. We hypothesize that targeted signaling to the negative accessory element within the GRU may provide tight regulation of the glucocorticoid stimulation.

  9. Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts

    PubMed Central

    Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Lu, Xi; Lind, Thomas; Melhus, Håkan; Engstrand, Thomas; Karlsson-Ott, Marjam; Engqvist, Hakan

    2016-01-01

    Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100μM and 0.1μM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100μM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100μM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression. PMID:27701417

  10. Molecular Control of Acid Phosphatase Secretion into the Rhizosphere of Proteoid Roots from Phosphorus-Stressed White Lupin1

    PubMed Central

    Miller, Susan Stade; Liu, Junqi; Allan, Deborah L.; Menzhuber, Christopher J.; Fedorova, Maria; Vance, Carroll P.

    2001-01-01

    White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced Mr of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted Mr of 49,000 but migrates as a protein with Mr of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5′-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots. PMID:11598233

  11. Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola.

    PubMed

    Schadeck, R J; Leite, B; de Freitas Buchi, D

    1998-12-01

    Colletotrichum graminicola, a pathogen of sorghum and corn, was investigated prior and during germination as to certain aspects of acid phosphatase activity and lipid mobilization. Ungerminated conidia cytoplasm was filled with lipid deposits, which were mobilized during the germination process. Cytochemical ultrastructural examination showed that conidia vacuoles exhibit acid phosphatase activity, which is suggestive of lytic activity. Lipid bodies, stored in the ungerminated conidia cytoplasm, were internalized by vacuoles in a process analogous to microautophagy and were apparently digested inside them. The lipid bodies disappeared and vacuoles became enlarged in conidial cells during germination. Appressoria also showed acid phosphatase activity in multiple heterogeneous vesicles which were, in most cases, juxtaposed with lipid bodies. These results suggest that the vacuolar system plays an important role during C. graminicola germination and that the initial stages of lipid metabolization are taking place inside the vacuoles.

  12. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis.

    PubMed

    Xiong, L; Lee Bh; Ishitani, M; Lee, H; Zhang, C; Zhu, J K

    2001-08-01

    The plant hormone abscisic acid (ABA) plays a wide range of important roles in plant growth and development, including embryogenesis, seed dormancy, root and shoot growth, transpiration, and stress tolerance. ABA and various abiotic stresses also activate the expression of numerous plant genes through undefined signaling pathways. To gain insight into ABA and stress signal transduction, we conducted a genetic screen based on ABA- and stress-inducible gene transcription. Here we report the identification of an Arabidopsis mutation, fiery1 (fry1), which results in super-induction of ABA- and stress-responsive genes. Seed germination and postembryonic development of fry1 are more sensitive to ABA or stress inhibition. The mutant plants are also compromised in tolerance to freezing, drought, and salt stresses. Map-based cloning revealed that FRY1 encodes an inositol polyphosphate 1-phosphatase, which functions in the catabolism of inositol 1, 4, 5-trisphosphate (IP(3)). Upon ABA treatment, fry1 mutant plants accumulated more IP(3) than did the wild-type plants. These results provide the first genetic evidence indicating that phosphoinositols mediate ABA and stress signal transduction in plants and their turnover is critical for attenuating ABA and stress signaling.

  13. Arabidopsis phosphatase under-producer mutants pup1 and pup3 contain mutations in the AtPAP10 and AtPAP26 genes.

    PubMed

    Zhang, Ye; Wang, Xiaoyue; Liu, Dong

    2015-01-01

    Production and secretion of acid phosphatases (APases) is a hallmark adaptive response of plants to phosphate (Pi) deprivation. Researchers have long hypothesized that Pi starvation-induced APases are involved in internal Pi recycling and remobilization as well as in external Pi utilization. Two phosphatase under-producer (pup) mutants, pup1 and pup3, were previously isolated in Arabidopsis. Characterization of these 2 pup mutants provided the first genetic evidence for the above hypothesis. To date, however, the molecular lesions in these 2 pup mutants remain unknown. In this work, we demonstrate that pup1 and pup3 contain point mutations in the Arabidopsis purple acid phosphatase gene AtPAP10 and AtPAP26, respectively. Our results answer a long-standing question about the molecular identity of the PUP1 and PUP3 genes and corroborate the conclusions from previous studies regarding the function of AtPAP10 and AtPAP26 in plant acclimation to Pi deprivation.

  14. Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase.

    PubMed

    Bradshaw, Niels; Losick, Richard

    2015-10-14

    Formation of a division septum near a randomly chosen pole during sporulation in Bacillus subtilis creates unequal sized daughter cells with dissimilar programs of gene expression. An unanswered question is how polar septation activates a transcription factor (σ(F)) selectively in the small cell. We present evidence that the upstream regulator of σ(F), the phosphatase SpoIIE, is compartmentalized in the small cell by transfer from the polar septum to the adjacent cell pole where SpoIIE is protected from proteolysis and activated. Polar recognition, protection from proteolysis, and stimulation of phosphatase activity are linked to oligomerization of SpoIIE. This mechanism for initiating cell-specific gene expression is independent of additional sporulation proteins; vegetative cells engineered to divide near a pole sequester SpoIIE and activate σ(F) in small cells. Thus, a simple model explains how SpoIIE responds to a stochastically-generated cue to activate σ(F) at the right time and in the right place.

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

    PubMed

    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

    2015-08-03

    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.

  16. Purification and Properties of Acid Phosphatase from Plump and Shriveled Seeds of Triticale 1

    PubMed Central

    Ching, Te May; Lin, Tsan-Piao; Metzger, Robert J.

    1987-01-01

    A major triticale (X Triticosecale Wittmack) endosperm acid phosphatase (EC 3.1.2.2) (APase) from sib-lines producing plump and shriveled seed was purified 140- and 230-fold to a specific activity of 94 and 153 micromoles per minute per milligram protein respectively, by ammonium sulfate fractionation, ion-exchange chromatography, chromatofocusing, affinity column chromatography, and gel filtration. The purified enzyme from both materials is a monomeric glycoprotein with an apparent molecular weight of 45,700 ± 500 containing 12% carbohydrate and an apparent isoelectric point of pH 5.9. It hydrolyzes tri- and di-phosphate of nucleosides as well as phosphate esters and exhibits characteristics of ATP-hydrolase and phosphatase. About 2-fold more of the APase was isolated from shriveled seeds, and the purified enzyme exhibited 3- and 5-fold higher Vmax for p-nitrophenyl phosphate and ATP, respectively, than that of plump seed. The I50 for Pi concentration was 5.5-fold higher in APase of shriveled seed than the plump one. These varied quantitative and kinetic properties substantiate the role of APase in lines with shriveled seeds being reduction of starch accumulation by depleting substrates and energy supply in the cytosol. Images Fig. 1 PMID:16665523

  17. Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase.

    PubMed

    Wojciechowski, Cheryl L; Kantrowitz, Evan R

    2003-06-26

    Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp(51). The D153E enzyme had an increased k(cat) in the presence of high concentrations of Mg(2+), along with a decreased Mg(2+) affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn(1) site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn(2+), dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.

  18. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines.

    PubMed

    Latheef, S A A; Devanabanda, Mallaiah; Sankati, Swetha; Madduri, Ramanadham

    2016-02-01

    Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

  19. [Tartrate-resistant acid phosphatase in free-living Amoeba proteus].

    PubMed

    Sopina, V A

    2002-01-01

    Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

  20. MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch

    PubMed Central

    Westmeyer, Gil G.; Emer, Elena G.; Lintelmann, Jutta; Jasanoff, Alan

    2014-01-01

    SUMMARY The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with magnetic resonance imaging (MRI) could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by post mortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets. PMID:24613020

  1. Purification and biochemical characterisation of acid phosphatase-I from seeds of Nelumbo nucifera.

    PubMed

    Khan, Sanaullah; Khan, Shahnaz; Batool, Sajida; Ahmed, Mushtaq

    2016-01-01

    Acid phosphatase-I (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combination of ammonium sulfate precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kDa under reducing and non-reducing conditions, indicating that the enzyme was a monomer. The purified enzyme showed maximum activity at 50°C and at pH 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 μM, 10 μmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn(2+), W(2+); weakly inhibited by Cu(2+), Mo(2+) and Cr(6+) and moderately activated by Mg(2+). The enzyme was shown to be thermolabile as it lost 50% of its activity at 50°C after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase.

  2. Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

    PubMed Central

    Pigretti, M. M.; Milstein, C.

    1965-01-01

    1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn2+, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme. ImagesFig. 3.Fig. 4. PMID:14342215

  3. Purification and characterization of a novel acid phosphatase from the split gill mushroom Schizophyllum commune.

    PubMed

    Zhang, Guo-Qing; Chen, Qing-Jun; Sun, Jian; Wang, He-Xiang; Han, Chun-Hua

    2013-10-01

    A monomeric acid phosphatase (ACP) with a molecular mass of 72.5 kDa was purified from fresh fruiting bodies of cultured Schizophyllum commune mushroom. The isolation procedure entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. It demonstrated a unique N-terminal amino acid sequence of NAPWAQIDEV, which exhibited 60% amino acid identity to that of S. commune hypothetical histidine ACP based on its genome sequence, but less than 30% amino acid identity to that of other fungal ACPs previously reported. The ACP exhibited an optimum temperature at 50 °C, an optimum pH at pH 4.6, and was considerably stable at a pH range of 4.0 to 9.0, and a temperature range of 20-40 °C. The Km of the purified enzyme for ρ-nitrophenyl phosphate (ρNPP) was 0.248 mM and the Vmax was 9.093 × 10(-3)  μM/min. ACP activity was strongly inhibited by Al(3+) and Fe(3+) , but enhanced by Co(2+) , Mg(2+) , and Ca(2+) at a concentration of 0.5 mM. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Zhi, Ying; Gao, Li-Xin; Jin, Yi; Tang, Chun-Lan; Li, Jing-Ya; Li, Jia; Long, Ya-Qiu

    2014-07-15

    Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC50 values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2-10 fold selectivity over a panel of PTP's. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.

  5. Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase.

    PubMed

    Cavagis, Alexandre Donizeti Martins; Granjeiro, Paulo Afonso; Ferreira, Carmen Veríssima; Aoyama, Hiroshi

    2004-04-01

    In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.

  6. Estimation of biodiesel cytotoxicity by using acid phosphatase as a biomarker of lysosomal integrity.

    PubMed

    da Cruz, Andrea Cristina Santos; Leite, Maria Bernadete N L; Rodrigues, Luiz Erlon Araújo; Nascimento, Iracema Andrade

    2012-08-01

    Biodiesel is promoted as environmentally less harmful than diesel fuel. Nevertheless its water-soluble-fraction (WSF) may contain methanol, which appears by a reversion of the transesterification reaction, when biodiesel contacts water. This paper evaluated the loss of the lysosomal membrane integrity in liver homogenate of juvenils Tilapia exposed to biodiesels-WSF, through the increase of the acid phosphatase activity, as an evidence of citotoxicity. Differences in the enzyme activity levels (3.4, 2.3 and 0.8 mU mg(-1) total protein over the control value, which was 1.6 mU mg(-1) total protein), found for castor oil, waste cooking-oil and palm oil-biodiesels, respectively, were indicative of their toxicity according to this decreasing trend. WSF-chromatograms suggest the cytotoxicity as related to methanol.

  7. Regulators of PP2C phosphatase activity function as abscisic acid sensors.

    PubMed

    Ma, Yue; Szostkiewicz, Izabela; Korte, Arthur; Moes, Danièle; Yang, Yi; Christmann, Alexander; Grill, Erwin

    2009-05-22

    The plant hormone abscisic acid (ABA) acts as a developmental signal and as an integrator of environmental cues such as drought and cold. Key players in ABA signal transduction include the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, which act by negatively regulating ABA responses. In this study, we identify interactors of ABI1 and ABI2 which we have named regulatory components of ABA receptor (RCARs). In Arabidopsis, RCARs belong to a family with 14 members that share structural similarity with class 10 pathogen-related proteins. RCAR1 was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in planta. Other RCARs also mediated ABA-dependent regulation of ABI1 and ABI2, consistent with a combinatorial assembly of receptor complexes.

  8. Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots.

    PubMed

    Dreyer, Beatriz; Pérez-Gilabert, Manuela; Olmos, Enrique; Honrubia, Mario; Morte, Asunción

    2008-04-01

    Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.

  9. Tartrate-resistant acid phosphatase from human osteoclastomas is translated as a single polypeptide.

    PubMed Central

    Hayman, A R; Dryden, A J; Chambers, T J; Warburton, M J

    1991-01-01

    Tartrate-resistant acid phosphatases have been isolated from a number of sources. These enzymes consist of one subunit (Mr 30,000-40,000) or two dissimilar subunits (Mr 15,000-20,000). Previously we isolated the enzyme from human osteoclastomas, as a two-subunit protein. By Northern blotting and hybridization with radiolabelled oligonucleotides corresponding to the N-terminal sequences of the two subunits, we demonstrate here that the enzyme is transcribed as one mRNA which is translated in vitro to produce a single polypeptide of approx. Mr 33,000. Transcription as a single mRNA species is also the case in other tissues. These results suggest that the osteoclastoma enzyme undergoes post-translational modification in the form of cleavage of a single peptide bond to give a disulphide-bonded two-subunit protein. Images Fig. 1. Fig. 2. Fig. 3. PMID:1872798

  10. Crystallization and preliminary crystallographic data of purple acid phosphatase from red kidney bean.

    PubMed

    Sträter, N; Fröhlich, R; Schiemann, A; Krebs, B; Körner, M; Suerbaum, H; Witzel, H

    1992-03-20

    Purple acid phosphatase from red kidney bean has been crystallized from ammonium sulfate solutions in the pH range from 3.5 to 5.5. The crystal form is tetragonal bipyramidal and the largest crystals grew up to 2.0 mm long. Systematic absences indicate one of the enantiomorphic space groups P4(1)2(1)2 (92) or P4(3)2(1)2 (96) with cell dimensions a = b = 104.1(1) A and c = 308.7(2) A. The asymmetric unit contains one dimer with Mr of 110,700, determined by ultraviolet-laser desorption mass spectrometry. The crystals, with a salt-free density of 1.12 g/cm3 and a water content of 67%, diffract to 3.5 A.

  11. Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system.

    PubMed

    Kudryashova, E V; Bronza, V L; Levashov, A V

    2009-03-01

    The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.

  12. Regulation of acid phosphatase in reverse micellar system by lipids additives: structural aspects.

    PubMed

    Kudryashova, E V; Bronza, V L; Vinogradov, A A; Kamyshny, A; Magdassi, S; Levashov, A V

    2011-01-15

    Reverse micelles system is suggested as a direct tool to study the influence of membrane matrix composition on the activity and structure of membrane-associated enzymes with the use of acid phosphatase (AP) as an example. In reverse micelles the functioning of the monomeric and dimeric forms of AP could be separately observed by variation of the size of the micelles. We found that including the lipids into the micellar system can dramatically affect the enzyme functioning even at low lipid content (2% w/w), and this effect depends on the lipid nature. Structural studies using CD spectroscopy and DLS methods have shown that the influence of lipid composition on the enzyme properties might be caused by the interaction of lipids with the enzyme as well as by the influence of lipids on structure and properties of the micellar matrix. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2013-09-02

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

  14. Expression and distribution of tartrate-resistant purple acid phosphatase in the rat nervous system.

    PubMed

    Lång, P; Schultzberg, M; Andersson, G

    2001-03-01

    Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte-macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10-20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC(50) for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in alpha-motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.

  15. Comparative Evaluation of Efficacy of Three Different Herbal Toothpastes on Salivary Alkaline Phosphatase and Salivary Acid Phosphatase - A Randomized Controlled Trial

    PubMed Central

    Dodamani, Arun; Karibasappa, G. N.; Deshmukh, Manjiri; Naik, Rahul

    2016-01-01

    Introduction Very few researches in the past have tried to evaluate the effect of herbal toothpaste on saliva and salivary constituents like alkaline phosphatase and acid phosphatase which play an important role in maintaining oral health. Aim To evaluate and compare the effect of three different herbal toothpastes on Salivary Alkaline Phosphatase (ALP) and salivary Acid Phosphatase (ACP). Material and Methods The present study was a preliminary study conducted among 45 dental students (15 subjects in each group) in the age group of 19-21 years. Subjects in each group were randomly intervened with three different herbal toothpastes respectively (Group A – Patanjali Dant Kanti, Group B - Himalaya Complete Care and Group C – Vicco Vajradanti). Unstimulated saliva sample were collected before and after brushing and salivary ACP and salivary ALP levels were assessed at an interval of one week each for a period of four weeks starting from day one. Compiled data was analyzed using chi square test, paired t-test and ANOVA based on the nature of the obtained data. Results All the three toothpastes showed significant (p<0.001) reduction in ACP and ALP levels at each interval. For patanjali toothpaste, the mean reduction was in the range of 2.55 – 2.62 IU/L for ACP and 2.94 – 2.99 IU/L for ALP. For Himalaya toothpaste, the mean reduction was in the range of 1.39 – 1.47 IU/L for ACP and 1.55 – 1.61 IU/L for ALP. For Vicco toothpaste, the mean reduction was in the range of 2.46 – 2.50 IU/L for ACP and 2.64 – 2.77 IU/L for ALP. Patanjali and Vicco toothpaste were significantly effective in reducing the levels of salivary ACP and ALP more than Himalaya toothpaste (p<0.05). Conclusion Herbal toothpastes, especially Dant Kanti and Vicco Vajradanti, showed significant reduction in levels of ACP and ALP resulting in overall improvement towards the oral health. PMID:27790584

  16. Gene Expression Profiles of Human Phosphotyrosine Phosphatases Consequent to Th1 Polarisation and Effector Function

    PubMed Central

    Castro-Sánchez, Patricia; Ramirez-Munoz, Rocio

    2017-01-01

    Phosphotyrosine phosphatases (PTPs) constitute a complex family of enzymes that control the balance of intracellular phosphorylation levels to allow cell responses while avoiding the development of diseases. Despite the relevance of CD4 T cell polarisation and effector function in human autoimmune diseases, the expression profile of PTPs during T helper polarisation and restimulation at inflammatory sites has not been assessed. Here, a systematic analysis of the expression profile of PTPs has been carried out during Th1-polarising conditions and upon PKC activation and intracellular raise of Ca2+ in effector cells. Changes in gene expression levels suggest a previously nonnoted regulatory role of several PTPs in Th1 polarisation and effector function. A substantial change in the spatial compartmentalisation of ERK during T cell responses is proposed based on changes in the dose of cytoplasmic and nuclear MAPK phosphatases. Our study also suggests a regulatory role of autoimmune-related PTPs in controlling T helper polarisation in humans. We expect that those PTPs that regulate T helper polarisation will constitute potential targets for intervening CD4 T cell immune responses in order to generate new therapies for the treatment of autoimmune diseases. PMID:28393080

  17. Atypical Protein Phosphatase 2A Gene Families Do Not Expand via Paleopolyploidization1[OPEN

    PubMed Central

    2017-01-01

    Protein phosphatase 2A (PP2A) presents unique opportunities for analyzing molecular mechanisms of functional divergence between gene family members. The canonical PP2A holoenzyme regulates multiple eukaryotic signaling pathways by dephosphorylating target proteins and contains a catalytic (C) subunit, a structural/scaffolding (A) subunit, and a regulatory (B) subunit. Genes encoding PP2A subunits have expanded into multigene families in both flowering plants and mammals, and the extent to which different isoform functions may overlap is not clearly understood. To gain insight into the diversification of PP2A subunits, we used phylogenetic analyses to reconstruct the evolutionary histories of PP2A gene families in Arabidopsis (Arabidopsis thaliana). Genes encoding PP2A subunits in mammals represent ancient lineages that expanded early in vertebrate evolution, while flowering plant PP2A subunit lineages evolved much more recently. Despite this temporal difference, our data indicate that the expansion of PP2A subunit gene families in both flowering plants and animals was driven by whole-genome duplications followed by nonrandom gene loss. Selection analysis suggests that the expansion of one B subunit gene family (B56/PPP2R5) was driven by functional diversification rather than by the maintenance of gene dosage. We also observed reduced expansion rates in three distinct B subunit subclades. One of these subclades plays a highly conserved role in cell division, while the distribution of a second subclade suggests a specialized function in supporting beneficial microbial associations. Thus, while whole-genome duplications have driven the expansion and diversification of most PP2A gene families, members of functionally specialized subclades quickly revert to singleton status after duplication events. PMID:28034953

  18. Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases

    PubMed Central

    2012-01-01

    Background Gene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs), symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids’ hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs) has undergone spectacular expansion in several PDV genomes with up to 42 genes. Results Here, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication), by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity. Conclusion The results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in adaptation to host targets. PMID

  19. Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize, and Rice[W][OA

    PubMed Central

    Dionisio, Giuseppe; Madsen, Claus K.; Holm, Preben B.; Welinder, Karen G.; Jørgensen, Malene; Stoger, Eva; Arcalis, Elsa; Brinch-Pedersen, Henrik

    2011-01-01

    Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic 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 the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains. PMID:21220762

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

    SciTech Connect

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

    1988-03-01

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

  1. Effect of gingival application of melatonin on alkaline and acid phosphatase, osteopontin and osteocalcin in patients with diabetes and periodontal disease

    PubMed Central

    López-Valverde, Antonio; Gómez-de-Diego, Rafel; Arias-Santiago, Salvador; de Vicente-Jiménez, Joaquín

    2013-01-01

    Objectives: To assess the effect of topical application of melatonin to the gingiva on salivary fluid concentrations of acid phosphatase, alkaline phosphatase, osteopontin, and osteocalcin. Study Design: Cross-sectional study of 30 patients with diabetes and periodontal disease and 30 healthy subjects. Diabetic patients were treated with topical application of melatonin (1% orabase cream formula) once daily for 20 days and controls with a placebo formulation. Results: Before treatment with melatonin, diabetic patients showed significantly higher mean salivary levels of alkaline and acid phosphatase, osteopontin and osteocalcin than healthy subjects (P < 0.01). After treatment with melatonin, there was a statistically significant decrease of the gingival index (15.84± 10.3 vs 5.6 ± 5.1) and pocket depth (28.3 ± 19.5 vs 11.9 ± 9.0) (P < 0.001). Also, use of melatonin was associated with a significant reduction of the four biomarkers. Changes of salivary acid phosphatase and osteopontin correlated significantly with changes in the gingival index, whereas changes of alkaline phosphatase and osteopontin correlated significantly with changes in the pocket depth. Conclusions: Treatment with topical melatonin was associated with an improvement in the gingival index and pocket depth, a reduction in salivary concentrations of acid phosphatase, alkaline phosphatase, osteopontin and osteocalcin. Key words:Melatonin, diabetes mellitus, alkaline phosphatase, acid phosphatase, osteopontin, osteocalcin. PMID:23524437

  2. Substrate positioning by His92 is important in catalysis by purple acid phosphatase.

    PubMed

    Funhoff, Enrico G; Wang, Yunling; Andersson, Goran; Averill, Bruce A

    2005-06-01

    Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen-bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pK(es,1) values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.

  3. Lactate dehydrogenase and alkaline phosphatase isoenzymes and protein-bound sialic acid in regenerating rat liver.

    PubMed

    Allalouf, D; Schwarzman, S; Levinsky, H; Feller, N; Hart, J; Zoher, S; Menache, R

    1986-01-01

    Lactate dehydrogenase (LDH) and alkaline phosphatase (AP) isoenzyme patterns and protein-bound sialic acid content were compared between normal, regenerating rat liver 10 days after partial hepatectomy and fetal rat liver. For this purpose, liver from ten adult rats and two pools of ten fetal livers each were examined. Isoenzymes were separated by electrophoresis on cellulose acetate and their percent distribution calculated after quantitation by densitometry of the bands. LDH-5 and LDH-4 combined represented in all the tissues examined 90%-94% of the total activity. LDH-5/LDH-4 ratios were nearly equivalent in the normal and regenerated liver (7.14, 6.41), but substantially lower in fetal liver (2.50). Two bands of AP were visualized in electropherograms. AP-1/AP-2 ratio was lower in regenerated liver (1.57) as compared to normal liver (2.27) and still lower in fetal liver (1.06). Protein-bound sialic acid was, on protein basis, slightly but not significantly higher in regenerated liver (1.71 microgram/mg protein) than in normal liver (1.43), and significantly higher in fetal liver (1.87). The relatively small differences in isoenzyme patterns and in protein-bound sialic acid between regenerated and normal liver as compared to those between fetal and normal tissue add support to the view that the cells in regenerated liver are not of embryonic origin.

  4. Avirulence gene mapping in the Hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family.

    PubMed

    Zhao, Chaoyang; Shukle, Richard; Navarro-Escalante, Lucio; Chen, Mingshun; Richards, Stephen; Stuart, Jeffrey J

    2016-01-01

    The genetic tractability of the Hessian fly (HF, Mayetiola destructor) provides an opportunity to investigate the mechanisms insects use to induce plant gall formation. Here we demonstrate that capacity using the newly sequenced HF genome by identifying the gene (vH24) that elicits effector-triggered immunity in wheat (Triticum spp.) seedlings carrying HF resistance gene H24. vH24 was mapped within a 230-kb genomic fragment near the telomere of HF chromosome X1. That fragment contains only 21 putative genes. The best candidate vH24 gene in this region encodes a protein containing a secretion signal and a type-2 serine/threonine protein phosphatase (PP2C) domain. This gene has an H24-virulence associated insertion in its promoter that appears to silence transcription of the gene in H24-virulent larvae. Candidate vH24 is a member of a small family of genes that encode secretion signals and PP2C domains. It belongs to the fraction of genes in the HF genome previously predicted to encode effector proteins. Because PP2C proteins are not normally secreted, our results suggest that these are PP2C effectors that HF larvae inject into wheat cells to redirect, or interfere, with wheat signal transduction pathways.

  5. Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea.

    PubMed

    Poirier, T P; Holt, S C

    1983-10-01

    Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS-PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AlP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

  6. Characterization of a Soluble Phosphatidic Acid Phosphatase in Bitter Melon (Momordica charantia)

    PubMed Central

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

    2014-01-01

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg2+-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53–60°C and unaffected by up to 0.3 mM MgCl2. The Km and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na3VO4, Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants. PMID:25203006

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

    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

  9. Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls.

    PubMed

    Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H M; Gurjar, Suraj K; Gupta, I D; Verma, Archana

    2017-02-01

    This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Genomic DNA was isolated by phenol-chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5'UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible.

  10. Probing the interaction induced conformation transitions in acid phosphatase with cobalt ferrite nanoparticles: Relation to inhibition and bio-activity of Chlorella vulgaris acid phosphatase.

    PubMed

    Ahmad, Farooq; Zhou, Xing; Yao, Hongzhou; Zhou, Ying; Xu, Chao

    2016-09-01

    The present study explored the interaction and kinetics of cobalt ferrite nanoparticles (NPs) with acid phosphatase (ACP) by utilizing diverse range of spectroscopic techniques. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in ACP by static quenching mechanism. The negative values of van't Hoff thermodynamic expressions (ΔH=-0.3293Jmol(-1)K(-1) and ΔG=-3.960kJmol(-1)K(-1)) corroborate the spontaneity and exothermic nature of static quenching. The positive value of ΔS (13.2893Jmol(-1)K(-1)) corroborate that major contributors of higher and stronger binding affinity among CoFe2O4 NPs with ACP were electrostatic. In addition, FTIR, UV-CD, UV-vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding induces microenvironment perturbations leading to secondary and tertiary conformation changes in ACP to a great extent. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the comparatively significant changes in microenvironment around tryptophan (Trp) residue by CoFe2O4 NPs. The effect of CoFe2O4 NPs on the activation kinetics of ACP was further examined in Chlorella vulgaris. Apparent Michaelis constant (Km) values of 0.57 and 26.5mM with activation energy values of 0.538 and 3.428kJmol(-1) were determined without and with 200μM CoFe2O4 NPs. Apparent Vmax value of -7Umml(-1) corroborate that enzyme active sites were completely captured by the NPs leaving no space for the substrate. The results confirmed that CoFe2O4 NPs ceased the activity by unfolding of ACP enzyme. This suggests CoFe2O4 NPs perturbed the enzyme activity by transitions in conformation and hence the metabolic activity of ACP. This study provides the pavement for novel and simple approach of using sensitive biomarkers for sensing NPs in environment.

  11. Structural and Functional Assays of AtTLP18.3 Identify Its Novel Acid Phosphatase Activity in Thylakoid Lumen1[W][OA

    PubMed Central

    Wu, Hsin-Yi; Liu, Mao-Sen; Lin, Tsan-Piao; Cheng, Yi-Sheng

    2011-01-01

    The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four β-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-l-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-l-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain. PMID:21908686

  12. Protein phosphatase 2A subunit gene haplotypes and proliferative breast disease modify breast cancer risk

    PubMed Central

    Dupont, William D.; Breyer, Joan P.; Bradley, Kevin M.; Schuyler, Peggy A.; Plummer, W. Dale; Sanders, Melinda E.; Page, David L.; Smith, Jeffrey R.

    2009-01-01

    BACKGROUND Protein phosphatase 2A (PP2A) is a major cellular phosphatase and plays key regulatory roles in growth, differentiation, and apoptosis. Women diagnosed with benign proliferative breast disease are at increased risk for the subsequent development of breast cancer. METHODS We evaluated genetic variation of PP2A holoenzyme subunits for potential contribution to breast cancer risk. We performed a nested case-control investigation of a cohort of women with a history of benign breast disease. Subjects were followed for an average of 18 years; DNA prepared from the original archival benign breast biopsy (1954 – 1995) was available for 450 women diagnosed with breast cancer on follow-up, and for 890 of their 900 controls who were matched on race, age, and year of entry biopsy. RESULTS Single allele- and haplotype-based tests of association were conducted, with assessment of significance by permutation testing. We identified significant risk and protective haplotypes of PPP2R1A, giving odds ratios of 1.63 (95% CI 1.3 – 2.1) and 0.55 (95% CI 0.41 – 0.76), respectively. These odds ratios remained significant upon adjustment for multiple comparisons. Women with both the risk PPP2R1A haplotype and a history of proliferative breast disease had an odds ratio of 2.44 (95% CI 1.7 – 3.5) for the subsequent development of breast cancer. The effects of haplotypes for two regulatory subunit genes, PPP2R2A and PPP2R5E on breast cancer risk were nominally significant, but did not remain significant upon adjustment for multiple comparisons. CONCLUSION This evidence supports the previously hypothesized role of PP2A as a tumor suppressor gene in breast cancer. PMID:19890961

  13. Psy2 Targets the PP4 Family Phosphatase Pph3 To Dephosphorylate Mth1 and Repress Glucose Transporter Gene Expression

    PubMed Central

    Ma, Hui; Han, Bong-Kwan; Guaderrama, Marisela; Aslanian, Aaron; Yates, John R.; Hunter, Tony

    2014-01-01

    The reversible nature of protein phosphorylation dictates that any protein kinase activity must be counteracted by protein phosphatase activity. How phosphatases target specific phosphoprotein substrates and reverse the action of kinases, however, is poorly understood in a biological context. We address this question by elucidating a novel function of the conserved PP4 family phosphatase Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity is required for the timely dephosphorylation of the downstream transcriptional repressor Rgt1 upon glucose withdrawal, a critical event in the repression of HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1, an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein kinase activated in response to glucose, in vitro and regulates Mth1 function via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2 phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene expression by glucose. PMID:24277933

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

    PubMed

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

    2016-04-28

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

  15. The Jasper Ridge elevated CO{sub 2} experiment: Root acid phosphatase activity in Bromus hordeaceus and Avena barbata remains unchanged under elevated [CO{sub 2}

    SciTech Connect

    Cardon, Z.G.; Jackson, R.

    1995-06-01

    Root acid phosphatase activity increases phosphate available to plants by cleaving phosphate esters in soil organic matter. Because of increased plant growth potential under elevated [CO{sub 2}], we hypothesized that high [CO{sub 2}]-grown plants might exhibit higher phosphatase activity than low [CO{sub 2}]-grown plants. We assayed phosphatase activity in two species grown on two substrates (Bromus on serpentine soil and Bromus and Avena on sandstone soil) under high and low [CO{sub 2}] and under several nutrient treatments. Phosphatase activity was expressed per gram fresh weight of roots. Phosphatase activity of Bromus roots (on sandstone) was first assayed in treatments where only P and K, or only N, were added to soil. Bromus roots in this case showed strong induction of phosphatase activity when N only had been added to soil, indicating that Bromus regulated its phosphatase activity in response to phosphate availability. Both Bromus and Avena growing in sandstone, and Bromus growing in serpentine, showed enhanced phosphatase activity at high nutrient (N, P, and K) levels over that at low nutrient levels, but no differences between phosphatase activity were apparent between [CO{sub 2}] treatments. The increased phosphatase activity at high N, P, and K may indicate enhanced {open_quotes}growth demand{close_quotes} (reflected in higher biomass) in both Avena and Bromus. In contrast, though Bromus {open_quotes}growth demand{close_quotes} (biomass) increased under high [CO{sub 2}] on sandstone, phosphatase activity did not increase.

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

    PubMed

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

    2016-11-03

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

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

    PubMed

    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 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.

  18. Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase.

    PubMed

    Mohd-Pahmi, Siti Hajar; Hussein, Waleed M; Schenk, Gerhard; McGeary, Ross P

    2011-05-15

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl derivatives of α-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K(ic)=5 μM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K(ic)=8 μM, the most potent inhibitor of a mammalian PAP yet reported. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

  20. Evaluation of serum sialic acid, heat stable alkaline phosphatase and fucose as markers of breast carcinoma.

    PubMed

    Patel, P S; Baxi, B R; Adhvaryu, S G; Balar, D B

    1990-01-01

    Serum levels of total sialic acid (TSA), lipid bound sialic acid (LSA), heat stable alkaline phosphatase (HSAP) and fucose were measured in 39 patients with breast carcinoma, 14 patients with benign breast diseases and 35 healthy female individuals. Elevated levels of the four biomarkers in breast carcinoma were significant when compared with controls (p less than 0.001). Fucose levels were most sensitive (71.8%), while TSA levels were most specific (64.3%) for breast carcinoma. Sensitivity and specificity were 100% when combinations of LSA with fucose and TSA with HSAP were studied respectively. LSA was significantly elevated in infiltrating duct carcinoma patients compared with lobular carcinoma (p less than 0.001). TSA, HSAP and fucose also had lower mean values in lobular carcinoma as compared to infiltrating duct carcinoma. Increase in the levels of LSA and HSAP after surgical removal of the tumor in breast carcinoma occurred prior to the clinical evidence of the recurrence. The results indicate that the combination of the markers studied might be useful in breast cancer diagnosis and treatment monitoring.

  1. Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.

    PubMed

    Tate, R J; Tolan, D; Pyne, S

    1999-07-01

    Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.

  2. Overexpression of OsPAP10a, a root-associated acid phosphatase, increased extracellular organic phosphorus utilization in rice.

    PubMed

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

    2012-09-01

    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 organo-phosphates. In this study we characterized the rice (Oryza sativa) purple acid phosphatase 10a (OsPAP10a). OsPAP10a belongs to group Ia 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. © 2012 Institute of Botany, Chinese Academy of Sciences.

  3. Evidence for an indirect transcriptional regulation of glucose-6-phosphatase gene expression by liver X receptors

    SciTech Connect

    Grempler, Rolf . E-mail: rolfgrempler@yahoo.de; Guenther, Susanne; Steffensen, Knut R.; Nilsson, Maria; Barthel, Andreas; Schmoll, Dieter

    2005-12-16

    Liver X receptor (LXR) paralogues {alpha} and {beta} (LXR{alpha} and LXR{beta}) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXR{alpha} or LXR{beta} suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors.

  4. Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

    PubMed Central

    Galvão, M.J.; Santos, A. R.; Ribeiro, M.D.; Ferreira, A.; Nolasco, F.

    2011-01-01

    According to the new kidney disease improving global outcomes (KDIGO) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling, the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 µm were stained to identify TRACP at different incubation temperatures (37°, 45°, 60°, 70° and 80°C) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60°C and 70°C. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 min, the reaction should be carried out at 60

  5. Enzymatic study of tonsil tissue alkaline and acid phosphatase in children with recurrent tonsillitis and tonsil hypertrophy.

    PubMed

    Jesic, Snezana; Stojiljkovic, Ljuba; Stosic, Svetlana; Nesic, Vladimir; Milovanovic, Jovica; Jotic, Ana

    2010-01-01

    Indications for tonsillectomy in recurrent tonsillitis are defined according to the number of episodes of acute bacterial infections in a year. However, little is known about the tonsil immune competence status in patients presenting with recurrent tonsillitis with either hypertrophied or atrophied tonsils, or in patients presenting with obstructive sleep apnoea. In this study we examined the tonsil immune status in children with 3-5 acute recurrent infections a year and in children with obstructive sleep apnoea by comparing the activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase. Specific activity of tonsil and adenoid tissue nonspecific alkaline and acid phosphatase was investigated in children who underwent tonsillectomy and adenoidectomy for recurrent infection (72 children) and for obstructive sleep apnoea (10 children). Tissue enzyme activities were measured using p-nitrophenylphosphate as a substrate. Tissue samples were examined by the haematoxylin-eosin histological technique. Statistical analyses were performed using SPSS v. 16 software. The tissue nonspecific alkaline phosphatase activity was similar in hypertrophied tonsils in the recurrent infection group and in the obstructive sleep apnoea group (3.437+/-1.226 and 3.978+/-0.762 U/mg of protein, respectively). The enzyme activity in both hypertrophied tonsil groups was significantly higher as compared to atrophied tonsils in the recurrent tonsillitis group, p=0.021 and p=0.006, respectively. The enzyme activity was significantly higher in the adenoids compared to the tonsils from all three groups. Contrary to this, no significant differences were noticed for tonsil and adenoid acid phosphatase activities among the groups. Similar acid phosphatase activity in all three groups implies that all three groups have preserved antigen presenting cell activity. In patients with hypertrophied tonsils similar tissue nonspecific alkaline phosphatase activity suggests preserved B cell

  6. Light microscopical localization of enzymes by means of cerium-based methods. I.V. Optimization procedures for acid phosphatase.

    PubMed

    Halbhuber, K J; Zimmermann, N; Feuerstein, H

    1986-01-01

    The earlier described cerium based histochemical reaction for acid phosphatase [Ce-Pb-reaction, Zimmermann and Halbhuber (1985)] was optimized. The target tissues (kidney, intestine) were fixed by perfusion with glutaraldehyde in cacodylate or piperazine buffer in anesthetized animals. Postfixation of prefixed sections is not advantageous because of the detectable repressing of the enzyme activity. Moreover, the employment of unfixed cryostat sections, which were postfixed, was always connected with a complete abolition of the acid phosphatase activity. The optimal concentration of the primary capture cerium III chloride in the incubation medium is about 1 mmol. Lower concentrations lead to an incomplete histochemical detection of phosphatase activity in lysosomes. The treatment of cryostat sections of perfusion fixed tissue with borohydride (diminution of aldehyde induced cross links) or with dimethylsulfoxide (extraction of lysosomal materials or the well known vehicle property) brought about an improvement of the penetration capacity for cerium-III-cations into the target structures. After conversion of the cerium phosphate (primary specific reaction product) into cerium perhydroxide, oxalate or fluoride, the Ce-Pb-reaction was negative. Therefore, these blocking reactions represent specific inhibition controls, which indicates the formation and presence of cerium phosphate. On the basis of these reactions it is possible to check the specificity of the histochemical Ce-Pb-reaction for phosphatase activity in sections.

  7. Intrauterine growth restriction affects hippocampal dual specificity phosphatase 5 gene expression and epigenetic characteristics

    PubMed Central

    Ke, Xingrao; McKnight, Robert A.; Caprau, Diana; O'Grady, Shannon; Fu, Qi; Yu, Xing; Callaway, Christopher W.; Albertine, Kurt H.

    2011-01-01

    Intrauterine growth retardation (IUGR) predisposes humans toward hippocampal morbidities, such as impaired learning and memory. Hippocampal dual specificity phosphatase 5 (DUSP5) may be involved in these morbidities because DUSP5 regulates extracellular signal-regulated kinase phosphorylation (Erk). In the rat, IUGR causes postnatal changes in hippocampal gene expression and epigenetic characteristics. However, the impact of IUGR upon hippocampal DUSP5 expression and epigenetic characteristics is not known. We therefore hypothesized that IUGR affects hippocampal 1) DUSP5 expression, DNA CpG methylation, and histone code, and 2) erk1/2 phosphorylation in a well-characterized rat model of IUGR. We found that IUGR significantly decreased DUSP5 expression in the day of life (DOL) 0 and 21 male rat, while decreasing only DUSP5 protein levels in the DOL21 female rat. Fluorescent in situ hybridization and immunohistochemistry analyses localized the changes in DUSP5 mRNA and protein, many of which occurred in the dentate gyrus. IUGR also caused sex-specific differences in DNA CpG methylation and histone code in two sites of the hippocampal DUSP5 gene, a 5′-flanking specificity protein-1 (SP1) site and exon 2. Finally, when IUGR decreased DUSP5 protein levels, Erk phosphorylation increased. We conclude that IUGR affects hippocampal DUSP5 expression and epigenetic characteristics in a sex-specific manner. PMID:21828247

  8. HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma.

    PubMed

    Martellini, Julie A; Cole, Amy L; Svoboda, Pavel; Stuchlik, Olga; Chen, Li-Mei; Chai, Karl X; Gangrade, Bhushan K; Sørensen, Ole E; Pohl, Jan; Cole, Alexander M

    2011-01-20

    We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.

  9. Serum tissue polypeptide antigen (TPA) and prostatic acid phosphatase (PAP) in patients with prostatic cancer.

    PubMed

    Marczyńska, A; Kulpa, J; Leńko, J; Augustyn, M

    1988-01-01

    Concurrent measurements of serum TPA and PAP concentrations by double antibody radioimmunoassays were done in 49 patients with prostatic cancer in different clinical stages. The reference group comprised patients suffering from BPH. Positive TPA was found in 32.7% of cancer patients, the lowest percentage in stage A (11.1%) and the highest in stage D (55.6%). The additional value as a diagnostic aid of the TPA test was revealed on the basis of examination of the selected group of patients with not increased PAP. Positive TPA was found in 16.7% of patients: none in stage A, 22.2% in stage B, and 33.3% in stage D. Prostatic cancer remains the most common malignancy of the genitourinary tract. The improvement in the results of treatment involves not only a modernization of treatment modalities but also the introduction of laboratory tests which give the most ample information on the stage of tumour development and improve possibilities to control tumour therapy. Besides the refinement of the determination procedures of specific prostatic markers, prostatic acid phosphatase (PAP), through radio- and enzyme-immunological methods, there is a search for additional markers which might be helpful in diagnosis and follow-up of treatment.

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

    PubMed

    Retegan, M; Milet, A; Jamet, H

    2010-07-08

    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.

  11. Expression and proteolytic processing of mammalian purple acid phosphatase in CHO-K1 cells.

    PubMed

    Wang, Yunling; Andersson, Göran

    2007-05-01

    Rat recombinant purple acid phosphatase (PAP) stably expressed in fibroblast-like CHO-K1 cells was purified and characterized with respect to post-translational modifications such as N-glycosylation and proteolytic processing in order to elucidate subcellular and molecular pathways for proteolytic activation. In these cells, proteolytically processed PAP was more abundant than the monomeric form. PAP-transfected CHO-K1 cells were expressing active cathepsin K intracellularly, which was partially co-localized with PAP. However, neither cathepsin K nor trypsin digestion of the purified monomeric PAP in vitro did result in a two-subunit form with kinetic and electrophoretic properties resembling the endogenous cellular two-subunit form. Treatment of PAP-transfected CHO-K1 cells with the cysteine proteinase inhibitor E-64 suggested that only a minor fraction of secreted PAP is processed intracellularly by cysteine proteinases. These data do not support a dominant or critical role for cathepsins or trypsin-like serine proteinases in the proteolytic activation of PAP in CHO-K1 cells, implicating yet unidentified proteinases in the proteolytic processing of both intracellular and secreted PAP in this cell line.

  12. Dependence on the Lazaro phosphatidic acid phosphatase for the maximum light response.

    PubMed

    Kwon, Young; Montell, Craig

    2006-04-04

    The Drosophila phototransduction cascade serves as a paradigm for characterizing the regulation of sensory signaling and TRP channels in vivo . Activation of these channels requires phospholipase C (PLC) and may depend on subsequent production of diacylglycerol (DAG) and downstream metabolites . DAG could potentially be produced through a second pathway involving the combined activities of a phospholipase D (PLD) and a phosphatidic acid (PA) phosphatase (PAP). However, a role for a PAP in the regulation of TRP channels has not been described. Here, we report the identification of a PAP, referred to as Lazaro (Laza). Mutations in laza caused a reduction in the light response and faster termination kinetics. Loss of laza suppressed the severity of the phenotype caused by mutation of the DAG kinase, RDGA , indicating that Laza functions in opposition to RDGA. We also showed that the retinal degeneration resulting from overexpression of the PLD was suppressed by elimination of Laza. These data demonstrate a requirement for a PLD/PAP-dependent pathway for achieving the maximal light response. The genetic interactions with both rdgA and Pld indicate that Laza functions in the convergence of both PLC- and PLD-coupled signaling in vivo.

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

    PubMed

    Wan, H; Horvath, C

    1975-11-20

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

  14. Renal neuroendocrine tumour and synchronous pancreas metastasis: histopathological diagnosis using prostatic acid phosphatase.

    PubMed

    Kawasaki, Keishi; Kawaguchi, Yoshikuni; Suzuki, Yoshio; Tanaka, Nobutaka

    2016-11-01

    A woman aged 56 years developed 2 synchronous tumours: one, 1.2 cm in diameter at the head of the pancreas; and the other, 4.0 cm in diameter, at the left side of her horseshoe kidney. Preoperative differential diagnosis of these hypovascular lesions included pancreatic ductal carcinoma (PDC) with renal metastasis, PDC with renal angiomyolipoma, renal cell carcinoma with pancreatic metastasis or PDC and renal cell carcinoma. Following pancreaticoduodenectomy and left nephrectomy, both specimens were diagnosed as grade 2 neuroendocrine tumours (NETs). Immunohistochemistry revealed that both were positive for prostatic acid phosphatase (PAP), which is specific to hindgut-derived NET, including renal NET. Accordingly, the renal tumour was diagnosed as the primary lesion, and the pancreatic tumour as a metastasis. To the best of our knowledge, this is the first report of a renal NET with a synchronous pancreas metastasis. Immunohistochemical staining for PAP was a useful diagnostic marker for synchronous NETs in the kidney and pancreas. 2016 BMJ Publishing Group Ltd.

  15. Characterization of four monoclonal antibodies to recombinant human tartrate-resistant acid phosphatase.

    PubMed

    Miyazaki, Takashi; Matsunaga, Toshiyuki; Miyazaki, Shuichi; Hokari, Shigeru; Komoda, Tsugikazu

    2002-06-01

    In this study we produced a recombinant human Tartrate-resistant acid phosphatase (TRAP) enzyme from baculovirus-infected insect cells, generated four monoclonal antibodies (MAbs) 15A4, 13B9, 1C6 and 3G7, to the enzyme, and characterized these antibodies. In the human serum and lung specimen, all four antibodies appeared to have a high specificity for native TRAP enzyme in western blot analysis, immunohistochemical analysis and enzyme immunoassay. These antibodies may react with respective conformational determinants, therefore, they may be useful for detection of active TRAP. Only one of the antibodies, 15A4 also reacted with a denatured epitope, therefore, it is suitable for western blot analysis, enzyme immunoassay and for immunohistochemistry in the rat. Taken together, having characterized properties of four monoclonal antibodies against recombinant human TRAP enzyme may be useful for development of TRAP specific immunoassays in pathology and hematology of the bone. They will certainly be of use for the study of biosynthesis, regulation and function of the TRAP enzyme.

  16. Monoesterase activity of a purple acid phosphatase mimic with a cyclam platform.

    PubMed

    Comba, Peter; Gahan, Lawrence R; Hanson, Graeme R; Mereacre, Valeriu; Noble, Christopher J; Powell, Annie K; Prisecaru, Ion; Schenk, Gerhard; Zajaczkowski-Fischer, Marta

    2012-02-06

    The synthesis and characterization of a novel dinucleating ligand L (L=4,11-dimethyl-1,8-bis{2-[N-(di-2-pyridylmethyl)amino]ethyl}cyclam) and its μ-oxo-bridged diferric complex [(H(2)L){Fe(III)(2)(O)}(Cl)(4)](2+) are reported. This diiron(III) complex is the first example of a truly functional purple acid phosphatase (PAP) mimic as it accelerates the hydrolysis of the activated phosphomonoester 2,4-dinitrophenyl phosphate (DNPP). The spectroscopic and kinetic data indicate that only substrates that are monodentately bound to one of the two ferric ions can be attacked by a suitable nucleophile. This is, most probably, a terminal iron(III)-bound hydroxide. DFT calculations support this assumption and also highlight the importance of secondary interactions, exerted by the protonated cyclam platform, for the positioning and activation of the iron(III)-bound substrate. Similar effects are postulated in the native enzyme but addressed in PAP mimics for the first time. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Kinetics and optical spectroscopic studies on the purple acid phosphatase from beef spleen.

    PubMed

    Davis, J C; Lin, S S; Averill, B A

    1981-07-07

    A new purification scheme has been developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only five steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate, and [p-(acetylamino)-benzyl]phosphonate, a nonhydrolyzable substrate analogue; the last two of these show simple competitive inhibition. In contrast, cyanide, azide, tartrate, and p-nitrophenol show no inhibition at concentrations up to 10 mM. Molecular weight estimations by gel electrophoresis and gel permeation chromatography give a value of 40 000 for the native enzyme, which is shown to consist of two subunits of apparent molecular weight 24 000 and 15 000. Careful metal analyses indicate the presence of 2.1 +/- 0.1 iron atoms per enzyme molecule, and less than 0.1 copper, zinc, nickel, or manganese atom per enzyme. The purple enzyme (lambda max 550 nm) is reversibly converted to a pink, active form (lambda max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. Electron paramagnetic resonance spectroscopy at several temperatures showed only weak g = 4.3 signals (less than 0.1 spin/molecule) for the native, reduced, and inhibited forms of the enzyme.

  18. HIV-1 Enhancing Effect of Prostatic Acid Phosphatase Peptides Is Reduced in Human Seminal Plasma

    PubMed Central

    Martellini, Julie A.; Cole, Amy L.; Svoboda, Pavel; Stuchlik, Olga; Chen, Li-Mei; Chai, Karl X.; Gangrade, Bhushan K.; Sørensen, Ole E.; Pohl, Jan; Cole, Alexander M.

    2011-01-01

    We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called “SEVI” and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1∶200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity. PMID:21283773

  19. Acid phosphatase activity and color changes in consumer-style griddle-cooked ground beef patties.

    PubMed

    Lyon, B G; Davis, C E; Windham, W R; Lyon, C E

    2001-08-01

    The U.S. Department of Agriculture and the Food and Drug Administration have issued temperature requirements to help consumers cook beef patty products that are free of pathogens. Verification of end-point temperature (EPT) is needed in cooked meat products due to concerns over outbreaks of Escherichia coli O157:H7. Acid phosphatase (ACP) activity was studied as a potential method for determination of EPT in ground beef patties cooked nonfrozen, patties frozen 7 days and thawed at room temperature 4 h in a refrigerator or by microwave, and patties made from ground beef frozen in store packages, then thawed in a refrigerator overnight. Pressed-out meat juices were analyzed from patties (n = 314) cooked to 57.2 degrees C (135 degrees F). 65.6 degrees C (150 degrees F), 71.1 degrees C (160 degrees F), and 79.4 degrees C (175 degrees F) target EPTs. Expressed meat juice and internal meat patty color decreased in redness as EPT increased. Freezing whole packs with slow refrigerator or room temperature thawing caused significantly greater loss of redness in expressed cooked meat juice than did other handling methods. Log10 ACP had a significant linear (R2 = 0.99) response to EPT. Results show that the 3- to 5-min ACP test could be used to verify EPT in griddle-cooked hamburger patties.

  20. Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer.

    PubMed

    How, Joan; Brown, Jason R; Saylor, Sasha; Rimm, David L

    2014-08-01

    Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate-resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all sub-populations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22 % increase in 5-year survival (uncorrected log-rank p = 0.025) and a 47 % risk reduction in disease-specific death (p = 0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19 % increase in 5-year survival (log-rank p = 0.0041) and a 52 % risk reduction in death (p = 0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome and implicates TRAP as a potential biomarker in colon cancer.

  1. Macrophage expression of tartrate-resistant acid phosphatase as a prognostic indicator in colon cancer

    PubMed Central

    How, Joan; Brown, Jason R.; Saylor, Sasha; Rimm, David L.

    2014-01-01

    Recent research has indicated that separate populations of macrophages are associated with differing outcomes in cancer survival. In our study, we examine macrophage expression of tartrate resistant acid phosphatase (TRAP) and its effect on survival in colon cancer. Immunohistochemical analysis on colorectal adenocarcinomas confirmed macrophage expression of TRAP. Co-localization of TRAP with CD68, a pan-macrophage marker, revealed that TRAP is present in some but not all subpopulations of macrophages. Further co-localization of TRAP with CD163, an M2 marker, revealed that TRAP is expressed by both M2 and non-M2 macrophages. TRAP expression was then measured using the AQUA method of quantitative immunofluorescence in a tissue microarray consisting of 233 colorectal cancer patients seen at Yale-New Haven Hospital. Survival analysis revealed that patients with high TRAP expression have a 22% increase in 5-year survival (uncorrected log rank p=0.025) and a 47% risk reduction for disease specific death (p=0.02). This finding was validated in a second cohort of older cases consisting of 505 colorectal cancer patients. Patients with high TRAP expression in the validation set had a 19% increase in 5-year survival (log rank p=0.0041) and a 52% risk reduction of death (p=0.0019). These results provide evidence that macrophage expression of TRAP is associated with improved outcome, and implicates TRAP as a potential biomarker in colon cancer. PMID:24429833

  2. Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

    PubMed Central

    Jain, Varsha; Patel, Brijesh; Umar, Farhat Paul; Ajithakumar, H. M.; Gurjar, Suraj K.; Gupta, I. D.; Verma, Archana

    2017-01-01

    Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible. PMID:28344410

  3. Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

    PubMed

    Eaton, James M; Mullins, Garrett R; Brindley, David N; Harris, Thurl E

    2013-04-05

    The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

  4. A novel type heterozygous mutation in the glucose-6-phosphatase gene in a Chinese patient with glycogen storage disease Ia.

    PubMed

    Zhu, Jie; Xing, Yan; Xing, Xuenong; Ren, An; Ye, Shandong; He, Guoping

    2012-12-10

    Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSD Ia). By genotype analysis of the affected pedigree, we identified a novel type mutation in a Chinese patient with GSD Ia. Mutation analysis was performed for the coding region of G6Pase gene using DNA sequencing and TaqMan gene expression assay was used to further confirm the novel mutation. The proband was compound heterozygous for c.311A>T/c.648G>T. Our report expands the spectrum of G6Pase gene mutation in China. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes.

    PubMed

    Liu, Pan-Dao; Xue, Ying-Bin; Chen, Zhi-Jian; Liu, Guo-Dao; Tian, Jiang

    2016-07-01

    Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26 Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  6. Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes

    PubMed Central

    Liu, Pan-Dao; Xue, Ying-Bin; Chen, Zhi-Jian; Liu, Guo-Dao; Tian, Jiang

    2016-01-01

    Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26. Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo. PMID:27194738

  7. Spatial distribution and expression of intracellular and extracellular acid phosphatases of cluster roots at different developmental stages in white lupin.

    PubMed

    Tang, Hongliang; Li, Xiaoqing; Zu, Chao; Zhang, Fusuo; Shen, Jianbo

    2013-09-15

    Acid phosphatases (APases) play a key role in phosphorus (P) acquisition and recycling in plants. White lupin (Lupinus albus L.) forms cluster roots (CRs) and produces large amounts of APases under P deficiency. However, the relationships between the activity of intracellular and extracellular APases (EC 3.1.3.2) and CR development are not fully understood. Here, comparative studies were conducted to examine the spatial variation pattern of APase activity during CR development using the enzyme-labelled fluorescence-97 (ELF-97) and the p-nitrophenyl phosphate methods. The activity of intracellular and extracellular APases was significantly enhanced under P deficiency in the non-CRs and CRs at different developmental stages. These two APases exhibited different spatial distribution patterns during CR development, and these distribution patterns were highly modified by P deficiency. The activity of extracellular APase increased steadily with CR development from meristematic, juvenile, mature to senescent stages under P deficiency. In comparison, P deficiency-induced increase in the activity of intracellular APase remained relatively constant during CR development. Increased activity of intracellular and extracellular APases was associated with enhanced expression of LaSAP1 encoding intracellular APase and LaSAP2 encoding extracellular APase. The expression levels of these two genes were significantly higher at transcriptional level in both mature and senescent CRs. Taken together, these findings demonstrate that both activity and gene expression of intracellular or extracellular APases exhibit a differential response pattern during CR development, depending on root types, CR developmental stages and P supply. Simultaneous in situ determination of intracellular and extracellular APase activity has proved to be an effective approach for studying spatial variation of APases during CR development. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Purkinje cell compartmentation in the cerebellum of the lysosomal Acid phosphatase 2 mutant mouse (nax - naked-ataxia mutant mouse).

    PubMed

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation.

  9. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    PubMed Central

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  10. Enhancement of acid phosphatase secretion and Pi acquisition in Suaeda fruticosa on calcareous soil by high saline level.

    PubMed

    Labidi, Nehla; Snoussi, Sana; Ammari, Manel; Metoui, Wissal; Ben Yousfi, N; Hamrouni, Lamia; Abdelly, C

    2010-12-01

    The aim of this study was to identify the relationship between the adaptive processes of Suaeda fruticosa for Pi acquisition and the physic-chemical and biological characteristics of two soil types under moderate and high saline conditions. Four treatments were established in pots: namely SS100, SS600, CS100 and CS600 where SS stood for sandy soil and CS for calcareous soil, and the indexes 100 and 600 were NaCl concentrations (mM) in irrigation distilled water. Assuming that Pi per g of plant biomass is an indicator of plant efficiency for P acquisition, the results showed that Pi acquisition was easiest on SS100 and was difficult on CS100. The differences in Pi acquisition between plants on SS100 and CS100 could be attributed to the low root surface area (-30%) and to the low alkaline phosphatases (Pases) activities (-50%) in calcareous rhizospheric soil. The high salinity level had no effect on the efficiency of P acquisition on SS but increased this parameter on CS (+50%). In the latter soil type, high acid phosphatase activities were observed in rhizospheric soil at high salinity level. Acid phosphatase seemed to be secreted from the roots. The higher secretion of acid phosphatase in this soil was related to the root lipid peroxidation in response to elevated salinity associated with the augmentation of unsaturated acids which might induce an oxidative damage of the root membrane. Thus we can conclude that in deficient soil such as calcareous, the efficiency of P acquisition in S. fruticosa which was difficult at moderate salinity level can be enhanced by high salinity level.

  11. [Effect of aluminium and cAMP on acid phosphatase from the apoplast of barley and maize root cells].

    PubMed

    Fedorovskaia, M D; Tikhaia, N I

    2003-01-01

    Acid phosphatase activity inhibited by 1 mM sodium molybdate was detected at the surface of barley seedling roots and in the cell wall fraction isolated from barley and maize seedling roots. This enzyme hydrolyzed NPP, GP, and PPi at low pH (4.0 and below). NPP hydrolysis was stimulated by magnesium (but not calcium or manganese) ions, while PPi hydrolysis was independent of the presence of bivalent ions. The activity of phosphatase localized in the cell walls of the both crops increased in the presence of 100 microM AlCl3 or CuCl2. Stimulation of NPP hydrolysis by micromolar concentrations of aluminium and copper as well as by millimolar concentrations of magnesium decreased in the presence of 25 microM cAMP. This agrees with the previous data on the enzyme localized at the outer side of the properly oriented vesicles in the microscomal fraction of plasmalemma. The role of the root extracellular acid phosphatase loosely associated with various apoplast structures in plant adaptation to toxic effect of aluminium in the acidic soils as well as possible control of this process by cAMP secretion to the apoplast are discussed.

  12. Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

    PubMed Central

    Lei, K J; Pan, C J; Shelly, L L; Liu, J L; Chou, J Y

    1994-01-01

    Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type 1a patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type 1a patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K355SP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis. Images PMID:8182131

  13. Responses of phosphate transporter gene and alkaline phosphatase in Thalassiosira pseudonana to phosphine.

    PubMed

    Fu, Mei; Song, Xiuxian; Yu, Zhiming; Liu, Yun

    2013-01-01

    Phosphine, which is released continuously from sediment, can affect the eco-physiological strategies and molecular responses of phytoplankton. To examine the effects of phosphine on phosphorus uptake and utilization in Thalassiosira pseudonana, we examined the transcriptional level of the phosphate transporter gene (TpPHO) and the activity of alkaline phosphatase (AKP) in relation to supplement of various concentrations of phosphine. TpPHO expression was markedly promoted by phosphine in both the phosphate-deficient and phosphate-4 µM culture. However, high phosphine concentrations can inhibit TpPHO transcription in the declining growth phase. AKP activity was also higher in the phosphine treatment groups than that of the control. It increased with increasing phosphine concentration in the range of 0 to 0.056 µM but was inhibited by higher levels of phosphine. These responses revealed that phosphine can affect phosphate uptake and utilization in T. pseudonana. This result was consistent with the effect of phosphine on algal growth, while TpPHO expression and AKP were even more sensitive to phosphine than algal growth. This work provides a basic understanding for further research about how phosphine affects phytoplankton.

  14. Responses of Phosphate Transporter Gene and Alkaline Phosphatase in Thalassiosira pseudonana to Phosphine

    PubMed Central

    Fu, Mei; Song, Xiuxian; Yu, Zhiming; Liu, Yun

    2013-01-01

    Phosphine, which is released continuously from sediment, can affect the eco-physiological strategies and molecular responses of phytoplankton. To examine the effects of phosphine on phosphorus uptake and utilization in Thalassiosira pseudonana, we examined the transcriptional level of the phosphate transporter gene (TpPHO) and the activity of alkaline phosphatase (AKP) in relation to supplement of various concentrations of phosphine. TpPHO expression was markedly promoted by phosphine in both the phosphate-deficient and phosphate-4 µM culture. However, high phosphine concentrations can inhibit TpPHO transcription in the declining growth phase. AKP activity was also higher in the phosphine treatment groups than that of the control. It increased with increasing phosphine concentration in the range of 0 to 0.056 µM but was inhibited by higher levels of phosphine. These responses revealed that phosphine can affect phosphate uptake and utilization in T. pseudonana. This result was consistent with the effect of phosphine on algal growth, while TpPHO expression and AKP were even more sensitive to phosphine than algal growth. This work provides a basic understanding for further research about how phosphine affects phytoplankton. PMID:23544096

  15. PHO8 gene coding alkaline phosphatase of Saccharomyces cerevisiae is involved in polyphosphate metabolism.

    PubMed

    Kizawa, Keiko; Aono, Toshihiro; Ohtomo, Ryo

    2017-01-25

    It has been argued for a long time whether alkaline phosphatase (ALP) is involved in polyphosphate (polyP) metabolism in arbuscular mycorrhizal fungi. In the present study, we have analyzed the effects of disrupting the PHO8 gene, which encodes phosphate (Pi)-deficiency-inducible ALP, on the polyP contents of Saccharomyces cerevisiae. The polyP content of the Δpho8 mutant was higher than the wild type strain in the logarithmic phase under Pi-sufficient conditions. On the contrary, the chain length of polyP extracted from the Δpho8 mutant did not differ from the wild type strain. When cells in Pi-deficient conditions were supplemented with Pi, the increase of the polyP amounts in the Δpho8 mutant was similar to that in the wild type strain. These results suggest that ALP, which is encoded by PHO8, affects the polyP content, but not the chain length, and participates in polyP homeostasis in Pi-sufficient conditions.

  16. Protection of epithelial barrier function by the Crohn's disease associated gene protein tyrosine phosphatase n2.

    PubMed

    Scharl, Michael; Paul, Gisela; Weber, Achim; Jung, Barbara C; Docherty, Michael J; Hausmann, Martin; Rogler, Gerhard; Barrett, Kim E; McCole, Declan F

    2009-12-01

    Protein tyrosine phosphatase N2 (PTPN2) has been identified as a Crohn's disease (CD) candidate gene. However, a role for PTPN2 in the pathogenesis of CD has not been identified. Increased permeability of the intestinal epithelium is believed to contribute prominently to CD. The aim of this study was to determine a possible role for PTPN2 in CD pathogenesis. Intestinal epithelial cell (IEC) lines T(84) and HT29cl.19a were used in all studies. Protein analysis was performed by Western blotting, and protein knockdown was induced by small interfering RNA. Primary samples were from control and CD patients. Here, we demonstrate increased PTPN2 expression in CD intestinal biopsy specimens and that the proinflammatory cytokine interferon (IFN)-gamma increases PTPN2 expression and activity in IEC. Moreover, IFN-gamma-induced STAT1 and STAT3 phosphorylation in IEC is enhanced by PTPN2 knockdown. The cellular energy sensor adenosine monophosphate-activated protein kinase partially regulates the IFN-gamma-induced effects on PTPN2. Additionally, PTPN2 knockdown potentiates IFN-gamma-induced increases in epithelial permeability, accompanied by elevated expression of the pore-forming protein claudin-2. PTPN2 is activated by IFN-gamma and limits IFN-gamma-induced signalling and consequent barrier defects. These data suggest a functional role for PTPN2 in maintaining the intestinal epithelial barrier and in the pathophysiology of CD.

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

    PubMed

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that

  18. Development of immunoassays for serum tartrate-resistant acid phosphatase isoform 5a.

    PubMed

    Chao, Tsu-Yi; Lee, Su-Huei; Chen, Mary M; Neustadt, David H; Chaudhry, Uzma A; Yam, Lung T; Janckila, Anthony J

    2005-09-01

    Serum tartrate-resistant acid phosphatase (TRACP) consists of 2 structurally related isoforms, TRACP 5a and 5b. TRACP 5b is from bone-resorbing osteoclasts. TRACP 5a may be a macrophage product of inflammation. We used a novel antibody to TRACP 5a to standardize immunoassays for serum TRACP 5a activity and protein. Biotinylated anti-TRACP antibodies were used to immobilize serum TRACP isoforms. TRACP activity was measured using 4-nitrophenyl phosphate as substrate. TRACP 5a protein was measured with an independent peroxidase-conjugated anti-TRACP antibody. Immunoassays were standardized for linearity of serum dose response, sensitivity and precision. Reference ranges for TRACP 5a were established from serum of 50 healthy males and 50 healthy age-matched females. Serum TRACP 5a activity and protein were determined in 29 cases of rheumatoid arthritis. Serum matrix interference in both TRACP 5a assays required dilution to 10% serum to approach linearity. Intra-assay and inter-assay CV% were <10%. Mean serum TRACP 5a activity and protein were significantly higher in healthy men than women. There was a slight, but significant age related increase in both serum TRACP 5a and 5b among females, but not males, from age 20 to 70 years. TRACP 5a activity was positively correlated to TRACP 5a protein in healthy sera. Neither TRACP 5a activity nor protein was correlated strongly to TRACP-5b activity. TRACP 5a protein was significantly increased in 8/29 RA sera, whereas TRACP 5a and 5b activities were not. TRACP 5a activity and protein were not significantly correlated in RA sera. Although TRACP 5a and 5b are related biosynthetically, their circulating levels in healthy humans were independent, suggesting differential regulation of expression. In chronic diseases, increased TRACP 5a may represent pathological processes of inflammation unrelated to bone metabolism.

  19. Atomistic details of the Catalytic Mechanism of Fe(III)-Zn(II) Purple Acid Phosphatase.

    PubMed

    Alberto, Marta E; Marino, Tiziana; Ramos, Maria J; Russo, Nino

    2010-08-10

    In the present work, we performed a theoretical investigation of the reaction mechanism of the Fe(III)-Zn(II) purple acid phosphatase from red kidney beans (rkbPAP), using the hybrid density functional theory and employing different exchange-correlation potentials. Characterization of the transition states and intermediates involved and the potential energy profiles for the reaction in different environments (gas phase, protein environment, and water) are reported. Our results show that the Fe(III)-Zn(II)PAP catalyzes the hydrolysis of methylphosphate via direct attack by a bridging metals-coordinated hydroxide leading to the cleavage of the ester bond. From our study emerges that the rate-limiting step of the reaction is the nucleophilic attack followed by the less energetically demanding release of the leaving group. Furthermore, we provide insights into some important points of contention concerning the precatalytic complex and the substrate coordination mode into the active site prior to hydrolysis. In particular: (i) Two models of enzyme-substrate with different orientations of the substrate into the active site were tested to evaluate the possible roles played by the conserved histidine residues (His 202 and His 296); (ii) Different protonation states of the substrate were taken into account in order to reproduce different pH values and to verify its influence on the catalytic efficiency and on the substrate binding mode; (iii) The metals role in each step of the catalytic mechanism was elucidated. We were also able to ascertain that the activation of the leaving group by the protonated His 296 is decisive to reach an optimal catalytic efficiency, while the bond scission without activation requires higher energy to occur.

  20. Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient.

    PubMed

    Kubicz, A; Szalewicz, A; Fawcett, J S; Chrambach, A

    1990-02-01

    Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.

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

    SciTech Connect

    Lau, K.H.W.; Freeman, T.K.; Baylink, D.J.

    1986-05-01

    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 37/sup 0/C. 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.

  2. Cysteine proteases and acid phosphatases contribute to Tetrahymena spp. pathogenicity in guppies, Poecilia reticulata.

    PubMed

    Leibowitz, M Pimenta; Ofir, R; Golan-Goldhirsh, A; Zilberg, D

    2009-12-03

    Systemic tetrahymenosis caused by the protozoan parasite Tetrahymena spp. is a serious problem in guppy (Poecilia reticulata) farms worldwide. There is no therapeutic solution for the systemic form of this disease. Guppies severely infected with Tetrahymena spp. were imported by a commercial ornamental fish farm and brought to our laboratory. Tetrahymena sp. (Tet-NI) was isolated and in vitro cultured. Isolates maintained in culture for different time periods (as reflected by different numbers of passages in culture) were analyzed-Tet-NI 1, 4, 5 and 6, with Tet-NI 1 being cultured for the longest period (about 15 months, 54 passages) and Tet-NI 6 for the shortest (2.5 months, 10 passages). Controlled internal infection was successfully achieved by IP injection with most isolates, except for Tet-NI 1 which produced no infection. The isolate Tet-NI 6 induced the highest infection rates in internal organs (80% vs. 50% and 64% for Tet-NI 4 and 5, respectively) and mortality rates (67% vs. 20% and 27% for Tet-NI 4 and 5, respectively, and 6.7% for Tet-NI 1). The correlation between pathogenicity and Tetrahymena enzymatic activity was studied. Electrophoretic analyses revealed at least two bands of gelanolytic activity in Tet-NI 4 and 5, three bands in Tet-NI 6, and no activity in Tet-NI 1. Total inhibition of gelanolytic activity was observed after pretreatment of Tet-NI 6 with E-64, a highly selective cysteine protease inhibitor. Using hemoglobin as a substrate, Tet-NI 6 had two bands of proteolytic activity and no bands were observed in Tet-NI 1. A correlation was observed between pathogenicity and acid phosphatase activities (analyzed by commercial fluorescence kit) for Tet-NI 1 and Tet-NI 6.

  3. Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity.

    PubMed

    Hurt, Julie K; Fitzpatrick, Brendan J; Norris-Drouin, Jacqueline; Zylka, Mark J

    2012-01-01

    Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.

  4. FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

    PubMed Central

    Chen, Jia; Yu, Feng; Liu, Ying; Du, Changqing; Li, Xiushan; Zhu, Sirui; Wang, Xianchun; Lan, Wenzhi; Rodriguez, Pedro L.; Liu, Xuanming; Li, Dongping; Chen, Liangbi; Luan, Sheng

    2016-01-01

    Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals. PMID:27566404

  5. Molecular cloning of the type 5, iron-containing, tartrate-resistant acid phosphatase from human placenta.

    PubMed

    Ketcham, C M; Roberts, R M; Simmen, R C; Nick, H S

    1989-01-05

    The type 5, iron-containing, tartrate-resistant acid phosphatase (TR-AP) constitutes a relatively minor intracellular isozyme of acid phosphatase in the human that is immunologically related to uteroferrin, a secreted progesterone-induced protein of the porcine uterus. Here, the purification of small amounts of TR-AP from human placenta is described. When a placental lambda gt11 cDNA library was screened with two short 32P-labeled cDNA clones from within the coding region of uteroferrin, a 1412-base pair cDNA was identified that encodes the entire human TR-AP isozyme. This cDNA contains an open reading frame of 969 base pairs, corresponding to a protein of 323 amino acids. A putative signal sequence of 19 amino acids and two potential glycosylation sites are present. The deduced amino acid sequence of the human TR-AP is 85% identical to that of porcine uteroferrin (whose sequence is also reported here in complete form for the first time) and 82% identical to the corresponding regions of a partial amino acid sequence of a bovine spleen phosphoprotein phosphatase. Northern blotting techniques employing a labeled TR-AP cDNA probe revealed the presence of a 1.5-kilobase transcript in white cells from a patient with hairy cell leukemia, in human K562 erythroleukemic cells, and in Epstein-Barr virus-transformed human B-cells, but not in a human T-cell line. Culture of K562 cells in presence of 10(-8) M phorbol 12-myristate 13-acetate ester for 48-72 h enhanced TR-AP activity per cell about 30-fold and led to a corresponding increase in TR-AP mRNA levels.

  6. Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

    PubMed

    Bellazi, L; Mornet, E; Meurice, G; Pata-Merci, N; De Mazancourt, P; Dieudonné, M-N

    2011-10-01

    During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

  7. Associations between serum bone-specific alkaline phosphatase activity, biochemical parameters, and functional polymorphisms of the tissue-nonspecific alkaline phosphatase gene in a Japanese population.

    PubMed

    Sogabe, Natsuko; Tanabe, Rieko; Haraikawa, Mayu; Maruoka, Yutaka; Orimo, Hideo; Hosoi, Takayuki; Goseki-Sone, Masae

    2013-01-01

    We had demonstrated that single nucleotide polymorphism (787T>C) in the tissue-nonspecific ALP (TNSALP) gene was associated with the bone mineral density (BMD). BMD was the lowest among TNSALP 787T homozygotes (TT-type) and highest among TNSALP 787T>C homozygotes (CC-type) in postmenopausal women. In the present study, we investigated the effects of the TNSALP genotype on associations among serum bonespecific alkaline phosphatase (BAP), serum calcium, and phosphorus in healthy young Japanese subjects. Young healthy adult subjects (n=193) were genotyped for the polymorphism, and we measured the levels of serum BAP, serum calcium, and phosphorus. Dietary nutrient intakes were calculated based on 3-day food records before the day of blood examinations. Grouped by the TNSALP genotype, a significant negative correlation between serum BAP and phosphorus was observed in 787T>C homozygotes (CC-type), but not in heterozygotes (TCtype), nor in 787T homozygotes (TT-type). In the present study, we revealed that the single nucleotide polymorphism 787T>C in the TNSALP gene had effects on the correlation between serum BAP and phosphorus in young adult subjects. These results suggest that variation in TNSALP may be an important determinant of phosphate metabolism. Our data may be useful for planning strategies to prevent osteoporosis.

  8. N-glycosylation sites of plant purple acid phosphatases important for protein expression and secretion in insect cells.

    PubMed

    Olczak, Mariusz; Olczak, Teresa

    2007-05-15

    Analysis of plant purple acid phosphatases (PAPs) showed high conservation and different distribution of N-glycosylation sites. Oligosaccharide structures of Lupinus luteus acid phosphatase (Lu_AP) produced in insect cells were determined. Mutant Lu_AP and Phaseolus vulgaris (Ph_AP) phosphatases lacking possibility of N-glycosylation at highly conserved sites were generated and expressed in insect cells. A role for N-glycosylation in the stability of PAPs was indicated by unsuccessful attempts to secrete Ph_AP and Lu_AP mutants generated by replacing Asn residues of conserved glycosylation sequons by Ser residues either singly or in combination. We showed that Ph_AP belongs to the group of glycoproteins that require occupancy of all highly conserved glycosylation sites for secretion, whereas replacing of the third position of the glycosylation sequon indicated that Lu_AP may tolerate the absence of some N-glycans. However, the N-glycan located at the polypeptide C-terminus was crucial for secretion of both enzymes. PAP specific activity of glycosylation mutants successfully secreted was similar to the wild-type recombinant proteins.

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

    PubMed

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

    2013-10-01

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

  10. Root surface acid phosphatases and their role in phosphorus assimilation by Eriophorum vaginatum

    SciTech Connect

    Kroehler, C.J.; Linkins, A.E.

    1988-01-01

    Eriophorum vaginatum is a dominant plant in much of the arctic tundra ecosystem where phosphorus is frequently a limiting nutrient. The mineralization of this organic phosphorus was thought to be principally controlled by microbial respiration, however, more recent work shows that extracellular soil phosphatases are the principal regulators. The existence of plant root and mycorrhizal surface phosphatases which are capable of hydrolyzing organic phosphorus compounds, suggests that soil organic phosphorus may be directly utilized by plants. Since E. vaginatum is a tussock forming sedge with a very dense annually produced rooting system which can exploit most of the tussock soil volume, its surface phosphatases may play a dominant role in organic phosphorus hydrolysis into inorganic phosphorus. Of equal significance would be the potential for this activity to contribute to the phosphorus nutrition through the coupling of phosphorus hydrolysis on the root and root uptake of the resultant inorganic phosphorus. Phosphatase activity was investigated and found to be uniformly distributed along the surface of the root. Kinetic analysis of the enzyme gave estimates of 9.23 mM for the apparent Km and 1.61 * 10/sup -3/ ..mu..moles mm-2 hr/sup -1/ for the apparent Vmax. Saturation values for E. vaginatum phosphatases are about 3 times higher than average soil solution organic phosphorus concentrations. 12 refs., 4 figs.

  11. Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of ABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha.

    PubMed

    Tougane, Ken; Komatsu, Kenji; Bhyan, Salma Begum; Sakata, Yoichi; Ishizaki, Kimitsune; Yamato, Katsuyuki T; Kohchi, Takayuki; Takezawa, Daisuke

    2010-03-01

    Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the amino-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the beta -glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

  12. A model of the acid sphingomyelinase phosphoesterase domain based on its remote structural homolog purple acid phosphatase.

    PubMed

    Seto, Marian; Whitlow, Marc; McCarrick, Margaret A; Srinivasan, Subha; Zhu, Ying; Pagila, Rene; Mintzer, Robert; Light, David; Johns, Anthony; Meurer-Ogden, Janet A

    2004-12-01

    Sequence profile and fold recognition methods identified mammalian purple acid phosphatase (PAP), a member of a dimetal-containing phosphoesterase (DMP) family, as a remote homolog of human acid sphingomyelinase (ASM). A model of the phosphoesterase domain of ASM was built based on its predicted secondary structure and the metal-coordinating residues of PAP. Due to the low sequence identity between ASM and PAP (approximately 15%), the highest degree of confidence in the model resides in the metal-binding motifs. The ASM model predicts residues Asp 206, Asp 278, Asn 318, His 425, and His 457 to be dimetal coordinating. A putative orientation for the phosphorylcholine head group of the ASM substrate, sphingomyelin (SM), was made based on the predicted catalysis of the phosphorus-oxygen bond in the active site of ASM and on a structural comparison of the PAP-phosphate complex to the C-reactive protein-phosphorylcholine complex. These complexes revealed similar spatial interactions between the metal-coordinating residues, the metals, and the phosphate groups, suggesting a putative orientation for the head group in ASM consistent with the mechanism considerations. A conserved sequence motif in ASM, NX3CX3N, was identified (Asn 381 to Asn 389) and is predicted to interact with the choline amine moiety in SM. The resulting ASM model suggests that the enzyme uses an SN2-type catalytic mechanism to hydrolyze SM, similar to other DMPs. His 319 in ASM is predicted to protonate the ceramide-leaving group in the catalysis of SM. The putative functional roles of several ASM Niemann-Pick missense mutations, located in the predicted phosphoesterase domain, are discussed in context to the model.

  13. Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

    PubMed Central

    Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

    2004-01-01

    An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286]. PMID:14521509

  14. Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

    PubMed

    Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

    2004-01-15

    An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

  15. The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

    PubMed

    Valenti, G; Procino, G; Carmosino, M; Frigeri, A; Mannucci, R; Nicoletti, I; Svelto, M

    2000-06-01

    Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

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

    PubMed

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

    2015-02-01

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

  17. Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

    PubMed Central

    Tasnádi, Gábor; Lukesch, Michael; Zechner, Michaela; Jud, Wolfgang; Hall, Mélanie; Ditrich, Klaus; Baldenius, Kai; Hartog, Aloysius F.; Wever, Ron

    2015-01-01

    Abstract A set of phosphatases was evaluated for their potential to catalyze the regio‐ and stereoselective phosphorylation of alcohols using a high‐energy inorganic phosphate donor, such as di‐, tri‐ and polyphosphate. Parameters such as type and amount of phosphate donor and pH of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester. Diols were monophosphorylated with high selectivities. This biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale (6.86 g) in a flow setup with immobilized phosphatase.

  18. Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

    PubMed

    Oliveira, Danielle M P; Ramos, Isabela B; Reis, Flavia C G; Lima, Ana P C A; Machado, Ednildo A

    2008-05-01

    In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

  19. MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm

    SciTech Connect

    Masuda, Kouhei; Katagiri, Chiaki; Nomura, Miyuki; Sato, Masami; Kakumoto, Kyoko; Akagi, Tsuyoshi; Kikuchi, Kunimi; Tanuma, Nobuhiro; Shima, Hiroshi

    2010-03-05

    MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localized with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.

  20. Direct observation of multiple protonation states in recombinant human purple acid phosphatase.

    PubMed

    Funhoff, Enrico G; de Jongh, Thyra E; Averill, Bruce A

    2005-08-01

    To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5-6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pHpK( a,2)). The pK( a,1) values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme-phosphate complex (which should mimic the enzyme-substrate complex), the enzyme-fluoride complex, and the enzyme-fluoride-phosphate complex (which should mimic the ternary enzyme-substrate-hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.

  1. Androgen metabolism and regulation of rat ventral prostate growth and acid phosphatase during sexual maturation.

    PubMed

    Orlowski, J; Bird, C E; Clark, A F

    1988-01-01

    Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2 +/- 1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P less than 0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5 alpha-dihydrotestosterone (5 alpha-DHT) levels increased from 4.6 +/- 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6 +/- 2.3 pmol/mg DNA at 58 days. Prostatic 5 alpha-DHT concentrations were always higher than testosterone levels. Prostatic 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-Adiol) levels were lower than 5 alpha-DHT levels except on day 58 when levels peaked dramatically at 26.2 +/- 5.5 pmol/mg DNA. Changes in prostatic 5 alpha-DHT and 3 alpha-Adiol levels corresponded with changes in 5 alpha-reductase and 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities. The oxidative reaction of 3 alpha-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5 alpha-DHT. The plasma levels of testosterone, 5 alpha-DHT and 3 alpha-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.

  2. The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

    PubMed

    Del Vecchio, Hernan A; Ying, Sheng; Park, Joonho; Knowles, Vicki L; Kanno, Satomi; Tanoi, Keitaro; She, Yi-Min; Plaxton, William C

    2014-11-01

    Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 μm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  3. DNA polymorphism of alkaline phosphatase isozyme genes: Linkage disequilibria between placental and germ-cell alkaline phosphotase alleles

    SciTech Connect

    Beckman, G.; Beckman, L.; Sikstroem, C. ); Millan, J.L. )

    1992-11-01

    The use of human placental alkaline phosphatase (PLAP) cDNA as a probe allows the detection and identification of restriction DNA fragments derived from three homologous genes, i.e., intestinal alkaline phosphatase (AP), germ-cell AP (GCAP), and PLAP. In previous RFLP studies the authors have reported linkage disequilibria between an RsaI and two PstI (a and b) polymorphic restriction sites and electrophoretic types of PLAP. In this report they present evidence that, in spite of the strong correlation with PLAP types, PstI(b) is an RFLP of GCAP. The data indicate close linkage between the PLAP and GCAP loci. 18 refs., 2 figs., 3 tabs.

  4. Polyphosphate-mediated inhibition of tartrate-resistant acid phosphatase and suppression of bone resorption of osteoclasts.

    PubMed

    Harada, Kana; Itoh, Hiromichi; Kawazoe, Yumi; Miyazaki, Shuichi; Doi, Kazuya; Kubo, Takayasu; Akagawa, Yasumasa; Shiba, Toshikazu

    2013-01-01

    Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α'-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.

  5. A novel class of PTEN protein in Arabidopsis displays unusual phosphoinositide phosphatase activity and efficiently binds phosphatidic acid.

    PubMed

    Pribat, Anne; Sormani, Rodnay; Rousseau-Gueutin, Mathieu; Julkowska, Magdalena M; Testerink, Christa; Joubès, Jerôme; Castroviejo, Michel; Laguerre, Michel; Meyer, Christian; Germain, Véronique; Rothan, Christophe

    2012-01-01

    PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3' phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein-phosphoinositide interactions indicated that substitutions at the PTEN2 core catalytic site of the Lys267 and Gly268 residues found in animals, which are critical for animal PTEN activity, by Met267 and Ala268 found in the eudicot PTEN2 are responsible for changes in substrate specificity. Remarkably, the AtPTEN2a protein also displays strong binding activity for PA (phosphatidic acid), a major lipid second messenger in plants. Promoter::GUS (β-glucuronidase) fusion, transcript and protein analyses further showed the transcriptional regulation of the ubiquitously expressed AtPTEN2a and AtPTEN2b by salt and osmotic stress. The results of the present study suggest a function for this novel class of plant PTEN proteins as an effector of lipid signalling in plants.

  6. Phosphatase and tensin homolog (PTEN) gene mutations and autism: literature review and a case report of a patient with Cowden syndrome, autistic disorder, and epilepsy.

    PubMed

    Conti, Sara; Condò, Maria; Posar, Annio; Mari, Francesca; Resta, Nicoletta; Renieri, Alessandra; Neri, Iria; Patrizi, Annalisa; Parmeggiani, Antonia

    2012-03-01

    Phosphatase and tensin homolog (PTEN) gene mutations are associated with a spectrum of clinical disorders characterized by skin lesions, macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers. Autism has rarely been described in association with these variable clinical features. At present, 24 patients with phosphatase and tensin homolog gene mutation, autism, macrocephaly, and some clinical findings described in phosphatase and tensin homolog syndromes have been reported in the literature. We describe a 14-year-old boy with autistic disorder, focal epilepsy, severe and progressive macrocephaly, and multiple papular skin lesions and palmoplantar punctate keratoses, characteristic of Cowden syndrome. The boy has a de novo phosphatase and tensin homolog gene mutation. Our patient is the first case described to present a typical Cowden syndrome and autism associated with epilepsy.

  7. Lipin-1 phosphatidic phosphatase activity modulates phosphatidate levels to promote peroxisome proliferator-activated receptor γ (PPARγ) gene expression during adipogenesis.

    PubMed

    Zhang, Peixiang; Takeuchi, Kazuharu; Csaki, Lauren S; Reue, Karen

    2012-01-27

    Adipose tissue plays a key role in metabolic homeostasis. Disruption of the Lpin1 gene encoding lipin-1 causes impaired adipose tissue development and function in rodents. Lipin-1 functions as a phosphatidate phosphatase (PAP) enzyme in the glycerol 3-phosphate pathway for triglyceride storage and as a transcriptional coactivator/corepressor for metabolic nuclear receptors. Previous studies established that lipin-1 is required at an early step in adipocyte differentiation for induction of the adipogenic gene transcription program, including the key regulator peroxisome proliferator-activated receptor γ (PPARγ). Here, we investigate the requirement of lipin-1 PAP versus coactivator function in the establishment of Pparg expression during adipocyte differentiation. We demonstrate that PAP activity supplied by lipin-1, lipin-2, or lipin-3, but not lipin-1 coactivator activity, can rescue Pparg gene expression and lipogenesis during adipogenesis in lipin-1-deficient preadipocytes. In adipose tissue from lipin-1-deficient mice, there is an accumulation of phosphatidate species containing a range of medium chain fatty acids and an activation of the MAPK/extracellular signal-related kinase (ERK) signaling pathway. Phosphatidate inhibits differentiation of cultured adipocytes, and this can be rescued by the expression of lipin-1 PAP activity or by inhibition of ERK signaling. These results emphasize the importance of lipid intermediates as choreographers of gene regulation during adipogenesis, and the results highlight a specific role for lipins as determinants of levels of a phosphatidic acid pool that influences Pparg expression.

  8. Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection.

    PubMed

    Hanaoka, Nozomu; Takano, Yukie; Shibuya, Kazutoshi; Fugo, Hajime; Uehara, Yoshimasa; Niimi, Masakazu

    2008-10-01

    Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Delta, yvh1Delta, sit4Delta, and ptc1Delta) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Delta, yvh1Delta, and sit4Delta mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Delta revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.

  9. Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase.

    PubMed

    Medda, Rosaria; Pintus, Francesca; Spanò, Delia; Floris, Giovanni

    2011-01-01

    This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources.

  10. Toxic impact of aldrin on acid and alkaline phosphatase activity of penaeid prawn, Metapenaeus monoceros: In vitro study

    SciTech Connect

    Reddy, M.S.; Jayaprada, P.; Rao, K.V.R. )

    1991-03-01

    The increasing contamination of the aquatic environment by the indiscriminate and widespread use of different kinds of pesticides is a serious problem for environmental biologists. Organochlorine insecticides are more hazardous since they are not only more toxic but also leave residues in nature. The deleterious effects of aldrin on several crustaceans have been studied. But studies concerning the impact of aldrin on biochemical aspects of crustaceans are very much limited. The present study is aimed at probing the in vitro effects of aldrin on the acid and alkaline phosphatase activity levels in selected tissues of penaeid prawn, Metapenaeus monoceros (Fabricius).

  11. Molecular and biochemical characterization of AtPAP15, a purple acid phosphatase with phytase activity, in Arabidopsis.

    PubMed

    Kuang, Ruibin; Chan, Kam-Ho; Yeung, Edward; Lim, Boon Leong

    2009-09-01

    Purple acid phosphatase (PAP) catalyzes the hydrolysis of phosphate monoesters and anhydrides to release phosphate within an acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays a greater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) with phytase activity than the other AtPAPs. In this study, transgenic Arabidopsis that expressed an AtPAP15 promoterbeta-glucuronidase (GUS) fusion protein showed that AtPAP15 expression was developmentally and temporally regulated, with strong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissue specific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified from transgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion lines exhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination rate compared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves in plants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermal cells, it is unlikely to play any role in external phosphorus assimilation.

  12. Toxicology of cupric salts on honeybees. V. Gluconate and sulfate action on gut alkaline and acid phosphatases.

    PubMed

    Bounias, M; Kruk, I; Nectoux, M; Popeskovic, D

    1996-10-01

    Some aspects of putative nontarget effects of cupric ions systemically fed to honeybees against their parasite mite Varroa jacobsoni have been investigated on the host phosphatases. The alkaline and acid forms extracted from the guts of worker bees exhibited substrate-inhibition features. Upon detailed kinetic analysis, cupric organic salts indicate activation effects at concentrations of about 1 mM. Concentrations up to 10 mM (alkaline form) and 25 mM (acid form) induced no important changes, except a partial quenching of the substrate-inhibition process, characterized by a wide increase in the constant of apparent inhibitory binding of substrate to the enzyme-substrate complex. Partial purification gave a single alkaline form with quite similar kinetic behavior in the absence of natural ions as in crude extracts. Cupric gluconate and sulfate demonstrated similar patterns, except an increase of the apparent Hill coefficient by sulfate only. The substrate constant of acid phosphatases was decreased at high cupric gluconate doses while its maximum velocity was biphasically increased (with observed maximum at 1 mM), resulting in a sustained activation. Chemiluminescence studies revealed that cupric ion activation is counteracted by oxygen radicals generated by cupric ions and also, in vitro, by the artificial substrate para-nitrophenylphosphate. The para-nitrophenol molecules released from the reaction are therefore responsible for biphasic effects selectively observed with gluconate salts. In apicultural practice, neither blockade of activity nor dramatic changes are to be expected at doses administered to bees against the parasite.

  13. Copy number alterations of chromosomal regions enclosing protein tyrosine phosphatase receptor-like genes in colorectal cancer.

    PubMed

    Laczmanska, Izabela; Karpinski, Pawel; Kozlowska, Joanna; Bebenek, Marek; Ramsey, David; Sedziak, Tomasz; Ziolkowski, Piotr; Sasiadek, Maria M

    2014-12-01

    Protein tyrosine phosphatases that act in different cellular pathways are described most commonly as tumor suppressors, but also as oncogenes. Their role has previously been described in colorectal cancer, as well as in gastric, breast, thyroid, prostate, ovarian, pancreatic, glioma, liver, leukemia and many other cancers. In a previous study, we have described protein tyrosine phosphatase receptor type T, M, Z1 and Q genes (PTPRT, PTPRM, PTPRZ1 and PTPRQ) hypermethylated in sporadic colorectal cancer. Thus, in this study, we examined the relation of unbalanced chromosomal alterations within regions covering these four protein tyrosine phosphatase genes with this cancer. One hundred and two cancer tissues were molecularly characterized, including analysis of the BRAF and K-ras mutations and methylator phenotype. The analysis of chromosomal aberrations was performed using Comparative Genomic Hybridization. We observed amplification of three regions containing genes coding for PTPs, such as PTPRZ1 (7q31.3, amplified in 23.5% of cases), PTPRQ (12q21.2, amplified in 5.9% of cases), PTPRT (20q12, amplified in 29.4% of cases), along with deletions in the region of PTPRM (18p11.2, deleted in 21.6% of cases). These data may suggest that in sporadic colorectal cancer PTPRZ1, PTPRT, PTPRQ probably act as oncogenes, while PTPRM acts as a tumor suppressor gene. Our study also revealed that gains on chromosome 20q12 and losses on chromosome 18p11.2 are connected with the absence of the BRAF mutation and the conventional adenocarcinoma pathway. Copyright © 2014 Elsevier GmbH. All rights reserved.

  14. A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

    PubMed Central

    Chen, Yupeng; Zhang, Lirong; Estarás, Conchi; Choi, Seung H.; Moreno, Luis; Karn, Jonathan; Moresco, James J.; Yates, John R.

    2014-01-01

    HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P–RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P–S2P transition at the integrated HIV-1 promoter. PMID:25319827

  15. Association between Phosphatase Related Gene Variants and Coronary Artery Disease: Case-Control Study and Meta-Analysis

    PubMed Central

    Han, Xia; Zhang, Lijun; Zhang, Zhiqiang; Zhang, Zengtang; Wang, Jianchun; Yang, Jun; Niu, Jiamin

    2014-01-01

    Recent studies showed that the serum alkaline phosphatase is an independent predictor of the coronary artery disease (CAD). In this work, we aimed to summarize the association between three phosphatase related single nucleotide polymorphisms (rs12526453, rs11066301 and rs3828329) and the risk of CAD in Han Chinese. Our results showed that the rs3828329 of the ACP1 gene was closely related to the risk of CAD in Han Chinese (OR = 1.45, p = 0.0006). This significant association of rs3828329 with CAD was only found in the females (Additive model: OR = 1.80, p = 0.001; dominant model: OR = 1.69, p = 0.03; recessive model: OR = 1.96, p = 0.0008). Moreover, rs3828329 was likely to exert its effect in females aged 65 years and older (OR = 2.27, p = 0.001). Further meta-analyses showed that the rs12526453 of PHACTR11 gene (OR = 1.14, p < 0.0001, random-effect method) and the rs11066301 of PTPN11 gene (OR = 1.15, p < 0.0001, fixed-effects method) were associated with CAD risk in multiple populations. Our results showed that the polymorphisms rs12526453 and rs11066301 are significantly associated with the CAD risk in multiple populations. The rs3828329 of ACP1 gene is also a risk factor of CAD in Han Chinese females aged 65 years and older. PMID:25123136

  16. Purple Acid Phosphatase5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis.

    PubMed

    Ravichandran, Sridhar; Stone, Sophia L; Benkel, Bernhard; Prithiviraj, Balakrishnan

    2013-07-29

    Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants. Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.

  17. Purple Acid Phosphatase5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis

    PubMed Central

    2013-01-01

    Background Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Results In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants. Conclusion Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses. PMID:23890153

  18. Synergistic salubrious effect of ferulic acid and ascorbic acid on membrane-bound phosphatases and lysosomal hydrolases during experimental myocardial infarction in rats.

    PubMed

    Yogeeta, Surinder Kumar; Gnanapragasam, Arunachalam; Senthilkumar, Subramanian; Subhashini, Rajakannu; Devaki, Thiruvengadam

    2006-12-23

    Altered membrane integrity has been suggested as a major factor in the development of cellular injury during myocardial necrosis. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on lysosomal hydrolases and membrane-bound phosphatases during isoproterenol (ISO) induced myocardial necrosis in rats. Induction of rats with 1SO (150 mg/kg b.wt, i.p.) for 2 days resulted in a significant increase in the activities of lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase and cathepsin-D) in the heart and serum. A significant increase in plasma lactate level, cardiac levels of sodium, calcium and a decrease in cardiac level of potassium was also observed, which was paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) in the heart of ISO-administered rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt) and AA (80 mg/kg b.wt) orally for 6 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to individual drug treated groups. The combination of FA and AA preserved the membrane integrity by mitigating the oxidative stress and associated cellular damage more effectively when compared to individual treatment groups. In our study, the protection conferred by FA and AA might be through the nitric oxide pathway and by their ability of quenching free radicals. In conclusion, these findings indicate the synergistic modulation of lysosomal hydrolases and membrane phosphatases by the combination of FA and AA.

  19. Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A.

    PubMed

    Saino, Tomoyuki; Watson, Eileen L

    2009-01-01

    Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.

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

    USGS Publications Warehouse

    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.

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

    USGS Publications Warehouse

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

    2003-01-01

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

  2. Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT-isooctane-water reverse micelles.

    PubMed

    Luchter-Wasylewska, Ewa; Iciek, Małgorzata

    2004-05-15

    The kinetics of 1-naphthyl phosphate and phenyl phosphate hydrolysis, catalyzed by human prostatic acid phosphatase (PAP) entrapped in AOT-isooctane-water reverse micelles, has been studied over surfactant hydration degree (w0) range 5 to 35. Continuous spectrophotometric acid phosphatase assays, previously prepared, were employed. PAP was catalytically active over the whole w0 studied range. In order to determine steady-state reaction constants the experimental data were fitted to Hill rate equation. Positive cooperativity in substrate binding was observed, as it was earlier found in aqueous solutions. The extent of cooperativity (expressed as the value of the Hill cooperation coefficient h) increased from 1 to 4, when the micellar water-pool size was growing, at fixed enzyme concentration. In the plots of catalytic activity (kcat) versus w0, the maxima have been found at w0=10 (pH 5.6) and 23 (pH 3.8). It is suggested that catalytically active monomeric and dimeric PAP forms are entrapped in reverse micelles of w0=10 and 23, respectively.

  3. Identification of Purple Acid Phosphatases in Chickpea and Potential Roles of CaPAP7 in Seed Phytate Accumulation.

    PubMed

    Bhadouria, Jyoti; Singh, Ajit Pal; Mehra, Poonam; Verma, Lokesh; Srivastawa, Rishi; Parida, Swarup K; Giri, Jitender

    2017-09-08

    Purple acid phosphatases (PAPs) play important roles in phosphate (Pi) acquisition and utilization. These PAPs hydrolyze organic Phosphorus (P) containing compounds in rhizosphere as well as inside the plant cell. However, roles of PAPs in one of the most widely cultivated legumes, chickpea (Cicer arietnum L.), have not been unraveled so far. In the present study, we identified 25 putative PAPs in chickpea (CaPAPs) which possess functional PAP motifs and domains. Differential regulation of CaPAPs under different nutrient deficiencies revealed their roles under multiple nutrient stresses including Pi deficiency. Interestingly, most of the CaPAPs were prominently expressed in flowers and young pods indicating their roles in flower and seed development. Association mapping of SNPs underlying CaPAPs with seed traits revealed significant association of low Pi inducible CaPAP7 with seed weight and phytate content. Biochemical characterization of recombinant CaPAP7 established it to be a functional acid phosphatase with highest activity on most abundant organic-P substrate, phytate. Exogenous application of recombinant CaPAP7 enhanced biomass and Pi content of Arabidopsis seedlings supplemented with phytate as sole P source. Taken together, our results uncover the PAPs in chickpea and potential roles of CaPAP7 in seed phytate accumulation.

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

    PubMed Central

    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. The acid phosphatase test two minute cut-off: an insufficient time to detect some semen stains.

    PubMed

    Redhead, Paul; Brown, Melanie K

    2013-06-01

    The ability to detect semen in sexual offence cases is a crucial first step to locating stains which may be suitable for DNA profiling. Since the development of the acid phosphatase test in the late 1950s by Stuart S. Kind, the process undertaken to perform the test has gone largely unchanged. The method currently accepted by operational forensic science laboratories allows 2 min for a reaction to be obtained, and until relatively recently, this has not been challenged. In this research, samples of semen were obtained from three donors and a range of dilutions for each sample were prepared. Each dilution was subjected to acid phosphatase testing using both direct testing and the 'press test' method. The results showed that semen could be detected in excess of 15 min in dilutions up to 1 in 400 using the press test method and in dilutions up to 1 in 1000 using the direct method. Of further significance was the observation that using the press test method, the two minute cut-off was insufficient to detect the majority of stains and in some cases, semen stains as strong as 1 in 20 dilutions. This research provides compelling evidence for protocols currently utilised in forensic practice to be reviewed in order that forensic scientists do not overlook potential evidential material that may prove suitable for body fluid identification such as DNA STR profiling.

  6. Chromosomal mapping of the gene (INPP5A) encoding the 43-kDa membrane-associated inositol polyphosphate 5-phosphatase to 10q26.3 by fluorescence in situ hybridization

    SciTech Connect

    Mitchell, C.A.; Speed, C.J.; Nicholl, J.; Sutherland, G.R.

    1996-01-01

    This report discusses the localization of a membrane-associated inositol polyphosphate 5-phosphatase gene to human chromosome 10q26.3 using fluorescence in situ hybridization. This 43-kDa 5-phosphatase does not map to the same location as any other 5-phosphatase enzymes. 13 refs., 1 fig.

  7. Structural basis for the divergence of substrate specificity and biological function within HAD phosphatases in lipopolysaccharide and sialic acid biosynthesis.

    PubMed

    Daughtry, Kelly D; Huang, Hua; Malashkevich, Vladimir; Patskovsky, Yury; Liu, Weifeng; Ramagopal, Udupi; Sauder, J Michael; Burley, Stephen K; Almo, Steven C; Dunaway-Mariano, Debra; Allen, Karen N

    2013-08-13

    The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover

  8. Variation of Photosynthesis, Fatty Acid Composition, ATPase and Acid Phosphatase Activities, and Anatomical Structure of Two Tea (Camellia sinensis (L.) O. Kuntze) Cultivars in Response to Fluoride

    PubMed Central

    Wang, L. X.; Tang, J. H.; Xiao, B.; Yang, Y. J.; Liu, J.

    2013-01-01

    The changes of photosynthetic parameters, water use efficiency (WUE), fatty acid composition, chlorophyll (Chl) content, malondialdehyde (MDA) content, ATPase and acid phosphatase activities, fluoride (F) content, and leaf anatomical structure of two tea cultivars, “Pingyangtezao” (PY) and “Fudingdabai” (FD), after F treatments were investigated. The results show that net photosynthetic rate (P n), stomatal conductance (g s), and transpiration rate (E) significantly decreased in both cultivars after 0.3 mM F treatment, but FD had higher P n, g s, and WUE and lower E than PY. Chl content in PY significantly decreased after 0.2 and 0.3 mM F treatments, while no significant changes were observed in FD. The proportions of shorter chain and saturated fatty acids increased and those of longer chain and unsaturated fatty acids decreased in both cultivars under F treatments. The contents of MDA increased after F treatments but were higher in PY than in FD. In addition, F treatments decreased the activities of ATPase and acid phosphatase and increased F content in both cultivars; however, compared with PY, FD showed higher enzymatic activities and lower F content in roots and leaves. Leaf anatomical structure in FD indicated that cells in leaf midrib region were less injured by F than in PY. PMID:24023526

  9. X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)

    NASA Astrophysics Data System (ADS)

    Ahlers, F.; Zippel, F.; Klabunde, T.; Krebs, B.; Löcke, R.; Witzel, H.; Nolting, H.-F.

    1995-02-01

    Purple acid phosphatase from red kidney beans (KBP) catalyzes the hydrolysis of activated phosphoric acid monoesters and contains a heterodinuclear Fe(III)Zn(II) core in its active site. Iron K-edge X-ray absorption data have been obtained for the native enzyme and for a metal exchanged derivative, where Zn(II) was substituted by Fe(III). The environment of the native enzyme consists of 2.5 O/N at 1.91 Å, 3 O/N at 2.09 Å, and 1 Zn at 4.05 Å. For the metal exchanged form we obtained 2.5 O/N at 1.94 Å, 2.5 O/N at 2.09 Å, and 1 Fe at 3.79 Å.

  10. A novel cinnamic acid derivative that inhibits Cdc25 dual-specificity phosphatase activity.

    PubMed

    Aoyagi, Yoshimi; Masuko, Norio; Ohkubo, Shuichi; Kitade, Makoto; Nagai, Kentaro; Okazaki, Shinji; Wierzba, Konstanty; Terada, Tadafumi; Sugimoto, Yoshikazu; Yamada, Yuji

    2005-09-01

    The Cdc25 dual-specificity phosphatases are key regulators of cell cycle progression through activation of cyclin-dependent kinases (Cdk). Three homologs exist in humans: Cdc25A, Cdc25B, and Cdc25C. Cdc25A and Cdc25B have oncogenic properties and are overexpressed in some types of tumors. Compounds that inhibit Cdc25 dual-specificity phosphatase activity might thus be potent anticancer agents. We screened several hundred compounds in a library using an in vitro phosphatase assay, with colorimetric measurement of the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol by the catalytic domain of recombinant human Cdc25, and discovered TPY-835, which inhibits Cdc25A and Cdc25B activity (IC50 = 5.1 and 5.7 microM, respectively). TPY-835 had mixed inhibition kinetics for Cdc25A and Cdc25B. TPY-835 caused cell cycle arrest in the G1 phase in human lung cancer cells (A549 and SBC-5) but not cell cycle arrest in the G2/M phase. After treatment with TPY-835, the activation of Cdk2 was suppressed and phosphorylation of the retinoblastoma (Rb) protein was decreased in SBC-5 cells. In addition, TPY-835 induced an increase of the sub-G1 phase cell population after 48-72 h treatment. The growth inhibitory effects of TPY-835 against cisplatin (CDDP)-, camptothecin- and 5-FU-resistant cell lines are comparable to the growth inhibitory effect on their parental lines, thus indicating that TPY-835 did not show cross-resistance to these cell lines. These results suggest that TPY-835 is a promising candidate for constructing a novel class of antitumor agents that can control the cell cycle progression of cancer cells.

  11. The purple acid phosphatase GmPAP21 enhances internal phosphorus utilization and possibly plays a role in symbiosis with rhizobia in soybean.

    PubMed

    Li, Chengchen; Li, Caifeng; Zhang, Haiyan; Liao, Hong; Wang, Xiurong

    2017-02-01

    Induction of secreted and intracellular purple acid phosphatases (PAPs; EC 3.1.3.2) is widely recognized as an adaptation of plants to phosphorus (P) deficiency. The secretion of PAPs plays important roles in P acquisition. However, little is known about the functions of intracellular PAP in plants and nodules. In this study, we identified a novel PAP gene GmPAP21 in soybean. Expression of GmPAP21 was induced by P limitation in nodules, roots and old leaves, and increased in roots with increasing duration of P starvation. Furthermore, the induction of GmPAP21 in nodules and roots was more intensive than in leaves in both P-efficient genotype HN89 and P-inefficient genotype HN112 in response to P starvation, and the relative expression in the leaves and nodules of HN89 was significantly greater than that of HN112 after P deficiency treatment. Further functional analyses showed that over-expressing GmPAP21 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under P starvation condition, indicating that GmPAP21 plays an important role in P utilization. Moreover, GUS expression driven by GmPAP21 promoter was shown in the nodules besides roots. Overexpression of GmPAP21 in transgenic soybean significantly inhibited nodule growth, and thereby affected plant growth after inoculation with rhizobia. This suggests that GmPAP21 is also possibly involved in regulating P metabolism in nodules. Taken together, our results suggest that GmPAP21 is a novel plant PAP that functions in the adaptation of soybean to P starvation, possibly through its involvement in P recycling in plants and P metabolism in nodules. © 2016 Scandinavian Plant Physiology Society.

  12. Improvement in phosphate acquisition and utilization by a secretory purple acid phosphatase (OsPAP21b) in rice.

    PubMed

    Mehra, Poonam; Pandey, Bipin Kumar; Giri, Jitender

    2017-08-01

    Phosphate (Pi) deficiency in soil system is a limiting factor for rice growth and yield. Majority of the soil phosphorus (P) is organic in nature, not readily available for root uptake. Low Pi-inducible purple acid phosphatases (PAPs) are hypothesized to enhance the availability of Pi in soil and cellular system. However, information on molecular and physiological roles of rice PAPs is very limited. Here, we demonstrate the role of a novel rice PAP, OsPAP21b in improving plant utilization of organic-P. OsPAP21b was found to be under the transcriptional control of OsPHR2 and strictly regulated by plant Pi status at both transcript and protein levels. Biochemically, OsPAP21b showed hydrolysis of several organophosphates at acidic pH and possessed sufficient thermostability befitting for high-temperature rice ecosystems with acidic soils. Interestingly, OsPAP21b was revealed to be a secretory PAP and encodes a distinguishable major APase (acid phosphatase) isoform under low Pi in roots. Further, OsPAP21b-overexpressing transgenics showed increased biomass, APase activity and P content in both hydroponics supplemented with organic-P sources and soil containing organic manure as sole P source. Additionally, overexpression lines depicted increased root length, biomass and lateral roots under low Pi while RNAi lines showed reduced root length and biomass as compared to WT. In the light of these evidences, present study strongly proposes OsPAP21b as a useful candidate for improving Pi acquisition and utilization in rice. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

    PubMed

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

    2005-11-01

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

  14. Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins.

    PubMed

    Smienk, Henry G F; Calvo, Dolores; Razquin, Pedro; Domínguez, Elena; Mata, Luis

    2012-05-01

    A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.

  15. Enhanced degradation of five organophosphorus pesticides in skimmed milk by lactic acid bacteria and its potential relationship with phosphatase production.

    PubMed

    Zhang, Ying-Hua; Xu, Di; Liu, Jia-Qi; Zhao, Xin-Huai

    2014-12-01

    Skimmed milk spiked with five organophosphorus pesticides (OPPs), chlorpyrifos, diazinon, fenitrothion, malathion and methyl parathion, was fermented by ten lactic acid bacteria (LAB) and four strain combinations at 42°C for 24h. OPPs left in the samples at different times were extracted, purified, detected by gas chromatography and calculated for degradation rate constants, based on a first-order reaction model. OPPs degradation was enhanced by the inoculated LAB, resulting in 0.8-225.4% increase in the rate constants. Diazinon and methyl parathion were more stable whereas chlorpyrifos, fenitrothion and malathion were more labile. Lactobacillus brevis 1.0209 showed the strongest acceleration on OPPs degradation while strain combination could bring about a synergy between the strains of lower ability. Phosphatase production of the strains might be one of the key factors responsible for the enhanced OPPs degradation, as the detected phosphatase activities were positively correlated to the measured degradation rate constants of OPPs (r=0.636-0.970, P<0.05).

  16. Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

    PubMed

    Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

    2002-12-01

    Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively. SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

  17. Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase.

    PubMed

    Schenk, Gerhard; Gahan, Lawrence R; Carrington, Lyle E; Mitic, Natasa; Valizadeh, Mohsen; Hamilton, Susan E; de Jersey, John; Guddat, Luke W

    2005-01-11

    Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K(m)) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-A resolution (final R(free) value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to lambda protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

  18. TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco.

    PubMed

    Hu, Wei; Yan, Yan; Hou, Xiaowan; He, Yanzhen; Wei, Yunxie; Yang, Guangxiao; He, Guangyuan; Peng, Ming

    2015-01-01

    Group A protein phosphatases 2Cs (PP2Cs) are essential components of abscisic acid (ABA) signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA) and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS) accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process.

  19. TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco

    PubMed Central

    Hu, Wei; Yan, Yan; Hou, Xiaowan; He, Yanzhen; Wei, Yunxie; Yang, Guangxiao; He, Guangyuan; Peng, Ming

    2015-01-01

    Group A protein phosphatases 2Cs (PP2Cs) are essential components of abscisic acid (ABA) signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA) and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS) accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process. PMID:26057628

  20. Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian in segregation and localization of mutation site in the gene

    SciTech Connect

    Tsavaler, L.; Penhallow, R.C.; Sussman, H.H. )

    1988-10-01

    The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site.

  1. The relationship between the acid and alkaline phosphatase activity and the adherence of clinical isolates of Candida parapsilosis to human buccal epithelial cells.

    PubMed

    Fernanado, P H; Panagoda, G J; Samaranayake, L P

    1999-11-01

    Candida parapsilosis is an emerging fungal pathogen implicated in many diseases, especially in compromised hosts. Candidal colonization and infection depends on the initial ability to adhere to host surfaces, which in turn depends upon the cell wall components and the allied structures of both the host and the fungus. Examination of a miscellaneous collection of 24 C. parapsilosis isolates, from both superficial and deep infections, for their potential pathogenic traits displayed a relationship between the phosphatase activity measured with p-nitrophenol phosphate and adhesion of the yeasts to human buccal epithelial cells (BECs). Significant intraspecies differences were seen in both the alkaline and acid phosphatase activity as well as in their adhesion to BECs (p<0.0001). The acid phosphatase activity of the superficial isolates was significantly greater (152%) than that of the systemic isolates (p = 0.0352). A highly significant positive correlation was also established between the yeast adhesion to BECs and both the acid (r = 0.88, p<0.0001) and alkaline (r = 0.9, p<0.0001) phosphatase activity. These relationships, described here for the first time, imply that phosphatases of Candida species may play a crucial role in potentiating their virulence.

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

    SciTech Connect

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

    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.

  3. Over-expression of a Zea mays L. protein phosphatase 2C gene (ZmPP2C) in Arabidopsis thaliana decreases tolerance to salt and drought.

    PubMed

    Liu, Lixia; Hu, Xiaoli; Song, Jian; Zong, Xiaojuan; Li, Dapeng; Li, Dequan

    2009-03-15

    ZmPP2C (AY621066) is a protein phosphatase type-2c previously isolated from roots of Zea mays (LD9002). In this study, constitutive expression of ZmPP2C in Arabidopsis thaliana under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter decreased plant tolerance to salt and drought during seed germination and vegetative growth. When growing on media with NaCl or mannitol, the ZmPP2C-overexpressed plants displayed more severe damages, with weaker growth phenotypes corresponding to a series of physiological changes: lower net photosynthesis rate (Pn) and free proline content, higher malondialdehyde (MDA) level, higher relative membrane permeability (RMP), and water loss. Under these stress conditions, they also showed decreased transcription of the stress-related genes RD29A, RD29B, P5CS1, and P5CS2, and ABA-related genes ABI1 and ABI2. Further, the transgenic plants became less sensitive to abscisic acid (ABA). ZmPP2C over-expression significantly attenuated ABA inhibition on seed germination and root growth of the transgenic plants. These results demonstrate that ZmPP2C is involved in plant stress signal transduction, and ZmPP2C gene over-expression in Arabidopsis thaliana may be exploited to study its potential roles in stress-induced signaling pathway.

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

    PubMed

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

    2004-08-27

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

  5. Tartrate-resistant acid phosphatase facilitates hydroxyl radical formation and colocalizes with phagocytosed Staphylococcus aureus in alveolar macrophages.

    PubMed

    Räisänen, S R; Halleen, J; Parikka, V; Väänänen, H K

    2001-10-19

    Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed specifically in osteoclasts and activated macrophages, two phagocytosing cell types originating from the same hematopoietic stem cells. TRAP contains a binuclear iron centre which has been shown to generate reactive oxygen species (ROS). In this study murine macrophage like cell line RAW-264 overexpressing TRAP was shown to produce elevated levels of hydroxyl radicals compared to parental cells. TRAP transfected cells also had reduced growth rate indicating harmful effects of excessive intracellular ROS levels. Using TRAP specific antibody TRAP protein was shown in alveolar macrophages partially colocalize with late endosomal/lysosomal markers Rab7, Lamp 1 and MHC II molecules that bind antigenic peptides. TRAP also colocalized into compartments where Staphylococcus aureus were phagocytosed. These results suggest that TRAP may have an important biological function in the defence mechanism of macrophages by generating intracellular ROS which would be targeted to destroy phagocytosed foreign material. Copyright 2001 Academic Press.

  6. Macrophages overexpressing tartrate-resistant acid phosphatase show altered profile of free radical production and enhanced capacity of bacterial killing.

    PubMed

    Räisänen, Seija R; Alatalo, Sari L; Ylipahkala, Hannele; Halleen, Jussi M; Cassady, A Ian; Hume, David A; Väänänen, H Kalervo

    2005-05-27

    Activated macrophages and osteoclasts express high amounts of tartrate-resistant acid phosphatase (TRACP, acp5). TRACP has a binuclear iron center with a redox-active iron that has been shown to catalyze the formation of reactive oxygen species (ROS) by Fenton's reaction. Previous studies suggest that ROS generated by TRACP may participate in degradation of endocytosed bone matrix products in resorbing osteoclasts and degradation of foreign compounds during antigen presentation in activated macrophages. Here we have compared free radical production in macrophages of TRACP overexpressing (TRACP+) and wild-type (WT) mice. TRACP overexpression increased both ROS levels and superoxide production. Nitric oxide production was increased in activated macrophages of WT mice, but not in TRACP+ mice. Macrophages from TRACP+ mice showed increased capacity of bacterial killing. Recombinant TRACP enzyme was capable of bacterial killing in the presence of hydrogen peroxide. These results suggest that TRACP has an important biological function in immune defense system.

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

    PubMed

    Kubicz, A; Szalewicz, A; Chrambach, A

    1991-01-01

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

  8. Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase

    PubMed Central

    Medda, Rosaria; Pintus, Francesca; Spanò, Delia; Floris, Giovanni

    2011-01-01

    This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources. PMID:22013530

  9. Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.

    PubMed Central

    Vihko, P; Kurkela, R; Porvari, K; Herrala, A; Lindfors, A; Lindqvist, Y; Schneider, G

    1993-01-01

    Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A. Images PMID:8430088

  10. Identification of alkaline phosphatase genes for utilizing a flame retardant, tris(2-chloroethyl) phosphate, in Sphingobium sp. strain TCM1.

    PubMed

    Takahashi, Shouji; Katanuma, Hiroshi; Abe, Katsumasa; Kera, Yoshio

    2017-03-01

    Tris(2-chloroethyl) phosphate (TCEP) is a haloalkyl phosphate flame retardant and plasticizer that has been recognized as a global environmental contaminant. Sphingobium sp. strain TCM1 can utilize TCEP as a phosphorus source. To identify the phosphomonoesterase involved in TCEP utilization, we identified four putative alkaline phosphatase (APase) genes, named SbphoA, SbphoD1, SbphoD2, and SbphoX-II, in the genome sequence. Following expression of these genes in Escherichia coli, APase activity was confirmed for the SbphoA and SbphoX-II gene products but was not clearly observed for the SbphoD1 and SbphoD2 gene products, owing to their accumulation in inclusion bodies. The single deletion of either SbphoA or SbphoX-II retarded the growth and reduced the APase activity of strain TCM1 cells on medium containing TCEP as the sole phosphorus source; these changes were more marked in cells with the SbphoX-II gene deletion. In contrast, the deletion of either SbphoD1 or SbphoD2 had no effect on cell growth or APase activity. The double deletion of SbphoA and SbphoX-II resulted in the complete loss of cell growth on TCEP. These results show that SbPhoA and SbPhoX-II are involved in the utilization of TCEP as a phosphorus source and that SbPhoX-II is the major phosphomonoesterase involved in TCEP utilization.

  11. Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture.

    PubMed

    Kang, Yang J; Kim, Deuk-Su; Myung, Soon-Chul; Ko, Kisung

    2017-01-01

    In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

  12. Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture

    PubMed Central

    Kang, Yang J.; Kim, Deuk-Su; Myung, Soon-Chul; Ko, Kisung

    2017-01-01

    In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins. PMID:28293250

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

    PubMed

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

    1989-01-01

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

  14. Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants.

    PubMed

    Truong, Ngoc Thanh; Naseri, Joseph Itor; Vogel, Andreas; Rompel, Annette; Krebs, B

    2005-08-01

    Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.

  15. Isolation of a gene from Burkholderia cepacia IS-16 encoding a protein that facilitates phosphatase activity.

    PubMed

    Rodríguez, H; Rossolini, G M; Gonzalez, T; Li, J; Glick, B R

    2000-06-01

    A genomic library from Burkholderia cepacia IS-16 was constructed in Escherichia coli by partial Sau3AI digestion of the chromosomal DNA, with the plasmid vector Bluescript SK. This library was screened for clones able to grow as green stained colonies on selective medium developed