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)

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.
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PMID:Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase. 1205 14

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.
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PMID:Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro. 1223 93

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.
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PMID:Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock. 1236 74

Lymphocyte stimulation by immunoreceptors is achieved through the activation of multiple signaling pathways leading to cytokine gene transcription. Adapter proteins are critical signaling components that can integrate multiple pathways by allowing the assembly of multimolecular signaling complexes. We previously showed that the cytoplasmic adapter 3BP2 (also known as SH3BP2) promotes NFAT/AP-1 transcriptional activities in T cells through the activation of Ras- and calcineurin-dependent pathways. However, the molecular mechanisms by which 3BP2/SH3BP2 regulates cell signaling and activation remain poorly documented. In this study, using a combination of yeast two-hybrid analysis and biochemical approaches, we present evidence for a physical interaction between 3BP2 and the chaperone protein 14-3-3. This interaction was direct and constitutively detected in yeast and in mammalian cells. Phorbol ester, pervanadate, and forskolin/isobutylmethylxanthine stimulations enhanced this interaction, as well as co-expression of constitutive active mutants of serine/threonine kinases, including protein kinase C. We found that dephosphorylation of 3BP2 by alkaline phosphatase disrupted its interaction with 14-3-3 and that 3BP2 was a substrate of purified protein kinase C in vitro, suggesting that the phosphorylation of 3BP2 by upstream kinases was required for 14-3-3 binding. Using deletion mutants of 3BP2, two 14-3-3 binding domains were mapped to two proline-rich (residues 201-240 and 270-310) domains of 3BP2. These domains were shown to contain two 14-3-3 consensus binding motifs. We identified residues Ser(225) and Ser(277) of 3BP2 as being essential for interaction with 14-3-3 family proteins, optimal 3BP2 serine phosphorylation, and then for 3BP2-dependent function. Indeed, a 3BP2 mutant protein incapable of binding 14-3-3 showed increased capacity to stimulate NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2 adapter function in lymphocytes.
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PMID:The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function. 1250 Dec 43

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.
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PMID:Zinc inhibits calcineurin activity in vitro by competing with nickel. 1284 81

Neale, S. (Department of Botany, University College, London, England), and H. Tristram. Effect of O-methyl-dl-threonine and O-methyl-dl-serine on growth and protein synthesis in Escherichia coli. J. Bacteriol. 86:1241-1250. 1963.-Addition of either O-methyl-dl-threonine or O-methyl-dl-serine to exponentially growing cultures of Escherichia coli resulted in "linear" increases in optical density. The total cell count, however, remained constant, the increase in optical density being accompanied by a marked increase in cell length. In the presence of O-methyl-dl-serine, a phase of "linear" growth was followed by exponential growth, which was maintained during a second passage through analogue-containing medium but not after a subsequent passage through normal medium, suggesting phenotypic adaptation to the analogue. The differential rate of incorporation of amino acids into trichloroacetic acid-insoluble material was unaffected by growth in the presence of either O-methyl-dl-threonine or O-methyl-dl-serine. Neither analogue was incorporated into E. coli protein. The effect of the analogues on the production of alkaline phosphatase and beta-galactosidase was examined. The precise point and mode of action of the analogues have not been determined, but available evidence suggests that the growth-inhibitory effects of both substances are due to interference with the biosynthesis of threonine and methionine.
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PMID:EFFECT OF O-METHYL-DL-THREONINE AND O-METHYL-DL-SERINE ON GROWTH AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI. 1408 96

Increased expression of N-Myc and expression of the high mobility group protein A1a (HMGA1a) were observed in the nuclei of SK-N-SH cells following exposure to hypoxia. These observations were accompanied by the appearance of additional high molecular weight bands, which were eliminated by pretreatment with alkaline phosphatase. Immunoprecipitation showed phosphorylation of serine, threonine and tyrosine residues of N-Myc in the nucleus. These results suggest that hypoxia-induced signals in SK-N-SH cells lead to persistent expression of HMGA1a, which may induce expression of the transcription factor N-Myc, and that phosphorylation at serine, threonine and tyrosine residues of N-Myc occurs at an early stage after stimulation. Such signal consolidation processes could play a role in neuronal survival after hypoxia in neurons.
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PMID:Possible involvement of the expression and phosphorylation of N-Myc in the induction of HMGA1a by hypoxia in the human neuroblastoma cell line. 1563 95

Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.
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PMID:Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. 1580 71

High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.
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PMID:Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function. 1594 59

The aim of this work was to test whether growth factors such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) undergo autophosphorylation and whether this affects their biological activity. Incubation of those growth factors with [gamma-(32)P]ATP resulted in phosphorylation in vitro. The phosphate bond was resistant to alkaline pH, yet acid-labile. Addition of alkaline phosphatase resulted in time and protein dependent dephosphorylation. Concomitantly, alkaline phosphatase abolished the neuroprotective effect of those growth factors upon oxygen and glucose deprivation and upon staurosporine-induced cell death. For those studies, we were using primary cultures of cortical and hippocampal neurons from embryonic and neonatal rats. Incubation of bFGF with non-hydrolyzable ATP-gammaS resulted in phosphorylation and in neuroprotection resistant to alkaline phosphatase. We conclude that bFGF, NGF and BDNF undergo autophosphorylation on site(s) other than serine, threonine, tyrosine and/or ATP-binding, and that this binding of phosphate is essential for neuroprotection in vivo.
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PMID:Phosphorylation of the growth factors bFGF, NGF and BDNF: a prerequisite for their biological activity. 1624 15


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