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 phosphonate hexasodium N,N,N',N'-ethylenediamine-tetramethylenephosphonate (EDITEMP.Na6) reduced alkaline phosphatase (Alp) activity and cAMP response to parathyroid hormone (PTH) in primary cultures of foetal rat calvaria cells in a dose-dependent manner, while not affecting culture DNA content. EDITEMP.Na6 also inhibited the mineralization of three-dimensional bone nodules formed in vitro, but not the number of nodules formed. Bone cell culture DNA content was also reduced by EDITEMP.Na6 but at concentrations in excess of those needed to modulate osteoblastic cell function. Withdrawal of EDITEMP.Na6 led to slow but complete recovery of Alp activity. At EDITEMP.Na6 concentrations of 25 microM and higher, recovery of Alp activity appeared to be independent of protein and/or DNA synthesis. Cell culture acid phosphatase (Acp) activity was not affected by EDITEMP.Na6. The results indicate that EDITEMP.Na6 has a direct inhibitory effect on (mature) osteoblastic cell function. In the presence of bone tissue this inhibition also occurred, although not at a relatively low dose level.
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PMID:Direct modulatory effect of hexasodium N,N,N',N'-ethylenediamine-tetramethylene-phosphonate on bone cell function in vitro. 215 36

Some of the effects of nerve growth factor (NGF) may be mediated by changes in protein phosphorylation. We have identified a protein kinase from PC-12 cells that catalyzes the phosphorylation of pig brain microtubule-associated protein (MAP)-2 in vitro. This activity is stimulated 2-4-fold in extracts from cells treated with NGF or epidermal growth factor (EGF). The partial purification and characterization of this MAP kinase indicate that it is distinct from previously described NGF-stimulated protein kinases. The NGF-stimulated kinase activity is unaffected by direct addition to the assay of the heat-stable cAMP-dependent kinase peptide inhibitor, staurosporine, or K-252A, is slightly stimulated by heparin and is inhibited by sodium fluoride and calcium ions. Treatment of cells with NGF increases the activity of the kinase within 2 min. The activity declines after 10 min, and a second phase of activation is observed at 20-30 min. Comparison of its behavior on gel permeation and sucrose density gradients indicates a molecular mass in the range of 40,000 daltons. The kinase activity is specific for ATP as substrate with a Km of 12 microM. Although the pathway of activation of MAP kinase by NGF is unknown, the stimulation can be reversed by treatment of the enzyme with alkaline phosphatase, suggesting that activation involves phosphorylation of the kinase itself. The properties and hormone sensitivity of the PC-12 MAP kinase suggest that it is similar to the previously identified, growth factor-sensitive MAP kinase from 3T3-L1 adipocytes.
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PMID:Nerve growth factor stimulates a protein kinase in PC-12 cells that phosphorylates microtubule-associated protein-2. 215 37

The effect of alkaline phosphatase (3.1.3.1) on desensitization of beta-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of beta-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30 degrees C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the Gs-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.
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PMID:Difference in sensitivity to alkaline phosphatase treatment between rat reticulocyte membranes in which beta-adrenoceptor desensitization was induced by isoproterenol, dibutyryl cAMP and phorbol ester. 216 74

The pregnenolone-binding protein (PBP) in guinea pig adrenocortical cytosol is inactivated (converted to a nonsteroid-binding form) by incubation with calf intestinal alkaline phosphatase at pH 9. Previously bound pregnenolone does not prevent this inactivation, and dephosphorylation causes dissociation of bound ligand from the protein. Cytosolic PBP, partially purified PBP, and highly purified PBP are equally susceptible to alkaline phosphatase-mediated inactivation. No change in apparent molecular weight or immunoreactivity is evident by Western blot analysis. Loss of pregnenolone-binding capacity of cytosolic PBP (but not partially purified PBP) could be reversed by inhibiting the phosphatase, lowering the pH to approximately 7, and adding ATP to the incubation. Reactivation is absolutely and specifically dependent upon ATP, which restores binding capacity in a concentration-dependent manner. Other nucleoside triphosphates, including the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate, as well as cAMP and cGMP are ineffectual as cofactors for reactivation. These data strongly implicate a cytosolic kinase which is apparently inactivated or separated from PBP during purification. Preliminary investigations indicate that the reactivating kinase is not cAMP-dependent, but may have a requirement for calcium and/or calmodulin. The identification of phosphorylation/dephosphorylation as the regulatory mechanism for steroid binding should prove pivital in elucidating the functional role of PBP.
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PMID:Regulation of adrenocortical pregnenolone-binding protein activity by phosphorylation/dephosphorylation. Phosphatase-mediated inactivation is reversed by cytosolic kinase. 216 58

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.
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PMID:Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase. 216 96

We investigated the differentiation-inducing effects of dibutyryl cyclic AMP (dBc AMP) on several cultured osteosarcoma cell lines (DUNN, MOLONEY, OST, FBJ). 1. Cell growth rates of all the osteosarcoma cell lines were reduced by 3mM dBc AMP. 2. Both alkaline phosphatase activity and 45Ca2(+)-uptake were promoted by 3mM dBc AMP. 3. There was, in each cell line except for the OST cells, a marked enlargement of the cell processes under the light microscopic observation. At the electron microscopic level, there were also many findings indicating an increase in cell functions. These results suggest that the differentiation may be induced by cAMP in osteosarcoma cells, and that the differentiation therapy by cAMP-related drugs is promising for a clinical application.
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PMID:[Experimental study on maturational therapy of osteosarcoma cell]. 216 18

We have examined the influence of parathyroid hormone (PTH) and 1,25(OH)2 vitamin D (1,25(OH)2D) on cytoskeletal assembly and biosynthesis in relation with cAMP production and parameters of cell growth and differentiation in normal human osteoblastic cells. Untreated human bone cells showed elongated morphology associated with high levels of actin, vimentin, alpha- and beta-tubulins and alpha-actinin as determined by 2-dimensional-gel electrophoresis and [35S]methionine labelling of cytoskeletal proteins. PTH (20 nM, 24 h) decreased the de novo biosynthesis of vimentin and alpha-actinin in human bone cells, an effect associated with a rise in intracellular cyclic AMP. In addition, PTH induced cytoskeletal disassembly as shown by a 52-70% decrease in the Triton-insoluble fractions of actin, alpha-tubulins and alpha-actinin. 1,25(OH)2D (10 nM, 24 h) also induced a 40-64% decrease in the polymerized fractions of actin, alpha-tubulins and alpha-actinin. These changes were associated with an 83% increase in osteocalcin production. Under these conditions, neither PTH nor 1,25(OH)2D at the doses tested affected alkaline phosphatase activity or cell growth as assessed by [3H]thymidine incorporation into DNA. The results show that PTH and 1,25(OH)2D induce similar inhibition of cytoskeletal proteins assembly involving microfilaments and microtubules in human osteoblastic cells. These alterations of cytoskeletal arrangement in response to PTH and 1,25(OH)2D may contribute to the functional response of human osteoblastic cells to these bone-resorbing hormones.
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PMID:Changes in cytoskeletal proteins in response to parathyroid hormone and 1,25-dihydroxyvitamin D in human osteoblastic cells. 216 75

A number of studies have shown membrane phospholipid metabolism to have an important role in biological mineralization. We considered the effects of exogenously applied phosphatidic acid (PA), a minor component of membrane phospholipids, on an osteoblast-like cell line, MOB 3-4. Exogenous PA (10-40 micrograms/ml) raised the level of cytoplasmic free Ca2+ [( Ca2+]i), independent of the level of extracellular Ca2+, in a dose-dependent fashion, and this Ca2+ response to PA gradually fell on serial application of PA. In a dose-dependent manner, exogenous PA also increased inositol 1,4,5-trisphosphate (IP3) accumulation and cytoplasmic pH, but decreased basal cAMP level. This cytoplasmic alkalinization was inhibited by pretreatment with nonspecific protein kinase C (PKC) inhibitors, such as sphingosine or H-7. A long-term incubation with PA increased alkaline phosphatase (ALP) activity and cell proliferation. Exogenous PA thus appeared to increase IP3 accumulation by activating phospholipase C, raise [Ca2+] by releasing Ca2+ from intracellular stores, induce cytoplasmic alkalinization via a PKC-dependent mechanism, and simultaneously decrease basal cAMP level. We suggest that these initial responses may be responsible for the increase in ALP activity and the proliferation of PA-treated MOB 3-4 cells.
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PMID:Initial responses of a clonal osteoblast-like cell line, MOB 3-4, to phosphatidic acid in vitro. 216 76

A human osteosarcoma cell line, HuO9, was established from a tumor that was heterotransplanted into athymic nude mice. Antiserum against nude mouse spleen cells was added to the early passage cultures to eliminate the host fibroblastic cells. The cell line retained a high activity of liver/bone/kidney-type alkaline phosphatase (ALP) and secreted osteocalcin, i.e., bone gamma-carboxyglutamic acid-containing protein (BGP), into the medium. The addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increased the ALP activity as well as the level of BGP secreted into the medium. The ALP of 1,25(OH)2D3-treated cells has the same inhibition characteristics to heat and amino acids as that of untreated cells. Synthetic human parathyroid hormone stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) approximately 100-fold within five minutes. However, the stimulation was not observed with a synthetic human thyrocalcitonin. When HuO9 cells were transplanted into the back of a nude mouse, a tumor with an abundant osteoid formation and mineralization was produced. The results indicate that the HuO9 cell line expresses well-differentiated osteoblastic phenotypes. HuO9 is the first established human cell line to produce BGP, and it provides a useful model for the studies of osteoblasts and the regulatory mechanisms of BGP production.
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PMID:A newly established human osteosarcoma cell line with osteoblastic properties. 217 70

Bone cells derived from human trabecular explants display osteoblastic features. We examined the modulation of alkaline phosphatase activity and cAMP production as the result of exposing trabecular explants to physiologic concentrations of dexamethasone for 4 weeks during cellular outgrowth and subculture. Cells treated with dexamethasone were observed to grow generally more slowly than control cells. Cells appeared larger and more polygonal, and staining for alkaline phosphatase was more intense in the dexamethasone-exposed cultures. There was a progressive increase in cellular PTH responsiveness with increasing duration of exposure of cells to dexamethasone. Cells grown for 6 weeks in 3 x 10(-8) M dexamethasone had a 10-fold increase in PTH-stimulated cyclic AMP accumulation. Dexamethasone-treated cells also had a significantly increased alkaline phosphatase activity. 1,25-(OH)2D3-stimulated alkaline phosphatase activity was increased approximately 20-fold. cAMP responses were significantly increased to PTH (21.7-fold), PGE1 (2.67-fold), and forskolin (4.81-fold), but not to cholera toxin. Dexamethasone-treated cells also had a mean decrease in 1,25-(OH)2D3-stimulated osteocalcin production to 26.2% of control values (p less than 0.001). Hydrocortisone treatment gave rise to similar effects but of smaller magnitude than those of dexamethasone. Testosterone did not have a significant effect on alkaline phosphatase activity or cAMP production. Skin fibroblasts showed a significant enhancement of alkaline phosphatase activity in response to dexamethasone, but of a much smaller magnitude than in bone cells. The phenotypic changes induced by long-term culture in dexamethasone are consistent with the promotion of a more differentiated osteoblastic phenotype.
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PMID:Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells. 217 56


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