Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The involvement of protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and other phosphorylation mechanisms in the rapid desensitization of the [Ca2+]i response to parathyroid hormone (PTH) stimulation was investigated in osteoblast-like UMR-106 cells. A 5 minute preincubation of the cell suspension with phorbol 12,13-dibutyrate (PDB) decreased the response to PTH in a concentration-dependent manner. 1-Oleoyl-2-acetyl-r-glycerol (OAG) pretreatment likewise decreased the PTH response. Staurosporine, a potent protein kinase inhibitor, completely prevented the desensitization caused by PDB. These PDB and staurosporine effects were also observed in 3 mM EGTA-containing medium ([Ca2+]free < 10(-8) M). A 5 minute pretreatment of cells with 1 microM forskolin had no effect on the calcium response to PTH. Homologous and PDB-induced desensitizations differed in several respects. Staurosporine pretreatment resulted in only a slight restoration of the PTH response under conditions of homologous desensitization. Chronic treatment with phorbol ester prevented the desensitization of the PTH response by acute phorbol treatment but not the homologous desensitization. Both homologous and PDB-induced desensitization were relieved by alkaline phosphatase treatment, consistent with the involvement of phosphorylation in the desensitization. This alkaline phosphatase effect on desensitization was inhibited by L-phenylalanine. These results suggest that PTH receptor homologous desensitization involves phosphorylation process(es) other than or in addition to those of PKC.
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PMID:Studies on the mechanism of desensitization of the parathyroid hormone-stimulated calcium signal in UMR-106 cells: reversal of desensitization by alkaline phosphatase but not by protein kinase C downregulation. 807 54

Phosphatase activities were investigated in Morganella morganii, which is one of the few enterobacterial species producing high-level phosphate-irrepressible acid phosphatase activity (HPAP phenotype), and the gene encoding the major phosphate-irrepressible acid phosphatase was cloned, sequenced, and its product characterized. Using p-nitrophenyl phosphate as substrate, Morganella produced a major phosphate-irrepressible acid phosphatase (named PhoC) which is associated with the HPAP phenotype, a minor phosphate-irrepressible acid phosphatase, and a phosphate-repressible alkaline phosphatase. The presence of the PhoC activity prevented induction of alkaline phosphatase when a PhoC-hydrolysable organic phosphate ester, such as glycerol 2-phosphate, was the sole phosphate source. PhoC is a secreted nonspecific acid phosphatase apparently composed of four 25 kDa polypeptide subunits. The enzyme is resistant to EDTA, P(i), fluoride and tartrate. The M. morganii PhoC showed 84.6% amino acid sequence identity to the PhoN nonspecific acid phosphatase of Providencia stuartii, 45.3% to the PhoN nonspecific acid phosphatase of Salmonella typhimurium, and 37.8% to the principal acid phosphatase (PhoC) of Zymomonas mobilis. Comparison of sequence data and of regulation of these enzymes suggested a different phylogeny of members of this gene family within the Enterobacteriaceae.
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PMID:Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii. 808 99

We describe a continuous coupled spectrophotometric assay for alkaline phosphatase which uses alpha- or beta-glycerophosphate as substrate, and glycerol dehydrogenase as ancillary enzyme. The glycerol liberated by alkaline phosphatase is determined by measuring the increase in absorbance at 340 nm caused by NADH formation that is combined with glycerol oxidation by the ancillary enzyme. The assay procedure was optimized using a bovine bone extract as alkaline phosphatase source.
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PMID:A continuous spectrophotometric assay for alkaline phosphatase with glycerophosphate as substrate. 815 Oct 68

Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue. In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses.
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PMID:Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade. 823 77

Phosphatidic acid (PA) added to intact cells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of superoxide generation in intact human neutrophils by a short-chain (dioctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of superoxide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). Activation did not require extracellular Ca2+ and coincided with near quantitative conversion of diC8PA to dioctanoylglycerol (diC8-glycerol). diC8PA also activated cellular phospholipase D with release of long-chain PA and secondary production of long-chain diradylglycerol (sn-1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol). The metabolism of diC8PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive release of Pi into the extracellular medium. This enzyme also showed activity toward PA containing long-chain unsaturated fatty acids. The ecto-PA phosphohydrolase differed from the intracellular PA phosphohydrolase based on its relative insensitivity to desipramine and N-ethylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO) cells and its activity did not change in transfected CHO cells expressing the two membrane-associated isoforms of alkaline phosphatase, indicating that the PA phosphohydrolase was not alkaline phosphatase. Non-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superoxide production. Activation of superoxide generation by diC8PA was inhibited by staurosporine, suggesting a protein kinase C-dependent mechanism. We suggest that the action of a novel ecto-PA phosphohydrolase permits exogenously added short-chain PA to serve as "timed-release diacylglycerol" and that its biological effects in neutrophils are secondary to diacylglycerol-mediated protein kinase C activation.
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PMID:A novel ecto-phosphatidic acid phosphohydrolase activity mediates activation of neutrophil superoxide generation by exogenous phosphatidic acid. 824 61

We analyzed 11 H. pylori isolates from humans using the artificial chromogenic substrate paranitrophenylphosphorylcholine to detect phospholipase C (PLC) activity. The range of PLC in sonicates was 8.8-92.3 (Mean 56.9 +/- 6.5) nmol of substrate hydrolysed min-1 mg-1 protein; the amount of activity was not associated with urease or cytotoxin levels. Addition of sorbitol or glycerol enhanced PLC activity of H. pylori sonicate and purified PLC from C. perfringens (PLC1) but not purified PLC from B. cereus (PLC3). H. pylori sonicates had little acid phosphatase and no detectable alkaline phosphatase activity, and H. pylori PLC showed markedly different biochemical characteristics from either phosphatase. In total, these studies indicate that activity measured in H. pylori sonicate by PLC assay is due to PLC and not phosphatase activity. The temperature optimum for PLC activity of H. pylori sonicate was 56 degrees C and for PLC 1 was 65 degrees C. For H. pylori PLC and PLC1, optimal activity occurred at pH 8. Despite multiple similarities between H. pylori PLC and PLC1, known PLC inhibitors show different interactions with each enzyme. Although PLC activity is present in many subcellular constituents of H. pylori, including culture supernatants and water extracts, highest specific activity is associated with a membrane-enriched fraction.
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PMID:Identification and characterization of Helicobacter pylori phospholipase C activity. 828 Sep 31

A new DNA hybridization technique, based on chromatographic migration of DNA on a nitrocellulose strip passing through an immobilized probe area, is described. The new paper chromatography hybridization assay (PACHA) is faster and simpler to use than the conventional dot hybridization assay. In this assay, an aliquot of biotinylated, PCR-amplified target DNA is applied to one end of a nitrocellulose strip. The DNA migrates to the opposite end of the strip by capillary forces and hybridizes to a specific DNA probe immobilized in a reaction zone (RZ), located in the middle of the strip. Unhybridized DNA migrates away from the RZ. The biotinylated hybrid is visualized by a color reaction employing a streptavidin-alkaline phosphatase (SA-AP) conjugate and a specific chromogenic substrate. The new PACHA technique allows for detection of as little as 1-5 pg of specific human papilloma virus 16 (HPV16) DNA in 25 min of hybridization. In this system, the hybridization efficiency is controlled by the flow velocity of the hybridization solution (HS) and by the volume of the amplified labeled DNA migrating across the immobilized probe. Glycerol (30%) or polyvinyl pyrrolidone (PVP) (1%) reduces the flow rate by a factor of 2.5-3 and increases the sensitivity of the assay by a factor of 5.2 for glycerol and 2.6 for PVP. This novel method ensures efficient hybridization to multiple probes and appears to be superior to currently available solid-phase hybridization techniques.
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PMID:A novel rapid hybridization technique: paper chromatography hybridization assay (PACHA). 829 6

Chick embryo vertebral chondrocytes (CHECOV cells) grown in agarose gels form spherical colonies containing cells of hypertrophic morphology and a metachromatically staining matrix. Biochemical analysis of these cultures resulted in the following findings. (i) Calcification of CHECOV cultures can be induced by addition of Pi (at least 1.9 mM) or beta-glycerol phosphate (BGP). (ii) Alkaline phosphatase activity reaches a maximal value at the time when mineral deposition is initiated. (iii) Added BGP is converted to Pi; maximal production of Pi occurs at the time of maximal alkaline phosphatase activity. (iv) BGP-supplemented cultures produce a degree of calcification that corresponds to the amount of BGP conversion to Pi. It can be concluded that Pi is rate-limiting for the calcification of chondrocyte cultures. BGP promotes calcification of these cultures by acting as a substrate for the alkaline phosphatase-mediated production of inorganic phosphate.
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PMID:Calcification of chick vertebral chondrocytes grown in agarose gels: a biochemical and ultrastructural study. 831 88

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.
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PMID:Glycerol effects on protein flexibility: a tryptophan phosphorescence study. 836 22

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.
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PMID:Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli. 841 12


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