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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)

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.
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PMID:Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase. Use of an exogenous phosphatase to study substrate-mediated regulation of dephosphorylation. 681 91

Alkaline phosphatase (E.C.3.1.3.1.) from unerupted bovine pulp was extracted from the microsomal fraction with eta-butanol and purified 77-fold, using DEAE-cellulose chromatography, Sephadex G-200 gel-filtration and concanavalin-A affinity chromatography, to a final specific activity of 92.3 units/mg protein. Affinity chromatography confirmed the glycoprotein nature of the enzyme. The pH optimum for the purified enzyme was 10.0 with rho-nitrophenylphosphate, and 8.7 with phosphoserine. The apparent Km was estimated to be 0.7 mM, using rho-nitrophenylphosphate in glycine-NaOH buffer, pH 10.0. The enzyme was markedly inhibited by EDTA, bromotetramisole and homoarginine but was insensitive to phenylalanine, and therefore resembled the alkaline phosphatase of liver and bone, but not that of intestine and placenta. No protein phosphatase activity towards dentine phosphoprotein and phosvitin was observed.
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PMID:Purification and properties of bovine dental-pulp alkaline-phosphatase. 695 31

1. To clarify the nature of the inhibition of whole-cell inwardly rectifying K+ current (IK1) by isoprenaline (Iso) and its antagonism by acetylcholine (ACh), we studied the effects of Iso and ACh and their surrogates on single channel currents (iK1) carried by inwardly rectifying K+ channels in cell-attached and excised inside-out patches obtained from guinea-pig ventricular myocytes. 2. Bath application of Iso suppressed iK1 channel activity in cell-attached patches. This was inhibited by propranolol. Bath-applied forskolin or dibutyryl cAMP mimicked the effect of bath-applied Iso. 3. Exposure of the cytosolic face of inside-out patches to purified catalytic subunit of the cAMP-dependent protein kinase (PKA) also suppressed iK1 channel activity, mimicking the effect of bath-applied Iso on iK1 recorded from cell-attached patches. 4. When applied directly to cell-attached patches via the patch pipette solution, ACh antagonized Iso-induced (1 microM applied via the bath) suppression of iK1 channels. In contrast, bath-applied ACh (10 microM) partially antagonized the effect of low concentrations of Iso (e.g. < 50 nM) on iK1 channels in cell-attached patches but had no detectable effect when 1 microM or more Iso was used. 5. In myocytes pretreated with pertussis toxin (PTX), ACh failed to antagonize Iso-induced suppression of iK1 channels. When inside-out patches were used, bath-applied preactivated exogenous inhibitory G protein subunit, G1 alpha, antagonized the suppression of iK1 channels induced by bath-applied catalytic subunit of PKA (PKA-CS), suggesting that a PTX-sensitive G1 alpha mediates ACh-induced antagonism of Iso-induced suppression of iK1. 6. Neither GTP gamma S nor G1 alpha antagonized the suppression of iK1 produced by bath-applied PKA-CS in inside-out patches when okadaic acid was present in the bath. In addition, bath application of alkaline phosphatase also reactivated iK1 channels suppressed by PKA-CS. 7. Findings in guinea-pig ventricular myocytes suggest that iK1 can be suppressed by a PKA-mediated phosphorylation of the iK1 channel occurring in response to Iso-induced beta-adrenergic receptor activation and that ACh can antagonize the suppression by mechanisms that involve both intracellular and membrane-delimited pathways. The membrane-delimited pathway appears to involve M2-cholinergic receptors, their associated G protein, G1, and a protein phosphatase, all located in the sarcolemma in close proximity to the involved iK1 channels.
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PMID:Beta-adrenergic and cholinergic modulation of inward rectifier K+ channel function and phosphorylation in guinea-pig ventricle. 747 27

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stage-specific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a gene-transfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were established. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 cell-lines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue non-specific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.
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PMID:Changes in cell adhesion and cell proliferation are associated with expression of tissue non-specific alkaline phosphatase. 750 6

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.
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PMID:A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes. 753 13

It has been demonstrated that dephosphorylation of the ferredoxin component of the mitochondrial 25-hydroxyvitamin D3-1-hydroxylase, as a result of a PTH-cAMP mediated activation, involves a protein phosphatase activity. However, the nature and properties of this phosphatase are uncertain. It has been proved that alkaline phosphatase, a magnesium dependent enzyme, could dephosphorylate in vitro the ferredoxin component of the 25-hydroxyvitamin D3-1-hydroxylase. Moreover, some evidence of mitochondrial localization of some alkaline phosphatases has been published. Although the existence of a levamisole inhibitable alkaline phosphatase activity has been described in renal cells, its role remains to be elucidated. In the present work, the existence of an alkaline phosphatase in mitochondrial membrane preparations from LLC-PK1 cells has been described. This alkaline phosphatase is magnesium dependent and levamisole inhibitable. Preparations of mitochondrial membrane from LLC-PK1 cells also showed 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin D3-24R-hydroxylase (24-hydroxylase) activities being both enzymes responsive to the 8Br-cAMP mediated regulation. The 8Br-cAMP not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial alkaline phosphatase activity. In the same way, the levamisole (specific inhibitor of some alkaline phosphatases) inhibited the mitochondrial alkaline phosphatase and also the 1-hydroxylase activity. In addition, the inhibition of mitochondrial alkaline phosphatase by levamisole avoids the effect of 8Br-cAMP on the 1-hydroxylase and 24-hydroxylase activities. On the other hand, the mitochondrial alkaline phosphatase and the 1-hydroxylase activities showed similar behaviour with respect to the magnesium concentrations in the incubation medium. Taking these results together it could be possible to suggest the implication of the Mg(2+)-dependent mitochondrial alkaline phosphatase activity found in LLC-PK1 cells in the regulation of the 1,25(OH)2D3 and 24,25(OH)2D3 synthesis.
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PMID:Possible involvement of a magnesium dependent mitochondrial alkaline phosphatase in the regulation of the 25-hydroxyvitamin D3-1 alpha-and 25-hydroxyvitamin D3-24R-hydroxylases in LLC-PK1 cells. 754 Apr 7

To determine whether protein phosphatases can affect bone regulation, we examined the effects of okadaic acid (OA) and calyculin A (CA), specific inhibitors of protein phosphatases type 1 and type 2A, on alkaline phosphatase activity of mouse osteoblastic cells. Clone MC3T3-E1 cells were cultured with varying concentrations of OA and CA. OA and CA stimulated alkaline phosphatase (ALP) activity in the cells in dose-dependent fashion with a maximal effect at concentrations of 5 nM and 2 nM, respectively. The properties of OA-induced and native ALP in the cells were the same and they were liver-bone-kidney type. These results show that protein phosphatase inhibitors stimulate bone formation in vitro and that phosphorylation and dephosphorylation of specific proteins in the cells may be involved in bone regulation in vivo as well.
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PMID:Protein phosphatase inhibitors, okadaic acid and calyculin A, induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria. 766 40

Acid extracts of thapsigargin-activated Jurkat cells have been shown to have intracellular activity in inducing a dose-dependent rapid chloride current upon microinjection in Xenopus laevis oocytes. The extracts act by elevation of calcium through calcium entry. The factor(s) responsible for this activity have been termed calcium influx factor (CIF) and have been found to be small, relatively polar molecules (< 1000 daltons) whose activity is abolished by alkaline phosphatase treatment and potentiated by co-injection of okadaic acid (a protein phosphatase inhibitor). CIF is produced in a time-dependent manner following thapsigargin treatment of Jurkat cells, being first elevated above basal levels by 2 min. Intracellular CIF activity is completely absent from NG115-401L neuronal cells, which lack capacitative entry. On this basis, it appears that Jurkat cells, activated by stimuli that deplete internal calcium stores, produce one or more CIF activities acting intracellularly, and Xenopus oocytes may be a powerful tool to purify and characterize CIFs.
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PMID:Evaluation of calcium influx factors from stimulated Jurkat T-lymphocytes by microinjection into Xenopus oocytes. 789 75

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary zone electrophoresis (CZE) were evaluated for monitoring protein phosphatase and kinase reactions in vitro. Varying concentrations of peptide C (YIHLEKKYVRRDSG), peptide S (YLIEDNEYTARQGA) and kemptide (LARRSALG) mixed with their corresponding phosphorylated peptides, pC, pS and pkemptide, were analyzed. Comparison between the two techniques indicated that MALDI MS was less quantitative than CZE, showing a bias towards detection of the unphosphorylated peptide S and kemptide. In terms of sensitivity, the MALDI MS and CZE techniques are comparable. Protein kinase A phosphorylation of kemptide was monitored with both MALDI MS and CZE, whereas alkaline phosphatase dephosphorylation of pC could only be monitored with MALDI MS. The absence of inhibition with phosphatase or kinase buffers is a significant advantage of MALDI MS. In contrast to CZE, the MALDI spectra allow identification of the species analyzed by virtue of their mass. The results obtained emphasize the advantage of monitoring enzymatic reactions in buffer solutions using MALDI MS compared with CZE.
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PMID:Monitoring protein kinase and phosphatase reactions with matrix-assisted laser desorption/ionization mass spectrometry and capillary zone electrophoresis: comparison of the detection efficiency of peptide-phosphopeptide mixtures. 791 94

We previously reported that fetal calf serum-induced alkaline phosphatase activity is suppressed due to the activation of protein kinase C in osteoblast-like MC3T3-E1 cells (Miwa et al. (1991) Bone Miner. 14, 15-25; Kotoyori et al. (1994) Horm. Metab. Res. 26, 116-118). In the present study, we examined the effect of okadaic acid, a potent and specific inhibitor of protein phosphatase type 1 and 2A, on fetal calf serum-induced alkaline phosphatase activity in MC3T3-E1 cells. The pretreatment with okadaic acid enhanced the fetal calf serum-induced alkaline phosphatase activity in a dose-dependent manner in the range between 0.1 and 5 nM. 1-Norokadaone, a less potent analogue of okadaic acid, had little effect on the fetal calf serum-induced alkaline phosphatase activity. Okadaic acid partially reversed the suppression of fetal calf serum-induced alkaline phosphatase activity by 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator. The effect of okadaic acid was dose-dependent in the range between 0.1 and 5 nM. The patterns of the dose-dependency of both okadaic acid effects on fetal calf serum-induced alkaline phosphatase activity and on the suppression by 12-O-tetradecanoylphorbol-13-acetate were similar. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of alkaline phosphatase activity at a point downstream from protein kinase C in osteoblast-like cells.
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PMID:Okadaic acid reverses the inhibitory effect of protein kinase C on alkaline phosphatase activity in osteoblast-like cells. 795 88


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