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 FeZn derivative of purple acid phosphatase from porcine uterus (FeZnUf) and its phosphate complex (FeZnUf.PO4) have been characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges, to gain insight into the nature of the FeZn active site as well as the phosphate binding mode. Pre-edge data show that both FeZnUf and FeZnUf.PO4 have a 6-coordinate iron site. The iron site has an average Fe-O/N bond length of 2.01-2.02 A, which can be resolved into subshells of 1.92 and 2.11 A for FeZnUf.PO4. On the other hand, the zinc site has a shell of scatterers at 2.02-2.03 A plus one scatterer at ca. 2.4 A. These metal-ligand bond lengths are consistent with the nature of the ligands deduced from spectroscopic studies or identified in the crystal structure of the related kidney bean purple acid phosphatase (KBPAP). The outer-sphere analysis indicates an Fe-Zn separation of approximately 3.3 A in both FeZnUf and FeZnUf.PO4, consistent with the presence of an M2(mu-OR)2 diamond core as found in the crystal structures of KBPAP, calcineurin, and protein phosphatase 1. The Fe-P and Zn-P bond distances in FeZnUf.PO4 are determined to be 3.23 and 3.18 A, respectively, indicating that phosphate binds to the dinuclear center in a bidentate mode; such a mode has been observed in oxoanion complexes of KBPAP, calcineurin, and alkaline phosphatase, as well as in a number of synthetic FeFe and FeZn complexes. The implications of these structural results on the mechanism of phosphatase action are discussed.
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PMID:X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin. 890 92

Many of the hormone-regulated ion transport processes in distal nephron involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate molecular mechanisms underlying the ionic permeability of the distal tubule apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused onto a planar lipid bilayer. These experiments led to the identification of several ionic channels including a Cl(-)-permeable channel of 14 pS with a Na+ over Cl- permeability ratio, PNa/PCl < 0.09. The open channel probability (Po) showed a weak voltage dependency with Po increasing slightly at negative potential values (intracellular (trans) relative to extracellular (cis) for right-side-out vesicles). Channel activity was inhibited by NPPB at high concentrations (> 100 microM) and by DIDS (300 microM). A small inhibitory effect was also observed in the presence of DPC at concentrations ranging from 200 microM to 500 microM. The presence of SO4(2-) (32 mmol/l) in the trans solution caused a complete inhibition of channel activity, but no modification of channel behaviour was observed with the non-selective channel blocking agent gadolinium (Gd3+) at 100 microM. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to an increase in channel activity characterized by a greater number of active channels coupled to an increase of the individual channel open probability. The action of the protein kinase A could be cancelled by the addition of a non specific protein phosphatase, such as alkaline phosphatase. Our results suggest that the apical membrane of the rabbit distal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones linked to the adenylate cyclase pathway.
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PMID:Evidence from incorporation experiments for an anionic channel of small conductance at the apical membrane of the rabbit distal tubule. 897 99

We examined the effect of okadaic acid, an inhibitor of protein phosphatase type 1 and 2A, on prostaglandin E1 (PGE1)-induced alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells. PGE1 increased ALP activity dose dependently in the range between 10 nM and 0.3 microM in these cells. The pretreatment with okadaic acid enhanced the PGE1-induced ALP activity in a dose-dependent manner in the range between 0.1 and 5 nM. On the other hand, 1-norokadaone, a less potent analogue of okadaic acid, had no effect on the PGE1-induced ALP activity. Tautomycin, an another inhibitor of protein phosphatase type 1 and 2A, also enhanced the PGE1-induced ALP activity. PGE1 stimulated cAMP accumulation dose dependently in the range between 10 nM and 0.3 microM. However, PGE1 had no effect on the formation of inositol phosphates. Okadaic acid did not affect the PGE1-induced cAMP accumulation. Okadaic acid dose dependently enhanced the dibutyryl cAMP-induced ALP activity. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of ALP activity at a point downstream from protein kinase A in osteoblast-like cells.
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PMID:Okadaic acid enhances prostaglandin E1-induced alkaline phosphatase activity in osteoblast-like cells: regulation at a point downstream from protein kinase A. 898 33

The Ah receptor binds aryl hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high affinity. After binding aryl hydrocarbons, the receptor releases the 90-kDa heat shock protein and forms a dimer with the Arnt protein capable of binding at xenobiotic-responsive elements (XREs) and stimulating the transcription of genes involved in the metabolism of aryl hydrocarbons. The activity of the Ah receptor/ Arnt dimer can be decreased by treatments causing the down-regulation of protein kinase C and decreasing the nuclear accumulation of the receptor. Incubation with acid phosphatase or with alkaline phosphatase has been reported to block XRE binding. Thus the literature suggests that phosphorylation regulates Ah receptor activity by affecting DNA binding and/or nuclear transport. A reporter plasmid containing two XREs was used to investigate the effects of phosphatase inhibitors on TCDD-dependent transcription by the Hepa-1 mouse liver cell line. The inhibitors calyculin A and okadaic acid caused two- to threefold increases in TCDD-dependent transcription at concentrations capable of selectively inhibiting protein phosphatase 1 and protein phosphatase 2A. The inhibitor cyclosporin A doubled TCDD-dependent transcription at a concentration capable of selectively inhibiting protein phosphatase 2B. All three of the phosphatase inhibitors increased TCDD-dependent transcription without affecting transcription in the absence of TCDD. Nuclear extracts were prepared from cells treated with concentrations of okadaic acid or cyclosporin A which substantially stimulated TCDD-dependent transcription. Neither of the inhibitors significantly increased the level of TCDD-dependent XRE binding in the extracts. GAL4-Arnt fusion proteins were used to further investigate whether the phosphatase inhibitors affected a step other than DNA binding. Okadaic acid treatment specifically increased the ability of a GAL4 fusion protein containing the Arnt PAS and transactivation domains to stimulate transcription. These results suggest that serine/threonine-specific protein phosphatases can act at a level subsequent to XRE binding to inhibit the ability of the Ah receptor/Arnt dimer to stimulate transcription.
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PMID:Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding. 912 79

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
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PMID:cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. 914 51

The anabolic effect of 17beta-estradiol in osteoblastic MC3T3-E1 cells was investigated. The cells were cultured for 3 days in the medium containing either vehicle or 17beta-estradiol (10(-11)-10(-9) M). 17beta-Estradiol significantly increased alkaline phosphatase activity and protein concentration in the cells. The steroid (10(-9) M) also significantly elevated the cell numbers and the cellular DNA content. The anabolic effect by 17beta-estradiol was blocked by the presence of dipicolinate (10(-3) M), a chelator of zinc ion, suggesting a role of cellular zinc in osteoblastic cell function. The presence of zinc sulfate (10(-5) M) or beta-alanyl-L-histidinato zinc (AHZ) (10(-5) M) significantly enhanced the 17beta-estradiol (10(-10) or 10(-9) M)-induced increase of alkaline phosphatase activity and protein concentration in the cells; the effect of AHZ was greater than that of zinc sulfate. The enhancement by zinc compounds was not based on the augmentation of osteoblastic cell numbers. The co-addition of cycloheximide (10(-6) M), an inhibitor of protein synthesis, completely blocked the zinc compound (10(-5) M)-induced enhancement of 17beta-estradiol's (10(-9) M) effect to increase alkaline phosphatase activity and protein concentration in the cells. Moreover, the anabolic effect of 17beta-estradiol together with or without zinc compounds was abolished by the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or of okadaic acid (10(-7) M), an inhibitor of protein phosphatase. The present study demonstrates that the anabolic effect of 17beta-estradiol is enhanced by zinc-chelating dipeptide in osteoblastic MC3T3-E1 cells, and that the enhancing effect may involve protein synthesis and protein kinase activity.
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PMID:Zinc enhancement of 17beta-estradiol's anabolic effect in osteoblastic MC3T3-E1 cells. 916 27

Orthovanadate is a known inhibitor of phosphotyrosyl protein phosphatase and is reported to stimulate osteogenic cell proliferation and differentiation when administered during the logarithmic growth phase and to potentiate the mitogenic effects of several growth factors. There is little information concerning the effects of orthovanadate on bone matrix deposition and mineralization, although there is some evidence that it increases collagen synthesis by osteogenic cells. To test the effects of orthovanadate on bone nodule formation and mineralization, osteogenic cells were exposed to 5-50 microM orthovanadate or 10(-7) M insulin-like growth factor-1 for 3, 7, and 21 days after plating. Exposure to orthovanadate produced differential effects on cellular proliferation and alkaline phosphatase activity following completion of the logarithmic growth phase, and on resultant bone nodule formation and mineralization by these populations. The effects of orthovanadate on osteogenic cultures were concentration dependent: 5 microM concentrations produced by a relatively large quantity of poorly mineralized matrix, while 30-50 microM concentrations produced a smaller quantity of heavily mineralized matrix. Thus, orthovanadate could possibly be used as a growth factor for bone, if administered at the critical dosage at the proper stage of the life cycle of the osteoblast.
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PMID:Enhancement by sodium orthovanadate of the formation and mineralization of bone nodules by chick osteoblasts in vitro. 922 45

Here we show that brain-derived neurotrophic factor (BDNF) stimulates both the phosphorylation of the Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) and its kinase activity in rat hippocampal slices. In addition, we find that: (i) the time course of BDNF action is not accompanied by a change in the spectrum of either alpha- and beta-subunits of CaMK2 detected by immunoblotting; (ii) both treatment of solubilized CaMK2 with alkaline phosphatase and treatment of immunoprecipitated CaMK2 with protein phosphatase 1 reverse phosphorylation and activation of the kinase; (iii) phospholipase C inhibitor D609 and intracellular Ca2+ chelation by 1,2-bis-(o-aminophenoxy)ethane-N,N,N",N',-tetracetic acid tetra(acetoxymethyl)ester or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate but not omission of Ca2+ or Ca2+ chelation by EGTA, abolish the stimulatory effect of BDNF on phosphorylation and activation of CaMK2. These results strongly suggest that the conversion of CaMK2 into its active, autophosphorylated form, but not its concentration, is increased by BDNF via stimulation of phospholipase C and subsequent intracellular Ca2+ mobilization.
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PMID:Brain-derived neurotrophic factor increases Ca2+/calmodulin-dependent protein kinase 2 activity in hippocampus. 930 59

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5'-[gamma-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.
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PMID:Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. 937

We have found that modification of rat PC12 cells with pertussis toxin resulted in an approximately 50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca2+, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.
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PMID:Pertussis toxin modification of PC12 cells inhibits a protein phosphatase 2A-like phosphatase. 964 72


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