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Sample records for alkaline phosphatase induction

  1. 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.

  2. Head and neck cancer. Relationship of the prechemotherapy serum alkaline phosphatase levels to response rate of induction chemotherapy.

    PubMed

    Coker, D D; Morris, D; Elias, E G; Didolkar, M S; Zentai, T A

    1982-01-01

    Fifty-one patients with stage III or IV squamous cell carcinoma of the head and neck who received induction chemotherapy with cisplatin and bleomycin sulfate with and without high-dose methotrexate were studied. The relationship of the prechemotherapy levels of serum alkaline phosphatase, lactic dehydrogenase, SGOT, SGPT, BUN, creatinine, calcium, total protein, albumin, hemoglobin, uric acid, and bilirubin and the WBC and platelet counts was correlated with the response rate. The overall response rate was 65%. No notable relationship between any of the laboratory values and the response rate was found. In contrast to an earlier report, patients with a low alkaline phosphatase level responded as well as patients with an elevated serum alkaline phosphatase level.

  3. Bovine Intestinal Alkaline Phosphatase Reduces Inflammation After Induction of Acute Myocardial Infarction in Mice

    PubMed Central

    Fiechter, Danielle; Kats, Suzanne; Brands, Ruud; van Middelaar, Ben; Pasterkamp, Gerard; de Kleijn, Dominique; Seinen, Willem

    2011-01-01

    Background There has been increasing evidence suggesting that lipopolysaccharide or endotoxin may be an important activator of the innate immune system after acute myocardial infarction. Bovine intestinal alkaline phosphatase reduces inflammation in several endotoxin mediated diseases by dephosphorylation of the lipid A moiety of lipopolysaccharide. The aim of this study was to investigate the effect of bovine intestinal alkaline phosphatase on reducing inflammation after acute myocardial infarction. Methods Just before permanent ligation of the left anterior descending coronary (LAD) artery to induce acute myocardial infarction in Balb/c mice, bovine intestinal alkaline phosphatase (bIAP) was administrated intravenously. After 4 hours, mice were sacrificed and the inflammatory response was assessed. Acute myocardial infarction induced the production of different cytokines, which were measured in blood. Results Treatment with bovine intestinal alkaline phosphatase resulted in a significant reduction of the pro-inflammatory cytokines IL-6, IL-1β and the chymase mouse mast cell protease-1. No difference in the production of the anti-inflammatory cytokine IL-10 was observed between the control group and the bovine intestinal alkaline phosphatase treated group. Conclusion In a mouse model of permanent LAD coronary artery ligation, bIAP diminishes the pro-inflammatory responses but does not have an effect on the anti-inflammatory response in the acute phase after acute myocardial infarction.

  4. Ambroxol reduces LPS toxicity mediated by induction of alkaline phosphatases in rat lung.

    PubMed

    Koyama, Iwao; Matsunaga, Toshiyuki; Harada, Tsuyoshi; Kikuno, Akira; Hokari, Shigeru; Komoda, Tsugikazu

    2004-08-01

    Alkaline phosphatases (APs) have been suggested to detoxify lipopolysaccharide (LPS) by dephosphorylation. Ambroxol, a bronchial expectorant, is known to accelerate the secretion of pulmonary surfactant particles including AP molecules as a pharmacological action. In the present study, some beneficial effects of ambroxol on LPS toxicity in the rat lung were investigated. In an experiment using the rat lung organ culture, AP activities were enhanced in a time-dependent manner by incubation with 25 microM of ambroxol in both the tissue and the medium. Western blot analysis indicated that AP activity was elevated by the treatment with ambroxol, due to the induction of surfactant proteins (SPs) and AP molecules. In the in vivo experiment, the serum LPS content was markedly increased after LPS administration to rats by intratracheal instillation of 20 mg/kg. However, when the rats were pretreated with oral ambroxol (1.0 mg/kg) at 1 h before LPS challenge, the area under the concentration--time curve (AUC) of serum LPS was significantly decreased. These results suggest that ambroxol inhibits the translocation of LPS from the lung into the circulation as well as its detoxification effect via the elevation of AP activity. Bromhexine, another expectorant, is less effective than ambroxol as an LPS detoxificant. Maintenance of high AP activity level in the lung suggests APs to have physiological significant effects against the inflammatory events induced by LPS.

  5. [Alkaline phosphatase in Amoeba proteus].

    PubMed

    Sopina, V A

    2005-01-01

    In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

  6. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  7. Evolution of alkaline phosphatases in primates.

    PubMed Central

    Goldstein, D J; Rogers, C; Harris, H

    1982-01-01

    Alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] in placenta, intestine, liver, kidney, bone, and lung from a variety of primate species has been characterized by quantitative inhibition, thermostability, and immunological studies. Characteristic human placental-type alkaline phosphatase occurs in placentas of great apes (chimpanzee and orangutan) but not in placentas of other primates, including gibbon. It is also present in trace amounts in human lung but not in lung or other tissues of various Old and New World monkeys. However, a distinctive alkaline phosphatase resembling it occurs in substantial amounts in lungs from Old World monkeys but not New World monkeys. It appears that duplication of alkaline phosphatase genes and mutations of genetic elements controlling their tissue expression have occurred relatively recently in mammalian evolution. Images PMID:6950431

  8. Alkaline phosphatase of Physarum polycephalum is insoluble.

    PubMed

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

  9. [Leucocyte alkaline phosphatase in normal and pathological pregnancy (author's transl)].

    PubMed

    Stark, K H; Zaki, I; Sobolewski, K

    1981-01-01

    The activities of leucocyte alkaline phosphatase were determined in 511 patients with normal and pathological pregnancy. Mean values were compared and the enzyme followed up, and the conclusion was drawn that leucocyte alkaline phosphatase was no safe indicator of foetal condition. No direct relationship were found to exist between leucocyte alkaline phosphatase, total oestrogens, HSAP, HLAP, HPL, and oxytocinase.

  10. Desialylated alkaline phosphatase: activation by 4-nitrophenol.

    PubMed

    Nayudu, P R

    1984-01-01

    Mouse ileal alkaline phosphatase is a sialyl enzyme (12-14 moles per mole of enzyme). When partially desialylated by treatment with neuraminidase, the enzyme loses most of its activity, associated with reduced apparent Vmax and Km. Part of that loss, however, is recovered as the product 4-nitrophenol's concentration builds up in the cuvette. Experimental results are presented to demonstrate that the activation is due to the binding of 4-nitrophenol as a ligand by the partially desialylated enzyme and that both the loss of activity by sialic acid removal and activation by ligand-binding are correlated with changes in protein conformation.

  11. Inhibition of renal alkaline phosphatase by cimetidine.

    PubMed

    Minai-Tehrani, Dariush; Khodai, Somayeh; Aminnaseri, Somayeh; Minoui, Saeed; Sobhani-Damavadifar, Zahra; Alavi, Sana; Osmani, Raheleh; Ahmadi, Shiva

    2011-08-01

    Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.

  12. 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.

  13. Identification of human pulmonary alkaline phosphatase isoenzymes.

    PubMed

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

    1997-04-01

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

  14. Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages.

    PubMed

    Shioi, Atsushi; Katagi, Miwako; Okuno, Yasuhisa; Mori, Katsuhito; Jono, Shuichi; Koyama, Hidenori; Nishizawa, Yoshiki

    2002-07-12

    Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-gamma (IFN-gamma) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-gamma and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-gamma and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of beta-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-alpha (TNF-alpha) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-alpha and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-gamma and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-alpha and OSM.

  15. Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase.

    PubMed

    O'Brien, P J; Herschlag, D

    2001-05-15

    Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of

  16. 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.

  17. Quantitation of Alkaline Phosphatase Isoenzymes Using Agarose Containing Wheat Germ Lectin

    DTIC Science & Technology

    1989-07-01

    SIl Quantitation of Alkaline Phosphatase Isoenzymes Using Agarose Containing Wheat Germ Lectin A thesis submitted in partial fulfillment of the...16 Wheat Germ Lectin Electrophoresis to Quantitate Alkaline Phosphatase Isoenzymes ................ 16 Alkaline Phosphatase Isoenzyme...vs Polyacrylamide Gel Electrophoresis ......................... 40 Clinical Correlation Using Wheat Germ Lectin 45 Placental Alkaline Phosphatase

  18. Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.

    PubMed

    O'Brien, Patrick J; Herschlag, Daniel

    2002-03-05

    Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Brønsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of

  19. Characteristics of plasmalemma alkaline phosphatase of rat mesenteric artery.

    PubMed

    Kwan, C Y

    1983-01-01

    General characteristics of alkaline phosphatase activity of the plasma membrane-enriched fraction isolated from rat mesenteric arteries were investigated. The vascular smooth muscle plasmalemma alkaline phosphatase is a metalloenzyme which is strongly inhibited by chelating agents and this inhibition can be completely overcome by addition of Mg2+ or Ca2+. Zn2+ only partially reactivates the enzyme in the presence of low concentrations of EDTA. The enzymatic hydrolysis of p-nitrophenyl phosphate, beta-glycerophosphate, alpha-glycerophosphate, or 3'-adenosine monophosphate showed an optimal activity in the alkaline region between pH 9 and 11. The alkaline phosphatase activity is distinctly different from the plasmalemma ATPase and 5'-nucleotidase activities with respect to their pH dependence, influence by added divalent metal ions and stability against heat inactivation. Vanadate ion, being structurally similar to the transition state analog of the phosphoryl group, potently inhibits alkaline phosphatase with an apparent Ki of 1.5 microM. The altered alkaline phosphatase activity of vascular smooth muscle in relation to its possible physiological function and pathophysiological manifestation associated with hypertensive disease are discussed.

  20. Crystal structure of rat intestinal alkaline phosphatase--role of crown domain in mammalian alkaline phosphatases.

    PubMed

    Ghosh, Kaushik; Mazumder Tagore, Debarati; Anumula, Rushith; Lakshmaiah, Basanth; Kumar, P P B S; Singaram, Senthuran; Matan, Thangavelu; Kallipatti, Sanjith; Selvam, Sabariya; Krishnamurthy, Prasad; Ramarao, Manjunath

    2013-11-01

    Intestinal alkaline phosphatases (IAPs) are involved in the cleavage of phosphate prodrugs to liberate the drug for absorption in the intestine. To facilitate in vitro characterization of phosphate prodrugs, we have cloned, expressed, purified and characterized IAPs from rat and cynomolgus monkey (rIAP and cIAP respectively) which are important pre-clinical species for drug metabolism studies. The recombinant rat and monkey enzymes expressed in Sf9 insect cells (IAP-Ic) were found to be glycosylated and active. Expression of rat IAP in Escherichia coli (rIAP-Ec) led to ~200-fold loss of activity that was partially recovered by the addition of external Zn(2+) and Mg(2+) ions. Crystal structures of rIAP-Ec and rIAP-Ic were determined and they provide rationale for the discrepancy in enzyme activities. Rat IAP-Ic retains its activity in presence of both Zn(2+) and Mg(2+) whereas activity of most other alkaline phosphatases (APs) including the cIAP was strongly inhibited by excess Zn(2+). Based on our crystal structure, we hypothesized the residue Q317 in rIAP, present within 7 Å of the Mg(2+) at M3, to be important for this difference in activity. The Q317H rIAP and H317Q cIAP mutants showed reversal in effect of Zn(2+), corroborating the hypothesis. Further analysis of the two structures indicated a close linkage between glycosylation and crown domain stability. A triple mutant of rIAP, where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity.

  1. Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme.

    PubMed

    Halford, S E; Schlesinger, M J; Gutfreund, H

    1972-03-01

    1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P(i) to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

  2. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  3. [Granulocyte alkaline phosphatase--a biomarker of chronic benzene exposure].

    PubMed

    Khristeva, V; Meshkov, T

    1994-01-01

    In tracing the cellular population status in the peripheral blood of workers, exposed to benzene, was included and cytochemical determination of the alkaline phosphatase activity in leucocytes. This enzyme is accepted as marker of the neutrophilic granulocytes, as maturation of the cells and their antibacterial activity are parallel to the cytochemical activity of the enzyme. 78 workers from the coke-chemical production from state firm "Kremikovtsi" and 41 workers from the production "Benzene" and "Isopropylbenzene"--Oil Chemical Plant, Burgas are included. The benzene concentrations in the air of the working places in all productions are in the range of 5 to 50 mg/m3. For cytochemical determination of the alkaline phosphatase activity is used the method of L. Kaplow and phosphatase index was calculated. It was established that in 98.4% of all examined the alkaline phosphatase activity is inhibited to different rate, as from 46.5% [61 workers] it is zero. In considerably lower percentage of workers were established and other deviations: leucocytosis or leucopenia, neutropenia, increased percent of band neutrophils and toxic granules. The results of the investigation of the granulocyte population show that from all indices, the activity of granulocyte alkaline phosphatase demonstrates most convincing the early myelotoxic effect of benzene.

  4. The catalytic properties of alkaline phosphatases under various conditions

    NASA Astrophysics Data System (ADS)

    Atyaksheva, L. F.; Chukhrai, E. S.; Poltorak, O. M.

    2008-11-01

    A comparative study was performed to examine the catalytic properties of alkaline phosphatases from bacteria Escherichia coli and bovine and chicken intestines. The activity of enzyme dimers and tetramers was determined. The activity of the dimer was three or four times higher than that of the tetramer. The maximum activity and affinity for 4-nitrophenylphosphate was observed for the bacterial alkaline phosphatase ( K M = 1.7 × 10-5 M, V max = 1800 μmol/(min mg of protein) for dimers and V max = 420 μmol/(min mg of protein) for tetramers). The Michaelis constants were equal for two animal phosphatases in various buffer media (pH 8.5) ((3.5 ± 0.2) × 10-4 M). Five buffer systems were investigated: tris, carbonate, hepes, borate, and glycine buffers, and the lowest catalytic activity of alkaline phosphatases at equal pH was observed in the borate buffer (for enzyme from bovine intestine, V max = 80 μmol/(min mg of protein)). Cu2+ cations formed a complex with tris-(oxymethyl)-aminomethane ( tris-HCl buffer) and inhibited the intestine alkaline phosphatases by a noncompetitive mechanism.

  5. Inhibition of Alkaline Phosphatase by Several Diuretics

    DTIC Science & Technology

    1980-01-01

    August 20th, 1979) . . Summary , . Acetazolamide, furosemide, ethacrynic acid and chlorothiazide, diuretics of considerable structural diversity, inhibit...Ki is calculated to be 8.4, 7.0, 2.8 and 0.1 mmol/l for acetazolamide, furosemide, ethacrynic acid and chlorothiazide, respectively. Chlorothiazide...is a much more potent inhibitor of alkaline phos- phatase than the other three diuretics. The combination of ethacrynic acid and cysteine, itself an

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

    PubMed

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

    1985-01-01

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

  7. Repeated probing of Southwestern blots using alkaline phosphatase stripping.

    PubMed

    Jia, Yinshan; Jiang, Daifeng; Jarrett, Harry W

    2010-11-05

    Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized.

  8. Hybrids of chemical derivatives of Escherichia coli alkaline phosphatase.

    PubMed

    Meighen, E; Yue, R

    1975-12-15

    The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit.

  9. Low serum alkaline phosphatase activity in Kikuchi-Fujimoto disease

    PubMed Central

    Inamo, Yasuji

    2017-01-01

    Abstract Various laboratory findings are helpful in making a diagnosis of Kikuchi-Fujimoto disease (KFD); however, they are not specific. We found decreased serum alkaline phosphatase (SAP) activity in children with KFD. The levels of SAP fell in the acute phase and recovered during convalescence. We conclude that low SAP activity is a characteristic of KFD and may be an auxiliary diagnostic marker for the disease. PMID:28248884

  10. Dephosphorylation of endotoxin by alkaline phosphatase in vivo.

    PubMed Central

    Poelstra, K.; Bakker, W. W.; Klok, P. A.; Kamps, J. A.; Hardonk, M. J.; Meijer, D. K.

    1997-01-01

    Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin is a substance that contains phosphate groups and is usually present in the extracellular space, we studied whether AP is able to dephosphorylate this bacterial product at physiological pH levels. We tested this in intestinal cryostat sections using histochemical methods with endotoxin from Escherichia coli and Salmonella minnesota R595 as substrate. Results show that dephosphorylation of both preparations occurs at pH 7.5 by AP activity. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, we examined the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat. The results show that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus. In view of the endotoxin-dephosphorylating properties of AP demonstrated in vitro, we propose a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role. Images Figure 1 Figure 5 PMID:9327750

  11. Physiological aspects of alkaline phosphatase in selected cyanobacteria.

    PubMed

    Doonan, B B; Jensen, T E

    1980-01-01

    The alkaline phosphatase of Plectonema boryanum shows a considerable increase in activity following placement of the cells in a phosphate free medium. Five days of phosphate starvation result in a 14-fold increase of alkaline phosphatase activity. Growth in the presence of inhibitors of transcription and translation indicate that the synthesis of the enzyme is de novo. Orthophosphate causes an immediate inhibition of enzyme activity. Enzyme was extracted from P. boryanum with lysozyme or polymyxin B treatment in order to make comparative studies of cell bound and cell free enzyme. Of several enzyme specific inhibitors tested, mercuric chloride was the most effective. Temperature studies showed that the cell bound enzyme was most active at 40 degrees C while the cell free enzyme was most active at 70 degrees C. The pH optimum was 9 for the cell free enzyme, and 8.8 for the cell bound. The enzyme was tested to determine if it could hydrolyse a number of different organic compounds. It hydrolysed p-nitrophenol phosphate 100%, fructose-6-phosphate 45%, beta-glycerol phosphate 25% and other compounds to a lesser degree. Of seventeen other Cyanobacteria tested for alkaline phosphatase, all were positive, and of these eleven were inducible for the enzyme. Ten of the isolates released some of the enzyme into the culture medium. Michaelis constants for the enzyme were also determined.

  12. Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin-biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochemical staining of human testicular tissue.

    PubMed

    Davidoff, M S; Schulze, W; Holstein, A F

    1991-01-01

    An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP-combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker.

  13. Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase.

    PubMed

    Lorenz, B; Schröder, H C

    2001-06-11

    Recent results revealed that inorganic polyphosphates (polyP), being energy-rich linear polymers of orthophosphate residues known from bacteria and yeast, also exist in higher eukaryotes. However, the enzymatic basis of their metabolism especially in mammalian cells is still uncertain. Here we demonstrate for the first time that alkaline phosphatase from calf intestine (CIAP) is able to cleave polyP molecules up to a chain length of about 800. The enzyme acts as an exopolyphosphatase degrading polyP in a processive manner. The pH optimum is in the alkaline range. Divalent cations are not required for catalytic activity but inhibit the degradation of polyP. The rate of hydrolysis of short-chain polyP by CIAP is comparable to that of the standard alkaline phosphatase (AP) substrate p-nitrophenyl phosphate. The specific activity of the enzyme decreases with increasing chain length of the polymer both in the alkaline and in the neutral pH range. The K(m) of the enzyme also decreases with increasing chain length. The mammalian tissue non-specific isoform of AP was not able to hydrolyze polyP under the conditions applied while the placental-type AP and the bacterial (Escherichia coli) AP displayed polyP-degrading activity.

  14. phoD Alkaline Phosphatase Gene Diversity in Soil

    PubMed Central

    Kertesz, Michael A.; Bünemann, Else K.

    2015-01-01

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

  15. Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture.

    PubMed

    Boskey, A L; Ziecheck, W; Guidon, P; Doty, S B

    1993-02-01

    The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.

  16. Osseous plate alkaline phosphatase is anchored by GPI.

    PubMed

    Pizauro, J M; Ciancaglini, P; Leone, F A

    1994-02-01

    Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized enzyme exhibited "Michaelian" kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

  17. A description of alkaline phosphatases from marine organisms

    NASA Astrophysics Data System (ADS)

    Tian, Jiyuan; Jia, Hongbing; Yu, Juan

    2016-07-01

    Alkaline phosphatases (APs) are non-specific phosphohydrolases, and they are widely used in clinical diagnostics and biological studies. APs are widespread in nature and exhibit different structural formulations. Based on the diversity of biogenetic sources, APs exhibit temperature-propensity traits, and they are classified as psychrophilic, mesophilic, and thermophilic. In this article, the characteristics of psychrophilic APs from marine organisms were described, accompanied by a simple description of APs from other organisms. This review will facilitate better utilization of marine APs in the biotechnology field.

  18. Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP).

    PubMed

    Mehta, Bakul Dhagat; Jog, Sonali P; Johnson, Steven C; Murthy, Pushpalatha P N

    2006-09-01

    Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

  19. Effect of aluminum phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria.

    PubMed

    Ramalingam, N; Prasanna, B Gowtham

    2006-09-01

    The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h(-1) mg(-1) protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.

  20. Characterization of Schistosome Tegumental Alkaline Phosphatase (SmAP)

    PubMed Central

    Bhardwaj, Rita; Skelly, Patrick J.

    2011-01-01

    Schistosomes are parasitic platyhelminths that currently infect over 200 million people globally. The parasites can live for years in a putatively hostile environment - the blood of vertebrates. We have hypothesized that the unusual schistosome tegument (outer-covering) plays a role in protecting parasites in the blood; by impeding host immunological signaling pathways we suggest that tegumental molecules help create an immunologically privileged environment for schistosomes. In this work, we clone and characterize a schistosome alkaline phosphatase (SmAP), a predicted ∼60 kDa glycoprotein that has high sequence conservation with members of the alkaline phosphatase protein family. The SmAP gene is most highly expressed in intravascular parasite life stages. Using immunofluorescence and immuno-electron microscopy, we confirm that SmAP is expressed at the host/parasite interface and in internal tissues. The ability of living parasites to cleave exogenous adenosine monophosphate (AMP) and generate adenosine is very largely abolished when SmAP gene expression is suppressed following RNAi treatment targeting the gene. These results lend support to the hypothesis that schistosome surface enzymes such as SmAP could dampen host immune responses against the parasites by generating immunosuppressants such as adenosine to promote their survival. This notion does not rule out other potential functions for the adenosine generated e.g. in parasite nutrition. PMID:21483710

  1. Purification and characterization of Ulva pertusa Kjellm alkaline phosphatase.

    PubMed

    Yang, Dong; Wang, Jingyun; Bao, Yongming; An, Lijia

    2003-05-01

    The activity of alkaline phosphatase (ALP, EC 3.1.3.1.) was found in seaweeds, including five kinds of green alga, eighteen kinds of red alga, and six kinds of brown alga, collected from the seaside of Dalian in China. The enzyme was purified 1230-fold from Ulva pertusa Kjellm. It had a specific activity of 48.6 U/mg protein and was proven to be homogeneous by SDS-PAGE with a subunit molecular mass of 19.5 kDa. The activity of ALP peaked at pH9.8, and was completely inhibited by DTT and partly by NBS. The Michaelis-Menten constant Km and the maximum reaction velocity Vmax, at pH 9.8 and 37 degrees C were 0.950 mM and 5.00 microM/min, respectively.

  2. Dephosphorylation of bovine casein by milk alkaline phosphatase.

    PubMed

    Lorient, D; Linden, G

    1976-02-01

    The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.

  3. The influence of complexing pharmaceutical compositions on alkaline phosphatase

    NASA Astrophysics Data System (ADS)

    Atyaksheva, L. F.; Chukhrai, E. S.; Stepina, N. D.; Novikova, N. N.; Yur'eva, E. A.

    2011-06-01

    It is established that the pharmaceutical compositions xydiphon, medifon, succimer, and EDTA, which are used as complexing agents for accelerating the excretion of heavy metals from human organism, at certain concentrations inhibit enzyme alkaline phosphatase (AP). It is concluded that xydiphon and EDTA have a noticeable effect on AP activity at concentrations over 0.01 mM; medifon and succimer, at concentrations of over 0.3-0.5 mM. The enzyme's inhibition constants and type of inhibition are determined. Xydiphon is found to manifest the highest affinity to AP ( K I = 0.35 mM). It is shown by kinetic analysis that dissociative chemoinactivation of the enzyme takes place under the action of complexing agents. The corresponding kinetic parameters are calculated.

  4. Significantly Elevated Liver Alkaline Phosphatase in Congestive Heart Failure

    PubMed Central

    Shamban, Leonid; Patel, Brijesh; Williams, Michael

    2014-01-01

    Congestive hepatopathy can have a mildly elevated liver profile, which should normalize with appropriate therapy. Liver specific alkaline phosphatase (ALP) in decompensated heart failure (HF) can be mildly elevated. The levels exceeding beyond the expected rise should be a concern and lead to further investigation. The literature reports insubstantial number of cases regarding significantly elevated levels of ALP and congestive hepatopathy. We report a case of a 45-year-old female with known history of severe cardiomyopathy that had persistently elevated levels of ALP. The extensive workup was negative for any specific pathology. The liver biopsy was consistent with congestive hepatopathy. The patient’s ALP levels decreased with aggressive diuretic therapy but still remained elevated. PMID:27785272

  5. Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

    PubMed Central

    Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.

    1998-01-01

    The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor. PMID:9546188

  6. Activity of alkaline phosphatase adsorbed and grafted on "polydopamine" films.

    PubMed

    Ball, Vincent

    2014-09-01

    The oxidation of dopamine in slightly basic solutions and in the presence of oxygen as an oxidant allows for the deposition of dopamine-eumelanin ("polydopamine") films on almost all kinds of materials allowing for an easy secondary functionalization. Molecules carrying nucleophilic groups like thiols and amines can be easily grafted on those films. Herein we show that alkaline phosphatase (ALP), as a model enzyme, adsorbs to "polydopamine" films and part of the adsorbed enzyme is rapidly desorbed in contact with Tris buffer. However a significant part of the enzyme remains irreversibly adsorbed and keeps some enzymatic activity for at least 2 weeks whereas ALP adsorbed on quartz slides is rapidly and quantitatively deactivated. In addition we estimated the Michaelis constant Km of the enzyme irreversibly bound to the "polydopamine" film. The Michaelis constant, and hence the affinity constant between paranitrophenol phosphate and ALP are almost identical between the enzyme bound on the film and the free enzyme in solution. Complementarily, it was found that "polydopamine" films display some phosphatase like catalytic activity.

  7. Alkaline phosphatase levels in patients with coronary heart disease saliva and its relation with periodontal status

    NASA Astrophysics Data System (ADS)

    Yunita, Dina Suci; Masulili, Sri Lelyati C.; Tadjoedin, Fatimah M.; Radi, Basuni

    2017-02-01

    Coronary heart disease (CHD) is a disease that causes narrowing of the coronary arteries. Currently, there is a hypothesis regarding periodontal infection that increases risk for heart disease. Alkaline phosphatase (ALP) as a marker of inflammation will increase in atherosclerosis and periodontal disease. The objective of this research is analyzing the relationship between the levels of alkaline phosphatase in saliva with periodontal status in patients with CHD and non CHD. Here, saliva of 104 subjects were taken, each 1 ml, and levels of Alkaline Phosphatase was analyzed using Abbott ci4100 architect. We found that no significant difference of Alkaline Phosphatase levels in saliva between CHD patients and non CHD. Therefore, it can be concluded that Alkaline Phosphatase levels in patients with CHD saliva was higher than non CHD and no association between ALP levels with periodontal status.

  8. Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

    PubMed Central

    Peter, A T; Bosu, W T; MacWilliams, P; Gallagher, S

    1987-01-01

    Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum. PMID:3453274

  9. The measurement of alkaline phosphatase at nanomolar concentration within 70 s using a disposable microelectrochemical transistor.

    PubMed

    Astier, Y; Bartlett, P N

    2004-08-01

    We report a new approach to the measurement of alkaline phosphatase concentration based on the use of a disposable poly(aniline) microelectrochemical transistor. The measurement is carried out in a two cell configuration in which the poly(aniline) microelectrochemical transistor operates in acid solution and is connected to the alkaline buffer solution containing the analyte by a salt bridge. Disposable microelectrochemical transistors were reproducibly fabricated by electrochemical deposition of poly(aniline) onto photolithographically fabricated gold microband arrays. Using these devices alkaline phosphatase was detected by employing p-aminophenyl phosphate as the substrate for the enzyme and using glucose and glucose oxidase to recycle the p-aminophenol generated upon enzyme catalysed hydrolysis of the phosphate. Recycling the p-aminophenol with glucose and glucose oxidase amplified the detection of alkaline phosphatase approximately tenfold. Using this approach we obtain linear calibration curves for alkaline phosphatase up to 5 nM within 70 s on single use devices.

  10. Promiscuity in alkaline phosphatase superfamily. Unraveling evolution through molecular simulations.

    PubMed

    López-Canut, Violeta; Roca, Maite; Bertrán, Juan; Moliner, Vicent; Tuñón, Iñaki

    2011-08-10

    We here present a theoretical study of the alkaline hydrolysis of a phosphodiester (methyl p-nitrophenyl phosphate or MpNPP) in the active site of Escherichia coli alkaline phosphatase (AP), a monoesterase that also presents promiscuous activity as a diesterase. The analysis of our simulations, carried out by means of molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials, shows that the reaction takes place through a D(N)A(N) or dissociative mechanism, the same mechanism employed by AP in the hydrolysis of monoesters. The promiscuous activity observed in this superfamily can be then explained on the basis of a conserved reaction mechanism. According to our simulations the specialization in the hydrolysis of phosphomonoesters or phosphodiesters, developed in different members of the superfamily, is a consequence of the interactions established between the protein and the oxygen atoms of the phosphate group and, in particular, with the oxygen atom that bears the additional alkyl group when the substrate is a diester. A water molecule, belonging to the coordination shell of the Mg(2+) ion, and residue Lys328 seem to play decisive roles stabilizing a phosphomonoester substrate, but the latter contributes to increase the energy barrier for the hydrolysis of phosphodiesters. Then, mutations affecting the nature or positioning of Lys328 lead to an increased diesterase activity in AP. Finally, the capacity of this enzymatic family to catalyze the reaction of phosphoesters having different leaving groups, or substrate promiscuity, is explained by the ability of the enzyme to stabilize different charge distributions in the leaving group using different interactions involving either one of the zinc centers or residues placed on the outer side of the catalytic site.

  11. Alkaline Phosphatase Assay for Freshwater Sediments: Application to Perturbed Sediment Systems

    PubMed Central

    Sayler, Gary S.; Puziss, Marla; Silver, Martin

    1979-01-01

    The p-nitrophenyl phosphate hydrolysis-phosphatase assay was modified for use in freshwater sediment. Laboratory studies indicated that the recovery of purified alkaline phosphatase activity was 100% efficient in sterile freshwater sediments when optimized incubation and sonication conditions were used. Field studies of diverse freshwater sediments demonstrated the potential use of this assay for determining stream perturbation. Significant correlations between phosphatase and total viable cell counts, as well as adenosine triphosphate biomass, suggested that alkaline phosphatase activity has utility as an indicator of microbial population density and biomass in freshwater sediments. PMID:16345464

  12. Alkaline phosphatase in stallion semen: characterization and clinical applications.

    PubMed

    Turner, R M O; McDonnell, S M

    2003-06-01

    Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additionally, accessory glands, epididymides, testicles and other components of the urogenital tract of normal stallions were assayed to determine which tissues contain SPAP activity. The results indicated that levels of AP activity are low in pre-ejaculatory fluid, but significantly higher in ejaculatory fluid from normal stallions. Spermatozoa were not a significant source of SPAP activity. High levels of SPAP activity were found in the testes and epididymides. These findings suggest that SPAP activity is a candidate for a sperm-independent marker for ejaculation in the stallion. Finally, AP activity was determined in ejaculatory fluid from a stallion with bilaterally blocked ampullae, both before and after relief of the blockage. While the blockage was present, AP activity in ejaculatory fluid was low. However, following relief of the blockage, AP activity in ejaculatory fluid rose dramatically, thus suggesting that AP activity will be useful as an inexpensive, simple clinical assay for differentiating ejaculatory failure or excurrent duct blockages from testicular origin azoospermia and oligospermia.

  13. Responses of alkaline phosphatase activity in Daphnia to poor nutrition.

    PubMed

    Wagner, Nicole D; Frost, Paul C

    2012-09-01

    The use of biochemical and molecular indices of nutritional stress have recently been promoted for their potential ability to assess the in situ nutritional state of zooplankton. The development and application of these indicators should at least consider the cross-reactivity with other nutritional stressors. We examined the potential usefulness of body alkaline phosphatase activity (APA) as an indicator of dietary phosphorus (P) stress in Daphnia. We measured growth rate, body P-content, and body APA of two species of Daphnia (D. magna, D. pulex) grown for different periods under diverse dietary conditions. We found P-poor food reduced daphnid growth rates and body P-content, while body APA increased in both species. However, body APA increased in P-sufficient D. magna and D. pulex that were feeding on cyanobacterial compared to green algal food, despite no differences in animal body P content. Body APA increased in D. magna fed P-poor food whether cyanobacterial or algal. Body APA also varied with age and other nutritional stresses (low food quantity, nitrogen-poor algae) in both daphnid species. Our results demonstrate that whole body homogenate APA in Daphnia is not singularly responsive to P-poor food, which will complicate or limit its future usefulness and application as an indicator of dietary P-stress in metazoans.

  14. Changes of serum alkaline phosphatase isoenzymes in fasted rats.

    PubMed

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1996-10-01

    Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

  15. Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.

    PubMed

    Mori, S; Okamoto, M; Nishibori, M; Ichimura, M; Sakiyama, J; Endo, H

    1999-06-01

    Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 micromol p-nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p-nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60-70 degrees C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N-terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.

  16. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  17. Interplay between intestinal alkaline phosphatase, diet, gut microbes and immunity.

    PubMed

    Estaki, Mehrbod; DeCoffe, Daniella; Gibson, Deanna L

    2014-11-14

    Intestinal alkaline phosphatase (IAP) plays an essential role in intestinal homeostasis and health through interactions with the resident microbiota, diet and the gut. IAP's role in the intestine is to dephosphorylate toxic microbial ligands such as lipopolysaccharides, unmethylated cytosine-guanosine dinucleotides and flagellin as well as extracellular nucleotides such as uridine diphosphate. IAP's ability to detoxify these ligands is essential in protecting the host from sepsis during acute inflammation and chronic inflammatory conditions such as inflammatory bowel disease. Also important in these complications is IAP's ability to regulate the microbial ecosystem by forming a complex relationship between microbiota, diet and the intestinal mucosal surface. Evidence reveals that diet alters IAP expression and activity and this in turn can influence the gut microbiota and homeostasis. IAP's ability to maintain a healthy gastrointestinal tract has accelerated research on its potential use as a therapeutic agent against a multitude of diseases. Exogenous IAP has been shown to have beneficial effects when administered during ulcerative colitis, coronary bypass surgery and sepsis. There are currently a handful of human clinical trials underway investigating the effects of exogenous IAP during sepsis, rheumatoid arthritis and heart surgery. In light of these findings IAP has been marked as a novel agent to help treat a variety of other inflammatory and infectious diseases. The purpose of this review is to highlight the essential characteristics of IAP in protection and maintenance of intestinal homeostasis while addressing the intricate interplay between IAP, diet, microbiota and the intestinal epithelium.

  18. Alkaline phosphatase activity in salivary gland cells of Rhodnius neglectus and R. prolixus (Hemiptera, Triatominae).

    PubMed

    Lima-Oliveira, A P M; Alevi, K C C; Anhê, A C B; Azeredo-Oliveira, M T V

    2016-07-29

    Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease.

  19. Rapidly diverging evolution of an atypical alkaline phosphatase (PhoAaty) in marine phytoplankton: insights from dinoflagellate alkaline phosphatases

    PubMed Central

    Lin, Xin; Wang, Lu; Shi, Xinguo; Lin, Senjie

    2015-01-01

    Alkaline phosphatase (AP) is a key enzyme that enables marine phytoplankton to scavenge phosphorus (P) from dissolved organic phosphorus (DOP) when inorganic phosphate is scarce in the ocean. Yet how the AP gene has evolved in phytoplankton, particularly dinoflagellates, is poorly understood. We sequenced full-length AP genes and corresponding complementary DNA (cDNA) from 15 strains (10 species), representing four classes of the core dinoflagellate lineage, Gymnodiniales, Prorocentrales, Suessiales, and Gonyaulacales. Dinoflagellate AP gene sequences exhibited high variability, containing variable introns, pseudogenes, single nucleotide polymorphisms and consequent variations in amino acid sequence, indicative of gene duplication events and consistent with the “birth-and-death” model of gene evolution. Further sequence comparison showed that dinoflagellate APs likely belong to an atypical type AP (PhoAaty), which shares conserved motifs with counterparts in marine bacteria, cyanobacteria, green algae, haptophytes, and stramenopiles. Phylogenetic analysis suggested that PhoAaty probably originated from an ancestral gene in bacteria and evolved divergently in marine phytoplankton. Because variations in AP amino acid sequences may lead to differential subcellular localization and potentially different metal ion requirements, the multiple types of APs in algae may have resulted from selection for diversifying strategies to utilize DOP in the P variable marine environment. PMID:26379645

  20. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    SciTech Connect

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-08-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G (IgG) complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table.

  1. Cloning and sequencing of human intestinal alkaline phosphatase cDNA

    SciTech Connect

    Berger, J.; Garattini, E.; Hua, J.C.; Udenfriend, S.

    1987-02-01

    Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme.

  2. Dephosphorylation of purine mononucleotides by alkaline phosphatases. Substrate specificity and inhibition patterns.

    PubMed

    Jensen, M H

    1979-11-09

    Three purine mononucleotides, adenosine-, inosine- and guanosine monophosphate, were used as substrates at pH 7.4 and at 10.4 for three alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.1) containing similar phosphate-binding serine groups at their esteratic sites. Substrate specificity was found for the enzymes from calf intestine and bovine liver. Alkaline phosphatase from Escherichia coli was nonspecific. A substrate-dependent and pronounced inhibition with the purine analogue 1,3-dimethyl xanthine was found for the enzymes from intestine and liver, but not for alkaline phosphatase from E. coli. A substrate-independent and pronounced inhibition was found for all three enzymes with the phosphomonoester p-nitrophenol phosphate as the inhibitor. Alkaline phosphatases may play an important role in the regulation of the intracellular content of purine mononucleotides.

  3. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    PubMed

    Lallès, Jean-Paul

    2016-07-01

    The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

  4. Serum alkaline phosphatase negatively affects endothelium-dependent vasodilation in naïve hypertensive patients.

    PubMed

    Perticone, Francesco; Perticone, Maria; Maio, Raffaele; Sciacqua, Angela; Andreucci, Michele; Tripepi, Giovanni; Corrao, Salvatore; Mallamaci, Francesca; Sesti, Giorgio; Zoccali, Carmine

    2015-10-01

    Tissue nonspecific alkaline phosphatase, promoting arterial calcification in experimental models, is a powerful predictor of total and cardiovascular mortality in general population and in patients with renal or cardiovascular diseases. For this study, to evaluate a possible correlation between serum alkaline phosphatase levels and endothelial function, assessed by strain gauge plethysmography, we enrolled 500 naïve hypertensives divided into increasing tertiles of alkaline phosphatase. The maximal response to acetylcholine was inversely related to alkaline phosphatase (r=−0.55; P<0.001), and this association was independent (r=−0.61; P<0.001) of demographic and classical risk factors, body mass index, estimated glomerular filtration rate, serum phosphorus and calcium, C-reactive protein, and albuminuria. At multiple logistic regression analysis, the risk of endothelial dysfunction was ≈3-fold higher in patients in the third tertile than that of patients in the first tertile. We also tested the combined role of alkaline phosphatase and serum phosphorus on endothelial function. The steepness of the alkaline phosphatase/vasodilating response to acetylcholine relationship was substantially attenuated (P<0.001) in patients with serum phosphorus above the median value when compared with patients with serum phosphorus below the median (−5.0% versus −10.2% per alkaline phosphatase unit, respectively), and this interaction remained highly significant (P<0.001) after adjustment of all the previously mentioned risk factors. Our data support a strong and significant inverse relationship between alkaline phosphatase and endothelium-dependent vasodilation, which was attenuated by relatively higher serum phosphorus levels.

  5. 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.

  6. Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir

    NASA Astrophysics Data System (ADS)

    Tseng, Y.

    2011-12-01

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

  7. Modeling catalytic promiscuity in the alkaline phosphatase superfamily

    PubMed Central

    Duarte, Fernanda; Amrein, Beat Anton

    2013-01-01

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein

  8. Cinacalcet Lowers Serum Alkaline Phosphatase in Maintenance Hemodialysis Patients

    PubMed Central

    Belozeroff, Vasily; Goodman, William G.; Ren, Lulu; Kalantar-Zadeh, Kamyar

    2009-01-01

    Background and objectives: Studies suggest an association between elevated serum alkaline phosphatase (AP) and increased mortality in hemodialysis patients, but the effect of existing therapies on AP is not fully understood. We assessed the effects of cinacalcet on AP in a secondary analysis of controlled trial data. Design, setting, participants, & measurements: This was a post hoc analysis of data from three 26-wk randomized, double-blind, placebo-controlled, phase 3 trials and a 26-wk double-blind, placebo-controlled extension trial that investigated cinacalcet in secondary hyperparathyroidism treatment in dialysis patients. Hemodialysis patients (n = 890) with intact parathyroid hormone ≥300 pg/ml and serum calcium ≥8.4 mg/dl received cinacalcet plus standard therapy or standard therapy alone for up to 52 wk. Total, not bone-specific, AP was assessed (proportion of cinacalcet/control subjects achieving a ≥20% or any AP reduction from baseline; the proportion of subjects with AP ≥120 U/L) at baseline; the end of titration; and study weeks 26, 42, and 52. Results: At 52 wk, a greater proportion of cinacalcet-treated patients had either a ≥20% (39 versus 18%) or any (58 versus 36%) AP reduction compared with control subjects, respectively. The likelihood of achieving either a ≥20% or any AP reduction (determined by relative proportion) was 2.33 (95% confidence interval 1.50 to 3.61) and 1.74 (95% confidence interval 1.31 to 2.31), respectively, at week 52. Cinacalcet treatment tended toward a decreased percentage of patients with AP ≥120 U/L (baseline, 42.6%; week 52, 30.6%) compared with control (35.0 to 48.6%, respectively). Conclusions: In this combined analysis of controlled trials of patients who were receiving hemodialysis, cinacalcet lowered total serum AP. PMID:19261825

  9. Small interfering RNA of alkaline phosphatase inhibits matrix mineralization.

    PubMed

    Kotobuki, Noriko; Matsushima, Asako; Kato, Youichi; Kubo, Yoko; Hirose, Motohiro; Ohgushi, Hajime

    2008-05-01

    To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.

  10. Imaging of Alkaline Phosphatase Activity in Bone Tissue

    PubMed Central

    Gade, Terence P.; Motley, Matthew W.; Beattie, Bradley J.; Bhakta, Roshni; Boskey, Adele L.; Koutcher, Jason A.; Mayer-Kuckuk, Philipp

    2011-01-01

    The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications. PMID:21799916

  11. Alkaline Phosphatase: A Biomarker of Cardiac Function in Pediatric Patients.

    PubMed

    Makil, Elizabeth S; Tang, Xinyu; Frazier, Elizabeth A; Collins, R Thomas

    2017-02-09

    Myocardial dysfunction and heart failure are common in pediatric patients with congenital and acquired heart disease. Alkaline phosphatase (AP) has been suggested as a biomarker for myocardial dysfunction after Fontan operation. We hypothesized that pediatric patients with myocardial dysfunction requiring orthotopic heart transplant (OHT) have diminished AP compared to normal. A retrospective review was performed in all patients who underwent OHT at Arkansas Children's Hospital between January 2007 and October 2012. Anatomic diagnoses, therapeutic interventions, and ventricular ejection fraction (EF) were recorded. Z scores for AP levels in the study group were determined by comparing the observed AP levels to age- and gender-matched normative values. T tests were performed to compare the mean AP Z score prior to and after OHT. p values <0.05 were considered statistically significant. During the study period, 124 OHTs were performed. Complete study data were available and analyzed from 71/124 patients (mean age at OHT 3.9 years; 51% female). The mean AP Z score was significantly lower in the study group prior to OHT compared to normal (p < 0.0001). The initiation of ACE inhibitor therapy prior to OHT was associated with a significant increase in AP and the ventricular EF (p < 0.001 for both). Treatment with milrinone was associated with an increase in EF. AP is significantly lower in pediatric patients with myocardial dysfunction prior to OHT compared to normal. AP increases significantly after the initiation of therapies to improve myocardial function. Diminished AP is an indicator of myocardial dysfunction in pediatric patients.

  12. Alkaline phosphatase encapsulated in gellan-chitosan hybrid capsules.

    PubMed

    Fujii, Toshihiro; Ogiwara, Daisuke; Ohkawa, Kousaku; Yamamoto, Hiroyuki

    2005-05-23

    Alkaline phosphatase (ALP) was encapsulated in gellan-chitosan polyion complex (PIC) capsules using a convenient procedure. The recovery of ALP was about 50% when the capsules were prepared by dropping a solution of ALP and gellan mixture (ALP/gellan) into a chitosan solution. When p-nitrophenyl phosphate (p-NPP) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were incubated with ALP/gellan-chitosan capsules as substrates for ALP, the transparent colorless capsules changed to yellow and blue, respectively. The encapsulation of ALP into the PIC capsules was also confirmed by SDS-PAGE and immunoblot analyses. The ALP and polypeptides of more than 30 kDa remained without release even after incubation at 4 degrees C for 14 d. The biochemical properties of the encapsulated ALP activity were similar to those of the intact enzyme. When the solution containing p-NPP was loaded on a column packed with ALP/gellan-chitosan capsules at 27 degrees C, approximately 75% of p-NPP was hydrolyzed by passing through the column. No significant leakage of ALP was observed during the procedure, indicating that the capsules were resistant to pressure in the chromatographic operation. Furthermore, 70% of the hydrolytic activity of the packed capsules remained after storage at 4 degrees C for one month. These results suggest that the polyion complex capsules could be useful materials for protein fixation without chemical modification. [Diagram: see text] Encapsulation of ALP into PIC capsules and the morphological changes seen in the absence of the ALP substrate and in the presence of p-NPP and BICP.

  13. Alkaline Phosphatase Revisited:  Hydrolysis of Alkyl Phosphates (†).

    PubMed

    O'Brien, Patrick J; Herschlag, Daniel

    2002-03-05

    Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound ∼4 Å apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (Pi) in excess of its inhibition constant (K i ≈ 1 μM). This tight binding by Pi has affected the majority of published kinetic constants. Furthermore, binding limits k cat/K m for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k cat/K m. The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK a of ≤5.5 in the free enzyme. An inactivating pK a of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k cat/K m on the pK a of the leaving group follows a Brønsted correlation with a slope of βlg = -0.85 ± 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P

  14. Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

    PubMed Central

    Thompson, Linda M. M.; MacLeod, Robert A

    1974-01-01

    The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO4, and 0.01 M KCl) or 0.05 M MgSO4 appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg2+ in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place. PMID:4811547

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

    PubMed

    Franzén, A; Hasselgren, G

    1978-01-01

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

  16. Separation of Alkaline Phosphatase Isoenzymes from Various Rat Tissues Using Flat-Bed Acrylamide Gel Isoelectric Focusing,

    DTIC Science & Technology

    1981-12-22

    Technical Bulletin No. 104. Alkaline phosphatase activity was expressed as 1 micromole of p- nitrophenol hydrolyzed per hour and specific alkaline... phosphatase activity was defined as the number of micromoles of p- nitrophenol hydrolyzed per hour per microgram of protein. The total protein was determined... phosphatase activity is known or suspected. Importantly, the procedure is easily adapted for acid phosphatase examination by merely changing the pH and

  17. Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

    PubMed Central

    Prinz, W A; Beckwith, J

    1994-01-01

    To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm. PMID:7929016

  18. Alkaline phosphatase: a possible treatment for sepsis-associated acute kidney injury in critically ill patients.

    PubMed

    Peters, Esther; Heemskerk, Suzanne; Masereeuw, Rosalinde; Pickkers, Peter

    2014-06-01

    Acute kidney injury (AKI) is a common disease in the intensive care unit and accounts for high morbidity and mortality. Sepsis, the predominant cause of AKI in this setting, involves a complex pathogenesis in which renal inflammation and hypoxia are believed to play an important role. A new therapy should be aimed at targeting both these processes, and the enzyme alkaline phosphatase, with its dual mode of action, might be a promising candidate. First, alkaline phosphatase is able to reduce inflammation through dephosphorylation and thereby detoxification of endotoxin (lipopolysaccharide), which is an important mediator of sepsis. Second, adenosine triphosphate, released during cellular stress caused by inflammation and hypoxia, has detrimental effects but can be converted by alkaline phosphatase into adenosine with anti-inflammatory and tissue-protective effects. These postulated beneficial effects of alkaline phosphatase have been confirmed in animal experiments and two phase 2a clinical trials showing that kidney function improved in critically ill patients with sepsis-associated AKI. Because renal inflammation and hypoxia also are observed commonly in AKI induced by other causes, it would be of interest to investigate the therapeutic effect of alkaline phosphatase in these nephropathies as well.

  19. Plasma alkaline phosphatase activity: a screening test for rickets in preterm neonates.

    PubMed

    Kovar, I; Mayne, P; Barltrop, D

    1982-02-06

    Both rickets and raised plasma alkaline phosphatase activity are common in the preterm infant. Measurement of plasma alkaline phosphatase activity is valuable in screening for active disease and in diagnosis, but normal reference data are not available for preterm babies. In 30 consecutive preterm infants (birthweight 1580 +/- 410 g, gestational age 31 +/- 2.5 weeks) serial measurements of plasma alkaline phosphatase activity, plasma levels of calcium and inorganic phosphorus, and the pattern of alkaline phosphatase isoenzymes were made. Four patterns of changes in plasma alkaline phosphatase activity with time were seen. 4 of the 30 infants were shown to have rickets; these children and 14 of the 26 non-rachitic infants showed an increasing/peak/decreasing pattern with increasing age, but levels were much higher in the rachitic infants. The activity in all 30 was raised above the adult and childhood reference ranges at some point in time. The data suggest that an activity of five times the upper limit of the normal adult reference range is acceptable in the preterm infant but an activity higher than this may suggest rickets.

  20. An alkaline phosphatase transport mechanism in the pathogenesis of Alzheimer's disease and neurodegeneration.

    PubMed

    Pike, Adrianne F; Kramer, Nynke I; Blaauboer, Bas J; Seinen, Willem; Brands, Ruud

    2015-01-25

    Systemic inflammation is associated with loss of blood-brain barrier integrity and neuroinflammation that lead to the exacerbation of neurodegenerative diseases. It is also associated specifically with the characteristic amyloid-β and tau pathologies of Alzheimer's disease. We have previously proposed an immunosurveillance mechanism for epithelial barriers involving negative feedback-regulated alkaline phosphatase transcytosis as an acute phase anti-inflammatory response that hangs in the balance between the resolution and the progression of inflammation. We now extend this model to endothelial barriers, particularly the blood-brain barrier, and present a literature-supported mechanistic explanation for Alzheimer's disease pathology with this system at its foundation. In this mechanism, a switch in the role of alkaline phosphatase from its baseline duties to a stopgap anti-inflammatory function results in the loss of alkaline phosphatase from cell membranes into circulation, thereby decreasing blood-brain barrier integrity and functionality. This occurs with impairment of both amyloid-β efflux and tau dephosphorylating activity in the brain as alkaline phosphatase is replenished at the barrier by receptor-mediated transport. We suggest systemic alkaline phosphatase administration as a potential therapy for the resolution of inflammation and the prevention of Alzheimer's disease pathology as well as that of other inflammation-related neurodegenerative diseases.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

    Weinberg, R A; Zusman, D R

    1990-05-01

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

  3. The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

    PubMed

    Firth, J A; Marland, B Y

    1975-08-01

    The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

  4. Extracellular Ca2(+)-dependent inducible alkaline phosphatase from extremely halophilic archaebacterium Haloarcula marismortui.

    PubMed Central

    Goldman, S; Hecht, K; Eisenberg, H; Mevarech, M

    1990-01-01

    When starved of inorganic phosphate, the extremely halophilic archaebacterium Haloarcula marismortui produces the enzyme alkaline phosphatase and secretes it to the medium. This inducible extracellular enzyme is a glycoprotein whose subunit molecular mass is 160 kDa, as estimated by sodium dodecyl sulfate-gel electrophoresis. The native form of the enzyme is heterogeneous and composed of multiple oligomeric forms. The enzymatic activity of the halophilic alkaline phosphatase is maximal at pH 8.5, and the enzyme is inhibited by phosphate. Unlike most alkaline phosphatases, the halobacterial enzyme requires Ca2+ and not Zn2+ ions for its activity. Both calcium ions (in the millimolar range) and NaCl (in the molar range) are required for the stability of the enzyme. Images PMID:2123861

  5. L-Phenylalanine inhibition of human alkaline phosphatases with p-nitrophenyl phosphate as substrate.

    PubMed

    Komoda, T; Hokari, S; Sonoda, M; Sakagishi, Y; Tamura, T

    1982-12-01

    With p-nitrophenyl phosphate as the substrate, there reportedly is no organ-specific inhibition of alkaline phosphatase (EC 3.1.3.1) activity by L-phenylalanine. However, we found that at pH 10.0, with p-nitrophenyl phosphate as the substrate, L-phenylalanine obviously inhibits the alkaline phosphatase isoenzyme from human placenta, whereas there is little if any inhibition of the isoenzyme from human intestine. Because of the differing effects of substrates (p-nitrophenyl phosphate and phenyl phosphate) and their enzymic products (p-nitrophenol and phenol) for L-phenylalanine action on the placental alkaline phosphatase isoenzyme, we suggest that the isoenzyme--inhibitor--substrate complex and the effect of released phosphate on L-phenylalanine inhibition of the isoenzyme activity differ from each other.

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

    PubMed Central

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104−106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

  7. Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

    PubMed Central

    Shephard, M D; Peake, M J; Walmsley, R N

    1986-01-01

    A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made. PMID:3760234

  8. Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.

    PubMed Central

    Tommassen, J; Lugtenberg, B

    1980-01-01

    Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst. Images PMID:6995425

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

    PubMed

    Beckman, G; Beckman, L; Löfstrand, T

    1978-01-01

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

  10. Separation and purification of the alkaline phosphatase and a phosphodiesterase from Halobacterium cutirubrum.

    PubMed Central

    Fitt, P S; Baddoo, P

    1979-01-01

    1. Halobacterium cutirubrum alkaline phosphatase is associated in crude extracts with a phosphodiesterase. 2. The enzymes were stabilized in buffers containing both (NH4)2SO4 and 10 mM-Mn2+. 3. Adsorption chromatography on Sepharose 6B/agarose-gel columns in the presence of 1.4M-(NH4)2SO4 gave a phosphatase-free phosphodiesterase and the alkaline phosphatase associated with some phosphodiesterase activity. 4. Further chromatography of the separated enzymes gave a good recovery of greater than 600-fold purified phosphodiesterase and greater than 3000-fold purified alkaline phosphatase. 5. The requirements of these enzymes and their relationship to each other was examined. 6. A detailed study showed that the alkaline phosphatase was adsorbed at least partially to agarose and dextran columns at all (NH4)2SO4 concentrations from 0.25 to 2M. 7. In contrast, no adsorption of the enzyme or protein standards was evident in 2.5M-KCl/l M-NaCl or 0.25 M-KCl/0.1 M-NaCl, in agreement with previous studies by Louis, Peterkin & Fitt [(1971) Biochem. J. 121, 635-641], thus confirming the validity of gel filtration in 2.5 M-KCl/1 M-NaCl as a method for determining the approximate molecular weights of extremehalophile proteins. PMID:227360

  11. Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms.

    PubMed

    Ozkanca, R; Flint, K P

    1996-03-01

    Escherichia coli grown in high or low phosphate medium was inoculated into a lake water starvation medium. The viable count decreased at 37 degrees C but not at the lower temperatures over 70 d. Alkaline phosphatase was monitored using a colorimetric assay with pNPP as the substrate. Derepression of the enzyme occurred in cultures starved for > 30 d in the lake water and within 5 d in lake water microcosms supplemented with carbon and nitrogen sources where there was rarely an increase in viable count. Chloramphenicol prevented the synthesis of alkaline phosphatase suggesting that, even under starvation conditions, de novo synthesis of the enzyme occurs.

  12. Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase

    SciTech Connect

    Butler-Ransohoff, J.E.; Kendall, D.A.; Freeman, S.; Knowles, J.R.; Kaiser, E.T.

    1988-06-28

    The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using /sup 31/P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (R/sub P/)-/sup 17/O, /sup 16/O, /sup 18/O)phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

  13. 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.

  14. DNA polymorphism of alkaline phosphatase isozyme genes: Linkage disequilibria between placental and germ-cell alkaline phosphotase alleles

    SciTech Connect

    Beckman, G.; Beckman, L.; Sikstroem, C. ); Millan, J.L. )

    1992-11-01

    The use of human placental alkaline phosphatase (PLAP) cDNA as a probe allows the detection and identification of restriction DNA fragments derived from three homologous genes, i.e., intestinal alkaline phosphatase (AP), germ-cell AP (GCAP), and PLAP. In previous RFLP studies the authors have reported linkage disequilibria between an RsaI and two PstI (a and b) polymorphic restriction sites and electrophoretic types of PLAP. In this report they present evidence that, in spite of the strong correlation with PLAP types, PstI(b) is an RFLP of GCAP. The data indicate close linkage between the PLAP and GCAP loci. 18 refs., 2 figs., 3 tabs.

  15. Extreme Elevation of Alkaline Phosphatase in a Pregnancy Complicated by Gestational Diabetes and Infant with Neonatal Alloimmune Thrombocytopenia

    PubMed Central

    Healey, Michael

    2016-01-01

    There have been few case reports of isolated elevation of alkaline phosphatase beyond the normal physiologic amount with subsequent return to baseline after delivery. Here we present a similar case of extreme elevation of alkaline phosphatase in a pregnancy complicated by gestational diabetes and subsequently by neonatal alloimmune thrombocytopenia (NAIT). PMID:27610256

  16. Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

    PubMed

    Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

    2007-09-26

    Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

  17. Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

    PubMed

    Kumar, Mukesh; Khan, Imran; Sinha, Subrata

    2015-01-01

    Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

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

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

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

  19. Heat stable alkaline phosphatase from thermophiles. Final report, March-October 1993

    SciTech Connect

    Combie, J.D.; Runnion, K.N.; Williamson, M.L.

    1994-07-01

    Alkaline phosphatase has been the most widely used enzyme for colorimetric immunoassays. The current potential for this enzyme lies in biosensors, fieldable assay kits, biotechnology applications, degradation of certain nerve agents and pesticides and detoxification of heavy metal waste streams. While the commercial source of this enzyme is predominantly from mammalian tissues, expanded commercial application is restricted by the enzyme's instability at elevated temperatures. Although alkaline phosphatases are ubiquitous in nature, two isolates out of 44 alkaline phosphatase producing isolates occurring in habitats at 50 deg C and above have been isolated possessing extremely stable enzymes. One enzyme retained 98% of original activity following boiling for 1 hr. The secretion of the enzyme by the organism is an added benefit promoting efficient and economical production capability. Procedures for the screening, isolation, and optimal growth and fermentation of organisms acquired from geothermal sources located in Yellowstone National Park, WY are described. Purification was most effectively achieved using size exclusion chromatography where 101% of the activity and 33% of the crude mother liquor protein were recovered. Although the presence of manganese in the assay buffer was observed to significantly elevate the enzyme's catalytic activity, a precipitate incompatibility with calcium chloride, a requirement for high temperature stability, prohibits its use. Bacteria, Fermentation, Alkaline phosphatase, Biosensors, Biotechnology, Heat stable enzymes, Biochemistry, Bioremediation, Thermophilic microorganisms.

  20. Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase.

    PubMed

    Simão, Ana Maria S; Beloti, Marcio M; Rosa, Adalberto L; de Oliveira, Paulo T; Granjeiro, José Mauro; Pizauro, João M; Ciancaglini, Pietro

    2007-11-01

    The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.

  1. Effect of polyphenols on calcium content and alkaline phosphatase activity in rat femoral tissues in vitro.

    PubMed

    Yamaguchi, M; Jie, Z

    2001-12-01

    The effect of various polyphenols on calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues of young rats in vitro was investigated. Bone tissues were cultured for 24 h in serum-free Dulbecco's modified Eagle's medium containing either vehicle or various polyphenols (10(-7) - 10(-4) M). The presence of genistein (10(-6) - 10(-4) M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. Resveratrol (10(-4) m) decreased metaphyseal calcium content significantly, and it (10(-6) - 10(-4) M) had a significant inhibitory effect on diaphyseal enzyme activity. Epigallocatechin gallate (EGCg; 10(-4) M) significantly inhibited alkaline phosphatase activity in the diaphyseal and metaphyseal tissues. EGCg (10(-7) - 10(-4) M) had no effect on bone calcium content. Meanwhile, glycitein, quercetin, or catechin in the range of 10(-7) to 10(-4) ml did not have an effect on calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. The present study suggests that a phytoestrogen genistein has a unique anabolic effect on bone calcification in vitro.

  2. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 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 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 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 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 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 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 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 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

    PubMed

    Pilar, María C; Ortega, Natividad; Perez-Mateos, Manuel; Busto, María D

    2009-03-11

    An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

  8. Immobilized E. coli alkaline phosphatase. Its properties, stability, and utility in studying the dephosphorylation of proteins.

    PubMed

    Basheeruddin, K; Rothman, V; Margolis, S

    1985-04-01

    We have immobilized E. coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg2+. The insoluble and soluble phosphatases removed 75 and 77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA:cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.

  9. Role of bone-type tissue-nonspecific alkaline phosphatase and PHOSPO1 in vascular calcification.

    PubMed

    Bobryshev, Yuri V; Orekhov, Alexander N; Sobenin, Igor; Chistiakov, Dimitry A

    2014-01-01

    Matrix vesicle (MV)-mediated mineralization is important for bone ossification. However, under certain circumstances such as atherosclerosis, mineralization may occur in the arterial wall. Bone-type tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes inorganic pyrophosphate (PPi) and generates inorganic phosphate (Pi), which is essential for MV-mediated hydroxyapatite formation. MVs contain another phosphatase, PHOSPHO1, that serves as an additional supplier of Pi. Activation of bone-type tissue-nonspecific alkaline phosphatase (TNAP) in vascular smooth muscle cells precedes vascular calcification. By degrading PPi, TNAP plays a procalcific role changing the Pi/PPi ratio toward mineralization. A pathologic role of bone-type TNAP and PHOSPHO1 make them to be attractive targets for cardiovascular therapy.

  10. Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods.

    PubMed

    Albillos, S M; Reddy, R; Salter, R

    2011-07-01

    Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.

  11. Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

    PubMed

    Simão, Ana Maria S; Beloti, Márcio M; Cezarino, Rodrigo M; Rosa, Adalberto Luiz; Pizauro, João M; Ciancaglini, Pietro

    2007-04-01

    Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    PubMed

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

    1982-08-01

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

  14. [Changes in the activity of alkaline phosphatase and the level of thiamine diphosphatase in simulations of B1 avitaminosis].

    PubMed

    Pron'ko, P S; Gritsenko, E A; Ostrovskiĭ, Iu M

    1975-01-01

    Alimentary B1 avitaminosis was attended by a declining activity of alkaline phosphatase in the brain and spleen of rats. A single administration of oxythiamine to rats in a dose of 400 mg/kg produced during the first hours an increased activity of alkaline phosphatase in the liver, brain and, to a lesser extent, in a number of other organs and reduced the thiamine-diphosphate content in the liver, brain and other tissues. The thiamin-diphosphate level in the brain returned back to normal in 12 hours, and in other tissues-towards the 3--5th day. In 24 hours after introduction of oxythiamine the activity of alkaline phosphatase was up in the liver alone, while in the brain the activity of the enzyme decreased. Thiamine, used in a dose of 400 mg/kg, exerted on the activity of alkaline phosphatase in a number of tissues an action similar to that of oxythiamine. It is suggested that the activation of alkaline phosphatase 3--12 hours following adminstration of a large dose of thiamine or oxythiamine is of a non-specific nature. Subcutaneous introduction of a commercial alkaline phosphatase preparation to rats brought down the thiamine-diphosphate level in all of the tissues under investigation. The presumed mechanisms accounting for the fall of thiamine-diphosphate and the possible part played in this process by alkaline phosphatase are discussed.

  15. Isolation, transcription, and inactivation of the gene for an atypical alkaline phosphatase of Synechococcus sp. strain PCC 7942.

    PubMed Central

    Ray, J M; Bhaya, D; Block, M A; Grossman, A R

    1991-01-01

    The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake. Images PMID:1712356

  16. Development of conductometric biosensors based on alkaline phosphatases for the water quality control

    NASA Astrophysics Data System (ADS)

    Berezhetskyy, A.

    2008-09-01

    Researches are focused on the elaboration of enzymatic microconductometric device for heavy metal ions detection in water solutions. The manuscript includes a general introduction, the first chapter contains bibliographic review, the second chapter described the fundamentals of conductometric transducers, the third chapter examining the possibility to create and to optimize conductometric biosensor based on bovine alkaline phosphatase for heavy metals ions detection, the fourth chapter devoted to creation and optimization of conductometric biosensor based on alkaline phosphatase active microalgae and sol gel technology, the last chapter described application of the proposed algal biosensor for measurements of heavy metal ions toxicity of waste water, general conclusions stating the progresses achieved in the field of environmental monitoring

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

    PubMed Central

    Sarthy, A; Michaelis, S; Beckwith, J

    1981-01-01

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

  18. Nutrient Limitation Dynamics of a Coastal Cape Cod Pond: Seasonal Trends in Alkaline Phosphatase Activity

    DTIC Science & Technology

    2000-11-13

    106C: 16N: 1P) and alkaline phosphatase activity (APA) were utilized in tandem as nutrient deficiency indicators (NDIs) for phytoplankton . The study...nutrient enrichment incubation re-affirmed the use of APA as a robust indicator of phosphate limitation in phytoplankton . APA data indicate that the system...nutrient deficiency indicators (NDIs) for phytoplankton . The study objective was to evaluate the limiting nutrient status of the pond throughout the

  19. Bio-nanocapsules for signal enhancement of alkaline phosphatase-linked immunosorbent assays.

    PubMed

    Iijima, Masumi; Yamamoto, Mikako; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi

    2013-01-01

    The bio-nanocapsules displaying about 240 molecules of immunoglobulin G Fc-binding Z domains (ZZ-BNCs) enhanced the signals of enzyme-linked immunosorbent assay by tethering the Fc regions of secondary antibodies (Abs), which were eliminated using high-molecular mass enzymes (e.g., alkaline phosphatase). By way of optimizing the distance between enzymes and Abs, ZZ-BNCs improved sensitivity independently of enzymes.

  20. Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization and defense against bacterial endotoxin in hamsters

    PubMed Central

    Lei, Wei; Nguyen, Heidi; Brown, Naoko; Ni, Hua; Kiffer-Moreira, Tina; Reese, Jeff; Millán, José Luis; Paria, Bibhash C.

    2013-01-01

    Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types, and their hormonal regulation, cell-type and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5 and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; and both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone is the major uterine Akp2 inducer, both progesterone and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. In contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection. PMID:23929901

  1. Promiscuous sulfatase activity and thio-effects in a phosphodiesterase of the alkaline phosphatase superfamily.

    PubMed

    Lassila, Jonathan K; Herschlag, Daniel

    2008-12-02

    The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn(2+) bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with k(cat)/K(M) value of 2 x 10(-5) M(-1) s(-1), similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these noncognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn(2+) ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 10(2) of the 10(7)-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn(2+) ions may play an important role in defining the selectivity of the enzymes.

  2. Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter.

    PubMed

    Smith, A A

    2016-08-01

    One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80(o) C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.

  3. Chemiluminescence-based pesticide biosensor utilizing the intelligent evolved properties of the enzyme alkaline phosphatase

    SciTech Connect

    Ayyagari, M.; Kamtekar, S.; Pande, R.; Marx, K.; Kumar, J.

    1994-12-31

    A methodology is described for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer, poly(3-undecylthiophene-co-3- methanoithiophene). A streptavidin conjugate of alkaline phosphatase is used in this study. The biotinylated polymer is attached to the silanized glass surface via hydrophobic interactions and the enzyme is interfaced with the polymer through the classical biotin- streptavidin interaction. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro) (1,2 dioxetane-3-2`-tricyclo-) (3.3.1.1 )-(decani-4-yl) phenyl phosphate, to a species which emits energy by chemiluminescence. This chemiluminescence signal can be detected with a photomultiplier tube for enzymatic catalysis with the biocatalyst both in solution and immobilized on a glass surface. The signal generation is inhibited by the organophosphorus based insecticides such as paraoxon as well as nerve agents. We demonstrate in this study that a number of organophosphorus based insecticides inhibit the enzyme-mediated generation of chemiluminescence signal. This is true for the enzyme conjugate both free in solution and immobilized on a glass surface. In solution, the inhibition resembles the case of a partially uncompetitive system. By this type of inhibition we are able to detect pesticides down to about 50 ppb for the enzyme in solution. The pesticide detection limit of immobilized enzyme is currently being investigated. The enzyme is capable of a number of measurement cycles without significant loss of signal level.

  4. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization.

    PubMed

    Hoemann, C D; El-Gabalawy, H; McKee, M D

    2009-06-01

    In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.

  5. Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.

    PubMed

    Basu, Swarna; Campagnola, Paul J

    2004-01-01

    We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 10(5)-10(6) M(-1) s(-1)and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix.

  6. A conserved domain of alkaline phosphatase expression in the Malpighian tubules of dipteran insects.

    PubMed

    Cabrero, Pablo; Pollock, Valerie P; Davies, Shireen A; Dow, Julian A T

    2004-09-01

    Malpighian (renal) tubules are key components of the insect osmoregulatory system and show correspondingly great diversity in both number and length. Recently, the organisation of the Drosophila melanogaster tubule has been elucidated by enhancer trapping, and an array for functional properties has been shown to align with the functional domains. In Drosophila, there is a lower tubule domain, which coincides with expression of alkaline phosphatase and delineates the absorptive region of the tubule. Here, these observations are extended to three dipteran vectors of disease (Aedes aegypti, Anopheles stephensii and Glossina morsitans) and a non-dipteran out-group, Schistocerca gregaria (Orthoptera). Despite a huge range in cell number and size, alkaline phosphatase was found on the apical surface of the lower 10% of each of the dipteran tubules but nowhere within the orthopteran tubule. An alkaline phosphatase lower tubule domain is thus conserved among Diptera. Cell counts are also provided for each species. As in Drosophila, stellate cells are not found in the lower tubule domain of Anopheles or Aedes tubules, confirming the unique genetic identity of this domain. As previously reported, we failed to find stellate cells in Schistocerca but, remarkably, also failed to find them in Glossina, the dipteran most closely related to Drosophila. The orthodoxy that stellate cells are unique to, and general among, Diptera may thus require revision.

  7. Synthesis, alkaline phosphatase inhibition studies and molecular docking of novel derivatives of 4-quinolones.

    PubMed

    Miliutina, Mariia; Ejaz, Syeda Abida; Khan, Shafi Ullah; Iaroshenko, Viktor O; Villinger, Alexander; Iqbal, Jamshed; Langer, Peter

    2017-01-27

    New and convenient methods for the functionalization of the 4-quinolone scaffold at positions C-1, C-3 and C-6 were developed. The 4-quinolone derivatives were evaluated for their inhibitory potential on alkaline phosphatase isozymes. Most of the compounds exhibit excellent inhibitory activity and moderate selectivity. The IC50 values on tissue non-specific alkaline phosphatase (TNAP) were in the range of 1.34 ± 0.11 to 44.80 ± 2.34 μM, while the values on intestinal alkaline phosphatase (IAP) were in the range of 1.06 ± 0.32 to 192.10 ± 3.78 μM. The most active derivative exhibits a potent inhibition on IAP with a ≈14 fold higher selectivity as compared to TNAP. Furthermore, molecular docking calculations were performed for the most potent inhibitors to show their binding interactions within the active site of the respective enzymes.

  8. Gly429 is the major determinant of uncompetitive inhibition of human germ cell alkaline phosphatase by L-leucine.

    PubMed Central

    Hummer, C; Millán, J L

    1991-01-01

    The catalytic activity of human placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (GCAP) can be inhibited, through an uncompetitive mechanism, by L-Phe. GCAP is also selectively inhibited by L-Leu. Site-directed mutagenesis of five of the 12 residues which are different in PLAP and GCAP revealed that Gly429 is the primary determinant of GCAP inhibition by L-Leu, and Ser84 and Leu297 play a modulatory role in the inhibition. PMID:2001256

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

    PubMed

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

    1993-06-01

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

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

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

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

  11. The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.

    PubMed

    Ahlers, J

    1975-09-01

    1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.

  12. The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.

    PubMed Central

    Ahlers, J

    1975-01-01

    1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector. PMID:995

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

    PubMed

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

    1998-10-01

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

  14. Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

    PubMed Central

    Behrens, C M; Enns, C A; Sussman, H H

    1983-01-01

    The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line. Images Fig. 1. PMID:6882358

  15. Roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated calcification. Effect of selective release of membrane-bound alkaline phosphatase and treatment with isosmotic pH 6 buffer.

    PubMed

    Register, T C; McLean, F M; Low, M G; Wuthier, R E

    1986-07-15

    The roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated mineralization have been studied by selectively releasing the enzyme from a wide variety of matrix vesicle preparations using treatment with a bacterial phosphatidylinositol-specific phospholipase C and by demineralization of the vesicles using isosmotic pH 6 buffer. Following depletion of 50-90% of the alkaline phosphatase activity or treatment with citrate buffer, the vesicles were tested for their ability to accumulate 45Ca2+ and 32Pi from a synthetic cartilage lymph. Removal of alkaline phosphatase by phospholipase C treatment caused two principal effects, depending on the matrix vesicle preparation. In rapidly mineralizing vesicle fractions which did not require organic phosphate esters (Po) to accumulate mineral ions, release of alkaline phosphatase had only a minor effect. In slowly mineralizing vesicles preparations or those dependent on Po substrates for mineral ion uptake, release of alkaline phosphatase caused significant loss of mineralizing activity. The activity of rapidly calcifying vesicles was shown to be dependent on the presence of labile internal mineral, as demonstrated by major loss in activity when the vesicles were decalcified by various treatments. Ion uptake by demineralized vesicles or those fractionated on sucrose step gradients required Po and was significantly decreased by alkaline phosphatase depletion. Uptake of Pi, however, was not coupled with hydrolysis of the Po substrate. These findings argue against a direct role for alkaline phosphatase as a porter in matrix vesicle Pi uptake, contrary to previous postulates. The results emphasize the importance of internal labile mineral in rapid uptake of mineral ions by matrix vesicles.

  16. Ozone inhalation in rats: effects on alkaline phosphatase and lactic dehydrogenase isoenzymes in lavage and plasma

    SciTech Connect

    Nachtman, J.P.; Moon, H.L.; Miles, R.C.

    1988-10-01

    Ozone is found in urban and rural atmospheres and is produced from a variety of natural and man-made sources. Animal studies conducted at typical ambient levels result in reproducible morphological, biochemical and functional effects. Ozone damages type I epithelial cells, induces proliferation of type II cells and produces inflammation of the terminal bronchiolar-alveolar duct region. Ozone increases lung oxygen utilization and increases glutathione metabolism. Ozone increases airway resistance. The authors measured lactic dehydrogenase (LD) isoenzymes to ascertain the tissue giving rise to the increased LD activity in lavage. They also assayed acid phosphatase, alkaline phosphatase, creatine kinase activities, and protein levels since these parameters were increased in rat lung lavage after particulate exposure. They determined white cell differential and red cell morphology parameters because previous investigators reported that ozone increased neutrophil/lymphocyte ratio.

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

    PubMed

    Todorov, I; Saprjanova, M

    1977-04-01

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

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

    PubMed

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

    1985-01-01

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

  19. Initial activity and inactivation of alkaline phosphatase in different lots of buffer.

    PubMed

    Pekelharing, J M; Leijnse, B

    1978-05-02

    Alkaline phosphatase activities were determined in six lots of 2-amino-2-methyl-1-propanol (AMP) and in six lots of diethanolamine (DEA) buffers without preincubation of the sample. There appeared to be differences between the lot numbers in both cases, resulting in a variation in initial activity. When serum samples are preincubated with buffer a loss of activity was observed in 4 out of the 6 AMP buffers. Four human isoenzymes showed varying inactivation during preincubation with AMP buffer. No loss of activity was observed when the preincubation was done with the six DEA buffers. These results indicate that the purity of the commercially-available buffers is quite unsatisfactory.

  20. Observations on the alkaline phosphatase isoenzyme distribution in maternal and amniotic fluid compartments in Nigerian parturients.

    PubMed

    Okpere, E; Okorodudu, A; Gbinigie, O

    1988-01-01

    Estimation of the alkaline phosphates isoenzymes in paired maternal serum and amniotic fluids in term uncomplicated pregnancies and in patients with pre-eclampsia, showed poor correlation coefficients between the levels of both heat stable and heat labile isoenzymes. There was a statistically significant fall in AF (P less than .05) HSAP in pre-eclampsia and a highly significant rise of HLAP in meconial liquor. It is concluded that the poor correlation between the levels of HSAP in maternal serum and amniotic fluid (despite their common source of origin), the normal levels of HLAP in maternal serum in the presence of significantly high levels of HSAP in maternal serum in the presence of significantly diminished levels in amniotic fluid point to a state of relatively diminished permeability of the chorioamniotic membranes to the alkaline phosphatase isoenzymes in Nigerians.

  1. Clinical utility of a wheat-germ precipitation assay for determination of bone alkaline phosphatase concentrations in patients with different metabolic bone diseases.

    PubMed

    Braga, V; Dorizzi, R; Brocco, G; Rossini, M; Zamberlan, N; Gatti, D; Adami, S

    1995-07-01

    Bone alkaline phosphatase was evaluated by wheat-germ lectin precipitation in several clinical conditions. The study included 33 premenopausal healthy women, 46 postmenopausal apparently healthy women, 19 growing children, 24 patients with Paget's disease, 31 patients with primary hyperparathyroidism and 66 patients with hepatobiliary diseases. In postmenopausal women the mean T score (i.e.: the number of SD below or above the mean for premenopausal women) was 2.6 +/- 1.3 (SD) for bone alkaline phosphatase and 1.61 +/- 1.21 for total alkaline phosphatase (p < 0.001). The T score for bone alkaline phosphatase provided a better discrimination from normals for both Paget's disease (22.1 +/- 27.8 versus 12.8 +/- 16 p < 0.001) and primary hyperparathyroidism (8.2 +/- 4.3 versus 4.6 +/- 3.7 p < 0.005 for bone alkaline phosphatase and total alkaline phosphatase respectively). After treatment with intravenous bisphosphonate the percent decrease of bone alkaline phosphatase was larger than that of total alkaline phosphatase both in patients with Paget's disease (-46% versus -72% p < 0.01) and in patients with primary hyperparathyroidism (-21% versus -47% p < 0.02) and an estimate of the precision (delta mean/SD of the delta mean) for bone alkaline phosphatase was 1.9-3.7 times higher than that of total alkaline phosphatase. In twelve osteoporotic patients treated for six months with oral alendronate the decrease in bone turnover was detected with significantly higher precision with bone alkaline phosphatase than with total alkaline phosphatase (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Diarylsulfonamides and their bioisosteres as dual inhibitors of alkaline phosphatase and carbonic anhydrase: Structure activity relationship and molecular modelling studies.

    PubMed

    Al-Rashida, Mariya; Ejaz, Syeda Abida; Ali, Sharafat; Shaukat, Aisha; Hamayoun, Mehwish; Ahmed, Maqsood; Iqbal, Jamshed

    2015-05-15

    The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, on alkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfonamides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a-1c, 2a-2d and their sulfonamide linked bioisosteres 3a-3d, 4a-4d was synthesized and evaluated for inhibitory activity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase (IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisosteric replacement of carboxamide linking group with a sulfonamide group. Some of these compounds were identified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were found to be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a competitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitive mode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carried out to rationalize important binding site interactions.

  3. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  4. Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

    PubMed

    Burtis, C A; Seibert, L E; Baird, M A; Sampson, E J

    1977-09-01

    The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed.

  5. Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.

    PubMed

    Hulett, F M; Schaffel, S D; Campbell, L L

    1976-11-01

    The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.

  6. Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

    PubMed

    Szumańska, G; Gadamski, R

    1992-01-01

    Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

  7. Transformation of glucocorticoid receptors bound to the antagonist RU 486: Effects of alkaline phosphatase

    SciTech Connect

    Gruol, D.J.; Wolfe, K.A. )

    1990-08-28

    RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. The studies have found that in vitro at 15{degree}C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition was significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Treatment of the Ru 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.

  8. Alkaline phosphatase in osteoblasts is down-regulated by pulsatile fluid flow

    NASA Technical Reports Server (NTRS)

    Hillsley, M. V.; Frangos, J. A.

    1997-01-01

    It is our hypothesis that interstitial fluid flow plays a role in the bone remodeling response to mechanical loading. The fluid flow-induced expression of three proteins (collagen, osteopontin, and alkaline phosphatase) involved in bone remodeling was investigated. Rat calvarial osteoblasts subjected to pulsatile fluid flow at an average shear stress of 5 dyne/cm2 showed decreased alkaline phosphatase (AP) mRNA expression after only 1 hour of flow. After 3 hours of flow, AP mRNA levels had decreased to 30% of stationary control levels and remained at this level for an additional 5 hours of flow. Steady flow (4 dyne/cm2 fluid shear stress), in contrast, resulted in a delayed and less dramatic decrease in AP mRNA expression to 63% of control levels after 8 hours of flow. The reduced AP mRNA expression under pulsatile flow conditions was followed by reduced AP enzyme activity after 24 hours. No changes in collagen or osteopontin mRNA expression were detected over 8 hours of pulsatile flow. This is the first time fluid flow has been shown to affect gene expression in osteoblasts.

  9. Ultrastructural localization of alkaline phosphatase in the eggs of Hydatigera taeniaeformis (Taenia taeniaeformis).

    PubMed

    Ajayi, S T; Smith, B F; LeFlore, W B

    1985-01-01

    Freshly shed gravid proglottids from a three-month-old infection of Hydatigera taeniaeformis collected from the faecal droppings of infected cats were used for this study. They were treated for transmission electron microscopy (TEM) followed by incubation using the lead precipitate method. Control sections were incubated in a substrate-free medium, a substrate medium containing 1.0 mM sodium fluoride (NaF) (an inhibitor), and the last sections were denatured at 90 degrees C for 1 min prior to incubation. Intensive alkaline phosphatase activity in the embryophoric blocks and the outer embryophoric membrane was revealed. The reaction products were also indicated in the oncospheral membrane. However, no enzyme activity was seen in any other part of the egg. The enzyme was also absent in the control sections. The presence of alkaline phosphatase activity in the outer embryophoric and oncospheral membranes suggested that this enzyme may be involved in carbohydrate metabolism and nutritional absorption, and also may play a role in the transport of nutrients and other substances from the adult to the developing embryo, respectively.

  10. A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited-state intramolecular proton transfer.

    PubMed

    Fan, Chunlei; Luo, Shengxu; Qi, Haiping

    2016-03-01

    A ratiometric fluorescent probe 2-(benzimidazol-2-yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited-state intramolecular proton transfer (ESIPT) process of 2-(2'-hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl3 , HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris-HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I430/I360) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP.

  11. Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection.

    PubMed

    Thompson, R Q; Barone, G C; Halsall, H B; Heineman, W R

    1991-01-01

    An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.

  12. Defective Multilayer Carbon Nanotubes Increase Alkaline Phosphatase Activity and Bone-Like Nodules in Osteoblast Cultures.

    PubMed

    Zancanela, Daniela Cervelle; Simaã, Ana Maria Sper; Matsubara, Elaine Yoshiko; Rosolen, José Maurício; Ciancaglini, Pietro

    2016-02-01

    Carbon nanotubes (CNT) is one of the most studied biomaterials, and issues about its cytotoxicity remain. The objective of our study was to investigate the in vitro influence of defective CNT on culture growth and on the formation of mineralized matrix nodules by primary osteoblastic cells grown in plastic or titanium (Ti) surfaces. Cellular viability, alkaline phosphatase activity and formation of mineral nodules were evaluated, besides the CNT characterization tests. The CNT studies showed better cell viability for osteoblasts incubated at stationary phase of culture in the presence of Ti (about 70%), but for the other phases, the cells suffered a significant reduction in viability. A peak of maximum alkaline phosphatase activity in the intermediate stage of growth (14 days of culture), which is characteristic for osteoblasts, was not affected, regardless of the presence of Ti or combination of CNT and Ti. Mineralized matrix nodules grew much more when the cells were incubated with CNT in the last 2 phases than when incubated in the first week, mainly when the cultures were grown on Ti discs. This study provides information for the application of CNT associated or not with Ti in processes of mineralization biostimulation.

  13. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN EWING'S SARCOMA

    PubMed Central

    Baptista, André Mathias; Zumárraga, Juan Pablo; dos Santos, Renan Pires Negrão; Haubert, Guilherme de Oliveira; de Camargo, Olavo Pires

    2016-01-01

    ABSTRACT Objective: To study the relationship between the serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) in patients with Ewing´s Sarcoma (ES). Methods: This is a case series with retrospective evaluation of patients with diagnosis of ES divided into 2 groups: Group 1, patients whose serum levels of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were obtained in the staging phase before preoperative chemotherapy (CT), and Group 2, patients whose values were measured after completion of the preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens extracted in surgery was also evaluated. Results: Eighty four medical records from 1995 to 2015 were included. Both AP as LDH decreased in the patients studied, the pre CT value being higher than the post CT value. The average decrease of LHD was 272.95 U/L and AP was 10.17 U/L. The average tumor necrosis was 65.12 %. There was no statistical correlation between serums levels and the tumor necrosis percentage. Conclusion: The serum levels values of AP and LDH are not predictors for chemotherapy-induced necrosis in patients with ES. Level of Evidence IV, Case Series. PMID:28243173

  14. [Changes of serum alkaline phosphatase and electrolytes during 21 d head down bed-rest].

    PubMed

    Yao, Yong-jie; Sun, Xi-qing; Wang, Zhong-bo; Zhao, Shuang-bao; Yang, Chang-bin; Wu, Xing-yu

    2002-06-01

    Objective. To investigate the effect of simulated weightlessness on serum alkaline phosphatase (ALP), calcium, magnesium, chlorine and phosphorus. Method. 6 healthy males, aged 24.8 +/- 6.1, were exposed to -6 degrees HDT bed rest for 21 d. Activity of serum alkaline phosphatase, serum contents of calcium (Ca), magnesium (Mg), chlorine (Cl) and phosphorus (P) ions were assayed before HDT (d-3), on the 3rd, 10th and 21st day during HDT and after HDT (d+2). Ca was measured by methyl thymol blue method, P was determined with ultraviolet spectrophotography, determination of Mg and Cl were made with enzyme method, ALP was examined with 4-nitrobenzene phosphate method. Result. Serum Ca2+ levels were significantly higher at d10, d2l and d+2 than the value of d-3 (P<0.01). P3+ levels declined significantly on d2l as compared with d-3 (P<0.01). During the HDT and after HDT, Mg2+ declined to a level below that before HDT (P<0.05 or P<0.01). Cl- were significantly higher at d2l and d+2 than the value of d-3 (P<0.01). ALP level was higher on d2l than on d-3 (P<0.01). Conclusion. 21 d HDT induced increase of Ca, Cl, ALP, and decline of Mg and P. The changes may reflect the imbalance of metabolism.

  15. Immobilization of Penaeus merguiensis alkaline phosphatase on gold nanorods for heavy metal detection.

    PubMed

    Homaei, Ahmad

    2017-02-01

    Biotechnology of enzyme has gained popularity due to the growing need for novel environmental technologies and the development of innovative mass-production. The work describes the original application of biosensors based on Penaeus merguiensis alkaline phosphatase (PM ALP) immobilized on gold nanorods (GNRs) to heavy metal determination. Penaeus merguiensis alkaline phosphatase (PM ALP) was immobilized on gold nanorods (GNRs) by ionic exchange and hydrophobic interactions. The optimum pH and temperature for maximum enzyme activity for the immobilized PM ALP are identified to be 11.0 and 60°C, respectively, for the hydrolysis of para-Nitrophenylphosphate (p-NPP). The kinetic studies confirm the Michaelis-Menten behavior and suggests overall slightly decrease in the performance of the immobilized enzyme with reference to the free enzyme. Km and Vmax values were 0.32µm and 54µm. min(-1) for free and 0.39µm and 48µmmin(-1) for immobilized enzymes, respectively. Similarly, the thermal stability, storage stability and stability at extreme pH of the enzyme is found to increase after the immobilization. The inhibitory effect heavy metal ions was studied on free and immobilized PM ALP. The bi-enzymatic biosensor were tested to study the influence of heavy metal ions and pesticides on the corresponding enzyme. The obtained high stability and lower decrease in catalytic efficiency suggested the great potential and feasibility of immobilized PM ALP nanobiocatalyst in efficient and apply the biosensor in total toxic metal content determination.

  16. Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase.

    PubMed

    Poe, R W; Sangadala, V S; Brewer, J M

    1993-05-15

    Seventeen salts were tested at various concentrations for their effects on E. coli alkaline phosphatase steady-state activity. Three effects were distinguished: a general ionic strength effect, and weaker cation and anion effects. 1. All salts tested, including those with "noninteracting" cations and anions, stimulate alkaline phosphatase activity usually ca. 100% at moderate (0.05-0.3 M) concentrations. 2. Cations such as Na+ and Li+ produce further increases in activity at concentrations up to 1 M. The noninteracting cations tetramethylammonium and tetrapropylammonium produce lower activities at these concentrations. These do not provide the secondary stimulatory effect of cations such as Na+ or Li+. 3. Anions associated with greater "salting in" effectiveness such as thiocyanate also reduce activity at ca. 1 M concentrations. These latter effects are not dependent on protein concentration so they probably do not involve subunit dissociation. There is little effect on the fluorescence or fluorescence-polarization spectrum of the enzyme so there is no general effect of 1 M salts on the conformation of the protein. The Michaelis constant for the substrate, p-nitrophenylphosphate, and inhibition constant for inorganic phosphate are increased to some extent by salts, but the increase in activity is due to an increase in Vmax. Our working hypothesis is that increased ionic strength weakens electrostatic interactions, enabling noncovalently bound phosphate to dissociate more rapidly.

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

    PubMed Central

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

    2016-01-01

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

  18. Flow screen-printed amperometric detection of p-nitrophenol in alkaline phosphatase-based assays.

    PubMed

    Fanjul-Bolado, Pablo; González-García, María Begoña; Costa-García, Agustín

    2006-08-01

    p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2x10(-8) mol L(-1), almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7x10(-14) mol L(-1) alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.

  19. Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

    PubMed

    Cano, R J; Torres, M J; Klem, R E; Palomares, J C; Casadesus, J

    1992-05-01

    This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.

  20. High Serum Alkaline Phosphatase, Hypercalcaemia, Race, and Mortality in South African Maintenance Haemodialysis Patients

    PubMed Central

    Duarte, Raquel; Naicker, Saraladevi

    2017-01-01

    Objective. To determine the association between serum total alkaline phosphatase (TAP) and mortality in African maintenance haemodialysis patients (MHD). Patients and Methods. The study enrolled a total of 213 patients on MHD from two dialysis centers in Johannesburg between January 2009 and March 2016. Patients were categorized into a low TAP group (≤112 U/L) versus a high TAP group (>112 U/L) based on a median TAP of 112 U/L. Results. During the follow-up period of 7 years, there were 55 (25.8%) deaths. After adjusting for cofounders such as age, other markers of bone disorder, and comorbidity (diabetes mellitus), patients in the high TAP group had significantly higher risk of death compared to patients in the low TAP group (hazard ratio, 2.50; 95% CI 1.24–5.01, P = 0.01). Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10–2.37 mmol/L (HR 6.34, 95% CI 1.40–28.76; P = 0.02). The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82–25.88; P = 0.004. Conclusion. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients. PMID:28168054

  1. High Serum Alkaline Phosphatase, Hypercalcaemia, Race, and Mortality in South African Maintenance Haemodialysis Patients.

    PubMed

    Waziri, Bala; Duarte, Raquel; Naicker, Saraladevi

    2017-01-01

    Objective. To determine the association between serum total alkaline phosphatase (TAP) and mortality in African maintenance haemodialysis patients (MHD). Patients and Methods. The study enrolled a total of 213 patients on MHD from two dialysis centers in Johannesburg between January 2009 and March 2016. Patients were categorized into a low TAP group (≤112 U/L) versus a high TAP group (>112 U/L) based on a median TAP of 112 U/L. Results. During the follow-up period of 7 years, there were 55 (25.8%) deaths. After adjusting for cofounders such as age, other markers of bone disorder, and comorbidity (diabetes mellitus), patients in the high TAP group had significantly higher risk of death compared to patients in the low TAP group (hazard ratio, 2.50; 95% CI 1.24-5.01, P = 0.01). Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10-2.37 mmol/L (HR 6.34, 95% CI 1.40-28.76; P = 0.02). The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82-25.88; P = 0.004. Conclusion. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients.

  2. 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.

  3. Acute decrease in alkaline phosphatase after brain injury: A potential mechanism for tauopathy.

    PubMed

    Arun, Peethambaran; Oguntayo, Samuel; Albert, Stephen Van; Gist, Irene; Wang, Ying; Nambiar, Madhusoodana P; Long, Joseph B

    2015-11-16

    Dephosphorylation of phosphorylated Tau (pTau) protein, which is essential for the preservation of neuronal microtubule assemblies and for protection against trauma-induced tauopathy and chronic traumatic encephalopathy (CTE), is primarily achieved in brain by tissue non-specific alkaline phosphatase (TNAP). Paired helical filaments (PHFs) and Tau isolated from Alzheimer's disease (AD) patients' brains have been shown to form microtubule assemblies with tubulin only after treatment with TNAP or protein phosphatase-2A, 2B and -1, suggesting that Tau protein in the PHFs of neurons in AD brain is hyperphosphorylated, which prevents microtubule assembly. Using blast or weight drop models of traumatic brain injury (TBI) in rats, we observed pTau accumulation in the brain as early as 6h post-injury and further accumulation which varied regionally by 24h post-injury. The pTau accumulation was accompanied by reduced TNAP expression and activity in these brain regions and a significantly decreased plasma total alkaline phosphatase activity after the weight drop. These results reveal that both blast- and impact acceleration-induced head injuries cause an acute decrease in the level/activity of TNAP in the brain, which potentially contributes to trauma-induced accumulation of pTau and the resultant tauopathy. The regional changes in the level/activity of TNAP or accumulation of pTau after these injuries did not correlate with the accumulation of amyloid precursor protein, suggesting that the basic mechanism underlying tauopathy in TBI might be distinct from that associated with AD.

  4. Influence of bone and soft-tissue operations on serum concentrations of growth hormone, somatomedin C and alkaline phosphatase.

    PubMed

    Casser, H R; Zilkens, K W; Forst, R; Brüggemann, A

    1990-01-01

    After animal experiments suggested there was an interaction between growth hormone and bone healing, our aim in this paper was to ascertain whether there were any changes or possible interaction between the serum level of growth hormone, somatomedin C and alkaline phosphatase while a fractured bone was healing. To this end, the serum concentrations of growth hormone, somatomedin C, alkaline phosphatase and calcium were ascertained both pre- and post-operatively in two groups of patients--one with bone operations, the other with soft-tissue operations--and the results were compared. Comparing the groups, we found that after bone operations there was no increase in the serum level of growth hormone, nor of somatomedin C. An increase would have implied that these two hormones are directly involved in bone regeneration. There was no change in the serum level of alkaline phosphatase or calcium after either bone or soft-tissue operations.

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

    SciTech Connect

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

    1988-09-01

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

  6. [Effect of different environmental factors on the activities of digestive enzymes and alkaline phosphatase of Macrobrochium nipponense].

    PubMed

    Wang, Weina; Sun, Ruyong; Wang, Anli; Bao, Lei; Wang, Peng

    2002-09-01

    The activities of digestive enzymes and alkaline phosphatase from the hepatopancreas of Macrobrochium nipponense were determined under different environmental factors (calcium concentrations 20 mg.L-1, 35 mg.L-1, 60 mg.L-1, 80 mg.L-1, 150 mg.L-1; salinity 7@1000, 14@1000, pH 7.6, 8.8, 9.8). The results showed that higher Ca2+ concentration could enhance the pepsin activity, but inhibit the trysin-like activity in hepatopancreas of M. nipponense. The activities of pepsin, trysin-like, alkaline phosphatase in hepatopancreas of M. nipponense were higher under salinity of 14@1000 than under salinity of 7@1000 and 20@1000. It showed that the activities of digestive enzymes and alkaline phosphatase of shrimp increased gradually with increasing pH value from 7.6 to 9.8.

  7. Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate.

    PubMed Central

    Rezende, A A; Pizauro, J M; Ciancaglini, P; Leone, F A

    1994-01-01

    Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in

  8. Fluoride stimulates ( sup 3 H)thymidine incorporation and alkaline phosphatase production by human osteoblasts

    SciTech Connect

    Khokher, M.A.; Dandona, P. )

    1990-11-01

    The effect of sodium fluoride on alkaline phosphatase (ALP) release and ({sup 3}H)thymidine uptake by human osteoblasts in culture was investigated. Sodium fluoride stimulated both ALP release and ({sup 3}H)thymidine uptake at concentrations of sodium fluoride greater than 250 mumol/L. This stimulation was similar in magnitude to that induced by 1,25-dihydroxycholecalciferol. The fluoride-induced increase in ALP was inhibited by verapamil, a calcium channel blocker. We conclude that sodium fluoride stimulates osteoblasts to proliferate and to release ALP. This stimulation by fluoride is dependent on calcium influx. Fluoride-induced stimulation of human osteoblasts may be relevant to its effect in enhancing bone formation in patients with osteoporosis.

  9. A disposable alkaline phosphatase-based biosensor for vanadium chronoamperometric determination.

    PubMed

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

    2014-02-24

    A chronoamperometric method for vanadium ion determination, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over 4-nitrophenyl phosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The developed method has a detection limit of 0.39 ± 0.06 µM, a repeatability of 7.7% (n = 4) and a reproducibility of 8% (n = 3). A study of the possible interferences shows that the presence of Mo(VI), Cr(III), Ca(II) and W(VI), may affect vanadium determination at concentration higher than 1.0 mM. The method was successfully applied to the determination of vanadium in spiked tap water.

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

    PubMed

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

    1989-01-01

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

  11. Quantum-Mechanical Study on the Catalytic Mechanism of Alkaline Phosphatases.

    PubMed

    Borosky, Gabriela L

    2017-02-21

    Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. The catalytic mechanism was examined by quantum-mechanical calculations using an active-site model based on the X-ray crystal structure of the human placental AP. Free energies of activation and of reaction for the catalytic steps were evaluated for a series of aryl and alkyl phosphate esters, and the computational results were compared with experimental values available in the literature. Mechanistic observations previously reported in experimental works were rationalized by the present theoretical study, particularly regarding the difference in the rate-determining step between aryl and alkyl phosphates. The formation rate of the covalent phosphoserine intermediate followed a linear free energy relationship (LFER) with the pKa of the leaving group. This LFER, which could be experimentally determined only for less reactive alkyl phosphates, was verified by the present calculations to apply for the entire set of aryl and alkyl phosphate substrates.

  12. Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner.

    PubMed

    Mikami, Yoshikazu; Tsuda, Hiromasa; Akiyama, Yuko; Honda, Masaki; Shimizu, Noriyoshi; Suzuki, Naoto; Komiyama, Kazuo

    2016-11-01

    Polyphosphate [Poly(P)] has positive effects on osteoblast mineralization; however, the underlying mechanism remains unclear. In addition, it is unknown whether Poly(P) promotes mineralization in soft tissues. We investigated this by using various cells. Poly(P) concentrations of 1 and 0.5 mg/mL yielded high levels of mineralization in ROS17/2.8 osteoblast cells. Similarly, Poly(P) induced mineralization in cell types expressing alkaline phosphatase (ALP), namely, ATDC5 and MC3T3-E1, but not in CHO, C3H10T1/2, C2C12, and 3T3-L1 cells. Furthermore, forced expression of ALP caused Poly(P)-induced mineralization in CHO cells. These results suggest that ALP determines Poly(P)-induced mineralization in a cell-type independent manner.

  13. Chronic Cadmium Exposure Lead to Inhibition of Serum and Hepatic Alkaline Phosphatase Activity in Wistar Rats.

    PubMed

    Treviño, Samuel; Andrade-García, Alejandra; Herrera Camacho, Irma; León-Chavez, Bertha Alicia; Aguilar-Alonso, Patricia; Flores, Gonzalo; Brambila, Eduardo

    2015-12-01

    Alkaline phosphatase (ALP) activity in the serum and liver from rats administered with cadmium (Cd) in drinking water was studied. After metal administration, Cd showed a time-dependent accumulation in the liver, meanwhile metallothionein had a maximum increase at 1 month, remaining in this level until the end of the study. On the other hand, serum and liver ALP activity was decreased after 3 months exposure. To determine if Cd produced an inhibition on enzyme, apo-ALP prepared from both nonexposed and exposed rats was reactivated with Zn, showing 60% more activity as compared with the enzyme isolated from nonexposed rats. In vitro assays showed that Cd-ALP was partially reactivated with Zn; however, in the presence of cadmium, Zn-ALP was completely inhibited. Kinetic studies indicate a noncompetitive inhibition by Cd; these results suggest that Cd can substitute Zn, and/or Cd can interact with nucleophilic ligands essential for the enzymatic activity.

  14. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

    PubMed

    Douglas, Timothy E L; Messersmith, Philip B; Chasan, Safak; Mikos, Antonios G; de Mulder, Eric L W; Dickson, Glenn; Schaubroeck, David; Balcaen, Lieve; Vanhaecke, Frank; Dubruel, Peter; Jansen, John A; Leeuwenburgh, Sander C G

    2012-08-01

    Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

  15. A smart fluorescence nanoprobe for the detection of cellular alkaline phosphatase activity and early osteogenic differentiation.

    PubMed

    Cao, Feng-Yi; Fan, Jin-Xuan; Long, Yue; Zeng, Xuan; Zhang, Xian-Zheng

    2016-07-01

    In the past decades, biomaterials were designed to induce stem cell toward osteogenic differentiation. However, conventional methods for evaluation osteogenic differentiation all required a process of cell fixation or lysis, which induce waste of a large number of cells. In this study, a fluorescence nanoprobe was synthesized by combining phosphorylated fluoresceinamine isomer I (FLA) on the surface of mesoporous silica-coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles. In the presence of alkaline phosphatase (ALP), the phosphorylated FLA on the nanoprobe would be hydrolyzed, resulting in a fluorescence recovery of FLA. During early osteogenic differentiation, a high-level expression of cellular ALP was induced, which accelerated the hydrolysis of phosphorylated FLA, resulting in an enhancement of cellular fluorescence intensity. This fluorescence nanoprobe provides us a rapid and non-toxic method for the detection of cellular ALP activity and early osteogenic differentiation.

  16. Fingerprint deposition on nitrocellulose and polyvinylidene difluoride membranes using alkaline phosphatase.

    PubMed

    Kurien, Biji T; Danda, Debashish; Scofield, R Hal

    2015-01-01

    Dactyloscopy or fingerprint identification is a vital part of forensic evidence. Identification with fingerprints has been known since the finding of finger impressions on the clay surface of Babylonian legal contracts almost 4,000 years ago. The skin on the fingers and palms appears as grooves and ridges when observed under a microscope. A unique fingerprint is produced by the patterns of these friction skin ridges. Visible fingerprints can be deposited on solid surfaces. Colored inks have been used to deposit fingermarks on documents. Herein, we show that alkaline phosphatase can be used to transfer prints from fingers or palm to nitrocellulose or polyvinylidene difluoride membranes. The prints can be detected by using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate method of detection.

  17. The Detection of Alkaline Phosphatase Using an Electrochemical Biosensor in a Single-Step Approach

    PubMed Central

    Wang, Joanne H.; Wang, Kevin; Bartling, Brandon; Liu, Chung-Chiun

    2009-01-01

    A one-step, single use, disposable Alkaline Phosphatase (ALP) biosensor has been developed. It is based on the detection of phenol produced by an ALP enzymatic reaction. It can operate at 25 °C in a pH 10 medium. It measures ALP of 0–300 IU/L. The permissible concentrations of glucose, ascorbic acid and urea without interference are 10 mM/L, 5 mg/L and 400 mg/L, respectively. Experimental results are compared to those obtained by spectrophotometric measurements in bovine serum. Excellent linearity between the biosensor outputs and the ALP concentrations exists. The agreement between the measurements of this biosensor and the spectrophotometer is also outstanding. PMID:22291532

  18. Alkaline phosphatase based amperometric biosensor immobilized by cysteamine-glutaraldehyde modified self-assembled monolayer.

    PubMed

    Yorganci, Emine; Akyilmaz, Erol

    2011-10-01

    Alkaline phosphatase (ALP) was immobilized with cross-linking agents glutaraldehyde and cysteamine by forming a self-assembled monolayer on a screen printed gold electrode. ALP converts p-nitrophenyl phosphate to p-nitrophenol and phosphate. p-Nitrophenol loses H(+) ion and turns into the negatively charged compound p-nitrophenolate at medium pH. As a result, the unstable product formed is measured chronoamperometrically at an application potential of + 0.95 V. The biosensor response depends linearly on p-nitrophenyl phosphate concentration between 0.05 - 0.6 mM with a response time of 40 seconds. Detection limit of the biosensor is 0.033 mM.

  19. Intestinal Alkaline Phosphatase Detoxifies Lipopolysaccharide and Prevents Inflammation in Response to the Gut Microbiota

    PubMed Central

    Bates, Jennifer M.; Akerlund, Janie; Mittge, Erika; Guillemin, Karen

    2009-01-01

    SUMMARY Vertebrates harbor abundant lipopolysaccharide (LPS) or endotoxin in their gut microbiota. Here we demonstrate that the brush border enzyme intestinal alkaline phosphatase (Iap), which dephosphorylates LPS, is induced during establishment of the microbiota and plays a crucial role in promoting mucosal tolerance to gut bacteria in zebrafish. We demonstrate that Iap deficient animals are hypersensitive to LPS toxicity through a mechanism mediated by Myd88 and Tumor Necrosis Factor Receptor (Tnfr). We further show that the endogenous microbiota establish the normal homeostatic level of neutrophils in the intestine through a process involving Myd88 and Tnfr. Iap deficient animals exhibit excessive intestinal neutrophil influx, similar to wild type animals exposed to LPS. When reared germ-free, however, the intestines of Iap deficient animals are devoid of neutrophils, demonstrating that Iap functions to prevent inflammatory responses to resident gut bacteria. PMID:18078689

  20. Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

    PubMed Central

    Vockley, J; Harris, H

    1984-01-01

    We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification. Images Fig. 2. PMID:6365087

  1. Phosphate monoester hydrolysis by trinuclear alkaline phosphatase; DFT study of transition States and reaction mechanism.

    PubMed

    Chen, Shi-Lu; Liao, Rong-Zhen

    2014-08-04

    Alkaline phosphatase (AP) is a trinuclear metalloenzyme that catalyzes the hydrolysis of a broad range of phosphate monoesters to form inorganic phosphate and alcohol (or phenol). In this paper, by using density functional theory with a model based on a crystal structure, the AP-catalyzed hydrolysis of phosphate monoesters is investigated by calculating two substrates, that is, methyl and p-nitrophenyl phosphates, which represent alkyl and aryl phosphates, respectively. The calculations confirm that the AP reaction employs a "ping-pong" mechanism involving two chemical displacement steps, that is, the displacement of the substrate leaving group by a Ser102 alkoxide and the hydrolysis of the phosphoseryl intermediate by a Zn2-bound hydroxide. Both displacement steps proceed via a concerted associative pathway no matter which substrate is used. Other mechanistic aspects are also studied. Comparison of our calculations with linear free energy relationships experiments shows good agreement.

  2. Activity of alkaline phosphatase in water-in-oil microemulsions containing vegetable oil.

    PubMed

    Gupta, S; Mukhopadhyay, L; Moulik, S P

    1995-10-01

    The hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase has been studied in vegetable oil containing water-in-oil (W/O) microemulsions of six different compositions at four different (water)/(surfactant) mole ratios of 10, 17.6, 24.7 and 37. The vegetable oils used are ricebran oil (RO) and clove oil (CO) and the amphiphiles used are Aerosol OT (AOT), cinnamic alcohol (CA) and Tween-20 (T-20). The hydrolytic process does not follow conventional Michaelis Menten equation normally observed for enzymatic process. In the water/vegetable oil microemulsions, the enzyme seems to lose its activity when AOT is the amphiphile. The amount of p-nitrophenol generated as a result of hydrolysis is independent of the presence of the enzyme. With Tween-20 as the amphiphile, the microemulsion produces an initial retarding effect which ultimately gets appreciably compensated.

  3. Canine serum thermostable alkaline phosphatase isoenzyme from a dog with hepatocellular carcinoma.

    PubMed

    Fukui, Yu-ichi; Sato, Jun; Sato, Reeko; Yasuda, Jun; Naito, Yoshihisa

    2006-10-01

    A dog histopathologically diagnosed with hepatocellular carcinoma (HCC) showed very high serum alkaline phosphatase (ALP) activity. A supernatant of ascitic fluid and tumor tissue extracted from the dog also showed much higher ALP activity than normal. ALP isoenzyme analysis of samples was performed using polyacrylamide gel disk electrophoresis, and a wide, broad abnormal band was observed. By various treatments, the abnormal band showed thermostability, which is a characteristic of tumor-associated ALP that has only been reported in humans. The thermostable ALP isoenzyme was not found in sera from 39 dogs with several types of tumor that originated from the liver, except for HCC, nor was it found in 10 dogs with hepatic diseases that did not include hepatic tumors. The thermostable ALP isoenzyme seemed to be associated with canine HCC.

  4. Spatial variability of dissolved phosphorous concentrations and alkaline phosphatase activity in the East China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Chang, J.; Ho, T.; Gong, G.

    2010-12-01

    The concentrations of dissolved inorganic phosphorus (DIP) and alkaline phosphatase activity (APA) have been determined at about 25 sampling stations in the East China Sea since 2003. The stations are mainly distributed from the Changjiang river mouth to northern Taiwan and east to the shelf break. In addition to the Changjiang discharge, we have found a specific nutrient source around a coastal site (122° 2’30’’ E, 28° 40’ N). Elevated DIP and nitrate concentrations have been constantly observed around the sampling station for 8 years, where the surface DIP concentrations are generally around 0.3 µM. The nutrient source may either originate from ground water discharge or coastal upwelling, where lower temperature has been observed in the water column around the station. In general, APA has been negatively correlated with DIP concentrations in the studies sites, with lowest APA around the high DIP station and the Changjiang river mouth.

  5. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase

    PubMed Central

    Douglas, Timothy E.L.; Messersmith, Philip B.; Chasan, Safak; Mikos, Antonios G.; de Mulder, Eric L.W.; Dickson, Glenn; Schaubroeck, David; Balcaen, Lieve; Vanhaecke, Frank; Dubruel, Peter; Jansen, John A.

    2013-01-01

    Alkaline Phosphatase (ALP), an enzyme involved in mineralization of bone, was incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These were collagen type I, a mussel protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG-fumaric acid copolymer OPF. After incubation in calcium glycerophosphate (Ca-GP) solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES and von Kossa staining confirmed CaP formation. The amount of mineral formed decreased in the order cPEG > collagen > OPF. Mineral:polymer ratio decreased in the order collagen > cPEG > OPF. Mineralization increased Young’s modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel-CaP composites could find application as bone regeneration materials. PMID:22648976

  6. A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination

    PubMed Central

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

    2014-01-01

    A chronoamperometric method for vanadium ion determination, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over 4-nitrophenyl phosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The developed method has a detection limit of 0.39 ± 0.06 μM, a repeatability of 7.7% (n = 4) and a reproducibility of 8% (n = 3). A study of the possible interferences shows that the presence of Mo(VI), Cr(III), Ca(II) and W(VI), may affect vanadium determination at concentration higher than 1.0 mM. The method was successfully applied to the determination of vanadium in spiked tap water. PMID:24569772

  7. Trypanosoma rangeli: an alkaline ecto-phosphatase activity is involved with survival and growth of the parasite.

    PubMed

    Dos-Santos, André L A; Dick, Claudia F; Silveira, Thaís S; Fonseca-de-Souza, André L; Meyer-Fernandes, José R

    2013-10-01

    The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when β-GP was the sole source of Pi and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

  8. β₂ adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

  9. 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…

  10. Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily

    PubMed Central

    2015-01-01

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design. PMID:26091851

  11. Reduced activity of alkaline phosphatase due to host-guest interactions with humic superstructures.

    PubMed

    Mazzei, Pierluigi; Oschkinat, Hartmut; Piccolo, Alessandro

    2013-11-01

    Nuclear Magnetic Resonance (NMR) spectroscopy was applied to directly study the interactions between the alkaline phosphatase enzyme (AP) and two different humic acids from a volcanic soil (HA-V) and a Lignite deposit (HA-L). Addition of humic matter to enzyme solutions caused signals broadening in (1)H-NMR spectra, and progressive decrease and increase of enzyme relaxation (T1 and T2) and correlation (τC) times, respectively. Spectroscopic changes were explained with formation of ever larger weakly-bound humic-enzyme complexes, whose translational and rotational motion was increasingly restricted. NMR diffusion experiments also showed that the AP diffusive properties were progressively reduced with formation of large humic-enzyme complexes. The more hydrophobic HA-L affected spectral changes more than the more hydrophilic HA-V. (1)H-NMR spectra also showed the effect of progressively greater humic-enzyme complexes on the hydrolysis of an enzyme substrate, the 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP). While AP catalysis concomitantly decreased NMR signals of p-NPP and increased those of nitrophenol, addition of humic matter progressively and significantly slowed down the rate of change for these signals. In agreement with the observed spectral changes, the AP catalytic activity was more largely inhibited by HA-L than by HA-V. Contrary to previous studies, in which humic-enzyme interactions were only indirectly assumed from changes in spectrophotometric behavior of enzyme substrates, the direct measurements of AP behavior by NMR spectroscopy indicated that humic materials formed weakly-bound host-guest complexes with alkaline phosphatase, and the enzyme catalytic activity was thereby significantly inhibited. These results suggest that the role of extracellular enzymes in soils may be considerably reduced when they come in contact with organic matter dissolved in the soil solution.

  12. Comparative Analysis of Salivary Alkaline Phosphatase in Post menopausal Women with and without Periodontitis

    PubMed Central

    Sophia, Khumukcham; Sudhakar, Uma; Jayakumar, Parvathee; Mathew, Danny

    2017-01-01

    Introduction Alkaline phosphatase is an intracellular destruction enzyme in the periodontium, and it takes part in the normal turnover of the periodontal ligament, alveolar bone, and root cementum formation and maintenance. Aim The aim of this case control study was to evaluate the enzyme Alkaline Phosphatase (ALP) level in saliva of post menopausal women with and without chronic periodontitis. Materials and Methods In this study, 40 individuals, satisfying the study inclusion and exclusion criteria, were recruited. They were categorically divided, on the basis of gingival index, probing pocket depth and clinical attachment level, into two groups: Group I (post menopausal women with a clinically healthy periodontium, n=20); and Group II (post menopausal women with generalized chronic periodontitis, n=20). Clinical parameters assessed were Plaque Index (PI), Gingival Index (GI), Clinical Attachment Level (CAL) and Probing Pocket Depth (PPD). Unstimulated salivary samples were obtained in which the ALP concentration was measured using p-Nitrophenylphosphate, and 2-amino-2-methyl-1-propanol reagents in Beckman and Coulter, AU 480 auto analyser. Mann-Whitney U test was used to find statistical difference with respect to all clinical parameters such as PI, GI, CAL, PPD and salivary ALP levels. Results The mean ALP in saliva was found to be higher in Group II compared to Group I and the difference was statistically significant with the p-value of 0.008. Conclusion A noteworthy increase in the ALP concentration was seen in saliva in our study (Group II) may be due to increased periodontal inflammation in post menopausal women. Thus salivary ALP can be taken as an additional biomarker to early diagnosis, development and progression of periodontitis especially among post menopausal women. PMID:28274061

  13. Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

    PubMed Central

    Hung, H C; Chang, G G

    2001-01-01

    Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally

  14. Alkaline phosphatase activity in the western English Channel: Elevations induced by high summertime rainfall

    NASA Astrophysics Data System (ADS)

    Rees, Andrew P.; Hope, Sam B.; Widdicombe, Claire E.; Dixon, Joanna L.; Woodward, E. Malcolm S.; Fitzsimons, Mark F.

    2009-03-01

    Alkaline phosphatase activity (APA) was determined in bulk particulate material and in a single-cell (ELF) assay at station L4 in the western English Channel during the summer of 2007. Throughout this period, the UK experienced its heaviest summertime rainfall since records began in 1914; with the result that riverine run-off into coastal waters was also elevated relative to long-term averages. Between May and August 2007, three distinct periods of elevated river run-off were observed which resulted in salinity minima at L4 on days 141, 190 and 232. An extended period of high river run-off between days 170 and 210 was responsible for decreases in near-surface salinity at L4 from 35.2068 to a minimum on day 190 of 34.7422. This contributed to the development of haline stratification which supported the development of an intense bloom of the centric diatom Chaetoceros debelis, with maximum observed chlorophyll a concentration of 8.69 μg l -1. Minima in salinity, and maxima in chlorophyll concentration on day 190 were coincident with a peak in river-derived dissolved inorganic nitrogen (DIN) of 1.9 μmol l -1 which was >5 times greater than the summertime mean and 24 times the concentrations experienced at L4 on weeks immediately before and after. There was no accompanying increase in dissolved inorganic phosphorus (DIP), and the DIN:DIP ratio increased to 49. With the inherent phosphorus stress that this caused, rates of APA increased from <4 to 42.4 nmolP l -1 h -1. ELF analysis on day 197 identified two taxa actively expressing alkaline phosphatase: the dinoflagellate Prorocentrum micans and ciliate Tiarana sp.

  15. Multiple unfolding intermediates of human placental alkaline phosphatase in equilibrium urea denaturation.

    PubMed

    Hung, H C; Chang, G G

    2001-12-01

    Alkaline phosphatase is an enzyme with a typical alpha/beta hydrolase fold. The conformational stability of the human placental alkaline phosphatase was examined with the chemical denaturant urea. The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme. None of these unfolding intermediates were observed in the presence of 83 mM NaCl, indicating the importance of ionic interactions in the stabilization of the unfolding intermediates. Guanidinium chloride, on the other hand, could stabilize one of the unfolding intermediates, which is not a salt effect. Some of the unfolding intermediates were also observed in circular dichroism spectroscopy, which clearly indicates steady loss of helical structure during unfolding, but very little change was observed for the beta strand content until the late stage of the unfolding process. The enzyme does not lose its phosphate-binding ability after substantial tertiary structure changes, suggesting that the substrate-binding region is more resistant to chemical denaturant than the other structural domains. Global analysis of the fluorescence spectral change demonstrated the following folding-unfolding process of the enzyme: N <--> I(1) <--> I(2) <--> I(3) <--> I(4) <--> I(5) <--> D. These discrete intermediates are stable at urea concentrations of 2.6, 4.1, 4.7, 5.5, 6.6, and 7.7 M, respectively. These intermediates are further characterized by acrylamide and/or potassium iodide quenching of the intrinsic fluorescence of the enzyme and by the hydrophobic probes, 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The stepwise unfolding process was interpreted by the folding energy landscape in terms of the unique structure of the enzyme. The rigid central beta-strand domain is surrounded by the peripheral alpha-helical and coil structures, which are marginally

  16. Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors.

    PubMed

    Jornet-Martínez, N; Campíns-Falcó, P; Hall, E A H

    2016-12-15

    A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L - compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at -20°C, so the zein film can preserve and deliver both the enzyme and substrates.

  17. Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

    PubMed

    Chung, Thomas D Y

    2013-01-01

    Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

  18. Changes in Bone Alkaline Phosphatase and Procollagen Type-1 C-Peptide after Static and Dynamic Exercises

    ERIC Educational Resources Information Center

    Kubo, Keitaro; Yuki, Kazuhito; Ikebukuro, Toshihiro

    2012-01-01

    We investigated the effects of two types of nonweight-bearing exercise on changes in bone-specific alkaline phosphatase (BAP) and pro-collagen type 1 C-peptide (P1P). BAP is a specific marker of bone synthesis, whereas P1P reflects synthesis of type 1 collagen in other organs as well as bone. Eight participants performed static and dynamic…

  19. On the influence of reaction conditions in activity determination of alkaline phosphatase on the molar absorptivity of 4-nitrophenol.

    PubMed

    Jung, K; Köhler, A

    1980-02-14

    In activity determination of alkaline phosphatase (AP), measuring temperature, type and concentration of buffer, and protein concentration in the test influence the molar absorptivity of 4-nitrophenol. Thus systematic errors of up to 3% may occur in activity determinations of AP if these influences are not taken into account.

  20. Cloning & Characterization of the Cry1Ac-binding Alkaline Phosphatase (HvALP) from Heliothis virescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Membrane bound alkaline phosphatases (mALPs) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac binding protein that is d...

  1. Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems.

    PubMed

    Pandey, S K; Banik, R M

    2011-03-01

    A study was made to find out maximum partitioning of Bacillus licheniformis alkaline phosphatase in different ATPSs composed of different molecular weight of PEG X (X=2000, 4000, 6000) with salts (magnesium sulphate, sodium sulphate, sodium citrate) and polymers (dextran 40, dextran T500). Physicochemical factors such as effect of system pH, system temperature and production media were evaluated for partitioning of alkaline phosphatase. PEG 4000 [9.0% (w/v)] and dextran T500 [9.6% (w/v)] were selected as most suitable system components for alkaline phosphatase production by B. licheniformis based on greater partition coefficient (k=5.23). The two-phase system produced fewer enzymes than the homogeneous fermentation (control) in early stage of fermentation, but after 72 h the enzyme produced in the control system was less than that in the ATPS. Total alkaline phosphatase yield in ATPS fermentation was 3907.01 U/ml and in homogeneous fermentation 2856.50 U/ml.

  2. Establishment of hybridomas secreting monoclonal antibodies to placental alkaline phosphatase and development of an enzyme immunoassay for its determination.

    PubMed

    Kinoshita, Y; Okamoto, T; Mano, H; Furuhashi, Y; Goto, S; Tomoda, Y

    1990-06-01

    We established seven hybridomas secreting murine IgG monoclonal antibodies (MoAbs) to placental alkaline phosphatase (PLAP). The seven hybridomas were designated (1) 7C6, (2) 6G10, (3) 5B9, (4) 6D5, (5) 6B5, (6) 11G6 and (7) 3E10, respectively. The characteristics of these hybridomas were evaluated by radioimmunoassay (RIA) with 125I-PLAP. Their reactivity with the intestinal alkaline phosphatase, one of the alkaline phosphatase isozymes, was (1) 0.04, (2) 0.2, (3) 1.4, (4) 1.8, (5) 0, (6) 4.0 and (7) 6.2(%), respectively. None of them showed signs of cross-reactivity with the liver-type alkaline phosphatase, also one of the alkaline phosphatase isozymes, within a PLAP concentration of 2,000 IU/l. The subtype of 5B9 was IgG1, and that of the others was IgG2a. We then used 7C6, to develop a sensitive, specific and convenient enzyme immunoassay (EIA) for the determination of PLAP, and assayed sera from patients with various gynecologic diseases. The incidence of increased PLAP was 6.4% in patients with benign diseases, 21.5% in cervical cancer, 36.4% in endometrial carcinoma, and 39.5% in malignant ovarian tumors. The specificity for malignant diseases seemed to be higher than that of CA125. Among endometrial carcinomas, well-differentiated adenocarcinoma had the highest incidence of an increased concentration. Among malignant ovarian tumors, serous cystadenocarcinoma, endometrioid carcinoma, dysgerminoma and Krukenberg's tumor showed a higher incidence than the other types.

  3. Biological Apatite Formed from Polyphosphate and Alkaline Phosphatase May Exchange Oxygen Isotopes from Water through Carbonate

    NASA Astrophysics Data System (ADS)

    Omelon, S. J.; Stanley, S. Y.; Gorelikov, I.; Matsuura, N.

    2011-12-01

    The oxygen isotopic composition in bone mineral phosphate is known to reflect the local water composition, environmental humidity, and diet1. Once ingested, biochemical processes presumably equilibrate PO43- with "body water" by the many biochemical reactions involving PO43- 2. Blake et al. demonstrated that enzymatic release of PO43- from organophosphorus compounds, and microbial metabolism of dissolved orthophosphate, significantly exchange the oxygen in precipitated apatite within environmental water3,4, which otherwise does not exchange with water at low temperatures. One of the enzymes that can cleave phosphates from organic substrates is alkaline phosphastase5, the enzyme also associated with bone mineralization. The literature often states that the mineral in bone in hydroxylapatite, however the mineral in bone is carbonated apatite that also contains some fluoride6. Deprotonation of HPO32- occurs at pH 12, which is impossibly high for biological system, and the predominate carbonate species in solution at neutral pH is HCO3-. To produce an apatite mineral without a significant hydroxyl content, it is possible that apatite biomineralization occurs through a polyphosphate pathway, where the oxygen atom required to transform polyphosphate into individual phosphate ions is from carbonate: [PO3-]n + CO32- -> [PO3-]n-1 + PO43- + CO2. Alkaline phosphatase can depolymerise polyphosphate into orthophosphate5. If alkaline phosphatase cleaves an oxygen atom from a calcium-carbonate complex, then there is no requirement for removing a hydrogen atom from the HCO3- or HPO43- ions of body water to form bioapatite. A mix of 1 mL of 1 M calcium polyphosphate hydogel, or nano-particles of calcium polyphosphate, and amorphous calcium carbonate were reacted with alkaline phosphatase, and maintained at neutral to basic pH. After two weeks, carbonated apatite and other calcium phosphate minerals were identified by powder x-ray diffraction. Orthophosphate and unreacted

  4. The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum.

    PubMed Central

    Bailyes, E M; Seabrook, R N; Calvin, J; Maguire, G A; Price, C P; Siddle, K; Luzio, J P

    1987-01-01

    1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the

  5. 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

  6. Reaction-Based Off-On Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice.

    PubMed

    Tan, Yi; Zhang, Ling; Man, Ka Ho; Peltier, Raoul; Chen, Ganchao; Zhang, Huatang; Zhou, Liyi; Wang, Feng; Ho, Derek; Yao, Shao Q; Hu, Yi; Sun, Hongyan

    2017-03-01

    Alkaline phosphatases are a group of enzymes that play important roles in regulating diverse cellular functions and disease pathogenesis. Hence, developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based near-infrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed an up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell-internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.

  7. Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593

    SciTech Connect

    Arai, Shigeki; Yonezawa, Yasushi; Ishibashi, Matsujiro; Matsumoto, Fumiko; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2014-03-01

    In order to clarify the structural basis of the halophilic characteristics of an alkaline phosphatase derived from the moderate halophile Halomonas sp. 593 (HaAP), the tertiary structure of HaAP was determined to 2.1 Å resolution by X-ray crystallography. The structural properties of surface negative charge and core hydrophobicity were shown to be intermediate between those characteristic of halophiles and non-halophiles, and may explain the unique functional adaptation to a wide range of salt concentrations. Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1–4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Å resolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded β-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique ‘crown’ domain containing an extended ‘arm’ structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C{sup α} r.m.s.d. of 0.82 Å for the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior

  8. Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea.

    PubMed

    Poirier, T P; Holt, S C

    1983-10-01

    Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS-PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AlP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

  9. Cloning and Overexpression of Alkaline Phosphatase PhoK from Sphingomonas sp. Strain BSAR-1 for Bioprecipitation of Uranium from Alkaline Solutions▿

    PubMed Central

    Nilgiriwala, Kayzad S.; Alahari, Anuradha; Rao, Amara Sambasiva; Apte, Shree Kumar

    2008-01-01

    Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn5 random mutagenesis. The corresponding gene, designated phoK, was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 ± 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in <2 h compared to only 1.5 g U/g dry weight in >7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions. PMID:18641147

  10. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

  11. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  12. Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

    PubMed

    Miao, Dengshun; Scutt, Andrew

    2002-03-01

    We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.

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

    PubMed

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

    2016-02-01

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

  14. Conjugated polyelectrolyte-based real-time fluorescence assay for alkaline phosphatase with pyrophosphate as substrate.

    PubMed

    Liu, Yan; Schanze, Kirk S

    2008-11-15

    The fluorescence of the anionic, carboxylate-substituted poly(phenylene ethynylene) polymer PPECO2 is quenched very efficiently via the addition of 1 equiv of Cu(2+). Addition of pyrophosphate (PPi) into the weakly fluorescent solution of PPECO2 and Cu(2+) induces recovery of the polymer's fluorescence; the recovery occurs because PPi complexes with Cu(2+), effectively sequestering the ion so it cannot bind to the carboxylate groups of the polymer. A calibration curve was developed that relates the extent of fluorescence recovery to [PPi], making the PPECO2-Cu(2+) system a sensitive and selective turn-on sensor for PPi. Using the PPECO2-Cu(2+) system as the signal transducer, a real-time fluorescence turn-off assay for the enzyme alkaline phosphatase (ALP) using PPi as the substrate is developed. The assay operates with [PPi] in the micromolar range, and it offers a straightforward and rapid detection of ALP activity with the enzyme present in the nanomolar concentration range, operating either in an end point or real-time format. Kinetic and product inhibition parameters are derived by converting time-dependent fluorescence intensity into PPi (substrate) concentration, thus allowing calculation of the initial reaction rates (v(o)). Weak, nonspecific fluorescence responses are observed concomitant to addition of other proteins to the assay solution; however, the signal response to ALP is demonstrated to arise from the ALP catalyzed hydrolysis of PPi to phosphate (Pi).

  15. Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability.

    PubMed

    Sekiguchi, Satoshi; Yasukawa, Kiyoshi; Inouye, Kuniyo

    2011-01-01

    In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 µM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.

  16. Screen-printed microsystems for the ultrasensitive electrochemical detection of alkaline phosphatase.

    PubMed

    Santiago, Luz M; Bejarano-Nosas, Diego; Lozano-Sanchez, Pablo; Katakis, Ioanis

    2010-06-01

    Screen printing technique has been used to manufacture a microsystem where the graphite-based electrodes hold both a functional and an architectural task. The thick film manufacturing technique has proved valid to develop a very low volume (ca. 20 microL) device where different electrochemical operations can be very efficiently performed. Biomolecule immobilisation within the microsystem for biosensors applications has been explored by inducing and optimizing the in situ generation of a potential pulse polypyrrole electropolymerised film entrapping either glucose oxidase or glucose dehydrogenase. This biomodified microsystem was applied to the ultrasensitive electrochemical detection of alkaline phosphatase yielding limits of detection below 10(-12) M for glucose oxidase and of 10(-15) M for glucose dehydrogenase modified systems, within 15 min of incubation time. The results obtained showed the advantages of using low volume microsystems in combination with an optimised polypyrrole-enzyme film, which displayed a good immobilisation efficiency in conjunction with a good diffusion of species through. Ultrasensitive detection of AP in combination with a stable and reproducible surface modification for entrapping of biomolecules opens the window for new electrochemical detection platform with great potential for integrated biosensor applications.

  17. Lectin affinity electrophoresis of serum alkaline phosphatase in metastasized breast cancer.

    PubMed

    Le Bricon, Thierry; Gay-Bellile, Cécile; Cottu, Paul; Benlakehal, Mourad; Guillon, Hélène; Houzé, Pascal

    2010-01-01

    The use of serum alkaline phosphatase (ALP) isoenzymes as markers of breast cancer metastases and treatment efficacy has received little attention. Twenty-six breast cancer women (56+/-13 years, all post-menopausal) were prospectively evaluated during their first and third course of chemotherapy (4-week interval). Serum samples were analyzed for ALP isoenzymes (bone, liver, and intestine) using a lectin affinity electrophoresis kit (Hydragel 15 ISO-PAL, Sebia) adapted on a semi-automated Hydrasys system (Sebia). Results were compared with imaging techniques for the presence of metastases; bone ALP isoenzyme (B-ALP) results were compared with C-Terminal degradation products of type I collagen (S-CTX) (CrossLaps, IDS Nordic). Serum B-ALP, but not S-CTX, confirmed the presence of bone metastases (BM) (n=15) with 67/100% sensitivity/specificity (using a 69 UI/L ROC cut-off); ROC AUC was 0.806 (P=0.0004) (NS for S-CTX). Chemotherapy reduced serum B-ALP by 24% over 4 weeks (P=0.0012); there was no change for S-CTX. There was no specific clinical pattern for other ALP isoenzymes (liver and intestine). In conclusion, serum B-ALP, but not S-CTX, could help confirm the presence of BM in breast cancer patients.

  18. Acceleration of gelation and promotion of mineralization of chitosan hydrogels by alkaline phosphatase.

    PubMed

    Douglas, Timothy E L; Skwarczynska, Agata; Modrzejewska, Zofia; Balcaen, Lieve; Schaubroeck, David; Lycke, Sylvia; Vanhaecke, Frank; Vandenabeele, Peter; Dubruel, Peter; Jansen, John A; Leeuwenburgh, Sander C G

    2013-05-01

    Thermosensitive chitosan hydrogels containing sodium beta-glycerophosphate (β-GP), whose gelation is induced by increasing temperature to body temperature, were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone. ALP incorporation led to acceleration of gelation upon increase of temperature for four different chitosan preparations of differing molecular weight, as demonstrated by rheometric time sweeps at 37 °C. Hydrogels containing ALP were subsequently incubated in calcium glycerophosphate (Ca-GP) solution to induce their mineralization with calcium phosphate (CaP) in order to improve their suitability as materials for bone replacement. Incorporated ALP retained its bioactivity and induced formation of CaP mineral, as confirmed by SEM, FTIR, Raman spectroscopy, XRD, ICP-OES, and increases in dry mass percentage, which rose with increasing ALP concentration and incubation time in Ca-GP solution. The results demonstrate that ALP accelerates formation of thermosensitive chitosan/β-GP hydrogels and induces their mineralization with CaP, which paves the way for applications as injectable bone replacement materials.

  19. Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase.

    PubMed

    Beloti, Marcio Mateus; de Oliveira, Paulo Tambasco; Tagliani, Marcela Martini; Rosa, Adalberto Luiz

    2008-02-01

    The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus communis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP+ALP was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBF. ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP.

  20. Enrichment of thermosensitive chitosan hydrogels with glycerol and alkaline phosphatase for bone tissue engineering applications.

    PubMed

    Douglas, Timothy E L; Krok-Borkowicz, Małgorzata; Macuda, Aleksandra; Pietryga, Krzysztof; Pamuła, Elżbieta

    2016-01-01

    Thermosensitive injectable chitosan hydrogels can be formed by neutralization of acidic chitosan solutions with sodium betaglycerophosphate (Na-β-GP) coupled with increasing temperature to body temperature. Such hydrogels have been considered for applications in bone regeneration. In this study, chitosan hydrogels were enriched with glycerol and the enzyme alkaline phosphatase (ALP) with a view to improving their suitability as materials for bone tissue engineering. Mineral formation was confirmed by infrared spectroscopy (FTIR) and increases in the mass fraction of the hydrogel not consisting of water. Incorporation of ALP in hydrogels followed by incubation in a solution containing calcium ions and glycerophosphate, a substrate for ALP, led to formation of calcium phosphate within the hydrogel. MG-63 osteoblast-like cells were cultivated in eluates from hydrogels containing ALP and without ALP at different dilutions and directly on the hydrogel samples. Hydrogels containing ALP exhibited superior cytocompatibility to ALP-free hydrogels. These results pave the way for the use of glycerol- and ALP-enriched hydrogels in bone regeneration.

  1. Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.

    PubMed

    Schmidt, Yvonne; Biniossek, Martin; Stark, G Björn; Finkenzeller, Günter; Simunovic, Filip

    2015-03-01

    Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications.

  2. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro.

    PubMed

    Sabokbar, A; Millett, P J; Myer, B; Rushton, N

    1994-10-01

    Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

  3. Purification and properties of molecular-weight variants of human placental alkaline phosphatase

    PubMed Central

    Ghosh, Nimai K.; Fishman, William H.

    1968-01-01

    1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8·6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4·2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined. ImagesFig. 3.Fig. 5.Fig. 7.Fig. 8.Fig. 9. PMID:4970595

  4. Intestinal Alkaline Phosphatase Prevents Antibiotic-Induced Susceptibility to Enteric Pathogens

    PubMed Central

    Alam, Sayeda Nasrin; Yammine, Halim; Moaven, Omeed; Ahmed, Rizwan; Moss, Angela K.; Biswas, Brishti; Muhammad, Nur; Biswas, Rakesh; Raychowdhury, Atri; Kaliannan, Kanakaraju; Ghosh, Sathi; Ray, Madhury; Hamarneh, Sulaiman; Barua, Soumik; Malo, Nondita S.; Bhan, Atul K.; Malo, Madhu S.; Hodin, Richard A.

    2013-01-01

    Objective To determine the efficacy of oral supplementation of the gut enzyme intestinal alkaline phosphatase (IAP) in preventing antibiotic-associated infections from Salmonella enterica serovar Typhimurium (S. Typhimurium) and Clostridium difficile. Summary background data The intestinal microbiota plays a pivotal role in human health and well-being. Antibiotics inherently cause dysbiosis, an imbalance in the number and composition of intestinal commensal bacteria, which leads to susceptibility to opportunistic bacterial infections. Previously, we have shown that IAP preserves the normal homeostasis of intestinal microbiota and that oral supplementation with calf IAP (cIAP) rapidly restores the normal gut flora. We hypothesized that oral IAP supplementation would protect against antibiotic-associated bacterial infections. Methods C57BL/6 mice were treated with antibiotic(s) +/− cIAP in the drinking water followed by oral gavage of S. Typhimurium or C. difficile. Mice were observed for clinical conditions and mortality. After a defined period of time mice were sacrificed and investigated for hematological, inflammatory and histological changes. Results We observed that oral supplementation with cIAP during antibiotic treatment protects mice from infections with S. Typhimurium as well as C. difficile. Animals given IAP maintained their weight, had reduced clinical severity and gut inflammation, and improved survival. Conclusion Oral IAP supplementation protected mice from antibiotic-associated bacterial infections. We postulate that oral IAP supplementation could represent a novel therapy to protect against antibiotic-associated diarrhea (AAD), C. difficile-associated disease (CDAD), and other enteric infections in humans. PMID:23598380

  5. Effects of macromolecular crowding on alkaline phosphatase unfolding, conformation and stability.

    PubMed

    Jia, Jiajia; Peng, Xin; Qi, Wei; Su, Rongxin; He, Zhimin

    2017-03-23

    The interior of the cell is tightly packed with various biological macromolecules, which affects physiological processes, especially protein folding process. To explore how macromolecular crowding may influence protein folding process, alkaline phosphatase (ALP) was chosen as a model protein, and the unfolding process of ALP induced by GdnHCl was studied in the presence of crowding agents such as PEG 4000, Dextran 70 and Ficoll 70. The effect of macromolecular crowding on the denatured state of ALP was directly probed by measuring enzyme activities, fluorescence spectroscopy and circular dichroism. From the results of circular dichroism, GdnHCl induced a biphasic change, suggesting that a three-state unfolding mechanism was involved in the denaturation process irrespective of the absence or presence of crowding agents. It was also found that crowding agents had a little impact on the unfolding process of ALP. The results of phase diagrams also demonstrated that the unfolding process of ALP induced by GdnHCl was three-state mechanism. Moreover, the results of fluorescence spectra demonstrated that with the increase of GdnHCl concentration, the structure of protein had changed, but existence of crowding agents can make protein structure more stable. Our results can provide valuable information for understanding the protein folding in vivo.

  6. Polyacrylamide gel disc electrophoresis of alkaline phosphatase isoenzymes in bone and liver disease.

    PubMed Central

    Warnes, T W; Hine, P; Kay, G

    1976-01-01

    Acrylamide gel disc electrophoresis provides a reliable and reasonably rapid method of differentiating the raised serum alkaline phosphatase (AP) of bone origin from that of liver origin. The technique has been placed for the first time on a semiquantitative basis. Measurement of both band width and band position effectively distinguishes the bone from the liver isoenzyme, but band width provides superior discrimination. An origin band was seen in none of the normal subjects and in only 7% of patients with bone disease but was present in 78% of patients with liver disease, a highly significant increase. Fifty percent of normal individuals had a small-intestinal band in serum taken two hours after a meal, as did 35% of patients with liver disease, but the incidence of intestinal bands in bone disease was only 11%, significantly less than in the other two groups. The genetic control of small-intestinal AP in serum has been confirmed, but it has been demonstrated that the decrease of intestinal AP in bone disorders is not genetically determined. Images PMID:977779

  7. Sensitive terbium probes for luminescent determination of both alkaline phosphatase and codeine phosphate.

    PubMed

    Duerkop, Axel; Aleksandrova, Darya; Scripinets, Yuliya; Yegorova, Alla; Vityukova, Ekateryna

    2008-01-01

    New terbium complexes of 2-oxo-4-hydroxy-quinoline-3-carboxylic acid derivatives are reported for determination of alkaline phosphatase (aPase) and codeine phosphate (CP). Complexation with terbium occurs by way of the oxygen atoms of the 4-hydroxyl group and the carbonyl group of the 3-carboxy function, respectively. The complexes are highly luminescent and do not require luminescence enhancers. The excitation maxima of the terbium chelates are at 320 nm. The assay is based on the quenching of the hypersensitive 545-nm luminescence of the terbium complexes with the ligands 4-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid (5-ethyl-[1,3,4]thiadiazol-2-yl)-amide (L(1)) and 1-ethyl-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid (4-trifluoromethyl-phenyl)-amide (L(2)) by phosphate ions. The assays require equimolar (1.0 microM) concentrations of Tb(3+) and of the ligand at pH 8.0 (Tris-HCl buffer). The luminescence quenching is proportional to the concentration of aPase and CP within the range of 0.1-70 mU mL(-1) and 0.3-20 microg mL(-1), respectively. The detection limits were 0.05 mU mL(-1)=40.0 pmol L(-1) of aPase and 0.120 microg mL(-1) of CP.

  8. Enzymatic kinetic parameters for polyfluorinated alkyl phosphate hydrolysis by alkaline phosphatase.

    PubMed

    Jackson, Derek A; Mabury, Scott A

    2012-09-01

    The hydrolysis kinetics of three polyfluorinated alkyl phosphate monoesters (monoPAPs), differing in fluorinated chain length, were measured using bovine intestinal alkaline phosphatase to catalyze the reaction. Kinetic values were also measured for analogous hydrogenated phosphate monoesters to elucidate the effects of the fluorinated chain on the rate of enzymatic hydrolysis. Michaelis constants (K(m)) were obtained by a competition kinetics technique in the presence of p-nitrophenyl phosphate (PNPP) using UV-vis spectroscopy. Compared with K(m) (PNPP), Michaelis constants for monoPAPs ranged from 0.9 to 2.1 compared with hydrogenated phosphates, which ranged from 4.0 to 13.0. Apparent bimolecular rate constants (k(cat)/K(m)) were determined by monitoring rates of product alcohol formation at low substrate concentrations using gas chromatography-mass spectrometry. The experimental values for k(cat)/K(m) averaged as 1.1 × 10(7) M(-1) s(-1) for monoPAPs compared with 3.8 × 10(5) M(-1) s(-1) for hexyl phosphate. This suggests that the electron-withdrawing nature of the fluorinated chain enhanced the alcohol leaving group ability. The results were used in a simple model to suggest that monoPAPs in a typical mammalian digestive tract would hydrolyze in approximately 100 s, supporting a previous study that showed its absence after a dosing study in rats.

  9. Reaction rate modeling in cryoconcentrated solutions: alkaline phosphatase catalyzed DNPP hydrolysis.

    PubMed

    Champion, D; Blond, G; Le Meste, M; Simatos, D

    2000-10-01

    The hydrolysis of disodium p-nitrophenyl phosphate catalyzed by alkaline phosphatase was chosen as a model to study the kinetics of changes in frozen food products. The initial reaction rate was determined in concentrated sucrose solutions down to -24 degrees C, and the enzymatic characteristics K(M) and V(max) were calculated. The experimental data were compared to the kinetics predicted by assuming that the reaction was viscosity dependent. Indeed, an analysis of the enzymatic reaction demonstrated that both the diffusion of the substrate and the flexibility of the enzyme segments were controlled by the high viscosity of the media. When the temperature was too low for the viscosity to be measured simply, the Williams-Landel-Ferry equation was used to predict the viscosity, taking, as reference temperature, the glass transition temperature (T(g)) corresponding to the concentration of the freeze-concentrated phase at the test temperature. Predicted values of the reaction rate were very close to the experimental ones in the studied temperature range.

  10. Purification and characterization of an extracellular alkaline phosphatase from Penicillium chrysogenum.

    PubMed

    Politino, M; Brown, J; Usher, J J

    1996-01-01

    An extracellular alkaline phosphatase from Penicillium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-nitrophenyl phosphate, alpha-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K(m) for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 degrees C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-linked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg+2 and Mn+2 and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound.

  11. Titanium dioxide nanotube films: Preparation, characterization and electrochemical biosensitivity towards alkaline phosphatase.

    PubMed

    Roman, Ioan; Trusca, Roxana Doina; Soare, Maria-Laura; Fratila, Corneliu; Krasicka-Cydzik, Elzbieta; Stan, Miruna-Silvia; Dinischiotu, Anca

    2014-04-01

    Titania nanotubes (TNTs) were prepared by anodization on different substrates (titanium, Ti6Al4V and Ti6Al7Nb alloys) in ethylene glycol and glycerol. The influence of the applied potential and processing time on the nanotube diameter and length is analyzed. The as-formed nanotube layers are amorphous but they become crystalline when subjected to subsequent thermal treatment in air at 550°C; TNT layers grown on titanium and Ti6Al4V alloy substrates consist of anatase and rutile, while those grown on Ti6Al7Nb alloy consist only of anatase. The nanotube layers grown on Ti6Al7Nb alloy are less homogeneous, with supplementary islands of smaller diameter nanotubes, spread across the surface. Better adhesion and proliferation of osteoblasts was found for the nanotubes grown on all three substrates by comparison to an unprocessed titanium plate. The sensitivity towards bovine alkaline phosphatase was investigated mainly by electrochemical impedance spectroscopy in relation to the crystallinity, the diameter and the nature of the anodization electrolyte of the TNT/Ti samples. The measuring capacity of the annealed nanotubes of 50nm diameter grown in glycerol was demonstrated and the corresponding calibration curve was built for the concentration range of 0.005-0.1mg/mL.

  12. The Role of Intestinal Alkaline Phosphatase in Inflammatory Disorders of Gastrointestinal Tract

    PubMed Central

    Wojcik, Dagmara; Zahradnik-Bilska, Janina; Mach, Tomasz

    2017-01-01

    Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients. PMID:28316376

  13. Growth and alkaline phosphatase activity of Chattonella marina and Heterosigma akashiwo in response to phosphorus limitation.

    PubMed

    Wang, Zhao-Hui; Liang, Yu

    2015-02-01

    The growth and alkaline phosphatase activity (APA) of two raphidophyceae species Chattonella marina and Heterosigma akashiwo were investigated in response to P-limitation and subsequent addition of dissolved inorganic phosphorus (DIP, NaH2PO4) and two dissolved organic phosphorus (DOP) compounds: guanosine 5-monophosphate (GMP) and triethyl phosphate (TEP). APA levels increased greatly after P-starvation as the decrease of the cellular phosphorus quotes (Qp). C. marina responded to P-limitation quickly and strongly, with 10-fold increase in APA within 24 hr after P-starvation. The larger difference between maximal and minimal QP values in C. marina indicated its high capacity in P storage. APA of H. akashiwo was maximally enlarged about 2.5 times at 48 hr of P-starvation. After the addition of nutrients, cell numbers of C. marina increased in all treatments including the P-free culture, demonstrating the higher endurance of C. marina to P-limitation. However, those of H. akashiwo increased only in DIP and GMP cultures. APA increased only after the addition of the monophosphate ester GMP. The results suggest that quick responses of C. marina to P-limitation, high capacity in P storage as well as endurance for P-depletion provide this species an ecological advantage in phytoplankton community competition under DIP-limited conditions.

  14. An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases.

    PubMed

    Iqbal, Jamshed

    2011-07-15

    A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases(EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of -15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K(m) and K(i) values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes.

  15. Fluorescent Biosensor for Phosphate Determination Based on Immobilized Polyfluorene-Liposomal Nanoparticles Coupled with Alkaline Phosphatase.

    PubMed

    Kahveci, Zehra; Martínez-Tomé, Maria José; Mallavia, Ricardo; Mateo, C Reyes

    2017-01-11

    This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.

  16. Evaluation of serum sialic acid, heat stable alkaline phosphatase and fucose as markers of breast carcinoma.

    PubMed

    Patel, P S; Baxi, B R; Adhvaryu, S G; Balar, D B

    1990-01-01

    Serum levels of total sialic acid (TSA), lipid bound sialic acid (LSA), heat stable alkaline phosphatase (HSAP) and fucose were measured in 39 patients with breast carcinoma, 14 patients with benign breast diseases and 35 healthy female individuals. Elevated levels of the four biomarkers in breast carcinoma were significant when compared with controls (p less than 0.001). Fucose levels were most sensitive (71.8%), while TSA levels were most specific (64.3%) for breast carcinoma. Sensitivity and specificity were 100% when combinations of LSA with fucose and TSA with HSAP were studied respectively. LSA was significantly elevated in infiltrating duct carcinoma patients compared with lobular carcinoma (p less than 0.001). TSA, HSAP and fucose also had lower mean values in lobular carcinoma as compared to infiltrating duct carcinoma. Increase in the levels of LSA and HSAP after surgical removal of the tumor in breast carcinoma occurred prior to the clinical evidence of the recurrence. The results indicate that the combination of the markers studied might be useful in breast cancer diagnosis and treatment monitoring.

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

    PubMed Central

    Pigretti, M. M.; Milstein, C.

    1965-01-01

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

  18. Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects.

    PubMed

    Roston, Daniel; Cui, Qiang

    2016-09-14

    Enzymes are powerful catalysts, and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction's transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around-as with classic "induced fit" models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.

  19. Neutrophil alkaline phosphatase score in chronic granulocytic leukaemia: effects of splenectomy and antileukaemic drugs.

    PubMed Central

    Spiers, A S; Liew, A; Baikie, A G

    1975-01-01

    Staining with naphthol AS phosphate and Fast Blue BB salt has been used for the estimation of neutrophil alkaline phosphatase (NAP) scores in patients with chronic granulocytic leukaemia (CGL). The very low scores found at diagnosis rise when the disease is treated, and there is some inverse correlation between the NAP score and the absolute neutrophil count. Patients treated intensively developed high NAP scores. Elective splenectomy performed during the chronic phase of CGL is followed by a pronounced but transient neutrophilia and a concurrent striking rise in the NAP score. Similar changes were observed in patients without CGL who underwent splenectomy. These observations can be explained by assuming that newly formed neutrophils in CGL have a normal content of NAP but are rapidly sequestered in non-circulating extramedullary pools, whereas the circulating neutrophil with a typically low NAP content is a relatively aged cell which has lost enzyme activity. In subjects with or without CGL, removal of the spleen, a major site of such pooling, temporarily permits the circulation of newly formed neutrophils but eventually other organs assume the sequestering functions of the spleen. Thus the aberrations of NAP score seen in CGL might be attributable not to an intrinsic cellular defect but to an exaggeration of the granulocyte storage phenomena which also occur in subjects without CGL. PMID:1056940

  20. Rat serum alkaline phosphatase electrophoretic fractions: variations with feeding, starvation and cellulose fibre ingestion.

    PubMed

    Martins, M J; Dias, P O; Hipólito-Reis, C

    1998-12-01

    The effect of feeding, starvation and fibre ingestion on alkaline phosphatase (ALP) activity (E.C. 3.1.3.1) was studied in Wistar rat serum. Using identical assay conditions for total ALP activity determination and for electrophoretic ALP isoenzymes/fractions activity calculation, alpha- and beta-naphthyl phosphates and p-nitrophenyl phosphate were used as substrates and 2-amino-2-methyl-1-propanol/HCI was used as buffer, respectively. Total activity with beta-naphthyl phosphate was significantly higher than with alpha-naphthyl phosphate and p-nitrophenyl phosphate; with alpha-naphthyl phosphate it was significantly higher than with p-nitrophenyl phosphate. With all substrates, fed animals had significantly higher total activity than starving ones. Electrophoresis allowed the separation of two fractions. The second fraction activity was significantly higher in the fed group than in the starving ones, irrespective of the substrate used. Starving animals with fibre showed higher values of this fraction than starving animals without fibre, the difference reaching statistical significance with alpha-naphthyl phosphate. The first fraction predominated in both starved groups and the second in the fed group. The second fraction was identified as intestinal ALP. We conclude that the mechanical stimulation of the digestive tract appears to influence the passage of intestinal ALP to serum. The experimental conditions used enable quantification of electrophoretic fractions based on total activity. Activity depends on the substrate used.

  1. Rat enterocytes secrete SLPs containing alkaline phosphatase and cubilin in response to corn oil feeding.

    PubMed

    Mahmood, Akhtar; Shao, Jian-su; Alpers, David H

    2003-08-01

    Surfactant-like particles (SLP) are unilamellar secreted membranes associated with the process of lipid absorption and isolated previously only from the apical surface of enterocytes. In this paper, the intracellular membrane has been isolated from corn oil-fed animals, identified by its content of the marker protein intestinal alkaline phosphatase (IAP). Another brush-border protein, cubilin, and its anchoring protein megalin have been identified as components of extracellular SLP, but only cubilin is present to any extent in intracellular SLP. During fat absorption, IAP is modestly enriched in intracellular SLP, but full-length cubilin (migrating at 210 kDa in fat-fed mucosal fractions) falls by one-half, although fragments of cubilin are abundant in the intracellular SLP. Both IAP and cubilin colocalize to the same cells during corn oil absorption and colocalize around lipid droplets. This localization is more intense during feeding of corn oil with Pluronic L-81, a detergent that allows uptake of fatty acids and monoglycerides from the lumen, but blocks chylomicron secretion. Confocal microscopy confirms the colocalization of IAP and the ligand for cubilin, intrinsic factor. Possible roles for cubilin in intracellular SLP include facilitating movement of the lipid droplet through the cell and binding to the basolateral membrane before reverse endocytosis.

  2. Alkaline phosphatase as a treatment of sepsis-associated acute kidney injury.

    PubMed

    Peters, Esther; van Elsas, Andrea; Heemskerk, Suzanne; Jonk, Luigi; van der Hoeven, Johannes; Arend, Jacques; Masereeuw, Rosalinde; Pickkers, Peter

    2013-01-01

    Currently there are no pharmacological therapies licensed to treat sepsis-associated acute kidney injury (AKI). Considering the high incidence and mortality of sepsis-associated AKI, there is an urgent medical need to develop effective pharmacological interventions. Two phase II clinical trials recently demonstrated beneficial effects of the enzyme alkaline phosphatase (AP). In critically ill patients with sepsis-associated AKI, treatment with AP reduced the urinary excretion of tubular injury biomarkers and plasma markers of inflammation, which was associated with improvement of renal function. The dephosphorylating enzyme, AP, is endogenously present in the renal proximal tubule apical membrane but becomes depleted during ischemia-induced AKI, thereby possibly contributing to further renal damage. The exact mechanism of action of AP in AKI is unknown, but might be related to detoxification of circulating lipopolysaccharide and other proinflammatory mediators that lose their proinflammatory effects after dephosphorylation. Alternatively, tissue damage associated with systemic inflammation might be attenuated by an AP-mediated effect on adenosine metabolism. Adenosine is a signaling molecule that has been shown to protect the body from inflammation-induced tissue injury, which is derived through dephosphorylation of ATP. In this Perspectives article, we discuss the clinical activity of AP and its putative molecular modes of action, and we speculate on its use to treat and possibly prevent sepsis-associated AKI.

  3. Genome wide expression profile in human HTR-8/Svneo trophoblastic cells in response to overexpression of placental alkaline phosphatase gene.

    PubMed

    Bellazi, L; Mornet, E; Meurice, G; Pata-Merci, N; De Mazancourt, P; Dieudonné, M-N

    2011-10-01

    During pregnancy, placental growth allows the adaptation of the feto-maternal unit to fetal requirements. Placental alkaline phosphatase (PLAP) is a phosphomonoesterase produced increasingly until term by the placenta and also ectopically in some tumors. To precise the role of this enzyme in the placenta, we analyzed the genome wide expression profile of HTR-8/Svneo trophoblastic cells after overexpression of the alkaline phosphatase gene (ALPP). We showed that ALPP overexpression mainly altered expression of genes implicated in cellular growth and proliferation. These results were confirmed by the study of cellular effects in HTR-8/Svneo cells overexpressing ALPP and in HTR-8/Svneo cells in which ALPP expression was suppressed by siRNA. We showed that PLAP exerts a positive effect on DNA replication and acts as a proliferative factor in trophoblastic cells.

  4. Effects of parathyroid hormone and calcitonin on alkaline phosphatase activity and matrix calcification in rabbit growth-plate chondrocyte cultures

    SciTech Connect

    Kato, Y.; Shimazu, A.; Nakashima, K.; Suzuki, F.; Jikko, A.; Iwamoto, M. )

    1990-07-01

    The effects of PTH and calcitonin (CT) on the expression of mineralization-related phenotypes by chondrocytes were examined. In cultures of pelleted growth-plate chondrocytes. PTH caused 60-90% decreases in alkaline phosphatase activity, the incorporation of {sup 45}Ca into insoluble material, and the calcium content during the post-mitotic stage. These effects of PTH were dose-dependent and reversible. In contrast, CT increased alkaline phosphatase activity, {sup 45}Ca incorporation into insoluble material, and the calcium content by 1.4- to 1.8-fold. These observations suggest that PTH directly inhibits the expression of the mineralization-related phenotypes by growth-plate chondrocytes, and that CT has the opposite effects.

  5. Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease

    SciTech Connect

    Garnero, P.; Delmas, P.D.

    1993-10-01

    The authors measured serum bone alkaline phosphatase (B-ALP) with a new immunoradiometric assay (IRMA) in a large sample of healthy controls comprising 173 women and 180 men, 20-88 yr of age, and in patients with metabolic bone disease. Using serum samples from patients with liver disease and patients with Paget's disease with elevated total alkaline phosphatase (T-ALP) as a source of, respectively, liver and bone isoenyzmes, they determined a liver cross-reactivity of the IRMA of 16% that was confirmed by electrophoresis of the circulating alkaline phosphatase isoenzymes. The IRMA was linear for serial sample dilutions, the recovery ranged from 89-110%, and the intra- and interassay variations were below 7% and 9%, respectively. B-ALP increased linearly with age in both sexes, and the mean B-ALP serum levels were not significantly different for women and men (11.3 [+-] 4.8 ng/mL for women; 11.0 [+-] 4.0 ng/mL for men). The increase in B-ALP after the menopause was significantly higher than that in T-ALP (+77% vs. +24%; P<0.001). When the values of postmenopausal women were expressed as the SD from the mean of premenopausal women, the mean Z scores were 2.2[+-] 1.8 for B-ALP and 0.9 [+-] 1.3 for T-ALP (P<0.001 between the two).

  6. Effect of sex and age on the activities of lactate dehydrogenase and alkaline phosphatase in the lungs of rats.

    PubMed Central

    Lopez, A; Yong, S; Sharma, A; Morwood-Clark, M; Lillie, L E; Albassam, M

    1986-01-01

    Since toxicity studies among different laboratories generally involve rats of different sex and age, this study was conducted to investigate the effect of sex, age and animal to animal variation in the activities of lactate dehydrogenase and alkaline phosphatase from bronchoalveolar lavage fluid, bronchoalveolar cell lysate and lung homogenate. Correlation between numbers of bronchoalveolar cells recovered from lungs and enzyme activity in bronchoalveolar cell lysate or lung homogenate supernatant were also investigated. Male rats showed significantly (p less than 0.05) higher activities of alkaline phosphatase in the bronchoalveolar lavage fluid and lung homogenate. Animal to animal variation for lactate dehydrogenase and alkaline phosphatase was higher in lungs than in serum. The number of bronchoalveolar cells recovered from lungs revealed a significant (p less than 0.01) positive correlation with the activities of both enzymes in the supernatant of cell lysates but not in the bronchoalveolar fluid. These results indicated that in an inhalation study interindividual variation in the levels of pulmonary enzymes should be considered in order to minimize the numerous possible sources of experimental error. PMID:3742377

  7. Investigation of the enzyme hydrolysis products of the substrates of alkaline phosphatase in electrochemical immunosensing.

    PubMed

    Preechaworapun, Anchana; Dai, Zong; Xiang, Yun; Chailapakul, Orawon; Wang, Joseph

    2008-07-15

    In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to

  8. Pathophysiological role of vascular smooth muscle alkaline phosphatase in medial artery calcification†

    PubMed Central

    Sheen, Campbell R.; Kuss, Pia; Narisawa, Sonoko; Yadav, Manisha C.; Nigro, Jessica; Wang, Wei; Chhea, T. Nicole; Sergienko, Eduard A.; Kapoor, Kapil; Jackson, Michael R.; Hoylaerts, Marc. F.; Pinkerton, Anthony B.; O'Neill, W. Charles; Millán, Jose Luis

    2015-01-01

    Medial vascular calcification (MVC) is a pathological phenomenon common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases, that causes vascular stiffening and can lead to heart failure. These conditions share the common feature of tissue-nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X-linked manner. Hemizygous overexpressor male mice (Tagln-Cre+/-; HprtALPL/Y, or TNAP-OE) show extensive vascular calcification, high blood pressure, cardiac hypertrophy and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln-Cre+/-; HprtALPL/-) female TNAP-OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP-OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug-like pharmacokinetic characteristics. TNAP-OE mice were treated with the prototypical TNAP inhibitor SBI-425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle-treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target. This article is protected by copyright. All rights reserved PMID:25428889

  9. Alkaline phosphatase inhibition based conductometric biosensor for phosphate estimation in biological fluids.

    PubMed

    Upadhyay, Lata Sheo Bachan; Verma, Nishant

    2015-06-15

    Determination of phosphate ions concentration is very important from both, environmental and clinical point of view. In this study, a simple and novel conductometric biosensor for indirect determination of the phosphate ions in aqueous solution has been developed. The developed biosensor is based on the inhibition of immobilized alkaline phosphatase activity, in the presence of the phosphate ions. This is the first time we developed a mono-enzymatic biosensor for indirect estimation of phosphate ions. The developed biosensor showed a broad linear response (as compared to other reported biosensors) for phosphate ions in the range of 0.5-5.0 mM (correlation coefficient=0.995), with a detection limit of 50 µM. Different optimized parameters were obtained as the buffer concentration of 30 mM, substrate concentration of 1.0mM, and a pH of 9.0. All the optimized parameters were analyzed by analysis of variance, and were found to be statistically significant at a level of α=0.05. The developed biosensor is also suitable to determine the serum phosphate concentration, with a recovery of 86-104%, while a recovery of 102% was obtained from the water samples that were spiked with 500 µM phosphate. A relative standard deviation in the conductance response for five successive measurements (n=5) did not exceed 7%, with a shelf life of 30 days. With a lower detection limit and a higher recovery, the biosensor provides a facile approach for phosphate estimation in biological fluids.

  10. Sensitive and selective colorimetric assay of alkaline phosphatase activity with Cu(II)-phenanthroline complex.

    PubMed

    Hu, Qiong; He, Minhui; Mei, Yaqi; Feng, Wenjie; Jing, Su; Kong, Jinming; Zhang, Xueji

    2017-01-15

    Alkaline phosphatase (ALP) plays a vital role in dephosphorylation- and phosphorylation-related cellular regulation and signaling processes. Accordingly, the development of efficient methods for ALP activity assay is of significant importance in clinical diagnosis. In this work, a simple and practical method is reported for the first time for the sensitive and selective colorimetric assay of ALP activity by exploiting a water-soluble Cu(II)-phenanthroline complex as the probe, on the basis of the distinctive metal-to-ligand charge-transfer (MLCT) properties. This method is simply built on a two-step chromogenic reaction: the enzymatic hydrolysis of the substrate ascorbic acid 2-phosphate to ascorbic acid (AA), followed by the reduction of the colorimetric probe Cu(BPDS)2(2-) (BPDS=bathophenanthroline disulfonate) by AA to its cuprous form. The latter process triggers a turn-on spectral absorption at 424nm and a striking color change of the solution from colorless to blackish-green. Needless of complicated protocols and instrumentation, this method allows a sensitive readout of ALP activity within a wide linear range of 0-200mUmL(-)(1), with a detection limit down to 1.25mUmL(-1). Results also reveal that it is highly selective and holds great potential in ALP inhibitor efficiency evaluation. In addition, quantitative analysis of ALP activity in spiked serum samples has been realized successfully in the linear range of 0-200mUmL(-1), with a detection limit of 1.75mUmL(-1). Advantages of simplicity, wide linear range, high sensitivity and selectivity, low cost, and little background interference render this method great potential in practical applications.

  11. Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter.

    PubMed

    Hu, Qiong; Zhou, Baojing; Dang, Pengyun; Li, Lianzhi; Kong, Jinming; Zhang, Xueji

    2017-01-15

    We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe(3+) to Fe(2+). The complexation of Fe(2+) with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS)3(4-) reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0-220 mU mL(-1) with a detection limit of 0.94 mU mL(-1). Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL(-1) being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing.

  12. Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes.

    PubMed Central

    Nakata, A; Yamaguchi, M; Izutani, K; Amemura, M

    1978-01-01

    Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides. Images PMID:348683

  13. Human Placental Alkaline Phosphatase as a Tracking Marker for Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Balmayor, Elizabeth Rosado; Flicker, Magdalena; Käser, Tobias; Saalmüller, Armin

    2013-01-01

    Abstract Currently, adult mesenchymal stem cells (MSCs) are being evaluated for a wide variety of therapeutic approaches. It has been suggested that MSCs possess regenerative properties when implanted or injected into damaged tissue. However, the efficacy of MSCs in several of the proposed treatments is still controversial. To further explore the therapeutic potential of these cells, it is necessary to trace the fate of individual donor or manipulated cells in the host organism. Recent studies from our lab showed that human placental alkaline phosphatase (hPLAP) is a marker with great potential for cell tracking. However, a potential concern related to this marker is its enzymatic activity, which might alter cell behavior and differentiation by hydrolyzing substrates in the extracellular space and thereby changing the cellular microenvironment. Therefore, the aim of this study was to characterize bone marrow MSCs (BMSCs) derived from hPLAP-transgenic inbred F344 rats (hPLAP-tg) in comparison to wild type (wt) BMSCs. Here, we show that BMSCs from wt and hPLAP-tg donors are indistinguishable in terms of cell morphology, viability, adhesion, immune phenotype, and proliferation as well as in their differentiation capacity over six passages. The expression of the hPLAP marker enzyme was not impaired by extensive in vitro cultivation, osteogenic, adipogenic, or chondrogenic differentiation, or seeding onto two- or three-dimensional biomaterials. Thus, our study underscores the utility of genetically labeled BMSCs isolated from hPLAP-tg donors for long-term tracking of the fate of transplanted MSCs in regenerative therapies. PMID:24083090

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  15. Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

    PubMed

    Aminian, Alieh; Shirzadi, Bahareh; Azizi, Zahra; Maedler, Kathrin; Volkmann, Eike; Hildebrand, Nils; Maas, Michael; Treccani, Laura; Rezwan, Kurosch

    2016-12-01

    Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

  16. Detection, purification and characterisation of a secretory alkaline phosphatase from Onchocerca species.

    PubMed

    Cho-Ngwa, Fidelis; Mbua, Eric Ngalle; Nchamukong, Kingsley G; Titanji, Vincent P K

    2007-12-01

    An Onchocerca secretory alkaline phosphatase (E.C. 3.1.3.1) of molecular weight 90 kDa when in crude extract, but which dimerises to about 180 kDa upon purification, was detected, purified and characterised. The enzyme was found to be secreted by both O. ochengi and O. volvulus worms. It was shown to be of Onchocerca origin by Western blotting with bovine onchocerciasis sera and by its time-dependent release in cultures. The O. ochengi enzyme was purified to near homogeneity by a combination of polyethylene glycol precipitation, DEAE-cellulose chromatography and preparative electrophoresis. About 0.96 mg of the active enzyme was purified from 48.4 mg of the crude parasite-released products, giving a purification fold of 71.45 and a yield of 8.7%. The purified enzyme exhibited a typical Michaelis-Menten kinetics with optimum activity on p-nitrophenylphosphate (p-NPP) at pH 10.2. Its apparent K(m) for p-NPP was 0.56+/-0.03 mM and it required Mg(2+) and dithiothreitol (DTT) for stability throughout its purification. Sodium dodecyl sulphate at 2% (w/v) did not inhibit the enzyme activity, but apparently stabilised it during freezing. Inorganic phosphate inhibited the enzyme competitively with an apparent inhibition constant (K(i)) of 3.33+/-0.04 mM, whereas l-phenylalanine inhibited it in a mixed way with a K(i) of 3.18+/-0.03 mM. While contributing to the understanding of metabolism in Onchocerca, the present apparently unique enzyme which is likely to serve in the nutrition of the parasite could be further characterised as a macrofilaricide target or diagnostic marker in onchocerciasis.

  17. Colocation and role of polyphosphates and alkaline phosphatase in apatite biomineralization of elasmobranch tesserae.

    PubMed

    Omelon, Sidney; Georgiou, John; Variola, Fabio; Dean, Mason N

    2014-09-01

    Elasmobranchs (e.g. sharks and rays), like all fishes, grow continuously throughout life. Unlike other vertebrates, their skeletons are primarily cartilaginous, comprising a hyaline cartilage-like core, stiffened by a thin outer array of mineralized, abutting and interconnected tiles called tesserae. Tesserae bear active mineralization fronts at all margins and the tesseral layer is thin enough to section without decalcifying, making this a tractable but largely unexamined system for investigating controlled apatite mineralization, while also offering a potential analog for endochondral ossification. The chemical mechanism for tesserae mineralization has not been described, but has been previously attributed to spherical precursors, and alkaline phosphatase (ALP) activity. Here, we use a variety of techniques to elucidate the involvement of phosphorus-containing precursors in the formation of tesserae at their mineralization fronts. Using Raman spectroscopy, fluorescence microscopy and histological methods, we demonstrate that ALP activity is located with inorganic phosphate polymers (polyP) at the tessera-uncalcified cartilage interface, suggesting a potential mechanism for regulated mineralization: inorganic phosphate (Pi) can be cleaved from polyP by ALP, thus making Pi locally available for apatite biomineralization. The application of exogenous ALP to tissue cross-sections resulted in the disappearance of polyP and the appearance of Pi in uncalcified cartilage adjacent to mineralization fronts. We propose that elasmobranch skeletal cells control apatite biomineralization by biochemically controlling polyP and ALP production, placement and activity. Previous identification of polyP and ALP shown previously in mammalian calcifying cartilage supports the hypothesis that this mechanism may be a general regulating feature in the mineralization of vertebrate skeletons.

  18. Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics.

    PubMed

    Simão, Ana Maria S; Yadav, Manisha C; Narisawa, Sonoko; Bolean, Mayte; Pizauro, Joao Martins; Hoylaerts, Marc F; Ciancaglini, Pietro; Millán, José Luis

    2010-03-05

    We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

  19. Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

    PubMed

    Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

    2016-03-01

    Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

  20. Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves.

    PubMed

    Maranto, A R; Schoen, F J

    1988-10-01

    Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.

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

    SciTech Connect

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

    2015-08-01

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

  2. Bone-specific alkaline phosphatase activity is inhibited by bisphosphonates: role of divalent cations.

    PubMed

    Vaisman, Diego N; McCarthy, Antonio D; Cortizo, Ana M

    2005-05-01

    Bisphosphonates (BPs) are drugs widely used in the treatment of various bone diseases. BPs localize to bone mineral, and their concentration in resorption lacunae could reach almost millimolar levels. Bone alkaline phosphatase (ALP) is a membrane-bound exoenzyme that has been implicated in bone formation and mineralization. In this study, we investigated the possible direct effect of three N-containing BPs (alendronate, pamidronate, and zoledronate) on the specific activity of bone ALP obtained from an extract of UMR106 rat osteosarcoma cells. Enzymatic activity was measured by spectrophotometric detection of p-nitrophenol product and by in situ visualization of ALP bands after an electrophoresis on cellulose acetate gels. Because ALP is a metalloprotein that contains Zn2+ and Mg2+, both of which are necessary for catalytic function, we also evaluated the participation of these divalent cations in the possible effect of BPs on enzymatic activity. All BPs tested were found to dose-dependently inhibit spectrophotometrically measured ALP activity (93-42% of basal) at concentrations of BPs between 10-5 M and 10-4 M, the order of potency being zoledronate approximately equals alendronate > pamidronate. However, coincubation with excess Zn2+ or Mg2+ completely abolished this inhibitory effect. Electrophoretic analysis rendered very similar results: namely a decrease in the enzymatic activity of the bone-ALP band by BPs and a reversion of this inhibition by divalent cations. This study shows that N-containing BPs directly inhibit bone-ALP activity, in a concentration range to which this exoenzyme is probably exposed in vivo. In addition, this inhibitory effect is most possibly the result of the chelation of Zn2+ and Mg2+ ions by BPs.

  3. Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

    PubMed

    Oliveira, Flávia Amadeu de; Matos, Adriana Arruda; Matsuda, Sandra Satiko; Buzalaf, Marília Afonso Rabelo; Bagnato, Vanderley Salvador; Machado, Maria Aparecida de Andrade Moreira; Damante, Carla Andreotti; Oliveira, Rodrigo Cardoso de; Peres-Buzalaf, Camila

    2017-04-01

    Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm(2) or 3.8J/cm(2), respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm(2) promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

  4. Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily.

    PubMed

    Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem Y; Ressl, Susanne; Wiersma-Koch, Helen; Borland, Jamar; Brown, Clayton L; Johnson, Tory A; Singh, Zorawar; Herschlag, Daniel

    2016-11-02

    Naively one might have expected an early division between phosphate monoesterases and diesterases of the alkaline phosphatase (AP) superfamily. On the contrary, prior results and our structural and biochemical analyses of phosphate monoesterase PafA, from Chryseobacterium meningosepticum, indicate similarities to a superfamily phosphate diesterase [Xanthomonas citri nucleotide pyrophosphatase/phosphodiesterase (NPP)] and distinct differences from the three metal ion AP superfamily monoesterase, from Escherichia coli AP (EcAP). We carried out a series of experiments to map out and learn from the differences and similarities between these enzymes. First, we asked why there would be independent instances of monoesterases in the AP superfamily? PafA has a much weaker product inhibition and slightly higher activity relative to EcAP, suggesting that different metabolic evolutionary pressures favored distinct active-site architectures. Next, we addressed the preferential phosphate monoester and diester catalysis of PafA and NPP, respectively. We asked whether the >80% sequence differences throughout these scaffolds provide functional specialization for each enzyme's cognate reaction. In contrast to expectations from this model, PafA and NPP mutants with the common subset of active-site groups embedded in each native scaffold had the same monoesterase:diesterase specificities; thus, the >10(7)-fold difference in native specificities appears to arise from distinct interactions at a single phosphoryl substituent. We also uncovered striking mechanistic similarities between the PafA and EcAP monoesterases, including evidence for ground-state destabilization and functional active-site networks that involve different active-site groups but may play analogous catalytic roles. Discovering common network functions may reveal active-site architectural connections that are critical for function, and identifying regions of functional modularity may facilitate the design of new enzymes

  5. Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis.

    PubMed

    Hatayama, Kazuhisa; Nishihara, Yoshito; Kimura, Sayaka; Goto, Ken; Nakamura, Daichi; Wakita, Atsushi; Urasoko, Yoshinaka

    2011-10-01

    Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

  6. Measurement of alkaline phosphatase in canine seminal plasma--an update.

    PubMed

    Schäfer-Somi, S; Fröhlich, T; Schwendenwein, I

    2013-02-01

    In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut-off value of 5000 IU/l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre-analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre-analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at -18 °C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107,328 IU/l. After 24 h of frozen storage, activities did not differ significantly (96,844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1:100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU/l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24h before analysis.

  7. Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation

    PubMed Central

    2012-01-01

    Background Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches. Methods Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects. Results AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS. Conclusions Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients. PMID:23231745

  8. Association between alendronate, serum alkaline phosphatase level, and heterotopic ossification in individuals with spinal cord injury

    PubMed Central

    Ploumis, Avraam; Donovan, Jayne M.; Olurinde, Mobolaji O.; Clark, Dana M.; Wu, Jason C.; Sohn, Douglas J.; O'Connor, Kevin C.

    2015-01-01

    Context/objective Only sparse evidence exists regarding the effectiveness of oral alendronate (ALN) in the prevention of heterotopic ossification (HO) in patients with spinal cord injury (SCI). The objective of this study is to investigate the protective effect of oral ALN intake on the appearance of HO in patients with SCI. Study design Retrospective database review. Setting A Spinal Cord Unit at a Rehabilitation Hospital. Participants Two hundred and ninety-nine patients with SCI during acute inpatient rehabilitation. Interventions Administration of oral ALN. Outcome measures The incidence of HO during rehabilitation was compared between patients with SCI receiving oral ALN (n = 125) and patients with SCI not receiving oral ALN (n = 174). The association between HO and/or ALN intake with HO risk factors and biochemical markers of bone metabolism were also explored. Results HO developed in 19 male patients (6.35%), however there was no significant difference in the incidence of HO in patients receiving oral ALN or not. The mean odds ratio of not developing versus developing HO given ALN exposure was 0.8. Significant correlation was found between abnormal serum alkaline phosphatase (ALP) levels and HO appearance (P < 0.001) as well as normal serum ALP and ALN intake (P < 0.05). Conclusion Even though there was no direct prevention of HO in patients with SCI by oral ALN intake, abnormal serum ALP was found more frequently in patients with HO development and without oral ALN intake. This evidence could suggest that ALN may play a role in preventing HO, especially in patients with acute SCI with increasing levels of serum ALP. PMID:24820653

  9. Alkaline phosphatase level in gingival crevicular fluid during treatment with Quad-Helix.

    PubMed

    Delli Mauri, A; Petrini, M; Vitale, D; Tecco, S; Festa, F; Barbato, E; Spoto, G

    2015-01-01

    The aim of this work is to assess the level of the human alkaline phosphatase enzyme (ALP) during palatal expansion with Quad-Helix (QH) appliance. A total of twenty-two orthodontic patients characterized by contraction of the upper jaw, that needed application of a QH in order to treat their condition, were included in this study. Gingival crevicular fluid (GCF) was collected at four different times: before cementation (T0), after two weeks (T1), after four weeks (T2) and after one year (T3) from application of QH. In each patient maxillary first molars, right (UM-right) and left (UM-left), which were connected with bands to QH, were used for testing; first lower molars were used as Controls (LM-right, LM-left). Data show that ALP level in tension sites was proportional to the average increase of the inter-molar distance; on the contrary, the enzymatic level in compression sites was characterized by an inverse trend. The only exception to this phenomenon was recorded after one year (T3), when the increase of ALP level in both sites of tension and compression was ascribed to a mild inflammation due to bacterial plaque accumulation. The level of ALP in control sites was constant for the whole period of observation. The described ALP fluctuations in accordance with the inter-molar distance increment, shows that the main action of QH on bone remodelling was exerted during the fourth week (T2); for this reason, the monitoring of this enzyme could be used as a marker of effective function of the QH appliance.

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

    PubMed

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

    2013-10-01

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

  11. Toxicology of cupric salts on honeybees. V. Gluconate and sulfate action on gut alkaline and acid phosphatases.

    PubMed

    Bounias, M; Kruk, I; Nectoux, M; Popeskovic, D

    1996-10-01

    Some aspects of putative nontarget effects of cupric ions systemically fed to honeybees against their parasite mite Varroa jacobsoni have been investigated on the host phosphatases. The alkaline and acid forms extracted from the guts of worker bees exhibited substrate-inhibition features. Upon detailed kinetic analysis, cupric organic salts indicate activation effects at concentrations of about 1 mM. Concentrations up to 10 mM (alkaline form) and 25 mM (acid form) induced no important changes, except a partial quenching of the substrate-inhibition process, characterized by a wide increase in the constant of apparent inhibitory binding of substrate to the enzyme-substrate complex. Partial purification gave a single alkaline form with quite similar kinetic behavior in the absence of natural ions as in crude extracts. Cupric gluconate and sulfate demonstrated similar patterns, except an increase of the apparent Hill coefficient by sulfate only. The substrate constant of acid phosphatases was decreased at high cupric gluconate doses while its maximum velocity was biphasically increased (with observed maximum at 1 mM), resulting in a sustained activation. Chemiluminescence studies revealed that cupric ion activation is counteracted by oxygen radicals generated by cupric ions and also, in vitro, by the artificial substrate para-nitrophenylphosphate. The para-nitrophenol molecules released from the reaction are therefore responsible for biphasic effects selectively observed with gluconate salts. In apicultural practice, neither blockade of activity nor dramatic changes are to be expected at doses administered to bees against the parasite.

  12. Comparative Evaluation of Efficacy of Three Different Herbal Toothpastes on Salivary Alkaline Phosphatase and Salivary Acid Phosphatase - A Randomized Controlled Trial

    PubMed Central

    Dodamani, Arun; Karibasappa, G. N.; Deshmukh, Manjiri; Naik, Rahul

    2016-01-01

    Introduction Very few researches in the past have tried to evaluate the effect of herbal toothpaste on saliva and salivary constituents like alkaline phosphatase and acid phosphatase which play an important role in maintaining oral health. Aim To evaluate and compare the effect of three different herbal toothpastes on Salivary Alkaline Phosphatase (ALP) and salivary Acid Phosphatase (ACP). Material and Methods The present study was a preliminary study conducted among 45 dental students (15 subjects in each group) in the age group of 19-21 years. Subjects in each group were randomly intervened with three different herbal toothpastes respectively (Group A – Patanjali Dant Kanti, Group B - Himalaya Complete Care and Group C – Vicco Vajradanti). Unstimulated saliva sample were collected before and after brushing and salivary ACP and salivary ALP levels were assessed at an interval of one week each for a period of four weeks starting from day one. Compiled data was analyzed using chi square test, paired t-test and ANOVA based on the nature of the obtained data. Results All the three toothpastes showed significant (p<0.001) reduction in ACP and ALP levels at each interval. For patanjali toothpaste, the mean reduction was in the range of 2.55 – 2.62 IU/L for ACP and 2.94 – 2.99 IU/L for ALP. For Himalaya toothpaste, the mean reduction was in the range of 1.39 – 1.47 IU/L for ACP and 1.55 – 1.61 IU/L for ALP. For Vicco toothpaste, the mean reduction was in the range of 2.46 – 2.50 IU/L for ACP and 2.64 – 2.77 IU/L for ALP. Patanjali and Vicco toothpaste were significantly effective in reducing the levels of salivary ACP and ALP more than Himalaya toothpaste (p<0.05). Conclusion Herbal toothpastes, especially Dant Kanti and Vicco Vajradanti, showed significant reduction in levels of ACP and ALP resulting in overall improvement towards the oral health. PMID:27790584

  13. Alkaline phosphatase activity in gingival crevicular fluid during human orthodontic tooth movement.

    PubMed

    Perinetti, Giuseppe; Paolantonio, Michele; D'Attilio, Michele; D'Archivio, Domenico; Tripodi, Domenico; Femminella, Beatrice; Festa, Felice; Spoto, Giuseppe

    2002-11-01

    Bone remodeling that occurs during orthodontic tooth movement is a biologic process involving an acute inflammatory response in periodontal tissues. A sequence characterized by periods of activation, resorption, reversal, and formation has been recently described as occurring in both tension and compression tooth sites during orthodontic tooth movement. We used a longitudinal design to investigate alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) to assess whether it can serve as a diagnostic aid in orthodontics. Sixteen patients (mean age, 15.5 years) participated in the study. The maxillary first molars under treatment served as the test teeth (TT) in each patient; in particular, 1 first molar was to be retracted and hence was considered the distalized molar (DM), whereas the contralateral molar (CM) was included in the fixed orthodontic appliance but was not subjected to the distal forces. The DM antagonist first molar (AM), free from any orthodontic appliance, was used as the baseline control. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance activation, 1 hour after, and weekly over the following 4 weeks. The clinical gingival condition was evaluated at the baseline and at the end of the experimental term. ALP activity was determined spectrophotometrically at 30 degrees C, and the results were expressed as total ALP activity (mUnits/sample). GCF ALP activity was significantly elevated in the DMs and the CMs as compared with the AMs at 1, 2, 3, and 4 weeks; conversely, in the AMs, GCF ALP activity remained at baseline levels throughout the experiment. Moreover, the enzyme activity in the DMs was significantly greater than in the CMs. In the DMs, a significantly greater ALP activity was observed in sites of tension compared with sites of compression. This difference was not seen with the CMs, in which the enzyme activity increased to the same extent in tension and compression

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

    PubMed

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that

  15. Goblet cells and intestinal Alkaline phosphatase expression (IAP) during the development of the rat small intestine.

    PubMed

    Gomes, José Rosa; Ayub, Laís Costa; Dos Reis, Camila Audrey; Machado, Miriam Joice; da Silva, Jéssica; Omar, Nádia Fayez; de Miranda Soares, Maria Albertina

    2017-01-01

    This study aimed to evaluate the temporal and spacial distribution of the mucins produced by goblet cells and intestinal alkaline phosphatase (IAP) expression during the development of the small intestine of the rat. Intestines were removed from rats on the 15th, 17th and 18th days of intratuterine life (i.u.) and on the 3rd, 10th, 17th and 25th days after birth (a.b.). Intestines were processed for routine histological procedures and sections were submitted to histochemistry using PAS to stain neutral glycoproteins and Alcian blue for acidic glycoproteins, as well as immunohistochemistry to detect IAP. In rats, glycoprotein production was seen to begin in the intestinal epithelium cell at around the 17th day of i.u. life; however, this production was not accompanied by morphological indications of the presence of goblet cells. By the 18th i.u. day, the villus epithelium was undergoing differentiation and the first goblet cells could be identified from this time. At around the 10th day a.b., both compartments of the small intestine were detected; i.e. the villi and the crypts. At this timepoint, goblet cells were present in the villi, and also in the upper regions of the crypts. On the 3rd, 10th 17th and 25th days a.b., the presence of the goblet cells increased and presented regional differences in the sections evaluated. IAP was not detected during i.u. life, but was weakly detected in the cells of the villi from the 3rd day a.b., along the entire extension of the villi. On the 10th day, IAP was detected at the tip of the villi, while on the 25th day, it was detected along the extension of the villi, but with a weaker intensity. In conclusion, a temporal and spacial distribution of goblet cells and IAP activity occurs during the development of the small intestine, suggesting a possible regulatory control in accordance with the suckling and weaning phases of food intake in the rat's life.

  16. Tissue Nonspecific Alkaline Phosphatase (TNAP) Regulates Cranial Base Growth and Synchondrosis Maturation

    PubMed Central

    Nam, Hwa K.; Sharma, Monika; Liu, Jin; Hatch, Nan E.

    2017-01-01

    Hypophosphatasia is a rare heritable disorder caused by inactivating mutations in the gene (Alpl) that encodes tissue nonspecific alkaline phosphatase (TNAP). Hypophosphatasia with onset in infants and children can manifest as rickets. How TNAP deficiency leads to bone hypomineralization is well explained by TNAP's primary function of pyrophosphate hydrolysis when expressed in differentiated bone forming cells. How TNAP deficiency leads to abnormalities within endochondral growth plates is not yet known. Previous studies in hypophosphatemic mice showed that phosphate promotes chondrocyte maturation and apoptosis via MAPK signaling. Alpl−/− mice are not hypophosphatemic but TNAP activity does increase local levels of inorganic phosphate. Therefore, we hypothesize that TNAP influences endochondral bone development via MAPK. In support of this premise, here we demonstrate cranial base bone growth deficiency in Alpl−/− mice, utilize primary rib chondrocytes to show that TNAP influences chondrocyte maturation, apoptosis, and MAPK signaling in a cell autonomous manner; and demonstrate that similar chondrocyte signaling and apoptosis abnormalities are present in the cranial base synchondroses of Alpl−/− mice. Micro CT studies revealed diminished anterior cranial base bone and total cranial base lengths in Alpl−/− mice, that were prevented upon injection with mineral-targeted recombinant TNAP (strensiq). Histomorphometry of the inter-sphenoidal synchondrosis (cranial base growth plate) demonstrated significant expansion of the hypertrophic chondrocyte zone in Alpl−/− mice that was minimized upon treatment with recombinant TNAP. Alpl−/− primary rib chondrocytes exhibited diminished chondrocyte proliferation, aberrant mRNA expression, diminished hypertrophic chondrocyte apoptosis and diminished MAPK signaling. Diminished apoptosis and VEGF expression were also seen in 15 day-old cranial base synchondroses of Alpl−/− mice. MAPK signaling was

  17. Isozyme profile and tissue-origin of alkaline phosphatases in mouse serum

    PubMed Central

    Linder, Cecilia Halling; Englund, Ulrika H; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

    2013-01-01

    Mouse serum alkaline phosphatase (ALP) is frequently measured and interpreted in mammalian bone research, however; little is known about the circulating ALPs in mice and their relation to human ALP isozymes and isoforms. Mouse ALP was extracted from liver, kidney, intestine, and bone from vertebra, femur and calvaria tissues. Serum from mixed strains of wild-type (WT) mice and from individual ALP knockout strains were investigated, i.e., Alpl−/− (a.k.a. Akp2 encoding tissue-nonspecific ALP or TNALP), Akp3−/− (encoding duodenum-specific intestinal ALP or dIALP), and Alpi−/− (a.k.a. Akp6 encoding global intestinal ALP or gIALP). The ALP isozymes and isoforms were identified by various techniques and quantified by high-performance liquid chromatography. Results from the WT and knockout mouse models revealed identical bone-specific ALP isoforms (B/I, B1, and B2) as found in human serum, but in addition mouse serum contains the B1x isoform only detected earlier in patients with chronic kidney disease and in human bone tissue. The two murine intestinal isozymes, dIALP and gIALP, were also identified in mouse serum. All four bone-specific ALP isoforms (B/I, B1x, B1, and B2) were identified in bone tissues from mice, in good correspondence with those found in human bones. All mouse tissues, except liver and colon, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Histochemical staining, Northern and Western blot analysis confirmed undetectable ALP expression in liver tissue. ALP activity staining showed some positive staining in the bile canaliculi for BALB/c and FVB/N WT mice, but not in C57Bl/6 and ICR mice. Taken together, while the main source of ALP in human serum originates from bone and liver, and a small fraction from intestine (<5%), mouse serum consists mostly of bone ALP, including all four isoforms, B/I, B1x, B1, and B2, and two intestinal ALP isozymes dIALP and gIALP. We suggest that the

  18. Pre-treatment serum lactate dehydrogenase and alkaline phosphatase as predictors of metastases in extremity osteosarcoma

    PubMed Central

    Marais, Leonard C.; Bertie, Julia; Rodseth, Reitze; Sartorius, Benn; Ferreira, Nando

    2015-01-01

    Background The prognosis of patients with metastatic osteosarcoma remains poor. However, the chance of survival can be improved by surgical resection of all metastases. In this study we investigate the value of serum alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in predicting the presence of metastatic disease at time of diagnosis. Methods Sixty-one patients with histologically confirmed conventional osteosarcoma of the extremity were included in the study. Only 19.7% of cases presented without evidence of systemic spread of the disease. Pre-treatment serum ALP and LDH were analysed in patients with and without skeletal or pulmonary metastases. Results Serum LDH and ALP levels were not significantly different in patients with or without pulmonary metastases (p=0.88 and p=0.47, respectively). The serum LDH and ALP levels did however differ significantly in patients with or without skeletal metastases (p<0.001 and p=0.02, respectively). The optimal breakpoint for serum LDH as a marker of skeletal metastases was 849 IU/L (AUC 0.839; Sensitivity=0.88; Specificity=0.73). LDH >454 IU/L equated to 100% sensitivity for detected bone metastases (positive diagnostic likelihood ratio (DLR)=1.32). With a cut-off of 76 IU/L a sensitivity of 100% was reached for serum ALP predicting the presence of skeletal metastases (positive DLR=1.1). In a multivariate analysis both LDH ≥850 IU/L (odds ratio [OR]=9; 95% confidence interval (CI) 1.8–44.3) and ALP ≥280 IU/L (OR=10.3; 95% CI 2.1–50.5) were predictive of skeletal metastases. LDH however lost its significance in a multivariate model which included pre-treatment tumour volume. Conclusion In cases of osteosarcoma with LDH >850 IU/L and/or ALP >280 IU/L it may be prudent to consider more sensitive staging investigations for detection of skeletal metastases. Further research is required to determine the value and the most sensitive cut-off points of serum ALP and LDH in the prediction of skeletal metastases. PMID

  19. Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase.

    PubMed Central

    Hoylaerts, M F; Manes, T; Millán, J L

    1992-01-01

    Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its

  20. Toxic impact of aldrin on acid and alkaline phosphatase activity of penaeid prawn, Metapenaeus monoceros: In vitro study

    SciTech Connect

    Reddy, M.S.; Jayaprada, P.; Rao, K.V.R. )

    1991-03-01

    The increasing contamination of the aquatic environment by the indiscriminate and widespread use of different kinds of pesticides is a serious problem for environmental biologists. Organochlorine insecticides are more hazardous since they are not only more toxic but also leave residues in nature. The deleterious effects of aldrin on several crustaceans have been studied. But studies concerning the impact of aldrin on biochemical aspects of crustaceans are very much limited. The present study is aimed at probing the in vitro effects of aldrin on the acid and alkaline phosphatase activity levels in selected tissues of penaeid prawn, Metapenaeus monoceros (Fabricius).

  1. Determination of urinary and serum beta-glucuronidase and alkaline phosphatase in various renal disease and kidney rejection transplanted patients.

    PubMed

    Refaie, M O; Abo-Zaid, H; Gomma, N A; Aboul-Enein, H Y

    2000-05-01

    Beta-glucuronidase (beta-Glu) and alkaline phosphatase (ALP) were evaluated in serum and urine in 50 subjects classified into five equal groups. Group I was control healthy subjects, while groups II, III, IV, and V were patients with nephritic syndrome, pyelonephritis, kidney rejection, and end-stage renal disease, respectively. Urinary beta-Glu was significantly elevated in all four groups; while serum enzyme showed no change. On the other hand, serum ALP showed a significant elevation in all abnormal groups. Accordingly, urinary beta-Glu and serum ALP could be used as diagnostic markers for various renal diseases.

  2. Poly(vinyl chloride) tubing with covalently bound alkaline phosphatase and alternative approach for investigations of open-tubular bioreactors.

    PubMed

    Rozum, Beata; Gajownik, Kamil; Tymecki, Lukasz; Koncki, Robert

    2010-05-01

    A one-step carbodiimide method was found to allow covalent binding of enzymes to the inner wall of poly(vinyl chloride) (PVC) tubing. The immobilization is performed under mild conditions without laborious pretreatment or activation of reactor surface. In these preliminary studies, alkaline phosphatase (ALP, EC 3.1.3.1) and p-nitrophenyl phosphate (NPP) were applied as a model enzyme and substrate, respectively. The resulting open-tubular bioreactor exhibits satisfactory operational and storage stability. In addition, a novel and very simple instrumental concept for optical monitoring of the biocatalytic process directly inside the microbioreactor using a system of paired emitter-detector diodes is presented.

  3. Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles.

    PubMed Central

    Huang, T M; Hung, H C; Chang, T C; Chang, G G

    1998-01-01

    Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2, 4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (pi) for deuterium for the transition state (piT) increased when

  4. Tumour-derived alkaline phosphatase regulates tumour growth, epithelial plasticity and disease-free survival in metastatic prostate cancer

    PubMed Central

    Rao, S R; Snaith, A E; Marino, D; Cheng, X; Lwin, S T; Orriss, I R; Hamdy, F C; Edwards, C M

    2017-01-01

    Background: Recent evidence suggests that bone-related parameters are the main prognostic factors for overall survival in advanced prostate cancer (PCa), with elevated circulating levels of alkaline phosphatase (ALP) thought to reflect the dysregulated bone formation accompanying distant metastases. We have identified that PCa cells express ALPL, the gene that encodes for tissue nonspecific ALP, and hypothesised that tumour-derived ALPL may contribute to disease progression. Methods: Functional effects of ALPL inhibition were investigated in metastatic PCa cell lines. ALPL gene expression was analysed from published PCa data sets, and correlated with disease-free survival and metastasis. Results: ALPL expression was increased in PCa cells from metastatic sites. A reduction in tumour-derived ALPL expression or ALP activity increased cell death, mesenchymal-to-epithelial transition and reduced migration. Alkaline phosphatase activity was decreased by the EMT repressor Snail. In men with PCa, tumour-derived ALPL correlated with EMT markers, and high ALPL expression was associated with a significant reduction in disease-free survival. Conclusions: Our studies reveal the function of tumour-derived ALPL in regulating cell death and epithelial plasticity, and demonstrate a strong association between ALPL expression in PCa cells and metastasis or disease-free survival, thus identifying tumour-derived ALPL as a major contributor to the pathogenesis of PCa progression. PMID:28006818

  5. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

    PubMed Central

    Chen, L; Rhoads, D; Tai, P C

    1985-01-01

    We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins. Images PMID:3882674

  6. Ultrasensitive detection of amifostine and alkaline phosphatase based on the growth of CdS quantum dots.

    PubMed

    Na, Weidan; Liu, Siyu; Liu, Xiaotong; Su, Xingguang

    2015-11-01

    In this study, we reported a simple and sensitive fluorescence nanosensor for rapid detection of amifostine and alkaline phosphatase (ALP). The novel nanosensor was based on the fluorescence "turn on-off" of CdS quantum dots (QDs). Firstly, Cd(2+) cation could react with S(2-) anion to generate fluorescent CdS QDs in the presence of amifostine. The fluorescence (FL) intensity of amifostine-capped CdS QDs (Amifostine-CdS QDs) was increased with the increasing amounts of amifostine, and could be used for amifostine detection. However, amifostine could be converted to 2-(3-aminopropylamino) ethanethiol (WR1065) in the presence of ALP based on the dephosphorylation of ALP. Under the optimum conditions, the affinity of WR1065 to CdS QDs was weaker than that of amifostine. Therefore the new generation of WR1065-CdS QDs would reduce the FL intensity with the increase of ALP concentration, and the fluorescence of CdS QDs was turn off. The metabolic process of amifostine in the presence of alkaline phosphatase could be also studied via the change of FL intensity of CdS QDs. The present method was cost-effective, convenient, and does not require any complicated synthetic procedures.

  7. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle*

    PubMed Central

    Panosian, Timothy D.; Nannemann, David P.; Watkins, Guy R.; Phelan, Vanessa V.; McDonald, W. Hayes; Wadzinski, Brian E.; Bachmann, Brian O.; Iverson, Tina M.

    2011-01-01

    Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-d-ribose 5-phosphate (ribose 5-phosphate) and α-d-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-d-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn2+-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[18O3]phosphate and [U-13C5]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle. PMID:21193409

  8. Use of solid phase extraction for the sequential injection determination of alkaline phosphatase activity in dynamic water systems.

    PubMed

    Santos, Inês C; Mesquita, Raquel B R; Bordalo, Adriano A; Rangel, António O S S

    2012-08-30

    In this work, a solid phase extraction sequential injection methodology for the determination of alkaline phosphatase activity in dynamic water systems was developed. The determination of the enzymatic activity was based on the spectrophotometric detection of a coloured product, p-nitrophenol, at 405 nm. The p-nitrophenol is the product of the catalytic decomposition of p-nitrophenyl phosphate, a non-coloured substrate. Considering the low levels expected in natural waters and exploiting the fact of alkaline phosphatase being a metalloprotein, the enzyme was pre-concentrated in-line using a NTA Superflow resin charged with Zn(2+) ions. The developed sequential injection method enabled a quantification range of 0.044-0.441 unit mL(-1) of enzyme activity with a detection limit of 0.0082 unit mL(-1) enzyme activity (1.9 μmol L(-1) of pNP) and a determination rate of 17 h(-1). Recovery tests confirmed the accuracy of the developed sequential injection method and it was effectively applied to different natural waters and to plant root extracts.

  9. Ultrasensitive electrochemical DNAzyme sensor for lead ion based on cleavage-induced template-independent polymerization and alkaline phosphatase amplification.

    PubMed

    Liu, Shufeng; Wei, Wenji; Sun, Xinya; Wang, Li

    2016-09-15

    In this article, a simple, highly sensitive and selective electrochemical DNAzyme sensor for Pb(2+) was developed on the basis of a 8-17 DNAzyme cleavage-induced template-independent polymerization and alkaline phosphatase amplification strategy. The hairpin-like substrate strand (HP DNA) of 8-17 DNAzyme was firstly immobilized onto the electrode. In the presence of Pb(2+) and the catalytic strand of 8-17 DNAzyme, the HP DNA could be cleaved to expose the free 3'-OH terminal, which could be then utilized for the cascade operation by terminal deoxynucleotidyl transferase (TdTase) for the base extension to incorporate biotinylated dUTP (dUTP-biotin). The further conjugated streptavidin-labeled alkaline phosphatase (SA-ALP) then catalyzed conversion of electrochemically inactive 1-naphthyl phosphate (1-NP) for the generation of electrochemical response signal. The currently fabricated Pb(2+) sensor effectively combines triply cascade amplification effects including cyclic Pb(2+)-dependent DNAzyme cleavage, TdTase-mediated base extension and enzymatic catalysis of ALP. An impressive detection limit of 0.043nM toward Pb(2+) with an excellent selectivity could be ultimately obtained, which was superior than most of the electrochemical methods. Thus, the developed amplification strategy opens a promising avenue for the detection of metal ions and may extend for the detection of other nucleic acid-related analytes.

  10. Maltol complexes of vanadium (IV) and (V) regulate in vitro alkaline phosphatase activity and osteoblast-like cell growth.

    PubMed

    Barrio, D A; Braziunas, M D; Etcheverry, S B; Cortizo, A M

    1997-06-01

    Vanadium compounds have been found to possess insulin- and growth factor-mimetic effects. In consequence, these derivatives are potentially useful as effective oral therapeutic agents in diabetic patients. However, their use has been limited by various toxic side-effects and by the low solubility of different derivatives. Recently, vanadium complex with maltol, a sugar used as a common food additive, have been synthesised and investigated in animals, showing possible insulin-mimetic effects with low toxic side-effects. In the present study we have investigated the effect of bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) on bone cells in culture as well as their direct effect on alkaline phosphatase in vitro. A comparison was also made with the action of vanadate and vanadyl cation. Vanadium compounds regulated cell proliferation in a biphasic manner with similar potencies. Osteoblast differentiation, assessed by alkaline phosphatase activity, was found to be dose-dependent, with the inhibitory effect being stronger for vanadate and BMOV than for vanadyl and BMV. All vanadium compounds directly inhibited bovine intestinal ALP with a similar potency. Thus, maltol vanadium derivatives behave in a similar way to vanadate and vanadyl in osteoblast-like UMR 106 cells in culture.

  11. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle

    SciTech Connect

    Panosian, Timothy D.; Nannemann, David P.; Watkins, Guy R.; Phelan, Vanessa V.; McDonald, W. Hayes; Wadzinski, Brian E.; Bachmann, Brian O.; Iverson, Tina M.

    2011-09-15

    Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert {alpha}-D-ribose 5-phosphate (ribose 5-phosphate) and {alpha}-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator {alpha}-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn{sup 2+}-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[{sup 18}O{sub 3}]phosphate and [U-{sup 13}C{sub 5}]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

  12. Chemical redox modulated fluorescence of nitrogen-doped graphene quantum dots for probing the activity of alkaline phosphatase.

    PubMed

    Liu, JingJing; Tang, Duosi; Chen, Zhitao; Yan, Xiaomei; Zhong, Zhou; Kang, Longtian; Yao, Jiannian

    2017-03-08

    Alkaline phosphatase (ALP) as an essential enzyme plays an important role in clinical diagnoses and biomedical researches. Hence, the development of convenient and sensitivity assay for monitoring ALP is extremely important. In this work, on the basis of chemical redox strategy to modulate the fluorescence of nitrogen-doped graphene quantum dots (NGQDs), a novel label-free fluorescent sensing system for the detection of alkaline phosphatase (ALP) activity has been developed. The fluorescence of NGQDs is firstly quenched by ultrathin cobalt oxyhydroxide (CoOOH) nanosheets, and then restored by ascorbic acid (AA), which can reduce CoOOH to Co(2+), thus the ALP can be monitored based on the enzymatic hydrolysis of L-ascorbic acid-2-phosphate (AAP) by ALP to generate AA. Quantitative evaluation of ALP activity in a range from 0.1 to 5U/L with the detection limit of 0.07U/L can be realized in this sensing system. Endowed with high sensitivity and selectivity, the proposed assay is capable of detecting ALP in biological system with satisfactory results. Meanwhile, this sensing system can be easily extended to the detection of various AA-involved analytes.

  13. A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

    PubMed Central

    2013-01-01

    Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has

  14. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  15. Application of alkaline phosphatases from different sources in pharmaceutical and clinical analysis for the determination of their cofactors; zinc and magnesium ions.

    PubMed

    Muginova, Svetlana V; Zhavoronkova, Anna M; Polyakov, Alexei E; Shekhovtsova, Tatyana N

    2007-03-01

    Prospects of using different alkaline phosphatases bearing zinc and magnesium ions in their catalytic and allosteric sites, respectively, in pharmaceutical and clinical analysis were demonstrated. Also their application for the determination of zinc in insulin to control injection quality and magnesium in human urine for the diagnosis and treatment of magnesium deficiency was shown. The reaction of p-nitrophenyl phosphate hydrolysis was chosen as an indicator. The choice of appropriate alkaline phosphatase was substantiated, the influence of the nature of buffer solutions on the behavior of the enzyme-metal systems was studied, and the conditions of the indicator reaction proceeding in the presence of sample matrixes were optimized. Simple, rapid, sensitive, and selective enzymatic procedures for determining zinc and magnesium based on their inhibiting and activating effects on the catalytic activity of alkaline phosphatases from seal and chicken intestine, respectively, were developed.

  16. Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation.

    PubMed

    Cathala, G; Brunel, C

    1975-08-10

    Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly

  17. Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

    PubMed

    Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

    2014-11-04

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar

  18. Levels of transaminases, alkaline phosphatase, and protein in tissues of Clarias gariepienus fingerlings exposed to sublethal concentrations of cadmium chloride.

    PubMed

    Velmurugan, Babu; Selvanayagam, Mariadoss; Cengiz, Elif I; Uysal, Ersin

    2008-12-01

    The freshwater fish, Clarias gariepienus fingerlings, were exposed to sublethal concentrations (1.7 and 3.4 mg/L) of cadmium chloride for 12 days. Aspartate aminotransferase (AAT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total protein levels were assayed in the gill, brain, and muscle of the fish at regular intervals of 6 and 12 days. The activities of AAT, ALT, and ALP of the treated fishes increased significantly in all the tissues compared with the control fish. Protein level in all the tissues showed a significant decrease in comparison to unexposed controls throughout the experimental periods. These results revealed that cadmium chloride effects the intermediary metabolism of C. gariepienus fingerlings and that the assayed enzymes can work as good biomarkers of contamination.

  19. Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe.

    PubMed

    Wang, Xiaobo; Zhang, Zhiyang; Ma, Xiaoyan; Wen, Jinghan; Geng, Zhirong; Wang, Zhilin

    2015-05-01

    An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn(2+) in aqueous medium was synthesized. L-Zn(2+) complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L-Zn(2+) was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

  20. Effect of copper on levels of collagen and alkaline phosphatase activity from chondrocytes in newborn piglets in vitro.

    PubMed

    Yuan, Xue; Wang, Jianguo; Zhu, Xiaoyan; Zhang, Zhigang; Ai, Yongxing; Sun, Guoquan; Wang, Zhe; Liu, Guowen

    2011-12-01

    The effects of different concentrations of copper on collagen content and alkaline phosphatase (AKP) activity from chondrocytes in newborn piglets were measured. Chondrocytes were cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 μmol/L copper in a 12-well culture plate. Collagen content and AKP activity from the chondrocyte extracellular matrix increased significantly in the culture media with 15.6, 31.2, and 62.5 μmol/L copper and was the highest at 31.2 μmol/L copper (P < 0.05). Thus, the results indicated that copper could promote AKP activity and collagen production by chondrocytes.

  1. Grazing-incidence small-angle X-ray scattering from alkaline phosphatase immobilized in atmospheric plasmapolymer coatings

    NASA Astrophysics Data System (ADS)

    Ortore, M. G.; Sinibaldi, R.; Heyse, P.; Paulussen, S.; Bernstorff, S.; Sels, B.; Mariani, P.; Rustichelli, F.; Spinozzi, F.

    2008-06-01

    Grazing-incidence small-angle X-ray scattering (GISAXS) has been used to study proteins embedded in thin polymer films obtained by a new cold, atmospheric-pressure plasma technique. In order to test the efficiency of the technology, four samples of alkaline phosphatase incorporated in organic polymer coatings in different plasma conditions have been investigated. Data have been analysed in the framework of the distorted-wave Born approximation (DWBA), by using a new method for the simultaneous fitting of the two-dimensional diffuse scattering from each sample. As a result, protein film concentration and aggregation state as well as a set of parameters describing the polymer coatings have been obtained.

  2. A continuous-flow method for the determination of the activity of serum alkaline phosphatase in diethanolamine buffer.

    PubMed

    Viitala, A J; Jokela, H A; Penttilä, I M; Nummi, S

    1975-05-01

    A procedure for determination of serum alkaline phosphatase activity (EC 3.1.3.1) in diethanolamine (DEA) buffer with an AutoAnalyzer II apparatus was designed. The buffer used was 1.0 mol/l DEA-HC buffer, pH 9.8 at 37 degree C, containing 0.5 mmol/l of MgCl2 and 10 mmol/l of substrate 4-nitrophenyl-phosphate. The reaction time was about 3 min at 37 degree C. The enzyme activity (U/l) was calculated by determining the amount of 4-nitrophenol formed in reaction. A sampling rate of 70 samples per hour can be used with good linearity up to 1000 U/l. The results obtained by the new continuous-flow system were compared with those measured by the kinetic method according to the Scandinavian recommendation (10). A close correlation between the two methods was observed.

  3. Aloe-emodin induces in vitro G2/M arrest and alkaline phosphatase activation in human oral cancer KB cells.

    PubMed

    Xiao, Bingxiu; Guo, Junming; Liu, Donghai; Zhang, Shun

    2007-10-01

    Aloe-emodin is a natural anthraquinone compound from the root and rhizome of Rheum palmatum. In this study, KB cells were treated with 2.5, 5, 10, 20, and 40 microM aloe-emodin for 1 to 5 days. The results showed that aloe-emodin inhibited cancer cells in a dose-dependent manner. Treatment with aloe-emodin at 10 to 40 microM resulted in cell cycle arrest at G2/M phase. The alkaline phosphatase (ALP) activity in KB cells increased upon treatment with aloe-emodin when compared to controls. This is one of the first studies to focus on the expression of ALP in human oral carcinomas cells treated with aloe-emodin. These results indicate that aloe-emodin has anti-cancer effect on oral cancer, which may lead to its use in chemotherapy and chemopreventment of oral cancer.

  4. Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay

    PubMed Central

    Zuliani-Alvarez, Lorena; Piccinini, Anna M.; Midwood, Kim S

    2017-01-01

    An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-Blue™ cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.

  5. Radionuclide bone scan, radiographic bone survey, and alkaline phosphatase: studies of limited value in asymptomatic patients with ovarian carcinoma

    SciTech Connect

    Metler, F.A. Jr.; Christie, J.H.; Crow, N.E. Jr.; Garcia, J.F.; Wicks, J.D.; Bartow, S.A.

    1982-10-15

    Bone scans or skeletal surveys were obtained in 104 patients with ovarian carcinoma. No metastases were identified at staging in the 43 patients with Stage I or II disease. Four patients in the entire series had osseous metastases. Three of the 40 patients with Stage III epithelian ovarian carcinoma has osseous metastases at the time of staging. All of these were Grade III lesions. One Stage I, Grade III patient demonstrated osseous metastases two years after initial diagnosis. None of the four patients with osseous metastases had an elevated alkaline phosphatase; three of the four had bone pain. Based on these results, it is suggested that radiographic bone survey and radionuclide bone scans are not indicated as screening procedures in asymptomatic patients with ovarian carcinoma.

  6. Skeletal alkaline phosphatase as a serum marker of bone metastases in the follow-up of patients with breast cancer.

    PubMed

    Reale, M G; Santini, D; Marchei, G G; Manna, A; Del Nero, A; Bianco, V; Marchei, P; Frati, L

    1995-01-01

    Immunoradiometric determination of the bone isoenzyme of alkaline phosphatase with a method provided by Hybritech Inc., San Diego CA (USA) was carried out in 145 female patients, 97 of whom with radically operated breast cancer and 48 with benign mammary cysts, in order to evaluate the correlation of serum levels with the metabolic process of bone rearrangement in patients with bone metastases. This study shows that skeletal ALP, having high specificity (86.48%) and sensitivity (78.6%) for early progression (the average anticipation time compared to scintigraphic detection was 101 days) could represent a valid marker for bone metastases in association with mucinous markers in the follow-up of patients operated for breast cancer. In addition, dynamic serum determination of skeletal ALP could be a valid help in monitoring the efficacy of therapy in patients with bone progression.

  7. Copper sulfide nanoparticle-decorated graphene as a catalytic amplification platform for electrochemical detection of alkaline phosphatase activity.

    PubMed

    Peng, Juan; Han, Xiao-Xia; Zhang, Qing-Chun; Yao, Hui-Qin; Gao, Zuo-Ning

    2015-06-09

    Copper sulfide nanoparticle-decorated graphene sheet (CuS/GR) was successfully synthesized and used as a signal amplification platform for electrochemical detection of alkaline phosphatase activity. First, CuS/GR was prepared through a microwave-assisted hydrothermal approach. The CuS/GR nanocomposites exhibited excellent electrocatalytic activity toward the oxidation of ALP hydrolyzed products such as 1-naphthol, which produced a current response. Thus, a catalytic amplification platform based on CuS/GR nanocomposite for electrochemical detection of ALP activity was designed using 1-naphthyl phosphate as a model substrate. The current response increased linearly with ALP concentration from 0.1 to 100 U L(-1) with a detection limit of 0.02 U L(-1). The assay was applied to estimate ALP activity in human serum samples with satisfactory results. This strategy may find widespread and promising applications in other sensing systems that involves ALP.

  8. THE OOGONIA OF MACROALGA UNDARIA PINNATIFIDA ARE ALKALINE-PHOSPHATASE POSITIVE AND CONTAIN GERMINAL BODY-LIKE STRUCTURES(1).

    PubMed

    Alexandrova, Ya N; Reunov, A A

    2008-06-01

    It was observed that in the female gametophyte of Undaria pinnatifida (Harv.) Suringar (Phaeophyta, Laminariales) gametangial initials and maturing oogonia demonstrated different levels of alkaline-phosphatase activity (APA). The oogonia exhibited a higher level of APA than in its initials. Electron-dense granular ovoid structures ∼0.5-0.6 μm were present in the cytoplasm of oogonia. These inclusions were not membrane bound and do not appear to be associated with any particular organelles. The number of the inclusions was 1 to 2 in a single section of the cell. In essential details, the specific APA and subcellular germinal body-like structure of the developing female gamete in U. pinnatifida were very similar to those in metazoan oocytes.

  9. Detection of zeptomolar concentrations of alkaline phosphatase based on a tyrosinase and horse-radish peroxidase bienzyme biosensor.

    PubMed

    Ruan, C; Li, Y

    2001-07-06

    A bienzyme biosensor based on tyrosinase and horse-radish peroxidase is described in a flow injection analysis and cyclic voltammetry for measurement of phenol. Tyrosinase and horse-radish peroxidase were immobilized on the surface of a glassy carbon electrode by bovine serum albumin and glutaric dialdehyde. Phenol was oxidized by tyrosinase and horse-radish peroxidase via catechol to o-quinone in the presence of oxygen and hydrogen peroxide. The o-quinone was reduced to produce catechol (the substrate recycling) on the electrode surface. The enhanced sensitivity of the bienzyme electrode to phenol was observed in the flow injection system comparing with tyrosinase and horse-radish peroxidase monoenzyme electrodes. The mechanisms for enhanced amperometric response to phenol of bienzyme electrode were discussed. The biosensor was used to detect alkaline phosphatase (ALP). A detection limit of 1.4x10(-15) M ALP (140 zmol/100 mul) was obtained after 1 h incubation with phenyl phosphate.

  10. A Further Investigation of the Effects of Extremely Low Frequency Magnetic Fields on Alkaline Phosphatase and Acetylcholinesterase

    PubMed Central

    Silkstone, Gary; Wilson, Michael T.

    2016-01-01

    Using a custom build spectrophotometer equipped with Helmholtz coils and designed to study the effects of magnetic fields on enzyme reactions in real-time we have investigated the influence of fields, from 100 μT to 10 mT and at a variety of field frequencies, on the membrane bound enzymes alkaline phosphatase and acetylcholinesterase. We have also employed other methods to apply a magnetic field, e.g. Biostim. In contrast to earlier reports we have been unable to detect any field effects on these enzymes under any field/frequency regime. We discuss possible reasons for the discrepancy between this and earlier work and note the particularly complex influence of small temperature changes that may confound analysis. PMID:26963611

  11. INFLUENCE OF EXPERIMENTAL KIDNEY DAMAGE ON HISTOCHEMICALLY DEMONSTRABLE LIPASE ACTIVITY IN THE RAT. COMPARISON WITH ALKALINE PHOSPHATASE ACTIVITY

    PubMed Central

    Wachstein, M.

    1946-01-01

    Lipase activity was found in the cytoplasm of the proximal convoluted tubules in tissue sections of rat, rabbit, dog, mouse, hamster, and guinea pig, stained according to Gomori's method. Uranium and mercury poisoning do not inactivate the enzyme in necrotic cells of the proximal convoluted tubules. Its activity diminished in the atrophic and regenerating cells of the kidneys of rats, surviving the acute phase of the intoxication. In the acute stage of choline deficiency marked reduction in enzymatic activity was seen in the necrotic tubules, and in the atrophied and regenerating tubules in the subacute stage. Lipase activity was markedly diminished in hydronephrotic kidneys 10 to 12 days after ligation of the ureter. In sections stained for alkaline phosphatase activity nearly identical alterations were found. Experimental damage influences both histochemically demonstrable enzymes in a similar manner. PMID:19871551

  12. Ratiometric detection of copper ions and alkaline phosphatase activity based on semiconducting polymer dots assembled with rhodamine B hydrazide.

    PubMed

    Sun, Junyong; Mei, Han; Gao, Feng

    2017-05-15

    The rational surface functionalization of semiconducting polymer dots (Pdots) has attracted much attention to extend their applications in fabricating chemo/biosensing platform. In this study, a novel ratiometric fluorescent sensing platform using functionalized Pdots as probes for fluorescence signal transmission has been designed for sensing Cu(Ⅱ) and activity of alkaline phosphatase (ALP) with high selectivity and enhanced sensitivity. The highly fluorescent Pdots were firstly prepared with Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT) via nanoprecipitation method, and then assembled with non-fluorescent rhodamine B hydrazide (RB-hy), which shows special binding activity to Cu(Ⅱ), through adsorption process to obtain functionalized nanohybrids, Pdots@RB-hy. As thus, a FRET donors/acceptors pair, in which PFBT Pdots act as energy donors while RB-hy-Cu(II) complexes act as energy acceptors were constructed. On the basis of the varies in fluorescence intensities of donors/acceptors in the presence of different amounts of Cu(II), a ratiometric method for sensing Cu(II) has been proposed. The proposed ratiometric Cu(II) sensor shows a good linear detection range from 0.05 to 5μM with a detection limit of 15nM. Furthermore, using the Pdots@RB-hy-Cu(II) system as signal transducer, a ratiometric sensing for alkaline phosphatase (ALP) activity has also been established with pyrophosphate (PPi) as substrates. The constructed ratiometric sensor of ALP activity displays a linear detection range from 0.005 to 15UL(-1) with a detection limit of 0.0018UL(-1). The sensor was further successfully used for ALP activity detection in human serum with satisfactory results.

  13. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    SciTech Connect

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J. )

    1991-05-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

  14. Quantification and comparison of bone-specific alkaline phosphatase with two methods in normal and paget’s specimens

    PubMed Central

    Masrour Roudsari, Jila; Mahjoub, Soleiman

    2012-01-01

    Background: Bone-specific alkaline phosphatase (BAP) is synthesized by the osteoblasts and is presumed to be involved in the calcification of bone matrix, though its precise role in the formation process is unknown. The aim of the present study was to measure the BAP activity in Paget's and normal specimens by two different techniques. Methods: Total ALP (TAP) as well as BAP activity-measuring tests were repeatedly undertaken at different times during the day and different days on the serum samples (inter and intra assay). Precision and repeatability of the phenylalanine inhibition (PHI) and heat inactivation (HI) techniques were approved during ten times repetition of all the tests on two normal samples besides one sample from Paget's disease of bone. The measurement of TAP and BAP activities was also carried out on 50 serum samples from normal adults using the standard IFCC-AACC and the established methods, respectively. Results: Coefficients of Variation (CV) for intra-assay of BAP were 2.33% and 3.16% by HI and PHI methods, respectively. Also, the inter-assay CV of BAP was 2.87% and 3.49% for mentioned methods in Paget's sample, respectively. In addition, the correlation of HI and PHI methods was found to be r= +0.873 for bone-specific isoenzyme. Conclusion: Regarding the appropriate precision, repeatability and correlation of HI and PHI techniques, as well as their cost effectiveness can be of use in the quantification of bone alkaline phosphatase isoenzyme activity, especially when bone is involved. PMID:24009918

  15. Sex-dependent, zinc-induced dephosphorylation of phospholamban by tissue-nonspecific alkaline phosphatase in the cardiac sarcomere

    PubMed Central

    Wang, Yuan; Bishop, Nicole M.; Taatjes, Douglas J.; Narisawa, Sonoko; Millán, José Luis

    2014-01-01

    We have previously reported that Zn2+ infused into the coronary arteries of isolated rat hearts leads to the potent dephosphorylation of phospholamban (PLB) as well as a noticeable but less potent dephosphorylation of the ryanodine receptor 2. We hypothesized in the present study that a Zn2+-activated phosphatase is located in the vicinity of the sarcoplasmic reticulum (SR) where PLB and ryanodine receptor 2 reside. We report here the novel finding of tissue-nonspecific alkaline phosphatase (TNAP), a zinc-dependent enzyme, localized to the SR in the cardiac sarcomere of mouse myocardium. TNAP activity was enhanced by injection of Zn acetate into a tail vein before harvesting the heart and imaged using electron microscopy of electron dense deposits indicative of the hydrolysis of exogenous β-glycerophosphate. TNAP activity was observed localized to the ends of the Z-line corresponding to SR and was qualitatively more visible in myocardium of males compared with females. Correspondingly, PLB phosphorylation status was potently reduced in myocardium of males injected with Zn acetate, whereas there was no apparent effect of Zn acetate injection on PLB phosphorylation in females. Surprisingly, Western blot analysis of TNAP content suggested a significantly lower TNAP content in males compared with females. These data suggest that TNAP plays a role in governing the phosphorylation status of calcium handling proteins in the SR. Furthermore, the content and activity of TNAP are differentially regulated between the sexes and thus may account for some sex differences in cardiopathologies associated with calcium handling. PMID:25015959

  16. A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

    PubMed

    Baranov, Konstantin; Volkova, Olga; Chikaev, Nikolai; Mechetina, Ludmila; Laktionov, Pavel; Najakshin, Alexander; Taranin, Alexander

    2008-03-20

    We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

  17. Sex-dependent, zinc-induced dephosphorylation of phospholamban by tissue-nonspecific alkaline phosphatase in the cardiac sarcomere.

    PubMed

    Wang, Yuan; Bishop, Nicole M; Taatjes, Douglas J; Narisawa, Sonoko; Millán, José Luis; Palmer, Bradley M

    2014-09-15

    We have previously reported that Zn(2+) infused into the coronary arteries of isolated rat hearts leads to the potent dephosphorylation of phospholamban (PLB) as well as a noticeable but less potent dephosphorylation of the ryanodine receptor 2. We hypothesized in the present study that a Zn(2+)-activated phosphatase is located in the vicinity of the sarcoplasmic reticulum (SR) where PLB and ryanodine receptor 2 reside. We report here the novel finding of tissue-nonspecific alkaline phosphatase (TNAP), a zinc-dependent enzyme, localized to the SR in the cardiac sarcomere of mouse myocardium. TNAP activity was enhanced by injection of Zn acetate into a tail vein before harvesting the heart and imaged using electron microscopy of electron dense deposits indicative of the hydrolysis of exogenous β-glycerophosphate. TNAP activity was observed localized to the ends of the Z-line corresponding to SR and was qualitatively more visible in myocardium of males compared with females. Correspondingly, PLB phosphorylation status was potently reduced in myocardium of males injected with Zn acetate, whereas there was no apparent effect of Zn acetate injection on PLB phosphorylation in females. Surprisingly, Western blot analysis of TNAP content suggested a significantly lower TNAP content in males compared with females. These data suggest that TNAP plays a role in governing the phosphorylation status of calcium handling proteins in the SR. Furthermore, the content and activity of TNAP are differentially regulated between the sexes and thus may account for some sex differences in cardiopathologies associated with calcium handling.

  18. Evidence of associations between feto-maternal vitamin D status, cord parathyroid hormone and bone-specific alkaline phosphatase, and newborn whole body bone mineral content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In spite of a high prevalence of vitamin D inadequacy in pregnant women and neonates, relationships among vitamin D status [25(OH)D], parathyroid hormone (PTH), bone specific alkaline phosphatase (BALP), and whole body bone mineral content (WBBMC) in the newborn are poorly characterized. The purpose...

  19. Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

    PubMed

    Goncharenko, A V; Malyuchenko, N V; Moisenovich, A M; Kotlyarova, M S; Arkhipova, A Yu; Kon'kov, A S; Agapov, I I; Molochkov, A V; Moisenovich, M M; Kirpichnikov, M P

    2016-09-01

    3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

  20. Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane.

    PubMed

    Azam, Md Golam; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2011-09-01

    In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27-31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14-20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.

  1. Comparative analysis of alkaline phosphatase-encoding genes (phoX) in two contrasting zones of Lake Taihu.

    PubMed

    Dai, Jiangyu; Chen, Dan; Wu, Shiqiang; Wu, Xiufeng; Zhou, Jie; Tang, Xiangming; Shao, Keqiang; Gao, Guang

    2015-03-01

    Limnetic habitats that are dominated by either algae or macrophytes represent the 2 dominant ecosystems in shallow lakes. We assessed seasonal variations in the diversity and abundance of alkaline phosphate-encoding genes (phoX) in these 2 zones of Lake Taihu, which is a large, shallow, eutrophic lake in China. There was no significant difference in seasonal mean phoX diversity between the 2 zones, whereas the seasonal mean phoX abundance in the macrophyte-dominated region was higher than that in the algae-dominated region. The bulk of the genotypes in the 2 regions were most similar to the alphaproteobacterial and betaproteobacterial phoX. Genotypes most similar to phoX affiliated with Betaproteobacteria were present with greater diversity in the macrophyte-dominated zone than in the algae-dominated zone. In the algae-dominated zone, the relative proportion of genotypes most similar to cyanobacterial phoX was highest (38.8%) in summer. In addition to the different genotype structures and environmental factors between the 2 stable states, the lower gene abundances and higher alkaline phosphatase activities in Meiliang Bay in summer than those in Xukou Bay reveals different organophosphate-mineralizing modes in these 2 contrasting habitats.

  2. Serum alkaline phosphatase and bilirubin are early surrogate markers for ischemic cholangiopathy and graft failure in liver transplantation from donation after circulatory death.

    PubMed

    Halldorson, J B; Rayhill, S; Bakthavatsalam, R; Montenovo, M; Dick, A; Perkins, J; Reyes, J

    2015-03-01

    Liver transplantation with the use of donation after circulatory death (DCD) is associated with ischemic cholangiopathy (IC) often leading to graft loss. We hypothesized that serial postoperative analysis of alkaline phosphatase and bilirubin might identify patients who would later on develop ischemic cholangiopathy and/or graft loss, allowing early recognition and potentially retransplantation. The University of Washington DCD experience totals 89 DCD liver transplantations performed from 2003 to 2011 with Kaplan-Meier estimated 5-year patient and graft survival rates of 81.6% and 75.6%, respectively; 84/89 patients transplanted with DCD livers lived ≥ 60 days after transplantation and were analyzed. Serum bilirubin and alkaline phosphatase levels at 1 week, 2 week, 1 month, and 2 months after transplantation were analyzed. Two-month serum bilirubin and alkaline phosphatase proved to have the strongest associations with development of IC and graft failure. Two-month alkaline phosphatase of <100 U/L had a negative predictive value of 97% for development of IC. Two-month alkaline phosphatase demonstrated an inflection starting at >300 U/L strongly associated with development of IC (P < .0001). Serum bilirubin at 2 months was most strongly associated with graft failure within the 1st year with a strong inflection point at 2.5 mg/dL (P = .0001). All jaundiced recipients at 60 days after transplantation (bilirubin >2.5 mg/dL) developed graft failure within the 1st year (P < .0001). Use of these early surrogate markers could facilitate prioritization and early retransplantation for DCD liver recipients with allografts destined for failure.

  3. A new member of the alkaline phosphatase superfamily with a formylglycine nucleophile: structural and kinetic characterisation of a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum.

    PubMed

    Jonas, Stefanie; van Loo, Bert; Hyvönen, Marko; Hollfelder, Florian

    2008-12-05

    The alkaline phosphatase superfamily comprises a large number of hydrolytic metalloenzymes such as phosphatases and sulfatases. We have characterised a new member of this superfamily, a phosphonate monoester hydrolase/phosphodiesterase from Rhizobium leguminosarum (R/PMH) both structurally and kinetically. The 1.42 A crystal structure shows structural homology to arylsulfatases with conservation of the core alpha/beta-fold, the mononuclear active site and most of the active-site residues. Sulfatases use a unique formylglycine nucleophile, formed by posttranslational modification of a cysteine/serine embedded in a signature sequence (C/S)XPXR. We provide mass spectrometric and mutational evidence that R/PMH is the first non-sulfatase enzyme shown to use a formylglycine as the catalytic nucleophile. R/PMH hydrolyses phosphonate monoesters and phosphate diesters with similar efficiency. Burst kinetics suggest that substrate hydrolysis proceeds via a double-displacement mechanism. Kinetic characterisation of active-site mutations establishes the catalytic contributions of individual residues. A mechanism for substrate hydrolysis is proposed on the basis of the kinetic data and structural comparisons with E. coli alkaline phosphatase and Pseudomonas aeruginosa arylsulfatase. R/PMH represents a further example of conservation of the overall structure and mechanism within the alkaline phosphatase superfamily.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  5. Assessment of the colorimetric and fluorometric assays for alkaline phosphatase activity in cow's, goat's, and sheep's milk.

    PubMed

    Klotz, V; Hill, Art; Warriner, K; Griffiths, M; Odumeru, J

    2008-09-01

    Raw milk is a well-established vehicle for the carriage of human pathogens, and many regulatory bodies have consequently mandated compulsory pasteurization as a food safety intervention. The residual activity of alkaline phosphatase (ALP) has historically been used to verify the adequacy of pasteurization of cow's milk. However, there is uncertainty on how the current ALP standards and methods of analysis can be applied to sheep's and goat's milk, which naturally contain different levels of the enzyme than that found in cow's milk. The official ALP methods applied in Canada (colorimetric assay; MFO-3) and in the United States (Fluorophos) were assessed for their ability to detect enzyme activity in raw and pasteurized milk derived from cows, sheep, and goats. The detection limit and the limit of quantitation were 0.8 and 2.02 microg/ml phenol, respectively, for the MFO-3 method and 43 and 85 mU/liter, respectively, for the Fluorophos method. The average ALP levels in raw goat's, cow's, and sheep's milk were 165, 1,562, and 3,512 microg/ml phenol, respectively. Raw milk detection limits, which correspond to raw milk phosphatase levels, were 0.051, 0.485, and 0.023% in cow's, goat's, and sheep's milk, respectively, for the MFO-3 method and 0.007, 0.070, and 0.004%, respectively, for the Fluorophos method. Although both methods can be used for ALP determination in cow's, goat's, and sheep's milk, the Fluorophos assay was superior to the colorimetric MFO-3 method based on sensitivity and time required to complete the analysis.

  6. The relationship between the acid and alkaline phosphatase activity and the adherence of clinical isolates of Candida parapsilosis to human buccal epithelial cells.

    PubMed

    Fernanado, P H; Panagoda, G J; Samaranayake, L P

    1999-11-01

    Candida parapsilosis is an emerging fungal pathogen implicated in many diseases, especially in compromised hosts. Candidal colonization and infection depends on the initial ability to adhere to host surfaces, which in turn depends upon the cell wall components and the allied structures of both the host and the fungus. Examination of a miscellaneous collection of 24 C. parapsilosis isolates, from both superficial and deep infections, for their potential pathogenic traits displayed a relationship between the phosphatase activity measured with p-nitrophenol phosphate and adhesion of the yeasts to human buccal epithelial cells (BECs). Significant intraspecies differences were seen in both the alkaline and acid phosphatase activity as well as in their adhesion to BECs (p<0.0001). The acid phosphatase activity of the superficial isolates was significantly greater (152%) than that of the systemic isolates (p = 0.0352). A highly significant positive correlation was also established between the yeast adhesion to BECs and both the acid (r = 0.88, p<0.0001) and alkaline (r = 0.9, p<0.0001) phosphatase activity. These relationships, described here for the first time, imply that phosphatases of Candida species may play a crucial role in potentiating their virulence.

  7. The phosphatase SHP2 regulates the spacing effect for long-term memory induction.

    PubMed

    Pagani, Mario R; Oishi, Kimihiko; Gelb, Bruce D; Zhong, Yi

    2009-10-02

    A property of long-term memory (LTM) induction is the requirement for repeated training sessions spaced over time. This augmentation of memory formation with spaced resting intervals is called the spacing effect. We now show that in Drosophila, the duration of resting intervals required for inducing LTM is regulated by activity levels of the protein tyrosine phosphatase corkscrew (CSW). Overexpression of wild-type CSW in mushroom body neurons shortens the inter-trial interval required for LTM induction, whereas overexpression of constitutively active CSW proteins prolongs these resting intervals. These gain-of-function csw mutations are associated with a clinical condition of mental retardation. Biochemical analysis reveals that LTM-inducing training regimens generate repetitive waves of CSW-dependent MAPK activation, the length of which appears to define the duration of the resting interval. Constitutively active CSW proteins prolong the resting interval by altering the MAPK inactivation cycle. We thus provide insight into the molecular basis of the spacing effect.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  9. 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.

  10. In vitro response to alkaline phosphatase coatings immobilized onto titanium implants using electrospray deposition or polydopamine-assisted deposition.

    PubMed

    Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Jansen, John A; Leeuwenburgh, Sander C G

    2014-04-01

    Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioral analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behavior (i.e., cell spreading) and osteogenic differentiation (i.e., proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium.

  11. Steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer and pyrene for alkaline phosphatase fluorescent sensing.

    PubMed

    Song, Chunxia; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Jianbo; Huang, Jin; Zhou, Maogui; Guo, Xiaochen

    2016-03-05

    We herein report a strategy for sensitive alkaline phosphatase (ALP) fluorescent sensing based on steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer (polyβ-CD) and pyrene. The fluorescence of pyrene was enhanced more than 10 times through supramolecular assembly with polyβ-CD. The 5'-phosphorylated dsDNA probe with pyrene attached on the 3'-terminal could be cleaved by λ exonuclease (λ exo), yielding pyrene attached on mononucleotides. Pyrene attached on mononucleotides could easily enter the cavity of polyβ-CD, resulting in fluorescence enhancement. When ALP was introduced, it could remove 5'-phosphate groups from dsDNA and then prevented the cleavage of dsDNA. Pyrene attached on dsDNA was difficult to enter the cavity of polyβ-CD because of steric hindrance, resulting in an inconspicuous fluorescence enhancement. Owing to the excellent fluorescence enhancement during steric hindrance regulated supramolecular assembly, excellent performance of the assay method was achieved for ALP with a detection limit of 0.04 Um L(-1). The detection limit was superior or comparable with the reported methods. Besides, this method was simple in design, avoiding double-labeling of probe.

  12. Shifting nutrient-mediated interactions between algae and bacteria in a microcosm: evidence from alkaline phosphatase assay.

    PubMed

    Liu, Huali; Zhou, Yiyong; Xiao, Wenjuan; Ji, Lei; Cao, Xiuyun; Song, Chunlei

    2012-05-20

    The impacts of different nutrient additions (N + P, N + P + C, 4N + P, 4N + P + C, N + 2P) on the growth of algae and bacteria were studied in a microcosm experiment. Since alkaline phosphatase activity (APA) provides an indication of phosphorus deficiency, the higher value for algal APA in the treatments with excess nitrogen and for bacterial APA in the treatments with excess carbon suggested that, algal and bacterial phosphorus-limited status were induced by abundant nitrogen and carbon input, respectively. Bacterial phosphorus-limited status was weakened due to higher bacterial competition for phosphorus, compared to algae. In comparison with the bacterial and specific bacterial APA, higher values of algal and specific algal APA were found, which showed a gradual increase that coincided with the increase of chlorophyll a concentration. This fact indicated not only a stronger phosphorus demand by algae than by bacteria, but also a complementary relationship for phosphorus demand between algae and bacteria. However, this commensalism could be interfered by glucose input resulting in the decline of chlorophyll a concentration. Furthermore, the correlation between bacterial numbers and chlorophyll a concentration was positive in treatments without carbon and blurry in treatments with carbon. These observations validate a hypothesis that carbon addition can stimulate bacterial growth justifying bacterial nutrient demand, which decreases the availability of nutrients to algae and affects nutrient relationship between algae and bacteria. However, this interference would terminate after algal and bacterial adaption to carbon input.

  13. Steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer and pyrene for alkaline phosphatase fluorescent sensing

    NASA Astrophysics Data System (ADS)

    Song, Chunxia; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Jianbo; Huang, Jin; Zhou, Maogui; Guo, Xiaochen

    2016-03-01

    We herein report a strategy for sensitive alkaline phosphatase (ALP) fluorescent sensing based on steric hindrance regulated supramolecular assembly between β-cyclodextrin polymer (polyβ-CD) and pyrene. The fluorescence of pyrene was enhanced more than 10 times through supramolecular assembly with polyβ-CD. The 5‧-phosphorylated dsDNA probe with pyrene attached on the 3‧-terminal could be cleaved by λ exonuclease (λ exo), yielding pyrene attached on mononucleotides. Pyrene attached on mononucleotides could easily enter the cavity of polyβ-CD, resulting in fluorescence enhancement. When ALP was introduced, it could remove 5‧-phosphate groups from dsDNA and then prevented the cleavage of dsDNA. Pyrene attached on dsDNA was difficult to enter the cavity of polyβ-CD because of steric hindrance, resulting in an inconspicuous fluorescence enhancement. Owing to the excellent fluorescence enhancement during steric hindrance regulated supramolecular assembly, excellent performance of the assay method was achieved for ALP with a detection limit of 0.04 U mL- 1. The detection limit was superior or comparable with the reported methods. Besides, this method was simple in design, avoiding double-labeling of probe.

  14. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

    SciTech Connect

    O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

    2009-05-22

    Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by {approx}3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 {angstrom} X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

  15. Electrochemical sandwich-type biosensors for α-1 antitrypsin with carbon nanotubes and alkaline phosphatase labeled antibody-silver nanoparticles.

    PubMed

    Zhu, Gangbing; Lee, Hye Jin

    2017-03-15

    A novel sandwich-type biosensor was developed for the electrochemical detection of α-1 antitrypsin (AAT, a recognized biomarker for Alzheimer's disease). The biosensor was composed of 3, 4, 9, 10-perylene tetracarboxylic acid/carbon nanotubes (PTCA-CNTs) as a sensing platform and alkaline phosphatase-labeled AAT antibody functionalized silver nanoparticles (ALP-AAT Ab-Ag NPs) as a signal enhancer. CNTs offer high surface area and good electrical conductivity. Importantly, Ag NPs could increase the amount of ALP on the sensing surface and the ALP could dephosphorylate 4-amino phenyl phosphate (APP) enzymatically to produce electroactive species 4-aminophenol (AP). For detecting AAT based on the sandwich-type biosensor, the results show that the peak current value of AP using ALP-AAT Ab-Ag NPs as signal enhancer is much higher than that by using ALP-AAT Ab bioconjugate (without Ag NPs), the biosensor exhibited desirable performance for AAT determination with a wide linearity in the range from 0.05 to 20.0pM and a low detection limit of 0.01pM. Finally, the developed sensor was successfully applied to the analysis of AAT concentration in serum samples.

  16. Molecular defect of tissue-nonspecific alkaline phosphatase bearing a substitution at position 426 associated with hypophosphatasia.

    PubMed

    Al-Shawafi, Hiba A; Komaru, Keiichi; Oda, Kimimitsu

    2017-03-01

    Mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP) cause hypophosphatasia (HPP), a genetic disorder characterized by deficiency of serum ALP and hypomineralization of bone and teeth. Three missense mutations for glycine 426 (by standard nomenclature) of TNSALP have been reported: cysteine (p.G426C), serine (p.G426S), and aspartate (p.G426D). We expressed TNSALP mutants carrying each missense mutation in mammalian cells. All three TNSALP mutants appeared on the cell surface like the wild-type (WT) TNSALP, although the cells expressing each TNSALP mutant exhibited markedly reduced ALP activity. TNSALP (WT) was mainly present as a 140 kDa catalytically active dimeric form, whereas ~80 kDa monomers were the predominant molecular species in the cells expressing TNSALP (p.G426D) or TNSALP (p.G426S), suggesting that aspartate or serine at position 426 may hamper the subunit assembly essential for the enzymatic function of TNSALP. Alternatively, the subunits of TNSALP (p.G426C) were found to be aberrantly cross-linked by disulfide bonds, giving rise to a 200 kDa form lacking ALP activity. Taken together, our results reveal that the amino acid substitutions at position 426 of TNSALP differentially affect the structure and function of TNSALP, leading to understanding of the molecular and cellular basis of HPP.

  17. Identification of alkaline phosphatase genes for utilizing a flame retardant, tris(2-chloroethyl) phosphate, in Sphingobium sp. strain TCM1.

    PubMed

    Takahashi, Shouji; Katanuma, Hiroshi; Abe, Katsumasa; Kera, Yoshio

    2017-03-01

    Tris(2-chloroethyl) phosphate (TCEP) is a haloalkyl phosphate flame retardant and plasticizer that has been recognized as a global environmental contaminant. Sphingobium sp. strain TCM1 can utilize TCEP as a phosphorus source. To identify the phosphomonoesterase involved in TCEP utilization, we identified four putative alkaline phosphatase (APase) genes, named SbphoA, SbphoD1, SbphoD2, and SbphoX-II, in the genome sequence. Following expression of these genes in Escherichia coli, APase activity was confirmed for the SbphoA and SbphoX-II gene products but was not clearly observed for the SbphoD1 and SbphoD2 gene products, owing to their accumulation in inclusion bodies. The single deletion of either SbphoA or SbphoX-II retarded the growth and reduced the APase activity of strain TCM1 cells on medium containing TCEP as the sole phosphorus source; these changes were more marked in cells with the SbphoX-II gene deletion. In contrast, the deletion of either SbphoD1 or SbphoD2 had no effect on cell growth or APase activity. The double deletion of SbphoA and SbphoX-II resulted in the complete loss of cell growth on TCEP. These results show that SbPhoA and SbPhoX-II are involved in the utilization of TCEP as a phosphorus source and that SbPhoX-II is the major phosphomonoesterase involved in TCEP utilization.

  18. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts.

  19. Ingestion of potato starch containing esterified phosphorus increases alkaline phosphatase activity in the small intestine in rats.

    PubMed

    Mineo, Hitoshi; Morikawa, Nao; Ohmi, Sayako; Ishida, Kyo; Machida, Ayaka; Kanazawa, Takumi; Chiji, Hideyuki; Fukushima, Michihiro; Noda, Takahiro

    2010-05-01

    Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters and plays an important role in phosphorus (P) metabolism. Several nutrients in food have been reported to affect intestinal ALP activity in animal models. Previous reports indicated that high levels of P or phosphate in diets decreased intestinal ALP activity in rats. Because potato starch contains considerable amounts of esterified P, unlike other starch-derived plants, we hypothesized that the feeding of potato starch would decrease ALP activity in the intestinal tract. Male Sprague-Dawley rats (7 weeks old) were fed 3 different types of diet containing 60% corn starch or 1 of 2 types of potato starch with different esterified P content for 1 or 5 weeks. Body weight and food intake of each rat were measured every day throughout the experimental periods. At the end of the feeding periods, the small intestine was removed to determine ALP activity in the mucosal tissues. Significant differences were observed in ALP activity in the small intestine between the 2 feeding periods, among the 4 segments of the small intestine, and among the 3 diet groups. Significant positive linear correlations between the amount of P derived from the starch and mucosal ALP activity were obtained in the jejunum and jejunoileum in rats after feeding for 5 weeks. We concluded, contrary to our hypotheses, that the ingestion of potato starch adaptively increases ALP activity in the upper part of the small intestine of growing rats in an esterified P content-dependent manner.

  20. Low molecular weight silk fibroin increases alkaline phosphatase and type I collagen expression in MG63 cells.

    PubMed

    Kim, Jwa-Young; Choi, Je-Yong; Jeong, Jae-Hwan; Jang, Eun-Sik; Kim, An-Sook; Kim, Seong-Gon; Kweon, Hae Yong; Jo, You-Young; Yeo, Joo-Hong

    2010-01-01

    Silk fibroin, produced by the silkworm Bombyx mori, has been widely studied as a scaffold in tissue engineering. Although it has been shown to be slowly biodegradable, cellular responses to degraded silk fibroin fragments are largely unknown. In this study, silk fibroin was added to MG-63 cell cultures, and changes in gene expression in the MG-63 cells were screened by DNA microarray analysis. Genes showing a significant (2-fold) change were selected and their expression changes confirmed by quantitative RT-PCR and western blotting. DNA microarray results showed that alkaline phosphatase (ALP), collagen type-I alpha-1, fibronectin, and transforming growth factor-beta1 expressions significantly increased. The effect of degraded silk fibroin on osteoblastogenic gene expression was confirmed by observing up-regulation of ALP activity in MG-63 cells. The finding that small fragments of silk fibroin are able to increase the expression of osteoblastogenic genes suggests that controlled degradation of silk fibroin might accelerate new bone formation. [BMB reports 2010; 43(1): 52-56].

  1. Ratiometric fluorescent probe for alkaline phosphatase based on betaine-modified polyethylenimine via excimer/monomer conversion.

    PubMed

    Zheng, Fangyuan; Guo, Sihua; Zeng, Fang; Li, Jun; Wu, Shuizhu

    2014-10-07

    Alkaline phosphatase (ALP) is an important diagnostic indicator for a number of human diseases since abnormal level of ALP is closely related to a variety of pathological processes; hence, the development of convenient and reliable assay methods for monitoring ALP is of great significance for medical sciences as well as biological diagnostics. Herein, we report the first ratiometric fluorescent sensing system for ALP. This sensing system consists of two components: the betaine-modified and positively charged polyethylenimine (PEI) and the negatively charged pyrene derivative containing one ALP-responsive phosphate group (Py-P, an aliphatic phosphate ester). In the absence of ALP, the two-component sensing system shows the excimer's emission of Py-P, since Py-P molecules complex with the positively charged polyelectrolyte via electrostatic interactions, leading to the formation of pyrene excimers. While in the presence of ALP, the phosphate moieties are cleaved from Py-P molecules due to the enzymatic reaction, thereby destroying the electrostatic interactions; as a result, the system displays the monomer emission of Py-P. This assay system is operable in aqueous media with a very low detection limit of 0.1 U/mL. The system is capable of detecting ALP in such biological fluid as serum, and this strategy may provide a new and effective approach for designing ratiometric sensing systems for detecting other biomolecules.

  2. Bioconjugation of alkaline phosphatase to mechanically processed, aqueous suspendible electrospun polymer nanofibers for use in chemiluminescent detection assays.

    PubMed

    Mark, Sonny S; Stolper, Samuel I; Baratti, Carla; Park, Jason Y; Taku, Maria A; Santiago-Avilés, Jorge J; Kricka, Larry J

    2008-06-11

    Aqueous suspendible polymer nanostructures were prepared by simple microtome processing of electrospun nylon 6 nanofibers and were used to immobilize calf intestinal alkaline phosphatase (ALP) by either covalent or noncovalent bioconjugation chemistries. It was found that noncovalent immobilization of ALP to the mechanically cut nanofibers (mean length approximately 4 microm; mean diameter approximately 80 nm) using a multi-stacked, layer-by-layer (LBL) approach with the cationic polymer Sapphire II resulted in the highest enzyme loading (48.1 +/- 0.4 microg . mg(-1) nanofiber) when compared to other covalent immobilization methods based on glutaraldehyde crosslinking. The biofunctionalized nanofibers were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD. The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme were influenced by the particular types of immobilization methods employed. In terms of the overall catalytic performance of the various immobilized ALP systems, a single-stacked LBL assembly approach resulted in the highest level of enzymatic activity per unit mass of nanofiber support. To the best of our knowledge, this study represents the first report examining the preparation of mechanically shortened, aqueous dispersed electrospun polymer nanofibers for potential application as enzyme scaffolds in chemiluminescent-based assay systems.

  3. Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    1995-01-01

    A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

  4. Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination.

    PubMed

    Neagu, D; Micheli, L; Palleschi, G

    2006-07-01

    This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4-15 ng mL(-1) with a limit of detection (LOD) of 2 ng mL(-1) (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2-50 ng mL(-1) and the LOD was 1 ng mL(-1).

  5. Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate in frozen model systems.

    PubMed

    Terefe, Netsanet Shiferaw; Mokwena, Kereilemang Khanah; Loey, Ann Van; Hendrickx, Marc E

    2002-01-01

    The alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC-lactose in a temperature range of -28 to 20 degrees C. In the maltodextrin and CMC-lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC-lactose systems with low initial solute concentration, as a consequence of freeze-concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the "rubbery" state of the CMC and CMC-lactose systems. The WLF equation with system-dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.

  6. Serum total and bone alkaline phosphatase levels and their correlation with serum minerals over the lifespan of sheep.

    PubMed

    Sousa, Cristina P; Azevedo, Jorge T; Silva, Amélia M; Viegas, Carlos A; Reis, Rui L; Gomes, Manuela E; Dias, Isabel R

    2014-06-01

    This study aimed to assess serum total alkaline phosphatase (ALP) and its bone isoform (BALP) levels during the ageing and in different physiologic states of sheep, in order to expand the knowledge about the variation of these biomarkers over the sheep lifespan. Ninety female sheep were divided into nine groups of various ages and physiological states (dry, lactation and pregnancy). Serum ALP, BALP and mineral levels were determined by commercial immunoassay, molecular absorbance spectrophotometry and chemical luminescence for BALP determination. Serum ALP and BALP decreased as sheep aged, and no statistically significant differences were obtained between ewes in different physiologic states. The continuous decline of serum BALP concentration along the sheep lifespan, namely in mature and old sheep, is a sign of decreasing bone turnover associated with ageing. Serum calcium concentrations increased slightly until 2 years of age and then showed a tenuous but statistically significant decrease in mature sheep, while serum phosphorus maintained an uninterrupted decrease as sheep matured. The knowledge of serum values of bone biomarkers throughout the sheep lifespan may be useful in preclinical orthopaedic research studies and for animal science studies using sheep.

  7. Matrix gla protein and alkaline phosphatase are differently modulated in human dermal fibroblasts from PXE patients and controls.

    PubMed

    Boraldi, Federica; Annovi, Giulia; Vermeer, Cees; Schurgers, Leon J; Trenti, Tommaso; Tiozzo, Roberta; Guerra, Deanna; Quaglino, Daniela

    2013-04-01

    Mineralization of elastic fibers in pseudoxanthoma elasticum (PXE) has been associated with low levels of carboxylated matrix gla protein (MGP), most likely as a consequence of reduced vitamin K (vit K) availability. Unexpectedly, vit K supplementation does not exert beneficial effects on soft connective tissue mineralization in the PXE animal model. To understand the effects of vit K supplementation and in the attempt to interfere with pathways leading to the accumulation of calcium and phosphate within PXE-mineralized soft connective tissues, we have conducted in vitro studies on dermal fibroblasts isolated from control subjects and from PXE patients. Cells were cultured in standard conditions and in calcifying medium (CM) in the presence of vit K1 and K2, or levamisole, an alkaline phosphatase (ALP) inhibitor. Control and PXE fibroblasts were characterized by a similar dose-dependent uptake of both vit K1 and vit K2, thus promoting a significant increase of total protein carboxylation in all cell lines. Nevertheless, MGP carboxylation remained much less in PXE fibroblasts. Interestingly, PXE fibroblasts exhibited a significantly higher ALP activity. Consistently, the mineralization process induced in vitro by a long-term culture in CM appeared unaffected by vit K, whereas it was abolished by levamisole.

  8. A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.

    PubMed

    Obayashi, Yusuke; Iino, Ryota; Noji, Hiroyuki

    2015-08-07

    Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

  9. A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

    PubMed

    Speel, E J; Schutte, B; Wiegant, J; Ramaekers, F C; Hopman, A H

    1992-09-01

    We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.

  10. The pharmacological role of phosphatases (acid and alkaline phosphomonoesterases) in snake venoms related to release of purines - a multitoxin.

    PubMed

    Dhananjaya, Bhadrapura L; D'Souza, Cletus J M

    2011-02-01

    Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.

  11. Comparative studies with penicillinase, horseradish peroxidase, and alkaline phosphatase as enzyme labels in developing enzyme immunoassay of cortisol.

    PubMed

    Kumari, G Lakshmi; Dhir, Ravindra N

    2003-01-01

    Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market.

  12. Changes in Stoichiometry, Cellular RNA, and Alkaline Phosphatase Activity of Chlamydomonas in Response to Temperature and Nutrients

    PubMed Central

    Hessen, Dag O.; Hafslund, Ola T.; Andersen, Tom; Broch, Catharina; Shala, Nita K.; Wojewodzic, Marcin W.

    2017-01-01

    Phytoplankton may respond both to elevated temperatures and reduced nutrients by changing their cellular stoichiometry and cell sizes. Since increased temperatures often cause increased thermal stratification and reduced vertical flux of nutrients into the mixed zone, it is difficult to disentangle these drivers in nature. In this study, we used a factorial design with high and low levels of phosphorus (P) and high and low temperature to assess responses in cellular stoichiometry, levels of RNA, and alkaline phosphatase activity (APA) in the chlorophyte Chlamydomonas reinhardtii. Growth rate, C:P, C:N, N:P, RNA, and APA all responded primarily to P treatment, but except for N:P and APA, also temperature contributed significantly. For RNA, the contribution from temperature was particularly strong with higher cellular levels of RNA at low temperatures, suggesting a compensatory allocation to ribosomes to maintain protein synthesis and growth. These experiments suggest that although P-limitation is the major determinant of growth rate and cellular stoichiometry, there are pronounced effects of temperature also via interaction with P. At the ecosystem level, nutrients and temperature will thus interact, but temperatures would likely exert a stronger impact on these phytoplankton traits indirectly via its force on stratification regimes and vertical nutrient fluxes. PMID:28167934

  13. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    PubMed

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

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  14. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity.

    PubMed

    Malo, Madhu S

    2015-12-01

    Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/- SEM: 67.4 +/- 3.2 vs 35.3 +/- 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have 'the incipient metabolic syndrome', including 'incipient diabetes', and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a 'temporal IAP profile' might be a valuable tool for predicting 'the incipient metabolic syndrome', including 'incipient diabetes'.

  15. Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses

    PubMed Central

    Evangelista, Monica; Baroudi, Mariama El; Rizzo, Milena; Tuccoli, Andrea; Poliseno, Laura; Pellegrini, Marco; Rainaldi, Giuseppe

    2015-01-01

    In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP+) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP+ I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP+ and AP− I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP+ I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP+ I-MEFs. Together with sestrin 1 upregulation, we found that AP+ I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP+ I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP+ phenotype and achieve a quiescent state characterized by a new transcriptional network. PMID:26740745

  16. Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis.

    PubMed

    Dziedziejko, Violetta; Safranow, Krzysztof; Slowik-Zylka, Dorota; Machoy-Mokrzynska, Anna; Millo, Barbara; Machoy, Zygmunt; Chlubek, Dariusz

    2009-03-01

    Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.

  17. Changes in Stoichiometry, Cellular RNA, and Alkaline Phosphatase Activity of Chlamydomonas in Response to Temperature and Nutrients.

    PubMed

    Hessen, Dag O; Hafslund, Ola T; Andersen, Tom; Broch, Catharina; Shala, Nita K; Wojewodzic, Marcin W

    2017-01-01

    Phytoplankton may respond both to elevated temperatures and reduced nutrients by changing their cellular stoichiometry and cell sizes. Since increased temperatures often cause increased thermal stratification and reduced vertical flux of nutrients into the mixed zone, it is difficult to disentangle these drivers in nature. In this study, we used a factorial design with high and low levels of phosphorus (P) and high and low temperature to assess responses in cellular stoichiometry, levels of RNA, and alkaline phosphatase activity (APA) in the chlorophyte Chlamydomonas reinhardtii. Growth rate, C:P, C:N, N:P, RNA, and APA all responded primarily to P treatment, but except for N:P and APA, also temperature contributed significantly. For RNA, the contribution from temperature was particularly strong with higher cellular levels of RNA at low temperatures, suggesting a compensatory allocation to ribosomes to maintain protein synthesis and growth. These experiments suggest that although P-limitation is the major determinant of growth rate and cellular stoichiometry, there are pronounced effects of temperature also via interaction with P. At the ecosystem level, nutrients and temperature will thus interact, but temperatures would likely exert a stronger impact on these phytoplankton traits indirectly via its force on stratification regimes and vertical nutrient fluxes.

  18. Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-1.

    PubMed

    Nilgiriwala, Kayzad S; Bihani, Subhash C; Das, Amit; Prashar, Vishal; Kumar, Mukesh; Ferrer, Jean Luc; Apte, Shree Kumar; Hosur, M V

    2009-09-01

    Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87.37, c = 168.16 A, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 A resolution at the ESRF.

  19. A simple-potentiometric method for determination of acid and alkaline phosphatase enzymes in biological fluids and dairy products using a nitrophenylphosphate plastic membrane sensor.

    PubMed

    Hassan, Saad S M; Sayour, Hossam E M; Kamel, Ayman H

    2009-04-27

    A novel poly(vinyl chloride) matrix membrane sensor responsive to 4-nitrophenylphosphate (4-NPP) substrate is described, characterized and used for the potentiometric assay of acid (ACP) and alkaline (ALP) phosphatase enzymes. The sensor is based on the use of the ion-association complex of 4-NPP anion with nickel(II)-bathophenanthroline cation as an electroactive material and nitrophenyloctyl ether (NPOE) as a solvent mediator. The sensor displays good selectivity and stability and demonstrates a near-Nernstian response for 4-NPP over the concentration range 9.6x10(-6) to 1.0x10(-2) M with an anionic slope of 28.6+/-0.3 mV decade(-1) and a detection limit of 6.3x10(-6) M over the pH range 4.5-10. The sensor is used to measure the decrease of a fixed concentration of 4-NPP substrate as a function of acid and alkaline phosphatase enzyme activities at optimized conditions of pH and temperature. A linear relationship between the initial rate of 4-NPP substrate hydrolysis and enzyme activity holds over 0.05-3.0 and 0.03-3.4 IU L(-1) of ACP and ALP enzymes, respectively. Validation of the method by measuring the lower detection limit, range, accuracy, precision, within-day repeatability and between-day-variability reveals good performance characteristics of the proposed sensor. The sensor is used for the determination of acid and alkaline phosphatase enzyme activities in biological fluids of some patients suffering from alcoholic cirrhosis, acute myelocytic leukemia, pre-eclampsia and prostatic cancer. The sensor is also utilized for assessment of alkaline phosphatase enzyme in milk and dairy products. The results obtained agree fairly well with data obtained by the standard spectrophotometric methods.

  20. [Some enzymatic activities of the amniotic fluid in human beings (LAP, GGTP, SGOT, SGPT, acid and alkaline phosphatases, 5' nucleotidase, amylase, beta-glucuronidase and aldolase)].

    PubMed

    Galerne, D; Baudon, J; Bruhat, M; Dastugue, G

    1973-10-01

    Quantitative analyses of 10 enzymes (LAP, GGTP, SGOT, SGPT. acid and alkaline phosphatases, 5' nucleotidase, amylase. beta-glucuronidase and aldolase) in a series of 50 samples of amniotic fluid gave widely-scattered results. In some cases, it was possible to relate high enzymatic activity to a pathological condition, in other cases, the amniotic fluid examined seemed to come from normal, full-term or almost full-term pregnancies without particular signs.

  1. Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Movahedi Najafabadi, Bent-al-hoda; Abnosi, Mohammad Hussein

    2016-01-01

    Objective Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). Materials and Methods In this experimental study, BMSCs were extracted and expanded to the 3rdpassage, then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 µg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. Results Although 6 µg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. Conclusion Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture. PMID:27054120

  2. Pst I restriction fragment length polymorphism of human placental alkaline phosphatase gene: Mendelian in segregation and localization of mutation site in the gene

    SciTech Connect

    Tsavaler, L.; Penhallow, R.C.; Sussman, H.H. )

    1988-10-01

    The pattern of inheritance of a Pst I restriction fragment length polymorphism (RFLP) of the human placental alkaline phosphatase gene was studied in nine nuclear families by Southern blot hybridization analysis of genomic DNA. The dimorphic RFLP is defined by the presence of allelic fragments 1.0 kilobase and 0.8 kilobase long. The results of this study show that the two alleles of the Pst I RFLP of the placental alkaline phosphatase gene segregate as codominant traits according to Mendelian expectations. For a polymorphism to be useful as a genetic marker the probability that an offspring is informative (PIC) must be at least 0.15. The allelic frequency of the 1.0-kilobase allele is 0.21, which correlates to a probability that an offspring is informative of 0.275 and is indicative of a useful polymorphism. By using probes derived from different regions of the placental alkaline phosphatase cDNA, the mutated Pst I site causing the RFLP was located in the penultimate intron 2497 base pairs downstream from the transcriptional initiation site.

  3. Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum.

    PubMed

    Raba, J; Mottola, H A

    1994-05-01

    Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).

  4. Serum Alkaline Phosphatase Level as a Prognostic Tool in Colorectal Cancer: A Study of 105 patients.

    PubMed

    Saif, M Wasif; Alexander, Dominik; Wicox, Charles M

    2005-01-01

    BACKGROUND: Serum alkaline phosphotase (ALP) levels are frequently elevated in patients with metastatic colorectal cancer (CRC). However, the significance of ALP in terms of detecting hepatic metastasis or prognosis is not well established. MATERIALS AND METHODS: Medical records of patients with CRC seen at University of Alabama at Birmingham (UAB) (1998-2002) were reviewed and statistical analysis was done to evaluate the significance of ALP as a prognostic tool. The normal range for ALP was quantified at 39 U/L to 117 U/L. Change in ALP levels over time (defined as time interval between two cycles; such as 4 weeks for Mayo regimen, 8 weeks for Roswell Park regimen and 6 weeks for IFL regimen) was categorized as large (120+ U/L), medium (20-119 U/L), and minimal (< 20 U/L). RESULTS: A total of 105 patients with eligible medical records were identified (Mean age: 59 yrs; 53% male; Staging: II: 43 patients, III: 31 patients, IV: 32 patients). Increasing ALP levels correlated with increasing stage (Mean: I = 116, II = 219, III = 302; P = 0.0003). ALP levels were elevated in 74% of patients with liver metastases (Mean, 290) and in 33% without liver metastases (Mean, 122) (P = 0.001). Patients with elevated ALP levels at the most recent time of progression were 5.7 (95% CI, 2.4-13.3) times more likely to have a liver metastases compared to patients with normal levels. Additionally, patients with elevated ALP levels at their most recent visit were 4.2 (95% CI, 1.7-10.7) times more likely to have a worse prognosis compared to patients with normal levels. However, after controlling for the effects of liver metastases, the association between elevated levels and prognosis was no longer significant. After controlling for the effects of age, sex, and liver metastases, large changes in AP levels were associated with a 4.4 (95% CI, 1.0-19.1) times greater odds of having a worse prognosis compared to patients with a minimal change. Patients with an ALP level greater than 160

  5. Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

    PubMed

    Farley, J R; Magnusson, P

    2005-01-01

    Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P <0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the

  6. Deficiency of lipid phosphatase SHIP enables long-term reconstitution of hematopoietic inductive bone marrow microenvironment.

    PubMed

    Liang, Olin D; Lu, Jiayun; Nombela-Arrieta, César; Zhong, Jia; Zhao, Li; Pivarnik, Gregory; Mondal, Subhanjan; Chai, Li; Silberstein, Leslie E; Luo, Hongbo R

    2013-05-28

    A dysfunctional bone marrow (BM) microenvironment is thought to contribute to the development of hematologic diseases. However, functional replacement of pathologic BM microenvironment through BM transplantation has not been possible. Furthermore, the study of hematopoietic inductive BM microenvironment is hampered by the lack of a functional nonhematopoietic reconstitution system. Here, we show that a deficiency of SH2-containing inositol-5'-phosphatase-1 (SHIP) in a nonhematopoietic host microenvironment enables its functional reconstitution by wild-type donor cells. This microenvironment reconstitution normalizes hematopoiesis in peripheral blood and BM and alleviates pathology of spleen and lung in the SHIP-deficient recipients. SHIP-deficient BM contains a significantly smaller population of multipotent stromal cells with distinct properties, which may contribute to the reconstitution by wild-type cells. We further demonstrate that it is the nonhematopoietic donor cells that are responsible for the reconstitution. Thus, we have established a nonhematopoietic BM microenvironment reconstitution system to functionally study specific cell types in hematopoietic niches.

  7. Effect of redox environment on the in vitro and in vivo folding of RTEM-1 beta-lactamase and Escherichia coli alkaline phosphatase.

    PubMed

    Walker, K W; Gilbert, H F

    1994-11-11

    The oxidative folding mechanisms of two Escherichia coli periplasmic proteins, alkaline phosphatase and RTEM-1 beta-lactamase, have been examined in vitro and in vivo. In contrast to eukaryotic proteins, which require a relatively reducing environment for optimal folding rates, both alkaline phosphatase and beta-lactamase fold fastest under very oxidizing conditions. For example, bovine pancreatic ribonuclease exhibits an optimal folding rate in a redox buffer consisting of 1 mM GSH and 0.2 mM GSSG (Lyles, M. M., and Gilbert, H. F. (1991) Biochemistry 30, 613-619); however, both E. coli alkaline phosphatase and beta-lactamase exhibit optimal in vitro folding rates at low concentrations of GSH (< 0.4 mM) and very high concentrations of GSSG (4-8 mM). For both bacterial proteins, GSH inhibits oxidative folding. Under optimal redox conditions, the rate-limiting step for the in vitro oxidative folding of alkaline phosphatase depends on the concentration of the protein, consistent with a mechanism involving rapid oxidation followed by slow dimerization. With beta-lactamase, the oxidative folding mechanism involves a competition between disulfide bond formation and folding of the molecule into a catalytically active conformation that buries the 2 reduced cysteines in the core of the enzyme. The effects of including a thiol reductant in the growth medium on the in vivo folding of alkaline phosphatase and beta-lactamase are similar to the effects observed during in vitro folding of these enzymes. The levels of both oxidized proteins are decreased by GSH in the growth medium. However, addition of a disulfide oxidant to the growth medium does not positively affect the production of either enzyme. These observations are consistent with the idea that the oxidative folding mechanisms of E. coli periplasmic proteins and, by inference, proteins of the eukaryotic endoplasmic reticulum have evolved to accommodate constraints placed on the folding reaction by the folding environment

  8. Molecular Evolution of the Tissue-nonspecific Alkaline Phosphatase Allows Prediction and Validation of Missense Mutations Responsible for Hypophosphatasia*

    PubMed Central

    Silvent, Jérémie; Gasse, Barbara; Mornet, Etienne; Sire, Jean-Yves

    2014-01-01

    ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction. PMID:25023282

  9. 1-step versus 2-step immobilization of alkaline phosphatase and bone morphogenetic protein-2 onto implant surfaces using polydopamine.

    PubMed

    Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Boerman, Otto C; Jansen, John A; Leeuwenburgh, Sander C G

    2013-08-01

    Immobilization of biomolecules onto implant surfaces is highly relevant in many areas of biomaterial research. Recently, a 2-step immobilization procedure was developed for the facile conjugation of biomolecules onto various surfaces using self-polymerization of dopamine into polydopamine. In the current study, a 1-step polydopamine-based approach was applied for alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2) immobilization, and compared to the conventional 2-step polydopamine-based immobilization and plain adsorption. To this end, ALP and BMP-2 were immobilized onto titanium and polytetrafluoroethylene (PTFE) substrates. The absolute quantity and biological activity of immobilized ALP were assessed quantitatively to compare the three types of immobilization. Plain adsorption of both ALP and BMP-2 was inferior to both polydopamine-based immobilization approaches. ALP was successfully immobilized onto titanium and PTFE surfaces via the 1-step approach, and the immobilized ALP retained its enzymatic activity. Using the 1-step approach, the amount of immobilized ALP was increased twofold to threefold compared to the conventional 2-step immobilization process. In contrast, more BMP-2 was immobilized using the conventional 2-step immobilization approach. Retention of ALP and BMP-2 was measured over a period of 4 weeks and was found to be similar for the 1-step and 2-step methods and far superior to the retention of adsorbed biomolecules due to the formation of covalent linkages between catechol moieties and immobilized proteins. The biological behavior of ALP and BMP-2 coatings immobilized using polydopamine (1- and 2-step) as well as adsorption was assessed by culturing rat bone marrow cells, which revealed that the cell responses to the various experimental groups were not statistically different. In conclusion, the 1-step polydopamine-based immobilization method was shown to be more efficient for immobilization of ALP, whereas the conventional 2

  10. Joint effect of phosphorus limitation and temperature on alkaline phosphatase activity and somatic growth in Daphnia magna.

    PubMed

    Wojewodzic, Marcin W; Kyle, Marcia; Elser, James J; Hessen, Dag O; Andersen, Tom

    2011-04-01

    Alkaline phosphatase (AP) is a potential biomarker for phosphorus (P) limitation in zooplankton. However, knowledge about regulation of AP in this group is limited. In a laboratory acclimation experiment, we investigated changes in body AP concentration for Daphnia magna kept for 6 days at 10, 15, 20 and 25 °C and fed algae with 10 different molar C:P ratios (95-660). In the same experiment, we also assessed somatic growth of the animals since phosphorus acquisition is linked to growth processes. Overall, non-linear but significant relationships of AP activity with C:P ratio were observed, but there was a stronger impact of temperature on AP activity than of P limitation. Animals from the lowest temperature treatment had higher normalized AP activity, which suggests the operation of biochemical temperature compensation mechanisms. Body AP activity increased by a factor of 1.67 for every 10 °C decrease in temperature. These results demonstrate that temperature strongly influences AP expression. Therefore, using AP as a P limitation marker in zooplankton needs to consider possible confounding effects of temperature. Both temperature and diet affected somatic growth. The temperature effect on somatic growth, expressed as the Q (10) value, responded non-linearly with C:P, with Q(10) ranging between 1.9 for lowest food C:P ratio and 1.4 for the most P-deficient food. The significant interaction between those two variables highlights the importance of studying temperature-dependent changes of growth responses to food quality.

  11. Coupling technique of self-ordered ring and phosphorimetry for the determination of alkaline phosphatase and diseases prediction

    NASA Astrophysics Data System (ADS)

    Zhang, Li Hong; Zheng, Zhi Yong; Jiang, Shu-Lian; Cui, Ma-Lin; Jiao, Li; Lin, Xuan; Cai, Wen-Lian; Lin, Shao-Qin; liu, Jia-Ming

    2012-11-01

    Rhodamine S could emit strong and stable room temperature phosphorescence (RTP) on polyamide membrane (PAM) in the presence of heavy atom perturber Pb2+. When Rhodamine S-piperidine solution was dropped on PAM, the red (Rhod.S)n-P-SOR (Rhod.S, (Rhod.S)n, P and SOR refer to alizarin red S, multiple Rhod.S molecules, piperidine and self-ordered ring, respectively) formed on PAM, leading to the enhancement of room temperature phosphorimetry (RTP) intensity (Ip, 117.2) of (Rhod.S)n-P-SOR system, which was 2.4 times higher than that without SOR (Ip, 48.1). Wheat germ agglutinin (WGA) was labelled with (Rhod.S)n-P-SOR by the -NH- of Rhod.S reacting with the -COOH of WGA to form WGA-(Rhod.S)n-P-SOR. The formation of WGA-AP-WGA-(Rhod.S)n-P-SOR in the affinity adsorption (AA) reaction carried out between the -COOH of WGA in WGA-(Rhod.S)n-P-SOR and the -NH2 of alkaline phosphatase (AP) caused the RTP intensity (ΔIp) of the WGA-AP-WGA-(Rhod.S)n-P-SOR system 7.8 times larger than that without (Rhod.S)n-P-SOR. Therefore, the coupling technique of SOR and solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace AP has been established. This method possessed good selectivity, high sensitivity (Detection limit (L.D) was 3.4 × 10-16 g mL-1) and accuracy, and it has been applied to the determination of trace AP in human serum and the forecast of human diseases, and the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Besides, the mechanism of the coupling technique for the determination of AP was discussed.

  12. Dephosphorylation of sodium caseinate, enzymatically hydrolyzed casein and casein phosphopeptides by intestinal alkaline phosphatase: implications for iron availability.

    PubMed

    Yeung, A C.; Glahn, R P.; Miller, D D.

    2001-05-01

    Clusters of phosphoserine residues in casein bind iron with high affinity. Casein inhibits iron absorption in humans but partial hydrolysis of casein prior to ingestion diminishes this inhibition. The objective of this study was to test two hypotheses: 1. Partial hydrolysis of the peptide bonds in casein exposes phosphoserine residues to attack by intestinal alkaline phosphatase (IAP). 2. Hydrolysis of the phospho-ester linkage in phosphoserine residues in casein by IAP releases bound iron or inhibits iron chelation, thereby allowing its absorption. Test of hypothesis 1: Suspensions of sodium caseinate (SC), enzymatically hydrolyzed casein (EHC), and casein phosphopeptides (CPP) were subjected to an in vitro pepsin/pancreatin digestion and subsequently incubated in the presence of calf IAP. The rate of release of inorganic phosphate was measured with the following results (expressed as &mgr;mol phosphate released/unit of IAP/min): 0.081, 0.104, 0.139 for SC, EHC, and CPP, respectively. These results are consistent with hypothesis 1. Test of hypothesis 2: (59)Fe-citrate or (59)Fe-citrate + CPP in minimum essential media were spiked with a Na(2)WO(4) solution or water (Na(2)WO(4) is a known inhibitor of IAP) and placed on Caco-2 cell monolayers. Uptake of (59)Fe by the cells was used as an index of iron bioavailability. Na(2)WO(4) did not affect (59)Fe uptake from samples containing only iron but did slightly inhibit (by 10%) uptake from samples containing iron + CPP. These results are consistent with hypothesis 2 and provide a possible explanation for the observation that partial hydrolysis of casein improves iron bioavailability.

  13. The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase.

    PubMed

    Helland, Ronny; Larsen, Renate Lie; Asgeirsson, Bjarni

    2009-02-01

    Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.

  14. Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

    PubMed

    Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.

  15. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  16. Tissue-nonspecific alkaline phosphatase is required for the calcification of collagen in serum: a possible mechanism for biomineralization.

    PubMed

    Price, Paul A; Toroian, Damon; Chan, Wai Si

    2009-02-13

    Previous studies have shown that the type I collagen of tendon and demineralized bone both calcify rapidly in serum. The speed, collagen matrix-type specificity, and extent of the re-calcification of demineralized bone in serum suggest that the serum calcification activity identified in these studies may participate in normal biomineralization. Because of its presence in serum and its long history of association with the normal mineralization of the collagen matrix of bone, tissue-nonspecific alkaline phosphatase (TNAP) is an obvious candidate for a protein that could be a component of serum calcification activity, and experiments were therefore carried out to test this possibility. These experiments show that the inactivation of TNAP in serum prevents collagen calcification, and that the addition of physiological levels of purified TNAP restores the ability of TNAP-deficient serum to calcify collagen. Additional experiments show that the role of TNAP in collagen calcification is to activate a serum nucleator of apatite crystal formation. Based on these and earlier studies, the mechanism of collagen calcification in serum requires at least four elements as follows. 1) A matrix (collagen fibrils) that is accessible to small apatite crystals but not large molecules ( Toroian, D., Lim, J. E., and Price, P. A. (2007) J. Biol. Chem. 282, 22437-22447 ). 2) A large serum nucleator that generates small crystals, some of which diffuse into the fibrils. 3) A source of TNAP to activate the serum nucleator. 4) A large protein (fetuin) that selectively inhibits growth of crystals remaining in solution, thereby ensuring that only crystals within fibrils grow ( Toroian, D. T., and Price, P. A. (2008) Calcif. Tissue Int. 82, 116-126 ).

  17. Pueraria mirifica extract and puerarin enhance proliferation and expression of alkaline phosphatase and type I collagen in primary baboon osteoblasts.

    PubMed

    Tiyasatkulkovit, Wacharaporn; Malaivijitnond, Suchinda; Charoenphandhu, Narattaphol; Havill, Lorena M; Ford, Allen L; VandeBerg, John L

    2014-10-15

    Phytoestrogen-rich Pueraria mirifica (PM) tuberous extract is a promising candidate for the development of anti-osteoporosis drugs for postmenopausal women, but its action has never been validated in humans or in non-human primates, which are more closely related to humans than rodents. In vitro study of non-human primate osteoblasts is thus fundamental to prepare for in vivo studies of phytoestrogen effects on primate bone. This study aimed to establish a culture system of baboon primary osteoblasts and to investigate the effects of PM extract and its phytoestrogens on these cells. Primary osteoblasts from adult baboon fibulae exhibited osteoblast characteristics in regard to proliferation, differentiation, mineralization, and estrogen receptor expression. They responded to 17β-estradiol by increased proliferation rate and mRNA levels of alkaline phosphatase (ALP), type I collagen, and osteocalcin. After being exposed for 48 h to 100 μg/ml PM extract, 1000 nM genistein, or 1000 nM puerarin, primary baboon osteoblasts markedly increased the rate of proliferation and mRNA levels of ALP and type I collagen without changes in Runx2, osterix, or osteocalcin expression. PM extract, genistein, and puerarin also decreased the RANKL/OPG ratio, suggesting that they could decrease osteoclast-mediated bone resorption. However, neither PM extract nor its phytoestrogens altered calcium deposition in osteoblast culture. In conclusion, we have established baboon primary osteoblast culture, which is a new tool for bone research and drug discovery. Furthermore, the present results provide substantial support for the potential of PM extract and its phytoestrogens to be developed as therapeutic agents against bone fragility.

  18. Ratiometric fluorescent response of electrospun fibrous strips for real-time sensing of alkaline phosphatase in serum.

    PubMed

    Zhao, Long; Xie, Songzhi; Song, Xiaojie; Wei, Jiaojun; Zhang, Zhao; Li, Xiaohong

    2017-05-15

    The development of rapid, convenient and reliable assays for monitoring alkaline phosphatase (ALP) levels is valuable for clinical diagnoses and biomedical research. In the current study, a ratiometric assay of ALP activity has been realized by covalent immobilization of fluorescein onto polyethylene terephthalate (PET) fibers, followed by electrostatic adsorption of bisquaternary ammonium salt of tetraphenylethene (TPE-2N(+)). In the absence of ALP, the complex formation between phosphorylated fluorescein and TPE-2N(+) results in the aggregation-induced emission (AIE) of TPE at 471nm. While in the presence of ALP, the hydrolysis of phosphoesters leads to a gradual removal of TPE-2N(+) and the restoration of fluorescein emission at 514nm. Fibers with surface amine densities of 30 nmol/mg show the most significant and almost linear increases in I514/I471 ratios from 0.73 to 3.05 with increasing ALP concentrations from 0 to 100 mU/mL. The ratiometric fluorescence responses result in color changes of fibrous strips from blue (TPE-2N(+)) to green (fluorescein) under an ultraviolet lamp in a matter of minutes. The color changes are more suitable for an eyeball detection of ALP levels ranging from 0 to 80 mU/mL, which is included in the concentration range of ALP in human serum considering the dilution factor if necessary. The ALP detection indicates no apparent interference by serum components and good agreement with enzyme-linked immunosorbent assay (ELISA). Thus, this is the first study on ratiometric fluorescent assay of serum ALP levels by fibrous strips, which offers a capacity to exploit electrospun fibrous mats and ratiometric responses for real-time assays of bioactive substances as self-test devices.

  19. Effects of garlic and diallyl trisulfide on the growth, photosynthesis, and alkaline phosphatase activity of the toxic cyanobacterium Microcystis aeruginosa.

    PubMed

    Wang, Shoubing; Wang, Yuanan; Ma, Xiaoxue; Xu, Ziran

    2016-03-01

    To identify a botanical algicide and elucidate the response of cyanobacteria to the extract from higher plants, the effects of garlic and garlic-derived diallyl trisulfide on Microcystis aeruginosa were studied. Effects were evaluated by changes in cell density, chlorophyll a, maximum effective quantum yield (Fv/Fm), effective quantum yield (YII), non-photochemical quenching (NPQ), and rapid light curves of M. aeruginosa. In addition, alkaline phosphatase activity (APA) was measured when M. aeruginosa was incubated with diallyl trisulfide. Results indicated that the inhibition by garlic and diallyl trisulfide was significant. The 120-h 50 % effective concentrations of garlic and diallyl trisulfide (EC50) were 0.75 g L(-1) and 2.84 mg L(-1), respectively. Moreover, the inhibitory rate increased with increasing concentration and the growth of M. aeruginosa was inhibited by 90.0 % at the highest concentrations. We also show that the response of M. aeruginosa to stress could involve both impairment of the photosynthetic center PSII and alteration of APA. For example, at high garlic concentration (2.0 g L(-1)), Fv/Fm significantly decreased from 0.501 to 0.084 (p < 0.05) after 120 h of exposure. Furthermore, the total APA was significantly decreased by exposure to a high diallyl trisulfide concentration after 24 h exposure. As new algal inhibitors, there are several advantages for their utilization, such as being common, cheap, non-toxic, and with high efficiency. It would be meaningful to further research on garlic as an environmentally friendly algicide.

  20. Prognostic value of combined preoperative lactate dehydrogenase and alkaline phosphatase levels in patients with resectable pancreatic ductal adenocarcinoma

    PubMed Central

    Ji, Fei; Fu, Shun-Jun; Guo, Zhi-Yong; Pang, Hui; Ju, Wei-Qiang; Wang, Dong-Ping; Hua, Yun-Peng; He, Xiao-Shun

    2016-01-01

    Abstract Serum enzymes, including lactate dehydrogenase (LDH) and alkaline phosphatase (ALP), have recently been reported to play important roles in tumor growth. Increases in LDH and ALP have been confirmed to predict poor prognosis in patients with various cancers. However, their prognostic value in pancreatic cancer has not been well studied. Therefore, we reviewed the preoperative data on LDH and ALP in 185 pancreatic ductal adenocarcinoma (PDAC) patients who underwent surgery between July 2005 and December 2010 to explore the prognostic value of these markers. The cutoff points were determined based on the upper limit of their normal values. The Chi-square test was used to analyze the relationships between LDH/ALP and clinical characteristics. Univariate and multivariate analyses were performed to identify the predictive value of the above factors for disease-free survival (DFS) and overall survival (OS). We found that elevation of LDH was related to carbohydrate antigen 19-9 (CA19-9), lymph node involvement, tumor size, TNM, distant metastasis, and recurrence. Additionally, ALP was correlated to perineural invasion. After multivariate analysis, LDH and ALP were identified as independent prognostic factors for DFS and OS, and elevation of LDH/ALP was correlated with poor DFS and OS. Notably, there was a positive correlation between LDH and ALP. The predictive power of LDH combined with ALP was more sensitive than that of either one alone. Therefore, we conclude that the preoperative LDH and ALP values are prognostic factors for PADC, and the prognostic accuracy of testing can be enhanced by the combination of LDH and ALP PMID:27399091

  1. The preoperative alkaline phosphatase-to-platelet ratio index is an independent prognostic factor for hepatocellular carcinoma after hepatic resection

    PubMed Central

    Yu, Ya-Qun; Li, Jun; Liao, Yan; Chen, Qian; Liao, Wei-Jia; Huang, Jian

    2016-01-01

    Abstract A simple, inexpensive, and readily available prognostic index is highly needed to accurately predict the prognosis of hepatocellular carcinoma (HCC). This study aimed to develop a simple prognostic index using routine laboratory tests, alkaline phosphatase-to-platelet count ratio index (APPRI), to predict the likelihood of postoperative survival in HCC patients. A total of 246 patients with HCC undergoing curative resection were retrospectively analyzed. Cutoff point for APPRI was calculated using receiver operating characteristic curve analysis, and then the patients were divided into the low-APPRI group (APPRI ≤ 4.0) and the high-APPRI group (APPRI > 4.0). The influences of APPRI on disease-free survival (DFS) and overall survival (OS) were tested by the Kaplan–Meier method, and multivariate analysis using Cox regression. Elevated APPRI was associated with age, cirrhosis, and aspartate aminotransferase (AST) in HCC. Univariate analysis showed that APPRI > 4.0, tumor size >6 cm, multiple tumors, Barcelona-clinic liver cancer stages B to C, and AST > 40 U/L were significant predictors of worse DFS and OS. A multivariate analysis suggested that APPRI > 4.0 was an independent factor for DFS (hazard ratio [HR] = 1.689; 95% confidence interval [CI], 1.139–2.505; P = 0.009) and OS (HR = 1.664; 95% CI, 1.123–2.466; P = 0.011). Preoperative APPRI > 4.0 was a powerful prognostic predictor of adverse DFS and OS in HCC after surgery. The APPRI may be a promising prognostic marker for HCC after surgical resection. PMID:28002346

  2. TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation.

    PubMed

    Jansen, Jos C; Timal, Sharita; van Scherpenzeel, Monique; Michelakakis, Helen; Vicogne, Dorothée; Ashikov, Angel; Moraitou, Marina; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; van den Boogert, Marjolein A W; Porta, Francesco; Calvo, Pier Luigi; Mavrikou, Mersyni; Cenacchi, Giovanna; van den Bogaart, Geert; Salomon, Jody; Holleboom, Adriaan G; Rodenburg, Richard J; Drenth, Joost P H; Huynen, Martijn A; Wevers, Ron A; Morava, Eva; Foulquier, François; Veltman, Joris A; Lefeber, Dirk J

    2016-02-04

    Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.

  3. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae

    PubMed Central

    Fernandez, Luisa E.; Aimanova, Karlygash G.; Gill, Sarjeet S.; Bravo, Alejandra; Soberón, Mario

    2005-01-01

    A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI–ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI–ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI–ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI–ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae. PMID:16255715

  4. Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa

    PubMed Central

    Wang, Shoubing; Xu, Ziran

    2016-01-01

    Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice. PMID:27755566

  5. Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers

    PubMed Central

    Ahmadi-Motamayel, Fatemeh; Falsafi, Parisa; Goodarzi, Mohammad T.; Poorolajal, Jalal

    2016-01-01

    Objectives: Saliva contains alkaline phosphatase (ALP)—a key intracellular enzyme related to destructive processes and cellular damage—and has buffering capacity (BC) against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. Methods: This retrospective cohort study took place between August 2012 and December 2013. A total of 251 healthy male non-smokers and 259 male smokers from Hamadan, Iran, were selected. Unstimulated whole saliva was collected from each participant and pH and BC were determined using a pH meter. Salivary enzymes were measured by spectrophotometric assay. Results: Mean salivary pH (7.42 ± 0.48 and 7.52 ± 0.43, respectively; P = 0.018) and BC (3.41 ± 0.54 and 4.17 ± 0.71; P = 0.001) was significantly lower in smokers compared to non-smokers. Mean ALP levels were 49.58 ± 23.33 IU/L among smokers and 55.11 ± 27.85 IU/L among non-smokers (P = 0.015). Conclusion: Significantly lower pH, BC and ALP levels were observed among smokers in comparison to a healthy control group. These salivary alterations could potentially be utilised as biochemical markers for the evaluation of oral tissue function and side-effects among smokers. Further longitudinal studies are recommended to evaluate the effects of smoking on salivary components. PMID:27606111

  6. Probing the origin of the compromised catalysis of E. coli alkaline phosphatase in its promiscuous sulfatase reaction.

    PubMed

    Catrina, Irina; O'Brien, Patrick J; Purcell, Jamie; Nikolic-Hughes, Ivana; Zalatan, Jesse G; Hengge, Alvan C; Herschlag, Daniel

    2007-05-02

    The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.

  7. A study of the association between serum bone-specific alkaline phosphatase and serum phosphorus concentration or dietary phosphorus intake.

    PubMed

    Haraikawa, Mayu; Tanabe, Rieko; Sogabe, Natsuko; Sugimoto, Aoi; Kawamura, Yuka; Michigami, Toshimi; Hosoi, Takayuki; Goseki-Sone, Masae

    2012-01-01

    Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters into phosphoric acid and alcohol at a high optimum pH (pH 8-10). Human ALPs are classified into four types: tissue-non specific (TNSALP, liver/bone/kidney), intestinal, placental, and germ cell types. Based on studies of hypophosphatasia (HPP), which is a systemic bone disease caused by the presence of either one or two pathologic mutations in ALPL that encodes TNSALP, TNSALP was suggested to be indispensable for skeletal mineralization. In this study, we explored the possibility that dietary nutrients contribute to regulate serum bone-specific ALP (BAP) activity. Serum biochemical parameters, such as serum ALP, BAP, osteocalcin, and fibroblast growth factor 23 (FGF23), were measured in healthy young subjects (n=193). Dietary nutrient intakes were measured based on 3-d food records before the day of blood examinations. The presence of a carrier of the deletion of T at nucleotide 1559 (c.1559delT), which has been reported to be the most frequent in Japanese HPP, was not detected in any subject. By the analysis of BAP activity and other biochemical parameters or dietary nutrient intakes, we obtained significant correlations between BAP activity and serum phosphorus (r=-0.165, p=0.022), calcium intake (mg/1,000 kcal/d) (r=-0.186, p=0.010), or phosphorus intake (mg/1,000 kcal/d) (r=-0.226, p=0.002). Further study on the regulation of BAP activity and calcium and/or phosphorus homeostasis will provide useful data for improving skeletal health.

  8. One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.

    PubMed

    Wang, Jia; Majkova, Zuzana; Bever, Candace R S; Yang, Jun; Gee, Shirley J; Li, Ji; Xu, Ting; Hammock, Bruce D

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL(-1). Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

  9. Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities.

    PubMed

    Liu, C; Sanghvi, R; Burnell, J M; Howard, G A

    1987-01-01

    Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Von Willebrand factor and alkaline phosphatase predict re-transplantation-free survival after the first liver transplantation

    PubMed Central

    Wannhoff, Andreas; Rauber, Conrad; Friedrich, Kilian; Rupp, Christian; Stremmel, Wolfgang; Weiss, Karl Heinz; Schemmer, Peter

    2016-01-01

    Background After liver transplantation (LT), there are liver-related, infectious and cardiovascular complications that contribute to reduced graft survival. These conditions are associated with an increase in the Von Willebrand factor antigen (VWF-Ag), which was previously correlated with survival in cirrhotic patients. Objective Evaluate VWF-Ag as a predictive marker of re-transplantation-free survival in patients after LT. Methods We measured VWF-Ag in patients after first LT and then followed them prospectively with regard to the primary endpoint, namely re-transplantation-free survival. Results There were 6 out of 80 patients who died or received re-LT during follow-up. In these patients, the median VWF-Ag was 510.6%, which was significantly higher (p = 0.001) than in the patients who were alive at the end of follow-up (with a median VWF-Ag = 186.8%). At a cut-off of 286.8%, VWF-Ag was significantly correlated with re-transplantation-free survival (p < 0.001). VWF-Ag was independently associated with re-transplantation-free survival in a multivariate analysis; as was alkaline phosphatase (ALP), but not the model of end-stage liver disease (MELD) score, donor age, nor cold ischemia time. A score combining VWF-Ag and ALP showed an impressive capability in the receiver operating characteristic (ROC) analysis (with area under the curve (AUC) = 0.958) to distinguish between patients with regard to the primary endpoint. Conclusions VWF-Ag is a non-invasive marker that can predict outcome in patients after LT. Its diagnostic performance increased when combined with ALP in a newly developed scoring system.

  11. Amplified thrombin aptasensor based on alkaline phosphatase and hemin/G-quadruplex-catalyzed oxidation of 1-naphthol.

    PubMed

    Yang, Zhe-Han; Zhuo, Ying; Yuan, Ruo; Chai, Ya-Qin

    2015-05-20

    An alkaline phosphatase (ALP)-based biosensor can in situ generate an electroactive product by enzymatic hydrolysis of inactive substrates. To obtain a higher signal-to-background ratio, a chemical redox cycling signal-amplified strategy based on the addition of a strong reducing agent has often be applied in the construction of ALP-based biosensors. However, the strong reducing agent not only affects the activity of ALP but also readily reacts with dissolved oxygen, leading to inaccurate results. In this work, a new signal-amplified strategy for a thrombin (TB) aptasensor based on the catalytic oxidation of ALP-generated products, 1-naphthol (NP), using hemin/G-quadruplex DNAzymes was reported. We implemented gold-nanoparticle-decorated zinc oxide nanoflowers (Au-ZnO) as the matrix for immobilizing ALP and TB aptamer (TBA) and then labeled it with hemin to form hemin/G-quadruplex/ALP/Au-ZnO bioconjugates (TBA II bioconjugates). Through a "sandwich" reaction, TBA II bioconjugates were captured on the electrode surface. The amplified signal was carried out in two steps: (i) an ALP-catalyzed inactive substrate, 1-naphthyl phosphate (NPP), in situ produces NP on the surface of the electrode; (ii) on the one hand, NP as a new reactant could be directly electrooxidized and generated an electrochemical signal, but, on the other hand, NP could be oxidized by hemin/G-quadruplex in the presence of H2O2, resulting in amplification of the electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM to 30 nM with a detection limit of 0.37 pM (defined as S/N = 3).

  12. Pre-treatment serum alkaline phosphatase and lactate dehydrogenase as prognostic factors in triple negative breast cancer

    PubMed Central

    Chen, Bo; Dai, Danian; Tang, Hailin; Chen, Xi; Ai, Xiaohong; Huang, Xiaojia; Wei, Weidong; Xie, Xiaoming

    2016-01-01

    Background: Serum parameters as prognostic parameters are studied widely. We aim to examine the prognostic significance of the serum alkaline phosphatase (ALP) level and lactate dehydrogenase (LDH) level in triple negative breast cancer (TNBC). Methods: Total of 253 TNBC patients from Sun Yat-sen University Cancer Center who underwent treatment between January 2004 and December 2009 was conducted in this retrospective study. Before treatment serum ALP and LDH levels were routinely measured. We use the receiver operating characteristic (ROC) curve analysis to estimate the cutoff value of serum ALP and LDH levels. The Kaplan-Meier method and multivariable Cox regression analysis were used for Disease free survival (DFS) and overall survival (OS) assessed. Results: The ROC curves determined that the optimum cutoff point for ALP and LDH were 66.5u/L and 160.5u/L, respectively. The elevated ALP and LDH were both significantly associated with decreased DFS and OS (both P < 0.001). In addition, the entire cohort was stratified into three subgroups basis of ALP levels and LDH levels. TNBC Patients who with ALP >66.5 u/L and LDH >160.5u/L had the worst DFS and OS (both P < 0.001). In TNBC patients, univariate and multivariate Cox regression analyses conformed ALP and LDH were independent unfavorable prognostic factors for DFS and OS. Conclusions: The serum levels of ALP and LDH before treatment are independent prognostic parameters and may serve as complement to help predict survival in TNBC. PMID:27994669

  13. Distinct expression of alkaline phosphatase activity in epilimnetic bacteria: Implication for persistent DOC consumption in a P-limited reservoir

    NASA Astrophysics Data System (ADS)

    Tseng, Y.; Kao, S.; Shiah, F.

    2013-12-01

    In a P-deficient system, P availability usually controls the microbial activity and thus the ecosystem function. Thingstad et al. (1997) first addressed a 'Malfunctioning Microbial-loop' theory, which stated that low bacterial production (BP) caused by insufficient nutrient supply would result in DOC accumulation in an oligotrophic ecosystem. In this study we re-examined the theory by conducting seasonal patterns and correlations among soluble reactive phosphate (SRP) and DOC, microbial abundances (picocyanobacteria, bacteria, and heterotrophic nanoflagellate; HNF) and activities (primary production, bacterial production, and alkaline phosphatase activity; APA) coupled with enzyme-labeled fluorescence (ELF) assays on bacterioplankton in a subtropical reservoir sharing the common features, nitrate-replete and P-deficient, with most natural freshwater system during Oct 2007-Oct 2008. Persistently high APA was recorded during most of time, implying that the system was P-deficient. Size fractionated APA and ELF assay revealed that bacteria were the major APA contributor. However, significantly low epilimnion DOC was recorded during the stratified summer season accompanying with high BP and APA as well as high PP, implying that heterotrophic bacteria can well sustain in P-deficient system by utilizing DOP to rapidly lower down DOC under relatively high PP. Such findings oppose the 'Malfunctioning Microbial-loop' theory. On the other hand, strong epilimnetic DOC accumulation occurred in Oct 2007 under low light and low PP condition accompanying with high abundance of HNF, implying that HNF grazing may contribute to a certain degree of DOC accumulation. Correlation matrix supported our suggestions. This study testified the DOC dynamics in P-deficient ecosystem are tightly coupled with the source (PP and grazing) and sink (BP). We also suggested that in SRP-limited freshwater systems bacteria are capable of breaking down autochthonous DOC to reduce the chance of DOC

  14. High Sequence Variability, Diverse Subcellular Localizations, and Ecological Implications of Alkaline Phosphatase in Dinoflagellates and Other Eukaryotic Phytoplankton

    PubMed Central

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort

  15. Real-time ratiometric fluorescent assay for alkaline phosphatase activity with stimulus responsive infinite coordination polymer nanoparticles.

    PubMed

    Deng, Jingjing; Yu, Ping; Wang, Yuexiang; Mao, Lanqun

    2015-03-03

    This study demonstrates a novel ratiometric fluorescent method for real-time alkaline phosphatase (ALP) activity assay with stimulus responsive infinite coordination polymer (ICP) nanoparticles as the probe. The ICP nanoparticles used in this study are composed of two components; one is the supramolecular ICP network formed with guanine monophosphate (GMP) as the ligand and Tb(3+) as the central metal ion, and the other is a fluorescent dye, i.e., 7-amino-4-methyl coumarin (coumarin) encapsulated into the ICP network. Upon being excited at 315 nm, the ICP network itself emits green fluorescence at 552 nm. Coumarin dye encapsulated in the ICP network emits weak fluorescence at 450 nm upon excitation at the same wavelength (315 nm), and this fluorescence emission becomes strong when the encapsulated dye is released from the network into the solution phase. Hence, we develop a ratiometric fluorescent assay based on the ALP-induced destruction of the supramolecular ICP network and the release of coumarin. This mechanism can be used for real-time ratiometric fluorescent monitoring of ALP activity by continuously measuring the ratio of fluorescent intensity at the wavelength of 552 nm (F552) to that at 450 nm (F450) (F552/F450) in the time-dependent fluorescent spectra of the coumarin@Tb-GMP suspension containing ALP with different activities. Under the experimental conditions employed here, the F552/F450 value is linear with the ALP activity within a range from 0.025 U/mL to 0.2 U/mL. The detection limit is down to 0.010 U/mL (S/N = 3). Moreover, the assay developed here is employed for ALP inhibitor evaluation. This study offers a simple yet sensitive method for real-time ALP activity assay.

  16. A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity☆

    PubMed Central

    Malo, Madhu S.

    2015-01-01

    Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/− SEM: 67.4 +/− 3.2 vs 35.3 +/− 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have ‘the incipient metabolic syndrome’, including ‘incipient diabetes’, and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a ‘temporal IAP profile’ might be a valuable tool for predicting ‘the incipient metabolic syndrome’, including ‘incipient diabetes’. PMID:26844282

  17. One-step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody-Alkaline Phosphatase Fusion Protein

    PubMed Central

    Wang, Jia; Majkova, Zuzana; Bever, Candace R. S.; Yang, Jun; Gee, Shirley J.; Li, Ji; Xu, Ting; Hammock, Bruce D.

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environment and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. Ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL−1. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogs was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples by this one-step assay ranged from 96.7–109.9% and correlated well with an HPLC-MS/MS method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring. PMID:25849972

  18. Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases.

    PubMed

    Holtz, K M; Catrina, I E; Hengge, A C; Kantrowitz, E R

    2000-08-08

    It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

  19. The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

    PubMed

    Shin, Joongho; Carr, Azadeh; Corner, Georgia A; Tögel, Lars; Dávalos-Salas, Mercedes; Dávaos-Salas, Mercedes; Tran, Hoanh; Chueh, Anderly C; Al-Obaidi, Sheren; Chionh, Fiona; Ahmed, Naseem; Buchanan, Daniel D; Young, Joanne P; Malo, Madhu S; Hodin, Richard A; Arango, Diego; Sieber, Oliver M; Augenlicht, Leonard H; Dhillon, Amardeep S; Weber, Thomas K; Mariadason, John M

    2014-09-05

    The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

  20. The Intestinal Epithelial Cell Differentiation Marker Intestinal Alkaline Phosphatase (ALPi) Is Selectively Induced by Histone Deacetylase Inhibitors (HDACi) in Colon Cancer Cells in a Kruppel-like Factor 5 (KLF5)-dependent Manner*

    PubMed Central

    Shin, Joongho; Carr, Azadeh; Corner, Georgia A.; Tögel, Lars; Dávaos-Salas, Mercedes; Tran, Hoanh; Chueh, Anderly C.; Al-Obaidi, Sheren; Chionh, Fiona; Ahmed, Naseem; Buchanan, Daniel D.; Young, Joanne P.; Malo, Madhu S.; Hodin, Richard A.; Arango, Diego; Sieber, Oliver M.; Augenlicht, Leonard H.; Dhillon, Amardeep S.; Weber, Thomas K.; Mariadason, John M.

    2014-01-01

    The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect. PMID:25037223

  1. ALP (Alkaline Phosphatase) Test

    MedlinePlus

    PLEASE NOTE: Your web browser does not have JavaScript enabled. Unless you enable Javascript , your ability to navigate and access the features of this website will be limited. ... Proceeds from website advertising help sustain Lab Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising & Sponsorship: ...

  2. Occurrence of matrix-bound phosphine in polar ornithogenic tundra ecosystems: effects of alkaline phosphatase activity and environmental variables.

    PubMed

    Zhu, Renbin; Ma, Dawei; Ding, Wei; Bai, Bo; Liu, Yashu; Sun, Jianjun

    2011-09-01

    Phosphine (PH(3)), a reduced phosphorus compound, is a highly toxic and reactive atmospheric trace gas. In this study, a total of ten ornithogenic soil/sediment profiles were collected from tundra ecosystems of east Antarctica and Arctic, and matrix-bound phosphine (MBP), the phosphorus fractions and alkaline phosphatase activity (APA) were analyzed. High MBP concentrations were found in these profiles with the range from 39.59 ng kg(-1) dw to 11.77 μg kg(-1) dw. MBP showed a consistent vertical distribution pattern in almost all the soil profiles, and its concentrations increased at soil surface layers and then decreased with depths. MBP levels in the ornithogenic soils were two to three orders of magnitude lower than those in ornithogenic sediments. The yield of PH(3) as a fraction of total P in all the profiles ranged from 10(-5) to 10(-9) mgPH(3) mg(-1)P with higher mean PH(3) yield in the ornithogenic sediments. The ornithogenic soils showed high concentrations of total phosphorus (TP), organic phosphorus (OP), inorganic phosphorus (IP) and metal elements (Cu, Zn, Mn, Fe, Al and Ca) but low MBP levels, vice versa for the ornithogenic sediments. No correlation had been obtained between MBP concentrations and IP, OP and TP. There existed an exponential correlation (r=0.67, p<0.01) between MBP and soil/sediment moisture. MBP concentrations showed a significant positive correlation with APA (r=0.668, p<0.0001), total organic carbon (r=0.501, p<0.0001), total hydrogen (r=0.483, p<0.0001) and total sulfur (r=0.398, p<0.001), indicating that the production of MBP is associated with microbially mediated factors rather than the contents of TP, IP and OP in the P-enriched ornithogenic soils/sediments. Our results indicated that MBP is an important gaseous link in the phosphorus biogeochemical cycles of ornithogenic tundra ecosystems in Antarctica and Arctic.

  3. Enzymatically mediated bioprecipitation of heavy metals from industrial wastes and single ion solutions by mammalian alkaline phosphatase.

    PubMed

    Chaudhuri, Gouri; Shah, Gaurav A; Dey, Pritam; S, Ganesh; Venu-Babu, P; Thilagaraj, W Richard

    2013-01-01

    The study was aimed at investigating the potential use of calf intestinal alkaline phosphatase (CIAP) enzyme in the removal of heavy metals (Cd(2+), Ni(2+), Co(2+) and Cr(3+/6+)) from single ion solutions as well as tannery and electroplating effluents. CIAP mediated bioremediation (white biotechnology) is a novel technique that is eco-friendly and cost effective unlike the conventional chemical technologies. Typical reactions containing the enzyme (CIAP) and p-nitrophenyl phosphate (pNPP) as substrate in Tris-HCl buffer (pH 8 and 11) and either single ion metal solutions (250 ppm and 1000 ppm) or effluents from tannery or electroplating industry were incubated at 37°C for 30 min, 60 min and 120 min. The inorganic phosphate (P(i)) generated due to catalytic breakdown of pNPP complexes free metal ions as metal-phosphate and the amount of metal precipitated was derived by estimating the reduction in the free metal ion present in the supernatant of reactions employing atomic absorption spectrophotometer (AAS). Better precipitation of metal was obtained at pH 11 than at pH 8 and between the two concentrations of different metals tested, an initial metal concentration of 250 ppm in the reaction gave more precipitation than with 1000 ppm. Experimental data showed that at pH 11, the percentage of removal of metal ions (for an initial concentration of 250 ppm) was in the following order: Cd(2+) (80.99%) > Ni(2+) (64.78%) > Cr(3+) > (46.15%) > Co(2+) (36.47%) > Cr(6+) (32.33%). The overall removal of Cr(3+) and Cr(6+) from tannery effluent was 32.77% and 37.39% respectively in 120 min at pH 11. Likewise, the overall removal of Cd(2+), Co(2+) and Ni(2+) from electroplating effluent was 50.42%, 13.93% and 38.64% respectively in 120 min at pH 11. The study demonstrates that bioprecipitation by CIAP may be a viable and environmental friendly method for clean-up of heavy metals from tannery and electroplating effluents.

  4. Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia.

    PubMed

    Numa-Kinjoh, Natsuko; Komaru, Keiichi; Ishida, Yoko; Sohda, Miwa; Oda, Kimimitsu

    2015-08-01

    Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-β-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to

  5. In vivo overexpression of tissue-nonspecific alkaline phosphatase increases skeletal mineralization and affects the phosphorylation status of osteopontin.

    PubMed

    Narisawa, Sonoko; Yadav, Manisha C; Millán, José Luis

    2013-07-01

    Functional ablation of tissue-nonspecific alkaline phosphatase (TNAP) (Alpl⁻/⁻ mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl⁻/⁻ mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl⁻/⁻ mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast-specific Col1a1 promoter (Col1a1-Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1-Tnap are 10 to 20 times higher than those of wild-type mice. The Col1a1-Tnap animals are healthy and exhibit increased bone mineralization by micro-computed tomography (µCT) analysis. Crossbreeding of Col1a1-Tnap transgenic mice to Alpl⁻/⁻ mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1-Tnap] mice mineralize better than nontransgenic controls and osteoblasts from [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice are able to mineralize to the level of Alpl⁺/⁻ heterozygous osteoblasts, whereas Alpl⁻/⁻ osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl⁻/⁻ mice are comprised of phosphorylated forms of OPN whereas wild-type (WT) and [Col1a1-Tnap⁺/⁻; Alpl⁻/⁻] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1-Tnap] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide-liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl⁻/⁻ bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹⁷⁴FQVS¹⁷

  6. Inhibition of the gut enzyme intestinal alkaline phosphatase may explain how aspartame promotes glucose intolerance and obesity in mice.

    PubMed

    Gul, Sarah S; Hamilton, A Rebecca L; Munoz, Alexander R; Phupitakphol, Tanit; Liu, Wei; Hyoju, Sanjiv K; Economopoulos, Konstantinos P; Morrison, Sara; Hu, Dong; Zhang, Weifeng; Gharedaghi, Mohammad Hadi; Huo, Haizhong; Hamarneh, Sulaiman R; Hodin, Richard A

    2017-01-01

    Diet soda consumption has not been associated with tangible weight loss. Aspartame (ASP) commonly substitutes sugar and one of its breakdown products is phenylalanine (PHE), a known inhibitor of intestinal alkaline phosphatase (IAP), a gut enzyme shown to prevent metabolic syndrome in mice. We hypothesized that ASP consumption might contribute to the development of metabolic syndrome based on PHE's inhibition of endogenous IAP. The design of the study was such that for the in vitro model, IAP was added to diet and regular soda, and IAP activity was measured. For the acute model, a closed bowel loop was created in mice. ASP or water was instilled into it and IAP activity was measured. For the chronic model, mice were fed chow or high-fat diet (HFD) with/without ASP in the drinking water for 18 weeks. The results were that for the in vitro study, IAP activity was lower (p < 0.05) in solutions containing ASP compared with controls. For the acute model, endogenous IAP activity was reduced by 50% in the ASP group compared with controls (0.2 ± 0.03 vs 0.4 ± 0.24) (p = 0.02). For the chronic model, mice in the HFD + ASP group gained more weight compared with the HFD + water group (48.1 ± 1.6 vs 42.4 ± 3.1, p = 0.0001). Significant difference in glucose intolerance between the HFD ± ASP groups (53 913 ± 4000.58 (mg·min)/dL vs 42 003.75 ± 5331.61 (mg·min)/dL, respectively, p = 0.02). Fasting glucose and serum tumor necrosis factor-alpha levels were significantly higher in the HFD + ASP group (1.23- and 0.87-fold increases, respectively, p = 0.006 and p = 0.01). In conclusion, endogenous IAP's protective effects in regard to the metabolic syndrome may be inhibited by PHE, a metabolite of ASP, perhaps explaining the lack of expected weight loss and metabolic improvements associated with diet drinks.

  7. Utility of urinary alkaline phosphatase and γ-glutamyl transpeptidase in diagnosing acute kidney injury in dogs.

    PubMed

    Nivy, Ran; Avital, Yochai; Aroch, Itamar; Segev, Gilad

    2017-02-01

    The diagnostic utility of urinary alkaline phosphatase (uALP) and γ-glutamyl transpeptidase (uGGT) activities in naturally occurring acute kidney injury (AKI) was investigated in a heterogeneous group of dogs. The study included client-owned dogs with AKI (n = 32), chronic kidney disease (CKD, n = 13), lower urinary tract infection (LUTI, n = 15) and healthy controls (n = 24). uGGT and uALP activities were normalised to urinary creatinine (uCr) concentrations (uGGT/uCr and uALP/uCr, respectively). uALP/uCr and uGGT/uCr were positively and significantly correlated (r = 0.619, P <0.001), and differed significantly (P ≤ 0.001) among groups, as well as between AKI and LUTI or CKD groups (P < 0.05), but not between the AKI and control groups. Areas under the receiver operator characteristics (ROC) curve for uALP/uCr and uGGT/uCr as predictors of AKI were 0.75 and 0.65, respectively, with optimal cut-off points showing poor to moderate sensitivity (59% for uALP/uCr and 79% for uGGT/uCr) and specificity (59% for uALP/uCr and 75% for uGGT/uCr). Higher cut-off points, with 90% specificity, showed low sensitivity (41% for both uALP/uCr and uGGT/uCr). In conclusion, uALP/uCr is superior to uGGT/uCr as a marker of AKI, but both uGGT/uCr and uALP/uCr have unsatisfactory discriminatory power for diagnosing naturally occurring AKI in dogs and therefore cannot be recommended as sole screening tests for canine AKI. However, both may serve as ancillary, confirmatory, biomarkers for detecting AKI in dogs if appropriate cut-off points with high specificities are used.

  8. iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

    PubMed Central

    2014-01-01

    Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC

  9. Cycling of Dissolved Organic Phosphorus and Alkaline Phosphatase Activity in Euphotic Zone of the Western North Pacific

    NASA Astrophysics Data System (ADS)

    Suzumura, M.

    2010-12-01

    Phosphorus is an essential nutrient for marine organisms. In oligotrophic environments, concentrations of dissolved inorganic phosphate (SRP), the most bioavailable form of phosphorus, are low and have been hypothesized to constrain the primary productivity. Evidence has been found that dissolved organic phosphorus (DOP) supports a significant fraction of primary production through hydrolytic remineralization of DOP to SRP by alkaline phosphatase (APA). In this study, DOP biogeochemistry was investigated at three locations of the open-ocean environment in the Kuroshio region and at a semi-eutrophic coastal site of the western North Pacific. Concentrations of SRP, DOP and hydrolyzable ester-P were measured in the euphotic zone. Kinetic parameters of APA were determined using a fluorogenic substrate, including potential maximum velocity (Vmax), apparent Michaelis-Menten half-saturation constant (Km), and turnover time (TA) of APA hydrolyzable DOP. SRP concentrations were quite low (≤ 10 nM) in the surface seawater and rapidly increased below the chlorophyll a maximum layer (CML). DOP concentration ranged from 29 to 223 nM. Above the CML, DOP composed a major fraction accounting for 60-100% of dissolved total P. A significant linear relationship was found between the concentrations of SRP and hydrolyzable ester-P (R2 = 0.83, P < 0.01). This suggests active utilization of ester-P under phosphate-depleted conditions. In the Kuroshio region, Vmax of APA exhibited the highest value at the surface water (0 m) and decreased rapidly with depth, while at the coastal site the peak value was found at CML. TA of hydrolyzable DOP was quite variable among the locations and increased with depth especially below CML. The estimated values of in situ hydrolysis rate were much lower (2-34%) than the potential Vmax which was determined with the addition of an excess amount of the substrate. The results suggest that marine microbes can efficiently and rapidly utilize hydrolyzable DOP

  10. NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

    PubMed

    Biacchesi, Stéphane; Mérour, Emilie; Chevret, Didier; Lamoureux, Annie; Bernard, Julie; Brémont, Michel

    2017-03-09

    Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg(2+)/Mn(2+)-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

  11. NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction

    PubMed Central

    Biacchesi, Stéphane; Mérour, Emilie; Chevret, Didier; Lamoureux, Annie; Bernard, Julie; Brémont, Michel

    2017-01-01

    Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition. PMID:28276468

  12. Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells.

    PubMed

    Owen, R L; Bhalla, D K

    1983-10-01

    M cells in Peyer's patch follicle epithelium endocytose and transport luminal materials to intraepithelial lymphocytes. We examined (1) enzymatic characteristics of the epithelium covering mouse and rat Peyer's patches by using cytochemical techniques, (2) distribution of lectin-binding sites by peroxidase-labeled lectins, and (3) anionic site distribution by using cationized ferritin to develop a profile of M cell surface properties. Alkaline phosphatase activity resulted in deposits of dense reaction product over follicle surfaces but was markedly reduced over M cells, unlike esterase which formed equivalent or greater product over M cells. Concanavalin A, ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin reacted equally with M cells and with surrounding enterocytes over follicle surfaces. Cationized ferritin distributed in a random fashion along microvillus membranes of both M cells and enterocytes, indicating equivalent anionic site distribution. Staining for alkaline phosphatase activity provides a new approach for distinguishing M cells from enterocytes at the light microscopic level. Identical binding of lectins indicates that M cells and enterocytes share common glycoconjugates even though molecular groupings may differ. Lectin binding and anionic charge similarities of M cells and enterocytes may facilitate antigen sampling by M cells of particles and compounds that adhere to intestinal surfaces in non-Peyer's patch areas.

  13. Determination of trace alkaline phosphatase by solid-substrate room-temperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol.

    PubMed

    Liu, Jia-Ming; Gao, Fei; Huang, Hong-Hua; Zeng, Li-Qing; Huang, Xiao-Mei; Zhu, Guo-Hui; Li, Zhi-Ming

    2008-04-01

    Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

  14. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

    Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

    2012-01-15

    2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.

  15. Integrating biotinylated polyalkylthiophene thin films with biological macromolecules: biosensing organophosphorus pesticides and metal ions with surface immobilized alkaline phosphatase utilizing chemiluminescence measurements

    NASA Astrophysics Data System (ADS)

    Pande, Rajiv; Kamtekar, S.; Ayyagari, Madhu S. R.; Marx, Kenneth A.; Kumar, Jayant; Tripathy, Sukant K.; Kaplan, David L.

    1995-05-01

    We describe a methodology for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer poly (3-undecylthiophene-co-3- thiophenecarboxaldehyde) 6-biotinamido hexanohydrazide attached hydrophobically to silanized glass. The biotin-streptavidin protein interaction is used to carry out this immobilization. Alkaline phosphatase catalyzes the dephosphorylation of a class of macrocyclic compounds: including CSPD {chloro 3-[4-methoxy spiro(1,2 dioxetane-3-2-trichloro-(3.3.1.1)-decan]-4 yl}phenyl phosphate to a product species which emits energy by chemiluminescence. We can detect this chemiluminescence signal with a photomultiplier tube for both enzymatic catalysis in solution and the surface immobilized enzyme (streptavidin conjugate). This enzyme is inhibited by the organophosphorus class of pesticides as well as nerve agents. The enzyme is also inhibited by Be(II), Bi(III) as well as excess Zn(II), while the apoenzyme is reactivated by Zn(II). We demonstrate in this study that two representative organophosphorus pesticides inhibit the enzymatic production of chemiluminescent products. This is true for the enzyme conjugate both free in solution and immobilized. We can detect pesticides down to about 50 ppb for the enzyme in solution and 500 ppb for surface immobilized enzyme in a 100 (mu) l capillary. Detection of Zn(II) by apoenzyme reactivation occurs down to 3 ppb. Be(II) and Bi(III) are detected by inhibition down to 1 ppm.

  16. Alkaline phosphatase activity and its relationship to inorganic phosphorus in the transition zone of the North-western African upwelling system

    NASA Astrophysics Data System (ADS)

    Sebastián, Marta; Arístegui, Javier; Montero, María F.; Escanez, Jose; Xavier Niell, F.

    2004-08-01

    The enzymatic activity of alkaline phosphatase (APA) was studied in the transition zone between the African upwelling system and the open ocean waters of the Canary Islands region. This region is recurrently dominated by the presence of upwelling filaments that may transport nutrient-enriched waters out into the open ocean before nutrients become exhausted by plankton. Turnover rates by APA were generally low in the whole region, but detectable in all the measurements carried out. On average, turnover rates were higher in the upwelling stations, and APA in those waters seemed to be mainly generated by heterotrophic bacteria to supply easily assimilable organic C. APA outside the upwelling area showed an inverse hyperbolic relationship with increasing phosphate, suggesting the presence of both constitutive and Pi-inducible APA. In these offshore waters, a threshold of 0.1 μM of phosphate could be defined for the regulatory function of Pi on APA. Thus, APA in nutrient-poor waters seemed to be induced to compensate for Pi-deficiency. Turnover rates in the filaments showed basal (probably constitutive) levels, whereas they increased in the surrounding waters, where phosphate concentration presumably did not satisfy plankton P-demands. The fertilising effect of the filaments and associated cyclonic eddies extended to at least 175 km offshore, where basal alkaline phosphatase activities were still found. The magnitude of this effect depends probably on the intensity of upwelling events and the degree of recirculation of filament water back to the coastal jet.

  17. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  18. Variation of alkaline phosphatase activity in sediments of shrimp culture ponds and its relationship with the contents of C, N and P

    NASA Astrophysics Data System (ADS)

    Su, Yuepeng; Ma, Shen; Dong, Shuanglin

    2005-01-01

    Nine enclosures (5 m × 5 m) were built in a Fenneropenaeus chinensis culture pond of Rushan Gulf in April, 2001. The probiotics and BIO ENERGIZER solution were applied for disparate treatments. Variations of alkaline phosphatase activity (APA) and its relationship with the contents of C, N and P in sediments were studied. Results show that APA of sediments increases from 3.096 nmol g-1min-1 to 5.407nmol g-1min-1 in culture period; the bacteria biomass is not the only factor to determine APA; the contents of total P and total organic carbon have a significant positive correlation with APA, while that of total nitrogen has a negative correlation. In addition, the contents of inorganic P and organic P are not regular with APA. By comparison, TOC shows a more significant coherence with APA, meaning that organic pollution in sediments affects APA remarkably.

  19. Using a Personal Glucose Meter and Alkaline Phosphatase for Point-of-Care Quantification of Galactose-1-Phosphate Uridyltransferase in Clinical Galactosemia Diagnosis.

    PubMed

    Zhang, Jingjing; Xiang, Yu; Novak, Donna E; Hoganson, George E; Zhu, Junjie; Lu, Yi

    2015-10-01

    The personal glucose meter (PGM) was recently shown to be a general meter to detect many targets. Most studies, however, focus on transforming either target binding or enzymatic activity that cleaves an artificial substrate into the production of glucose. More importantly, almost all reports exhibit their methods by using artificial samples, such as buffers or serum samples spiked with the targets. To expand the technology to even broader targets and to validate its potential in authentic, more complex clinical samples, we herein report expansion of the PGM method by using alkaline phosphatase (ALP) that links the enzymatic activity of galactose-1-phosphate uridyltransferase to the production of glucose, which allows point-of-care galactosemia diagnosis in authentic human clinical samples. Given the presence of ALP in numerous enzymatic assays for clinical diagnostics, the methods demonstrated herein advance the field closer to point-of-care detection of a wide range of targets in real clinical samples.

  20. Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC.

    PubMed

    Zekri, M; Harb, J; Bernard, S; Poirier, G; Devaux, C; Meflah, K

    1989-01-01

    1. We have compared the effect of phosphatidyl inositol specific phospholipase C (PI-PLC) on the attachment of both 5'-nucleotidase and alkaline phosphatase to the liver plasma membrane from different species. 2. Our results demonstrate differences in the susceptibilities of both enzymes to PI-PLC treatment in relation to their origin. 3. These results were confirmed by immunoblotting using polyclonal anti-5'-nucleotidase antibodies. 4. In addition, in a single animal, susceptibility of both enzymes to PI-PLC treatment is different from one tissue to another. 5. The different percentages of released enzymes could be explained either by a polymorphism in the anchoring of these proteins at the cell surface membrane, or by a different steric hindrance or environment at the cleavage site itself.

  1. Electron micrographic study of precipitates formed by interaction of silicic acid and alkaline phosphatase: contribution to a study of silica urolithiasis in cattle.

    PubMed

    Bailey, C B; Cheng, K J; Costerton, J W

    1982-12-01

    Association of alkaline phosphatase with silicic acid in precipitates formed in dilute solution was studied as a model for the nonspecific reaction between silicic acid and protein. Precipitates contained 68-83% of the silicic acid and 52-83% of the enzyme in the original mixture and were in the form of aggregates of roundish particles 150-800 nm in diameter. Enzyme protein formed a tightly bound layer on the surface of particles formed in solutions of freshly prepared silicic acid. The similarity between the ultrastructural features of precipitates from solutions of silicic acid and of internal portions of siliceous urinary calculi from cattle suggests that deposition of silica during development of such calculi is due, at least in part, to the interaction of protein with silicic acid in urine.

  2. Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase.

    PubMed

    Kechadi, Mohammed; Sotta, Bruno; Gamby, Jean

    2015-01-01

    This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics.

  3. 4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.

    PubMed

    Culbreth, P H; Duncan, I W; Burtis, C A

    1977-12-01

    We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.

  4. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

    PubMed Central

    Micanovic, R; Bailey, C A; Brink, L; Gerber, L; Pan, Y C; Hulmes, J D; Udenfriend, S

    1988-01-01

    A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan. PMID:3422741

  5. Effects of carbamazepine on serum parathormone, 25- hydroxyvitamin D, bone specific alkaline phosphatase, C-telopeptide, and osteocalcin levels in healthy rats.

    PubMed

    Kir, Hale Maral; Garip, Sebnem; Sahin, Deniz; Öztaş, Berrin

    2012-11-01

    It is still not completely clear whether carbamazepine causes alterations in vitamin D status and in bone metabolism. The objective of this study was to investigate the effects of carbamazepine on serum levels of 25-hydroxyvitamin D and on biomarkers of bone formation and resorption in healthy rats. Levels of calcium, 25- hydroxyvitamin D, parathormone, C-telopeptide, bone specific alkaline phosphatase and osteocalcin were measured in 3 groups of rats consisting of controls (n=10), isotonic saline solution group (n=10) and carbamazepine group (n=10). Mean calcium levels were found to be significantly lower in healthy controls in comparison to isotonic saline solution and carbamazepine groups (10.0±0.24, 10.81±0.16, 10.93±0.22 mg/dL, respectively, p<0.05). Mean levels of 25- hydroxyvitamin D, were found to be significantly higher in control group compared to isotonic saline solution group (25- hydroxyvitamin D; 25.91±1.12, 19.99±0.99 ng/mL, respectively, p<0.01). Mean levels of parathormone and osteocalcin were found to be significantly higher in control group compared to isotonic saline solution group and carbamazepine group. Parathormone levels were measured as 3.46±0.83, 1.08±0.08, 0.94±0.02 pg/mL, respectively (p<0.01). Osteocalcine levels were measured as 1.66±0.001, 1.32±0.002, 1.32±0.001 ng/mL, respectively (p<0.001). A significant difference in terms of mean serum bone specific alkaline phosphatase and C-telopeptide levels among groups was not observed. The main outcome of this prospective study in healthy rats showed no change in biochemical parameters of bone turnover during treatment with carbamazepine.

  6. Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway.

    PubMed

    Ho, Ming-Hua; Yao, Chih-Jung; Liao, Mei-Hsiu; Lin, Pei-I; Liu, Shing-Hwa; Chen, Ruei-Ming

    2015-01-01

    Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline

  7. Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

    PubMed

    Hodson, A W; Skillen, A W

    1988-03-01

    Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrophenyl phosphate: effects of carbohydrate additives, low temperature, and freezing.

    PubMed

    Terefe, Netsanet Shiferaw; Arimi, Joshua Mbaabu; Van Loey, Ann; Hendrickx, Marc

    2004-01-01

    The kinetics of the alkaline phosphatase catalyzed hydrolysis of disodium p-nitrphenyl phosphate was studied at 25 degrees C in the presence of the carbohydrates sucrose, fructose, lactose, maltodextrin (DE = 13-17), carboxymethylcellulose (CMC), and CMC-lactose (in 1:1 proportion) at different concentrations and in the presence of sucrose at two different concentrations in a temperature range between 25 and -10 degrees C in subcooled and frozen systems. The objective was to determine whether the reaction is diffusion-controlled, to gain an insight about the factors that determine the diffusion of the reaction species, to understand the mechanism through which the different carbohydrate additives affect the kinetics of the reaction, and to determine the effect of low temperature and freezing on the structural conformation of the enzyme. It was found that the alkaline phosphatase catalyzed hydrolysis of DNPP under the condition studied is at least partially diffusion-controlled. The results also indicate that the diffusion is not controlled by the macroviscosity of the reaction media. The concentration and type of the molecules that constitute the background matrix seem to be the main factors governing the reaction. The results indicate that the different carbohydrates affect the kinetics of the reaction through the excluded volume effect of molecular crowding and decreased substrate and product diffusion rate and not through nonspecific solute effects, which may cause protein denaturation and alteration in enzyme activity. Low temperature does not seem to affect the structural conformation of the enzyme in the temperature range studied, whereas freezing affected the catalytic properties of the enzyme perhaps through its effect on the structural conformation of the enzyme.

  9. Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway

    PubMed Central

    Ho, Ming-Hua; Yao, Chih-Jung; Liao, Mei-Hsiu; Lin, Pei-I; Liu, Shing-Hwa; Chen, Ruei-Ming

    2015-01-01

    Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline

  10. Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

    PubMed Central

    Gasque, Kellen; Foster, Brian L.; Kuss, Pia; Yadav, Manisha C.; Liu, Jin; Kiffer-Moreira, Tina; van Elsas, Andrea; Hatch, Nan; Somerman, Martha J.; Millán, JoséLuis

    2014-01-01

    Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl−/− mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP. PMID:25433339

  11. Calcium-phosphate biomineralization induced by alkaline phosphatase activity in Escherichia coli: localization, kinetics and potential signatures in the fossil record

    NASA Astrophysics Data System (ADS)

    Cosmidis, Julie; Benzerara, Karim; Guyot, François; Skouri-Panet, Fériel; Duprat, Elodie; Férard, Céline; Guigner, Jean-Michel; Babonneau, Florence; Coelho, Cristina

    2015-12-01

    Bacteria are thought to play an important role in the formation of calcium-phosphate minerals composing marine phosphorites, as supported by the common occurrence of fossil microbes in these rocks. Phosphatase enzymes may play a key role in this process. Indeed, they may increase the supersaturation with respect to Ca-phosphates by releasing orthophosphate ions following hydrolysis of organic phosphorus. However, several questions remain unanswered about the cellular-level mechanisms involved in this model, and its potential signatures in the mineral products. We studied Ca-phosphate precipitation by different strains of Escherichia coli which were genetically modified to differ in the abundance and cellular localization of the alkaline phosphatase (PHO A) produced. The mineral precipitated by either E. coli or purified PHO A was invariably identified as a carbonate-free non-stoichiometric hydroxyapatite. However, the bacterial precipitates could be discriminated from the ones formed by purified PHO A at the nano-scale. PHO A localization was shown to influence the pattern of Ca-phosphate nucleation and growth. Finally, the rate of calcification was proved to be consistent with the PHO A enzyme kinetics. Overall, this study provides mechanistic keys to better understand phosphogenesis in the environment, and experimental references to better interpret the microbial fossil record in phosphorites.

  12. Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) from Pasteurella multocida strain X-73.

    PubMed

    Wu, Jin-Ru; Shien, Jui-Hung; Shieh, Happy K; Hu, Chung-Chi; Gong, Shuen-Rong; Chen, Ling-Yun; Chang, Poa-Chun

    2007-02-01

    We have identified a new phoX gene encoding the monomeric alkaline phosphatase from Pasteurella multocida X-73. This gene was not found in the published genome sequence of Pasteurella multocida pm70. Characterization of the recombinant PhoX of Pasteurella multocida X-73 showed that it is a monomeric enzyme, activated by Ca(2+) and possibly secreted by the Tat pathway. These features distinguish phosphatases of the PhoX family from those of the PhoA family. All proteins of the PhoX family were found to contain a conserved motif that shares significant sequence homology with the calcium-binding site of a phosphotriesterase known as diisopropylfluorophosphatase. Site-directed mutagenesis revealed that D527 of PhoX might be the ligand bound to the catalytic calcium. This is the first report on identification of homologous sequences between PhoX and the phosphotriesterase and on the potential calcium-binding site of PhoX.

  13. Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius.

    PubMed

    Menzorova, Natalie I; Seitkalieva, Alexandra V; Rasskazov, Valerу A

    2014-02-15

    A new salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius (StAP) has been shown to have a unique property to hydrolyze substrate in seawater without loss of enzymatic activity. The enzyme has pH optimum at 8.0-8.5. Model experiments showed various concentrations of copper, zinc, cadmium and lead added to seawater or a standard buffer mixture to inhibit completely the enzyme activity at the concentrations of 15-150 μg/l. StAP sensitivity to the presence in seawater of metals, pesticides, detergents and oil products appears to be considerably less. Samples of seawater taken from aquatic areas of the Troitsy Bay of the Peter the Great Bay, Japan Sea have been shown to inhibit the enzyme activity; the same was shown for the samples of fresh waters. The phosphatase inhibition assay developed proved to be highly sensitive, technically easy-to use allowing to test a great number of samples.

  14. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2011-04-10

    The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.

  15. Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification.

    PubMed

    Yadav, Manisha C; Simão, Ana Maria Sper; Narisawa, Sonoko; Huesa, Carmen; McKee, Marc D; Farquharson, Colin; Millán, José Luis

    2011-02-01

    Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP(i)). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1(-/-) mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1(-/-) tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1(-/-) mice show reduced levels of TNAP and elevated plasma PP(i) concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1(-/-) mice despite normalization of their plasma PP(i) levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P(i) influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization.

  16. Characterization of the Proteomic Profiles of the Brown Tide Alga Aureoumbra lagunensis under Phosphate- and Nitrogen-Limiting Conditions and of Its Phosphate Limitation-Specific Protein with Alkaline Phosphatase Activity

    PubMed Central

    Sun, Ming-Ming; Sun, Jin; Qiu, Jian-Wen; Jing, Hongmei

    2012-01-01

    The persistent bloom of the brown tide alga Aureoumbra lagunensis has been reported in coastal embayments along southern Texas, but the molecular mechanisms that sustain such algal bloom are unknown. We compared the proteome and physiological parameters of A. lagunensis grown in phosphate (P)-depleted, P- and nitrogen (N)-depleted, and nutrient-replete cultures. For the proteomic analysis, samples from three conditions were subjected to two-dimensional electrophoresis and tandem mass spectrometry analysis. Because of the paucity of genomic resources in this species, a de novo cross-species protein search was used to identify the differentially expressed proteins, which revealed their involvement in several key biological processes, such as chlorophyll synthesis, antioxidative protection, and protein degradation, suggesting that A. lagunensis may adopt intracellular nutrient compensation, extracellular organic nutrient regeneration, and damage protection to thrive in P-depleted environments. A highly abundant P limitation-specific protein, tentatively identified as a putative alkaline phosphatase, was further characterized by enzyme activity assay on nondenaturing gel and confocal microscopy, which confirmed that this protein has alkaline phosphatase activity, is a cytoplasmic protein, and is closely associated with the cell membrane. The abundance, location, and functional expression of this alkaline phosphatase all indicate the importance of organic P utilization for A. lagunensis under P limitation and the possible role of this alkaline phosphatase in regenerating phosphate from extra- or intracellular organic phosphorus. PMID:22247172

  17. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

  18. Human osteoblasts express functional CXC chemokine receptors 3 and 5: activation by their ligands, CXCL10 and CXCL13, significantly induces alkaline phosphatase and beta-N-acetylhexosaminidase release.

    PubMed

    Lisignoli, Gina; Toneguzzi, Stefania; Piacentini, Anna; Cattini, Luca; Lenti, Anna; Tschon, Matilde; Cristino, Sandra; Grassi, Francesco; Facchini, Andrea

    2003-01-01

    Osteoblasts (OBs) contribute to the maintenance of bone homeostasis and their activity can be influenced by immune cells localized in bone lacunae. We investigated the expression of the chemokine receptors in isolated human OBs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, and report a novel finding, namely, that OBs express high levels of CXC chemokine receptor 3 (CXCR3) and 5 (CXCR5). Functional assays to evaluate CXCR3 and CXCR5 demonstrated that their ligands-CXCL10 and CXCL13, respectively-significantly induce the release of beta-N-acetylhexosaminidase, an enzyme involved in endochondral ossification and bone remodeling able to degrade important extracellular matrix components. Alkaline phosphatase activity, a useful index of matrix formation was also up-regulated by CXCL10 and CXCL13. However, OB activation by these ligands does not affect OB proliferation. Both Bordetella pertussis toxin and neutralizing anti-CXCR3/anti-CXCR5 monoclonal antibodies block CXCL10 and CXCL13 induction, respectively. We also demonstrated the expression of CXCL10 and CXCL13 in human bone tissue biopsies. These results indicate that both CXCR3/CXCL10 and CXCR5/CXCL13 receptor-ligand pairs may play an important role in OB activity through the specific up-regulation of two enzymes, which are involved in the bone remodeling process. Moreover, our data suggest that OBs may play a role in the modulation of bone formation through the combined action of these two enzymes.

  19. [Effect of low intensity pulse-modulated electromagnetic radiation on activity of alkaline phosphatase in blood serum].

    PubMed

    Pashovkina, M S; Akoev, I G

    2001-01-01

    The change in alkaline phosphotase activity in vitro with frequencies modulation at low intensity of pulse-modulated electromagnetic radiation was experimentally shown (EMR, 2375 MHz, intensity: 0.8, 8.0; 40.0 microW/cm2; range modulation: 30-310 Hz; time of interaction: 1-3 min). Revealed effects could be regarded as an evidence of informative character of interaction of modulated EMR.

  20. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.

    PubMed

    Baykov, A A; Evtushenko, O A; Avaeva, S M

    1988-06-01

    An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).

  1. Production of placental alkaline phosphatase (PLAP) and PLAP-like material by epithelial germ cell and non-germ cell tumours in vitro.

    PubMed Central

    Iles, R. K.; Ind, T. E.; Chard, T.

    1994-01-01

    Placental and placental-like alkaline phosphatase (PLAP) levels in the culture media of 87 cell lines of neoplastic and 'normal' origin were measured by a conventional immunosorbent enzymatic assay (IAEA) and by a new immunoradiometric assay (IRMA). The IRMA detected immunoreactive PLAP in 37 of 80 (46%) human epithelial and germ cell cultures, while the IAEA detected PLAP in only 25 (33%). Of the 52 non-germ cell tumour cultures, the IRMA detected expression in 24 (46%) and the IAEA in only 16 (31%). In 17 cases (21%) the IRMA recorded levels double that of the IAEA, while in five cultures (6%) the reverse was true. The IRMA was much more robust than the IAEA and had considerably lower inter- and intra-assay coefficients of variation (3.75-8.5% vs 5.2-46%). Detection of PLAP(-like) expression by IAEA is dependent on neoplastic expression of enzymatically functional molecules and quantification assumes constant enzyme kinetics. PLAP-like material has a higher catalytic rate constant than PLAP and thus will give higher values on a stoichiometric basis in an IAEA. The higher detection rate and levels of PLAP-like material in neoplastic cultures when measured by the IRMA clearly demonstrate ectopic expression of non-enzymatic PLAP and PLAP-like genes. The incidence of PLAP(-like) expression by non-germ cell and possible germ cell tumours has been underestimated and its utility as a tumour marker should be re-examined using assays which measure antigen mass rather than phosphatase activity. PMID:8297725

  2. Ultrasensitive detection of cancer cells and glycan expression profiling based on a multivalent recognition and alkaline phosphatase-responsive electrogenerated chemiluminescence biosensor

    NASA Astrophysics Data System (ADS)

    Chen, Xiaojiao; He, Yao; Zhang, Youyu; Liu, Meiling; Liu, Yang; Li, Jinghong

    2014-09-01

    A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening.A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode

  3. Extracellular Matrix Proteins, Alkaline Phosphatase and Pyrophosphate as Molecular Determinants of Bone, Tooth, Kidney and Vascular Calcification

    NASA Astrophysics Data System (ADS)

    McKee, Marc D.

    2008-09-01

    Progress in biomineralization research in recent years has identified, characterized and described functions for key noncollagenous extracellular matrix proteins regulating crystal growth in the skeleton and dentition. Some of these same proteins expressed in soft tissues undergoing pathologic calcification also inhibit ectopic crystal growth. In addition to extracellular matrix proteins regulating matrix mineralization, the enzyme tissue-nonspecific alkaline phosphatase—which is highly expressed by cells in mineralized tissues—cleaves pyrophosphate, an anionic small-molecule inhibitor of mineralization. Together with the required mineral ion availability necessary for crystal growth, these molecular determinants appear to function in limiting the spread of pathologic calcification seen in soft tissues such as blood vessels and kidneys. Osteopontin, in particular, is a potent calcification inhibitor that accumulates in mineralized tissues and in calcified deposits during vascular calcification and nephrolithiasis/urolithiasis. Additional research is required to establish the exact temporal sequence in which the molecular determinants of pathologic calcification appear relative to mineral crystal growth in different tissues, and to establish their relationship (if any) to the activation of osteogenic differentiation programs.

  4. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    NASA Astrophysics Data System (ADS)

    Zheng, Dong; Neoh, Koon Gee; Kang, En-Tang

    2016-01-01

    In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  5. Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.

    PubMed

    Liu, Jia-Ming; Huang, Xiao-Mei; Liu, Zhen-Bo; Lin, Shao-Qin; Li, Fei-Ming; Gao, Fei; Li, Zhi-Ming; Zeng, Li-Qing; Li, Lian-Ying; Ouyang, Ying

    2009-08-26

    A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.

  6. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    PubMed

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  7. Alkaline phosphatases are involved in the response of Aedes aegypti larvae to intoxication with Bacillus thuringiensis subsp. israelensis Cry toxins.

    PubMed

    Stalinski, Renaud; Laporte, Frédéric; Després, Laurence; Tetreau, Guillaume

    2016-03-01

    Bacillus thuringiensis subsp. israelensis (Bti) is a natural pathogen of dipterans widely used as a biological insecticide for mosquito control. To characterize the response of mosquitoes to intoxication with Bti, the transcriptome profile of Bti-exposed susceptible Aedes aegypti larvae was analysed using Illumina RNA-seq. Gene expression of 11 alkaline phosphatases (ALPs) was further investigated by real time quantitative polymerase chain reaction and ALP activity was measured in the susceptible strain and in four strains resistant to a single Bti Cry toxin or to Bti. These strains were unexposed or exposed to their toxin of selection. Although all resistant strains constitutively exhibited a higher level of transcription of ALP genes than the susceptible strain, they showed a lower total ALP activity. The intoxication with different individual Cry toxins triggered a global pattern of ALP gene under-transcription in all the one-toxin-resistant strains but involving different specific sets of ALPs in each resistant phenotype. Most of the ALPs involved are not known Cry-binding proteins. RNA interference experiment demonstrated that reducing ALP expression conferred increased the survival of larvae exposed to Cry4Aa, confirming the involvement of ALP in Cry4Aa toxicity.

  8. Growth inhibitory effects of gastric cancer cells with an increase in S phase and alkaline phosphatase activity repression by aloe-emodin.

    PubMed

    Guo, Junming; Xiao, Bingxiu; Zhang, Shun; Liu, Donghai; Liao, Yiping; Sun, Qian

    2007-01-01

    Aloe-emodin is a novel active compound found in the root and rhizome of Rheum palmatum. To investigate the effects and mechanisms of aloe-emodin on human gastric cancer, MGC-803 cells were treated with 2.5, 5, 10, 20 and 40 microM aloe-emodin for 1-5 d. The results showed that aloe-emodin inhibited the growth of cancer cells in a dose-dependent manner with an increase in S phase and in the proportion of cells cycling at a higher ploidy level (>G2/M). Moreover, the alkaline phosphatase (ALP) activity, an indicator of cell differentiation, was found decreased. This is one of the first to focus on the effect of ALP activity in human gastric carcinomas cells treated by aloe-emodin. These results indicate that aloe-emodin has a potential value for the treatment of gastric cancer and its mechanisms are by means of cell cycle interruption and induce differentiation.

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

    PubMed

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

    2016-09-15

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

  10. Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

    PubMed

    Tibbitts, T T; Xu, X; Kantrowitz, E R

    1994-11-01

    Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

  11. Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase.

    PubMed

    Mölders, Martina; Felix, Joachim; Bingmann, Dieter; Hirner, Alfred; Wiemann, Martin

    2007-11-01

    Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.

  12. Evaluation of Anti-aging Compounds Using the Promoters of Elastin and Fibrillin-1 Genes Combined with a Secreted Alkaline Phosphatase Reporter in Normal Human Fibroblasts.

    PubMed

    Lin, Chih-Chien; Yang, Chao-Hsun; Kuo, Wan-Ting; Chen, Cheng-Yu

    2015-01-01

    Elastic fibers are major constituents of the extracellular matrix (ECM) in dynamic tissues in the human body, and regulation of elastin and fibrillin-1 expression mediates the formation of these fibers. Traditional assays for the measurement of elastin and fibrillin-1, such as western blotting, Luna staining and immunostaining, are relatively complex and time-consuming. Thus, a relatively simple assay system that also provides rational results is urgently needed. In the study, we aimed to develop a human cell-based assay system that can be used to analyze functional compounds using the promoters of elastin (ELN) and fibrillin-1 (FBN1) genes integrated with a secreted alkaline phosphatase (SEAP) reporter in normal human fibroblast cells. We used this system to assess anti-aging compounds. We used several regulators of elastinogenesis, including retinol, coenzyme Q10, deoxyArbutin and Elestan(TM) (Manilkara multinervis leaf extract), to verify the efficacy of this assay system. Our results demonstrate that this assay system can be used as a fast and realistic method for identifying anti-aging components for future use in foods, cosmetics and drugs.

  13. Copper(II) complexes with cyanoguanidine and o-phenanthroline: Theoretical studies, in vitro antimicrobial activity and alkaline phosphatase inhibitory effect

    NASA Astrophysics Data System (ADS)

    Martínez Medina, Juan J.; Islas, María S.; López Tévez, Libertad L.; Ferrer, Evelina G.; Okulik, Nora B.; Williams, Patricia A. M.

    2014-01-01

    Calculations based on density functional methods are carried out for two Cu(II) complexes with cyanoguanidine (cnge) and o-phenanthroline (o-phen): [Cu(o-phen)2(cnge)](NO3)2ṡ2H2O (1) and [Cu(o-phen)(cnge)(H2O)(NO3)2] (2). The calculated geometrical parameters are in agreement with the experimental values. The results of Atoms in Molecules (AIM) topological analysis of the electron density indicate that the Cu-N(phen) bonds in complex (1) have lower electron density, suggesting that those bonds are stronger in complex (2). Moreover, the ionic character of the Cu-N bond in the complex (1) is slightly stronger than that in the complex (2) and this situation would explain the fact that only complex (2) was stable in water solution. For this reason, the antimicrobial and enzymatic assays were performed using complex (2). It is well known that the increased use of antibiotics has resulted in the development of resistant bacterial and fungal strains. In this context, the study of novel antimicrobial agents has an enormous importance and metal complexes represent an interesting alternative for the treatment of infectious diseases. The aim of this work is to prove the modification of some biological properties like antimicrobial activity or alkaline phosphatase inhibitory activity upon copper complexation.

  14. Prokaryotic Responses to Ammonium and Organic Carbon Reveal Alternative CO2 Fixation Pathways and Importance of Alkaline Phosphatase in the Mesopelagic North Atlantic

    PubMed Central

    Baltar, Federico; Lundin, Daniel; Palovaara, Joakim; Lekunberri, Itziar; Reinthaler, Thomas; Herndl, Gerhard J.; Pinhassi, Jarone

    2016-01-01

    To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates—assumed to be related to autotrophic metabolisms—were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention. PMID:27818655

  15. Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

    SciTech Connect

    Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.; Courtenay-Luck, N.; Dhokia, B.; Snook, D.; Sivolapenko, G.B.; Hooker, G.R.; McKenzie, C.G.; Lavender, P.J. )

    1989-10-01

    Twenty-seven patients with brain glioma were scanned using {sup 123}I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of {sup 131}I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.

  16. A single electrochemical biosensor for detecting the activity and inhibition of both protein kinase and alkaline phosphatase based on phosphate ions induced deposition of redox precipitates.

    PubMed

    Shen, Congcong; Li, Xiangzhi; Rasooly, Avraham; Guo, Linyan; Zhang, Kaina; Yang, Minghui

    2016-11-15

    Protein kinase (PKA) and alkaline phosphatase (ALP) are clinically relevant enzymes for a number of diseases. In this work, we developed a new simple electrochemical biosensor for the detection of the activity and inhibition of both PKA and ALP. One common feature of the PKA and ALP catalyzing process is that PKA can hydrolysis adenosine-5'-triphosphate (ATP) and ALP can hydrolysis pyrophosphate, both reactions produce phosphate ions, and the amount of phosphate ion produced is proportional to enzyme activity. Our assay is based on the principle that phosphate ions react with molybdate to form redox molybdophosphate precipitates on the electrode surface, thus generating electrochemical current. The detection limit for PKA and ALP were much lower than existing assays. The biosensor has good specificity and was used to measure drug-stimulated PKA from lysates of HeLa cells. We also evaluated the use of the biosensor as a screening tool for enzyme inhibitors. To the best of our knowledge, this is the first report of a biosensor capable of detecting the activity of both PKA and ALP. This tool has the potential to simplify PKA and ALP clinical measurement, thereby improving diagnostics of relevant diseases. It also may serve as the basis for a simple screening method for new enzyme inhibitors for disease treatment.

  17. Expanding the spectrum of phenotypes associated with germline PIGA mutations: a child with developmental delay, accelerated linear growth, facial dysmorphisms, elevated alkaline phosphatase, and progressive CNS abnormalities.

    PubMed

    van der Crabben, Saskia N; Harakalova, Magdalena; Brilstra, Eva H; van Berkestijn, Frédérique M C; Hofstede, Floris C; van Vught, Adrianus J; Cuppen, Edwin; Kloosterman, Wigard; Ploos van Amstel, Hans Kristian; van Haaften, Gijs; van Haelst, Mieke M

    2014-01-01

    Phosphatidyl inositol glycan (PIG) enzyme subclasses are involved in distinct steps of glycosyl phosphatidyl inositol anchor protein biosynthesis. Glycolsyl phosphatidyl inositol-anchored proteins have heterogeneous functions; they can function as enzymes, adhesion molecules, complement regulators and co-receptors in signal transduction pathways. Germline mutations in genes encoding different members of the PIG family result in diverse conditions with (severe) developmental delay, (neonatal) seizures, hypotonia, CNS abnormalities, growth abnormalities, and congenital abnormalities as hallmark features. The variability of clinical features resembles the typical diversity of other glycosylation pathway deficiencies such as the congenital disorders of glycosylation. Here, we report the first germline missense mutation in the PIGA gene associated with accelerated linear growth, obesity, central hypotonia, severe refractory epilepsy, cardiac anomalies, mild facial dysmorphic features, mildly elevated alkaline phosphatase levels, and CNS anomalies consisting of progressive cerebral atrophy, insufficient myelinization, and cortical MRI signal abnormalities. X-exome sequencing in the proband identified a c.278C>T (p.Pro93Leu) mutation in the PIGA gene. The mother and maternal grandmother were unaffected carriers and the mother showed 100% skewing of the X-chromosome harboring the mutation. These results together with the clinical similarity of the patient reported here and the previously reported patients with a germline nonsense mutation in PIGA support the determination that this mutation caused the phenotype in this family.

  18. Acute effects of microcystins MC-LR and MC-RR on acid and alkaline phosphatase activities and pathological changes in intraperitoneally exposed tilapia fish (Oreochromis sp.).

    PubMed

    Atencio, Loyda; Moreno, Isabel; Prieto, Ana I; Moyano, Rosario; Molina, Ana M; Cameán, Ana M

    2008-04-01

    Microcystins (MC) are frequently present in cyanobacterial blooms in rivers and lakes, increasing the risk of toxicity to both animals and humans. There more than eighty reported microcystins, and the present study was undertaken to determine whether MC-LR and MC-RR can induce different enzyme alterations and histopathological changes in tilapia fish (Oreochromis sp.) exposed to a single intraperitoneal (i.p.) injection of the pure standards (MC-LR and MC-RR) at a dose of 500 mug/kg; the tilapia fish were then observed for seven days. The two MC variants caused significant changes in the activities of acid and alkaline phosphatases (ACP and ALP) in vital organs, showing a different response pattern. The livers and kidneys of fish injected with MC-LR were particularly affected. MC-RR induced a very pronounced increase of ACP in the kidney and a significant increase of ALP in the liver. Both MC variants caused pathological lesions in hepatic tissues, such as megalocytosis, necrotic process, and microvesicular steatosis, particularly in fish treated with MC-LR, and degenerative renal changes, glomerulopathy, were more severe in tilapias exposed to MC-RR. In addition, both microcystins also caused significant myopathy in the heart. In contrast, the gills did not show any change in enzyme activity or histopathological injury.

  19. Partial Enteral Nutrition Preserves Elements of Gut Barrier Function, Including Innate Immunity, Intestinal Alkaline Phosphatase (IAP) Level, and Intestinal Microbiota in Mice

    PubMed Central

    Wan, Xiao; Bi, Jingcheng; Gao, Xuejin; Tian, Feng; Wang, Xinying; Li, Ning; Li, Jieshou

    2015-01-01

    Lack of enteral nutrition (EN) during parenteral nutrition (PN) leads to higher incidence of infection because of gut barrier dysfunction. However, the effects of partial EN on intestina linnate immunity, intestinal alkaline phosphatase (IAP) and microbiota remain unclear. The mice were randomized into six groups to receive either standard chow or isocaloric and isonitrogenous nutritional support with variable partial EN to PN ratios. Five days later, the mice were sacrificed and tissue samples were collected. Bacterial translocation, the levels of lysozyme, mucin 2 (MUC2), and IAP were analyzed. The composition of intestinal microbiota was analyzed by 16S rRNA pyrosequencing. Compared with chow, total parenteral nutrition (TPN) resulted in a dysfunctional mucosal barrier, as evidenced by increased bacterial translocation (p < 0.05), loss of lysozyme, MUC2, and IAP, and changes in the gut microbiota (p < 0.001). Administration of 20% EN supplemented with PN significantly increased the concentrations of lysozyme, MUC2, IAP, and the mRNA levels of lysozyme and MUC2 (p < 0.001). The percentages of Bacteroidetes and Tenericutes were significantly lower in the 20% EN group than in the TPN group (p < 0.001). These changes were accompanied by maintained barrier function in bacterial culture (p < 0.05). Supplementation of PN with 20% EN preserves gut barrier function, by way of maintaining innate immunity, IAP and intestinal microbiota. PMID:26247961

  20. An association between time-varying serum alkaline phosphatase concentrations and mortality rate in patients undergoing peritoneal dialysis: a five-year cohort study

    PubMed Central

    Liu, Ying; Zhu, Jin-Gang; Cheng, Ben-Chung; Liao, Shang-Chih; Lee, Chih-Hsiung; Chang, Wen Xiu; Chen, Jin-Bor

    2017-01-01

    The relationship between serum alkaline phosphatase (ALP) concentrations and mortality in peritoneal dialysis (PD) patients is rarely reported. We enrolled 667 PD patients in one PD centre in Taiwan to retrospectively examine the association between three ALP concentrations (baseline, time-averaged, time-dependent) and mortality over a 5-year period (2011–2015). Baseline data collection included demographics, clinical, and laboratory parameters. Multivariable-adjusted Cox models were used to analyse the association. Four ALP quartiles were defined at the baseline: ≤62, 63–82, 83–118, and ≥119 U/L. Of 667 patients, 65 patients died, of which 8 patients died due to cardiovascular disease. Females were predominant in the higher ALP quartiles, and 24-h urine volume was significantly proportionately decreased in the higher ALP quartiles. ALP quartiles expressed by the three models were not associated with all-cause or cardiovascular mortalities after adjusting for demographics, liver function, bone metabolism, mortality, hemoglobin, and 24-h urine volume. In conclusion, ALP concentrations were not associated with death risk in PD patients over the 5-year period. PMID:28256582

  1. Inhibition of dsDNA-templated copper nanoparticles by pyrophosphate as a label-free fluorescent strategy for alkaline phosphatase assay.

    PubMed

    Zhang, Liangliang; Zhao, Jingjin; Duan, Min; Zhang, Hua; Jiang, Jianhui; Yu, Ruqin

    2013-04-16

    On the basis of the inhibition of double strand DNA (dsDNA)-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions has been developed. This method relies on the strong interaction between PPi and Cu(2+), which would hamper the effective formation of fluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPi hydrolysis would disable the complexation between Cu(2+) and PPi, facilitating the formation of fluorescent CuNPs through the reduction by ascorbate in the presence of dsDNA templates. Thus, the fluorescence intensity was recovered, and the fluorescence enhancement was related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operations. The present strategy exhibits a high sensitivity and the turn-on mode provides a high selectivity for the ALP assay. Additionally, the inhibition effect of phosphate on the ALP activity was also studied. The proposed method using a PPi substrate may hold a potential application in diagnosis of ALP-related diseases or evaluation of ALP functions in biological systems.

  2. Effect of dietary caraway (Carum carvi L.) on aberrant crypt foci development, fecal steroids, and intestinal alkaline phosphatase activities in 1,2-dimethylhydrazine-induced colon carcinogenesis.

    PubMed

    Kamaleeswari, Muthaiyan; Deeptha, Kumaraswami; Sengottuvelan, Murugan; Nalini, Namasivayam

    2006-08-01

    Colon cancer is one of the most common malignancies in many regions of the world and is thought to arise from the accumulation of mutations in a single epithelial cell of the colon and rectum. Caraway (Carum carvi L. Umbelliferae) is a shrub with a long history as a medicinal plant since ancient times. The effect of different doses of caraway (CC) on the formation of aberrant crypt foci (ACF) and the levels of fecal bile acids, neutral sterols, and alkaline phosphatase (ALP) activities were studied in 1,2-dimethylhydrazine (DMH)-induced colon cancer in rats. Animals were randomized into 6 groups. Group 1 served as control, and group 2 received 90 mg/kg body weight caraway orally everyday. Groups 3-6 rats were given subcutaneous injections of DMH (20 mg/kg body weight) once a week for the first 4 weeks to induce ACF. Rats in groups 4-6, in addition to DMH injections, received caraway at 30, 60, and 90 mg/kg body weight respectively p.o. everyday until the end of whole experimental period of 15 weeks. Caraway supplementation significantly reduced ACF development and also decreased the levels of fecal bile acids, neutral sterols, and tissue ALP activities. The histological alterations induced by DMH were also significantly improved. Overall, our results showed that all 3 doses of caraway inhibited tumorigenesis though the effect of the intermediary dose of 60 mg/kg body weight was more pronounced.

  3. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    PubMed

    Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  4. Prognostic impact of alkaline phosphatase measured at time of presentation in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction

    PubMed Central

    Kim, Tae-Hoon; Moon, Jeonggeun; Park, Hyun Woo; Jang, Ho-Jun; Park, Sang-Don; Kwon, Sung Woo; Suh, Jon

    2017-01-01

    Background Serum alkaline phosphatase (ALP) has been shown to be a prognostic factor in several subgroups of patients due to its promotion of vascular calcification. However, the prognostic impact of serum ALP level in ST-segment elevation myoca