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Sample records for acid phosphatase acp

  1. Mannose 6 Dephosphorylation of Lysosomal Proteins Mediated by Acid Phosphatases Acp2 and Acp5

    PubMed Central

    Makrypidi, Georgia; Damme, Markus; Müller-Loennies, Sven; Trusch, Maria; Schmidt, Bernhard; Schlüter, Hartmut; Heeren, Joerg; Lübke, Torben; Saftig, Paul

    2012-01-01

    Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5−/−) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5−/− mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. PMID:22158965

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

    PubMed

    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

  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. A colorimetric and near-infrared fluorescent probe with high sensitivity and selectivity for acid phosphatase and inhibitor screening.

    PubMed

    Xu, Yongqian; Li, Benhao; Xiao, Liangliang; Ouyang, Jia; Sun, Shiguo; Pang, Yi

    2014-08-14

    A dual-channel including a colorimetric and fluorescent probe based on the aggregation-caused quenching (ACQ) and enzymolysis approach has been presented to screen acid phosphatase (ACP) and its inhibitor. Moreover, the ACP activity was determined by real time assay. PMID:24957006

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

    PubMed

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

    2012-01-01

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

  7. 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

  8. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  9. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. PMID:27540631

  10. Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases.

    PubMed

    Zhang, Xiujun; Li, Mai; Agrawal, Arpita; San, Ka-Yiu

    2011-11-01

    Microbial biosynthesis of fatty acid-like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Free fatty acids can be produced by introducing an acyl-acyl carrier protein thioesterase gene into Escherichia coli. The presence of the acyl-ACP thioesterase will break the fatty acid elongation cycle and release free fatty acid. Depending on their sequence similarity and substrate specificity, class FatA thioesterase is active on unsaturated acyl-ACPs and class FatB prefers saturated acyl group. Different acyl-ACP thioesterases have different degrees of chain length specificity. Although some of these enzymes have been characterized from a number of sources, information on their ability to produce free fatty acid in microbial cells has not been extensively examined until recently. In this study, we examined the effect of the overexpression of acyl-ACP thioesterase genes from Diploknema butyracea, Gossypium hirsutum, Ricinus communis and Jatropha curcas on free fatty acid production. In particular, we are interested in studying the effect of different acyl-ACP thioesterase on the quantities and compositions of free fatty acid produced by an E. coli strain ML103 carrying these constructs. It is shown that the accumulation of free fatty acid depends on the acyl-ACP thioesterase used. The strain carrying the acyl-ACP thioesterase gene from D. butyracea produced approximately 0.2g/L of free fatty acid while the strains carrying the acyl-ACP thioesterase genes from R. communis and J. curcas produced the most free fatty acid at a high level of more than 2.0 g/L at 48 h. These two strains accumulated three major straight chain free fatty acids, C14, C16:1 and C16 at levels about 40%, 35% and 20%, respectively. PMID:22001432

  11. 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. PMID:18334003

  12. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  13. Near-infrared fluorescence probe for the determination of acid phosphatase and imaging of prostate cancer cells.

    PubMed

    Lin, Zihan; Liu, Ziping; Zhang, Hao; Su, Xingguang

    2015-03-01

    In this paper, we developed a near-infrared mercaptopropionic acid (MPA)-capped CuInS2 quantum dot (QD) fluorescence probe for the detection of acid phosphatases (ACP), which is an important biomarker and indicator of prostate cancer. The fluorescence of CuInS2 QDs could be quenched by Cu(2+), and then the addition of adenosine-5'-triphosphate (ATP) could effectively turn on the quenched fluorescence due to the strong interaction between Cu(2+) and ATP. The ACP could catalyze the hydrolysis of ATP, which would disassemble the complex of Cu(2+)-ATP. Therefore, the recovered fluorescence could be quenched again by the addition of ACP. In our method, the limit of detection (LOD) is considerably low for ACP detection in solution. Using the CuInS2 QDs fluorescence probe, we successfully performed in vitro imaging of human prostate cancer cells. PMID:25632410

  14. 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. PMID:25996812

  15. Acid and Alkaline Phosphatase Levels in GCF during Orthodontic Tooth Movement

    PubMed Central

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

    2015-01-01

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

  16. Stacking of a stearoyl-ACP thioesterase with a dual-silenced palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean.

    PubMed

    Park, Hyunwoo; Graef, George; Xu, Yixiang; Tenopir, Patrick; Clemente, Tom E

    2014-10-01

    Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end-use applications. However, the hydrogenation process leads to the formation of trans-fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl-ACP thioesterase into soybean and the subsequent stacking with an event that is dual-silenced in palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase expression in a seed-specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl-ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%-19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%-40%, while the stearic level remained elevated. PMID:24909647

  17. Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

    PubMed

    Kremer, Laurent; Dover, Lynn G; Carrère, Séverine; Nampoothiri, K Madhavan; Lesjean, Sarah; Brown, Alistair K; Brennan, Patrick J; Minnikin, David E; Locht, Camille; Besra, Gurdyal S

    2002-06-01

    Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA. PMID:12023885

  18. 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. PMID:27209386

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

    PubMed

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

    2009-03-01

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

  20. Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

    PubMed

    Rodríguez-Rodríguez, Manuel Fernando; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2014-11-01

    Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results. PMID:25212866

  1. TbGT8 is a bifunctional glycosyltransferase that elaborates N-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in Trypanosoma brucei.

    PubMed

    Nakanishi, Masayuki; Karasudani, Moe; Shiraishi, Takahiro; Hashida, Kazunori; Hino, Mami; Ferguson, Michael A J; Nomoto, Hiroshi

    2014-06-01

    The procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110 kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively. PMID:24508870

  2. Acid phosphatase deactivation by a series mechanism.

    PubMed

    Gianfreda, L; Marrucci, G; Grizzuti, N; Greco, G

    1984-05-01

    Acid phosphatase (E.C.3.1.3.2.) thermal deactivation at pH 3.77 has been investigated by monitoring the enzyme activity as a function of time in the hydrolysis of p-nitrophenyl phosphate. The experimental curves obtained show a two-slope behavior in a log (activity)versus-time plot, which indicates that deactivation occurs via a complex mechanism. From the dependence of the kinetic parameters on both deactivation and hydrolysis temperatures, it is inferred that the deactivation mechanism involves intermediate, temperature-dependent, less-active forms of the enzyme. This interpretation is confirmed by the results of additional tests in which the temperature was suddenly changed during the deactivation process. PMID:18553349

  3. Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis.

    PubMed Central

    Shen, Z; Byers, D M

    1996-01-01

    We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II. PMID:8550484

  4. Unique structural features of red kidney bean purple acid phosphatase.

    PubMed

    Cashikar, A G; Rao, M N

    1995-06-01

    Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes. PMID:7590853

  5. Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

    PubMed Central

    Jiang, Yanfang; Morgan-Kiss, Rachael M.; Campbell, John W.; Chan, Chi Ho; Cronan, John E.

    2010-01-01

    Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously-supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function. PMID:20028080

  6. 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.

  7. Reversible Fluorescent Nanoswitch Based on Carbon Quantum Dots Nanoassembly for Real-Time Acid Phosphatase Activity Monitoring.

    PubMed

    Qian, Zhaosheng; Chai, Lujing; Zhou, Qian; Huang, Yuanyuan; Tang, Cong; Chen, Jianrong; Feng, Hui

    2015-07-21

    A reversible fluorescence nanoswitch by integrating carbon quantum dots nanoassembly and pyrophosphate ion is developed, and a reliable real-time fluorescent assay for acid phosphatase (ACP) activity is established on the basis of the fluorescence nanoswitch. Carbon quantum dots (CQDs) abundant in carboxyl groups on the surface, nickel(II) ion and pyrophosphate ion comprise the fluorescent nanoswitch, which operates in the following way: the nanoassembly consisting of CQDs and nickel ions can be triggered by pyrophosphate ion serving as an external stimulus. At the same time, the fluorescence nanoswitch switches between two fluorescence states (OFF and ON) accompanying shifts in their physical states aggregation and disaggregation. Based on the nanoswitch, the introduction of ACP leads to breakdown of pyrophosphate ions into phosphate ions and resultant fluorescence quenching due to catalytic hydrolysis of ACP toward pyrophosphate ions (PPi). Quantitative evaluation of ACP activity in a broad range from 18.2 U/L to 1300 U/L, with a detection limit of 5.5 U/L, can be achieved in this way, which endows the assay with sufficiently high sensitivity for practical detection in human serum and seminal plasma. PMID:26115095

  8. Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

    PubMed

    De Marchis, Francesca; Valeri, Maria Cristina; Pompa, Andrea; Bouveret, Emmanuelle; Alagna, Fiammetta; Grisan, Simone; Stanzione, Vitale; Mariotti, Roberto; Cultrera, Nicolò; Baldoni, Luciana; Bellucci, Michele

    2016-02-01

    Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3. PMID:26560313

  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. 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. PMID:24722417

  11. 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…

  12. Enhanced free fatty acid production by codon-optimized Lactococcus lactis acyl-ACP thioesterase gene expression in Escherichia coli using crude glycerol.

    PubMed

    Lee, Sunhee; Park, Soohyun; Park, Chulhwan; Pack, Seung Pil; Lee, Jinwon

    2014-12-01

    Fatty acid production and composition are determined by the type of acyl-acyl carrier protein thioesterases (acyl-ACP TEs) expressed in Escherichia coli. Bacterial acyl-ACP TEs from Lactococcus lactis (SGJS47), Enterococcus faecalis (SGJS49), and Burkholderia cepacia (SGJS50) were codon-optimized and expressed in E. coli for enhanced fatty acid production. Samples were extracted at the lag, log, and stationary phases of cell growth, and gene expression levels of the codon optimized acy-ACP TEs as well as fatty acid production were monitored. At 24h after initiation of gene expression, the OPLlTE expression level and fatty acid production in SGJS47 increased up to 15.8-fold and 3.2-fold compared to the control and other recombinant strains, respectively. Additionally, in SGJS47, improvement in free fatty acid (FFA) composition, high-specificity production of short-chain fatty acids (C8, C10) and unsaturated fatty acids (C16:1) was achieved in crude glycerol medium condition. Compared with control strain, the percentage of FFAs (C8 and C10) was enhanced by approximately 16- to 21-fold, C16:1 FFA ratio increased approximately 18-fold. Observation of codon-optimized acyl-ACP TE genes expression level in E. coli may be useful for understanding mechanisms towards improving fatty acid production. Engineered strains have the potential to overproduce specific FFAs and thereby reduce the cost of fatty acid production by using industrially inexpensive carbon sources. PMID:25442943

  13. Leishmanial phosphatase hydrolyzes phosphoproteins and inositol phosphates

    SciTech Connect

    Saha, A.K.; Das, S.; Glew, R.H.

    1986-05-01

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

  14. Campylobacter jejuni fatty acid synthase II: Structural and functional analysis of [beta]-hydroxyacyl-ACP dehydratase (FabZ)

    SciTech Connect

    Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2009-08-14

    Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide a framework for investigation on roles of FAS II in C. jejuni virulence

  15. 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.

  16. Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons.

    PubMed

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

    2002-03-01

    The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]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 K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, 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

  17. 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

  18. 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.

  19. Testicular acid phosphatase induces odontoblast differentiation and mineralization.

    PubMed

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

    2016-04-01

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

  20. Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

    SciTech Connect

    Zhang, P.; Shanklin, J.; Burton, J. W.; Upchurch, R. G.; Whittle, E.; Dewey, R. E.

    2008-11-01

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoform of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.

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

    PubMed

    Robles, L J; Fisher, S K

    1975-01-01

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

  2. Deactivation of free and stabilized acid phosphatase by urea.

    PubMed

    Gianfreda, L; Marrucci, G; Greco, G

    1986-11-01

    Tests on acid phosphatase (E.G. 3.1.3.2) deactivation by urea have been performed at two pH values. Two conditions have been used: native enzyme operating batch-wise in dilute solution and stabilized enzyme in continuous flow ultrafiltration membrane reactor. Stabilization is achieved by confining the enzyme within a concentrated solution of a linear chain polymer that forms a polarization layer over the membrane. The results provide significant information on the kinetics and thermodynamics of the complex phenomena taking place during deactivation. Deactivation by urea is also compared with thermal deactivation. PMID:18555278

  3. 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.

  4. Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

    PubMed

    Zhao, Na; Zhang, Yuan; Li, Qiuqi; Li, Rufang; Xia, Xinli; Qin, Xiaowei; Guo, Huihong

    2015-02-01

    Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants. PMID:25528221

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

    PubMed

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

    2014-06-01

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

  6. The effect of sorbitol on acid phosphatase deactivation.

    PubMed

    Gianfreda, L; Toscano, G; Pirozzi, D; Greco, G

    1991-12-01

    Acid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity-versus-time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed. PMID:18600710

  7. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

    1998-01-06

    Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  8. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

  9. Human Prostatic Acid Phosphatase: Structure, Function and Regulation

    PubMed Central

    Muniyan, Sakthivel; Chaturvedi, Nagendra K.; Dwyer, Jennifer G.; LaGrange, Chad A.; Chaney, William G.; Lin, Ming-Fong

    2013-01-01

    Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy. PMID:23698773

  10. 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

  11. Follow-up on the Berg acid phosphatase test.

    PubMed

    Schiff, A F

    1998-03-01

    Approximately 42 years ago, the Berg acid phosphatase (AP) test (1) was accepted in most rape treatment centers nationally as the standard to determine whether sexual intercourse or related actions in any form had occurred. More specifically, the test was designed to determine the presence of a certain enzyme. In October 1969, I published an article making the test simpler (2) and reviewing the history of various tests for the detection of AP, an enzyme found in great abundance in seminal fluid. Both AP-impregnated material and refrigerated reagents had been saved along with a quantity of seminal fluid used in the original tests. The objectives of this study were to determine whether 25-year-old seminal fluid in any form can still be identified by the AP test and whether 25-year-old chemicals have remained stable and are still usable. PMID:9539395

  12. Effects of cadmium alone and in combination with low molecular weight chitosan on metallothionein, glutathione-S-transferase, acid phosphatase, and ATPase of freshwater crab Sinopotamon yangtsekiense.

    PubMed

    Li, Ruijin; Zhou, Yanying; Wang, Lan; Ren, Guorui; Zou, Enmin

    2014-03-01

    Cadmium (Cd) is an environmental contaminant showing a variety of deleterious effects, including the potential threat for the ecological environment and human health via food chains. Low molecular weight chitosan (LMWC) has been demonstrated to be an effective antioxidant. Metallothionein (MT) mRNA levels and activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), acid phosphatase (ACP), Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as malondialdehyde (MDA) contents in the gills of the freshwater crab Sinopotamon yangtsekiense were analyzed in vivo in order to determine the injury of Cd exposure on the gill tissues as well as the protective effect of LMWC against this injury. The results showed that there was an apparent accumulation of Cd in the gills, which was lessened by the presence of LMWC. Moreover, Cd(2+) significantly increased the gill MT mRNA levels, ACP activity and MDA content while decreasing the activities of SOD, GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in the crabs relative to the control. Cotreatment with LMWC reduced the levels of MT mRNA and ACP but raised the activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase in gill tissues compared with the crabs exposed to Cd(2+) alone. These results suggest that LMWC may exert its protective effect through chelating Cd(2+) to form LMWC-Cd(2+) complex, elevating the antioxidative activities of GST, Na(+),K(+)-ATPase, and Ca(2+)-ATPase as well as alleviating the stress pressure on MT and ACP, consequently protecting the cell from the adverse effects of Cd. PMID:22331632

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

    PubMed

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

    1999-06-01

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

  14. Okadaic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sustained hypoxia.

    PubMed

    Wilkerson, Julia E R; Satriotomo, Irawan; Baker-Herman, Tracy L; Watters, Jyoti J; Mitchell, Gordon S

    2008-03-12

    Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined. PMID:18337426

  15. Anti-thyroid and antifungal activities, BSA interaction and acid phosphatase inhibition of methimazole copper(II) complexes.

    PubMed

    Urquiza, Nora M; Islas, María S; Ariza, Santiago T; Jori, Nadir; Martínez Medina, Juan J; Lavecchia, Martín J; López Tévez, Leonor L; Lezama, Luis; Rojo, Teófilo; Williams, Patricia A M; Ferrer, Evelina G

    2015-03-01

    It has been reported that various metal coordination compounds have improved some biological properties. A high activity of acid phosphatase (AcP) is associated to several diseases (osteoporosis, Alzheimer's, prostate cancer, among others) and makes it a target for the development of new potential inhibitors. Anti-thyroid agents have disadvantageous side effects and the scarcity of medicines in this area motivated many researchers to synthesize new ones. Several copper(II) complexes have shown antifungal activities. In this work we presented for a first time the inhibition of AcP and the anti-thyroid activity produced by methimazole-Cu(II) complexes. Cu-Met ([Cu(MeimzH)2(H2O)2](NO3)2·H2O) produces a weak inhibition action while Cu-Met-phen ([Cu(MeimzH)2(phen)(H2O)2]Cl2) shows a strong inhibition effect (IC50 = 300 μM) being more effective than the reported behavior of vanadium complexes. Cu-Met-phen also presented a fairly good anti-thyroid activity with a formation constant value, Kc=1.02 × 10(10)M(-1) being 10(6) times more active than methimazole (Kc = 4.16 × 10(4)M(-1)) in opposition to Cu-Met which presented activity (Kc=9.54 × 10(3)M(-1)) but in a lesser extent than that of the free ligand. None of the complexes show antifungal activity except Cu-phen (MIC = 11.71 μgmL(-1) on Candidaalbicans) which was tested for comparison. Besides, albumin interaction experiments denoted high affinity toward the complexes and the calculated binding constants indicate reversible binding to the protein. PMID:25641192

  16. 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

  17. Modified Acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.

    1999-03-30

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.

  18. Modified acyl-ACP desaturase

    DOEpatents

    Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.

    1998-01-06

    Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.

  19. 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

  20. 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.

  1. Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae

    PubMed Central

    Nishida, Kentaro; Kubota, Teruyo; Matsumoto, Saki; Kato, Junki; Watanabe, Yu; Yamamoto, Atsuko; Furui, Mari; Ohishi, Akihiro; Nagasawa, Kazuki

    2016-01-01

    ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5′-nucleotidase (NT5E) and prostatic acid phosphatase (PAP) were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience). On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice. PMID:27348306

  2. 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.

  3. 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. PMID:20071031

  4. Effects of precipitation on soil acid phosphatase activity in three successional forests in Southern China

    NASA Astrophysics Data System (ADS)

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

    2011-01-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of P supply to ecosystems. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment of precipitation treatments (no precipitation, natural precipitation and doubled precipitation) in three forests of early-, mid- and advanced-successional stages in Southern China was carried out. Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, no precipitation treatment depressed soil acid phosphatase activity, while doubled precipitation treatment exerted no positive effects on it, and even significantly lowered it in the advanced forest. These indicate the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. The negative responses of soil acid phosphatase activity to precipitation suggest that P supply in subtropical ecosystems might be reduced if there was a drought in a whole year or more rainfall in the wet season in the future. NP, no precipitation; Control, natural precipitation; DP, double precipitation.

  5. A single domain of human prostatic acid phosphatase shows antibody-mediated restoration of catalytic activity.

    PubMed Central

    Choe, B K; Dong, M K; Walz, D; Gleason, S; Rose, N R

    1982-01-01

    By limited proteolysis with mouse submaxillaris protease, human prostatic acid phosphatase (EC 3.1.3.2) was cleaved into three fragments, Sp1, Sp2, and Sp3, which individually had no enzymatic activity. One of the fragments, Sp3, regained enzymatic activity after interaction with rabbit antibody to prostatic acid phosphatase. The Sp3 fragment was purified and characterized as to its molecular weight, amino acid composition, and carbohydrate content. The Sp3 fragment behaved like the parent molecule in L(+)-tartrate affinity and in trapping of a phosphoryl intermediate. The same Sp3 fragment also bears the most prominent antigenic determinants. This evidence suggest that Sp3 is the enzymatically active domain of prostatic acid phosphatase. Images PMID:6193513

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

    PubMed

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

    1990-06-01

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

  7. Histochemical localization and characterization of AKP, ACP, NSE, and POD from cultured Apostichopus japonicus

    NASA Astrophysics Data System (ADS)

    Li, Jiye; Sun, Xiuqin; Zheng, Fengrong; Sun, Hushan

    2009-09-01

    We investigated the distribution of four enzymes involved in the immune response of Apostichopus japonicus. We collected samples of the tentacles, papillate podium, integument, respiratory tree, and digestive tract and stained them for acid phosphatase (ACP), alkaline phosphatase (AKP), non-specific esterase (NSE) and peroxidase (POD) activity. The distribution and content of ACP, AKP, NSE, and POD differed among the tissues. The coelomic epithelium of the tentacle, papillate podium, and integument and the mucous layer of respiratory tree were positive for ACP activity. The coelomic epithelium and cuticular layer of the tentacle, papillate podium, and integument and the mucous layer and tunica externa of the respiratory tree and digestive tract stained positive or weakly positive for AKP activity. Almost all the epithelial tissues stained positive, strongly positive, or very strongly positive for NSE activity. The cuticular layer of the tentacle and integument and the mucous layer, tunica submucosa, and tunica externa of the respiratory tree and digestive tract stained positive for POD activity. We hypothesize that these enzymes play a role in the immune response in A. japonicus.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

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

    1990-03-01

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

  10. 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.

  11. Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.

    PubMed Central

    Damuni, Z; Merryfield, M L; Humphreys, J S; Reed, L J

    1984-01-01

    Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml. PMID:6589597

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

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

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

  13. An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs.

    PubMed Central

    Lorca, T; Fesquet, D; Zindy, F; Le Bouffant, F; Cerruti, M; Brechot, C; Devauchelle, G; Dorée, M

    1991-01-01

    Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor activity. Images PMID:1846666

  14. Autosomal dominant aniridia: probable linkage to acid phosphatase-1 locus on chromosome 2.

    PubMed Central

    Ferrell, R E; Chakravarti, A; Hittner, H M; Riccardi, V M

    1980-01-01

    Maximum likelihood analysis for linkage between autosomal dominant aniridia and 12 biochemical and serological markers in a single large family showed a probable linkage between autosomal dominant aniridia and the enzyme acid phosphatase-1. The presence of an autosomal dominant aniridia gene linked to acid phosphatase-1 on chromosome arm 2p and the existence of an aniridia syndrome resulting from deletion of band 13 of the short arm of chromosome 11 establishes a chromosome basis for genetic heterogeneity of aniridia phenotypes. PMID:6929510

  15. Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

    DOE PAGESBeta

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; Sisson, Lyle A.; Schneider, Philip E.; Posewitz, Matthew C.

    2016-05-26

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

  16. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase

    PubMed Central

    Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; Sisson, Lyle A.; Schneider, Philip E.; Posewitz, Matthew C.

    2016-01-01

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. PMID:27303412

  17. Modulation of Medium-Chain Fatty Acid Synthesis in Synechococcus sp. PCC 7002 by Replacing FabH with a Chaetoceros Ketoacyl-ACP Synthase.

    PubMed

    Gu, Huiya; Jinkerson, Robert E; Davies, Fiona K; Sisson, Lyle A; Schneider, Philip E; Posewitz, Matthew C

    2016-01-01

    The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novo assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase III increased MCFA synthesis up to fivefold. The level of increase is dependent on promoter strength and culturing conditions. PMID:27303412

  18. Immunochemical detection of serum prostatic acid phosphatase. Methodology and clinical evaluation.

    PubMed

    Chu, T M; Wang, M C; Scott, W W; Gibbons, R P; Johnson, D E; Schmidt, J D; Loening, S A; Prout, G R; Murphy, G P

    1978-01-01

    An immunochemical method for detection of prostatic acid prosphatase is described. Purified acid phosphatase was isolated from cancerous human prostate. A specific antiserum to the purified enzyme was produced in rabbits. The antiserum to postatic acid phosphatase did not react with acid phosphatase originating from other tissues. A counter immunolectrophoresis, utilizing the specific antibodies and a chemical staining technique, has been developed and clinically evaluated. Sera from patients with prostatic carcinoma (6/20 of stage B, 27/49 of stage C, and 98/125 of stage D) gave positive results. Sera from 19 patients with benign prostatic hypertrophy, from 89 patients with other tumors, from 12 patients with Gaucher's disease, from 107 healthy volunteers, and from 50 normal age-matched men all gave negative results. The sensitivity of this method was 0.4 IU of enzyme activity or 20 ng per ml of prostatic acid phosphatase protein. Further clinical evaluation of patients in the early stage of prostatic cancer and of patients undergoing chemotherapy is in progress. PMID:75196

  19. 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 ...

  20. Development of markers for Delta9-Stearoyl-ACP-Desaturase (SAD) to screen for cold acclimation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delta 9-Stearoyl-acyl carrier protein (ACP) desaturase (SAD) is an important enzyme of fatty acid biosynthesis in higher plants. Located in the plastid stroma, SAD catalyzes the desaturation of stearoyl-ACP to oleyl-ACP. SAD plays a key role in determining the ratio of saturated fatty acids to unsat...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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 ...

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

    PubMed

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

    2003-10-31

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

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

    PubMed Central

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

    2008-01-01

    Background 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. Results 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 Å) and fluoride (2.2 Å) 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 μ-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this μ-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity. Conclusion 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. PMID:18234116

  10. Mutations in a novel 9-stearoyl-ACP-desaturase gene are associated with enhanced stearic acid levels in soybean seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2-4 % of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process...

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

    PubMed Central

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

    2010-01-01

    Summary The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD+ utilization pathway by dephosphorylating NMN to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases, 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 NMN, 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 hydrogen-bonding edge of the base. The span between the hydrophobic box and phosphoryl site is optimal for recognizing nucleoside monophosphates, which explains the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, which is consistent with observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5′- 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 and C acid phosphatases share a common strategy for nucleotide recognition. PMID:20934434

  12. 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.

  13. 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. PMID:20934434

  14. 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.

  15. 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. PMID:9784397

  16. Physiological function of mycobacterial mtFabD, an essential malonyl-CoA:AcpM transacylase of type 2 fatty acid synthase FASII, in yeast mct1Delta cells.

    PubMed

    Gurvitz, Aner

    2009-01-01

    Mycobacterium tuberculosis mtFabD is an essential malonyl-CoA:AcpM transacylase and is important for vital protein-protein interactions within type 2 fatty acid synthase FASII. mtFabD contacts KasA, KasB, FabH, InhA, and possibly also HadAB, HadBC, and FabG1/MabA. Disruption of mtFabD's interactions during FASII has been proposed for drug development. Here, the gene for a mitochondrially targeted mtFabD was ectopically expressed in Saccharomyces cerevisiae mct1Delta mutant cells lacking the corresponding mitochondrial malonyl-CoA transferase Mct1p, allowing the mutants to recover their abilities to respire on glycerol and synthesize lipoic acid. Hence, mtFabD could physiologically function in an environment lacking holo-AcpM or other native interaction partners. PMID:19859569

  17. The prostatic acid phosphatase (ACPP) gene is localized to human chromosome 3q21-q23

    SciTech Connect

    Li, S.S.L.; Sharief, F.S. )

    1993-09-01

    Human prostatic acid phosphatase (ACPP) has been used as a diagnostic marker for prostate cancer. It is synthesized under androgen regulation and secreted by the epithelial cells of the prostate gland. The authors have confirmed the previous assignment of the ACPP gene to chromosome 3 by probing a panel of 25 human-Chinese hamster somatic cell hybrids, and they have further localized the ACPP gene to chromosome 3q21-q23 by fluorescence in situ hybridization. 10 refs., 1 fig.

  18. Purification and properties of catalytic subunit of branched-chain -keto acid dehydrogenase phosphatase

    SciTech Connect

    Reed, L.J.; Damuni, Z.

    1987-05-01

    The catalytic subunit of the branched-chain -keto acid dehydrogenase (BCKDH) phosphatase has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent M/sub r/ of about 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with apparent M/sub r/ of 460,000 was dissociated to its catalytic subunit, with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1500-2500 units/mg. The catalytic subunit exhibited approx.10% maximal activity with TSP-labeled pyruvate dehydrogenase complex, but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the M/sub r/ 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates, but not by nucleoside monophosphates.

  19. 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.

  20. 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. PMID:15797237

  1. 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. PMID:26380880

  2. Biochemical Characterization and Subcellular Localization of the Red Kidney Bean Purple Acid Phosphatase.

    PubMed Central

    Cashikar, A. G.; Kumaresan, R.; Rao, N. M.

    1997-01-01

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the non-specificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination. PMID:12223752

  3. Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells1[W

    PubMed Central

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

    2010-01-01

    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 α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-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 α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls. PMID:20357138

  4. Development of an ACP facility

    SciTech Connect

    Gil-Sung You; Won-Myung Choung; Jeong-Hoe Ku; il-Je Cho; Dong-Hak Kook; Kie-Chan Kwon; Eun-Pyo Lee; Ji-Sup Yoon; Seong-Won Park; Won-Kyung Lee

    2007-07-01

    KAERI has been developing an advanced spent fuel conditioning process (ACP). The ACP facility for a process demonstration consists of two air-sealed type hot cells. The safety analysis results showed that the facility was designed safely. The relevant integrated performance tests were also carried out successfully. (authors)

  5. 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

  6. 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

  7. Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

    PubMed Central

    2011-01-01

    Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

  8. Shewanella oneidensis FabB: A β-ketoacyl-ACP Synthase That Works with C16:1-ACP

    PubMed Central

    Luo, Qixia; Li, Meng; Fu, Huihui; Meng, Qiu; Gao, Haichun

    2016-01-01

    It is established that Escherichia coli β-ketoacyl-ACP synthase (KAS) I (encoded by EcfabB) is the primary, if not exclusive, factor for elongation of the cis-3-decenoyl-ACP (C10:1-ACP) but not effective with C16:1- or longer-chain-ACPs. To test the extent to which these features apply to KAS I proteins in other species, in this study, we examined the physiological role of FabB in Shewanella oneidensis, an excellent model for researching type II fatty acid synthetic (FAS) system and its regulation. We showed that the loss of either FabA (the enzyme that introduces double bond) or FabB, in the absence of DesA which desaturizes C16 and C18 to generate respective C16:1 and C18:1, leads to a UFA auxotroph. However, fatty acid profiles of membrane phospholipid of the fabA and fabB mutants are significantly different, suggesting that FabB participates in steps beyond elongation of C10:1-ACP. Further analyses demonstrated that S. oneidensis FabB differs from EcFabB in that (i) it is not the only enzyme capable of catalyzing elongation of the cis-3-decenoyl-ACP produced by FabA, (ii) it plays a critical role in elongation of C16:1- and longer-chain-ACPs, and (iii) its overproduction is detrimental. PMID:27014246

  9. 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

  10. 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

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

    PubMed

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

    2003-12-01

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

  12. 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

    2007-01-01

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

  13. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    DOE PAGESBeta

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products inmore » BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.« less

  14. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxgenase with a bacterial type-I fatty acid synthase in E. coli

    SciTech Connect

    Coursolle, Dan; Shanklin, John; Lian, Jiazhang; Zhao, Huimin

    2015-06-23

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg/L long chain alcohol/alkane products including a 57 mg/L titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

  15. Production of long chain alcohols and alkanes upon coexpression of an acyl-ACP reductase and aldehyde-deformylating oxygenase with a bacterial type-I fatty acid synthase in E. coli.

    PubMed

    Coursolle, Dan; Lian, Jiazhang; Shanklin, John; Zhao, Huimin

    2015-09-01

    Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L(-1) long chain alcohol/alkane products including a 57 mg L(-1) titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system. PMID:26135500

  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. 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

  18. 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. PMID:26105827

  19. Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro.

    PubMed

    Yajima, T

    1986-01-01

    Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues. PMID:3742560

  20. Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou.

    PubMed

    Cai, Xia; Li, Wei; Yin, Lingfang

    2009-12-01

    Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER. PMID:19649693

  1. 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. PMID:26938873

  2. 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

  3. 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

    2015-08-01

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

  4. Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae).

    PubMed Central

    Dormann, P.; Frentzen, M.; Ohlrogge, J. B.

    1994-01-01

    This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide. PMID:12232130

  5. Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

    PubMed Central

    Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

    2014-01-01

    The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

  6. 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. PMID:25887488

  7. [Secretory acid phosphatase of Mycobacterium tuberculosis inhibits the autophagy of murine macrophages].

    PubMed

    Hu, Dong; Wang, Wan; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Xu, Congjing; Zhang, Rongbo; Wu, Jing

    2016-06-01

    Objective To investigate the effect of secretory acid phosphatase as a virulence factor of Mycobacterium tuberculosis (SapM) on the autophagy of murine macrophages. Methods GFP-LC3-Raw264.7 cells were treated with SapM, wortmannin, or starvation. Then the formation of autophagosomes was observed under a fluorescence microscope. The level of microtubule-associated protein 1 light chain 3 (LC3) II was detected using Western blotting. After chloroquine was added in the SapM-treated cells, LC3II level was again tested by Western blotting. Results Both starvation and SapM increased the number of GFP-LC3 puncta and the level of LC3 II. There was no further increase of LC3 II level in SapM-treated cells after chloroquine addition. Conclusion SapM can block autophagosome-lysosome fusion and inhibit autophagy of murine macrophages. PMID:27371835

  8. Trichomonas vaginalis: determination of acid phosphatase activity as a pharmacological screening procedure.

    PubMed

    Martínez-Grueiro, M M; Montero-Pereira, D; Giménez-Pardo, C; Nogal-Ruiz, J J; Escario, J A; Gómez-Barrio, A

    2003-10-01

    A simple method to screen trichomonacides, based on the quantification of acid phosphatase (AP) activity, has been designed. Using p-nitrophenyl phosphate as chromogenic substrate, we first determined the optimal conditions for enzyme reaction. After seeding, a linear correlation between number of trichomonads and optical densities at 405 nm was obtained at 24 hr but not at 48 hr. Then, the inhibitory effect of metronidazole was assessed both by microscope counts and by AP determination. Similar values for 50% inhibitory concentrations (2.6 microM), with 95% confidence limits of 1.91-3.33 for microscopic and 2.21-3.05 for colorimetric method, were obtained. We concluded that the colorimetric method described in this investigation is suitable for pharmacological studies and for the screening of new, potential antitrichomonal agents. PMID:14627165

  9. 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. PMID:22717620

  10. Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1 [corrected].

    PubMed

    Takemiya, Atsushi; Shimazaki, Ken-ichiro

    2010-08-01

    Stomata open in response to blue light under a background of red light. The plant hormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening, an effect essential for promoting stomatal closure in the daytime to prevent water loss. However, the mechanisms and molecular targets of this inhibition in the blue light signaling pathway remain unknown. Here, we report that phosphatidic acid (PA), a phospholipid second messenger produced by ABA in guard cells, inhibits protein phosphatase 1 (PP1), a positive regulator of blue light signaling, and PA plays a role in stimulating stomatal closure in Vicia faba. Biochemical analysis revealed that PA directly inhibited the phosphatase activity of the catalytic subunit of V. faba PP1 (PP1c) in vitro. PA inhibited blue light-dependent stomatal opening but did not affect red light- or fusicoccin-induced stomatal opening. PA also inhibited blue light-dependent H(+) pumping and phosphorylation of the plasma membrane H(+)-ATPase. However, PA did not inhibit the autophosphorylation of phototropins, blue light receptors for stomatal opening. Furthermore, 1-butanol, a selective inhibitor of phospholipase D, which produces PA via hydrolysis of phospholipids, diminished the ABA-induced inhibition of blue light-dependent stomatal opening and H(+) pumping. We also show that hydrogen peroxide and nitric oxide, which are intermediates in ABA signaling, inhibited the blue light responses of stomata and that 1-butanol diminished these inhibitions. From these results, we conclude that PA inhibits blue light signaling in guard cells by PP1c inhibition, accelerating stomatal closure, and that PP1 is a cross talk point between blue light and ABA signaling pathways in guard cells. PMID:20498335

  11. 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

  12. Directed Evolution of Metabolic Pathways in Microbial Populations. I. Modification of the Acid Phosphatase Ph Optimum in S. CEREVISIAE

    PubMed Central

    Francis, J. C.; Hansche, P. E.

    1972-01-01

    An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.—The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 109 cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (Km) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.—The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.—The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained. PMID:4552227

  13. 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.

  14. 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

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

    PubMed

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

    2015-05-01

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

  16. 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. PMID:25203006

  17. 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

  18. 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.

  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. PMID:27235981

  20. Purification and Properties of Acid Phosphatase-1 from a Nematode Resistant Tomato Cultivar

    PubMed Central

    Paul, Elizabeth M.; Williamson, Valerie M.

    1987-01-01

    In tomato the acid phosphatase-1 isozyme (Apase-1) is inherited as a single locus linked to the nematode resistance gene (Mi). The Apase-11 electrophoretic variant has been purified from a tomato cell suspension culture using ion exchange and concanavalin A sepharose affinity chromatography. A cellulose acetate electrophoresis method was used to distinguish Apase-11 rapidly from other Apase isozymes in tomato. The subunit molecular weight of the purified enzyme was estimated to be 31,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme, which is reported to be a dimer, was determined to be approximately 51,000 by high performance liquid chromatography gel filtration. Apase-11 has a lower pH optimum and a distinct substrate specificity as compared to Apases extracted from tomato fruit or from other plant species. The amino acid composition of Apase-11 is similar to that of a potato Apase. Images Fig. 1 Fig. 2 Fig. 4 PMID:16665451

  1. 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. PMID:25672855

  2. Co-detection of PTH/PTHrP receptor and tartrate resistant acid phosphatase in osteoclasts.

    PubMed

    Gay, Carol V; Zheng, Betty; Gilman, Virginia R

    2003-08-01

    Serial sections of rat metaphyses were prepared from paraffin embedded tissue blocks and analyzed in sets of three. The central section was stained for tartrate resistant acid phosphatase (TRAP) in order to identify osteoclasts, one adjacent section was immunostained with an affinity purified antibody to a 15 amino acid sequence unique to rat PTH/PTHrP receptor, and the other adjacent section in the set served as an immunostaining control. This allowed each of the 110 osteoclasts examined to be identified by TRAP and to be tested for the presence or absence of PTH/PTHrP receptor. All antibody solutions and rinses contained 1% donkey serum and 0.5% Tween 20 to ensure antibody integrity and good rinsing procedure. Confocal microscopy was used to evaluate fluorescence intensity of the immunostained osteoclasts. Pixel intensities of 58 osteoclasts from young (4 month) rats and 52 osteoclasts from old (15 month) rats were obtained. Pixel intensities were similar (P = 0.89) for both young and old animals. However, the number of PTH/PTHrP receptor deficient osteoclasts was greater for the older animals (14.29% vs. 7.24%). This provides direct evidence of PTH/PTHrP receptors in osteoclasts. PMID:12874824

  3. Substrate Recognition by β-Ketoacyl-ACP Synthases†

    PubMed Central

    Borgaro, Janine G.; Chang, Andrew; Machutta, Carl A.; Zhang, Xujie; Tonge, Peter J.

    2011-01-01

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14 residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the kcat/Km for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  4. Substrate recognition by β-ketoacyl-ACP synthases.

    PubMed

    Borgaro, Janine G; Chang, Andrew; Machutta, Carl A; Zhang, Xujie; Tonge, Peter J

    2011-12-13

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

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

    PubMed

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

    1995-03-15

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

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

    PubMed

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

    2003-12-01

    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

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

    PubMed

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

    2003-03-01

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

  8. Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture

    PubMed Central

    1979-01-01

    Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0- tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage- like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA- treated cells, the isoenzymes 3a and 3b were present only in TPA- induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes. PMID:291600

  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. PMID:24012521

  10. Mice Deficient in Transmembrane Prostatic Acid Phosphatase Display Increased GABAergic Transmission and Neurological Alterations

    PubMed Central

    Myöhänen, Timo T.; Voikar, Vootele; Mijatovic, Jelena; Segerstråle, Mikael; Herrala, Annakaisa M.; Kulesskaya, Natalia; Pulkka, Anitta E.; Kivinummi, Tanja; Abo-Ramadan, Usama; Taira, Tomi; Piepponen, T. Petteri; Rauvala, Heikki; Vihko, Pirkko

    2014-01-01

    Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP−/− mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype. PMID:24846136

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

    PubMed

    Retegan, M; Milet, A; Jamet, H

    2010-07-01

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

  12. Hydrolysis of phosphodiesters by diiron complexes: design of nonequivalent iron sites in purple acid phosphatase models.

    PubMed

    Verge, François; Lebrun, Colette; Fontecave, Marc; Ménage, Stéphane

    2003-01-27

    New mu-oxo-diferric complexes have been designed for hydrolysis of phosphodiesters. To mimic the diiron active site of purple acid phosphatase, a combinatorial method has been used to select complexes containing two distinct iron coordination spheres. The introduction of a bidentate ligand, a substituted phenanthroline (L) into complex 1, [Fe2O(bipy)4(OH2)2](NO3)4, generates in solution the complex [Fe2O(bipy)3(L)(OH2)2](NO3)4 as shown by ESI/MS and 1H NMR studies. The latter complex was found to be 20-fold more active than complex 1. On the basis of kinetic studies, we demonstrated that the complex [Fe2O(bipy)3(L)(OH)(OH2)](NO3)3 was the active species and the reaction proceeded through the formation of a ternary complex in which one iron binds a hydroxide and the second, the substrate. At nonsaturating concentrations of the substrate, the increased activity with increased methyl substituents in L was due to an increased affinity of the complex for the substrate. The activity of [Fe2O(bipy)3(33'44'Me2-Phen)(OH2)2](NO3)4 [33'44'Me2Phen = 3,3',4,4'-dimethyl-1,10-phenanthroline] was found to be comparable to that reported for Co(III) or Ce(IV) complexes. PMID:12693232

  13. Association of Tartrate-Resistant Acid Phosphatase-Expressed Macrophages and Metastatic Breast Cancer Progression.

    PubMed

    Chen, Yu-Guang; Janckila, Anthony; Chao, Tsu-Yi; Yeh, Ren-Hua; Gao, Hong-Wei; Lee, Su-Huei; Yu, Jyh-Cherng; Liao, Guo-Shiou; Dai, Ming-Shen

    2015-12-01

    Infiltrating neutrophils, lymphocytes, macrophages, and cytokines constitute a state of chronic inflammation within the tumor microenvironment. Tartrate-resistant acid phosphatase 5a (TRACP5a) protein, a novel product of activated macrophage, is postulated to be a biomarker for systemic inflammatory burden in states of chronic inflammation. We aimed to investigate the clinical significance of TRACP5a expression in tumor-infiltrating macrophages and serum TRACP5a in patients with metastatic breast cancer (BC). We retrospectively analyzed the clinical data from 34 BC patients with confirmed skeletal/visceral metastasis upon or during first-line palliative treatment. Patients were stratified into 3 groups based on the therapeutic responses and follow-up disease course. The association of TRACP5a protein with other inflammatory and cancer biomarkers was assessed among the clinically distinct group of patients. Higher TRACP5a protein was significantly correlated with earlier disease progression and survival (P = 0.0045) in comparison to other inflammatory markers, CRP or IL-6. Patients with higher serum TRACP5a level and shorter survival and treatment refractoriness also had more TRACP+ tumor-infiltrating macrophages. Our data support a hypothesis that serum TRACP5a protein can potentially be a predictive and prognostic marker to evaluate disease progression and therapeutic response in BC patients with bone/visceral metastasis. The associations between overall survival and TRACP expression by macrophages require further prospective investigation. PMID:26632898

  14. 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. PMID:27194738

  15. Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.

    PubMed Central

    Jones, A; Davies, H M; Voelker, T A

    1995-01-01

    Acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the two lipid biosynthesis pathways in plants. We have discovered that there are two distinct but related thioesterase gene classes in higher plants, termed FatA and FatB, whose evolutionary divergence appears to be ancient. FatA encodes the already described 18:1-ACP thioesterase. In contrast, FatB representatives encode thioesterases preferring acyl-ACPs having saturated acyl groups. We unexpectedly obtained a 16:0-ACP thioesterase cDNA from Cuphea hookeriana seed, which accumulate predominantly 8:0 and 10:0. The 16:0 thioesterase transcripts were found in non-seed tissues, and expression in transgenic Brassica napus led to the production of a 16:0-rich oil. We present evidence that this type of FatB gene is ancient and ubiquitous in plants and that specialized plant medium-chain thioesterases have evolved independently from such enzymes several times during angiosperm evolution. Also, the ubiquitous 18:1-ACP thioesterase appears to be a derivative of a 16:0 thioesterase. PMID:7734968

  16. Cellular prostatic acid phosphatase, a PTEN-functional homologue in prostate epithelia, functions as a prostate-specific tumor suppressor

    PubMed Central

    Muniyan, Sakthivel; Ingersoll, Matthew A.; Batra, Surinder K.; Lin, Ming-Fong

    2014-01-01

    The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to Phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases’ roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis. PMID:24747769

  17. An acpXL Mutant of Rhizobium leguminosarum bv. phaseoli Lacks 27-Hydroxyoctacosanoic Acid in Its Lipid A and Is Developmentally Delayed during Symbiotic Infection of the Determinate Nodulating Host Plant Phaseolus vulgaris ▿

    PubMed Central

    Brown, Dusty B.; Huang, Yu-Chu; Kannenberg, Elmar L.; Sherrier, D. Janine; Carlson, Russell W.

    2011-01-01

    Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures. PMID:21764936

  18. [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. PMID:12712579

  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. PMID:26613496

  20. Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection.

    PubMed

    Sassolas, Audrey; Catanante, Gaëlle; Hayat, Akhtar; Marty, Jean-Louis

    2011-09-30

    Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample. In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L(-1) using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L(-1) and 0.123 μg L(-1) with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC(50) value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L(-1). This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish. PMID:21839207

  1. Characterization of soluble acyl-ACP desaturases from Camelina sativa, Macadamia tetraphylla and Dolichandra unguis-cati.

    PubMed

    Rodríguez, Manuel Fernando Rodríguez; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-04-15

    Acyl-acyl carrier protein (ACP) desaturases (EC 1.14.19.2) are soluble enzymes that catalyse the insertion of a double bond into saturated fatty acid bound in saturated acyl chains bound to ACP in higher plants, producing cis-monounsaturated fatty acids. Three types of soluble acyl-ACP desaturases have been described: Δ(9)-acyl-ACP, Δ(6)-acyl-ACP and Δ(4)-acyl-ACP desaturases, which differ in the substrate specificity and the position in which the double bond is introduced. In the present work, Camelina sativa (CsSAD), Macadamia tetraphylla (MtSAD) and Dolichandra unguis-cati (DuSAD) desaturases were cloned, sequenced and characterized. Single copies of CsSAD, MtSAD and DuSAD with three, one and two different alleles, respectively, were found. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization in protein extracts. The recombinant CsSAD enzyme showed 300-fold higher specificity towards 18:0-ACP than 16:0-ACP. Similar profile exhibited MtSAD although the differences in the specificity were lower, around 170-fold higher for 18:0-ACP than 16:0-ACP. Furthermore, DuSAD presented a profile showing preference towards 16:0-ACP against 18:0-ACP, around twice more, being so a Δ(9) palmitoyl-ACP desaturase. Also, we reported the expression profile of CsSAD, which showed the highest levels of expression in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm. Moreover, the possibility to express a new desaturase in C. sativa (oilseed crop that store high levels of oil and is easy to transform) to create a new line rich in short monounsaturated fatty acid is discussed. PMID:25765361

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

    PubMed

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

    1993-10-15

    Leishmania promastigotes, the stage of the parasite characteristic for the sandfly vector, express an abundant glycoconjugate, called lipophosphoglycan, at their surface. Lipophosphoglycan consists of lysoalkyl-sn-glycerophosphoinositol linked to a phosphosaccharide core conserved in all species, which is connected to PO4-6Gal beta 1,4Man alpha 1 repeats with species-specific substitutions at the Gal residue; the repeats are capped by conserved and species-specific oligosaccharides. Most Leishmania species also secrete an acid phosphatase, which, in Leishmania mexicana, is a filamentous complex composed of a phosphorylated glycoprotein and non-covalently associated proteo-(high-molecular-mass)phosphoglycan. The secreted acid phosphatase complex was used as an antigen to derive a panel of monoclonal antibodies (mAbs). A total of 25 mAbs (17 novel and 8 previously described) were tested by different techniques for their specificity against lipophosphoglycan and secreted acid phosphatase from several Leishmania species. This comparison and the modification of the antigens by chemical or enzymic treatments allowed a classification of the mAbs into several groups. First, from 25 mAbs examined, 22 recognize lipophosphoglycan and the enzyme complex of L. mexicana; only three are specific for secreted acid phosphatase. Two of the latter group are also directed against carbohydrate structures, whereas the third mAb recognizes the 100-kDa polypeptide of the complex. The secreted acid-phosphatase-specific class detects antigen in the flagellar pocket of promastigotes while all anti-lipophosphoglycan mAbs bind to the cell surface. Second, all 15 anti-lipophosphoglycan mAbs investigated in detail appear to be directed against the phosphosaccharide repeats or the cap structure rather than the phosphosaccharide core. Two mAbs recognize terminal cap-structures containing Man alpha 1,2Man residues. Four antibodies are specific for L. mexicana and are probably directed against PO4

  3. 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.

  4. 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

  5. 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.

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

    PubMed Central

    2013-01-01

    Background 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. Results 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. Conclusions 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

  7. Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

    PubMed Central

    Pandey, Manoj K.; Sung, Bokyung; Aggarwal, Bharat B.

    2009-01-01

    STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents. PMID:19937797

  8. A MUTATION IN A 3-KETO-ACYL-ACP SYNTHASE II GENE IS ASSOCIATED WITH ELEVATED PALMITIC ACID LEVELS IN SOYBEAN SEEDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Palmitic acid is the major saturated fatty acid component of soybean [Glycine max, (L.) Merr.] oil, typically accounting for ~11 % of total seed oil content. Several genetic loci have been shown to control the seed palmitate content of soybean. One such locus, fap2, mediates an elevated seed palmit...

  9. High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

    PubMed Central

    Wu, Zhi-Qi; Zhang, Yan; Xie, Erfu; Song, Wei-Juan; Yang, Rui-Xia; Yan, Cheng-Jing; Zhang, Bing-Feng; Xu, Hua-Guo

    2016-01-01

    Background Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. Methods A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. Results The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). Conclusions High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases. PMID:26800211

  10. Chronological changes in acid phosphatase activity within neurons and perineuronal satellite cells of the inferior vagal ganglion of the cat induced by vagotomy.

    PubMed Central

    Glover, R A

    1982-01-01

    The hexazonium pararosaniline method was employed to describe the distribution of acid phosphatase activity, chronologically, within neurons and their investing satellite cells of the inferior vagal ganglion of the cat after vagotomy. In control ganglia, acid phosphatase activity was invariably confined to the cytoplasm of neurons and satellite cells. Reaction product was visible as distinct granules within neuronal perikarya. The cytoplasm of perineuronal satellite cells also contained reaction product but, in most instances, activity was weak and granules were difficult to distinguish. No reaction product was observed in myelin or axonal processes; nuclear staining was absent. Acid phosphatase activity was increased in ganglionic neurons as early as 24 hours after vagotomy. Increased activity in perineuronal satellite cells was not evident until 3 days post-operatively. By 15 days, activity was ubiquitously increased in the cytoplasm of both neurons and satellite cells. Evidence suggesting neuronophagia was also apparent. Between 30 and 60 days post-operatively acid phosphatase activity gradually decreased in both neurons and satellite cells until a picture comparable with that seen in control tissue sections was visible. The functional significance of these changes in acid phosphatase activity within an altered metabolic environment induced by vagotomy is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:7076551

  11. 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.

  12. 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

  13. 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. PMID:26042133

  14. Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

    PubMed Central

    Miyata, M; Miyata, H

    1978-01-01

    By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation. PMID:711673

  15. 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

  16. 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.

  17. Solution Structure of 4'-Phosphopantetheine - GmACP3 from Geobacter Metallireducens: A Specialized Acyl Carrier Protein with Atypical Structural Features and a Putative Role in Lipopolysaccharide Biosyntheses

    SciTech Connect

    Ramelot, Theresa A.; Smola, Matthew J.; Lee, Hsiau-Wei; Ciccosanti, Colleen; Hamilton, Keith; Acton, Thomas; Xiao, Rong; Everett, John K.; Prestegard, James H.; Montelione, Gaetano; Kennedy, Michael A.

    2011-03-08

    GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by 15NNMRrelaxation measurements. Addition of 4'-phosphopantetheine (4'-PP) via enzymatic conversion by E. coli holo-ACP synthase resulted in stable >95% holo-GmACP3 that was characterized as monomeric by 15N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4'-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4'-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conservedWDSLxH/N motif found in GmACP3 and its orthologs. The helix locations and the large hydrophobic cavity are more similar tomediumand long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggests that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially is involved in synthesis of secondary metabolites.

  18. Single Laboratory Validation of A Ready-to-Use Phosphatase Inhibition Assay for Detection of Okadaic Acid Toxins

    PubMed Central

    Smienk, Henry G. F.; Calvo, Dolores; Razquin, Pedro; Domínguez, Elena; Mata, Luis

    2012-01-01

    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 IC50 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. PMID:22778904

  19. 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). PMID:24996321

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

    PubMed

    Cook, Naomi L; Moeke, Cassidy H; Fantoni, Luca I; Pattison, David I; Davies, Michael J

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation. PMID:26616646

  1. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal.

    PubMed

    Cronan, John E

    2014-06-01

    ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4'-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP-enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4'-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping. PMID:24825445

  2. Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

    PubMed

    Shen, Z; Fice, D; Byers, D M

    1992-07-01

    A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis. PMID:1514693

  3. Spontaneous circadian fluctuations of prostate specific antigen and prostatic acid phosphatase serum activities in patients with prostatic cancer.

    PubMed

    Mannini, D; Maver, P; Aiello, E; Corrado, G; Vecchi, F; Bellanova, B; Marengo, M

    1988-01-01

    Spontaneous circadian variations of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), determined simultaneously by radioimmunoassay (RIA), were investigated by multiple sampling, over a 24-hour period, in 32 patients with prostatic cancer. In 29/32 patients (91%), the coefficient of variation of 24-hour values, for either marker, was greater than that of the RIA method at the same range of values; stage D patients showed the greatest spontaneous variability. Fluctuations around the mean of 24-hour values ranged from -65% to +85% for PAP, from -72% to +190% for PSA, occurring random and independently for each marker. Variability was about 20% greater for PSA than for PAP. The existence of spontaneous fluctuations should be considered in multiple marker evaluation of prostatic cancer patients. PMID:2449758

  4. Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

    PubMed

    Cejková, J; Lojda, Z; Havránková, E

    1975-09-29

    Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase. PMID

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

    EPA Science Inventory

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

  6. 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. PMID:16009331

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initia...

  8. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library.

    PubMed

    Huang, Ziliang; Li, Gang; Zhang, Chong; Xing, Xin-Hui

    2016-02-01

    Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes. PMID:26777244

  9. Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid.

    PubMed Central

    Félix, M A; Cohen, P; Karsenti, E

    1990-01-01

    In Xenopus embryos, the cell cycle is abbreviated to a rapid alternation between interphase and mitosis. The onset of each M phase is induced by the periodic activation of the cdc2 kinase which is triggered by a threshold level of cyclins and apparently involves dephosphorylation of p34cdc2. We have prepared post-ribosomal supernatants from eggs sampled during interphase (interphase extracts) and just before the first mitosis of the early embryonic cell cycle (prophase extracts). In 'interphase extracts', the cdc2 kinase never activates spontaneously upon incubation at room temperature whereas in 'prophase extracts' it does. We show here that in 'interphase extracts', specific inhibition of type 2A phosphatase by okadaic acid induces cdc2 kinase activation. This requires a subthreshold level of cyclin and the presence of a particulate factor in the extract. Inhibition of type 1 phosphatases by inhibitor 1 and inhibitor 2 never results in cdc2 kinase activation. These results demonstrate that during the period of cyclin accumulation, cdc2 kinase activation is inhibited by a type 2A phosphatase. In 'prophase extracts', spontaneous activation of the cdc2 kinase is inhibited by beta-glycerophosphate and NaF, but not by okadaic acid, inhibitor 1 and inhibitor 2 or divalent cation chelation. This demonstrates that when enough cyclin has accumulated, cdc2 kinase activation involves a protein phosphatase which must be distinct from the type 1 and 2A phosphatases, and from the calcium-dependent (type 2B) and magnesium-dependent (type 2C) phosphatases. Images Fig. 4. PMID:2155777

  10. Assessment the levels of tartrate-resistant acid phosphatase (TRAP) on mice fed with eggshell calcium citrate malate.

    PubMed

    Yu, Yiding; Zhang, Mingdi; Lin, Songyi; Wang, Liyan; Liu, Jingbo; Jones, Gregory; Huang, Hsiang-Chi

    2013-07-01

    Optimized conditions were obtained by one-factor-at-a-time test (OFAT) and ternary quadratic regression orthogonal composite design (TQROCD) respectively. By pulse electric fields (PEF) technology, the process of eggshell calcium citrate malate (ESCCM), eggshell calcium citrate (ESCC) and eggshells calcium malate (ESCM) were comprehensive compared. The levels of tartrate-resistant acid phosphatase (TRAP) and the bioavailability on mice fed with eggshell calcium citrate malate (ESCCM) treated by pulsed electric field (PEF) were evaluated. Results showed that the rates of calcium dissolution of the different acids studied can be arranged as ESCCM (7.90 mg/mL)>ESCC (7.12 mg/mL)>ESCM (7.08 mg/mL) from highest to lowest rate of dissolution. At the same dose 133.0 mg kg(-1) d(-1), the levels of TRAP in the ESCCM treatment groups were significantly lower than those in ESCM and ESCC (P<0.05). Bone calcium content in the mice fed with ESCCM was generally higher than fed with ESCM and ESCC. PMID:23603074

  11. Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin.

    PubMed

    Miller, S S; Liu, J; Allan, D L; Menzhuber, C J; Fedorova, M; Vance, C P

    2001-10-01

    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 M(r) 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 M(r) of 49,000 but migrates as a protein with M(r) 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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. p53 Codon 72 Genetic Polymorphism in Asthmatic Children: Evidence of Interaction With Acid Phosphatase Locus 1.

    PubMed

    Saccucci, Patrizia; Verrotti, Alberto; Giannini, Cosimo; Verini, Marcello; Chiarelli, Francesco; Neri, Anna; Magrini, Andrea

    2014-05-01

    Several lines of evidence are implicating an increased persistence of apoptotic cells in patients with asthma. This is largely due to a combination of inhibition, or defects in the apoptotic process and/or impaired apoptotic cell removal mechanisms. Among apoptosis-inducing genes, an important role is played by p53. In the present study, we have investigated the possible relationship between p53 codon 72 polymorphism and asthma and the interaction with ACP1, a genetic polymorphism involved in the susceptibility to allergic asthma. We studied 125 asthmatic children and 123 healthy subjects from the Caucasian population of Central Italy. p53 codon 72 and ACP1 polymorphisms were evaluated using a restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method. There is a statistically significant association between p53 codon 72 polymorphism and allergic asthma: Arg/Arg genotype is more represented in asthmatic patients than in controls (P=0.018). This association, however, is present in subjects with low ACP1 activity A/A and A/B only (P=0.023). The proportion of children with A/A and A/B genotype carrying Arg/Arg genotype is significantly high in asthmatic children than in controls (OR=1.941; 95% C.I. 1.042-3.628). Our finding could have important clinical implications since the subjects with A/A and A/B genotypes of ACP1 carrying Arg/Arg genotype are more susceptible to allergic asthma than Pro/Pro genotype. PMID:24843801

  15. Cytoplasmic Tyrosine Phosphatase Shp2 Coordinates Hepatic Regulation of Bile Acid and FGF15/19 Signaling to Repress Bile Acid Synthesis

    PubMed Central

    Li, Shuangwei; Hsu, Diane D.F.; Li, Bing; Luo, Xiaolin; Alderson, Nazilla; Qiao, Liping; Ma, Lina; Zhu, Helen H.; He, Zhao; Suino-Powell, Kelly; Ji, Kaihong; Li, Jiefu; Shao, Jianhua; Xu, H. Eric; Li, Tiangang; Feng, Gen-Sheng

    2015-01-01

    Summary Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR), and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that non-receptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through orchestration of multiple signals in hepatocytes. PMID:24981838

  16. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  17. Tartrate resistant acid phosphatase positive splenic lymphoma: a relatively benign condition occurring in a time-space cluster?

    PubMed Central

    Kettle, P; Morris, T C; Markey, G M; Alexander, H D; Curry, R C; Hayes, D; Cameron, C H; Toner, P G

    1990-01-01

    Conventional light and electron microscopic studies, together with cytochemical and immunocytochemical staining procedures, were carried out to ascertain whether the lymphomata of four elderly female patients living within 10 kilometers of each other, who presented within a short space of time with massive splenomegaly and varying cytopenia, belonged to any particular subgroup of lymphoma. In each case the lymphoma had a diffuse pattern and mature B cell phenotype. The malignant cells were of uniform cell type, slightly larger than admixed polymorphonuclear leucocytes, and showed minimal nuclear irregularity and positivity for tartrate resistant acid phosphatase (TRAP) staining. Their clinical and morphological features were compared with those of other lymphoproliferative disorders, but while sharing some features in common with each condition, this small group of patients seemed to have a unique combination of findings. The cytopenias of all four responded well after removal of the spleen and their disease has not been aggressive. It is concluded that these patients have a distinct subgroup of lymphoma, which it is important to recognise so that inappropriate use of aggressive cytotoxic drugs can be avoided. Images PMID:1698823

  18. Sensitivity and specificity of acid phosphatase to detect prostate cancer using data from a hospital information system.

    PubMed

    Zwetsloot-Schonk, J H; Hermans, J; Frolich, M; Snitker, P; Souverijn, J H; Zwartendijk, J

    1990-07-01

    Indices of diagnostic tests, such as sensitivity and specificity, should be determined using diagnostic test results of patients tested in clinical practice. Hospital information systems that store data on diagnostic tests and diagnoses might be used for sampling the desired study population and in the actual process of collecting the data. This paper presents, as an example, a study calculating the sensitivity and specificity of the prostate-specific acid phosphatase test. All data needed in the study were obtained from the hospital information system of Leiden University Hospital. The final health status of each patient was assessed by the cancer registry of the system. The reason for ordering the test was deduced from data on histopathological examinations of prostatic tissue. The actual selections made from the central database are described in dataflow diagrams. The sensitivity of the test was found to be 0.34 and the specificity 0.88, using a discrimination value of 1.00 U/l. The impact of the reason for ordering the test on the specificity is illustrated. Possible biases of these measured values are discussed. PMID:2215263

  19. Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid.

    PubMed

    Léran, Sophie; Edel, Kai H; Pervent, Marjorie; Hashimoto, Kenji; Corratgé-Faillie, Claire; Offenborn, Jan Niklas; Tillard, Pascal; Gojon, Alain; Kudla, Jörg; Lacombe, Benoît

    2015-05-01

    Living organisms sense and respond to changes in nutrient availability to cope with diverse environmental conditions. Nitrate (NO3-) is the main source of nitrogen for plants and is a major component in fertilizer. Unraveling the molecular basis of nitrate sensing and regulation of nitrate uptake should enable the development of strategies to increase the efficiency of nitrogen use and maximize nitrate uptake by plants, which would aid in reducing nitrate pollution. NPF6.3 (also known as NRT1.1), which functions as a nitrate sensor and transporter; the kinase CIPK23; and the calcium sensor CBL9 form a complex that is crucial for nitrate sensing in Arabidopsis thaliana. We identified two additional components that regulate nitrate transport, sensing, and signaling: the calcium sensor CBL1 and protein phosphatase 2C family member ABI2, which is inhibited by the stress-response hormone abscisic acid. Bimolecular fluorescence complementation assays and in vitro kinase assays revealed that ABI2 interacted with and dephosphorylated CIPK23 and CBL1. Coexpression studies in Xenopus oocytes and analysis of plants deficient in ABI2 indicated that ABI2 enhanced NPF6.3-dependent nitrate transport, nitrate sensing, and nitrate signaling. These findings suggest that ABI2 may functionally link stress-regulated control of growth and nitrate uptake and utilization, which are energy-expensive processes. PMID:25943353

  20. Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions.

    PubMed Central

    Cantarella, M; Remy, M H; Scardi, V; Alfani, F; Iorio, G; Greco, G

    1979-01-01

    1. An analysis of the kinetic behaviour of immobilized acid phosphatase (EC 3.1.3.2) layers, gelled on the active surface of an ultrafiltration membrane, was carried out. 2. Two possible forms of such immobilized-enzyme systems were dealt with, namely enzyme-polyalbumin co-gelation through an ultrafiltration process, and enzyme co-polymerization to the same albumin polymers and subsequent gelation. 3. A preliminary analysis was also performed on both the corresponding homogeneous-phase (soluble systems to provide reference kinetics. 4. The main conclusions drawn are: (i) the enzyme-albumin co-polymers show a decrease in specific activity compared with the corresponding free enzyme in both soluble and immobilized forms; (ii) in the homogeneous phase a slight increase in the apparent Michaelis constant was measured for the co-polymerized enzyme compared with the free one, which suggests a decrease in affinity towards substrate; (iii) the activation energy in the immobilized phase is halved, compared with that in the homogeneous phase, which indicates that the combined mass-transfer/reaction step is rate-controlling. PMID:475752

  1. Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.

    PubMed Central

    Takai, A; Ohno, Y; Yasumoto, T; Mieskes, G

    1992-01-01

    As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses. PMID:1329723

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

    PubMed Central

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

    2004-01-01

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

  3. 7 CFR 701.44 - Agricultural Conservation Program (ACP) contracts.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... be subject to, the regulations for ACP contracts and the ACP program that were contained in the 7 CFR... 7 Agriculture 7 2012-01-01 2012-01-01 false Agricultural Conservation Program (ACP) contracts. 701... AGENCY, DEPARTMENT OF AGRICULTURE AGRICULTURAL CONSERVATION PROGRAM EMERGENCY CONSERVATION...

  4. 7 CFR 701.44 - Agricultural Conservation Program (ACP) contracts.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... be subject to, the regulations for ACP contracts and the ACP program that were contained in the 7 CFR... 7 Agriculture 7 2014-01-01 2014-01-01 false Agricultural Conservation Program (ACP) contracts. 701... AGENCY, DEPARTMENT OF AGRICULTURE AGRICULTURAL CONSERVATION PROGRAM EMERGENCY CONSERVATION...

  5. 7 CFR 701.44 - Agricultural Conservation Program (ACP) contracts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... be subject to, the regulations for ACP contracts and the ACP program that were contained in the 7 CFR... 7 Agriculture 7 2011-01-01 2011-01-01 false Agricultural Conservation Program (ACP) contracts. 701... AGENCY, DEPARTMENT OF AGRICULTURE AGRICULTURAL CONSERVATION PROGRAM EMERGENCY CONSERVATION...

  6. 7 CFR 701.44 - Agricultural Conservation Program (ACP) contracts.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... be subject to, the regulations for ACP contracts and the ACP program that were contained in the 7 CFR... 7 Agriculture 7 2013-01-01 2013-01-01 false Agricultural Conservation Program (ACP) contracts. 701... AGENCY, DEPARTMENT OF AGRICULTURE AGRICULTURAL CONSERVATION PROGRAM EMERGENCY CONSERVATION...

  7. 7 CFR 701.44 - Agricultural Conservation Program (ACP) contracts.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... regulations for ACP contracts and the ACP program that were contained in the 7 CFR, parts 700 to 899, edition... 7 Agriculture 7 2010-01-01 2010-01-01 false Agricultural Conservation Program (ACP) contracts. 701... AGENCY, DEPARTMENT OF AGRICULTURE AGRICULTURAL CONSERVATION PROGRAM EMERGENCY CONSERVATION PROGRAM...

  8. Activation of protein phosphatase 2A is responsible for increased content and inactivation of respiratory chain complex i induced by all-trans retinoic acid in human keratinocytes.

    PubMed

    Papa, F; Sardaro, N; Lippolis, R; Panelli, D; Scacco, S

    2016-01-01

    This study presents the effect of all-trans retinoic acid (ATRA) on cell growth and respiratory chain complex I in human keratinocyte cultures. Keratinocyte treatment results in increased level of GRIM-19 and other subunits of complex I, in particular of their carbonylated forms, associated with inhibition of its enzymatic activity. The results show that in keratinocytes ATRA-promoted phosphatase activity controls the proteostasis and activity of complex I. PMID:27358125

  9. Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori

    PubMed Central

    Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.

    2010-01-01

    Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera. PMID:20874583

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  12. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

    PubMed

    Moreno-Pérez, Antonio J; Sánchez-García, Alicia; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2011-01-01

    The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results. PMID:21071236

  13. Lipid phosphate phosphatase inhibitors locally amplify lysophosphatidic acid LPA1 receptor signalling in rat brain cryosections without affecting global LPA degradation

    PubMed Central

    2012-01-01

    Background Lysophosphatidic acid (LPA) is a signalling phospholipid with multiple biological functions, mainly mediated through specific G protein-coupled receptors. Aberrant LPA signalling is being increasingly implicated in the pathology of common human diseases, such as arteriosclerosis and cancer. The lifetime of the signalling pool of LPA is controlled by the equilibrium between synthesizing and degradative enzymatic activity. In the current study, we have characterized these enzymatic pathways in rat brain by pharmacologically manipulating the enzymatic machinery required for LPA degradation. Results In rat brain cryosections, the lifetime of bioactive LPA was found to be controlled by Mg2+-independent, N-ethylmaleimide-insensitive phosphatase activity, attributed to lipid phosphate phosphatases (LPPs). Pharmacological inhibition of this LPP activity amplified LPA1 receptor signalling, as revealed using functional autoradiography. Although two LPP inhibitors, sodium orthovanadate and propranolol, locally amplified receptor responses, they did not affect global brain LPA phosphatase activity (also attributed to Mg2+-independent, N-ethylmaleimide-insensitive phosphatases), as confirmed by Pi determination and by LC/MS/MS. Interestingly, the phosphate analog, aluminium fluoride (AlFx-) not only irreversibly inhibited LPP activity thereby potentiating LPA1 receptor responses, but also totally prevented LPA degradation, however this latter effect was not essential in order to observe AlFx--dependent potentiation of receptor signalling. Conclusions We conclude that vanadate- and propranolol-sensitive LPP activity locally guards the signalling pool of LPA whereas the majority of brain LPA phosphatase activity is attributed to LPP-like enzymatic activity which, like LPP activity, is sensitive to AlFx- but resistant to the LPP inhibitors, vanadate and propranolol. PMID:22686545

  14. Antitumor effects of methotrexate-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor

    SciTech Connect

    Deguchi, T.; Chu, T.M.; Leong, S.S.; Horoszewicz, J.S.; Lee, C.L.

    1986-03-01

    Methotrexate (MTX) was conjugated to an IgG/sub 1/ monoclonal antibody (MCA) specific for human prostatic acid phosphatase (PAP) by an active ester method, resulting in a molar ratio of MTX to IgG/sub 1/ of 14. MTX-MCA conjugate retained 94% of free antibody activity and preserved 90% of dihydrofolate reductase inhibitory activity of free MTX. MTX-MCA conjugate was shown to be accumulated in vitro by prostate tumor cells (LNCaP) 1.3 times higher than that of MTX conjugate to normal mouse IgG (NIgG) and 6.2 times higher than that of free MTX. Antitumor activity in vitro exhibited that MTX-MCA conjugate is more effective on inhibition (52%) of /sup 3/H-deoxyuridine incorporation into LNCaP cells than that of MTX-NIgG (39%), but both were less effective than free MTX (70%). The in vivo distribution of /sup 3/H-MTX-MCA conjugate in human prostate tumor xenograft (tumor: blood ratio 5.1) was higher than those of /sup 3/H-MTX-NIgG conjugate (1.1) and of free /sup 3/H-MTX (1.5). Anti-tumor activity in vivo demonstrated that MTX-MCA conjugate retarded the growth of xenografted human prostate tumor greatly and persistently, as compared with the control groups. These results suggested that MTX-monoclonal anti-PAP antibody conjugate represents a potential reagent for immunochemotherapy of human prostate tumor (NIH CA-34536, CA-15437 and ACS CH-269.

  15. NGF-trkA signaling modulates the analgesic effects of prostatic acid phosphatase in resiniferatoxin-induced neuropathy

    PubMed Central

    Wu, Chieh-Hsin; Ho, Wan-Yi; Lee, Yi-Chen

    2016-01-01

    Background Neuropathic pain in small-fiber neuropathy results from injury to and sensitization of nociceptors. Functional prostatic acid phosphatase (PAP) acts as an analgesic effector. However, the mechanism responsible for the modulation of PAP neuropathology, which leads to loss of the analgesic effect after small-fiber neuropathy, remains unclear. Results We used a resiniferatoxin (RTX)-induced small-fiber neuropathy model to examine whether functional PAP(+) neurons are essential to maintain the analgesic effect. PAP(+) neurons were categorized into small to medium neurons (25th–75th percentile: 17.1–23.7 µm); these neurons were slightly reduced by RTX (p = 0.0003). By contrast, RTX-induced activating transcription factor 3 (ATF3), an injury marker, in PAP(+) neurons (29.0% ± 5.6% vs. 0.2% ± 0.2%, p = 0.0043), indicating PAP neuropathology. Moreover, the high-affinity nerve growth factor (NGF) receptor (trkA) colocalized with PAP and showed similar profiles after RTX-induced neuropathy, and the PAP/trkA ratios correlated with the degree of mechanical allodynia (r = 0.62, p = 0.0062). The NGF inducer 4-methylcatechol (4MC) normalized the analgesic effects of PAP; specifically, it reversed the PAP and trkA profiles and relieved mechanical allodynia. Administering 2.5S NGF showed similar results to those of administering 4MC. This finding suggests that the analgesic effect of functional PAP is mediated by NGF-trkA signaling, which was confirmed by NGF neutralization. Conclusions This study revealed that functional PAP(+) neurons are essential for the analgesic effect, which is mediated by NGF-trkA signaling. PMID:27306411

  16. Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize.

    PubMed

    Wang, Ying-Ge; Yu, Hao-Qiang; Zhang, Yuan-Yuan; Lai, Cong-Xian; She, Yue-Hui; Li, Wan-Chen; Fu, Feng-Ling

    2014-10-01

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively. PMID:25091169

  17. Solution Structure of 4′-Phosphopantetheine - GmACP3 from Geobacter metallireducens: A Specialized Acyl Carrier Protein with Atypical Structural Features and a Putative Role in Lipopolysaccharide Biosynthesis†

    PubMed Central

    Ramelot, Theresa A.; Smola, Matthew J.; Lee, Hsiau-Wei; Ciccosanti, Colleen; Hamilton, Keith; Acton, Thomas B.; Xiao, Rong; Everett, John K.; Prestegard, James H.; Montelione, Gaetano T.; Kennedy, Michael A.

    2011-01-01

    GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by 15N NMR relaxation measurements. Addition of 4′-phosphopantetheine (4′-PP) via enzymatic conversion by E. coli holo-ACP synthase, resulted in stable >95% holo-GmACP3 that was characterized as monomeric by 15N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4′-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4′-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and it’s orthologs. The helix locations and the large hydrophobic cavity are more similar to medium- and long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggest that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially involved in synthesis of secondary metabolites. PMID:21235239

  18. Phosphorylation of Lipin 1 and Charge on the Phosphatidic Acid Head Group Control Its Phosphatidic Acid Phosphatase Activity and Membrane Association*

    PubMed Central

    Eaton, James M.; Mullins, Garrett R.; Brindley, David N.; Harris, Thurl E.

    2013-01-01

    The lipin gene family encodes a class of Mg2+-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. PMID:23426360

  19. The Arabidopsis mitogen-activated protein kinase phosphatase PP2C5 affects seed germination, stomatal aperture, and abscisic acid-inducible gene expression.

    PubMed

    Brock, Anita K; Willmann, Roland; Kolb, Dagmar; Grefen, Laure; Lajunen, Heini M; Bethke, Gerit; Lee, Justin; Nürnberger, Thorsten; Gust, Andrea A

    2010-07-01

    Abscisic acid (ABA) is an important phytohormone regulating various cellular processes in plants, including stomatal opening and seed germination. Although protein phosphorylation via mitogen-activated protein kinases (MAPKs) has been suggested to be important in ABA signaling, the corresponding phosphatases are largely unknown. Here, we show that a member of the Protein Phosphatase 2C (PP2C) family in Arabidopsis (Arabidopsis thaliana), PP2C5, is acting as a MAPK phosphatase. The PP2C5 protein colocalizes and directly interacts with stress-induced MPK3, MPK4, and MPK6, predominantly in the nucleus. Importantly, altered PP2C5 levels affect MAPK activation. Whereas Arabidopsis plants depleted of PP2C5 show an enhanced ABA-induced activation of MPK3 and MPK6, ectopic expression of PP2C5 in tobacco (Nicotiana benthamiana) resulted in the opposite effect, with the two MAPKs salicylic acid-induced protein kinase and wound-induced protein kinase not being activated any longer after ABA treatment. Moreover, depletion of PP2C5, whose gene expression itself is affected by ABA treatment, resulted in altered ABA responses. Loss-of-function mutation in PP2C5 or AP2C1, a close PP2C5 homolog, resulted in an increased stomatal aperture under normal growth conditions and a partial ABA-insensitive phenotype in seed germination that was most prominent in the pp2c5 ap2c1 double mutant line. In addition, the response of ABA-inducible genes such as ABI1, ABI2, RD29A, and Erd10 was reduced in the mutant plants. Thus, we suggest that PP2C5 acts as a MAPK phosphatase that positively regulates seed germination, stomatal closure, and ABA-inducible gene expression. PMID:20488890

  20. Measuring phosphatidic acid phosphatase (EC 3.1.3.4) activity using two phosphomolybdate-based colorimetric methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), which is also known as PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol (DAG) and inorganic phosphate. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacyl...

  1. Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid.

    PubMed

    Mdluli, K; Slayden, R A; Zhu, Y; Ramaswamy, S; Pan, X; Mead, D; Crane, D D; Musser, J M; Barry, C E

    1998-06-01

    Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug. PMID:9616124

  2. Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

    PubMed Central

    Kuo, A; Cappelluti, S; Cervantes-Cervantes, M; Rodriguez, M; Bush, D S

    1996-01-01

    The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling. PMID:8742711

  3. Differential therapeutic responses of thiol compounds in the reversal of methylmercury inhibited acid phosphatase and cathepsin E in the central nervous system of rat

    SciTech Connect

    Vinay, S.D.; Raghu, K.G.; Sood, P.P.

    1992-07-01

    Though considerable headway has been made in elucidating the effect of methylmercury on the biochemical machinery of nervous system, the studies on the alterations in the levels of acid hydrolases received less attention. Being a lysosomal marker, acid phosphatase is one of the most extensively studies enzymes amongst the acid hydrolases. Its significance in various key physiological as well as pathological processes is well preserved in literature. Cathepsin E, an aspartic proteinase, has been demonstrated in a number of cells and tissues within the human body, rat, E. coli where its role is implicated in a number of important metabolic processes. In the present paper, we report the results of the differential levels of inhibition of these enzymes with methylmercury as well as their differential recoveries with two thiols (N-acetyl-DL-homocysteine thiolactone and glutathione) in various neuroanatomical areas (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata and spinal cord) of rat. 22 refs., 5 figs.

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

    PubMed Central

    Antonyuk, Svetlana Vladimirovna; Olczak, Mariusz; Olczak, Teresa; Ciuraszkiewicz, Justyna; Strange, Richard William

    2014-01-01

    Phosphatases function in the production, transport and recycling of inorganic phosphorus, which is crucial for cellular metabolism and bioenergetics, as well as in bacterial killing, since they are able to generate reactive oxygen species via Fenton chemistry. Diphosphonucleotide phosphatase/phosphodiesterase (PPD1), a glycoprotein plant purple acid phosphatase (PAP) from yellow lupin seeds, contains a bimetallic Fe–Mn catalytic site which is most active at acidic pH. Unlike other plant PAPs, PPD1 cleaves the pyrophosphate bond in diphosphonucleotides and the phosphodiester bond in various phosphodiesters. The homohexameric organization of PPD1, as revealed by a 1.65 Å resolution crystal structure and confirmed by solution X-ray scattering, is unique among plant PAPs, for which only homodimers have previously been reported. A phosphate anion is bound in a bidentate fashion at the active site, bridging the Fe and Mn atoms in a binding mode similar to that previously reported for sweet potato PAP, which suggests that common features occur in their catalytic mechanisms. The N-terminal domain of PPD1 has an unexpected and unique fibronectin type III-like fold that is absent in other plant PAPs. Here, the in vitro DNA-cleavage activity of PPD1 is demonstrated and it is proposed that the fibronectin III-like domain, which ‘overhangs’ the active site, is involved in DNA selectivity, binding and activation. The degradation of DNA by PPD1 implies a role for PPD1 in plant growth and repair and in pathogen defence. PMID:25075326

  5. OsPAP10c, a novel secreted acid phosphatase in rice, plays an important role in the utilization of external organic phosphorus.

    PubMed

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

    2016-10-01

    Under phosphate (Pi ) starvation, plants increase the secretion of purple acid phosphatases (PAPs) into the rhizosphere to scavenge organic phosphorus (P) for plant use. To date, only a few members of the PAP family have been characterized in crops. In this study, we identified a novel secreted PAP in rice, OsPAP10c, and investigated its role in the utilization of external organic P. OsPAP10c belongs to a monocotyledon-specific subclass of Ia group PAPs and is specifically expressed in the epidermis/exodermis cell layers of roots. Both the transcript and protein levels of OsPAP10c are strongly induced by Pi starvation. OsPAP10c overexpression increased acid phosphatase (APase) activity by more than 10-fold in the culture media and almost fivefold in both roots and leaves under Pi -sufficient and Pi -deficient conditions. This increase in APase activity further improved the plant utilization efficiency of external organic P. Moreover, several APase isoforms corresponding to OsPAP10c were identified using in-gel activity assays. Under field conditions with three different Pi supply levels, OsPAP10c-overexpressing plants had significantly higher tiller numbers and shorter plant heights. This study indicates that OsPAP10c encodes a novel secreted APase that plays an important role in the utilization of external organic P in rice. PMID:27411391

  6. Derepressed 2-deoxyglucose-resistant mutants of Aspergillus niger with altered hexokinase and acid phosphatase activity in hyperproduction of beta-fructofuranosidase.

    PubMed

    Ashokkumar, B; Senthilkumar, S R; Gunasekaran, P

    2004-01-01

    Aspergillus niger NRRL330 produces extracellular beta-fructofuranosidase (Ffase), and its production is subject to repression by hexoses in the medium. After ultraviolet mutagenization and selection, seven derepressed mutants resistant to 2-deoxyglucose (2-DG) were isolated on Czapek's minimal medium containing glycerol. One of the mutants, designated DGRA-1, produced higher levels of Ffase. A considerable difference occurred in the mutants with reference to hexokinase and intracellular acid phosphatase activities. The hexokinase activity of the mutant DGRA-1 (0.69 U/mg) was 1.8-fold higher than the wild type (0.38 U/mg). Intracellular acid phosphatase activity of the mutant DGRA-1 (0.83 U/g of mycelia) was twofold higher than that of the wild type (0.42 U/g of mycelia), suggesting that phosphorylation and dephosphorylation steps could attribute to the 2-DG resistance of A. niger. However, additional mutations could account for the increased production of Ffase in the mutant DGRA-1. PMID:15304742

  7. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

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

    PubMed Central

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

    2015-01-01

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

  9. FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis.

    PubMed

    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

    2016-09-13

    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

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

    SciTech Connect

    Parente, J.E.; Risley, J.M.; Van Etten, R.L.

    1984-12-26

    The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixture of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.

  11. Contribution of chlorogenic acids to the inhibition of human hepatic glucose-6-phosphatase activity in vitro by Svetol, a standardized decaffeinated green coffee extract.

    PubMed

    Henry-Vitrac, Caroline; Ibarra, Alvin; Roller, Marc; Mérillon, Jean-Michel; Vitrac, Xavier

    2010-04-14

    Glucose-6-phosphatase (Glc-6-Pase) is a multicomponent system that exists primarily in the liver and catalyzes the terminal step in gluconeogenesis and glycogenolysis. Several studies have attempted to identify synthetic or natural compounds that inhibit this enzyme complex for therapeutic use in regulating blood glucose and type 2 diabetes. For this paper an in vitro structure-activity relationship study of several natural chlorogenic acids was conducted, and the active components of the natural decaffeinated green coffee extract Svetol were identified. Glucose-6-phosphate (Glc-6-P) hydrolysis was measured in the presence of Svetol or chlorogenic acids in intact human liver microsomes. Svetol significantly inhibited Glc-6-P hydrolysis in intact human liver microsomes in a competitive manner, and it was determined that chlorogenic acids (caffeoylquinic acids and dicaffeoylquinic acids) were the chief compounds mediating this activity. In addition, the structure-activity analysis showed that variation in the position of the caffeoyl residue is an important determinant of inhibition of Glc-6-P hydrolysis. This inhibition by Svetol contributes to its antidiabetic, glucose-lowering effects by reducing hepatic glucose production. PMID:20302380

  12. 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

  13. A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells*

    PubMed Central

    Ramesh, Mathangi; Krishnan, Navasona; Muthuswamy, Senthil K.; Tonks, Nicholas K.

    2015-01-01

    We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function. PMID:25681440

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. [The cellular acid phosphatase activity in yeast-like fungi of the genus Candida exposed to ultrasound, polyene antibiotics and dyes].

    PubMed

    Sergeev, P V; Romanenko, I M; Ukhina, T V

    1993-09-01

    The activity of one of the lysosomal membrane marker enzymes--acid phosphatase from the Candida yeast fungi on their exposure to ultrasound (US), polyenic antibiotics (amphotericin B and nystatin) dye antiseptics (ethacridine lactate, methylene blue), and their combinations was assayed. The impact of US and the drugs, in particular their combination, was found to be followed by activation of the fungal lysosomal apparatus function and increases in their catabolic processes. The highest rise in lysosomal catabolic activity was found when the polyenic antibiotics were used in combination with US, which reflects the higher damaging effect of this combination against Candida lysosomal membranes than the dyes and of these antibiotics and US alone. The studies provide strong evidence for the preference of the combined use of US and the polyenic antibiotics in candidiasis as a factor enhancing their fungicidal effect against Candida yeast fungi. PMID:8118000

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

    PubMed

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

    2009-04-01

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

  18. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  19. Three-dimensional structure of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27: a new member of the haloalcanoic acid dehalogenase superfamily.

    PubMed

    Gonçalves, Susana; Esteves, Ana M; Santos, Helena; Borges, Nuno; Matias, Pedro M

    2011-11-01

    Mannosyl-3-phosphoglycerate phosphatase (MpgP) is a key mediator in the physiological response to thermal and osmotic stresses, catalyzing the hydrolysis of mannosyl-3-phosphoglycerate (MPG) to the final product, α-mannosylglycerate. MpgP is a metal-dependent haloalcanoic acid dehalogenase-like (HAD-like) phosphatase, preserving the catalytic motifs I-IV of the HAD core domain, and classified as a Cof-type MPGP (HAD-IIB-MPGP family; SCOP [117505]) on the basis of its C2B cap insertion module. Herein, the crystallographic structures of Thermus thermophilus HB27 MpgP in its apo form and in complex with substrates, substrate analogues, and inhibitors are reported. Two distinct enzyme conformations, open and closed, are catalytically relevant. Apo-MpgP is primarily found in the open state, while holo-MpgP, in complex with the reaction products, is found in the closed state. Enzyme activation entails a structural rearrangement of motifs I and IV with concomitant binding of the cocatalytic Mg(2+) ion. The closure motion of the C2B domain is subsequently triggered by the anchoring of the phosphoryl group to the cocatalytic metal center, and by Arg167 fixing the mannosyl moiety inside the catalytic pocket. The results led to the proposal that in T. thermophilus HB27 MpgP the phosphoryl transfer employs a concerted D(N)S(N) mechanism with assistance of proton transfer from the general acid Asp8, forming a short-lived PO(3)(-) intermediate that is attacked by a nucleophilic water molecule. These results provide new insights into a possible continuum of phosphoryl transfer mechanisms, ranging between those purely associative and dissociative, as well as a picture of the main mechanistic aspects of phosphoryl monoester transfer catalysis, common to other members of the HAD superfamily. PMID:21961705

  20. A major root-associated acid phosphatase in Arabidopsis, AtPAP10, is regulated by both local and systemic signals under phosphate starvation

    PubMed Central

    Zhang, Ye; Wang, Xiaoyue; Lu, Shan; Liu, Dong

    2014-01-01

    The induction and secretion of acid phosphatases (APases) is a universal response of plants to phosphate (Pi) starvation. AtPAP10 (Arabidopsis purple acid phosphatase 10) is a major Pi starvation-induced APase that is associated with the root surface in Arabidopsis. So far, the roles of local and systemic signalling in regulating root-associated AtPAP10 activity remain largely unknown. In this work, we show that a decrease of local, external Pi availability is sufficient to induce AtPAP10 transcription in roots in the presence of sucrose, a systemic signal from shoots, whereas the magnitude of the induction is affected by the Pi status of the whole plant. Once the AtPAP10 mRNAs are synthesized in roots, subsequent accumulation of AtPAP10 proteins in root cells and increase in AtPAP10 activity on the root surface are mainly controlled by local signalling. Previously, ethylene has been demonstrated to be a positive regulator of AtPAP10 activity. In this study, we provide evidence that under Pi deficiency ethylene mainly modulates enzymatic activity of AtPAP10 on the root surface, but not AtPAP10 transcription and protein accumulation, suggesting that it functions as a local signal. Furthermore, our work indicates that the effect of ethylene on the induction of root-associated AtPAP10 activity depends on sucrose, but that the effect of sucrose does not depend on ethylene. These results reveal new insights into the distinct roles of local and systemic signalling in the regulation of root-associated AtPAP10 activity under Pi starvation. PMID:25246445

  1. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

    PubMed

    Shane, Michael W; Stigter, Kyla; Fedosejevs, Eric T; Plaxton, William C

    2014-11-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  2. Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.

    PubMed

    Sánchez-García, Alicia; Moreno-Pérez, Antonio J; Muro-Pastor, Alicia M; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2010-06-01

    Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed. PMID:20382402

  3. Rhamnolipid and poly (hydrozyalkanoate) biosynthesis in 3-hydrozyacyl-ACP:COA transacylase (phaG) - knockouts of pseudomonas chloroaphis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 3-hydroxyacyl-ACP:CoA transacylase gene (phaG(Pc30761)) of P. chlororaphis NRRL B-30761 was cloned and analyzed. The nucleotide and translated amino-acid sequences of phaG(Pc30761) had 99% identities (at 100% query coverage) with the phaG gene of P. fluorescens O6. Two phaG-knockout strains of...

  4. Design and Synthesis of 2-Pyridones as Novel Inhibitors of the Bacillus Anthracis Enoyl–ACP Reductase

    PubMed Central

    Tipparaju, Suresh K.; Joyasawal, Sipak; Forrester, Sara; Mulhearn, Debbie C.; Pegan, Scott; Johnson, Michael E.; Mesecar, Andrew D.; Kozikowski, Alan P.

    2008-01-01

    Enoyl-ACP reductase (ENR), the product of the FabI gene, from Bacillus anthracis (BaENR) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis. A number of novel 2-pyridone derivatives were synthesized and shown to be potent inhibitors of BaENR. PMID:18499454

  5. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    PubMed

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  6. The ACP (Advanced Computer Program) Branch bus and real-time applications of the ACP multiprocessor system

    SciTech Connect

    Hance, R.; Areti, H.; Atac, R.; Biel, J.; Cook, A.; Fischler, M.; Gaines, I.; Husby, D.; Nash, T.; Zmuda, T.

    1987-05-08

    The ACP Branchbus, a high speed differential bus for data movement in multiprocessing and data acquisition environments, is described. This bus was designed as the central bus in the ACP multiprocessing system. In its full implementation with 16 branches and a bus switch, it will handle data rates of 160 MByte/sec and allow reliable data transmission over inter rack distances. We also summarize applications of the ACP system in experimental data acquisition, triggering and monitoring, with special attention paid to FASTBUS environments.

  7. The Interactions of CPP–ACP with Saliva

    PubMed Central

    Huq, Noorjahan Laila; Myroforidis, Helen; Cross, Keith J.; Stanton, David P.; Veith, Paul D.; Ward, Brent R.; Reynolds, Eric C.

    2016-01-01

    The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP–ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP–ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP–ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP–ACP complexes are localised at the tooth surface to promote remineralisation. PMID:27294918

  8. The Interactions of CPP-ACP with Saliva.

    PubMed

    Huq, Noorjahan Laila; Myroforidis, Helen; Cross, Keith J; Stanton, David P; Veith, Paul D; Ward, Brent R; Reynolds, Eric C

    2016-01-01

    The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP-ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP-ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP-ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP-ACP complexes are localised at the tooth surface to promote remineralisation. PMID:27294918

  9. Selection on the Drosophila seminal fluid protein Acp62F

    PubMed Central

    Wong, Alex; Rundle, Howard

    2013-01-01

    Sperm competition and sexual conflict are thought to underlie the rapid evolution of reproductive proteins in many taxa. While comparative data are generally consistent with these hypotheses, few manipulative tests have been conducted and those that have provided contradictory results in some cases. Here, we use both comparative and experimental techniques to investigate the evolution of the Drosophila melanogaster seminal fluid protein Acp62F, a protease inhibitor for which extensive functional tests have yielded ambiguous results. Using between-species sequence comparisons, we show that Acp62F has been subject to recurrent positive selection. In addition, we experimentally evolved populations polymorphic for an Acp62F null allele over eight generations, manipulating the opportunities for natural and sexual selection. We found that the Acp62F null allele increased in frequency in the presence of natural selection, with no effect of sexual selection. PMID:23919141

  10. Spatial structure of heptapeptide Glu-Ile-Leu-Asn-His-Met-Lys, a fragment of the HIV enhancer prostatic acid phosphatase, in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Bloсhin, Dmitri S.; Aganova, Oksana V.; Yulmetov, Aidar R.; Filippov, Andrei V.; Gizatullin, Bulat I.; Afonin, Sergii; Antzutkin, Oleg N.; Klochkov, Vladimir V.

    2013-02-01

    Prostatic acid phosphatase (PAP) is a protein abundantly present in human seminal fluid. PAP plays important role in fertilization. Its 39-amino-acid fragment, PAP(248-286), is effective in enhancing infectivity of HIV virus. In this work, we determined the spatial structure in aqueous solution of a heptapeptide within the PAP fragment, containing amino acid residues 266-272 (Glu-Ile-Leu-Asn-His-Met-Lys). We also report the structure of the complex formed by this heptapeptide with sodium dodecyl sulfate micelles, a model of a biological membrane, as determined by 1H NMR spectroscopy and 2D NMR (TOCSY, HSQC-HECADE, NOESY) spectroscopy. Complex formation was confirmed by chemical shift alterations in the 1H NMR spectra of the heptapeptide, as well as by the signs and values of NOE effects. We also present a comparison of the spatial structure of Glu-Ile-Leu-Asn-His-Met-Lys in water and in complex with sodium dodecyl sulfate.

  11. 26 CFR 1.401(m)-2 - ACP test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... satisfy the ACP test, but not in excess of 100%. In this case, an increase in the match rate from 50% to... under the ACP test only to the extent they do not exceed 25.00% of compensation. In this case, all of... determined under § 1.401(m)-2(b)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1)....

  12. The β-ketoacyl-ACP synthase from Mycobacterium tuberculosis as potential drug targets.

    PubMed

    Singh, V; Mani, I; Chaudhary, D K; Somvanshi, P

    2011-01-01

    The continuous preventive measures and control of tuberculosis are often hampered by re-emergence of multi-drug-resistant (MDR) strains of Mycobacterium tuberculosis. A novel drug approach is desperately needed to combat the global threat posed by MDR strains. In spite of current advancement in biological techniques viz. microarray and proteomics data for tuberculosis, no such potent drug has been developed in the past decades yet. Therefore, mycolic acid is an essential constituent which is involved in the formation of cell wall of Mycobacterium species. The biosynthesis of mycolic acid is involved in two fatty acid synthase systems, the multifunctional polypeptide fatty acid synthase I (FASI) which performs de novo fatty acid synthesis and dissociate FASII system. FASII system consists of monofunctional enzymes and acyl carrier protein (ACP), elongating FASI products to long chain mycolic acid precursor. In this review, the β-ketoacyl-ACP synthases (fadH, kasA and kasB) are distinct and play a vital role in mycolic acid synthesis, cell wall synthesis, biofilm formation and also pathogenesis. On the basis of substantial observation we suggest that these enzymes may be used as promising and attractive targets for novel anti-TB drugs designing and discovery. PMID:21370994

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

    PubMed

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  14. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  15. Abscisic Acid Promotion of Arbuscular Mycorrhizal Colonization Requires a Component of the PROTEIN PHOSPHATASE 2A Complex1[W][OPEN

    PubMed Central

    Charpentier, Myriam; Sun, Jongho; Wen, Jiangqi; Mysore, Kirankumar S.; Oldroyd, Giles E.D.

    2014-01-01

    Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB′1. Mutations in PP2AB′1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB′1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB′1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization. PMID:25293963

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

    PubMed

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

    2015-01-01

    Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase indole-3-butyric acid-response5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways. PMID:26147117

  17. O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

    PubMed

    Ilg, T; Overath, P; Ferguson, M A; Rutherford, T; Campbell, D G; McConville, M J

    1994-09-30

    The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of

  18. ALP (Alkaline Phosphatase) Test

    MedlinePlus

    ... known as: ALK PHOS; Alkp Formal name: Alkaline Phosphatase Related tests: AST ; ALT ; GGT ; Bilirubin ; Liver Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP All content on Lab ...

  19. Biochemical study on the protective role of folic acid in rabbits treated with chromium (VI).

    PubMed

    El-Demerdash, Fatma M; Yousef, Mokhtar I; Elaswad, Fathia A M

    2006-01-01

    Deleterious effects of chromium (VI) compounds are diversified affecting almost all the organ systems in a wide variety of animals. Therefore, the present study was carried out to determine the effectiveness of folic acid (FA) in alleviating the toxicity of chromium (VI) on certain biochemical parameters, lipid peroxidation, and enzyme activities of male New Zealand white rabbits. Six rabbits per group were assigned to one of four treatment groups: 0 mg FA and 0 mg Cr(VI)/kg BW (control); 8.3 microg FA/kg BW; 5 mg Cr(VI)/kg BW; 5 mg Cr(VI) plus 8.3 microg FA/kg BW, respectively. Rabbits were orally administered their respective doses every day for 10 weeks. Results obtained showed that Cr(VI) significantly (P < 0.05) increased the levels of free radicals and the activity of glutathione S-transferase (GST), and decreased the content of sulfhydryl groups (SH groups) in liver, testes, brain, kidney, and lung. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AlP), acid phosphatase (AcP), and lactate dehydrogenase (LDH) were significantly decreased in liver and testes due to Cr(VI) administration. Also, AlP and AcP activities were significantly decreased in kidney and lung. The activity of acetylcholinesterase (AChE) was significantly decreased in brain and plasma. Contrariwise, the activities of AST and ALT were significantly increased in plasma, while AlP and AcP decreased. Chromium (VI) treatment caused a significant decrease in plasma total protein (TP) and globulin, and increased total lipids (TL), cholesterol, glucose, urea, creatinine, and bilirubin concentrations. Folic acid alone significantly decreased the levels of free radicals in liver, brain, and kidney, and increased the content of SH-group. The activities of AST, ALT, and LDH in liver; AST, ALT, AlP, AcP, and LDH in testes; AcP in kidney; AlP and AcP in lung, and LDH in brain were significantly increased. Plasma TP and albumin were increased, while

  20. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    SciTech Connect

    Einstein, R.; Gabel, C.A. )

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

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

    ERIC Educational Resources Information Center

    Grunwald, Sandra K.; Krueger, Katherine J.

    2008-01-01

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

  2. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  3. Mode of action of anticancer peptides (ACPs) from amphibian origin.

    PubMed

    Oelkrug, Christopher; Hartke, Martin; Schubert, Andreas

    2015-02-01

    Although cancer belongs to one of the leading causes of death around the world, fortunately studies have shown that tumor cells have various targets that are susceptible to attack. Interestingly, tumor cells are comprised of cellular membranes, which are altered in chemical composition relative to non-neoplastic cells, giving them an increased net negative charge. These altered membranes are ideal targets for antimicrobial peptides (AMPs) shown to have additional tumoricidal properties and, hence, named anticancer peptides (ACPs). Several hundred ACPs have been explored in vitro and in vivo on various types of cancer. Novel anticancer agents are supposed not to cause serious side effects and the formation of multidrug-resistant tumor cells. During the quest for potent ACPs, promising candidates were isolated from skin secretions of amphibians, such as the granular glands of the Chinese brown frog, Rana chensinensis. ACPs have to be selective to cancer cells and should not induce strong immune responses or be cleared from the body rapidly. Several modifications can improve ACPs either by optimizing the primary structure rationally or randomly or even by introducing other chemical modifications. PMID:25667440

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

    PubMed

    Pavelka, M; Ellinger, A

    1981-04-01

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

  5. A salicylic acid-based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2)

    PubMed Central

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-01-01

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias and solid tumors. Thus there is considerable interest in SHP2 as a potential target for anti-cancer and anti-leukemia therapy. We report a salicylic acid-based combinatorial library approach aimed to bind both active site and unique nearby sub-pockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anti-cancer and anti-leukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors. PMID:20170098

  6. Small activating ribonucleic acid reverses tyrosine kinase inhibitor resistance in epidermal growth factor receptor‐mutant lung cancer by increasing the expression of phosphatase and tensin homolog

    PubMed Central

    Li, Meng; Peng, Zhongmin; Ren, Wangang

    2016-01-01

    Background Epidermal growth factor receptor‐tyrosine kinase inhibitors (TKI‐EGFRs) present a new prospect for the treatment of lung cancer. However, in clinical application, the majority of patients become TKI resistant within a year. More and more studies have shown that a loss of phosphatase and tensin homolog (PTEN) expression is associated with TKI resistance. An alternative method of upregulating PTEN expression may reverse TKI resistance. Methods We designed five candidate small activating ribonucleic acids (saRNAs) to target PTEN, and transfected them into H‐157 cells to screen out functional saRNA. We used reverse transcriptase‐polymerase chain reaction and Western blot to evaluate the effect of saRNA to PTEN expression. We then analyzed the growth and apoptosis of cells transfected with saRNA under the treatment of TKI to investigate whether saRNAs can reverse TKI resistance by upregulating PTEN expression. Results The functional saRNA we designed could upregulate PTEN expression. The H‐157 cells transfected with saRNA grew slower in the presence of TKI drugs than the cells that were not transfected with saRNA. The apoptosis rate was also obviously higher. Conclusions Our study proves that loss of PTEN expression is an important mechanism of TKI resistance. It is possible to control TKI resistance by upregulating PTEN expression using RNA activation technology. PMID:27385992

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

    PubMed Central

    Chang, Heng-Kwei

    2015-01-01

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

  8. Comparison of the effects of eldecalcitol with either raloxifene or bisphosphonate on serum tartrate resistant acid phosphatase-5b, a bone resorption marker, in postmenopausal osteoporosis

    PubMed Central

    Takada, Junichi; Ikeda, Satoshi; Kusanagi, Tetsuya; Mizuno, Satoshi; Wada, Hiroshi; Iba, Kousuke; Yoshizaki, Takashi; Yamashita, Toshihiko

    2016-01-01

    Summary Objective This study analyzes whether concomitant raloxifene (RLX) or bisphosphonates (BP) plus eldecalcitol (ELD) has excessive suppressive effects on a bone resorption marker during the first 6 months of treatment in postmenopausal women in real-world setting. Methods 285 postmenopausal osteoporotic patients who had been treated with RLX or BP plus ELD were evaluated the bone resorption marker, serum tartrate resistant acid phosphatase-5b (TRACP-5b), during the first 6 months of treatment. Results In drug-naïve group (not received osteoporosis medications before the administration, n=70), the concomitant RLX or BP with ELD significantly decreased levels of TRACP-5b without severe suppression. In vitamin D switch group [RLX or BP plus alfacalcidol (ALF) and then switched to RLX or BP plus ELD, n=215], the replacing ALF with ELD further and significantly decreased TRACP-5b and tertile analyses based on baseline values were significantly decreased far more in the highest, compared with the lowest tertile in the ELD+RLX and ELD+BP groups. Conclusion ELD combined with RLX or BP administered for 6 months to postmenopausal women with osteoporosis who were drug-naïve or who had switched medications significantly reduced and maintained TRACP-5b values within the reference range. PMID:27252739

  9. Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2015-01-01

    Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer. PMID:25591398

  10. [Clinical significance of tumor markers in prostatic carcinoma--comparative study of prostatic acid phosphatase, prostate specific antigen and gamma-seminoprotein].

    PubMed

    Yoshiki, T; Okada, K; Oishi, K; Yoshida, O

    1987-12-01

    We measured the prostatic acid phosphatase (PAP), gamma-Seminoprotein (gamma-Sm) and prostate specific antigen (PA) in the serum of 862 patients with various urologic diseases including 89 patients with prostatic cancer. We used a PAP radioimmunoassay kit, gamma-Sm enzyme immunoassay kit, Markit-F-PA enzyme immunoassay kit and PA test Wako enzyme immunoassay kit. Serum PA level in advanced prostatic carcinoma (stage C, D) tended to be higher than that in early stage cancer (stage A, B). The Wako kit gave a higher PA than the Markit-F in each stage. The sensitivity rate of Wako PA test was the highest (81%) of all kits. The specificity rate of PAP was the highest (83%), and the accuracy rate of Markit-F PA was the highest (79%). The positive rate in the combined assay of PAP, gamma-Sm and PA in prostatic cancer was higher than that in the single assay of each tumor marker. We regarded PAP, gamma-Sm and PA as clinically different tumor markers, because their serum level did not correlate definitely. No apparent correlation was found between histopathological grade and the level of each tumor marker. The level of PAP, gamma-Sm and PA in the reactivated patients was significantly higher than that of the well-controlled patients. In the reactivated patients, the positive rate of Markit-F PA was the highest (89%) of all the kits. PMID:2452559

  11. [Measurement of serum prostatic acid phosphatase (PAP) by Delfia PAP Kit using europium and clinical evaluation in patients with prostate cancer].

    PubMed

    Akimoto, S; Ohki, T; Ichikawa, T; Akakura, K; Shimazaki, J

    1994-11-01

    Fundamental and clinical studies of serum prostatic acid phosphatase (PAP) detected by a Delfia PAP kit were performed. The system is a time-resolved fluoroimmunoassay using europium as a tracer. The lower limit of detection was 0.2 ng/ml. Sera from 54 patients with prostate cancer, 20 with benign prostatic hypertrophy, 20 with urological malignancies other than prostate cancer and 140 adult males over 46 years old were determined. From the mean + 2 S.D. of serum PAP values obtained on the adult males, 1.5 ng/ml was considered as the upper normal level of adult males. By calculating the efficiency and ROC curve using the PAP values of prostate cancer and benign prostatic cancer, 2.5 ng/ml was decided as a cut-off value of this kit. The positive rates of adult males, prostate cancer, benign prostatic cancer and urological malignancies other than prostate cancer were 0.7%, 65%, 20% and 10%, respectively. The sensitivity of stage A2, B2, C and D1 + D2 was, 0%, 0%, 64% and 83%, respectively. The efficiency of the Delfia PAP kit was 52% and that of the Markit M PA kit was 71%. The correlation between the values assayed with the Delfia PAP kit and the Dinabot PAP kit was very high; the value obtained with the Delfia PAP kit was about 80% of that obtained with the Dinabot PAP kit. PMID:7530404

  12. ACP research and scientific coordination: Annual performance report

    SciTech Connect

    Hales, J.M.

    1992-09-17

    This research grant addresses two primary objectives: 1. Conduct of specific research tasks under the DOE Atmospheric Chemistry Program (ACP), and 2. Provision of scientific coordination for the Atmospheric Chemistry Program. In summary, the project is progressing essentially as expected, with some minor exceptions. Higher than anticipated demands within Objective 2, associated primarily with the ACP Ozone Project and the recompetition of the National Laboratories protion of ACP, have resulted in a somewhat higher proportionate emphasis on the coordination activities associated with this objective. Also, the immediate need for pre-campaign modeling of the North Pacific has resulted in an acceleration of the Pacific modeling task relative to other tasks, particularly the Atlantic modeling task. Modeling activities in general have been delayed somewhat because of the noted Objective 2 demands and because of the extended time that was necessary to complete financial agreements for this grant.

  13. Biochemical effect of a histidine phosphatase acid (phytase) of Aspergillus japonicus var. Saito on performance and bony characteristics of broiler.

    PubMed

    Maller, Alexandre; de Quadros, Thays Cristina Oliveira; Junqueira, Otto M; Graña, Alfredo Lora; de Lima Montaldi, Ana Paula; Alarcon, Ricardo Fernandes; Jorge, João Atílio; de Lourdes T M Polizeli, Maria

    2016-01-01

    Phytases are enzymes that hydrolyze the ester linkage of phytic acid, releasing inositol and inorganic phosphate. The phytic acid (phytate) is a major form of phosphorus in plant foods. Knowing that diet for animal of production has the cereal base (corn and soybean), primarily, broilers need for an alternative to use of the phosphate present in these ingredients, since it does not naturally produce the enzyme phytase, which makes it available. The aims of this work was studding the safe supplementation of Aspergillus japonicus var. Saito crude phytase in feeding broilers and check the biochemical effect on performance and bones of these animals. The enzymatic extract did not have aflatoxins B1, B2, G2 and G1 and zearalenone and ochratoxin, and low concentrations of this extract did not have cytotoxic effects on cells derived from lung tissue. The in vivo experiments showed that the phytase supplied the available phosphate reduction in the broiler feed formulation, with a live weight, weight gain, feed intake, feed conversion, viability, productive efficiency index and carcass yield similar to the control test. Furthermore, the phytase supplementation favored the formation of bone structure and performance of the broilers. The results show the high biotechnological potential of A. japonicus phytase on broiler food supplementation to reduce phosphorus addition in the food formulation. So, this enzyme could be used as a commercial alternative to animal diet supplementation. PMID:27625972

  14. Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice

    PubMed Central

    Schweitzer, George G.; Chen, Zhouji; Gan, Connie; McCommis, Kyle S.; Soufi, Nisreen; Chrast, Roman; Mitra, Mayurranjan S.; Yang, Kui; Gross, Richard W.; Finck, Brian N.

    2015-01-01

    Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are deficient in lipin-1-encoded PAP activity in a liver-specific manner (Alb-Lpin1−/− mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1−/− mouse liver. Hepatic PAP activity was significantly reduced in mice with liver-specific lipin 1 deficiency. However, hepatocytes from Alb-Lpin1−/− mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1−/− mice were not protected from intrahepatic accumulation of diacylglyerol and TG after chronic feeding of a diet rich in fat and fructose. Collectively, these data demonstrate that marked deficits in hepatic PAP activity do not impair TG synthesis and accumulation under acute or chronic conditions of lipid overload. PMID:25722343

  15. 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.

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

    PubMed Central

    Li, Lin-Jie; Kim, So-Nam

    2016-01-01

    PURPOSE In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P<.05). Furthermore, ALP expression levels of MSLA and LAT surfaces were significantly higher than expression levels of LT surface-adherent cells at 7, 14, and 21 days, respectively (P<.05). However, ALP expression levels between MSLA and LAT surface were equal at 7, 14, and 21 days (P>.05). CONCLUSION This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. PMID:27350860

  17. Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor.

    PubMed

    Wu, Jingjing; Zhang, Mingzhi; Liu, Delong

    2016-01-01

    More and more targeted agents become available for B cell malignancies with increasing precision and potency. The first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has been in clinical use for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom's macroglobulinemia. More selective BTK inhibitors (ACP-196, ONO/GS-4059, BGB-3111, CC-292) are being explored. Acalabrutinib (ACP-196) is a novel irreversible second-generation BTK inhibitor that was shown to be more potent and selective than ibrutinib. This review summarized the preclinical research and clinical data of acalabrutinib. PMID:26957112

  18. Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity.

    PubMed

    Bommineni, Gopal R; Kapilashrami, Kanishk; Cummings, Jason E; Lu, Yang; Knudson, Susan E; Gu, Chendi; Walker, Stephen G; Slayden, Richard A; Tonge, Peter J

    2016-06-01

    β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection. PMID:27187871

  19. Cell death-inducing stresses are required for defense activation in DS1-phosphatidic acid phosphatase-silenced Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Yoshioka, Hirofumi; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-07-20

    We previously identified DS1 plants that showed resistance to compatible Ralstonia solanacearum with accelerated defense responses. Here, we describe activation mechanisms of defense responses in DS1 plants. After inoculation with incompatible R. solanacearum 8107, DS1 plants showed hyperinduction of hypersensitive response (HR) and reactive oxygen species (ROS) generation. Transient expression of PopP1 and AvrA induced hyperinduction of HR and ROS generation. Furthermore, Pseudomonas cichorii (Pc) and a type III secretion system (TTSS)-deficient mutant of P. cichorii showed accelerated induction of HR and ROS generation. Chitin and flg22 did not induce either HR or ROS hyperaccumulation; however, INF1 accelerated HR and ROS in DS1 plants. Activation of these defense responses was closely associated with increased phosphatidic acid (PA) content. Our results show that DS1 plants exhibit PA-mediated sensitization of plant defenses and that cell death-inducing stress is required to achieve full activation of defense responses. PMID:26188395

  20. A Community-oriented CEOS Atmospheric Composition Portal (ACP)

    NASA Astrophysics Data System (ADS)

    Bernonville, S.; Goussev, O.; Falke, S.; Lindsay, F.; Lynnes, C. S.; Yang, W.; Zhao, P.; Johnson, J.

    2012-04-01

    The Atmospheric Composition Constellation (ACC) and the Workgroup for Information Systems and Services (WGISS) within the Committee on Earth Observation Satellites (CEOS) is developing a portal to support interoperability among the atmospheric composition research and applications communities. The CEOS Atmospheric Composition Portal (ACP) is defining approaches for providing data access, tools and contextual guidance for an international suite of remote sensing datasets. An initial prototype provides access to data services and analysis tools hosted by the World Data Center for Remote Sensing of the Atmosphere (WDC-RSAT), NASA's Goddard Earth Sciences Data and Information Services Center (GES DISC) and DataFed. Distributed access to data is implemented via interoperability standards, including the Open Geospatial Consortium's (OGC) Web Map Service (WMS) and Web Coverage Service (WCS). A fundamental aspect to the design, implementation and evolution of the ACP is community collaboration. The portal is intended as a community resource that is created through collaboration across remotely sensed atmospheric composition data organizations and used by a variety of groups across the climate, air quality, and stratospheric ozone domains. The implementation of interoperability standards in the ACP has involved coordination on identifying the most applicable standards and the definition of community-specific conventions to ensure consistent adoption of standards. This presentation includes an overview of the ACP, its community oriented approach, and use of community-conventions in achieving standards-based interoperability.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-09-01

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

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

    PubMed

    Sopina, V A

    2007-01-01

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

  4. The THO/TREX Complex Active in miRNA Biogenesis Negatively Regulates Root-Associated Acid Phosphatase Activity Induced by Phosphate Starvation1[OPEN

    PubMed Central

    Tao, Sibo; Zhang, Ye; Wang, Xiaoyue; Xu, Le; Fang, Xiaofeng; Lu, Zhi John

    2016-01-01

    Induction and secretion of acid phosphatases (APases) is an adaptive response that plants use to cope with P (Pi) deficiency in their environment. The molecular mechanism that regulates this response, however, is poorly understood. In this work, we identified an Arabidopsis (Arabidopsis thaliana) mutant, hps8, which exhibits enhanced APase activity on its root surface (also called root-associated APase activity). Our molecular and genetic analyses indicate that this altered Pi response results from a mutation in the AtTHO1 gene that encodes a subunit of the THO/TREX protein complex. The mutation in another subunit of this complex, AtTHO3, also enhances root-associated APase activity under Pi starvation. In Arabidopsis, the THO/TREX complex functions in mRNA export and miRNA biogenesis. When treated with Ag+, an inhibitor of ethylene perception, the enhanced root-associated APase activity in hps8 is largely reversed. hpr1-5 is another mutant allele of AtTHO1 and shows similar phenotypes as hps8. ein2 is completely insensitive to ethylene. In the hpr1-5ein2 double mutant, the enhanced root-associated APase activity is also greatly suppressed. These results indicate that the THO/TREX complex in Arabidopsis negatively regulates root-associated APase activity induced by Pi starvation by inhibiting ethylene signaling. In addition, we found that the miRNA399-PHO2 pathway is also involved in the regulation of root-associated APase activity induced by Pi starvation. These results provide insight into the molecular mechanism underlying the adaptive response of plants to Pi starvation. PMID:27329222

  5. Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts.

    PubMed

    Nakayama, Takahiro; Mizoguchi, Toshihide; Uehara, Shunsuke; Yamashita, Teruhito; Kawahara, Ichiro; Kobayashi, Yasuhiro; Moriyama, Yoshinori; Kurihara, Saburo; Sahara, Noriyuki; Ozawa, Hidehiro; Udagawa, Nobuyuki; Takahashi, Naoyuki

    2011-12-01

    Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts. PMID:21983021

  6. The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana

    PubMed Central

    Plaxton, William C.

    2012-01-01

    Orthophosphate (Pi) is an essential but limiting macronutrient for plant growth. Extensive soil P reserves exist in the form of organic P (Po), which is unavailable for root uptake until hydrolysed by secretory acid phosphatases (APases). The predominant purple APase (PAP) isozymes secreted by roots of Pi-deficient (–Pi) Arabidopsis thaliana were recently identified as AtPAP12 (At2g27190) and AtPAP26 (At5g34850). The present study demonstrated that exogenous Po compounds such as glycerol-3-phosphate or herring sperm DNA: (i) effectively substituted for Pi in supporting the P nutrition of Arabidopsis seedlings, and (ii) caused upregulation and secretion of AtPAP12 and AtPAP26 into the growth medium. When cultivated under –Pi conditions or supplied with Po as its sole source of P nutrition, an atpap26/atpap12 T-DNA double insertion mutant exhibited impaired growth coupled with >60 and >30% decreases in root secretory APase activity and rosette total Pi concentration, respectively. Development of the atpap12/atpap26 mutant was unaffected during growth on Pi-replete medium but was completely arrested when 7-day-old Pi-sufficient seedlings were transplanted into a –Pi, Po-containing soil mix. Both PAPs were also strongly upregulated on root surfaces and in shoot cell-wall extracts of –Pi seedlings. It is hypothesized that secreted AtPAP12 and AtPAP26 facilitate the acclimation of Arabidopsis to nutritional Pi deficiency by: (i) functioning in the rhizosphere to scavenge Pi from the soil’s accessible Po pool, while (ii) recycling Pi from endogenous phosphomonoesters that have been leaked into cell walls from the cytoplasm. Thus, AtPAP12 and AtPAP26 are promising targets for improving crop P-use efficiency. PMID:23125358

  7. Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.

    PubMed

    Allen, G J; Kuchitsu, K; Chu, S P; Murata, Y; Schroeder, J I

    1999-09-01

    Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt). PMID:10488243

  8. 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

  9. Modeling holo-ACP:DH and holo-ACP:KR complexes of modular polyketide synthases: a docking and molecular dynamics study

    PubMed Central

    2012-01-01

    Background Modular polyketide synthases are multifunctional megasynthases which biosynthesize a variety of secondary metabolites using various combinations of dehydratase (DH), ketoreductase (KR) and enoyl-reductase (ER) domains. During the catalysis of various reductive steps these domains act on a substrate moiety which is covalently attached to the phosphopantetheine (P-pant) group of the holo-Acyl Carrier Protein (holo-ACP) domain, thus necessitating the formation of holo-ACP:DH and holo-ACP:KR complexes. Even though three dimensional structures are available for DH, KR and ACP domains, no structures are available for DH or KR domains in complex with ACP or substrate moieties. Since Ser of holo-ACP is covalently attached to a large phosphopantetheine group, obtaining complexes involving holo-ACP by standard protein-protein docking has been a difficult task. Results We have modeled the holo-ACP:DH and holo-ACP:KR complexes for identifying specific residues on DH and KR domains which are involved in interaction with ACP, phosphopantetheine and substrate moiety. A novel combination of protein-protein and protein-ligand docking has been used to first model complexes involving apo-ACP and then dock the phosphopantetheine and substrate moieties using covalent connectivity between ACP, phosphopantetheine and substrate moiety as constraints. The holo-ACP:DH and holo-ACP:KR complexes obtained from docking have been further refined by restraint free explicit solvent MD simulations to incorporate effects of ligand and receptor flexibilities. The results from 50 ns MD simulations reveal that substrate enters into a deep tunnel in DH domain while in case of KR domain the substrate binds a shallow surface exposed cavity. Interestingly, in case of DH domain the predicted binding site overlapped with the binding site in the inhibitor bound crystal structure of FabZ, the DH domain from E.Coli FAS. In case of KR domain, the substrate binding site identified by our simulations

  10. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

    PubMed Central

    2012-01-01

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

  11. Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor

    SciTech Connect

    Chemler, Joseph A.; Buchholz, Tonia J.; Geders, Todd W.; Akey, David L.; Rath, Christopher M.; Chlipala, George E.; Smith, Janet L.; Sherman, David H.

    2012-08-10

    Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 {angstrom} germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.

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

    PubMed

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

    2016-05-31

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

  13. Effect of a mutagenized acyl-ACP thioesterase FATA allele from sunflower with improved activity in tobacco leaves and Arabidopsis seeds.

    PubMed

    Moreno-Pérez, Antonio Javier; Venegas-Calerón, Mónica; Vaistij, Fabián E; Salas, Joaquin J; Larson, Tony R; Garcés, Rafael; Graham, Ian A; Martínez-Force, Enrique

    2014-03-01

    The substrate specificity of the acyl-acyl carrier protein (ACP) thioesterases significantly determines the type of fatty acids that are exported from plastids. Thus, designing acyl-ACP thioesterases with different substrate specificities or kinetic properties would be of interest for plant lipid biotechnology to produce oils enriched in specialty fatty acids. In the present work, the FatA thioesterase from Helianthus annuus was used to test the impact of changes in the amino acids present in the binding pocket on substrate specificity and catalytic efficiency. Amongst all the mutated enzymes studied, Q215W was especially interesting as it had higher specificity towards saturated acyl-ACP substrates and higher catalytic efficiency compared to wild-type H. annuus FatA. Null, wild type and high-efficiency alleles were transiently expressed in tobacco leaves to check their effect on lipid biosynthesis. Expression of active FatA thioesterases altered the composition of leaf triacylglycerols but did not alter total lipid content. However, the expression of the wild type and the high-efficiency alleles in Arabidopsis thaliana transgenic seeds resulted in a strong reduction in oil content and an increase in total saturated fatty acid content. The role and influence of acyl-ACP thioesterases in plant metabolism and their possible applications in lipid biotechnology are discussed. PMID:24327259

  14. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

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

    PubMed

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

    2011-03-20

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

  16. 26 CFR 1.401(m)-2 - ACP test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... determined under § 1.401(m)-2(b)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (E... determined under § 1.401(m)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). If... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ACP test. 1.401(m)-2 Section 1.401(m)-2...

  17. Biogeochemical drivers of phosphatase activity in salt marsh sediments

    NASA Astrophysics Data System (ADS)

    Freitas, Joana; Duarte, Bernardo; Caçador, Isabel

    2014-10-01

    Although nitrogen has become a major concern for wetlands scientists dealing with eutrophication problems, phosphorous represents another key element, and consequently its biogeochemical cycling has a crucial role in eutrophication processes. Microbial communities are a central component in trophic dynamics and biogeochemical processes on coastal systems, since most of the processes in sediments are microbial-mediated due to enzymatic action, including the mineralization of organic phosphorus carried out by acid phosphatase activity. In the present work, the authors investigate the biogeochemical sediment drivers that control phosphatase activities. Authors also aim to assess biogeochemical factors' influence on the enzyme-mediated phosphorous cycling processes in salt marshes. Plant rhizosediments and bare sediments were collected and biogeochemical features, including phosphatase activities, inorganic and organic phosphorus contents, humic acids content and pH, were assessed. Acid phosphatase was found to give the highest contribution for total phosphatase activity among the three pH-isoforms present in salt marsh sediments, favored by acid pH in colonized sediments. Humic acids also appear to have an important role inhibiting phosphatase activity. A clear relation of phosphatase activity and inorganic phosphorous was also found. The data presented reinforces the role of phosphatase in phosphorous cycling.

  18. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein.

    PubMed Central

    Takai, A; Sasaki, K; Nagai, H; Mieskes, G; Isobe, M; Isono, K; Yasumoto, T

    1995-01-01

    Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values. PMID:7702557

  19. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    SciTech Connect

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

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

    PubMed Central

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

    2015-01-01

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

  1. Mechanistic studies on the reactions of molybdenum(VI), tungsten(VI), vanadium(V), and arsenic(V) tetraoxo anions with the Fe{sup II}Fe{sup III} form of purple acid phosphatase from porcine uteri (Uteroferrin)

    SciTech Connect

    Lim, J.S.; Aquino, M.A.S.; Skyes, A.G.

    1996-01-31

    The Fe{sup II}-Fe{sup III} form of purple acid phosphatase (PAP{sub r}) from porcine uteri (uteroferrin) catalyses the hydrolysis of phosphate esters. Here, kinetic studies have been extended to include the complexing of tetraoxo XO{sub 4} anions of molybdate(VI), tungstate(VI), vanadate(V), and arsenate(V) with PAP{sub r}. UV-vis absorbance changes are small and the range of concentrations is restricted by the need to maximise monomer XO{sub 4} forms. Rate constants k{sub obs}(25{degrees}C) were determined by stopped-flow monitoring of the reactions at {approximately}520 nm.

  2. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

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

  3. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    PubMed

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  4. Zn-exchange and Mössbauer studies on the [Fe-Fe] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans.

    PubMed

    Suerbaum, H; Körner, M; Witzel, H; Althaus, E; Mosel, B D; Müller-Warmuth, W

    1993-05-15

    In order to perform Mössbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with delta of 0.51 mm/s and 0.53 mm/s and delta E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublets with delta values of 0.55 mm/s and 1.14 mm/s and delta E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and with delta E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system

  5. Cloning of Acyl-ACP Thioesterase FatA from Arachis hypogaea L. and Its Expression in Escherichia coli

    PubMed Central

    Chen, Gao; Peng, Zhen-ying; Shan, Lei; Xuan, Ning; Tang, Gui-ying; Zhang, Yan; Li, Lan; He, Qing-fang; Bi, Yu-ping

    2012-01-01

    In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli. PMID:23093853

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

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

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

  7. Enzymatic and Functional Analysis of a Protein Phosphatase, Pph3, from Myxococcus xanthus ▿

    PubMed Central

    Kimura, Yoshio; Mori, Yumi; Ina, Youhei; Takegawa, Kaoru

    2011-01-01

    A protein phosphatase, designated Pph3, from Myxococcus xanthus showed the enzymatic characteristics of PP2C-type serine/threonine protein phosphatases, which are metal ion-dependent, okadaic acid-insensitive protein phosphatases. The pph3 mutant under starvation conditions formed immature fruiting bodies and reduced sporulation. PMID:21398555

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

    PubMed

    Alonso, Andrés; Pulido, Rafael

    2016-04-01

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

  9. Phosphatase activities as biosignatures of extant life

    NASA Astrophysics Data System (ADS)

    Kobayashi, K.; Itoh, Y.; Edazawa, Y.; Moroi, A.; Takano, Y.

    It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere high temperature hot springs and stratosphere Possible extraterrestrial biospheres in Mars Europa and Titan are being discussed Many biosignatures or biomarkers have been proposed to detect microbial activities in such extreme environments Phosphate esters are essential for the terrestrial life since they are constituents of nucleic acids and cell mebranes Thus all the terrestrial organisms have phosphatases that are enzymes catalyzing hydrolysis of phosphate esters We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and discussed whether they can be used as biosignatures for extant life Core samples and chimney samples were collected at the Suiyo Seamount Izu-Bonin Arc the Pacific Ocean in 2001 and 2002 and in South Mariana hydrothermal systems the Pacific Oceanas in 2003 both in a part of the Archaean Park Project Phosphatase activity in solid rock samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate pH 8 0 or pH 6 5 as a substrate as follows Pulverized samples were incuvated with substrate solution for an hour and then production rate of p-nitrophenol was calculated with absorbance at 410 nm Phosphatase activity in extracts was measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate Concentration of amino acids and their enantiomeric ratio were determined by HPLC after HF digestion of the

  10. Walking Together: A Narrative/Pastoral Approach to the ACPE Accreditation Site Visit

    ERIC Educational Resources Information Center

    Oertli, Karrie

    2010-01-01

    Walking together is a strong theme in religious literature: God companions persons, and persons companion one another. All share and hear stories in that action. The Association for Clinical Pastoral Education, Inc. (ACPE) began to walk together in 1967, as it gathered four organizations into one. ACPE has grown and changed, becoming world's…

  11. Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean.

    PubMed Central

    Battistuzzi, G; Dietrich, M; Löcke, R; Witzel, H

    1997-01-01

    The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives. PMID:9169589

  12. Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

    DOE PAGESBeta

    Wang, Hui; Liu, Li; Lu, Yang; Pan, Pan; Hooker, Jacob M.; Fowler, Joanna S.; Tonge, Peter J.

    2015-07-14

    PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

  13. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  14. Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH. PMID:24015914

  15. Alkaline Phosphatase in Stem Cells

    PubMed Central

    Štefková, Kateřina; Procházková, Jiřina; Pacherník, Jiří

    2015-01-01

    Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells. PMID:25767512

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

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes. PMID:25785438

  17. Modulators of intestinal alkaline phosphatase.

    PubMed

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

    2013-01-01

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

  18. A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

    PubMed

    Dassa, J; Fsihi, H; Marck, C; Dion, M; Kieffer-Bontemps, M; Boquet, P L

    1991-10-01

    The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants. PMID:1658595

  19. Enhancing Potato System Sustainability: Crop Rotation Impacts on Soil Phosphatase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is a species with a low efficiency of acquiring soil P. Rotation crops may potentially influence P uptake by potato by increasing soil organic acids, phosphatase activity, and microbial biomass. However, this kind of information is very limited. We measured the activities of acid phosphatase,...

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

    PubMed

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

    2015-01-01

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

  1. A Theileria parva type 1 protein phosphatase activity.

    PubMed

    Cayla, X; Garcia, A; Baumgartner, M; Ozon, R; Langsley, G

    2000-09-01

    The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1. PMID:10989153

  2. Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea.

    PubMed

    Jha, Saheli Sinha; Jha, Jyoti K; Chattopadhyaya, Banani; Basu, Asitava; Sen, Soumitra K; Maiti, Mrinal K

    2010-06-01

    Four types of cDNAs corresponding to the fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) enzyme were isolated from the developing seeds of Brassica juncea, a widely cultivated species amongst the oil-seed crops. The mature polypeptides deduced from the cDNAs showed sequence identity with the FatB class of plant thioesterases. Southern hybridization revealed the presence of at least four copies of BjFatB gene in the genome of this amphidiploid species. Western blot and RT-PCR analyses showed that the BjFatB class thioesterase is expressed poorly in flowers and leaves, but significantly in seeds at the mid-maturation stage. The enzymatic activities of different BjFatB isoforms were established upon heterologous expression of the four BjFatB CDSs in Escherichia coli K27fadD88, a mutant strain of fatty acid beta-oxidation pathway. The substrate specificity of each BjFatB isoform was determined in vivo by fatty acid profile analyses of the culture supernatant and membrane lipid of the recombinant K27fadD88 and E. coli DH10B (fadD(+)) clones, respectively. The BjFatB1 and BjFatB3 were predominantly active on C18:0-ACP substrate, whereas BjFatB2 and BjFatB4 were specific towards C18:0-ACP as well as C16:0-ACP. These novel FatB genes may find potential application in metabolic engineering of crop plants through their over-expression in seed tissues to generate stearate-rich vegetable fats/oils of commercial importance. PMID:20356753

  3. Cloning, characterization, and expression analysis of acyl-acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis).

    PubMed

    Dong, Shubin; Huang, Jiacong; Li, Yannan; Zhang, Jing; Lin, Shanzhi; Zhang, Zhixiang

    2014-05-25

    Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)△FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches. PMID:24631366

  4. Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans.

    PubMed

    Sá-Pessoa, Joana; Amillis, Sotiris; Casal, Margarida; Diallinas, George

    2015-03-01

    AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate, butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of 16.89±2.12, 9.25±1.01, 12.06±3.29 and 1.44±0.13mM, respectively. AcpA is also shown not to be directly involved in ammonia export, as proposed for its Saccharomyces cerevisiae homologue Ady2p. In the second part of this work, we search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the closest AcpA homologue (AN1839), named AcpB, is the 'missing' secondary acetate transporter in mycelia. We also identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA (AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework for further exploiting acetate and carboxylate transport in filamentous ascomycetes. PMID:25708319

  5. The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation.

    PubMed

    Molle, Virginie; Brown, Alistair K; Besra, Gurdyal S; Cozzone, Alain J; Kremer, Laurent

    2006-10-01

    Phosphorylation of proteins by Ser/Thr protein kinases (STPKs) has recently become of major physiological importance because of its possible involvement in virulence of bacterial pathogens. Although Mycobacterium tuberculosis has eleven STPKs, the nature and function of the substrates of these enzymes remain largely unknown. In this work, we have identified for the first time STPK substrates in M. tuberculosis forming part of the type II fatty acid synthase (FAS-II) system involved in mycolic acid biosynthesis: the malonyl-CoA::AcpM transacylase mtFabD, and the beta-ketoacyl AcpM synthases KasA and KasB. All three enzymes were phosphorylated in vitro by different kinases, suggesting a complex network of interactions between STPKs and these substrates. In addition, both KasA and KasB were efficiently phosphorylated in M. bovis BCG each at different sites and could be dephosphorylated by the M. tuberculosis Ser/Thr phosphatase PstP. Enzymatic studies revealed that, whereas phosphorylation decreases the activity of KasA in the elongation process of long chain fatty acids synthesis, this modification enhances that of KasB. Such a differential effect of phosphorylation may represent an unusual mechanism of FAS-II system regulation, allowing pathogenic mycobacteria to produce full-length mycolates, which are required for adaptation and intracellular survival in macrophages. PMID:16873379

  6. Effect of the Algaecide Palmitoleic Acid on the Immune Function of the Bay Scallop Argopecten irradians.

    PubMed

    Chi, Cheng; Giri, Sib Sankar; Jun, Jin Woo; Kim, Hyoun Joong; Kim, Sang Guen; Yun, Saekil; Park, Se Chang

    2016-01-01

    Palmitoleic acid (PA), an algicidal compound, is used against the toxin producing dinofagelate Alexandrium tamarense, however, its impact on the edible bay scallop (Argopecten irradians) is still unclear. Therefore, we investigated the impacts of effective algicidal concentrations (20, 40, and 80 mg/L) of PA on immune responses in A. irradians. Various immune parameters including acid phosphatase (ACP) activity, superoxide dismutase (SOD), lysozyme, phagocytic activity, total protein, malondialdehyde (MDA) level, and reactive oxygen species (ROS) production and the expression of immune-related genes (PrxV, CLT-6, MT, and BD) were measured at 3, 6, 12, 24, and 48 h post-exposure (hpe) to PA. Lysozyme activity was lower in scallops at 12-48 hpe to 80 mg/L. SOD, ACP activity, ROS production, the total protein, and MDA level was higher at 12 to 48 hpe with different concentrations of PA. Phagocytic activity increased at 6-12 hpe to 40-80 mg/L of PA, but decreased at 24-48 hpe. The expressions of genes PrxV, CLT-6, MT and BD down-regulated at 3 hpe were observed, while differential expressions from 6-48 hpe with different concentrations of PA. The present study demonstrated that immersing A. irradians in PA at effective concentrations could result in differential effects on non-specific immune responses and expressions of immune-related genes. PMID:27171074

  7. A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid.

    PubMed

    Kuwahara, Takashi; Homma, Toshimasa; Kondo, Mizuki; Shimomura, Masato

    2011-03-15

    An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction. PMID:21247749

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

    PubMed Central

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

    2000-01-01

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

  9. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  10. Cloning and characterization of a novel human phosphatidic acid phosphatase type 2, PAP2d, with two different transcripts PAP2d_v1 and PAP2d_v2.

    PubMed

    Sun, Liyun; Gu, Shaohua; Sun, Yaqiong; Zheng, Dan; Wu, Qihan; Li, Xin; Dai, Jianfeng; Dai, Jianliang; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2005-04-01

    This study reports the cloning and characterization of a novel human phosphatidic acid phosphatase type 2 isoform cDNAs (PAP2d) from the foetal brain cDNA library. The PAP2d gene is localized on chromosome 1p21.3. It contains six exons and spans 112 kb of the genomic DNA. By large-scale cDNA sequencing we found two splice variants of PAP2d, PAP2d_v1 and PAP2d_v2. The PAP2d_v1 cDNA is 1722 bp in length and spans an open reading frame from nucleotide 56 to 1021, encoding a 321aa protein. The PAP2d_v2 cDNA is 1707 bp in length encoding a 316aa protein from nucleotide 56-1006. The PAP2d_v1 cDNA is 15 bp longer than the PAP2d_v2 cDNA in the terminal of the fifth exon and it creates different ORF. Both of the proteins contain a well-conserved PAP2 motif. The PAP2d_v1 is mainly expressed in human brain, lung, kidney, testis and colon, while PAP2d_v2 is restricted to human placenta, skeletal muscle, and kidney. The two splice variants are co-expressed only in kidney. PMID:16010976

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

    PubMed

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

    2015-02-01

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

  12. Spatial structure of oligopeptide PAP(248-261), the N-terminal fragment of the HIV enhancer prostatic acid phosphatase peptide PAP(248-286), in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Blokhin, Dmitriy S.; Filippov, Andrei V.; Antzutkin, Oleg N.; Karataeva, Farida Kh.; Klochkov, Vladimir V.

    2014-07-01

    Prostatic acid phosphatase (PAP) is an enzyme that facilitates infection of cells by HIV. Its peptide fragment PAP(248-286) forms amyloid fibrils known as SEVI, which enhance attachment of the virus by viral adhesion to the host cell prior to receptor-specific binding via reducing the electrostatic repulsion between the membranes of the virus and the target cell. The secondary structure of PAP(248-286) in aqueous and SDS solutions can be divided into an N-terminal disordered region, an α-helical central part and an α/310-helical C-terminal region (Nanga et al., 2009). In this work, we used NMR spectroscopy to study the spatial structure of the isolated N-terminal fragment of PAP(248-286), PAP(248-261) (GIHKQKEKSRLQGG), in aqueous and SDS micelle solutions. Formation of a PAP(248-261)-SDS complex was confirmed by chemical shift alterations in the 1H NMR spectra of the peptide, as well as by the signs and values of Nuclear Overhauser Effect (NOE). In addition, the PAP(248-261) peptide does not form any specified secondary structure in either aqueous or SDS solutions.

  13. A chronoamperometric screen printed carbon biosensor based on alkaline phosphatase inhibition for W(IV) determination in water, using 2-phospho-L-ascorbic acid trisodium salt as a substrate.

    PubMed

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver-Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  14. The number of tartrate-resistant acid phosphatase-positive osteoclasts on neonatal mouse parietal bones is decreased when prostaglandin synthesis is inhibited and increased in response to prostaglandin E2, parathyroid hormone, and 1,25 dihydroxyvitamin D3.

    PubMed

    Marshall, M J; Holt, I; Davie, M W

    1995-03-01

    The culture of parietal bones from 4-day old mice in indomethacin (Ind) for 1 day caused a large reduction in the number of tartrate-resistant acid phosphatase positive osteoclasts (TRAP + OC) relative to both control bones and to freshly isolated bones. This reduction did not occur if prostaglandin E2 (PGE2) was present. When 5-bromo-2'-deoxyuridine (BDU) was injected into 4-day old mice, newly formed TRAP + OC nuclei became labeled 1 day later; these bones were then cultured with Ind for 1 day. TRAP + OC and newly labeled TRAP+OC nuclei were commensurately decreased in number. This suggests an active down-regulation rather than merely the inhibition of new TRAP+OC formation. Incubation of bones with Ind and either PGE2, parathyroid hormone, or 1,25 dihydroxyvitamin D3 for 6 hours following a 1-day preincubation in Ind, resulted in an increase in TRAP + OC compared with Ind alone. Using BDU labeling in vitro and in vivo, we show that this increase in number of TRAP+OC is not the result of cell proliferation, but rather differentiation of postmitotic precursors. PMID:7538445

  15. A Chronoamperometric Screen Printed Carbon Biosensor Based on Alkaline Phosphatase Inhibition for W(VI) Determination in Water, Using 2-Phospho-l-Ascorbic Acid Trisodium Salt as a Substrate

    PubMed Central

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 μM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 μM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  16. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  17. Interaction between infectious diseases and personality traits: ACP1*C as a potential mediator.

    PubMed

    Napolioni, Valerio; Murray, Damian R; Comings, David E; Peters, Warren R; Gade-Andavolu, Radhika; MacMurray, James

    2014-08-01

    In geographical regions characterized by high pathogen prevalence, it has been shown that human populations tend to be characterized by lower levels of extraversion (E) and openness to experience (OtE). According to the "behavioral immune system" hypothesis, the reduction of extraversion and openness levels represents a behavioral defense against infections. Like the 'classical' immune system, the "behavioral immune system" could also be shaped by its underlying genetic background. Previous studies have shown that the *C allele of the ACP1 gene confers increased susceptibility to infectious/parasitic diseases. We hypothesized that carriers of the ACP1*C allele should likewise be associated with reduced E and OtE. We tested this hypothesis using two samples comprised of 153 students from Southern California (Group 1), and 162 female subjects recruited from an executive health program (Group 2), genotyped for ACP1 polymorphism and evaluated by the NEO Five-Factor Inventory (NEO-FFI). ACP1 was significantly associated with E: we found that carriers of ACP1*C showed reduced scores for E (Group 1: β=-4.263, P=0.027; Group 2: β=-8.315, P=0.003; Group 1+Group 2: β=-5.366, P=0.001). Across groups, ACP1 was only marginally associated with OtE. In conclusion, the present study found that the ACP1*C allele, previously associated with an increased vulnerability to infectious/parasitic diseases may also be able to shape behavioral immune defenses by interaction with the level of E. PMID:24933463

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

    PubMed

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

    2016-07-01

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

  19. Structural Genomics of Protein Phosphatases

    SciTech Connect

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  20. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  1. Protein tyrosine and serine–threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: Identification and potential functions

    PubMed Central

    Byrum, C.A.; Walton, K.D.; Robertson, A.J.; Carbonneau, S.; Thomason, R.T.; Coffman, J.A.; McClay, D.R.

    2011-01-01

    Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine–threonine (ser–thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser–thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser–thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser–thr phosphatases. For several expressed NRPTPs, MKPs, and ser–thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity. PMID:17087928

  2. 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. PMID:26896939

  3. Effect of CPP/ACP on initial bioadhesion to enamel and dentin in situ.

    PubMed

    Grychtol, Susann; Basche, Sabine; Hannig, Matthias; Hannig, Christian

    2014-01-01

    The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer's recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining). Additionally, the colony forming units (CFU) were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management. PMID:25386603

  4. Effect of CPP/ACP on Initial Bioadhesion to Enamel and Dentin In Situ

    PubMed Central

    2014-01-01

    The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturer's recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining). Additionally, the colony forming units (CFU) were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management. PMID:25386603

  5. Efficacious long-term cooling and freezing of Sapajus apella semen in ACP-118(®).

    PubMed

    Leão, D L; Miranda, S A; Brito, A B; Lima, J S; Santos, R R; Domingues, S F S

    2015-08-01

    The objectives of the present study were to test the effect of coconut water solution (CWS), TES-TRIS and ACP-118(®) on the seminal cooling and cryopreservation of semen from capuchin monkeys (Sapajus apella). Semen was collected from six males by electro-ejaculation, diluted in TES-TRIS, CWS or ACP-118(®), and maintained at 4°C for 28h. Semen was subsequently evaluated (Experiment I) or cryopreserved in the presence of different glycerol concentrations (3%, 5% or 7%) (Experiment II). ACP-118(®) was the preferred extender to preserve sperm motility and viability after 28h incubation at 4°C. Cooled sperm were successfully frozen-thawed in a medium containing 3% glycerol. After thawing, sperm retained the capacity to fertilize oocytes and zygotes were obtained. In conclusion, ACP-118(®) can be effectively and efficiently used as extender for the cooling of S. apella semen. Furthermore, cryopreservation using ACP-118(®) by adding 3% glycerol is suitable to maintain sperm morphology and the capacity of these cells to fertilize in vitro. PMID:26071650

  6. iACP: a sequence-based tool for identifying anticancer peptides

    PubMed Central

    Chen, Wei; Ding, Hui; Feng, Pengmian; Lin, Hao; Chou, Kuo-Chen

    2016-01-01

    Cancer remains a major killer worldwide. Traditional methods of cancer treatment are expensive and have some deleterious side effects on normal cells. Fortunately, the discovery of anticancer peptides (ACPs) has paved a new way for cancer treatment. With the explosive growth of peptide sequences generated in the post genomic age, it is highly desired to develop computational methods for rapidly and effectively identifying ACPs, so as to speed up their application in treating cancer. Here we report a sequence-based predictor called iACP developed by the approach of optimizing the g-gap dipeptide components. It was demonstrated by rigorous cross-validations that the new predictor remarkably outperformed the existing predictors for the same purpose in both overall accuracy and stability. For the convenience of most experimental scientists, a publicly accessible web-server for iACP has been established at http://lin.uestc.edu.cn/server/iACP, by which users can easily obtain their desired results. PMID:26942877

  7. [Constructing an ACP Simulation-Situation Communication Training Program for Patients With Chronic Kidney Disease].

    PubMed

    Chen, Jui-O; Lin, Chiu-Chu

    2016-06-01

    The aging population and changing lifestyles have lead to the increased general risk of chronic kidney disease. Taiwan currently has the highest incidence and prevalence of end-stage renal disease (ESRD) of any country or region in the world. Hemodialysis patients must endure comorbidities and face the uncertainties of death. The best way to achieve a good death is for patients to sign advance care planning (ACP). However, the key factors contributing to low ACP signature rates have been the lack of communication skills and related training among medical staffs. This article explores the dilemma of ACP using an example of chronic kidney disease (CKD) and proposes a theory-based approach to develop a theoretical framework for an ACP simulation-situation communication training program that integrates the simulation situation model, PREPARED model, and scaffolding theory. Readers may use this framework to design ACP simulation-situation communication training programs that conform to their own conditions and then test the effectiveness and feasibility of these programs in clinical settings. PMID:27250964

  8. Characterization of the PEST family protein tyrosine phosphatase BDP1.

    PubMed

    Kim, Y W; Wang, H; Sures, I; Lammers, R; Martell, K J; Ullrich, A

    1996-11-21

    Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues. PMID:8950995

  9. Glucose-6-phosphatase deficiency

    PubMed Central

    2011-01-01

    Glucose-6-phosphatase deficiency (G6P deficiency), or glycogen storage disease type I (GSDI), is a group of inherited metabolic diseases, including types Ia and Ib, characterized by poor tolerance to fasting, growth retardation and hepatomegaly resulting from accumulation of glycogen and fat in the liver. Prevalence is unknown and annual incidence is around 1/100,000 births. GSDIa is the more frequent type, representing about 80% of GSDI patients. The disease commonly manifests, between the ages of 3 to 4 months by symptoms of hypoglycemia (tremors, seizures, cyanosis, apnea). Patients have poor tolerance to fasting, marked hepatomegaly, growth retardation (small stature and delayed puberty), generally improved by an appropriate diet, osteopenia and sometimes osteoporosis, full-cheeked round face, enlarged kydneys and platelet dysfunctions leading to frequent epistaxis. In addition, in GSDIb, neutropenia and neutrophil dysfunction are responsible for tendency towards infections, relapsing aphtous gingivostomatitis, and inflammatory bowel disease. Late complications are hepatic (adenomas with rare but possible transformation into hepatocarcinoma) and renal (glomerular hyperfiltration leading to proteinuria and sometimes to renal insufficiency). GSDI is caused by a dysfunction in the G6P system, a key step in the regulation of glycemia. The deficit concerns the catalytic subunit G6P-alpha (type Ia) which is restricted to expression in the liver, kidney and intestine, or the ubiquitously expressed G6P transporter (type Ib). Mutations in the genes G6PC (17q21) and SLC37A4 (11q23) respectively cause GSDIa and Ib. Many mutations have been identified in both genes,. Transmission is autosomal recessive. Diagnosis is based on clinical presentation, on abnormal basal values and absence of hyperglycemic response to glucagon. It can be confirmed by demonstrating a deficient activity of a G6P system component in a liver biopsy. To date, the diagnosis is most commonly confirmed

  10. Inhibitors of fatty acid biosynthesis in sunflower seeds.

    PubMed

    Pleite, Rafael; Martínez-Force, Enrique; Garcés, Rafael

    2006-09-01

    During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds. PMID:16500723

  11. Advanced spent fuel conditioning process (ACP) progress with respect to remote operation and maintenance

    SciTech Connect

    Lee, Hyo Jik; Lee, Jong Kwang; Park, Byung Suk; Yoon, Ji Sup

    2007-07-01

    Korea Atomic Energy Research Institute (KAERI) has been developing an Advanced Spent Fuel Conditioning Process (ACP) to reduce the volume of spent fuel, and the construction of the ACP facility (ACPF) for a demonstration of its technical feasibility has been completed. In 2006 two inactive demonstrations were performed with simulated fuels in the ACPF. Accompanied by process equipment performance tests, its remote operability and maintainability were also tested during that time. Procedures for remote operation tasks are well addressed in this study and evaluated thoroughly. Also, remote maintenance and repair tasks are addressed regarding some important modules with a high priority order. The above remote handling test's results provided a lot of information such as items to be revised to improve the efficiency of the remote handling tasks. This paper deals with the current status of ACP and the progress of remote handling of ACPF. (authors)

  12. Acyl-ACP Substrate Recognition in Burkholderia mallei BmaI1 Acyl-Homoserine Lactone Synthase

    PubMed Central

    2015-01-01

    The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme. PMID:25215658

  13. USGS tethered ACP platforms: New design means more safety and accuracy

    USGS Publications Warehouse

    Morlock, S.E.; Stewart, J.A.; Rehmel, M.S.

    2004-01-01

    The US Geological Survey has developed an innovative tethered platform that supports an Acoustic Current Profiler (ACP) in making stream-flow measurements (use of the term ACP in this article refers to a class of instruments and not a specific brand name or model). The tethered platform reduces the hazards involved in conventional methods of stream-flow measurement. The use of the platform reduces or eliminates time spent by personnel in streams and boats or on bridges and cableway and stream-flow measurement accuracy is increased.

  14. Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase.

    PubMed

    Wang, Hui; Liu, Li; Lu, Yang; Pan, Pan; Hooker, Jacob M; Fowler, Joanna S; Tonge, Peter J

    2015-11-01

    PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography. PMID:26227776

  15. Phosphatase activity in the limb bones of monkeys (Lagothrix humboldti) with hyperparathyroidism

    PubMed Central

    Jeffree, Grace M.

    1962-01-01

    The paper reports a study of the distribution of phosphatases in the femora of three specimens of Humboldt's woolly monkey (Lagothrix humboldti) suffering from chronic hyperparathyroidism. Bone structure ranged from the apparently normal to extreme osteitis fibrosa. Most marked changes were found in the distribution of alkaline phosphatase, which reached at least 10 times the normal levels in the bone of the second monkey in the series, dropping to levels still well above normal in that of the most severely affected animal. Very high concentrations were found in the deeper layers of hypertrophied growth cartilage and in the osteoblasts lining poorly calcified trabeculae, and high concentrations in the fibre bone of the third animal. Lack of mineralization and the development of osteitis fibrosa are thus associated with a marked increase in alkaline phosphatase activity. Osteoclasts reacted strongly for acid phosphatase but were negative for alkaline phosphatase. Acid phosphatase levels were comparatively high in fibre bone, but overall levels ranged from 1/20 to less than 1/100 those of alkaline phosphatase. Some slow staining for acid phosphatase probably represents residual activity at acid pH of the markedly increased alkaline phosphatase. There may be some association between a failure of mineralization and the presence of acid phosphatase in osteoclasts and osteoid. The aetiology of the monkeys' condition is discussed. It seems likely that the parathyroid hypertrophy and rachitic changes were caused by low blood calcium dependent on a low calcium diet and lack of vitamin D, in which the requirements of New World monkeys are reputedly high. Images PMID:14451521

  16. Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

    PubMed

    Gupta, Manju; DeKelver, Russell C; Palta, Asha; Clifford, Carla; Gopalan, Sunita; Miller, Jeffrey C; Novak, Stephen; Desloover, Daniel; Gachotte, Daniel; Connell, James; Flook, Josh; Patterson, Thomas; Robbins, Kelly; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Petolino, Joseph F

    2012-09-01

    Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species. PMID:22520333

  17. X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

    SciTech Connect

    Dyer, David H.; Lyle, Karen S.; Rayment, Ivan; Fox, Brian G.

    2010-07-13

    Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X{sub 2}HX{sub {approx}100}(D/E)X{sub 2}H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 {angstrom} resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP {Delta}9 desaturase from castor plant with an rms difference 1.42 {angstrom}. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.

  18. Crystallization and preliminary X-ray crystallographic analysis of enoyl-ACP reductase III (FabL) from Bacillus subtilis

    SciTech Connect

    Kim, Kook-Han; Park, Joon Kyu; Ha, Byung Hak; Moon, Jin Ho; Kim, Eunice EunKyeong

    2007-03-01

    Enoyl-ACP reductase III (FabL) from B. subtilis has been overexpressed, purified and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120°, and data were collected to 2.5 Å resolution using synchrotron radiation. Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120° and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 Å resolution.

  19. Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

    NASA Astrophysics Data System (ADS)

    Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

    2010-09-01

    The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule

  20. Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites

    SciTech Connect

    Zhang, Fan; Allen, Andrew J.; Levine, Lyle E.; Vaudin, Mark D.; Skrtic, Drago; Antonucci, Joseph M.; Hoffman, Kathleen M.; Giuseppetti, Anthony A.; Ilavsky, Jan

    2014-07-28

    Our objective was to investigate the complex structural and dynamical conversion process of the amorphous-calcium-phosphate (ACP)-to-apatite transition in ACP based dental composite materials. Composite disks were prepared using zirconia hybridized ACP fillers (0.4 mass fraction) and photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction). We performed an investigation of the solution-mediated ACP-to-apatite conversion mechanism in controlled acidic aqueous environment with in situ ultra-small angle X-ray scattering based coherent X-ray photon correlation spectroscopy and ex situ X-ray diffraction, as well as other complementary techniques. We established that the ACP-to-apatite conversion in ACP composites is a two-step process, owing to the sensitivity to local structural changes provided by coherent X-rays. Initially, ACP undergoes a local microstructural rearrangement without losing its amorphous character. We established the catalytic role of the acid and found the time scale of this rearrangement strongly depends on the pH of the solution, which agrees with previous findings about ACP without the polymer matrix being present. In the second step, ACP is converted to an apatitic form with the crystallinity of the formed crystallites being poor. Separately, we also confirmed that in the regular Zr-modified ACP the rate of ACP conversion to hydroxyapatite is slowed significantly compared to unmodified ACP, which is beneficial for targeted slow release of functional calcium and phosphate ions from dental composite materials. Significantly, for the first time, we were able to follow the complete solution-mediated transition process from ACP to apatite in this class of dental composites in a controlled aqueous environment. A two-step process, suggested previously, was conclusively identified.

  1. Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites

    DOE PAGESBeta

    Zhang, Fan; Allen, Andrew J.; Levine, Lyle E.; Vaudin, Mark D.; Skrtic, Drago; Antonucci, Joseph M.; Hoffman, Kathleen M.; Giuseppetti, Anthony A.; Ilavsky, Jan

    2014-07-28

    Our objective was to investigate the complex structural and dynamical conversion process of the amorphous-calcium-phosphate (ACP)-to-apatite transition in ACP based dental composite materials. Composite disks were prepared using zirconia hybridized ACP fillers (0.4 mass fraction) and photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction). We performed an investigation of the solution-mediated ACP-to-apatite conversion mechanism in controlled acidic aqueous environment with in situ ultra-small angle X-ray scattering based coherent X-ray photon correlation spectroscopy and ex situ X-ray diffraction, as well as other complementary techniques. We established that the ACP-to-apatite conversion in ACP composites is a two-step process, owing to the sensitivity to localmore » structural changes provided by coherent X-rays. Initially, ACP undergoes a local microstructural rearrangement without losing its amorphous character. We established the catalytic role of the acid and found the time scale of this rearrangement strongly depends on the pH of the solution, which agrees with previous findings about ACP without the polymer matrix being present. In the second step, ACP is converted to an apatitic form with the crystallinity of the formed crystallites being poor. Separately, we also confirmed that in the regular Zr-modified ACP the rate of ACP conversion to hydroxyapatite is slowed significantly compared to unmodified ACP, which is beneficial for targeted slow release of functional calcium and phosphate ions from dental composite materials. Significantly, for the first time, we were able to follow the complete solution-mediated transition process from ACP to apatite in this class of dental composites in a controlled aqueous environment. A two-step process, suggested previously, was conclusively identified.« less

  2. Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites

    PubMed Central

    Zhang, Fan; Allen, Andrew J.; Levine, Lyle E.; Vaudin, Mark D.; Skrtic, Drago; Antonucci, Joseph M.; Hoffman, Kathleen M.; Giuseppetti, Anthony A.; Ilavsky, Jan

    2014-01-01

    Objective To investigate the complex structural and dynamical conversion process of the amorphous-calcium-phosphate (ACP) -to-apatite transition in ACP based dental composite materials. Methods Composite disks were prepared using zirconia hybridized ACP fillers (0.4 mass fraction) and photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction). We performed an investigation of the solution-mediated ACP-to-apatite conversion mechanism in controlled acidic aqueous environment with in situ ultra-small angle X-ray scattering based coherent X-ray photon correlation spectroscopy and ex situ X-ray diffraction, as well as other complementary techniques. Results We established that the ACP-to-apatite conversion in ACP composites is a two-step process, owing to the sensitivity to local structural changes provided by coherent X-rays. Initially, ACP undergoes a local microstructural rearrangement without losing its amorphous character. We established the catalytic role of the acid and found the time scale of this rearrangement strongly depends on the pH of the solution, which agrees with previous findings about ACP without the polymer matrix being present. In the second step, ACP is converted to an apatitic form with the crystallinity of the formed crystallites being poor. Separately, we also confirmed that in the regular Zr-modified ACP the rate of ACP conversion to hydroxyapatite is slowed significantly compared to unmodified ACP, which is beneficial for targeted slow release of functional calcium and phosphate ions from dental composite materials. Significance For the first time, we were able to follow the complete solution-mediated transition process from ACP to apatite in this class of dental composites in a controlled aqueous environment. A two-step process, suggested previously, was conclusively identified. PMID:25082155

  3. Yeast Acid Phosphatase in a Student Laboratory.

    ERIC Educational Resources Information Center

    Barbaric, Sloeodan; Ries, Blanka

    1988-01-01

    Examines the influence of enzyme and substrate concentrations, pH, temperature, and inhibitors on catalytic activity. Follows the influence of different phosphate concentrations in the growth medium on enzyme activity. Studies regulation of enzyme synthesis by repression. Includes methodology for six experiments. (MVL)

  4. What's in a Number? Estimating numbers of viable Candidatus Liberibacter asiaticus cells in citrus and ACP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of CLas viability is critical for answering questions about how the organism is proliferating within citrus and ACP, what is the response to therapeutic treatments, and for understanding disease development. Because CLas cannot be grown in culture, estimating cell viability has not been po...

  5. Introducing an Absolute Cavity Pyrgeometer (ACP) for Improving the Atmospheric Longwave Irradiance Measurement (Poster)

    SciTech Connect

    Reda, I.; Stoffel, T.

    2012-03-01

    Advancing climate change research requires accurate and traceable measurement of the atmospheric longwave irradiance. Current measurement capabilities are limited to an estimated uncertainty of larger than +/- 4 W/m2 using the interim World Infrared Standard Group (WISG). WISG is traceable to the Systeme international d'unites (SI) through blackbody calibrations. An Absolute Cavity Pyrgeometer (ACP) is being developed to measure absolute outdoor longwave irradiance with traceability to SI using the temperature scale (ITS-90) and the sky as the reference source, instead of a blackbody. The ACP was designed by NREL and optically characterized by the National Institute of Standards and Technology (NIST). Under clear-sky and stable conditions, the responsivity of the ACP is determined by lowering the temperature of the cavity and calculating the rate of change of the thermopile output voltage versus the changing net irradiance. The absolute atmospheric longwave irradiance is then calculated with an uncertainty of +/- 3.96 W/m2 with traceability to SI. The measured irradiance by the ACP was compared with the irradiance measured by two pyrgeometers calibrated by the World Radiation Center with traceability to the WISG.

  6. Phosphatase regulation of macrophage activation.

    PubMed

    Kozicky, Lisa K; Sly, Laura M

    2015-08-01

    Macrophages are innate immune cells that play critical roles in tissue homeostasis and the immune response to invading pathogens or tumor cells. A hallmark of macrophages is their "plasticity," that is, their ability to respond to cues in their local microenvironment and adapt their activation state or phenotype to mount an appropriate response. During the inflammatory response, macrophages may be required to mount a profound anti-bacterial or anti-tumor response, an anti-inflammatory response, an anti-parasitic response, or a wound healing response. To do so, macrophages express cell surface receptors for growth factors, chemokines and cytokines, as well pathogen and danger associated molecular patterns. Downstream of these cell surface receptors, cell signalling cascades are activated and deactivated by reversible and competing activities of lipid and protein kinases and phosphatases. While kinases drive the activation of cell signalling pathways critical for macrophage activation, the strength and duration of the signalling is regulated by phosphatases. Hence, gene knockout mouse models have revealed critical roles for lipid and protein phosphatases in macrophage activation. Herein, we describe our current understanding and the key roles of specific cellular phosphatases in the regulation of the quality of macrophage polarization as well as the quantity of cytokines produced by activated macrophages. PMID:26216598

  7. Human osteoclast and giant cell differentiation: the apparent switch from nonspecific esterase to tartrate resistant acid phosphatase activity coincides with the in situ expression of osteopontin mRNA.

    PubMed

    Connor, J R; Dodds, R A; James, I E; Gowen, M

    1995-12-01

    Animal model and in vitro cultures suggest that osteoclasts and cells of the mononuclear phagocyte system share a common precursor. However, the human osteoclast precursor has not been positively identified. We attempted to identify the precursor in situ by using a number of osteoclast- and macrophage-selective markers, together with the expression of osteopontin mRNA, previously shown to be abundant in human osteoclasts. Sections of osteophytic bone and a panel of inflammatory connective tissues were processed for in situ hybridization; serial sections were analyzed for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, selective cytochemical markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. The murine anti-human osteoclast monoclonal antibodies 23C6 (vitronectin receptor) and C35 (osteoclast-selective) were used to further identify the osteoclast phenotype. We compared osteoclasts, giant cells, and their respective putative mononuclear precursors. At resorption sites within osteophytic bone, osteopontin mRNA was expressed in osteoclasts and a distinct population of TRAP+, NSE- mononuclear cells. Adjacent clusters of mononuclear cells were TRAP- and NSE+ or were active for both enzymes; these cells demonstrated variable expression of osteopontin mRNA. In the inflammatory connective tissues, abundant macrophage-like cells (NSE+/TRAP-) did not express osteopontin mRNA. However, TRAP+ mononuclear cells observed among clusters of NSE+ cells did express osteopontin mRNA. At these sites, clusters of putative macrophage polykaryons removing fragments of bone debris were observed. These giant cells and associated mononuclear cells were NSE- and distinctly TRAP+, and expressed osteopontin mRNA, C35, and 23C6 (human osteoclast) reactivity. Therefore, cells involved in the remodeling (resorption) of bone or the removal of bone debris, together with their immediate precursors, switch from being NSE

  8. The Crystal Structure of the Ivy delta4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

    SciTech Connect

    Guy,J.; Whittle, E.; Kumaran, D.; Lindqvist, Y.; Shanklin, J.

    2007-01-01

    The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the {Delta}{sup 4} desaturation of 16:0-ACP and the{Delta}{sup 9} desaturation of 18:0-ACP and further desaturates{Delta}{sup 9}-16:1 or {Delta}{sup 9}-18:1 to the corresponding {Delta}{sup 4,9} dienes. The crystal structure of the enzyme has been solved to 1.95{angstrom} resolution, and both the iron-iron distance of 3.2{angstrom} and the presence of a {mu}-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His{sup 227} coordination to Fe2 is lost, and the side chain of Glu{sup 224}, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.

  9. Physiological and Histopathological Investigations on the Effects of α-Lipoic Acid in Rats Exposed to Malathion

    PubMed Central

    Al-Attar, Atef M.

    2010-01-01

    The present study was designed to evaluate the influence of α-lipoic acid treatment in rats exposed to malathion. Forty adult male rats were used in this study and distributed into four groups. Animals of group 1 were untreated and served as control. Rats of group 2 were orally given malathion at a dose level of 100 mg/kg body weight (BW) for a period of one month. Experimental animals of group 3 were orally given α-lipoic acid at a dose level of 20 mg/kg BW and after 3 hours exposed to malathion at the same dose given to group 2. Rats of group 4 were supplemented with α-lipoic acid at the same dose given to group 3. The activities of serum glutamic oxaloacetic acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), alkaline phosphatase (ALP), and acid phosphatase (ACP), and the values of creatinine, urea, and uric acid were statistically increased, while the values of total protein and total albumin were significantly decreased in rats exposed to malathion. Moreover, administration of malathion for one month resulted in damage of liver and kidney structures. Administration of α-lipoic acid before malathion exposure to rat can prevent severe alterations of hematobiochemical parameters and disruptions of liver and kidney structures. In conclusion, this study obviously demonstrated that pretreatment with α-lipoic acid significantly attenuated the physiological and histopathological alterations induced by malathion. Also, the present study identifies new areas of research for development of better therapeutic agents for liver, kidney, and other organs' dysfunctions and diseases. PMID:20454535

  10. Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.

    PubMed

    Jha, J K; Maiti, M K; Bhattacharjee, A; Basu, A; Sen, P C; Sen, S K

    2006-01-01

    A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way. PMID:17092734

  11. Phosphatase acitivity as biosignatures in terrestrial extreme environments

    NASA Astrophysics Data System (ADS)

    Kawai, Jun; Nakamoto, Saki; Hara, Masashi; Obayashi, Yumiko; Kaneko, Takeo; Mita, Hajime; Yoshimura, Yoshitaka; Takano, Yoshinori; Kobayashi, Kensei

    Since phosphate esters are essential for the terrestrial life, phosphatase activity can be a can-didate for biosignatures of biological activity. It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere, high temperature hot springs and stratosphere. We analyzed phosphatase activities in the samples obtained in ex-treme environments such as submarine hydrothermal systems and Antarctica , and discussed whether they can be used as biosignatures for extant life. Core samples and chimney samples were collected at Tarama Knoll in Okinawa Trough in 2009, both in a part of the Archaean Park Project. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Alkaline Phosphatase activ-ity in sea water and in soil was measured spectrometrically by using 25 mM p-nitrophenyl phosphate (pH 8.0) as a substrate. Phosphatase activities in extracts were measured fluoro-metrically by using 4-methylumberyferryl phosphate as a substrate. Concentration of amino acids and their enantiomeric ratios were also determined by HPLC . Significant enzymatic ac-tivities were revealed in both some of the hydrothermal sub-vent systems and Antarctica soils, which is crucial evidence of vigorous microbial oasis. It is consistent with the fact that large enantiomeric excess of L-form amino acids were found in the same core sequences. Optimum temperatures of ALP in the chimney, Antarctica soil and YNU campus soil were 353 K, 313 K, and 333 K, respectively. The present results suggested that phosphatase activities,, together with amino acids, can be used as possible biosignatures for extant life.

  12. Results of First Outdoor Comparison Between Absolute Cavity Pyrgeometer (ACP) and Infrared Integrating Sphere (IRIS) Radiometer at PMOD (Presentation)

    SciTech Connect

    Reda, I.; Grobner, J.; Wacker, S.; Stoffel, T.

    2013-03-01

    The ACP and IRIS are developed to establish a world reference for calibrating pyrgeometers with traceability to SI units. The two radiometers are unwindowed with negligible spectral dependence, and traceable to SI units through the temperature scale (ITS-90). The first outdoor comparison between the two designs was held from January 28 to February 8, 2013 at the Physikalisch-Metorologisches Observatorium Davos (PMOD). The difference between the irradiance measured by ACP and that of IRIS was within 1 W/m2. A difference of 5 W/m2 was observed between the irradiance measured by ACP&IRIS and that of the interim World Infrared Standard Group (WISG).

  13. Determination of liver microsomal glucose-6-phosphatase.

    PubMed

    Zak, B; Epstein, E; Baginski, E S

    1977-01-01

    A procedure for the determination of liver microsomal glucose-6-phosphatase is described. Homogenization and ultracentrifrigation were used to prepare a precipitate whose character was defined by monitoring the desire enzyme activity which serves as a marker. Activity of the enzyme was determined by means of a sensitive colorimetric reaction for the product, inorganic phosphate. Non-enzymatic hydrolysis problems with the substrate are minimized in this procedure by the masking action of citrate. The final heteropoly blue color appears to be considerably sensitized by interaction of phosphomolybdous ion with arsenite. The stability of the relatively labile enzyme was ensured by chelating any metals present with ethylene diamine tetraacetic acid. The overall results obtained by the procedure appear to be useful as an aid in the diagnosis of Type I glycogenosis, a glycogen storage disease called Von Gierke's disease. PMID:192125

  14. The glucose-6-phosphatase system.

    PubMed Central

    van Schaftingen, Emile; Gerin, Isabelle

    2002-01-01

    Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and the kidneys, plays the important role of providing glucose during starvation. Unlike most phosphatases acting on water-soluble compounds, it is a membrane-bound enzyme, being associated with the endoplasmic reticulum. In 1975, W. Arion and co-workers proposed a model according to which G6Pase was thought to be a rather unspecific phosphatase, with its catalytic site oriented towards the lumen of the endoplasmic reticulum [Arion, Wallin, Lange and Ballas (1975) Mol. Cell. Biochem. 6, 75--83]. Substrate would be provided to this enzyme by a translocase that is specific for glucose 6-phosphate, thereby accounting for the specificity of the phosphatase for glucose 6-phosphate in intact microsomes. Distinct transporters would allow inorganic phosphate and glucose to leave the vesicles. At variance with this substrate-transport model, other models propose that conformational changes play an important role in the properties of G6Pase. The last 10 years have witnessed important progress in our knowledge of the glucose 6-phosphate hydrolysis system. The genes encoding G6Pase and the glucose 6-phosphate translocase have been cloned and shown to be mutated in glycogen storage disease type Ia and type Ib respectively. The gene encoding a G6Pase-related protein, expressed specifically in pancreatic islets, has also been cloned. Specific potent inhibitors of G6Pase and of the glucose 6-phosphate translocase have been synthesized or isolated from micro-organisms. These as well as other findings support the model initially proposed by Arion. Much progress has also been made with regard to the regulation of the expression of G6Pase by insulin, glucocorticoids, cAMP and glucose. PMID:11879177

  15. Effects of the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on mechanical properties of luting and lining glass ionomer cement

    NASA Astrophysics Data System (ADS)

    Heravi, Farzin; Bagheri, Hossein; Rangrazi, Abdolrasoul; Mojtaba Zebarjad, Seyed

    2016-07-01

    Recently, the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into glass ionomer cements (GICs) has attracted interest due to its remineralization of teeth and its antibacterial effects. However, it should be investigated to ensure that the incorporation of CPP-ACP does not have significant adverse effects on its mechanical properties. The purpose of this study was to evaluate the effects of the addition of CPP-ACP on the mechanical properties of luting and lining GIC. The first step was to synthesize the CPP-ACP. Then the CPP-ACP at concentrations of 1%, 1.56% and 2% of CPP-ACP was added into a luting and lining GIC. GIC without CPP-ACP was used as a control group. The results revealed that the incorporation of CPP-ACP up to 1.56%(w/w) increased the flexural strength (29%), diametral tensile strength (36%) and microhardness (18%), followed by a reduction in these mechanical properties at 2%(w/w) CPP-ACP. The wear rate was significantly decreased (23%) in 1.56%(w/w) concentration of CPP-ACP and it was increased in 2%(w/w). Accordingly, the addition of 1.56%(w/w) CPP-ACP into luting and lining GIC had no adverse effect on the mechanical properties of luting and lining GIC and could be used in clinical practice.

  16. Myosin light-chain phosphatase.

    PubMed Central

    Morgan, M; Perry, S V; Ottaway, J

    1976-01-01

    1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain. Images PLATE 1 PMID:186030

  17. 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.

  18. Transition metal HE`s - VII ACP. Progress report, August 1971--October 1971

    SciTech Connect

    Clink, G.L.

    1998-12-31

    Physical and physicochemical properties of hexaamminechromium III perchlorate (ACP) were investigated to determine its potentiality for HE and/or HE component application. Physicochemical properties obtained through investigation of isothermally aged ACP under conditions of various system and boundary restrictions (open and closed; 50 and 80 C; 0.65 and 0.85 g/cc) show the material to be substantially stable under these simulated isothermal storage conditions from a standpoint of infrared and DTA thermal pattern behavior and chemical analysis of aged residues. Spark sensitivity was 0.25 joules at 5 kv (by LASL type test) and impact sensitivity was about 23 and 14 cm on sandpaper (12A) and steel (12B), respectively.

  19. Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi.

    PubMed Central

    Shen, Z; Byers, D M

    1994-01-01

    To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with [9,10-3H]myristic acid, and labeled proteins were analyzed by gel electrophoresis. Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V. harveyi ACP serum and comigration with acyl-ACP standards and [3H]beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels. Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total [3H]14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V. harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain. 14:0- and 12:0-ACP were also the predominant species labeled in complex medium. In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V. harveyi was incubated with [3H]acetate, indicating that acyl-ACP labeling with [3H]14:0 in vivo is not due to the total degradation of [3H]14:0 to [3H]acetyl coenzyme A followed by resynthesis. Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with [3H]beta-alanine fivefold, while total incorporation of [3H]14:0 was not affected, although a shift to shorter chain lengths was noted. Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography. The levels of incorporation of [3H] 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min. Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl

  20. Effect of CPP-ACP Paste on Dental Caries in Primary Teeth

    PubMed Central

    Sitthisettapong, T.; Phantumvanit, P.; Huebner, C.; DeRouen, T.

    2012-01-01

    This clinical trial tested the effect of daily application of 10% w/v calcium phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste for 1 yr when added to regular toothbrushing with fluoridated toothpaste to prevent dental caries in pre-school children. High-caries-risk children aged 2½ to 3½ yrs in a suburban area of central Thailand were assigned to receive either CPP-ACP (n = 150) or a placebo control (n = 146) in addition to fluoridated toothpaste. The International Caries Detection and Assessment System (ICDAS) was recorded at baseline, 6 mos, and 1 yr. At 1 yr, a significant increase in mean numbers of enamel and dentin caries lesions, as well as dmfs, was found in both groups (p < 0.001). No significant difference was observed between groups on these 3 outcome measures (p = 0.23, 0.84, and 0.91, respectively). The odds of enamel caries lesion transitions to a state of regression or stability, compared with progression from baseline, was also not different between groups [OR = 1.00, 95% CI (0.86, 1.17)]. This trial found that daily application of 10% w/v CPP-ACP paste on school days for 1 yr, when added to regular toothbrushing with a fluoride toothpaste, had no significant added effect in preventing caries in the primary dentition of these pre-school children (ClinicalTrials.gov number CT01 604109). PMID:22805294

  1. Effect of CPP-ACP paste on dental caries in primary teeth: a randomized trial.

    PubMed

    Sitthisettapong, T; Phantumvanit, P; Huebner, C; Derouen, T

    2012-09-01

    This clinical trial tested the effect of daily application of 10% w/v calcium phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste for 1 yr when added to regular toothbrushing with fluoridated toothpaste to prevent dental caries in pre-school children. High-caries-risk children aged 2½ to 3½ yrs in a suburban area of central Thailand were assigned to receive either CPP-ACP (n = 150) or a placebo control (n = 146) in addition to fluoridated toothpaste. The International Caries Detection and Assessment System (ICDAS) was recorded at baseline, 6 mos, and 1 yr. At 1 yr, a significant increase in mean numbers of enamel and dentin caries lesions, as well as dmfs, was found in both groups (p < 0.001). No significant difference was observed between groups on these 3 outcome measures (p = 0.23, 0.84, and 0.91, respectively). The odds of enamel caries lesion transitions to a state of regression or stability, compared with progression from baseline, was also not different between groups [OR = 1.00, 95% CI (0.86, 1.17)]. This trial found that daily application of 10% w/v CPP-ACP paste on school days for 1 yr, when added to regular toothbrushing with a fluoride toothpaste, had no significant added effect in preventing caries in the primary dentition of these pre-school children (ClinicalTrials.gov number CT01 604109). PMID:22805294

  2. Type III polyketide synthase beta-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining.

    PubMed

    Song, Lijiang; Barona-Gomez, Francisco; Corre, Christophe; Xiang, Longkuan; Udwary, Daniel W; Austin, Michael B; Noel, Joseph P; Moore, Bradley S; Challis, Gregory L

    2006-11-22

    Polyketide synthases (PKSs) are involved in the biosynthesis of many important natural products. In bacteria, type III PKSs typically catalyze iterative decarboxylation and condensation reactions of malonyl-CoA building blocks in the biosynthesis of polyhydroxyaromatic products. Here it is shown that Gcs, a type III PKS encoded by the sco7221 ORF of the bacterium Streptomyces coelicolor, is required for biosynthesis of the germicidin family of 3,6-dialkyl-4-hydroxypyran-2-one natural products. Evidence consistent with Gcs-catalyzed elongation of specific beta-ketoacyl-ACP products of the fatty acid synthase FabH with ethyl- or methylmalonyl-CoA in the biosynthesis of germicidins is presented. Selectivity for beta-ketoacyl-ACP starter units and ethylmalonyl-CoA as an extender unit is unprecedented for type III PKSs, suggesting these enzymes may be capable of utilizing a far wider range of starter and extender units for natural product assembly than believed until now. PMID:17105255

  3. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

    PubMed

    Chen, Xi'en; Lü, Shumin; Zhang, Yalin

    2014-01-01

    Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests. PMID:24823652

  4. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

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

  5. Characterization of the threonine-phosphatase of mouse eyes absent 3.

    PubMed

    Sano, Teruyuki; Nagata, Shigekazu

    2011-09-01

    Eyes absent (EYA) has tyrosine- and threonine-phosphatase activities in their C-terminal and N-terminal regions, respectively. Using various mutants of mouse EYA3, we showed that the 68-amino acid domain between positions 53 and 120 was necessary and sufficient for its threonine-phosphatase activity. Point mutations were then introduced, and residues Cys-56, Tyr-77, His-79, and Tyr-90 were essential for the EYA3s threonine-phosphatase. The 68-amino acid domain is not well conserved among the four mouse EYA members, but is evolutionally highly conserved in the orthologous EYA members of different species, suggesting that the threonine-phosphatase of EYA3 has a function distinct from that of the other EYAs. PMID:21821028

  6. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  7. 3-Oxoacyl-ACP Reductase from Schistosoma japonicum: Integrated In Silico-In Vitro Strategy for Discovering Antischistosomal Lead Compounds

    PubMed Central

    Liu, Jian; Dyer, Dave; Wang, Jipeng; Wang, Shuqi; Du, Xiaofeng; Xu, Bin; Zhang, Haobing; Wang, Xiaoning; Hu, Wei

    2013-01-01

    Background Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR. Methodology/Principal Findings After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells. Conclusions/Significance The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells. PMID:23762275

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

    PubMed

    Hofmann, Kerstin; Heuck, Christine; Spohn, Marie

    2016-06-01

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

  9. A Mg(2+)-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate.

    PubMed

    Fonseca-de-Souza, André L; Dick, Claudia Fernanda; Dos Santos, André Luiz Araújo; Meyer-Fernandes, José Roberto

    2008-08-01

    In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi). PMID:18599005

  10. Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Šmidák, Roman; Mayer, Rupert Laurenz; Bileck, Andrea; Gerner, Christopher; Mechtcheriakova, Diana; Stork, Oliver; Lubec, Gert; Li, Lin

    2016-04-15

    A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms. PMID:26748257

  11. [Phosphoprotein phosphatase nonspecifically hydrolyzes CoA].

    PubMed

    Reziapkin, V I; Moiseenok, A G

    1988-01-01

    CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate). PMID:2849829

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

    PubMed

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans. PMID:27611587

  13. Protein phosphatases in pancreatic islets

    PubMed Central

    Ortsäter, Henrik; Grankvist, Nina; Honkanen, Richard E.; Sjöholm1, Åke

    2014-01-01

    The prevalence of diabetes is increasing rapidly world-wide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis. PMID:24681827

  14. Development of the ACP safeguards neutron counter for PWR spent fuel rods

    NASA Astrophysics Data System (ADS)

    Lee, Tae-Hoon; Menlove, Howard O.; Lee, Sang-Yoon; Kim, Ho-Dong

    2008-04-01

    An advanced neutron multiplicity counter has been developed for measuring spent fuel in the Advanced spent fuel Conditioning Process (ACP) at the Korea Atomic Energy Research Institute (KAERI). The counter uses passive neutron multiplicity counting to measure the 244Cm content in spent fuel. The input to the ACP process is spent fuel from pressurized water reactors (PWRs), and the high intensity of the gamma-ray exposure from spent fuel requires a careful design of the counter to measure the neutrons without gamma-ray interference. The nuclear safeguards for the ACP facility requires the measurement of the spent fuel input to the process and the Cm/Pu ratio for the plutonium mass accounting. This paper describes the first neutron counter that has been used to measure the neutron multiplicity distribution from spent fuel rods. Using multiple samples of PWR spent fuel rod-cuts, the singles (S), doubles (D), and triples (T) rates of the neutron distribution for the 244Cm nuclide were measured and calibration curves were produced. MCNPX code simulations were also performed to obtain the three counting rates and to compare them with the measurement results. The neutron source term was evaluated by using the ORIGEN-ARP code. The results showed systematic difference of 21-24% in the calibration graphs between the measured and simulation results. A possible source of the difference is that the burnup codes have a 244Cm uncertainty greater than ±15% and it would be systematic for all of the calibration samples. The S/D and D/T ratios are almost constant with an increment of the 244Cm mass, and this indicates that the bias is in the 244Cm neutron source calculation using the ORIGEN-ARP source code. The graphs of S/D and D/T ratios show excellent agreement between measurement and MCNPX simulation results.

  15. Combining CPP-ACP with fluoride: a synergistic remineralization potential of artificially demineralized enamel or not?

    NASA Astrophysics Data System (ADS)

    El-Sayad, I. I.; Sakr, A. K.; Badr, Y. A.

    2008-08-01

    Background and objective: Minimal intervention dentistry (MID) calls for early detection and remineralization of initial demineralization. Laser fluorescence is efficient in detecting changes in mineral tooth content. Recaldent is a product of casein phosphopeptide-amorphous calcium phosphate (CPP- ACP) which delivers calcium and phosphate ions to enamel. A new product which also contains fluoride is launched in United States. The remineralizing potential of CPP- ACP per se, or when combined with 0.22% Fl supplied in an oral care gel on artificially demineralised enamel using laser fluorescence was investigated. Methods: Fifteen sound human molars were selected. Mesial surfaces were tested using He-Cd laser beam at 441.5nm with 18mW power as excitation source on a suitable set-up based on Spex 750 M monochromator provided with PMT for detection of collected auto-fluorescence from sound enamel. Mesial surfaces were subjected to demineralization for ten days. The spectra from demineralized enamel were measured. Teeth were then divided according to the remineralizing regimen into three groups: group I recaldent per se, group II recaldent combined with fluoride gel and group III artificial saliva as a positive control. After following these protocols for three weeks, the spectra from remineralized enamel from the three groups were measured. The spectra of enamel auto-fluorescence were recorded and normalized to peak intensity at about 540 nm to compare between spectra from sound, demineralized and remineralized enamel surfaces. Results: A slight red shift was noticed in spectra from demineralized enamel, while a blue shift may occur in remineralized enamel. Group II showed the highest remineralizing potential. Conclusions: Combining fluoride with CPP-ACP had a synergistic effect on enamel remineralization. In addition, laser auto-fluorescence is an accurate technique for assessment of changes in tooth enamel minerals.

  16. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  17. HuPho: the human phosphatase portal.

    PubMed

    Liberti, Susanna; Sacco, Francesca; Calderone, Alberto; Perfetto, Livia; Iannuccelli, Marta; Panni, Simona; Santonico, Elena; Palma, Anita; Nardozza, Aurelio P; Castagnoli, Luisa; Cesareni, Gianni

    2013-01-01

    Phosphatases and kinases contribute to the regulation of protein phosphorylation homeostasis in the cell. Phosphorylation is a key post-translational modification underlying the regulation of many cellular processes. Thus, a comprehensive picture of phosphatase function and the identification of their target substrates would aid a systematic approach to a mechanistic description of cell signalling. Here we present a website designed to facilitate the retrieval of information about human protein phosphatases. To this end we developed a search engine to recover and integrate information annotated in several publicly available web resources. In addition we present a text-mining-assisted annotation effort aimed at extracting phosphatase related data reported in the scientific literature. The HuPho (human phosphatases) website can be accessed at http://hupho.uniroma2.it. PMID:22804825

  18. Phosphoinositide Phosphatases in Cell Biology and Disease

    PubMed Central

    Liu, Yang; Bankaitis, Vytas A.

    2010-01-01

    Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

  19. Insights from computational analysis of full-length β-ketoacyl-[ACP] synthase-II cDNA isolated from American and African oil palms

    PubMed Central

    Bhore, Subhash J.; Cha, Thye S.; Amelia, Kassim; Shah, Farida H.

    2014-01-01

    Background: Palm oil derived from fruits (mesocarp) of African oil palm (Elaeis guineensis Jacq. Tenera) and American oil palm (E. oleifera) is important for food industry. Due to high yield, Elaeis guineensis (Tenera) is cultivated on commercial scale, though its oil contains high (~54%) level of saturated fatty acids. The rate-limiting activity of beta-ketoacyl-[ACP] synthase-II (KAS-II) is considered mainly responsible for the high (44%) level of palmitic acid (C16:0) in the oil obtained from E. guineensis. Objective: The objective of this study was to annotate KAS-II cDNA isolated from American and African oil palms. Materials and Methods: The full-length E. oleifera KAS-II (EoKAS-II) cDNA clone was isolated using random method of gene isolation. Whereas, the E. guineensis KAS-II (EgTKAS-II) cDNA was isolated using reverse transcriptase polymerase chain reaction (RT-PCR) technique; and missing ends were obtained by employing 5’and 3’ RACE technique. Results: The results show that EoKAS-II and EgTKAS-II open reading frames (ORFs) are of 1689 and 1721 bp in length, respectively. Further analysis of the both EoKAS-II and EgTKAS-II predicted protein illustrates that they contains conserved domains for ‘KAS-I and II’, ‘elongating’ condensing enzymes, ‘condensing enzymes super-family’, and ‘3-oxoacyl-[ACP] synthase II’. The predicted protein sequences shows 95% similarity with each other. Consecutively, the three active sites (Cys, His, and His) were identified in both proteins. However, difference in positions of two active Histidine (His) residues was noticed. Conclusion: These insights may serve as the foundation in understanding the variable activity of KAS-II in American and African oil palms; and cDNA clones could be useful in the genetic engineering of oil palms. PMID:24678202

  20. Short-term storage of canine preantral ovarian follicles using a powdered coconut water (ACP)-based medium.

    PubMed

    Lima, G L; Costa, L L M; Cavalcanti, D M L P; Rodrigues, C M F; Freire, F A M; Fontenele-Neto, J D; Silva, A R

    2010-07-01

    The objective was to investigate the use of powdered coconut water (ACP)-based medium for short-term preservation of canine preantral follicles. Pairs of ovaries from mongrel bitches (n=9) were divided into fragments. One ovarian fragment, treated as a fresh control, was immediately fixed for histological analysis, whereas the other six ovarian fragments were stored either in phosphate-buffered saline (PBS; control group) or ACP medium in isothermal Styrofoam boxes containing biological ice packs. The boxes were sealed and opened only after 12, 24, or 36h. After opening each box, the ovarian fragments were submitted to histological analysis. In total, 12,302 preantral follicles were evaluated, with 64.5% primordial, 33.3% primary, and 2.3% secondary follicles. There were multiple oocytes in 1.3% of the follicles analyzed. At 24h, ACP was more efficient in preserving follicular morphology than PBS (P<0.05). Compared with the fresh control group, a significant reduction in the percentage of morphologically normal ovarian follicles was observed for PBS, starting at 24h; however, the decline started only at 36h for the ACP medium. During the experiment, the temperature inside the isothermal boxes increased from 3 to 9 degrees C (P<0.05), despite a constant room temperature. In conclusion, powdered coconut water (ACP) was an appropriate medium for short-term storage of canine preantral ovarian follicles. PMID:20207405

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  2. Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    PubMed Central

    Signorelli, Janetti R.; Díaz, Emilce S.; Fara, Karla; Barón, Lina; Morales, Patricio

    2013-01-01

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

  3. Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

    PubMed Central

    Cohen, P; Nimmo, G A; Antoniw, J F

    1977-01-01

    A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian

  4. Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

    PubMed

    Daum, G; Schmid, B; MacKintosh, C; Cohen, P; Hofer, H W

    1992-07-13

    In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a. PMID:1321672

  5. Redirection of metabolic flux for high levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds.

    PubMed

    Nguyen, Huu Tam; Park, Hyunwoo; Koster, Karen L; Cahoon, Rebecca E; Nguyen, Hanh T M; Shanklin, John; Clemente, Thomas E; Cahoon, Edgar B

    2015-01-01

    Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1∆9) and cis-vaccenic acid (18:1∆11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ∆9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ∆9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ∆9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ∆9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications. PMID:25065607

  6. New functional aspects of the atypical protein tyrosine phosphatase VHZ

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.

    2013-01-01

    LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date, and was originally misclassified as an atypical dual specificity phosphatase (DSP). Kinetic isotope effects with steady state and pre-steady state kinetics of VHZ and mutants with para-nitrophenol phosphate (pNPP) have revealed several unusual properties. VHZ is significantly more active than previously reported, but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations to Q-loop residues affect this phosphotransferase activity, mutations on the IPD-loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer. PMID:24073992

  7. Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase.

    PubMed

    Neckles, Carla; Pschibul, Annica; Lai, Cheng-Tsung; Hirschbeck, Maria; Kuper, Jochen; Davoodi, Shabnam; Zou, Junjie; Liu, Nina; Pan, Pan; Shah, Sonam; Daryaee, Fereidoon; Bommineni, Gopal R; Lai, Cristina; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2016-05-31

    The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156. PMID:27136302

  8. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  9. Phosphatidate phosphatase, a key regulator of lipid homeostasis.

    PubMed

    Pascual, Florencia; Carman, George M

    2013-03-01

    Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The cellular amounts of these lipid intermediates influence the synthesis of triacylglycerol and the pathways by which membrane phospholipids are synthesized. Physiological functions affected by PAP activity include phospholipid synthesis gene expression, nuclear/endoplasmic reticulum membrane growth, lipid droplet formation, and vacuole homeostasis and fusion. Yeast lacking Pah1p PAP activity are acutely sensitive to fatty acid-induced toxicity and exhibit respiratory deficiency. PAP is distinguished in its cellular location, catalytic mechanism, and physiological functions from Dpp1p and Lpp1p lipid phosphate phosphatases that utilize a variety of substrates that include phosphatidate. Phosphorylation/dephosphorylation is a major mechanism by which Pah1p PAP activity is regulated. Pah1p is phosphorylated by cytosolic-associated Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A and is dephosphorylated by the endoplasmic reticulum-associated Nem1p-Spo7p phosphatase. The dephosphorylation of Pah1p stimulates PAP activity and facilitates the association with the membrane/phosphatidate allowing for its reaction and triacylglycerol synthesis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. PMID:22910056

  10. Searching for the role of protein phosphatases in eukaryotic microorganisms.

    PubMed

    da-Silva, A M; Zapella, P D; Andrioli, L P; Campanhã, R B; Fiorini, L C; Etchebehere, L C; da-Costa-Maia, J C; Terenzi, H F

    1999-07-01

    Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism. PMID:10454741

  11. AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei—Crystal structure, mode of action, and biological activity

    PubMed Central

    Narasimha Rao, Krishnamurthy; Lakshminarasimhan, Anirudha; Joseph, Sarah; Lekshmi, Swathi U; Lau, Ming-Seong; Takhi, Mohammed; Sreenivas, Kandepu; Nathan, Sheila; Yusof, Rohana; Abd Rahman, Noorsaadah; Ramachandra, Murali; Antony, Thomas; Subramanya, Hosahalli

    2015-01-01

    Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors. PMID:25644789

  12. AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei--Crystal structure, mode of action, and biological activity.

    PubMed

    Rao, Krishnamurthy Narasimha; Lakshminarasimhan, Anirudha; Joseph, Sarah; Lekshmi, Swathi U; Lau, Ming-Seong; Takhi, Mohammed; Sreenivas, Kandepu; Nathan, Sheila; Yusof, Rohana; Abd Rahman, Noorsaadah; Ramachandra, Murali; Antony, Thomas; Subramanya, Hosahalli

    2015-05-01

    Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors. PMID:25644789

  13. Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

    PubMed

    Etchebehere, L C; Simon, M N; Campanhã, R B; Zapella, P D; Véron, M; Maia, J C

    1993-08-01

    Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases. PMID:8394312

  14. Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

    PubMed

    Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

    2014-10-01

    Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. PMID:25038276

  15. Multiple Functions of the Eya Phosphotyrosine Phosphatase

    PubMed Central

    2015-01-01

    Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling. PMID:26667035

  16. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes...

  17. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes...

  18. Analysis of Smad Phosphatase Activity In Vitro.

    PubMed

    Shen, Tao; Qin, Lan; Lin, Xia

    2016-01-01

    Phosphorylation of Smad1/5/8 at the C-terminal SXS motif by BMP type I receptors is one of the most critical events in BMP signaling. Conversely, protein phosphatases that dephosphorylate phospho-Smad1/5/8 can consequently prevent or terminate BMP signaling. PPM1H is an undercharacterized phosphatase in the PPM family. We recently demonstrated that PPM1H can dephosphorylate Smad1 in the cytoplasm and block BMP signaling responses in cellular assays. Here we describe in vitro method showing that PPM1H is a bona fide phosphatase for Smad1/5/8. PPM1H is produced as GST fusion protein in E. coli, and purified against glutathione sepharose beads. Bacterially purified recombinant PPM1H possesses phosphatase activity toward artificial substrate para-nitrophenyl phosphate (pNPP). Recombinant PPM1H also dephosphorylates immuno-purified phosphorylated Smad1 in test tubes. These direct in vitro phosphatase assays provide convincing evidence demonstrating the role of PPM1H as a specific phosphatase for P-Smad1. PMID:26520120

  19. Chaplain Documentation and the Electronic Medical Record: A Survey of ACPE Residency Programs.

    PubMed

    Tartaglia, Alexander; Dodd-McCue, Diane; Ford, Timothy; Demm, Charles; Hassell, Alma

    2016-01-01

    This study explores the extent to which chaplaincy departments at ACPE-accredited residency programs make use of the electronic medical record (EMR) for documentation and training. Survey data solicited from 219 programs with a 45% response rate and interview findings from 11 centers demonstrate a high level of usage of the EMR as well as an expectation that CPE residents document each patient/family encounter. Centers provided considerable initial training, but less ongoing monitoring of chaplain documentation. Centers used multiple sources to develop documentation tools for the EMR. One center was verified as having created the spiritual assessment component of the documentation tool from a peer reviewed published model. Interviews found intermittent use of the student chart notes for educational purposes. One center verified a structured manner of monitoring chart notes as a performance improvement activity. Findings suggested potential for the development of a standard documentation tool for chaplain charting and training. PMID:26168408

  20. A Project Management Approach to an ACPE Accreditation Self-study

    PubMed Central

    Iwanowicz, Susan L.; Bailie, George R.; Clarke, David W.; McGraw, Patrick S.

    2007-01-01

    In preparation for an on-site evaluation and accreditation by the American Council on Pharmaceutical Education (ACPE), the Albany College of Pharmacy employed project management techniques to complete a comprehensive self-study. A project lifecycle approach, including planning, production, and turnover phases, was used by the project's Self-Study Steering Committee. This approach, with minimal disruption to college operations, resulted in the completion of the self-study process on schedule. Throughout the project, the Steering Committee maintained a log of functions that either were executed successfully or in hindsight, could have been improved. To assess the effectiveness of the project management approach to the the self-study process, feedback was obtained from the College community through a poststudy survey. This feedback, coupled with the Steering Committee's data on possible improvements, form the basis for the lessons learned during this self-study process. PMID:17533432

  1. Evaluation of the remineralization capacity of CPP-ACP containing fluoride varnish by different quantitative methods

    PubMed Central

    SAVAS, Selcuk; KAVRÌK, Fevzi; KUCUKYÌLMAZ, Ebru

    2016-01-01

    ABSTRACT Objective The aim of this study was to evaluate the efficacy of CPP-ACP containing fluoride varnish for remineralizing white spot lesions (WSLs) with four different quantitative methods. Material and Methods Four windows (3x3 mm) were created on the enamel surfaces of bovine incisor teeth. A control window was covered with nail varnish, and WSLs were created on the other windows (after demineralization, first week and fourth week) in acidified gel system. The test material (MI Varnish) was applied on the demineralized areas, and the treated enamel samples were stored in artificial saliva. At the fourth week, the enamel surfaces were tested by surface microhardness (SMH), quantitative light-induced fluorescence-digital (QLF-D), energy-dispersive spectroscopy (EDS) and laser fluorescence (LF pen). The data were statistically analyzed (α=0.05). Results While the LF pen measurements showed significant differences at baseline, after demineralization, and after the one-week remineralization period (p<0.05), the difference between the 1- and 4-week was not significant (p>0.05). With regards to the SMH and QLF-D analyses, statistically significant differences were found among all the phases (p<0.05). After the 1- and 4-week treatment periods, the calcium (Ca) and phosphate (P) concentrations and Ca/P ratio were higher compared to those of the demineralization surfaces (p<0.05). Conclusion CPP-ACP containing fluoride varnish provides remineralization of WSLs after a single application and seems suitable for clinical use. PMID:27383699

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

    PubMed

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

    2016-01-01

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

  3. Microleakage of Three Types of Glass Ionomer Cement Restorations: Effect of CPP-ACP Paste Tooth Pretreatment

    PubMed Central

    Doozandeh, Maryam; Shafiei, Fereshteh; Alavi, Mostafa

    2015-01-01

    Statement of the Problem Casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) increases the mineral content of tooth structure. This may enhance the chemical bonding of glass ionomer cements (GIC) and marginal sealing of their restorations. Purpose The aim of this study was to evaluate the effect of CPP-ACP paste pretreatment on the microleakage of three types of GIC. Materials and Method In this study, 72 Class V cavities were prepared on the buccal and lingual surfaces of molars with occlusal margins in enamel and gingival margins in root. The cavities were divided into 6 groups. Cavities in group 1 and 2 were restored with Fuji II, group 3 and 4 with Fuji II LC, and group 5 and 6 with Ketac N100 with respect to the manufacturers’ instructions. In groups 2, 4 and 6, CPP-ACP containing paste (MI paste) was placed into the cavities for 3 minutes before being filled with GIC. The teeth were thermocycled, stained with dye, sectioned, and scored for microleakage under stereomicroscope. Kruskall-Wallis and Chi-Square tests were used to analyze the data. Result There were no statistically significant differences between the control and the CPP-ACP pretreatment groups in enamel and dentin margins. In pairwise comparisons, there were no significant differences between the control and the experimental groups in enamel margin, and in dentin margins of G1 and 2, G5 and 6; however, a significant differences was detected in dentin margins between G3 and 4 (p= 0.041). Conclusion CPP-ACP paste pretreatment did not affect the microleakage of Fuji II and Ketac N100 in enamel or dentin, but decreased the microleakage in dentine margins of Fuji II LC when cavity conditioner was applied before surface treatment. PMID:26331147

  4. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    PubMed

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  5. Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Travis, Sue M.; Berger, Herbert A.; Welsh, Michael J.

    1997-01-01

    cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target. PMID:9380758

  6. An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag.

    PubMed

    Garcia, Francisco J; Carroll, Kate S

    2016-05-24

    Protein tyrosine phosphatases are crucial regulators of signal transduction and function as antagonists towards protein tyrosine kinases to control reversible tyrosine phosphorylation, thereby regulating fundamental physiological processes. Growing evidence has supported the notion that reversible oxidative inactivation of the catalytic cysteine residue in protein tyrosine phosphatases serves as an oxidative post-translational modification that regulates its activity to influence downstream signaling by promoting phosphorylation and induction of the signaling cascade. The oxidation of cysteine to the sulfenic acid is often transient and difficult to detect, thus making it problematic in understanding the role that this oxidative post-translational modification plays in redox-biology and pathogenesis. Several methods to detect cysteine oxidation in biological systems have been developed, though targeted approaches to directly detect oxidized phosphatases are still lacking. Herein we describe the development of a novel immunochemical approach to directly profile oxidized phosphatases. This immunochemical approach consists of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone (CDMD), a nucleophile that chemoselectively reacts with cysteine sulfenic acids to form a stable thioether adduct. Additionally, we provide biochemical and mass spectrometry workflows to be used in conjugation with this newly developed immunochemical approach to assist in the identification and quantification of basal and oxidized phosphatases. PMID:26757830

  7. Protein Phosphatase 1 β Paralogs Encode the Zebrafish Myosin Phosphatase Catalytic Subunit

    PubMed Central

    Jayashankar, Vaishali; Nguyen, Michael J.; Carr, Brandon W.; Zheng, Dale C.; Rosales, Joseph B.; Rosales, Joshua B.; Weiser, Douglas C.

    2013-01-01

    Background The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. Methodology/Principal Findings We observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. Conclusions/Significance Our work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required. PMID:24040418

  8. Bacillus licheniformis MC14 alkaline phosphatase I gene with an extended COOH-terminus.

    PubMed

    Kim, J W; Peterson, T; Bee, G; Hulett, F M

    1998-02-01

    Bacterial alkaline phosphatases (APases), except those isolated from Bacillus licheniformis, are approximately 45-kDa proteins while eucaryotic alkaline phosphatases are 60 kDa. To answer the question of whether the apparent 60-kDa alkaline phosphatase from Bacillus licheniformis accurately reflected the size of the protein, the entire gene was analyzed. DNA sequence analysis of the alkaline phosphatase I (APaseI) gene of B. licheniformis MC14 indicated that the gene could code for a 60-kDa protein of 553 amino acids. The deduced protein sequence of APaseI showed about 32% identity to those of B. subtilis APase III and IV and had apparent sequence homologies in the core structure and active sites that are conserved among APases of various sources. The extra carboxy-terminal sequence of APaseI, which made the enzyme bigger than other procaryotic APases, was not homologous to those of eucaryotic APases. The amino acid composition of APaseI was most similar to that of salt-dependent APase among the isozymes of B. licheniformis MC14. Another open reading frame of 261 amino acids was present 142 nucleotide upstream of the APaseI gene and its predicted amino acid sequence showed 68% identity to that of glucose dehydrogenase of B. megaterium. PMID:9485594

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

    PubMed

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

    2008-05-01

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

  10. Bacterial-like PPP protein phosphatases

    PubMed Central

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, “bacterial-like” enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the “eukaryotic-like” phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research. PMID:24675170

  11. Biomimetic remineralization of demineralized dentine using scaffold of CMC/ACP nanocomplexes in an in vitro tooth model of deep caries.

    PubMed

    Chen, Zhen; Cao, Shansong; Wang, Haorong; Li, Yanqiu; Kishen, Anil; Deng, Xuliang; Yang, Xiaoping; Wang, Yinghui; Cong, Changhong; Wang, Huajun; Zhang, Xu

    2015-01-01

    Currently, it is still a tough task for dentists to remineralize dentine in deep caries. The aim of this study was to remineralize demineralized dentine in a tooth model of deep caries using nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) based on mimicking the stabilizing effect of dentine matrix protein 1 (DMP1) on ACP in the biomineralization of dentine. The experimental results indicate that CMC can stabilize ACP to form nanocomplexes of CMC/ACP, which is able to be processed into scaffolds by lyophilization. In the single-layer collagen model, ACP nanoparticles are released from scaffolds of CMC/ACP nanocomplexes dissolved and then infiltrate into collagen fibrils via the gap zones (40 nm) to accomplish intrafibrillar mineralization of collagen. With this method, the completely demineralized dentine was partially remineralized in the tooth mode. This is a bottom-up remineralizing strategy based on non-classical crystallization theory. Since nanocomplexes of CMC/ACP show a promising effect of remineralization on demineralized dentine via biomimetic strategy, thereby preserving dentinal tissue to the maximum extent possible, it would be a potential indirect pulp capping (IPC) material for the management of deep caries during vital pulp therapy based on the concept of minimally invasive dentistry (MID). PMID:25587986

  12. Biomimetic Remineralization of Demineralized Dentine Using Scaffold of CMC/ACP Nanocomplexes in an In Vitro Tooth Model of Deep Caries

    PubMed Central

    Chen, Zhen; Cao, Shansong; Wang, Haorong; Li, Yanqiu; Kishen, Anil; Deng, Xuliang; Yang, Xiaoping; Wang, Yinghui; Cong, Changhong; Wang, Huajun; Zhang, Xu

    2015-01-01

    Currently, it is still a tough task for dentists to remineralize dentine in deep caries. The aim of this study was to remineralize demineralized dentine in a tooth model of deep caries using nanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) based on mimicking the stabilizing effect of dentine matrix protein 1 (DMP1) on ACP in the biomineralization of dentine. The experimental results indicate that CMC can stabilize ACP to form nanocomplexes of CMC/ACP, which is able to be processed into scaffolds by lyophilization. In the single-layer collagen model, ACP nanoparticles are released from scaffolds of CMC/ACP nanocomplexes dissolved and then infiltrate into collagen fibrils via the gap zones (40 nm) to accomplish intrafibrillar mineralization of collagen. With this method, the completely demineralized dentine was partially remineralized in the tooth mode. This is a bottom-up remineralizing strategy based on non-classical crystallization theory. Since nanocomplexes of CMC/ACP show a promising effect of remineralization on demineralized dentine via biomimetic strategy, thereby preserving dentinal tissue to the maximum extent possible, it would be a potential indirect pulp capping (IPC) material for the management of deep caries during vital pulp therapy based on the concept of minimally invasive dentistry (MID). PMID:25587986

  13. Structure of a Specialized Acyl Carrier Protein Essential for Lipid A Biosynthesis with Very Long-chain Fatty Acids in Open and Closed Conformations

    SciTech Connect

    Ramelot, Theresa A.; Rossi, Paolo M.; Forouhar, Farhad; Lee, Hsiau-Wei; Yang, Yunhuang; Ni, Shuisong; Unser, Sarah; Lew, Scott; Seetharaman, Jayaraman; Xiao, Rong; Acton, Thomas; Everett, John K.; Prestegard, James H.; Hunt, John F.; Montelione, Gaetano; Kennedy, Michael A.

    2012-09-18

    The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.

  14. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

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

    2005-04-05

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

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

    PubMed

    Humiczewska, M

    1989-01-01

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

  16. Concentrations of long-chain acyl-acyl carrier proteins during fatty acid synthesis by chloroplasts isolated from pea (Pisum sativum), safflower (Carthamus tinctoris), and amaranthus (Amaranthus lividus) leaves

    SciTech Connect

    Roughan, G.; Nishida, I. )

    1990-01-01

    Fatty acid synthesis from (1-14C)acetate by chloroplasts isolated from peas and amaranthus was linear for at least 15 min, whereas incorporation of the tracer into long-chain acyl-acyl carrier protein (ACP) did not increase after 2-3 min. When reactions were transferred to the dark after 3-5 min, long-chain acyl-ACPs lost about 90% of their radioactivity and total fatty acids retained all of theirs. Half-lives of the long-chain acyl-ACPs were estimated to be 10-15 s. Concentrations of palmitoyl-, stearoyl-, and oleoyl-ACP as indicated by equilibrium labeling during steady-state fatty acid synthesis, ranged from 0.6-1.1, 0.2-0.7, and 0.4-1.6 microM, respectively, for peas and from 1.6-1.9, 1.3-2.6, and 0.6-1.4 microM, respectively, for amaranthus. These values are based on a chloroplast volume of 47 microliters/mg chlorophyll and varied according to the mode of the incubation. A slow increase in activity of the fatty acid synthetase in safflower chloroplasts resulted in long-chain acyl-ACPs continuing to incorporate labeled acetate for 10 min. Upon re-illumination following a dark break, however, both fatty acid synthetase activity and acyl-ACP concentrations increased very rapidly. Palmitoyl-ACP was present at concentrations up to 2.5 microM in safflower chloroplasts, whereas those of stearoyl- and oleoyl-ACPs were in the lower ranges measured for peas. Acyl-ACPs were routinely separated from extracts of chloroplasts that had been synthesising long-chain fatty acids from labeled acetate by a minor modification of the method of Mancha et al. The results compared favorably with those obtained using alternative analytical methods such as adsorption to filter paper and partition chromatography on silicic acid columns.

  17. Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia.

    PubMed

    Hasegawa, S; Morioka, M; Goto, S; Korematsu, K; Okamura, A; Yano, S; Kai, Y; Hamada, J I; Ushio, Y

    2000-02-15

    We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration. PMID:10652442

  18. Highly conserved nucleotide phosphatase essential for membrane lipid homeostasis in Streptococcus pneumoniae.

    PubMed

    Kuipers, Kirsten; Gallay, Clement; Martínek, Václav; Rohde, Manfred; Martínková, Markéta; van der Beek, Samantha L; Jong, Wouter S P; Venselaar, Hanka; Zomer, Aldert; Bootsma, Hester; Veening, Jan-Willem; de Jonge, Marien I

    2016-07-01

    Proteins belonging to the DHH family, a member of the phosphoesterase superfamily, are produced by most bacterial species. While some of these proteins are well studied in Bacillus subtilis and Escherichia coli, their functions in Streptococcus pneumoniae remain unclear. Recently, the highly conserved DHH subfamily 1 protein PapP (SP1298) has been reported to play an important role in virulence. Here, we provide a plausible explanation for the attenuated virulence of the papP mutant. Recombinant PapP specifically hydrolyzed nucleotides 3'-phosphoadenosine-5'-phosphate (pAp) and 5'-phosphoadenylyl-(3'->5')-adenosine (pApA). Deletion of papP, potentially leading to pAp/pApA accumulation, resulted in morphological defects and mis-localization of several cell division proteins. Incubation with both polar solvent and detergent led to robust killing of the papP mutant, indicating that membrane integrity is strongly affected. This is in line with previous studies showing that pAp inhibits the ACP synthase, an essential enzyme involved in lipid precursor production. Remarkably, partial inactivation of the lipid biosynthesis pathway, by inhibition of FabF or depletion of FabH, phenocopied the papP mutant. We conclude that pAp and pApA phosphatase activity of PapP is required for maintenance of membrane lipid homeostasis providing an explanation how inactivation of this protein may attenuate pneumococcal virulence. PMID:26691161

  19. Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

    PubMed Central

    Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

    2015-01-01

    Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

  20. Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase.

    PubMed

    Inaba, Kazuo

    2002-10-01

    Sperm flagellar movements are regulated by cAMP-dependent protein phosphorylation. Tctex2-related light chain of outer arm dynein is a well-defined phosphorylated protein that is phosphorylated at activation of sperm motility. Here, the protein phosphatase that dephosphorylates Tctex2-related dynein light chain (LC2) has been characterized in salmonid fish sperm. Most of the phosphatase activity against LC2 is found in Triton-soluble fraction of flagella but trace extent of the activity is retained in the axoneme. The dephosphorylation of LC2 is inhibited by okadaic acid at more than 1nM, whereas that of dynein alpha heavy chain is inhibited at more than 10nM. The addition of Ca(2+) gives no direct effect on LC2 dephosphorylation, but it accelerates the dephosphorylation of the regulatory subunit of cAMP-dependent protein kinase, resulting in the decrease of LC2 phosphorylation. The activity to dephosphorylate the LC2 is separated by MonoQ ion-exchange column chromatography along with the immunoreactivity to the antibody against the catalytic subunit of type 2A protein phosphatase. These results suggest that LC2 is dephosphorylated by type 2A protein phosphatase and that dynein alpha heavy chain and the regulatory subunit of cAMP-dependent protein kinase are dephosphorylated by other types of protein phosphatases. PMID:12359223

  1. PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity.

    PubMed Central

    Iwanicki, Adam; Herman-Antosiewicz, Anna; Pierechod, Marcin; Séror, Simone J; Obuchowski, Michał

    2002-01-01

    Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265-2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine. PMID:12059787

  2. Structure of human PIR1, an atypical dual-specificity phosphatase.

    PubMed

    Sankhala, Rajeshwer Singh; Lokareddy, Ravi Kumar; Cingolani, Gino

    2014-02-11

    PIR1 is an atypical dual-specificity phosphatase (DSP) that dephosphorylates RNA with a higher specificity than phosphoproteins. Here we report the atomic structure of a catalytically inactive mutant (C152S) of the human PIR1 phosphatase core (PIR1-core, residues 29-205), refined at 1.20 Å resolution. PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5'-phosphatases such as the baculovirus RNA triphosphatase and the human mRNA capping enzyme. The PIR1 active site cleft is wider and deeper than that of VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the γ-phosphate of RNA, and ∼6 Å away, a chloride ion coordinates the general base R158. Two residues in the PIR1 phosphate-binding loop (P-loop), a histidine (H154) downstream of C152 and an asparagine (N157) preceding R158, make close contacts with the active site phosphate, and their nonaliphatic side chains are essential for phosphatase activity in vitro. These residues are conserved in all RNA 5'-phosphatases that, analogous to PIR1, lack a "general acid" residue. Thus, a deep active site crevice, two active site ions, and conserved P-loop residues stabilizing the γ-phosphate of RNA are defining features of atypical DSPs that specialize in dephosphorylating 5'-RNA. PMID:24447265

  3. [Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

    PubMed

    Sibirnyi, A A; Shavlovskii, G M

    1978-01-01

    The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed. PMID:208203

  4. Radiosynthesis and biological evaluation of a novel enoyl-ACP reductase inhibitor for Staphylococcus aureus

    DOE PAGESBeta

    Wang, Hui; Lu, Yang; Liu, Li; Kim, Sung Won; Hooker, Jacob M.; Fowler, Joanna S.; Tonge, Peter J.

    2014-09-06

    Here we evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) of PT119, a potent Staphylococcus aureus enoyl-ACP reductase (saFabI) inhibitor with a Ki value of 0.01 nM and a residence time of 750 min on the enzyme target in mice. PT119 was found to have promising antibacterial activity in two different S. aureus infection models: it caused a 3 log reduction in the CFU’s in a mouse thigh muscle infection model and increased the survival rate from 0% to 50% in a mouse systemic infection model. PT119 was then radiolabeled with carbon-11 to evaluate its biodistribution and PK in both healthymore » and S. aureus infected mice using positron emission tomography (PET). The biodistribution of [11C]PT119 and/or its labeled metabolites did not differ significantly between the healthy group and the infected group, and PT119 was found to distribute equally between serum and tissue during the ~1 h of analysis permitted by the carbon-11 half life. This approach provides important data for PK/PD modeling and is the first step in identifying radiotracers that can non-invasively image bacterial infection in vivo.« less

  5. Radiosynthesis and biological evaluation of a novel enoyl-ACP reductase inhibitor for Staphylococcus aureus

    SciTech Connect

    Wang, Hui; Lu, Yang; Liu, Li; Kim, Sung Won; Hooker, Jacob M.; Fowler, Joanna S.; Tonge, Peter J.

    2014-09-06

    Here we evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) of PT119, a potent Staphylococcus aureus enoyl-ACP reductase (saFabI) inhibitor with a Ki value of 0.01 nM and a residence time of 750 min on the enzyme target in mice. PT119 was found to have promising antibacterial activity in two different S. aureus infection models: it caused a 3 log reduction in the CFU’s in a mouse thigh muscle infection model and increased the survival rate from 0% to 50% in a mouse systemic infection model. PT119 was then radiolabeled with carbon-11 to evaluate its biodistribution and PK in both healthy and S. aureus infected mice using positron emission tomography (PET). The biodistribution of [11C]PT119 and/or its labeled metabolites did not differ significantly between the healthy group and the infected group, and PT119 was found to distribute equally between serum and tissue during the ~1 h of analysis permitted by the carbon-11 half life. This approach provides important data for PK/PD modeling and is the first step in identifying radiotracers that can non-invasively image bacterial infection in vivo.

  6. Probing Mechanistic Similarities between Response Regulator Signaling Proteins and Haloacid Dehalogenase Phosphatases.

    PubMed

    Immormino, Robert M; Starbird, Chrystal A; Silversmith, Ruth E; Bourret, Robert B

    2015-06-01

    Response regulator signaling proteins and phosphatases of the haloacid dehalogenase (HAD) superfamily share strikingly similar folds, active site geometries, and reaction chemistry. Proteins from both families catalyze the transfer of a phosphoryl group from a substrate to one of their own aspartyl residues, and subsequent hydrolysis of the phosphoprotein. Notable differences include an additional Asp that functions as an acid/base catalyst and an active site well-structured prior to phosphorylation in HAD phosphatases. Both features contribute to reactions substantially faster than those for response regulators. To investigate mechanisms underlying the functional differences between response regulators and HAD phosphatases, we characterized five double mutants of the response regulator CheY designed to mimic HAD phosphatases. Each mutant contained the extra Asp paired with a phosphatase-inspired substitution to potentially position the Asp properly. Only CheY DR (Arg as the anchor) exhibited enhanced rates of both autophosphorylation with phosphoramidate and autodephosphorylation compared to those of wild-type CheY. Crystal structures of CheY DR complexed with MoO4(2-) or WO4(2-) revealed active site hydrogen bonding networks similar to those in HAD·substrate complexes, with the extra Asp positioned for direct interaction with the leaving group (phosphorylation) or nucleophile (dephosphorylation). However, CheY DR reaction kinetics did not exhibit the pH sensitivities expected for acid/base catalysis. Biochemical analysis indicated CheY DR had an enhanced propensity to adopt the active conformation without phosphorylation, but a crystal structure revealed unphosphorylated CheY DR was not locked in the active conformation. Thus, the enhanced reactivity of CheY DR reflected partial acquisition of catalytic and structural features of HAD phosphatases. PMID:25928369

  7. Glycerol-3-phosphatase of Corynebacterium glutamicum.

    PubMed

    Lindner, Steffen N; Meiswinkel, Tobias M; Panhorst, Maren; Youn, Jung-Won; Wiefel, Lars; Wendisch, Volker F

    2012-06-15

    Formation of glycerol as by-product of amino acid production by Corynebacterium glutamicum has been observed under certain conditions, but the enzyme(s) involved in its synthesis from glycerol-3-phosphate were not known. It was shown here that cg1700 encodes an enzyme active as a glycerol-3-phosphatase (GPP) hydrolyzing glycerol-3-phosphate to inorganic phosphate and glycerol. GPP was found to be active as a homodimer. The enzyme preferred conditions of neutral pH and requires Mg²⁺ or Mn²⁺ for its activity. GPP dephosphorylated both L- and D-glycerol-3-phosphate with a preference for the D-enantiomer. The maximal activity of GPP was estimated to be 31.1 and 1.7 U mg⁻¹ with K(M) values of 3.8 and 2.9 mM for DL- and L-glycerol-3-phosphate, respectively. For physiological analysis a gpp deletion mutant was constructed and shown to lack the ability to produce detectable glycerol concentrations. Vice versa, gpp overexpression increased glycerol accumulation during growth in fructose minimal medium. It has been demonstrated previously that intracellular accumulation of glycerol-3-phosphate is growth inhibitory as shown for a recombinant C. glutamicum strain overproducing glycerokinase and glycerol facilitator genes from E. coli in media containing glycerol. In this strain, overexpression of gpp restored growth in the presence of glycerol as intracellular glycerol-3-phosphate concentrations were reduced to wild-type levels. In C. glutamicum wild type, GPP was shown to be involved in utilization of DL-glycerol-3-phosphate as source of phosphorus, since growth with DL-glycerol-3-phosphate as sole phosphorus source was reduced in the gpp deletion strain whereas it was accelerated upon gpp overexpression. As GPP homologues were found to be encoded in the genomes of many other bacteria, the gpp homologues of Escherichia coli (b2293) and Bacillus subtilis (BSU09240, BSU34970) as well as gpp1 from the plant Arabidosis thaliana were overexpressed in E. coli MG1655 and

  8. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  9. Results of Second Outdoor Comparison Between Absolute Cavity Pyrgeometer (ACP) and Infrared Integrating Sphere (IRIS) Radiometer at PMOD (Presentation)

    SciTech Connect

    Reda, I.; Grobner, J.; Wacker, S.

    2014-01-01

    The Absolute Cavity Pyrgeometer (ACP) and InfraRed Integrating Sphere radiometer (IRIS) are developed to establish a world reference for calibrating pyrgeometers with traceability to SI units. The two radiometers are un-windowed with negligible spectral dependence, and traceable to SI units through the temperature scale (ITS-90). The second outdoor comparison between the two designs was held from September 30 to October 11, 2013 at the Physikalisch-Metorologisches Observatorium Davos (PMOD). The difference between the irradiance measured by ACP and that of the IRIS was within 1 W/m2 (3 IRISs: PMOD + Australia + Germany). From the first and second comparisons, a difference of 4-6 W/m2 was observed between the irradiance measured by ACP&IRIS and that of the interim World Infrared Standard Group (WISG). This presentation includes results from the first and second comparison in an effort to establish the world reference for pyrgeometer calibrations, a key deliverable for the World Meteorological Organization (WMO), and the DOE-ASR.

  10. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

    Robert J. Martinez; Melanie J. Beazley; Samuel M. Webb; Martial Taillefert; and Patricia A. Sobecky

    2007-04-19

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

  11. Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

    PubMed Central

    Strom, M S; Lory, S

    1987-01-01

    Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein. Images PMID:2885309

  12. Elucidating the structural basis of diphenyl ether derivatives as highly potent enoyl-ACP reductase inhibitors through molecular dynamics simulations and 3D-QSAR study.

    PubMed

    Kamsri, Pharit; Punkvang, Auradee; Saparpakorn, Patchareenart; Hannongbua, Supa; Irle, Stephan; Pungpo, Pornpan

    2014-07-01

    Diphenyl ether derivatives are good candidates for anti-tuberculosis agents that display a promising potency for inhibition of InhA, an essential enoyl-acyl carrier protein (ACP) reductase involved in fatty acid biosynthesis pathways in Mycobacterium tuberculosis. In this work, key structural features for the inhibition were identified by 3D-QSAR CoMSIA models, constructed based on available experimental binding properties of diphenyl ether inhibitors, and a set of four representative compounds was subjected to MD simulations of inhibitor-InhA complexes for the calculation of binding free energies. The results show that bulky groups are required for the R1 substituent on the phenyl A ring of the inhibitors to favor a hydrophobic pocket formed by residues Phe149, Met155, Pro156, Ala157, Tyr158, Pro193, Met199, Val203, Leu207, Ile215, and Leu218. Small substituents with a hydrophilic property are required at the R3 and R4 positions of the inhibitor phenyl B rings to form hydrogen bonds with the backbones of Gly96 and Met98, respectively. For the R2 substituent, small substituents with simultaneous hydrophilic or hydrophobic properties are required to favor the interaction with the pyrophosphate moiety of NAD(+) and the methyl side chain of Ala198, respectively. The reported data provide structural guidance for the design of new and potent diphenyl ether-based inhibitors with high inhibitory activities against M. tuberculosis InhA. PMID:24935113

  13. Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase.

    PubMed

    Pato, M D; Sutherland, C; Winder, S J; Walsh, M P

    1993-07-01

    Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase

  14. Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

    PubMed

    Dong, Yu; Qiu, Xiaodi; Shaw, Neil; Xu, Yueyang; Sun, Yuna; Li, Xuemei; Li, Jun; Rao, Zihe

    2015-07-01

    Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs. PMID:26081470

  15. Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

    PubMed Central

    McGovern, K; Ehrmann, M; Beckwith, J

    1991-01-01

    We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them. Images PMID:1915262

  16. Association of phosphoenolpyruvate phosphatase activity with the cytosolic pyruvate kinase of germinating mung beans.

    PubMed

    Podestá, F E; Plaxton, W C

    1991-12-01

    The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP. PMID:16668551

  17. Association of Phosphoenolpyruvate Phosphatase Activity with the Cytosolic Pyruvate Kinase of Germinating Mung Beans 1

    PubMed Central

    Podestá, Florencio E.; Plaxton, William C.

    1991-01-01

    The procedure of Malhotra and Kayastha ([1990] Plant Physiology 93: 194-200) for the purification to homogeneity of a phosphoenolpyruvate-specific alkaline phosphatase (PEP phosphatase) from germinating mung beans (Vigna radiata) was followed. Although a higher specific activity of 1.4 micromoles pyruvate produced per minute per milligram protein was obtained, the final preparation was less than 10% pure as judged by polyacrylamide gel electrophoresis. Attempts to further purify the enzyme resulted in loss of activity. The partially purified enzyme contained significant pyruvate kinase activity (0.13 micromole pyruvate produced per minute per milligram protein) when assayed at pH 7.2, but not at pH 8.5. The PEP phosphatase activity of the final preparation exhibited hysteresis; a lag time of 5 to 6 minutes was required before a steady-state reaction rate was attained. A western blot of the final preparation revealed an immunoreactive 57 kilodalton polypeptide when probed with monospecific rabbit polyclonal antibodies prepared against germinating castor bean cytosolic pyruvate kinase. No antigenic cross-reaction of the final preparation was observed with antibodies against castor bean leucoplast pyruvate kinase, or black mustard PEP-specific acid phosphatase. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resulted in a single PEP phosphatase activity band; when this band was excised and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting, a 57 kilodalton silver-staining polypeptide was obtained that strongly cross-reacted with the anti-(cytosolic pyruvate kinase) immunoglobulin G. It is suggested that mung bean PEP-specific alkaline phosphatase activity is due to cytosolic pyruvate kinase, in which pyruvate and ortho-phosphate are formed in the absence of ADP. ImagesFigure 1Figure 2 PMID:16668551

  18. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    PubMed Central

    Chen, Xi’en; Lü, Shumin; Zhang, Yalin

    2013-01-01

    Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin. PMID:24351830

  19. Protein phosphatases and their regulation in the control of mitosis

    PubMed Central

    Mochida, Satoru; Hunt, Tim

    2012-01-01

    Cell cycle transitions depend on protein phosphorylation and dephosphorylation. The discovery of cyclin-dependent kinases (CDKs) and their mode of activation by their cyclin partners explained many important aspects of cell cycle control. As the cell cycle is basically a series of recurrences of a defined set of events, protein phosphatases must obviously be as important as kinases. However, our knowledge about phosphatases lags well behind that of kinases. We still do not know which phosphatase(s) is/are truly responsible for dephosphorylating CDK substrates, and we know very little about whether and how protein phosphatases are regulated. Here, we summarize our present understanding of the phosphatases that are important in the control of the cell cycle and pose the questions that need to be answered as regards the regulation of protein phosphatases. PMID:22482124

  20. Role of Protein Tyrosine Phosphatases in Plants

    PubMed Central

    Shankar, Alka; Agrawal, Nisha; Sharma, Manisha; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    Reversible protein phosphorylation is a crucial regulatory mechanism that controls many biological processes in eukaryotes. In plants, phosphorylation events primarily occur on serine (Ser) and threonine (Thr) residues, while in certain cases, it was also discovered on tyrosine (Tyr) residues. In contrary to plants, extensive reports on Tyr phosphorylation regulating a large numbers of biological processes exist in animals. Despite of such prodigious function in animals, Tyr phosphorylation is a least studied mechanism of protein regulation in plants. Recently, various chemical analytical procedures have strengthened the view that Tyr phosphorylation is equally prevalent in plants as in animals. However, regardless of Tyr phosphorylation events occuring in plants, no evidence could be found for the existence of gene encoding for Tyr phosphorylation i.e. the typical Tyr kinases. Various methodologies have suggested that plant responses to stress signals and developmental processes involved modifications in protein Tyr phosphorylation. Correspondingly, various reports have established the role of PTPs (Protein Tyrosine Phosphatases) in the dephosphorylation and inactivation of mitogen activated protein kinases (MAPKs) hence, in the regulation of MAPK signaling cascade. Besides this, many dual specificity protein phosphatases (DSPs) are also known to bind starch and regulate starch metabolism through reversible phosphorylation. Here, we are emphasizing the significant progress on protein Tyr phosphatases to understand the role of these enzymes in the regulation of post-translational modification in plant physiology and development. PMID:26962298

  1. Regulatory Roles of MAPK Phosphatases in Cancer

    PubMed Central

    Low, Heng Boon

    2016-01-01

    The mitogen-activated protein kinases (MAPKs) are key regulators of cell growth and survival in physiological and pathological processes. Aberrant MAPK signaling plays a critical role in the development and progression of human cancer, as well as in determining responses to cancer treatment. The MAPK phosphatases (MKPs), also known as dual-specificity phosphatases (DUSPs), are a family of proteins that function as major negative regulators of MAPK activities in mammalian cells. Studies using mice deficient in specific MKPs including MKP1/DUSP1, PAC-1/DUSP2, MKP2/DUSP4, MKP5/DUSP10 and MKP7/DUSP16 demonstrated that these molecules are important not only for both innate and adaptive immune responses, but also for metabolic homeostasis. In addition, the consequences of the gain or loss of function of the MKPs in normal and malignant tissues have highlighted the importance of these phosphatases in the pathogenesis of cancers. The involvement of the MKPs in resistance to cancer therapy has also gained prominence, making the MKPs a potential target for anti-cancer therapy. This review will summarize the current knowledge of the MKPs in cancer development, progression and treatment outcomes. PMID:27162525

  2. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

    NASA Technical Reports Server (NTRS)

    Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

    2001-01-01

    Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

  3. Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

    PubMed Central

    Desdouits, F; Siciliano, J C; Nairn, A C; Greengard, P; Girault, J A

    1998-01-01

    DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1. PMID:9461512

  4. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

    PubMed Central

    van Zyl, W; Huang, W; Sneddon, A A; Stark, M; Camier, S; Werner, M; Marck, C; Sentenac, A; Broach, J R

    1992-01-01

    We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase. Images PMID:1328868

  5. Oleic acid biosynthesis in cyanobacteria

    SciTech Connect

    VanDusen, W.J.; Jaworski, J.G.

    1986-05-01

    The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with /sup 14/CO/sub 2/. None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating /sup 14/CO/sub 2/ into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria.

  6. Detection of Extant Life in Extreme Environmentsby Phosphatase ActivitiesDetection of Extant Life in Extreme Environments by Measuring Phosphatase Activities

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kensei; Sato, Shuji; Naganawa, Kazuki; Itoh, Yuki; Kurihara, Hironari; Kaneko, Takeo; Takano, Yoshinori; Yoshimura, Yoshitaka; Kawasaki, Yukishige

    Since phosphate esters are essential for the terrestrial life, phosphatase activity can be a candidate for biosignatures of biological activity. It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere, high temperature hot springs and stratosphere. We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and Antarctica soils, and discussed whether they can be used as biosignatures for extant life. Core samples and chimney samples were collected at the Suiyo Seamount, Izu-Bonin Arc, the Pacific Ocean in 2001 and 2002, and in South Mariana hydrothermal systems, the Pacific Ocean in 2003, both in a part of the Archaean Park Project. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Alkaline (or acid) Phosphatase activity in solid samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate (pH 8.0 (or pH 6.5)) as a substrate. Phosphatase activities in extracts were measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate. Concentration of amino acids and their enantiomeric ratios were also determined by HPLC and GC/MS. Significant enzymatic activities were revealed in both some of the hydrothermal sub-vent systems and Antarctica soils, which is crucial evidence of vigorous microbial oasis. It is consistent with the fact that large enantiomeric excess of L-form amino acids were found in the same core sequences. The ALP activity was diminished with EDTA and was recovered with addition of zinc ion. The present results showed that zinc-containing metalloenzymes are present in such environments as hydrothermal vent chimneys and Antarctica soils. Optimum temperatures of ALP in the chimney, Antarctica soil and YNU campus soil were 353 K, 313 K, and 333 K, respectively. The present results suggested that phosphatase

  7. Can laboratory tholins mimic the chemistry producing Titan's aerosols? A review in light of ACP experimental results

    NASA Astrophysics Data System (ADS)

    Coll, P.; Navarro-González, R.; Szopa, C.; Poch, O.; Ramírez, S. I.; Coscia, D.; Raulin, F.; Cabane, M.; Buch, A.; Israël, G.

    2013-03-01

    The first results obtained by the ACP experiment onboard Huygens probe revealed that the main products obtained after thermolysis of Titan's collected aerosols, were ammonia (NH3) and hydrogen cyanide (HCN). Titan's aerosols, and their laboratory analogues named tholins, have been the subject of experimental or theoretical studies during the last four decades. These studies have been mainly devoted to understanding their origin and formation mechanisms, their physical, chemical and optical properties, and their role in the radiative equilibrium of the satellite. Before the arrival of the Cassini-Huygens mission, the dense layer of aerosols hid many aspects of the satellite's surface and precious information about its composition. If Titan's aerosols have been in the eye and mind of planetary scientists during such a long time, it is not surprising that a literature survey displays a good quantity of papers on aerosol analogues. With aerosol analogues we mean any material produced in a terrestrial laboratory under conditions that try to represent those of Titan's atmosphere. We present here a study aimed to understand the particularities of aerosol analogues synthesized in different laboratories around the world in order to determine some of their most representative chemical fingerprints and in some cases, to perform a direct comparison of the volatiles produced after a thermal treatment done in conditions similar to the ones used by the ACP experiment. From the information collected, we propose a broad classification of aerosol analogues highlighting the materials that can be more representative of Titan's aerosols in terms of their content of organic volatiles. We identify the laboratory analogs that best suit the ACP results; such identification is of prime importance to correctly predict the optical properties of Titan's aerosol and to accurately estimate their contribution in radiative equilibrium models and/or to assess their role in chemical reactions of

  8. Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum.

    PubMed

    Zhu, Jia-Ying; Yin Ye, Gong; Fang, Qi; Hu, Cui

    2010-01-01

    Using chromogenic substrates 5-bromo-4-chloro-3'-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperature dependent with bivalent cation effects. The full-length cDNA sequence of ALPase was amplified from the cDNA library of the venom apparatus of P. puparum, providing the first molecular characterization of ALPase in the venom of a parasitoid wasp. The cDNA consisted of 2645 bp with a 1623 bp open reading frame coding for 541 deduced amino acids with a predicted molecular mass of 59.83 kDa and pI of 6.98. Using multiple sequence alignment, the deduced amino acid sequence shared high identity to its counterparts from other insects. A signal peptide and a long conserved ALPase gene family signature sequence were observed. The amino acid sequence of this venom protein was characterized with different potential glycosylation, myristoylation, phosphorylation sites and metal ligand sites. The transcript of the ALPase gene was detected by RT-PCR in the venom apparatus with development related expression after adult wasp emergence, suggesting a possible correlation with the oviposition process. PMID:20575745

  9. Phosphoglycolate phosphatase of spinach acts as a phosphoenzyme

    SciTech Connect

    Rose, Z.B.; Seal, S.N.

    1987-05-01

    When /sup 32/P-glycolate and phosphoglycolate phosphatase from spinach are mixed, /sup 32/P is incorporated into acid precipitated protein. Properties that relate this phosphorylation to the enzyme are: The K/sub m/ value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the /sup 32/P appearing in the phosphoenzyme is diluted by unlabeled P-glycolate or the alternative substrate, ethyl-P; the activator Cl/sup -/ enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of /sup 32/P-enzyme; and /sup 32/P is lost from the enzyme when /sup 32/P-glycolate is consumed. The acid denatured phosphorylated protein is a molecule of 34,000 Da, which is half of the molecular weight of the native protein and is similar in size to the labeled band that is seen on SDS-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acyl-phosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by /sup 32/P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms.

  10. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    PubMed

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  11. Agronomic effects of mutations in two soybean Stearoyl-ACP-Desaturases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean [Glycine max (L.) Merr.] oil normally contains 2-4% stearic acid. Oil with higher levels of stearic acid is desired for use in the baking industry, for both its chemical properties and human health benefits. Several lines with increased stearic acid have been identified; however, the agronom...

  12. Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.

    PubMed

    Romá-Mateo, Carlos; Sacristán-Reviriego, Almudena; Beresford, Nicola J; Caparrós-Martín, José Antonio; Culiáñez-Macià, Francisco A; Martín, Humberto; Molina, María; Tabernero, Lydia; Pulido, Rafael

    2011-04-01

    Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including

  13. NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase

    PubMed Central

    Dynia, Diane W.; Steinmetz, Amy G.

    2010-01-01

    Na+/H+ exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites. PMID:20015946

  14. Possible protein phosphatase inhibition by bis(hydroxyethyl) sulfide, a hydrolysis product of mustard gas

    SciTech Connect

    Brimfield, A.A.

    1995-12-31

    Recently, the natural vesicant cantharidin was shown to bind exclusively to and inhibit protein phosphatase 2A (PP2A) in mouse tissue extracts (Li and Casida (1992) Proc. Nati. Acad. Sci. USA 89, 11867-11870). To explore the generality of this effect in vesicant action, we measured the protein serinelthreonine phosphatase activity in mouse liver cytosol (in the form of the okadaic acid inhibitable increment of p-nitrophenyl phosphate (p-NPP) phosphatase activity) in the presence of aqueous sulfur mustard or its hydrolysis product, bis(hydroxyethyl)sulfide (TDG). Sulfur mustard inhibited p-NPP hydrolysis. However, inhibition correlated with the time elapsed between thawing and the addition of mustard to the enzyme preparation, not with concentration. TDG exhibited a direct, concentration-related inhibition of p-NPP hydrolysis between 30 and 300 1LM. We conclude that sulfur mustard also has an inhibitory effect on protein serinelthreonine phosphatases. However, the inhibition is an effect of its non-alkykating hydrolysis product TDG, not of sulfur mustard itself.

  15. Crystal Structure of Colicin M, a Novel Phosphatase Specifically Imported by Escherichia coli*>

    PubMed Central

    Zeth, Kornelius; Römer, Christin; Patzer, Silke I.; Braun, Volkmar

    2008-01-01

    Colicins are cytotoxic proteins secreted by certain strains of Escherichia coli. Colicin M is unique among these toxins in that it acts in the periplasm and specifically inhibits murein biosynthesis by hydrolyzing the pyrophosphate linkage between bactoprenol and the murein precursor. We crystallized colicin M and determined the structure at 1.7Å resolution using x-ray crystallography. The protein has a novel structure composed of three domains with distinct functions. The N-domain is a short random coil and contains the exposed TonB box. The central domain includes a hydrophobic α-helix and binds presumably to the FhuA receptor. The C-domain is composed of a mixed α/β-fold and forms the phosphatase. The architectures of the individual modules show no similarity to known structures. Amino acid replacements in previously isolated inactive colicin M mutants are located in the phosphatase domain, which contains a number of surface-exposed residues conserved in predicted bacteriocins of other bacteria. The novel phosphatase domain displays no sequence similarity to known phosphatases. The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins. The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains. PMID:18640984

  16. A potent and selective inhibitor for the UBLCP1 proteasome